INTRODUCTION

espiratory diseases represent one of the largest health problems word wide. Diseases such as asthma and the smoking related diseases are already common and increasing so we urgently need better approaches to treat or cure these diseases. At the same time, new respiratory diseases such as those associated with viruses threaten pandemics that challenge our national health systems. With these continued challenges for new treatment with better patient care, clinical and respiratory researchers have sought better approaches to all aspects of patient care from improved diagnoses to superior therapies. This has lead to an explosion of new research with an increasingly better understanding of how to diagnose diseases and then develop new therapies. Thus, for example ever improving technologies for imaging lung disease have lead to increasingly better diagnoses, although challenges remain as we seek to further improve resolution. At the same time, the revolution in molecular biology, culminating with the publication of the complete human genome, has lead to hopes for nding more precise clues to disease susceptibility pathogenesis in genetic analysis. This is leading to new concepts in pharmacogenomics as we start to use new drugs, including those used for lung cancers, being directed at mutations associated with disease. This is the rst Encyclopaedia of Respiratory Medicine. It is our hope that it is comprehensive and captures the key aspects of current patient care, as well as the exciting developments in respiratory science that we all believe will eventually lead to better patient care in the twenty rst century. This encyclopaedia is comprehensive in scope and provides clinician and researcher with a snap shot of the current state of knowledge in respiratory medicine. All entries have adhered to a structured layout, starting with an abstract crystallizing the key facts and nishing with reading lists for those who want to delve further into the subject. In addition, most entries have a colour diagram designed to help understanding and provide a valuable aid for undergraduate and post-graduate teaching. These are exciting times for respiratory medicine. We hope this encyclopedia will become a valuble tool for clinicians and researches at all stages of their careers from those beginning their carreers to those established but wanting to update themselves on the new developments. Finally, we would like to thank our Advisory Editorial Board who helped so much in shaping the contents of this works, as well as the authors who wrote the articles and faced the challenge of condensing areas of respiratory medicine, often the subject of entire textbooks, into a short article of 4000 words or less.

GEOFFREY J. LAURENT STEVEN D. SHAPIRO

FOREWORD

Animals live by two principal things, food and breath. Of these, by far the most important is the respiration, for if it is stopped, the man will not endure long, but immediately dies. Aretaeus the Cappocian (150200 AD)

f course, not all medical specialists would agree with this statement, and those who disagree would be quick to posit that it is the failure of their particular organ that tends to cause immediate death. However, that is not the issue. The point of this quotation is to illustrate that the proper functioning of the lung has been a subject of great interest for centuries. The Greek physician Aretaeus devoted many of his observations to diabetes, but his manuscript On the Causes and Indications of Acute and Chronic Diseases also discussed lung diseases, such as pneumonia. Since his time, great numbers of physicians from all continents and cultures have contributed to our knowledge of respiratory diseases. While acknowledging our rich history of discoveries about pulmonary and respiratory medicine discoveries that were made by men and women whose names symbolize the great journey of this specialtyone must concede that the eld experienced an extraordinary growth spurt beginning in the 1940s. Knowledge of respiratory physiology, which developed very fast during World War II, created a tidal wave of interest that continued for years afterward. The ability to measure and understand respiratory physiology and its alterations became a diagnostic tool, and it opened the door to therapeutic or respiratory support procedures. But, then, in the 1950s and 1960s cell biology and subcellular research entered the scene. The potential of molecular biology and genetics was quickly recognized, and respiratory medicine appreciated that a better understanding of normal and disordered biological respiratory processes hinged on use of these new approaches. Lung and respiratory researchers, impelled in part by the ever-increasing public health burdens of respiratory diseases, seized the opportunity. The stage was set for progress to occur. The architects of this revolution in respiratory medicine are well known; it is our good fortune that many have contributed to these four volumes. Four volumes! y Encyclopedia! y Indeed, these four volumes truly constitute an encyclopedia of pulmonary biology and respiratory medicine! Respiratory medicine is still growing. Because it is such a dynamic and exciting eld, new investigators will almost surely want to be part of it. However, to do so they will need to know about the established state of knowledge that will be the basis of their work. New investigators in the science of respiratory medicine, whether interested in fundamental research or clinical research or application, will nd ideas and inspiration in these volumes. All of the tools of the trade are assembled therein. As noted, respiratory medicine has been a progressive and expanding eld but, as is the case with many elds of medicine, the transfer of what we know to the general practice of medicine has been slow and limited. Translation, as it is called, is an emerging discipline in need of assistance; fortunately, the breadth of the knowledge presented in these volumes provides tools to facilitate this translation process. This four-volume encyclopedia is, at once, both a tribute to the centuries of pioneering investigations in the eld of respiratory medicine and a foundation for even greater accomplishments in the future. The presentation of all this knowledge in these excellent and comprehensive volumes can only serve to stimulate further work of equal or surpassing significance. The editors and the authors are to be commended for their contributions to this singular effort. Because of their work, respiratory science and medicine will advance faster and patients worldwide will be the beneciaries. Claude Lenfant, MD Gaithersburg, Maryland

Notes on the Subject Index

To save space in the index, the following abbreviations have been used: ALI ARDS BAL BPD CAP CFTR COP COPD CWP G-CSF GERD GM-CSF HUVS IL IPF IPH MCP M-CSF MIP MMP NSCLC PPAR SCLC SP TGF TIMP TNF VEGF acute lung injury acute respiratory distress syndrome bronchoalveolar lavage bronchopulmonary dysplasia community-acquired pneumonia cystic fibrosis transporter regulation cryptogenic organizing pneumonia chronic obstructive pulmonary disease coal workers pneumoconiosis granulocyte colony-stimulating factor gastroesophageal reflux disease granulocyte-macrophage colony-stimulating factor hypocomplementemic urticarial vasculitis syndrome interleukin idiopathic pulmonary fibrosis idiopathic pulmonary hemosiderosis monocyte chemoattractant protein macrophage colony-stimulating factor macrophage inflammatory protein matrix metalloproteinase non-small cell lung carcinoma peroxisome proliferator-activated receptor small-cell lung carcinoma surfactant protein transforming growth factor tissue inhibitor of metalloproteinases tumor necrosis factor vascular endothelial growth factor

Editorial Advisory Board

Kenneth B. Adler, North Carolina State University, Raleigh, NC, USA Peter J. Barnes, Imperial College London, UK Paul Borm, Zuyd University, Heerlen, The Netherlands Arnold R. Brody, Tulane Medical School, New Orleans, LA, USA Rachel C. Chambers, University College London, UK Augustine M. K. Choi, University of Pittsburgh, PA, USA Jack A. Elias, Yale University School of Medicine, New Haven, CT, USA Patricia W. Finn, University of California San Diego, La Jolla, CA, USA Stephen T. Holgate, University of Southampton, Southampton, UK Steven Idell, The University of Texas Health Center at Tyler, TX, USA Sebastian L. Johnston, National Heart and Lung Institute, Imperial college London, UK Talmadge E. King, Jr, University of California, San Francisco, CA, USA Stella Kourembanas, Childrens Hospital Boston, Harvard Medical School, Boston, MA, USA Y. C. Gary Lee, University College London, UK Richard Marshall, University College London, UK Sadis Matalon, University of Alabama, Birmingham, AL, USA Joel Moss, National Institutes of Health, Bethesda, MD, USA William C. Parks, University of Washington, Seattle, WA, USA Charles G. Plopper, University of California, Davis, CA, USA Bruce W. S. Robinson, The University of Western Australia, Nedlands, Australia Neil Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA Edwin K. Silverman, Brigham and Womens Hospital Boston, MA, USA Eric S. Silverman, Brigham and Womens Hospital, Boston, MA, USA Peter Sly, Institute for Child Health Research, West Perth, Australia Kingman Strohl, Case Western Reserve University, Cleveland, OH, USA Teresa D. Tetley, Imperial College London, UK John B. West, University of California, San Diego, CA, USA

Editors
Geoffrey J Laurent, Royal Free and University College Medical School, London, UK Steven D Shapiro, Brigham and Womans Hospital, Boston, USA

Dedication
To my family, Lal, Guy, David and Gabrielle (GJL). My contribution to this work would not have been possible without the love and support from my wife Nicole and my daughters Calli, Tess, Skylar, and Ellery. I also thank my mentors and trainees for my continual education and the Division of Pulmonary and Critical Care Medicine at Brigham and Womens Hospital who took care of our patients allowing me the time to undertake this project (SDS)

Permission Acknowledgments
The following material is reproduced with kind permission of Lippincott Williams and Wilkins Figure 4 and 8 of ARTERIAL BLOOD GASES Table 1 of ARTERIES AND VEINS Figure 2 and 3 of BREATHING | Breathing in the Newborn Figure 2a, 2b, 3, 4 and 5 of DRUG-INDUCED PULMONARY DISEASE Table 1, 2 and 3 of DRUG-INDUCED PULMONARY DISEASE Figure 2 of ENVIRONMENTAL POLLUTANTS | Diesel exhaust particles Figure 4 of EXERCISE PHYSIOLOGY Figure 2 of FLUID BALANCE IN THE LUNG Figure 2 of GASTROESOPHAGEAL REFLUX Figure 2 of GENE REGULATION Figure 2 of HIGH ALTITUDE, PHYSIOLOGY AND DISEASES Figure 2 and 3 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Table 1 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Figure 1, 2 and 3 of OXYGEN-HEMOGLOBIN DISSOCIATION CURVE Figure 10a, 10b and 11a of SYSTEMIC DISEASE | Eosinophilic Lung Diseases http://www.lww.com

The following material is reproduced with kind permission of Nature Publishing Group Figure 2 of COAGULATION CASCADE | iuPA, tPA, uPAR Figure 1 of COAGULATION CASCADE | Tissue Factor Figure 1a of MATRIX METALLOPROTEINASES Figure 1 of MYOFIBROBLASTS

Figure 1 of VESICULAR TRAFFICKING http://www.nature.com/nature and http://www.nature.com/reviews

The following material is reproduced with kind permission of Taylor & Francis Ltd Figure 2 of AUTOANTIBODIES Table 1 of BASAL CELLS Figure 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Table 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Figure 1 and 2 of SURFACANT | Overview Tables 1, 2, 3 and 4 of SURFACANT | Overview http://www.tandf.co.uk/journals

A
ACETYLCHOLINE
J Zaagsma and H Meurs, University of Groningen, Groningen, The Netherlands
& 2006 Elsevier Ltd. All rights reserved.

Abstract
In the airways, acetylcholine is a neurotransmitter in parasympathetic ganglia and in postganglionic parasympathetic nerves, as well as a nonneural paracrine mediator in various cells in the airway wall. Ganglionic transmission by acetylcholine is mediated by nicotinic receptors, which are ligand-gated in channels, whereas postganglionic transmission is through G-protein-coupled muscarinic receptors. Of the ve mammalian muscarinic receptor subtypes, mainly M1, M2, and M3 receptors are involved in airway functions. Gq-coupled M1 receptors facilitate ganglionic transmission mediated by nicotinic receptors and modulate surfactant production and uid resorption in the alveoli. Prejunctional Gi/o-coupled M2 receptors in parasympathetic nerve terminals attenuate acetylcholine release upon nerve stimulation. M2 receptors are also abundantly present in airway smooth muscle; however, the major function of these postjunctional M2 receptors is unknown. Postjunctional Gq-coupled M3 receptors mediate airway smooth muscle contraction and mucus secretion. Dysfunction of the prejunctional M2 autoreceptor induced by allergic airway inammation has been implied in exaggerated vagal reex activity and airway hyperresponsiveness in asthma. Inammation-induced increased M3 receptor stimulation may be involved in airway remodeling in chronic asthma. Possible mechanisms include potentiation of growth factor-induced proliferation of airway smooth muscle cells and induction of a contractile phenotype of these cells. Exaggerated M3 receptor stimulation may also cause reduced responsiveness to b2-adrenoceptor agonists by transductional cross-talk between phosphoinositide metabolism and adenylyl cyclase, which involves protein kinase C-induced uncoupling of the b2adrenoceptor from the effector system. Muscarinic receptor antagonists have been shown to be effective in airway diseases like asthma and, especially, chronic obstructive pulmonary disease. Of these, tiotropium bromide is particularly useful, due to its long duration of action as well as its kinetic selectivity for the M3 receptor.

vagosympathetic trunk; when applied to a second, unstimulated heart, the perfusate slowed its rate, resembling the effect of vagus stimulation. In 1926 Loewi provided evidence for identication of Vagusstoff as acetylcholine. Acetylcholine is the neurotransmitter of all sympathetic and parasympathetic autonomic ganglia and of the postganglionic parasympathetic nerves. In the airways, the parasympathetic ganglia are located near or within the airway wall. Ganglionic transmission mediated by acetylcholine is through nicotinic receptors which belong to the family of ligand-gated ion channels. Postganglionic transmission by acetylcholine, released from parasympathetic nerve terminals, is through muscarinic receptors of which ve different subtypes have been identied, all being G-protein-coupled receptors. During periods of airway inammation vagal release of acetylcholine may be increased by various mechanisms. Hence, both in asthma and (particularly) in chronic obstructive pulmonary disease (COPD) blockade of postjunctional muscarinic receptors is the key to reversing airway obstructions.

Synthesis, Storage, and Release

Acetylcholine is synthesized from choline and acetylcoenzyme A (acetyl-CoA) in the cytoplasm of the nerve terminal through the enzyme choline acetyltransferase (ChAT). Choline is taken up by the nerve terminal from the extracellular uid through a sodium-dependent carrier; this transport is the ratelimiting process in acetylcholine synthesis. Acetyl-CoA is synthesized in mitochondria which are abundantly present in the nerve endings. Most of the synthesized acetylcholine is actively transported from the cytosol into synaptic vesicles by a specic transporter; this vesicular (quantal) package of acetylcholine reaches up to 50 000 molecules per vesicle. Release of acetylcholine is initiated by inux of Ca2 ions through voltage-operated N- or P-type calcium channels. The increased intracellular Ca2 ions bind to a vesicle-associated protein (synaptotagmin) which favors association of a second vesicle protein (synaptobrevin) with one or more proteins in the plasma membrane of the nerve terminal. Following

Introduction
Acetylcholine is a neurotransmitter in the central and peripheral nervous system where it plays a major role in the afferent neurons of both the autonomic and somatic (voluntary) branches. As a chemical transmitter, it has been identied as Vagusstoff in 1921 by Otto Loewi showing its release from an isolated frog heart following stimulation of the

2 ACETYLCHOLINE

this vesicle-docking process, fusion between vesicle membrane and plasma membrane occurs, followed by exocytosis. After the expulsion of acetylcholine the empty vesicle is recaptured by endocytosis and can be reused. In the synaptic cleft, the released acetylcholine will associate with post- and prejunctional receptors and is also subject to rapid hydrolysis by the enzyme acetylcholinesterase into choline and acetate. Over 50% of the choline formed will be taken up again by the nerve terminal and reused for neurotransmitter synthesis. Acetylcholine is also present in nonneuronal cells. In recent years it has become clear that in the airways the majority of cells express ChAT and contain acetylcholine, including epithelial cells, smooth muscle cells, mast cells, and migrated immune cells such as alveolar macrophages, granulocytes, and lymphocytes. However, the regulatory role of this nonneuronal acetylcholine in inammatory airways diseases has yet to be established.

Regulation of Synaptic Transmission and Activity

Ganglionic

Preganglionic nerves innervating the parasympathetic ganglia in the airways evoke action potentials during normal breathing with relatively high frequencies, in the range of 120 Hz. As a result, basal airway smooth muscle tone in vivo is mediated to a significant extent by cholinergic nerve activity. The pattern of ganglionic action potential bursts coincides with respiration, suggesting that the respiratory centers in the brainstem govern preganglionic nerve activity. However, in addition to this central drive, reex stimulation through mechanically sensitive afferent nerve terminals in the lungs during respiration is importantly involved as well. The delity by which preganglionic impulses are translated into action potentials in the postganglionic neurons is relatively low in parasympathetic airway ganglia, implying a ltering function of these ganglia. This ltering function can be diminished by various inammatory mediators. Thus, histamine, prostaglandin D2 (PGD2), and bradykinin are able to enhance ganglionic cholinergic transmission and the same is true for tachykinins (substance P, neurokinin A) released by nonmyelinated sensory C-bers in the airways.
Postganglionic

muscarinic M2 receptors, activated by acetylcholine itself, represent an important negative feedback, limiting further release, at higher ring rates in particular. In animal models of allergic airway inammation and asthma as well as in human asthma, dysfunction of these M2 autoreceptors has been found to contribute to exaggerated acetylcholine release from vagal nerve endings, to increased cholinergic reex activity in response to inhaled stimuli, and to contribute to airway hyperresponsiveness. Most of this receptor dysfunction is thought to be caused by activated eosinophils that migrate to cholinergic nerves and release major basic protein (MBP) which acts as an allosteric antagonist of muscarinic M2 receptors. Since eosinophilic inammation is far less prominent in COPD and since M2 autoreceptors are more prominent in larger airways, it is no surprise that these receptors are still functional in patients with stable COPD; however, this does not exclude a dysfunction during acute exacerbations. In addition to M2 autoreceptors, a variety of heteroreceptors modulating acetylcholine release have been identied on cholinergic nerve endings. Catecholamines may inhibit or facilitate acetylcholine overow through prejunctional a2- and b2-adrenoceptors, respectively. Neurokinins like substance P may enhance cholinergic transmission through facilitatory neurokinin 1 (NK1) and/or 2 (NK2) receptors. Interestingly, substance P may also induce MBP release from eosinophils, causing M2 receptor dysfunction, which could act synergistically to direct facilitation. Allergic inammation-derived prostanoids, including PGD2, PGF2a, and thromboxane A2, as well as histamine, can also augment acetylcholine release through prejunctional receptors. Taken together, the above observations indicate that parasympathetic acetylcholine release is governed by various regulatory systems, the set-point of which is subject to environmental modulations. During periods of airway inammation these modulations often result in enhanced cholinergic transmission.

Receptors and Biological Function

In the ganglia, acetylcholine interacts with nicotinic receptors. These receptors consist of ve polypeptide subunits, together forming a cylindric structure of about 8 nm diameter, which acts as an ion channel. Each subunit passes the membrane four times, so the central pore is surrounded by 20 membranespanning helices. The subunits have been subdivided into ve classes, designated a, b, g, d, and e. Of the a and b subunits, 10 and 4 different subtypes have been identied, respectively. Peripheral ganglionic

The release of acetylcholine from parasympathetic nerve terminals is regulated by a variety of prejunctional receptors, which may inhibit or facilitate transmitter outow. In the airways, autoinhibitory

ACETYLCHOLINE 3

receptors consist of only a and b subunits, the main subtype being (a3)2(b4)3. Each a subunit possesses a binding site for acetylcholine; they need to be occupied both to induce channel opening, which will enhance Na and K permeability. This results in an inward ux of mainly Na ions, causing depolarization and action potential generation in the postganglionic cell (provided the acetylcholine concentration is high enough). Acetylcholine released by postganglionic parasympathetic nerves may choose between ve muscarinic receptor subtypes, designated M1 to M5, to interact with. Most organs and tissues express more than one subtype and this is true for many individual cells as well. The ve subtypes can be subdivided into two main classes, the odd-numbered receptors (M1, M3, M5), which couple preferentially to heterotrimeric Gqproteins, and the even-numbered (M2, M4) receptors which show selectivity for Gi/o type of G-proteins. The principal signaling route of Gq-coupled receptors is the activation of phospholipase C, mediating hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (Figure 1, left). IP3 mobilizes Ca2 ions from intracellular stores, which generates a rapid and transient rise of the free Ca2 concentration in the cytosol. DAG triggers the translocation and activation of protein kinase C (PKC) which is able to phosphorylate a variety of different protein substrates. The main signal transduction of Gi/o-coupled receptors is to inhibit adenylyl cyclase activity, which reduces the intracellular cyclic AMP concentration.

In airway smooth muscle, activation of Gi/o by M2 receptors may also diminish opening of calcium-activated potassium channels (KCa or maxi-K channels) induced by (Gs-coupled) b-adrenoceptors. Thus, K efux, membrane hyperpolarization, and subsequent smooth muscle relaxation, initiated by b-agonist administration, is under restraint of acetylcholine through this mechanism. In the airways of most mammalian species, including human, M1, M2, and M3 receptors are the most important ones. So far, M4 receptors have only been detected convincingly in bronchiolar smooth muscle and alveolar walls of the rabbit, whereas muscarinic M5 receptors, now known to mediate dilatation of cerebral arteries and arterioles, are absent in the lungs. M1 receptors have been found in alveolar walls, parasympathetic ganglia, and submucosal glands. Rat and guinea pig lung studies have indicated their presence in type II alveolar cells, mediating surfactant production and uid reabsorption, respectively. In parasympathetic airway ganglia of several species, including human, M1 receptor stimulation is able to facilitate ganglionic transmission mediated by nicotinic receptors. Thus, vagal bronchoconstriction, induced by inhalation of SO2, has been found especially sensitive to inhaled pirenzepine, a M1-selective antagonist. In submucosal glands, M1 receptors are not involved in mucus secretion, which appears to be mediated solely by M3 receptors. M2 and M3 receptors represent the major receptor populations, both in intra- and extrapulmonary
Epinephrine, 2-agonists

Acetylcholine

M3 Gq PLC

PIP2
2

AC Gs
P

DAG

PKC
P P

IP3

ATP

Ca2+

ARK

cAMP

Contraction

Relaxation

Figure 1 Cross-talk between M3 muscarinic receptors and b2-adrenoceptors in airway smooth muscle. Generation of 1,2-diacylglycerol (DAG) by M3 receptor-induced phosphoinositide (PIP2) metabolism causes activation of protein kinase C (PKC). PKC may phosphorylate the b2-adrenoceptor as well as Gs, causing uncoupling of the receptor from the effector system. Moreover, PKC may phosphorylate b-adrenoceptor kinase(s) (bARK), which amplies b-agonist-induced desensitization mediated by bARK-induced phosphorylation of the receptor. AC, adenylyl cyclase; cAMP, cyclic adenosine 30 , 50 -monophosphate; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C.

4 ACETYLCHOLINE

airways. As already discussed, inhibitory M2 autoreceptors located at parasympathetic nerve terminals have an important regulatory role in limiting acetylcholine release. Postjunctional muscarinic receptor populations in airway smooth muscle are a mixture of M2 and M3 receptors, the M2 subtype being predominant, particularly in the large airways. Contraction, however, is primarily mediated by M3 receptors (even in those smooth muscle preparations where the ratio of M2 : M3 receptors is 90 : 10), the M2 receptor population having at most a minor supporting role. This is conrmed in airway preparations from M2 receptor knockout mice, in which carbachol, a muscarinic agonist, was hardly less potent than in preparations from wildtype mice. Cross-talk between Gi-coupled M2 receptors and Gs-coupled b-adrenoceptors (having opposing effects on cyclic AMP accumulation or maxi-K channel opening) has no major effects in modulating muscarinic agonist induced contraction or b-agonist induced relaxation, at least under physiological circumstances. However, in inammatory conditions such as asthma, in which Gi-proteins may be upregulated, the situation may change. In contrast to M2 receptors, Gq-coupled M3 receptors, generating IP3 and DAG by stimulating phosphoinositide metabolism, may have a major inuence on b2-adrenoceptor function, even in noninamed airways. This is due to DAG-induced activation of PKC which may (1) phosphorylate the b2-adrenoceptor as well as Gs, causing receptor uncoupling and desensitization, and (2) phosphorylate and activate b-adrenoceptor kinase(s) (bARKs, which are members of the G-protein receptor kinase (GRK) family), amplifying homologous, b-agonist induced desensitization (Figure 1). These processes may explain the well-known attenuation of b-agonist efcacy during episodes of severe bronchoconstriction, for example, during exacerbations.
Airway Remodeling

indicates that acetylcholine may contribute to airway remodeling as observed in asthma and COPD. Indeed, in an animal model of chronic asthma it was recently demonstrated that the long-acting muscarinic antagonist tiotropium bromide inhibited increased airway smooth muscle mass, enhanced airway smooth muscle contractility, and increased expression of contractile proteins in the lung upon repeated allergen challenge. This implies that, in addition to their bronchodilating properties, muscarinic receptor antagonists could be benecial in the treatment of asthma by preventing chronic airway hyperresponsiveness and decline of lung function. Although not fully established, a more extensive role of acetylcholine in airway remodeling, including airway smooth muscle proliferation, contractile protein expression, promitogenic signaling, and regulation of secretory functions as well as cell migration, has recently been proposed (Figure 2).

Acetylcholine in Respiratory Diseases

As indicated above, the parasympathetic nervous system represents a major constrictory pathway of the airways; even basal bronchomotor tone is partly governed by acetylcholine. Both its release and its postjunctional effects, including smooth muscle contraction and mucus secretion, are regulated and mediated, respectively, by muscarinic receptors. So far, no evidence for upregulation of postjunctional M3 and M2 receptors has been found in hyperresponsive airways of patients with asthma and COPD. In contrast, dysfunctional autoreceptors, leading to exaggerated vagal reexes in the airways, are well established in allergic asthma. M1 receptors, facilitating nicotinic neurotransmission in parasympathetic airway ganglia, do not appear to contribute significantly to bronchomotor tone in humans, either with or without obstructive airway diseases. Hence, the principle therapeutic muscarinic receptor target in asthma and COPD is the M3 receptor. In COPD, muscarinic receptor antagonists like ipratropium, a quarternary nonselective antagonist, are very effective in causing bronchodilatation. A recently introduced antagonist is tiotropium, which acutely occupies both M2 and M3 receptors; however, while it dissociates rapidly from M2 receptors during washout, blockade of M3 receptors persists for hours. This kinetically based M3 receptor subtype selectivity could be of therapeutic advantage since blockade of prejunctional M2 receptors enhances acetylcholine outow. Both in COPD and, particularly, in allergic asthma, in which M2 autoreceptors are already dysfunctional, this is an unwanted side effect.

In addition to phosphoinositide metabolism, inhibition of adenylyl cyclase and maxi K-channel activation, stimulation of M3 and/or M2 receptors in airway smooth muscle cells has been shown to activate different promitogenic signaling pathways, including the p42/p44 mitogen-activated protein kinase, Rho/Rho kinase, and PI3 kinase pathways. In vitro studies have revealed that muscarinic agonists do not induce airway smooth muscle cell proliferation by themselves, but enhance the proliferative response induced by peptide growth factors. This effect, which is solely mediated by the M3 receptor,

ACIDBASE BALANCE 5

ACh Extracellular matrix proteins Chemotaxis activation

Migration

Contraction

Hypercontractility

Hyperplasia

Figure 2 Proposed mechanisms by which acetylcholine (ACh) could affect airway smooth muscle remodeling. Acetylcholine has been shown to affect airway smooth muscle contractility, contractile protein expression, promitogenic signaling, and proliferation. In addition, like several other G-protein-coupled receptor agonists, acetylcholine could also be involved in airway smooth muscle cell migration, extracellular matrix protein production, and secretion of cytokines and chemokines. Altogether, these effects could contribute to airway remodeling in asthma and COPD. Reproduced from Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193201, with permission from Elsevier.

See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy; Neurons and Neuromuscular Transmission.

Further Reading
Berge RE ten, Santing RE, Hamstra JJ, Roffel AF, and Zaagsma J (1995) Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs. British Journal of Pharmacology 114: 881887. Billington CK and Penn RB (2002) M3 muscarinic acetylcholine receptor regulation in the airway. American Journal of Respiratory Cell and Molecular Biology 26: 269272. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology and Therapeutics 98: 5969. Gosens R, Bos ST, Zaagsma J, and Meurs H (2005) Protective effect of tiotropium bromide in the progression of airway

smooth muscle remodeling. American Journal of Respiratory and Critical Care Medicine 171: 10961102. Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193201. Racke K and Matthiesen S (2004) The airway cholinergic system: physiology and pharmacology. Pulmonary Pharmacology and Therapeutics 17: 181198. Wess J (2004) Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications. Annual Review of Pharmacology and Toxicology 44: 423450. Wessler I, Kilbinger H, Bittinger F, Unger R, and Kirkpatrick CJ (2003) The nonneural cholinergic system in humans: expression, function and pathophysiology. Life Sciences 72: 20552061. Zaagsma J, Meurs H, and Roffel AF (eds.) (2001) Muscarinic Receptors in Airways Diseases. Basel: Birkhauser Verlag. Zaagsma J, Roffel AF, and Meurs H (1997) Muscarinic control of airway function. Life Sciences 60: 10611068.

ACIDBASE BALANCE
O Siggaard-Andersen, University of Copenhagen, Copenhagen, Denmark
& 2006 Elsevier Ltd. All rights reserved. titratable hydrogen ion (ctH ). A pH, log pCO2 chart illustrates the acidbase status of the arterial blood. The chart shows normal values as well as values to be expected in typical acid base disturbances, i.e., acute and chronic respiratory acidosis and alkalosis, and acute and chronic nonrespiratory (metabolic) acidosis and alkalosis. The chart allows estimation of the concentration of titratable H of the extended extracellular uid (including erythrocytes), ctH Ecf. This quantity is also called standard base deficit but the term base does not directly indicate that the quantity refers to the excess or deficit of hydrogen ions. ctH Ecf is the preferred indicator of a nonrespiratory acidbase disturbance being independent of acute changes in pCO2 in vivo. While pH and pCO2 are directly measured, ctH Ecf

Abstract
The acidbase balance or neutrality regulation maintains a pH around 7.4 in the extracellular uid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acidbase status in blood and extracellular uid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of

6 ACIDBASE BALANCE
is calculated from pH and pCO2 using the HendersonHasselbalch equation and the Van Slyke equation.

Description
The acidbase balance or neutrality regulation maintains a pH around 7.4 in the extracellular uid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acidbase status in blood and extracellular uid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of titratable hydrogen ion (ctH ). Figure 1 illustrates the acidbase status of the blood, especially the relationships among the three key variables.
pH and the Hydrogen Ion Concentration (cH )

with cations) and does not directly indicate that the relevant chemical component is the hydrogen ion. The term hydrogen ion excess or acronym HX may also be used. Note: by definition, ctH of blood refers to the actual hemoglobin oxygen saturation, not the fully oxygenated blood. Acid and base are dened by the equilibrium: Acidz # H Basez1 where Acidz and Basez 1 is a conjugate acidbase pair. The charge number z may be positive, zero, or negative. A strong acid, e.g., HCl, dissociates completely: HCl-H Cl . The anion that follows the hydrogen ion is called an aprote, nonbuffering, or strong anion. At physiological pH, even lactic acid is a strong acid and lactate an aprote anion. A base is a molecule containing a hydrogen ion-binding group. A strong base, e.g. OH , associates completely with hydrogen ion: OH H -H2O. The cation that follows the hydroxyl ion is called an aprote, nonbuffering, or strong cation, e.g., Na or K . A weak acid (buffer acid) is in equilibrium with its conjugate weak base (buffer base), e.g.: H2 CO3 # H HCO 3 hemoglobinz # H hemoglobinz1 The concentration of titratable hydrogen ion may be determined for plasma (P), whole blood (B), or a model of the extended extracellular uid, i.e., blood plus interstitial uid (Ecf). The model consists of blood diluted threefold with its own plasma to get a hemoglobin concentration similar to the one obtained if the red cells were evenly distributed in the whole extracellular volume. An acute increase in pCO2 in vivo causes a rise in ctH B and a fall in ctH P while ctH Ecf remains constant. For example, an acute rise in pCO2 from 5.33 to 10.66 kPa (4080 mmHg) causes a rise in whole blood ctH of about 5 mmol l 1, a fall in plasma ctH of about 3 mmol l 1, while the extracellular ctH remains independent of acute changes in pCO2 . The cause is a redistribution of hydrogen ions within the extended extracellular volume. The hydrogen ion concentration increases more in the poorly buffered interstitial uid than in the blood plasma, where it increases more than inside the erythrocytes, where hemoglobin binds the hydrogen ions. Hydrogen ions diffuse from the poorly buffered interstitial uid into the blood plasma and further into the erythrocytes. Very little transfer of hydrogen ions occurs between the intracellular space and the extracellular space, so ctH Ecf remains virtually constant during acute

pH and cH of the plasma are both indicated on the abscissa of Figure 1. pH is the negative dacadic logarithm of molal hydrogen ion activity. Concentration of free hydrogen ion (cH ) is calculated as 109 pH nmol l 1. pH and pOH are closely related: pH pOH pKw 13.622 at 371C, where Kw is the ionization constant of water. If H is considered a key component of an aqueous solution, then OH is a derived component. Accounting for H and H2O indirectly accounts for OH as well. It is the authors conviction that the relevant component is the hydrogen ion, not hydrogen ion binding groups (base) nor hydroxyl ions.
The Carbon Dioxide Tension of the Blood (pCO2 )

pCO2 , i.e., the partial pressure of carbon dioxide in a gas phase in equilibrium with the blood, is shown on the ordinate on a logarithmic scale in Figure 1. When pCO2 increases, the concentration of dissolved carbon dioxide and carbonic acid increases, and hence the hydrogen ion concentration increases: CO2 H2 O-H2 CO3 -H HCO 3
The Concentration of Titratable Hydrogen Ion (ctH )

ctH is indicated on the scale in the upper left corner of Figure 1. ctH is a measure of added noncarbonic acid or base. The amount of hydrogen ion added or removed in relation to a reference pH of 7.40 may be determined by titration to pH 7.40 at pCO2 5:33 kPa ( 40 mmHg) and T 371C using strong acid or base, depending upon the initial pH. Titratable hydrogen ion is also called base deficit, or with the opposite sign base excess. Unfortunately, the term base is ambiguous (it has previously been associated

ACIDBASE BALANCE 7
p CO2 in arterial blood Concentration of titratable mmHg kPa hydrogen ion in extracellular fluid 20.0 150 mmol l1 19.0 Siggaard-Andersen 140 0 5 0 5 0 18.0 1 1 2 2 3 acidbase chart 130 17.0 16.0 120 15.0 110 14.0 100 13.0
r Ch
Ac

No

rm

al

H+ deficit

0 +5
ex ce ss

0 +1
5

H+

Hypercapnia

+1

e ut p hy ca er

ic on p hy er

90 80 70 60
ia

12.0 11.0 10.0 9.0 8.0 7.0

0 +2

ca

pn ia

pn

Concentration of in plasma +25 bicarbonate mmol l1

10
en rog ion ex s ces

it ic defic n ro n Ch n io e g o dr hy
Normal

50 6.0 Normal 40

15

20

30

40

50

Area

5.0 35 30 4.0 3.5 25 3.0

Acu

yd te h

Chronic hypo

Ac

exc ess

ut e

capnia

p hy

Hypocapnia

ion

oc ap
20 2.5

hyd roge n

nia

onic

+30
pH in arterial plasma 6.9 7.0 1.5

6.8

7.1

Chr

15

2.0

7.2

7.3

7.4

7.5

7.6

7.7

140 120 100 90 Concentration of free hydrogen ion in plasma nmol l1

80

70

60 50 Acidemia

40 35 Normal

30 25 Alkalemia

20

Figure 1 Acidbase chart for arterial blood with normal and pathophysiological reference areas. The acidbase status is shown as a point with three coordinates: pH (abscissa), p CO2 (ordinate), and c tH (oblique coordinate). The bands radiating from the normal area (the central ellipse) show reference areas for typical acute and chronic, respiratory and nonrespiratory, acidbase disturbances. Hyper- and hypocapnia are also called respiratory acidosis and alkalosis, respectively. Hydrogen ion excess and deficit, i.e., increased and decreased c tH , are also called nonrespiratory (or metabolic) acidosis and alkalosis, respectively. Reproduced from Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory kandevej 21, Brnshj, Denmark. Investigation 27: 239245. Copyright & 1970, 1974 by Radiometer Copenhagen A/S, A

changes in pCO2 in vivo. ctH Ecf is also called standard base deficit (SBD), or with the opposite sign standard base excess (SBE), but the term base is deprecated by the author. It is important to use ctH Ecf

rather than ctH B (whole blood titratable hydrogen ion) as a measure of a nonrespiratory acidbase disturbance, especially in neonatology where high hemoglobin concentrations and high pCO2 values may be

8 ACIDBASE BALANCE

encountered. The ctH B may then be considerably higher than ctH Ecf (as much as 4 mmol l 1) causing an erroneous diagnosis of metabolic acidosis, when the situation is merely a redistribution of hydrogen ions within the extended extracellular volume. Projections to the ctH scale in the upper left corner of Figure 1 should be made along the slanting so-called vivo-CO2 titration curves, which are virtually straight lines (slightly convex upwards). The slope of the lines depends on the concentration of nonbicarbonate buffers, i.e., mainly hemoglobin. In the chart, the slope corresponds to a hemoglobin concentration of 3 mmol l 1 corresponding to the hemoglobin concentration of the extended extracellular uid. Variations in the slope due to variations in blood hemoglobin concentration are small and generally without clinical significance. Variations in the concentration of other buffers, e.g., albumin, are even less significant. In summary, the hydrogen ion status of the blood is described by a point in the acidbase chart: the x,y coordinates indicate cH and pCO2 , the oblique coordinate is ctH Ecf.
The HendersonHasselbalch Equation

representing respiratory and metabolic disturbances. is shown in Figure 1 on a horizontal logcHCO3 arithmic scale along the pCO2 5:33 kPa line. Projections to the scale should be made at an angle of 451. However, cHCO3 is not independent of pCO2 . For this reason, standard bicarbonate was introduced, i.e., the bicarbonate concentration in plasma of whole blood equilibrated with a gas mixture with a normal pCO2 (5.33 kPa 40 mmHg) at 371C. However, even the standard bicarbonate is not completely independent of acute changes in pCO2 in vivo, decreasing slightly in acute hypercapnia. To be independent, the equilibration should be performed with a model of the extended extracellular uid. Projecting from a given point in the chart to the bicarbonate scale along the slanting vivo-CO2 equilibration lines gives the standard bicarbonate concentration of the extended extracellular uid.
The Van Slyke Equation

Often a description of acidbase balance is based on the HendersonHasselbalch equation, derived from the law of mass action: pH pK log10 cHCO 3 =aCO2 pCO2 where pK 6.10 and aCO2 0.23 mmol l 1 kPa 1 0.0306 mmol l 1 mmHg 1 (solubility coefcient of carbon dioxide in plasma at 371C). aCO2 pCO2 gives the concentration of H2CO3 plus CO2. pH is determined by two variables, pCO2 and cHCO3 ,

Blood gas analyzers measure pH with a glass electrode and pCO2 with a membrane-covered glass electrode (Stow-Severinghaus electrode). ctH Ecf is calculated from pH, pCO2 , and cHb (concentration of hemoglobin) using a model of the titration curve called the Van Slyke equation (Table 1). The equation calculates the change in buffer base concentration (bicarbonate plus protein anion plus phosphate) from the value at the reference point:
y  pHPy 7:40; Py CO2 5:33 kPa; and T 37 C

Buffer base (BB) is the difference between the concentrations of buffer anions and buffer cations (the latter being virtually zero at physiological pH). Strong ion difference (SID) is the difference between

Table 1 Van Slyke equation for calculation of the concentration of titratable hydrogen ion in the extended extracellular uid, c tH Ecf
c tH Ecf 1 c HbEcf=c Hby Dc HCO 3 P bH Ecf Dp HP c HbEcf c HbB V B/V Ecf concentration of hemoglobin in the extended extracellular uid V B/V Ecf 1/3 (default value) ratio between the volume of blood and volume of extended extracellular uid c Hby 43 mmol l 1 empirical parameter accounting for an unequal distribution of hydrogen ions between plasma and erythrocytes y Dc HCO3 P c HCO3 P c HCO3 P y y c HCO3 P 24.5 mmol l 1 concentration of bicarbonate in plasma at pHPy 7.40, PCO 5:33 kPa, T y 37:0 C 2 DpHP pHP pHPy bH Ecf bm Hby c HbEcf bP bm Hby 2.3 apparent molar buffer capacity of hemoglobin monomer in whole blood bP 7.7 mmol l 1 (default value) buffer value of nonbicarbonate buffers in plasma for a normal plasma protein (albumin) concentration c HbB rHbB/MmHb (substance) concentration of hemoglobin in blood (unit: mmol l 1) as a function of the mass concentration, rHbB (unit: g l 1) MmHb 16 114 g mol 1 molar mass of hemoglobin monomer

Ecf refers to the extended extracellular uid, B to whole blood, P to plasma. Replacing c HbEcf by c HbB gives c tH B; replacing c HbEcf by zero gives c tH P. Note: if c HbB 9.0 mmol l 1 3 rHbB 14.5 g dl 1, then the Van Slyke equation simplies to c tH Ecf 0.93 (Dc HCO3 P DpHP 14.6 mmol l 1).

ACIDBASE BALANCE 9

the concentrations of nonbuffer cations and nonbuffer anions (see Figure 2). According to the law of electroneutrality, the value of BB and SID must be identical. Buffer base is not a suitable indicator of a nonrespiratory acidbase disturbance; although independent of pCO2 , it varies with the albumin and hemoglobin concentrations, which are unrelated to acidbase disturbances.

Normal AcidBase Balance

Acidbase balance refers to the balance between input (intake and production) and output (elimination) of hydrogen ion. The body is an open system in equilibrium with the alveolar air where the partial pressure of carbon dioxide pCO2 is identical to the carbon dioxide tension in the blood. pCO2 is directly proportional to the CO2 production rate (at constant

Concentration of ions in arterial plasma (mmol l 1)

150

Mg2+ Ca2+ K+ SID+

HCO3 BB Pr

100

HPO42 + H2PO4 SO42 Organic anions

Na+

Cl

50

Cations

Anions

Figure 2 Electrolyte balance of arterial plasma showing columns of cations and anions of equal height (law of electroneutrality). The equality of the strong ion difference (SID) and buffer base (BB) is illustrated. The change in concentration of buffer base from normal (at pH 7.40, p CO2 5:3 kPa, and T 371C) with opposite sign equals the concentration of titratable hydrogen ion.

alveolar ventilation and CO2 free inspired air) and inversely proportional to the alveolar ventilation (at constant CO2 production rate and CO2 free inspired air). CO2 is constantly produced in the oxidative metabolism at a rate of about 10 mmol min 1 ( 224 ml min 1) and eliminated in the lungs at the same rate so that the pCO2 remains at about 5.33 kPa ( 40 mmHg). Hydrogen ions associated with any anion other than bicarbonate or exchanging with a cation are eliminated by the kidneys. In the oxidative metabolism of sulfur-containing amino acids, hydrogen ions are produced together with sulfate ions at a rate of about 70 mmol day 1 depending upon the protein intake. Amino acids are oxidized to carbon dioxide and water, and the amino nitrogen, liberated as NH3, combines with carbon dioxide in the liver via the Krebs urea cycle to form neutral urea. Therefore, there is no production of base (ammonia) except in the kidneys, where ammonia formed from glutamine diffuses into the urine where it binds a hydrogen ion (NH3 H -NH4 ) thereby preventing an excessively low urine pH. Normal values for the acidbase status of arterial blood are given in Table 2. The lower pCO2 in women than men is probably a progesterone effect on the respiratory center. The values are independent of age except at birth, where babies tend to have higher pCO2 , lower pH, and slightly increased ctH Ecf, approaching normal values for adults in the course of a few hours. In the last trimester of pregnancy, the pCO2 is lower (about 1 kPa 7.5 mmHg), compensated by a slightly increased ctH Ecf. A protein-rich diet causes a higher ctH Ecf (12 mmol l 1) and a slightly lower pH due to production of sulfuric acid from sulfur-containing amino acids. A diet rich in vegetables and fruit causes a lower (negative) ctH Ecf and a slightly higher pH due to organic anions binding H in the metabolism to carbon dioxide and water. High-altitude hypoxia stimulates ventilation; at 5 km above sea level, pCO2 is decreased to about 3.3 kPa 25 mmHg. The hypocapnia is compensated by increased ctH Ecf, so pH is only slightly elevated. The values fall in the area of chronic hypocapnia in the acidbase chart (Figure 1).

Table 2 Reference values for arterial blood Women cH P c tH Ecf p CO2 c HCO3 P

Men
1

36.341.7 nmol l (pH: 7.387.44) 2.3 to 2.7 mmol l 1 4.595.76 kPa (33.842.4 mmHg) 21.227.0 mmol l 1

37.242.7 nmol l 1 (pH: 7.377.43) 3.2 to 1.8 mmol l 1 4.916.16 kPa (36.846.2 mmHg) 22.228.3 mmol l 1

c H P: conc. of (free) hydrogen ion in plasma; c tH Ecf: conc. of titratable hydrogen ion in extracellular uid (also called standard base deficit, SBD); p CO2 : tension of carbon dioxide; c HCO3 P: conc. of bicarbonate in plasma.

10

ACIDBASE BALANCE

AcidBase Disturbances
Respiratory AcidBase Disturbances

Acute respiratory acidbase disturbances are characterized by an acute change in pCO2 associated with an acute change in pH but with unchanged ctH Ecf. The relationship between pCO2 and pH is illustrated by the oblique in vivo CO2 equilibration lines in the acidbase chart (Figure 1). Primary increase and decrease in pCO2 are compensated by secondary renal decrease and increase in ctH Ecf, respectively. The acidbase chart shows the expected values in chronic hypercapnia and chronic hypocapnia. The effect of the compensation is a return of pH about two-thirds towards normal, slightly more in acute hypocapnia.
Nonrespiratory AcidBase Disturbances

organic acidosis. A hyperchloremic acidosis may be a renal acidosis with retention of H and Cl or an intestinal loss of Na HCO3 with subsequent intake of saline (Na Cl ). A hypochloremic alkalosis may be due to loss of H and Cl by vomiting. Hypokalemic alkalosis is due to inability of the kidneys to retain hydrogen ions in the presence of potassium depletion.
See also: Arterial Blood Gases. Carbon Dioxide. Peripheral Gas Exchange. Ventilation: Overview.

Further Reading
Astup P and Severinghaus JW (1986) The History of Blood Gases Acids and Bases. Copenhagen: Munksgaard International Publishers. Davenport HW (1969) The ABC of AcidBase Chemistry, 5th edn. Chicago: The University of Chicago Press. Grogono AW (1986) Acidbase balance. International Anesthesiology Clinics, Problems and Advances in Respiratory Therapy 24(1). Halperin ML and Goldstein MB (1988) Fluid, Electrolyte, and AcidBase Emergencies. Philadelphia: Saunders. Hills AG (1973) AcidBase Balance. Chemistry, Physiology, and Pathophysiology. Baltimore: Williams & Wilkins. International Federation of Clinical Chemistry and International Union of Pure and Applied Chemistry (1987) Approved Recommendation (1984) on Physico-Chemical Quantities and Units in Clinical Chemistry. Journal of Clinical Chemistry and Clinical Biochemistry 25: 369391. Masoro EJ and Siegel PD (1971) AcidBase Regulation. Its Physiology and Pathophysiology. Philadelphia: Saunders. Nahas G and Schaefer KE (eds.) (1974) Carbon Dioxide and Metabolic Regulations. New York: Springer. Rooth G (1975) AcidBase and Electrolyte Balance. Lund: Studentlitteratur. Severinghaus JW and Astrup P (1987) History of Blood Gas Analysis. Boston: Little, Brown and Company. Shapiro BA, Peruzzi WT, and Templin R (1994) Clinical Application of Blood Gases, 5th edn. St Louis: Mosby Year Book. Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27: 239245. Siggaard-Andersen O (1974) The AcidBase Status of the Blood, 4th edn. Copenhagen: Munksgaard and Baltimore: Williams & Wilkins Company. Siggaard-Andersen O (1979) Hydrogen ions and blood gases. In: Brown SS, Mitchell FL, and Young DS (eds.) Chemical Diagnosis of Disease, pp. 181245. London: Elsevier, NorthHolland Biomedical Press. Siggaard-Andersen O and Fogh-Andersen N (1995) Base excess or buffer base (strong ion difference) as measure of a non-respiratory acidbase disturbance. Acta Anaesthesiologica Scandinavica 39(supplement 107): 123128. Thomson WST, Adams JF, and Cowan RA (1997) Clinical Acid Base Balance. New York: Oxford University Press. West JB (1974) Respiratory Physiology, the Essentials. Oxford: Blackwell. West JB (2001) Pulmonary Physiology and Pathophysiology. An Integrated, Case-Based Approach. Philadelphia: Lippincott Williams & Wilkins.

Primary increase and decrease in ctH Ecf are compensated by secondary decrease and increase in pCO2 . A very acute rise in ctH Ecf, for example, due to anaerobic exercise with lactic acid formation, is only partly compensated because only peripheral chemoreceptors react promptly to a fall in blood pH. It takes about an hour before H equilibrium between blood and brain extracellular uid is achieved and the central chemoreceptors are maximally stimulated. The acidbase values in acute nonrespiratory acidemia are illustrated in Figure 1 by the area labeled acute hydrogen ion excess. The outline of the area is dotted because it is less well-dened than the other areas of the chart. The compensations in more slowly developing nonrespiratory acidemia or alkalemia are illustrated by the areas labeled chronic hydrogen ion excess and deficit, respectively. The effect of the respiratory compensation is a return of pH one-third to halfway towards normal. Once an increase in ctH Ecf has been detected, the question is: what caused the metabolic acidosis? It may be a production of lactic acid due to anaerobic metabolism or acetoacetic acid (ketoacidosis) due to diabetes mellitus or starvation. In both cases the diagnosis may be veried by direct measurement of blood lactate or acetoacetate. When these analyses are unavailable, calculation of the concentration of undetermined anions may be useful, i.e., the sum of the concentrations of measured cations (Na and K ) minus the sum of the concentrations of meas ured and calculated anions (Cl and HCO3 ). This equals the sum of the concentrations of unmeasured 2 anions (mainly Protein , SO2 4 , HPO4 , fatty carboxylate, lactate, acetoacetate)minus the sum of the concentrations of unmeasured cations (Ca2 and Mg2 ). A metabolic acidosis with a major increase in undetermined anions usually indicates

ACUTE RESPIRATORY DISTRESS SYNDROME 11

Acute Exacerbations
Acute Exacerbations.

see Asthma: Acute Exacerbations. Chronic Obstructive Pulmonary Disease:

Acute Lung Injury

see Acute Respiratory Distress Syndrome.

ACUTE RESPIRATORY DISTRESS SYNDROME

G Bellingan, University College London, London, UK S J Finney, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved. Table 1 Clinical triggers of ARDS Etiology Sepsis (including pulmonary sepsis) Aspiration of gastric contents Pulmonary contusion Bacteremia Head injury Multiple bony fractures requiring ICU admission Blood transfusion exceeding 10 units in 24 h Cardiopulmonary bypass Burns (including smoke inhalation) Acute pancreatitis Lung reperfusion injury (e.g., posttransplant) Near-drowning Percentage 4352 2236 826 412 611 512 58 2 2 1

Abstract
The acute respiratory distress syndrome (ARDS) is the devastating manifestation of the diffuse pulmonary inammation that may occur following a wide range of life-threatening systemic illnesses. The rapid onset of inammation and bilateral nonhydrostatic alveolar edema results in severe hypoxemia and reduced pulmonary compliance often mandating mechanical ventilation. The clinical features, radiology, and pathogenesis are reviewed in this article. The management of patients comprises primarily of ventilatory support while the lung injury resolves. The techniques of ventilatory support can propagate the lung injury and adversely affect outcome; the techniques are discussed in detail here. By contrast, pharmacotherapy has a less clear role in ARDS. Corticosteroids may be benecial after the acute phases, whilst other anti-inammatory agents have not proved benecial. Mortality is determined primarily by the underlying trigger for ARDS, but is approximately 3040%. Follow-up of survivors has demonstrated that lung function often improves considerably, whereas nonpulmonary morbidities persist even 12 months after discharge from the intensive care unit.

The gures in this were based on studies at the university of Washington and Colorado.

Introduction
The acute respiratory distress syndrome (ARDS) and its less extreme manifestation, acute lung injury (ALI), are devastating conditions that result in sudden bilateral nonhydrostatic alveolar edema and severe hypoxemia. Pulmonary failure is usually so severe that patients require mechanical ventilation of their lungs. ARDS and ALI are the consequence of the diffuse pulmonary inammation that can be triggered by an insult either to the lung itself or more commonly at a distant site. As such, they form part of the spectrum of systemic inammation that can occur following many life-threatening insults (see Table 1). The constellation of severe respiratory distress, refractory hypoxemia, decreased pulmonary compliance,

and diffuse radiological changes was rst appreciated by Ashbaugh and co-workers in 1967. They referred to the clinical scenario as the adult respiratory distress syndrome. Since an identical condition can also occur in children, it is now referred to as the acute respiratory distress syndrome. Subsequently, the clinical syndrome has been increasingly recognized and estimates of the annual incidence of ARDS range from 8 to 70 cases per 100 000 population in developed countries. Overall, mortality in patients with ARDS is approximately 3040%, although death is usually attributable to the underlying etiology rather than pulmonary failure per se.

Etiology
The many possible triggers for ARDS are outlined in Table 1. Sepsis accounts for the majority of cases in general intensive care units (ICUs). Patients with multiple risk factors are more likely to develop ARDS. Intriguingly, ARDS only occurs in a subset of patients who suffer apparently similar insults. It is not clear what explains a particular individual going on

12

ACUTE RESPIRATORY DISTRESS SYNDROME

to develop ARDS. Although environmental factors such as age, sex, smoking history, and intercurrent pulmonary disease may be important, it is widely considered that genetic factors are critical. Candidate genes for which associations have been described include those that encode tumor necrosis factor alpha (TNF-a), angiotensin-converting enzyme, interleukin-6 (IL-6), surfactant protein B, and Toll-like receptors. Nevertheless, it has not been possible to draw denitive conclusions since these associations are complicated by tight linkage disequilibrium to other genes, lack of clear functional effects, and small patient numbers. Moreover, the heterogeneity of ARDS suggests that many genes with varying phenotypes and penetrance may underpin an individuals susceptibility. Functional genomic approaches may help elucidate these complex genotypes.

After the initial injury, myobroblasts are observed in the interstitium and then the airspace, and start to produce new matrix substance. Indeed, the lung collagen content can double by 2 weeks. With time, type II alveolar epithelial cells increase in number and may represent a stem cell population of the lung; they differentiate into type I alveolar epithelial cells and repopulate the denuded alveolar basement membrane during healing. Pulmonary function and computed tomography (CT) suggest that many patients subsequently return to have structurally normal lungs although this can take months. Some patients however develop severe brosis involving both the alveolar space and interstitium. At its most extreme, the honeycomb of advanced brotic lung disease may form. Many of these patients succumb to intercurrent infection associated with a prolonged ICU stay. A few can recover with incomplete resolution of pulmonary damage.

Pathology
Traditionally, the histological development of ARDS has been described as having three sequential phases which affect the lungs in a diffuse manner: exudative, proliferative, and brotic. It is now evident that these stages overlap considerably and brotic changes are initiated very early. Within the rst 24 h, the lungs appear macroscopically edematous and congested. At this point, light microscopy reveals edema within the airspaces, alveolar walls, and septae. Type I alveolar epithelial cells are swollen, necrotic, and often detached from the underlying basement membrane. Pulmonary endothelial cells may also be swollen, with brin thrombi occluding alveolar capillaries. Neutrophils are also initially observed within alveolar capillaries, but inammatory cells then accumulate rapidly in the edematous alveoli. Over the next few days, the lungs become more uniformly red as alveolar wall and airspace edema increase and red cells leak into the airspaces. Histologically, hyaline membranes form from brin-rich edema uid and line the alveoli. The number of neutrophils increases rapidly as they move via the interstitium into the airspace and there is increased disruption of vascular structures with further neutrophil and brin plugs occluding capillaries.

Clinical Features
Definition

The diagnosis of ARDS is based on the criteria developed at the 1992 AmericanEuropean consensus conference and is illustrated in Table 2. Since the definition does not consider current management strategies, the relevance of scenarios in which suboptimal mechanical ventilation and management can inuence whether criteria are met or not is not clear. The cutoffs for severity of hypoxemia are arbitrary, the definition of acute onset lacks clarity and the use of the chest X-ray opens the way for individual interpretation. Further limitations of the definition include the inclusion of other conditions that probably have different pathological processes (such as severe pneumocystis carinii infection, diffuse alveolar hemorrhage), and exclusion of unilateral disease that may occur following pulmonary lobectomy. Nevertheless, the definition is simple to use and is supported by extensive literature. Post-mortem studies have demonstrated that the definition is 84% sensitive and 94% specic for diffuse alveolar damage; its performance in patients who survive is not clear.

Table 2 AmericanEuropean consensus criteria for diagnosing ALI and ARDS ALI Chest radiography Clinical scenario Left atrial pressure Oxygenation Bilateral airspace shadowing Acute onset and associated with a condition known to cause ALI/ARDS No direct or clinical evidence of left atrial hypertension (PAOP o18 mmHg) PaO2/FiO2 o39.9 kPa (300 mmHg) ARDS

PaO2/FiO2 o26.6 kPa (200 mmHg)

PAOP, pulmonary artery occlusion pressure.

ACUTE RESPIRATORY DISTRESS SYNDROME 13 Natural History

The clinical picture of ARDS is dominated initially by severe hypoxemia due to mismatching of ventilation and perfusion. Indeed, intrapulmonary shunting may result in oxygen saturations that are relatively refractory to increases in the inspired oxygen content. Decreased pulmonary compliance increases the work of breathing and most patients require endotracheal intubation and mechanical ventilation. From a pulmonary perspective, the high oxygen requirements persist for some time. Further increases in the alveolararterial oxygen gradient occur with ongoing pulmonary inammation, particularly in the setting of a positive uid balance, but may also reect a superimposed ventilatory pneumonia or pneumothorax. Pneumothoraces tend to occur after the rst week and may tension rapidly. Since the inamed lung may tether to the chest wall, pneumothoraces can be loculated and anterior, and thus easily missed on plain chest radiography. Thoracic CT may be required to locate pneumothoraces accurately. Surprisingly, despite devastating pulmonary failure, oxygenation often slowly improves allowing withdrawal of mechanical ventilation; for many, this may take weeks or months and often necessitate temporary tracheostomy. Although disease can remain compartmentalized with isolated lung failure, ARDS is usually part of the spectrum of systemic inammation; hence, patients often demonstrate peripheral vasodilation, increased cardiac outputs, and systemic hypotension which may require the administration of vasopressors such as norepinephrine. Secondary pulmonary hypertension can occur and result in acute right ventricular failure. Renal dysfunction is also common as is the need for acute renal support. Other organs can also fail as part of this process and outcome is related to the number of failing organs. The course of ARDS is not a smooth wave of deterioration and recovery. Rather, it is interspersed by episodes of deterioration (commonly linked with intercurrent infection such as ventilator-associated pneumonia or line-related sepsis) and many patients need repeated episodes of inotrope and other organ support prior to nal recovery (or demise).

Chest CT demonstrates that the lungs are affected in a heterogenous manner. Typically, there is a gradient of opacication from apparently normally aerated lung, through ground-glass appearances, to densely consolidated lung. In the supine patient, this gradient occurs both in ventrodorsal and cephalocaudal directions. These gradients typically reverse within a few minutes if the patient is moved to the prone position. Since alveolar edema would not redistribute so quickly, some of these appearance are not due to increased edema in dependent zones but due to collapse of these areas due to the weight of the overlying lung. Thus ground-glass appearances most likely represent airspace edema, with densely opacied areas representing collapsed edematous lung. Regions of dense opacication in nondependent areas may signify collapse/consolidation due to infection or retained secretions. At later stages, groundglass appearances may be accompanied by bronchial dilatation which persists into recovery, suggesting established ne intralobular brosis (Figure 1).
Other Pulmonary Investigations

Bronchoalveolar lavage samples are dominated by the granulocytic cell population initially. As the disease evolves, the proportion of granulocytes declines and a greater proportion of macrophages is seen. Persistent neutrophilia often portends a poor prognosis. Since most patients are mechanically ventilated, there are few data about classical pulmonary function tests in patients with ARDS. Re-breathing techniques have been used during positive pressure ventilation and have demonstrated marked reductions in the functional residual capacity, carbon monoxide diffusing capacity (DLCO), and diffusing coefcient (KCO). Lower values of DLCO and KCO tend to be associated with nonsurvival.
Disease Severity

Radiology

The appearance of the plain chest radiograph, although forming part of the consensus definition for ARDS, can be relatively non-specic. Clues distinguishing ARDS from cardiogenic pulmonary edema include normal cardiac dimensions, a normal vascular pedicle width, a peripheral distribution of airspace shadowing, and the absence of septal lines.

There are several scoring systems that evaluate the severity of ARDS. Although these systems have only limited clinical utility in individuals, they describe well the degree of physiological disturbance and act as useful descriptors of disease severity in clinical studies. The scoring systems include the acute physiology and chronic health evaluation II and the lung injury score. The former is used to evaluate all critically ill patients whereas the latter is specifically designed for patients with ARDS.

Pathogenesis
Since neutrophils appear early in histological specimens and dominate in bronchoalveolar uid samples,

14

ACUTE RESPIRATORY DISTRESS SYNDROME

also key initiators of pulmonary inammation in ARDS. Studies of patient groups at risk of ARDS who do or do not progress to develop refractory hypoxemia suggest compartmentalized intrapulmonary inammatory changes (e.g., increased IL-8) may precede a systemic inammatory response. There is widespread activation and/or dysfunction of many cell types within the lung which results in the clinical manifestations of ARDS (Figure 2). Thus endothelial dysfunction and loss of epithelial integrity reduce the barrier function of the alveolar wall and result in alveolar edema. Alveolar edema is further exacerbated by the loss of epithelial cells which normally promote uid transport out of the alveolus through apical sodium pumps. Surfactant is lost early during ARDS due to reduced production by damaged epithelial cells along with neutralization of preexisting surfactant by the protein-rich edema uid. Surfactant loss contributes to alveolar collapse, intrapulmonary shunt, and hypoxemia. Endothelial and smooth muscle cell dysfunction result in impaired hypoxic pulmonary vasoconstriction and, along with microthrombi, contribute to the development of secondary pulmonary hypertension and also impacts on outcome.

Animal Models
Animal models allow the study of the pathophysiology of ARDS and the effects of interventions whilst tightly controlling both insults and genetic background. However, the multiple models that exist for ARDS/ALI illustrate that none are ideal. Models in which the lung is injured directly include hyperoxia, high tidal-volume mechanical ventilation, and instillation of oleic acid, bleomycin, or thiourea. Alternatively, models mimic systemic sepsis in which the lung is also injured; these include systemic injections of lipopolysaccharide and caecal ligation and puncture. Some authors suggest that two-hit models are more clinically appropriate and combine models of sepsis with hypovolemia, burns, or high tidal-volume ventilation.

Figure 1 Typical appearances of ARDS with (a) plain radiology and (b) computed tomography.

it has been considered that they are important in the pathogenesis of ARDS. It is possible that activated and thus rigid 7.5 mm neutrophils may get stuck in pulmonary capillaries where they release a plethora of inammatory mediators that include chemokines, cytokines, and proteases. Activation may occur at remote sites and/or by circulating cytokines. However, since ARDS can occur in neutropenic patients, neutrophils cannot be absolutely required for the development of ARDS. Circulating inammatory mediators (e.g., TNF-a, IL-1b, IL-6, IL-8, leukotrienes) along with the changes that occur within the coagulation system during systemic inammation are

Management and Current Therapy

The primary management aims for a patient with ARDS are to maintain adequate oxygenation and to support any other failing organs whilst the lung injury resolves. In general, respiratory failure is sufciently severe to mandate mechanical ventilation, the techniques of which significantly inuence mortality. By contrast, few, if any, therapies directed at modifying the pathogenesis and evolution of ARDS, have been demonstrated to effect outcome. A detailed

ACUTE RESPIRATORY DISTRESS SYNDROME 15

Inflammatory trigger

Mechanical ventilation

E.g., bacterial sepsis, massive transfusion Stretch-induced cell signaling

Activated neutrophils

Soluble mediators E.g., cytokines, chemokines, coagulation factors, eicosanoids, ROS

Stretch-induced cell damage

Smooth muscle cells

Endothelial cells

Epithelial cells

Vascular dysfunction

Barrier dysfunction

Production

Reduced HPV Pulmonary hypertension

Alveolar edema Neutralization

Surfactant

Alveolar collapse
Figure 2 Pathogenesis of ARDS and ALI. HPV, hypoxic pulmonary vasoconstriction; ROS, reactive oxygen species.

description of optimal mechanical ventilation along with the pharmacotherapeutic approaches that have been explored are outlined below.
Conventional Mechanical Ventilation

The manner in which the injured lung is ventilated inuences mortality and may perpetuate lung injury. The origins of such ventilator-induced lung injury (VILI) are multifactorial but include the shear stresses exerted on alveoli during overdistension and cyclical collapse/re-ination, and oxygen toxicity. VILI is illustrated by chest CT of those patients who have survived ARDS: relatively normal lung architecture is often present in previously densely consolidated areas, whereas a reticular pattern of brosis is seen in those regions (often nondependent) that were exposed to mechanical ventilation. VILI results in the ongoing release of inammatory mediators that may spill over into the circulation and propagate systemic inammation, thereby inuencing mortality. Indeed, increased plasma levels of cytokines have been demonstrated in animal models and in patients receiving injuriously large tidal-volume ventilation. This theory has stimulated the development of socalled protective strategies for mechanical ventilation which minimize VILI.

Tidal volume Delivery of a normal tidal volume to extensively consolidated lungs inevitably results in overdistension of the remaining lung units. Experimental work demonstrated that this may be a significant trigger for VILI, and resulted in the National Institutes of Health (NIH)-sponsored study of low tidal-volume ventilation in 861 patients. This landmark study showed that mortality could be reduced by the use of lower tidal volumes (46 ml kg 1, ideal body mass) or at least the avoidance of more traditional tidal volumes (1012 ml kg 1). The corresponding plateau inspiratory pressures were 25 and 35 cmH2O, respectively. Plasma and bronchoalveolar lavage cytokine and chemokines levels were greater in those patients receiving higher tidal volumes. The study protocol has been adopted by some as the denitive ventilatory strategy. More correctly, it provided excellent evidence for VILI and should form the basis of lung-protective strategies. Indeed, it has been suggested that targeting lower-end expiratory alveolar pressures may be more sensible, since the specic pulmonary compliance (compliance corrected for accessible lung volume) has been reported as normal in patients with ALI/ARDS. Smaller tidal volumes may reduce VILI by virtue of the reduced cyclical volume per se, or through a reduction in the end-inspiratory volume. It is not clear

16

ACUTE RESPIRATORY DISTRESS SYNDROME

which is the important factor although in vitro experiments on mechanically deformed epithelial layers have demonstrated that cyclical changes are more damaging than constant stretch to a high volume. How these results translate to the in vivo scenario is not clear. In the absence of increased respiratory rates, reductions in tidal volume will reduce alveolar ventilation and result in a hypercapnic acidosis. Permissive hypercapnia is generally well tolerated except in the setting of a marked metabolic acidosis and increased intracranial pressure. Hypercapnia may also reduce myocardial contractility, and possibly increase the need for sedation and/or paralysis. The effects on the immune response are still unclear. The degree of hypercapnic acidosis allowable is unclear although many clinicians accept arterial pH values not less than 7.20. When titrating respiratory rate to pCO2 , it must be remembered that increases in respiratory rate will have less inuence on alveolar ventilation in ARDS due to the increased dead space, and that reductions in respiratory rate can paradoxically increase CO2 clearance when inspiratory times are particularly prolonged. Tracheal insufation of oxygen and administration of weak bases are sometimes used to combat the acidosis. Fractional inspired oxygen High fractions of inspired oxygen (FiO2) cause absorption atelectasis and may be cytotoxic to the lung per se. The standard practice is to titrate the FiO2 to arterial saturations of 8892%, rather than a specic pO2 which may be less relevant to oxygen delivery. Lower targets may be appropriate in those patients whose cardiac output is high or where an improvement of SaO2 would require an unacceptably injurious pattern of ventilation (e.g., through a higher FiO2, positive end expiratory pressure (PEEP), or tidal volume). The benets of further reductions in FiO2 afforded by the use of inhaled nitric oxide (iNO), prone positioning, and related maneuvers are unknown and still subject to review (vide infra). Intrapulmonary shunting may result in arterial oxygen saturations being relatively independent of FiO2 between 0.8 and 1.0. Positive end expiratory pressure The application of PEEP during mechanical ventilation increases FRC and thus prevents the collapse of alveoli at end expiration. This will reduce intrapulmonary shunt and improve oxygenation. Furthermore, the shear forces required to repeatedly open collapsed alveoli during inspiration are considerable and most likely contribute to VILI. Thus, by keeping these alveoli open throughout the respiratory cycle, PEEP can limit

VILI. Since the lung is heterogeneously affected in ARDS, excessively high levels of PEEP can lead to overexpansion of those regions whose compliance is higher, an effect that may exacerbate VILI. Other detrimental effects of PEEP include reduced cardiac preload and cerebral perfusion. The method to determine the optimal level of PEEP in ARDS is still not established. A randomized multicenter trial in 549 patients, titrating PEEP according to the required FiO2, found no outcome differences between a lower and higher PEEP algorithm. Many clinicians set PEEP by inferring the region of maximal slope on the pressurevolume curve by assessing maximal tidal volume as PEEP is adjusted with a constant pressure control. Mathematical modeling suggests that this is best determined using a decremental rather than an incremental PEEP trial. Ventilatory modes The NIH trial of lower tidal-volume ventilation used a mandatory constant ow, volume control mode of mandatory ventilation. There is considerable interest in the role of descending ow/ pressure control modes since these may promote more even distribution of gas, as ow is slower at the end of expiration and thus more likely to be laminar. Pressure-control modes do not inuence peak alveolar pressure, although peak airway pressures may be reduced. The most likely advantage afforded by pressure control ventilation and inverse ratio ventilation is that these may be associated with a greater proportion of the respiratory cycle spent at plateau inspiratory pressure. Greater plateau times promote more homogenous ventilation by allowing slow-lling regions to inter-ll from areas with faster time constants. The recruitment of previously collapsed or partially collapsed slow-lling alveoli reduces shunt and improves oxygenation. Prolongation of inspiration can also improve CO2 clearance by forcing expiration to be delayed to a point where alveolar pCO2 has risen to a maximum. Faster respiratory rates reduce the efcacy of inverse ratio ventilation by shortening the inspiratory time. Excessive prolongation of inspiration may reduce expiratory time to a point at which expiration is not completed. This may result in dynamic hyperination and the generation of autoPEEP. Most clinicians consider auto-PEEP undesirable since levels may be unpredictable thus leading to cardiovascular compromise. There are theoretical advantages to modes that allow spontaneous ventilation such as biphasic positive airway pressure and airway pressure release ventilation. These modes allow continued diaphragmatic and intercostal function and the ventilation of dependent, better-perfused regions. Although pressure support modes also allow spontaneous ventilation,

ACUTE RESPIRATORY DISTRESS SYNDROME 17

some argue against them as they preclude any plateau time. No study to date, excluding those examining weaning, has demonstrated any outcome differences according to ventilatory mode. Drainage of pneumothoraces Multiple pneumothoraces may complicate mechanical ventilation of patients with ARDS. Pneumothoraces can be complex and localized as the inamed lung may tether to the chest wall. Indeed, localization can be difcult by conventional radiography and it may be necessary to insert drains under the guidance of CT. Intercostal drains are generally left in situ until the patient is well established on a spontaneous ventilatory weaning program.
Supplemental Techniques for Mechanical Ventilation

Prone positioning Ventilation in the prone position improves oxygenation in approximately 70% of patients. It is not possible to predict which patients will respond, and nonresponders may improve on subsequent turns. Experienced teams can easily turn patients, even with multiple intercostal drains and intravascular catheters, with few complications. Improvements in oxygenation continue in 50% of patients after they are returned to the supine position and are probably the consequence of expansion of previously collapsed lung. More homogenous ventilation may prevent regional overdistension and limit VILI. Improved secretion drainage and altered diaphragmatic mechanics may also contribute to clinical improvement. Despite improvements in oxygenation, prone positioning has not been demonstrated to improve outcome in ARDS. Recruitment maneuvers Sustained and high airway pressures of up to 60 cmH2O may be required to open (or recruit) some regions of collapsed lung. There has been considerable enthusiasm for the use of such recruitment maneuvers which, in combination with the application of PEEP, may keep these newly recruited lung units open. Techniques vary from sophisticated determination of recruitment and derecruitment by stepwise alteration of ventilatory pressures and repeated blood gas sampling through to simply temporarily increasing PEEP to 30 cmH2O for 30 s. Improvements in oxygenation have often been demonstrated, although these may not be sustained. Such high intrathoracic pressures are often accompanied by brief episodes of reduced cardiac output and hypotension, but rarely barotrauma. The role of recruitment maneuvers still has to be claried in ARDS.

Partial liquid ventilation Partial liquid ventilation has been proposed to improve oxygenation and limit VILI. Peruorocarbons are dense liquids that have little biological activity but very low surface tension and exceptionally high-solubility coefcients for oxygen and carbon dioxide. The lung is lled (partially or fully) with the peruorocarbon and then normal mechanical ventilation applied simultaneously. Since the peruorocarbon is volatile, it evaporates over time through the ventilator circuit. Animal studies suggest that peruorocarbons reduce atelectasis, improve perfusion matching, and improve secretion clearance. Some studies suggest they may also be anti-inammatory, reducing permeability rises and neutrophil activation. Human studies have revealed an increase in the incidence of pneumothoraces, mucus plugging, and a disruption of the normal surfactant system. They are ingested by macrophages and their effect on immune function is unclear. A phase II/III study of partial-liquid ventilation is underway in France currently. Extracorporeal gas exchange Traditionally, extracorporeal gas exchange (ECGE) has only been used as a last resort in patients in whom oxygenation cannot be maintained by any other means. Devices vary from those which oxygenate and support the whole cardiac output, to those that provide partial oxygenation, and carbon dioxide removal to supplement conventional ventilation at lower pressures (extracorporeal lung assist, ECLA). Hemorrhage and coagulopathies have been the most significant complications, occurring in about 67% of adult patients. Although no mortality advantage has ever been shown for ECGE, new technology such as heparinbonded circuits, nonocclusive roller pumps, pumpless circuits, and better anticoagulation control, that may reduce complications is leading to a re-evaluation of this modality. Surfactant administration Alveolar surfactant is lost early in ARDS. The administration of exogenous surfactant either via a bronchoscope or nebulizer may reduce surface tension within alveoli and prevent cyclical collapse, improving oxygenation, and ameliorating VILI. Multiple administrations appear necessary due to the neutralization of surfactant by edema uid. Despite improvements in oxygenation and phase I/II survival benets, no large studies of surfactant have demonstrated any mortality advantage and thus it is not administered to patients with ARDS. However, there are many outstanding questions regarding the optimal formulation, patient selection, and dosing regimen.

18

ACUTE RESPIRATORY DISTRESS SYNDROME

Inhaled vasodilators iNO and prostacyclin cause selective vasodilatation of the regions of the lung to which they are delivered by ventilation, thus improving ventilation/perfusion matching and oxygenation. Their short half-lives limit systemic vasodilatation and hypotension. Both agents typically improve arterial pO2 by about 20% in responders, and this may be enhanced by the administration of intravenous almitrine bismesylate, an agent that is reported to enhance hypoxic pulmonary vasoconstriction. iNO is usually maximally effective at inspired concentrations of less than 10 parts per million, although the doseresponse curve may vary considerably after 24 h. iNO and prostacyclin may also favorably inuence inammation, platelet activity, and vascular permeability. Although frequently used for their benecial effects on oxygenation, no survival benet has been shown for either agent in controlled studies. High-frequency ventilation The corollary of the fact that low tidal-volume ventilation is benecial is that high-frequency ventilation may further protect against VILI. Tidal volumes delivered by jet ventilators and oscillators are less than the anatomical dead space, and respiratory rates are greater than 1 Hz. Gas transport occurs via diffusion rather than bulk ow. To date, no mortality advantage has been demonstrated for these ventilatory techniques in adults. Noninvasive ventilation Noninvasive ventilation (NIV) avoids the need for endotracheal intubation and the associated risks of ventilator-associated pneumonias, sinusitis, and sedation. The use of NIV in hypoxemic respiratory failure is controversial and a consensus conference into the role of NIV in acute respirator failure found little data to support its use in ARDS. Indeed, the natural history of ARDS is often longer than the time for which many patients are able to tolerate tight-tting masks continuously. It may thus only delay endotracheal intubation.
Manipulation of the Alveolar Fluid Balance

evidence of organ hypoperfusion would seem inappropriate. The careful use of vasopressors may allow the limitation of uid administration. Apical sodium and probably chloride channels in alveolar epithelial cells play an important role in normal alveoli in maintaining a ux of water into the circulation. A subgroup of the sodium channels can be stimulated by b-agonists such as salbutamol and terbutaline which may be administered either as inhaled preparations or as continuous intravenous infusion. Recent data suggest that salbutamol infusions can reduce extravascular lung water. How this inuences outcome in ARDS is not known and thus these agents remain experimental.
Manipulation of the Inammatory Process

Corticosteroids Corticosteroids can reduce inammation by repressing transcription and destabilizing pro-collagen mRNA. Initially evaluated in large doses in patients considered at risk of ARDS or early in ARDS, they have no benecial effects on mortality. By contrast, their administration later when repair and remodeling have commenced, may be helpful. Possible negative effects include nosocomial infection, hyperglycemia and an exacerbation of critical illness poly(myo)neuropathy. In a study of only 24 patients, corticosteroids caused a significant improvement in oxygenation and reduced mortality in patients with unresolving ARDS. Methylprednisolone (2 mg kg 1 day 1) was administered from day 8 for the shorter of either the duration of mechanical ventilation or 14 days. Although the study was methodologically limited, many clinicians administer a similar regimen to patients with persistent ARDS after 7 days and in whom there is little evidence for systemic sepsis. The results of the North American Late Steroid Rescue Study (LaSRS) will shortly provide better evidence regarding the role of corticosteroids in ARDS. Other agents Other pharmacological agents that have been assessed in ARDS/ALI are outlined in Table 3. None have been demonstrated to improve mortality despite good rationale for their use and promising preliminary studies.

High pulmonary capillary pressures increase extravascular lung water and result in worsening oxygenation. Indeed, a persistently positive uid balance is associated with a worse outcome in ARDS. It is difcult to ascertain whether this association is due to detrimental effects of administered uids per se or simply a reection of the severity of the underlying illness requiring uids for cardiovascular support. Nevertheless, whilst it is critical to maintain organ perfusion with an adequate cardiac output, the administration of intravenous uids in the absence of

Outcome
Over the last ten years, mortality has fallen from around 60% to around 30% in the current major trials. Although multifactorial in origin, this is in part due to improvement in the whole critical-care process but a greater appreciation that inappropriate mechanical ventilation can further injure the inamed lung. Most patients succumb to multi-organ failure

ADAMs AND ADAMTSs 19

Table 3 Other Pharmacological agents assessed in ARDS/ALI Agent Prostaglandin E1 Rationale A direct pulmonary vasodilator, inhibitor of platelet aggregation, and inhibitor of neutrophil adhesion Effects Mortality unaltered More rapid improvement in oxygenation Mortality unaltered

Dazoxiben

Inhibitor of thomboxane synthase and thus acts as a pulmonary vasodilator and inhibitor of platelet aggregation Inhibitor of thomboxane synthase and 5-lipoxygenase, thus reducing production of leukotrienes, neutrophil chemokines Antioxidant that reduces damage by reactive oxygen species

Ketoconazole

Mortality unaltered

N-acetyl cysteine

Mortality unaltered Improved oxygenation and compliance Mortality unaltered

Lisofylline

Inhibitor of phosphatidic acid which increases cytokine production and activates neutrophils

or sepsis rather than specic pulmonary failure and an inability to maintain adequate oxygenation. Follow-up of patients who survive ARDS has demonstrated progressive improvement in their lung function that continues to improve even one year after discharge from the ICU. Lung volumes seem to improve more rapidly than diffusing capacity and the distance walked in six min. Radiologically, previously densely consolidated areas frequently appear normal, with brosis of nondependent regions. By contrast, survivors often have considerable long-term nonrespiratory morbidities, particularly related to physical strength and to psychosocial, functional, and stress indices. Indeed, less than 50% return to work within 12 months.
See also: Corticosteroids: Therapy. Extracorporeal Membrane-Gas Exchange. Fluid Balance in the Lung. Genetics: Gene Association Studies. Hypoxia and Hypoxemia. Leukocytes: Neutrophils. Lung Imaging. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading
Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. New England Journal of Medicine 351: 327336. Evans TW, Grifths MJD, and Keogh BF (eds.) (2002) European Respiratory Monograph: ARDS, vol. 70(20). Shefeld: ERS Journals. Herridge MS, Cheung AM, Tansey CM, et al. (2003) One-year outcomes in survivors of the acute respiratory distress syndrome. New England Journal of Medicine 348: 683693. Meduri GU, Headley AS, Golden E, et al. (1998) Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial. Journal of the American Medical Association 280: 182183. Pinhu L, Whitehead T, Evans T, and Grifths M (2003) Ventilatorassociated lung injury. Lancet 361: 332340. The Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342: 13011308. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 13341349.

ADAMs AND ADAMTSs

C P Blobel, Cornell University, New York, NY, USA S S Apte, Cleveland Clinic Foundation, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved. extracellular milieu. In principle, proteolysis can activate or inactivate a substrate, or can change its functional properties. ADAM (a disintegrin and metalloprotease) and ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats) proteases are related members of a superfamily of metalloendopeptidases that also includes matrix metalloproteinases (MMPs) and astacins. ADAMs are integral membrane proteins that typically cleave other membrane anchored proteins, whereas ADAMTS proteases lack a membrane anchor, and process both secreted and cell surface molecules.

Abstract
Proteolysis has emerged as a key posttranslational regulator of the function of molecules on the cell surface and in the

20

ADAMs AND ADAMTSs

ADAMs are implicated in fertilization, neurogenesis, regulation of the function of ligands for the epidermal growth factor receptor, and the release of proteins such as the proinammatory cytokine tumor necrosis factor alpha (TNF-a) from the plasma membrane. ADAMTS proteases have key roles in the molecular maturation of von Willebrand factor and procollagen and are implicated in the pathogenesis of osteoarthritis. Here, we provide a general overview of the biochemical properties and physiological functions of ADAMs and ADAMTS proteases and describe their relevance to lung and airway disorders.

sequence homology to fertilin, or based on functional assays, such as their ability to shed the proinammatory cytokine TNF-a or process and activate the cell surface receptor Notch.
Structure

ADAMs
Introduction

ADAMs (a disintegrin and metalloprotease) are a family of membrane anchored glycoproteins that have been implicated in cleaving and releasing proteins from the cell surface. This process, which is referred to as protein ectodomain shedding, affects the function of molecules with key roles in development and disease, including growth factors such as transforming growth factor alpha (TGF-a), heparinbinding epidermal growth factor (HB-EGF), cytokines such as tumor necrosis factor alpha (TNF-a), and receptors such as the tumor necrosis factor receptor 1(TNFR1). The rst recognized ADAMs were the two subunits of a sperm protein termed fertilin, which has a critical role in fertilization. Other ADAMs were subsequently identied based on their

The typical domain organization of an ADAM is shown in Figure 1. Of the over 30 ADAMs that have been identied to date, only about half contain a catalytic site consensus sequence and are therefore predicted to be catalytically active. The remaining ADAMs that are not catalytically active are thought to function mainly in cellcell or cellmatrix interactions.
Regulation of Production and Activity

All catalytically active ADAMs are synthesized with a pro-domain that helps the metalloprotease domain fold in the endoplasmic reticulum (ER), and keeps it inactive until the pro-domain is cleaved, usually just before reaching the trans-Golgi network. Once the pro-domain is removed, additional mechanisms, including phorbol esters, phosphatase inhibitors, calcium ionophores, and activation of G-protein-coupled receptors, regulate the catalytic activity of ADAMs. The disintegrin domain and cysteine-rich region of ADAMs are thought to have a role in cellcell interaction and potentially also in

Cell

ADAMs Pro Catalytic Disintegrin CRD EGF-like TM Cytosolic

TSR ADAMTSs Pro Catalytic Protease domain Disintegrin-like CRD

TSR

(014)

Spacer

Ancillary domain

Figure 1 All ADAMs are membrane-anchored glycoproteins that are characterized by a conserved domain structure: an N-terminal signal sequence followed by pro (Pro), metalloprotease, and disintegrin domains, a cysteine-rich region (CRD), usually containing an EGF repeat, and nally a transmembrane (TM) domain and cytoplasmic tail. Only about half of the known ADAMs have a catalytic site consensus sequence, and are therefore predicted to be catalytically active. The disintegrin domain was rst identied in snake venom toxins that bind to platelet integrins and function as anticoagulants, but is now known to be a characteristic feature of all ADAM and ADAMTS proteins. ADAMTS proteins are similar to ADAMs in the prometalloprotease domain, but unlike ADAMs, all ADAMTSs are catalytically active. ADAMTSs have a functionally critical ancillary domain containing specic modules not found in ADAMs, e.g., thrombospondin type 1 repeats (TSRs) (see text for details). A critical difference between these protease families is the absence of an integral membrane segment in ADAMTS proteases.

ADAMs AND ADAMTSs 21

substrate recognition. Finally, the cytoplasmic domain of catalytically active ADAMs usually contains signaling motifs such as potential phosphorylation sites and proline-rich Src-homology 3 (SH3) ligand domains, and several molecules that interact with the cytoplasmic domains of ADAMs and might regulate their maturation and function have been identied. The catalytic activity and substrate selectivity of ADAMs has been explored using both biochemical and cell biological approaches. One important conclusion from biochemical studies was that individual ADAMs do not have a clear consensus cleavage site in vitro. Since ADAMs and their substrates are both membrane anchored, cell-based assays are critical tools for understanding the substrate selectivity and regulation of ADAMs in the context of the plasma membrane. Studies using cells from ADAM knockout mice, or cells treated with small inhibitory RNA (siRNA) against different ADAMs, have revealed that these enzymes display substrate selectivity in cells, although the mechanism underlying this remains to be established. A common feature, however, is that ADAMs frequently cleave their substrates close to the plasma membrane, resulting in release or ectodomain shedding of the substrates soluble ectodomain.
Biological Function

ADAM17 resemble mice lacking TGF-a or HB-EGF or EGFR. Ectodomain shedding is also a key mediator of the role of TNF-a in autoimmune diseases such as rheumatoid arthritis. Interestingly, receptors can be either inactivated by ectodomain shedding, such as the TNFR1, or activated, such as Notch. Mutations in the cleavage site of the TNFR1 that decrease its shedding cause accumulation of the receptor, leading to increased susceptibility to TNF in patients with TNF-receptor associated periodic febrile syndrome (TRAPS).
Function of ADAMs in Lung Development and in Respiratory Diseases

As noted above, ADAM-dependent ectodomain shedding can profoundly affect the function of the released substrate protein. Ectodomain shedding can enable the released molecule to act at a distance from the cell that it was shed from, which is referred to as paracrine signaling. For example, processing of the EGF receptor (EGFR) ligands TGF-a and HB-EGF by ADAM17 is critical for activation of the EGFR during development, and therefore mice lacking

Currently, ADAM17 is the only ADAM with a clearly established role in lung development (Figure 2). Mice lacking ADAM17 are born with respiratory distress, presumably caused by abnormal alveoli with septation defects and thickened mesenchyme, as well as impaired branching morphogenesis and delayed vasculogenesis, and thus reduced surface for gas exchange. Since similar defects are observed in mice lacking HB-EGF or the EGFR, the abnormal lung development in Adam17 / mice is most likely explained by a lack of HB-EGF shedding. With respect to respiratory diseases, smoking has been implicated in the activation of ADAMs and the release of EGFR ligands such as amphiregulin. The resulting activation of the EGFR can presumably contribute to the pathogenesis of lung cancer by stimulating cell proliferation and DNA replication at the same time that mutagens are delivered in smoke. Moreover, Grampositive bacteria stimulate the G-protein-coupled platelet activating receptor (PAR) in patients with cystic brosis, which in turn activates ADAMdependent release of HB-EGF, and thus mucin production. Therefore, inhibitors of ADAMs, such as hydroxamic acid type metalloprotease inhibitors,

HB-EGF

HB-EGF ADAM17 Extracellular space

Cell membrane

Cytoplasm
Figure 2 Proteolytic processing of heparin-binding EGF-like growth factor (HB-EGF) by ADAM17 releases this growth factor from its membrane tether. The ADAM17-dependent ectodomain shedding of HB-EGF is thought to be critical for the function of this growth factor during lung and heart development.

22

ADAMs AND ADAMTSs

might be useful in the treatment of cystic brosis. Finally, mutations in the ADAM33 gene have been linked to asthma susceptibility, although the mechanism underlying the role of ADAM33 in asthma remains to be determined. In light of the key roles of ADAMs in regulating signaling via the EGF receptor and other cell surface signaling pathways, and the critical roles for ADAMs in lung development and in asthma and cystic brosis, it appears likely that further studies of this protein family in the context of respiratory disease will uncover novel functions, thus hopefully also providing new targets for drug design.

for example, ADAMTS7 and ADAMTS12, constituting one such subfamily, are the only metalloproteases known to have mucin modules and glycosaminoglycan attachment sites.
Regulation of Production and Activity

ADAMTSs
Introduction

ADAMTS (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type 1 repeats) comprises a family of 19 secreted metalloproteases that is distinct from the membrane-anchored ADAMs. The founding member of this family, ADAMTS1, was described very recently, in 1997. ADAMTS1 was so named because it resembled the ADAMs in the metalloprotease domain and disintegrin-like module and was thought to be a variant ADAM. Its main point of distinction from ADAMs, apart from the absence of a transmembrane segment, was the presence of three modules resembling thrombospondin type 1 repeats (TSRs). Soon afterwards, it became clear that all 19 ADAMTS proteases shared these major features.
Structure

A typical ADAMTS consists of prometalloprotease and ancillary domains. The prometalloprotease domains resemble ADAMs, since the active site sequence is of the reprolysin (snake venom) type. Basic amino acid rich sequences providing sites for removal of the pro-domain by subtilisin-like proprotein convertases (SPCs) are present in the prometalloprotease domain and at its junction with the catalytic domain. The ancillary domain (from N-terminus to C-terminus) consists of a disintegrinlike module, a central TSR, a cysteine-rich module, a cysteine-free spacer, and a variable number of additional TSRs, ranging from 0 (ADAMTS4) to 14 (ADAMTS9 and 20) (Figure 1). An interesting feature of ADAMTS proteases is their clear grouping into distinct subfamilies. Proteases within a subfamily have an identical modular organization, gene structure, and active site sequence, suggesting evolution by gene duplication from a common precursor. Each subfamily has a distinct modular organization,

Transcriptional regulation appears to be very important, since many ADAMTS mRNAs are highly regulated during embryogenesis, for example, ADAMTS2, 3, and 14, or induced in specic circumstances such as inammation, for example, ADAMTS1. ADAMTS proteases are synthesized as zymogens and undergo removal of the prometalloprotease domain by SPCs either within the secretory pathway or at the cell surface. Subsequent to SPC processing, proteolysis within the ancillary domain appears to be an important posttranslational regulator of activity, resulting in further activation or inactivation of the enzymes. Several ADAMTS proteases bind to the cell surface through unknown mechanisms that suggest activity as operational cell surface proteases, and indicate locations at which posttranslational processing may occur. Unlike ADAMs, the ADAMTSs typically attack specic cleavage sites in their substrates. For instance, ADAMTS4 is a glutamyl endopeptidase that processes GluXaa bonds in many proteoglycans and ADAMTS13 specifically cleaves von Willebrand factor (vWF) at the Tyr1605Met1606 peptide bond. An important concept in the ADAMTS family is that their catalytic domains alone do not retain activity towards natural substrates. The ancillary domain is critical for the recognition and binding of substrates. In many instances, this requires specic posttranslational modications in the substrates (such as glycosylation of proteoglycans, triplehelicity in the case of procollagens, and physical unfolding of vWF by uid shear force). In addition, the minimal substrate binding sites are quite extended in length and difcult to reproduce in synthetic peptides. This makes the development of assays for ADAMTS activity and inhibition quite challenging. The only known ADAMTS inhibitors are tissue inhibitor of metalloprotease 3 (TIMP-3) and a2-macroglobulin.
Biological Function

Unexpectedly diverse functions for ADAMTS proteases have been revealed through human genetic disorders and transgenic animals. All ADAMTS genetic disorders are recessive, as is typical of enzyme deciencies. Inherited thrombocytopenic purpura results from ADAMTS13 mutations, with retention of unusually large polymers of vWF and failure to

ADAMs AND ADAMTSs 23

process these into forms that are optimal for coagulative homeostasis. Acquired thrombocytopenic purpura may result from circulating anti-ADAMTS13 autoantibodies. EhlersDanlos syndrome (type VIIC or the dermatosparactic type) is a consequence of ADAMTS2 mutations. In this disorder, tissue (especially skin) fragility results from lack of complete procollagen processing and a decrease in structurally competent collagen brils. ADAMTS1 is needed for mouse urinary tract development and fertility, and inhibits angiogenesis in vitro and in vivo by binding to vascular endothelial growth factor (VEGF). ADAMTS4 and ADAMTS5 null mice are developmentally normal, but ADAMTS5 mice are resistant to both mechanically induced and immune arthritis, suggesting that ADAMTS5 is a major cartilage-degrading enzyme. ADAMTS10 mutations cause WeillMarchesani syndrome (WMS), comprising short stature, brachydactyly, cardiovascular defects, and ectopia lentis. Intriguingly, several aspects of WMS are the opposite of those seen in Marfan syndrome, a fairly common inherited connective tissue disorder. ADAMTS20 mediates melanoblast migration from the neural crest and is decient in a natural mouse mutant named Belted because of the belt of white fur across the torso.
Receptors

See also: Asthma: Overview. Epidermal Growth Factors. Matrix Metalloproteinases. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading
Apte SS (2004) A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. International Journal of Biochemistry and Cell Biology 36: 981985. Becherer JD and Blobel CP (2003) Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs). Current Topics in Developmental Biology 54: 101123. Blobel CP (2005) ADAMs: key players in EGFR-signaling, development and disease. Nature Reviews: Molecular Cell Biology 6: 3243. Dagoneau N, Benoist-Lasselin C, Huber C, et al. (2004) ADAMTS10 mutations in autosomal recessive WeillMarchesani syndrome. American Journal of Human Genetics 75: 801806. Gao G, Plaas AH, Thompson VP, et al. (2004) ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by GPI-anchored MT4-MMP and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. Journal of Biological Chemistry 279: 1004210051. Kheradmand F and Werb Z (2002) Shedding light on sheddases: role in growth and development. BioEssays 24: 812. Kuno K, Kanada N, Nakashima E, et al. (1997) Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inammation associated gene. Journal of Biological Chemistry 272: 556562. Lapiere CM and Nusgens BV (1993) EhlersDanlos type VII-C, or human dermatosparaxis: the offspring of a union between basic and clinical research. Archives of Dermatological Research 129: 13161319. Lemjabbar H and Basbaum C (2002) Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nature Medicine 8: 4146. Lemjabbar H, Li D, Gallup M, et al. (2003) Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin. Journal of Biological Chemistry 278: 2620226207. Levy GG, Nichols WC, Lian EC, et al. (2001) Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413: 488494. Porter S, Clark IM, Kevorkian L, and Edwards DR (2005) The ADAMTS metalloproteinases. Biochemical Journal 386: 1527. Shapiro SD and Owen CA (2002) ADAM-33 surfaces as an asthma gene. New England Journal of Medicine 347: 936938. Stanton H, Rogerson FM, East CJ, et al. (2005) ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 434: 648652. White JM (2003) ADAMs: modulators of cellcell and cellmatrix interactions. Current Opinion in Cell Biology 15: 598606. Zhao J, Chen H, Peschon JJ, et al. (2001) Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Developmental Biology 232: 204218.

Although some ADAMTS proteases can bind to the cell surface, a specic receptor, syndecan-1, has been identied only for ADAMTS4. Nevertheless, because of the afnity of many ADAMTS for heparin and chondroitin sulfate, cell surface proteoglycans may constitute a broad category of receptors for this family.
ADAMTS Proteases in Respiratory Diseases

Since thrombocytopenic purpura is a systemic coagulation disorder, patients can develop microthrombi in their lungs and have sometimes manifested with acute respiratory distress syndrome. Pneumothorax has occasionally been reported in EhlersDanlos syndrome type VIIC patients and Adamts2 null mice have widening of their distal air spaces. These mice show deciency of procollagen I as well as procollagen III processing. Both collagens are structurally major components of the lung. Although ADAMTS10 is highly expressed in the lung, WMS cases are not reported to have lung problems.

24

ADENOSINE AND ADENINE NUCLEOTIDES

ADENOSINE AND ADENINE NUCLEOTIDES

R Polosa, University of Catania, Catania, Italy D Zeng, CV Therapeutics, Palo Alto, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Adenosine is a purine nucleoside. A growing body of evidence has emerged in support of a proinammatory and immunomodulatory role for adenosine in the pathogenic mechanisms of chronic inammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). The fact that adenosine enhances mast cell allergen-dependent activation, that elevated levels of adenosine are present in chronically inamed airways, and that adenosine given by inhalation causes dose-dependent bronchoconstriction in subjects with asthma and COPD emphasizes the importance of adenosine in the initiation, persistence, and progression of these common inammatory disorders of the airways. These distinctive features of adenosine have been recently exploited in the clinical and research setting to identify innovative diagnostic applications for asthma and COPD. In addition, because adenosine exerts its multiple biological activities by interacting with four adenosine receptor subtypes, selective activation or blockade of these receptors may lead to the development of novel therapies for asthma and COPD. In this article, the evidence for the roles of adenosine receptors in the pathophysiology of chronic inammatory airway diseases such as asthma and COPD are reviewed.

IgE-dependent mediator release from isolated rodent mast cells. A few years later, adenosine (but not its metabolite inosine or the unrelated nucleoside guanosine) administered by inhalation was shown to be a powerful bronchoconstrictor of asthmatic but, importantly, not of normal airways. Asthma and chronic obstructive pulmonary disease (COPD) are complex syndromes sharing clinical heterogeneity and a number of pathogenic traits, which include variable degrees of airow obstruction, bronchial hyperresponsiveness (BHR), and chronic airway inammation. In addition to its known effect as a bronchoconstrictor, a growing body of evidence has emphasized the importance of adenosine in the initiation, progression, and control of chronic inammation and remodeling of the airways. The key evidence can be summarized as follows: 1. Adenosine is generated in high concentrations at sites of tissue injury (e.g., hypoxia and inammatory cell activation). 2. Elevated levels of adenosine are present in chronically inamed airways; they have been observed both in the bronchoalveolar lavage (BAL) uid and the exhaled breath condensate (EBC) of patients with asthma, and adenosine concentrations are also increased after specic allergen challenge in the plasma of atopic individuals and in the BAL uid obtained from sensitized rabbits. 3. The progressive accumulation of adenosine in the lungs of mice lacking the purine catabolic enzyme adenosine deaminase (ADA) is strongly associated with development of lung inammation, tissue eosinophilia, airway hyperresponsiveness mucus metaplasia, airway remodeling, and emphysemalike injury of the lung parenchyma. 4. Adenosine administration by inhalation induces concentration-dependent bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. 5. Adenosine augments allergen-induced mediator release from human mast cells in vitro, and potentiates the immediate response to allergen in asthmatics. 6. Airway hyperresponsiveness to adenosine better reects the inammatory status of the lung in asthma compared with directly acting bronchospasmogens including methacholine; the exquisite sensitivity of adenosine challenge to detect inammatory changes in human airways has been

Introduction
The cardiovascular actions of adenosine were rst described in a classic publication by Drury and SzentGyorgyi in 1929. It was shown that adenosine causes coronary vasodilation, hypotension, and bradycardia. In 1963, Berne proposed the hypothesis that adenosine mediates the metabolic regulation of coronary blood ow. Since then, a large body of literature has supported the critical role of adenosine in modulating numerous cardiovascular functions. Two well-characterized effects of adenosine have successfully found clinical applications. First, adenosine causes bradycardia, slows A-V nodal conduction, and reduces atrial contractility, and is used as a rapid intravenous bolus for the acute termination of re-entrant supraventricular arrhythmias. Second, adenosine causes coronary vasodilation and increased blood ow, and it is used with radionuclide imaging in the heart to detect underperfused areas of myocardium. The action of adenosine in pulmonary diseases was rst discovered in the late 1970s. Holgate and colleagues observed that adenosine and related synthetic analogs were potent agents in augmenting

ADENOSINE AND ADENINE NUCLEOTIDES 25

benecially exploited to evaluate modications in the level of airway inammation in a prospective and noninvasive manner. 7. Dipyridamole, a blocker of facilitated adenosine uptake, may precipitate asthma. 8. Theophylline, an established asthma therapy, is an adenosine receptor antagonist. Low dose of theophylline blocks AMP-induced bronchoconstriction in asthmatics; this effect is unlikely due to phosphodiesterase inhibition.

cell function and indirectly by providing a more favorable environment for parenchymal cells, the best example of which is adenosine-mediated augmentation of nutrient availability via vasodilatation. Moreover, adenosine helps to maintain tissue integrity by modulating the function of the immune system.

Adenosine Receptors
Adenosine elicits its biological activities by interacting with four adenosine receptor subtypes designated as A1, A2A, A2B, and A3 adenosine receptors. The genes for these receptors have been cloned from humans and several animal species. Tissue distributions of these receptors have been determined at the mRNA levels using Northern blot or in situ hybridization techniques or at the protein levels using subtype-selective radioligands or antibodies. In general, these receptors are widely expressed in many tissues. For example, high levels of A1 receptors are found in brain, adrenal gland, and adipose tissue whereas high levels of A2A receptors are found in spleen, thymus, striatum, and blood vessels. In addition, these receptors are often found to co-express in the same tissues or even on the same cells. The relative expression levels of these receptors have been found to be modulated by physiological and/or pathophysiological tissue environments. Although adenosine is the natural agonist for the four adenosine receptors, the ability of adenosine to activate these receptor subtypes varies. In many tissues, A1 and A2A receptors have relatively higher receptor reserves for adenosine, and can be activated by the physiological levels of adenosine and thus mediate the tonic action of adenosine. On the other hand, A2B and A3 receptors appear to have relatively lower afnities and/or receptor reserves for adenosine and require higher concentrations of adenosine for activation. However, the tissue adenosine levels in many pathophysiological conditions can reach significantly high levels to activate the A2B and A3 receptors. Numerous subtype-selective agonists and antagonists of adenosine receptors have been synthesized and are used as pharmacological tools. Although these ligands were classied as selective ligands based on their differential binding afnities for the four adenosine receptors, these compounds are often poorly characterized and not as selective as suggested. There are pharmacological reasons for the lack of functional selectivity. For example, potencies of an agonist for one given receptor could vary from one tissue to another depending on the receptor expression levels and the coupling efciencies of the cells. As indicated before, adenosine receptors are widely distributed and their expression levels may be modied during disease processes. This

In light of these observations, adenosine has been proposed to be an important mediator of asthma. The evidence for the role of adenosine receptor signaling in the pathogenesis of chronic inammatory disorders of the airways such as asthma and COPD is reviewed below.

Adenosine Metabolic Pathways

Adenosine is an endogenous nucleoside consisting of the purine base, adenine, in glycosidic linkage with the sugar ribose (Figure 1). Adenosine is present at low concentrations in the extracellular space and its levels are greatly increased under metabolically stressful conditions as a result of enzymatic cleavage of the nucleotide adenosine 50 -monophosphate (AMP) by the 50 -nucleotidase. This increase in adenosine formation may also occur during inammation when a large number of inltrating inammatory cells are competing for a limited oxygen supply. Intracellular levels of adenosine are kept low principally by its conversion to AMP by the enzyme adenosine kinase. Adenosine may also be degraded to inosine by ADA (Figure 2). It has been proposed that adenosine may function as a retaliatory metabolite. During injury, the imbalance of O2 supply and demand triggers the increased formation of adenosine. Adenosine may exert its protective effects by decreasing the energy demand of the tissue via a direct inhibitory effect on parenchymal

NH2 N N O N

HO

OH
Figure 1 Structure of adenosine.

OH

26

ADENOSINE AND ADENINE NUCLEOTIDES

Ecto-adenosine deaminase

Ecto-5-nucleotidase

AMP

Adenosine

Inosine

Extracellular space

Nucleoside transporter

5-nucleotidase

Adenosine deaminase

AMP
Adenosine kinase

Adenosine

Inosine

SAH hydrolase

SAH

Figure 2 Adenosine production and removal occur both intracellularly and extracellularly. Intracellularly, adenosine is formed by the action of 50 -nucleotidase, which dephosphorylates AMP, or by the action of S-adenosyl-homocysteine (SAH) hydrolase. When adenosine concentrations are high, it is phosphorylated to AMP by adenosine kinase (AK) or degraded to inosine by adenosine deaminase (ADA). Both 50 -nucleotidase and ADA are found in the extracellular space where they mediate the formation and degradation of adenosine. The intracellular and extracellular pools of adenosine are kept in equilibrium by the actions of bi-directional nucleoside transporters (NT). Inhibition of AK, ADA, or NT by drugs or genetic deletions raises the tissue adenosine level.

certainly adds complexity in predicting functional selectivity of agonists. In the case of antagonists, the functional blocking effects of competitive antagonists are dependent on the tissue levels of adenosine. If the levels of adenosine are too low to activate a given receptor, an antagonist for this receptor will not have any functional effects regardless of its binding afnity. In addition to these pharmacological issues, there are pharmacokinetic issues. In many cases, the half-life and tissue distributions of adenosine receptor ligands are poorly understood, making it difcult to draw conclusions on the role of receptor subtypes based on the absence of effects of selective ligands in animal models. In spite of these limitations, selective agonists and antagonists of adenosine receptors are commonly utilized to establish the functions mediated by adenosine receptor subtypes. The four adenosine receptors also differ in their ability to couple to G-proteins and activate various intracellular signaling pathways. In most cells, A1 and A3 receptors couple to Gi/o and inhibit adenylate cyclase activity whereas A2A and A2B receptors couple to Gs proteins and increase adenylate cyclase activity and intracellular cAMP levels. While the adenylate cyclasecAMPprotein kinase A axis is the most well-studied second messenger system involved in adenosine receptor function, it is clear that adenosine receptors utilize other signaling pathways as well. These include members of the mitogen-activated protein-kinase family, such as p38, p42/p44 (ERK 1/2), and c-jun terminal kinase, as well as various phospholipases, protein phosphatases, and ion channels.

Adenosine Biological Function

Many cell types that play important roles in the pathogenesis of chronic inammatory airway diseases are known to express adenosine receptors and to exhibit relevant effects through adenosine receptor signaling. These cell types include various inammatory cells, such as mast cells, eosinophils, lymphocytes, neutrophils, and macrophages, and the structural cells in the lung, such as bronchial epithelial cells, smooth muscle cells, lung broblasts, and endothelial cells. The role of the adenosine receptors in these inammatory and structural cells has been studied extensively using isolated cultured cells in vitro. In addition, numerous animal models have been developed and are extremely useful in determining the potential role of adenosine receptor subtypes in pulmonary functions.
A1 Adenosine Receptor

The A1 receptor has been implicated in both pro- and anti-inammatory aspects of disease processes. It has been shown that activation of the A1 receptor can promote activation of human neutrophils and monocytes and thus, their activation leads to proinammatory responses. On the other hand, A1 receptors have been reported to be involved in anti-inammatory and protective pathways in experimental models of injury in the heart, nerves, and kidney. The early evidence suggesting that the A1 receptor plays a role in asthma came from experimental work in rabbits rendered allergic by immunization

ADENOSINE AND ADENINE NUCLEOTIDES 27

protocols with allergen. These animals exhibited a bronchoconstrictor response to adenosine that was attenuated by pretreatment with A1 receptor blockers. In the same model, an antisense that targets the initiation codon of A1 receptor mRNA reduced the bronchoconstrictor response to adenosine and, more importantly, to the early response to allergen. However, the relevance of these observations to human asthma have been questioned due to the fundamental mechanistic difference between adenosine-induced bronchoconstriction in the allergic rabbit, which is due to activation of A1 receptors on the bronchial smooth muscle, and that in man, which appears to be dependent on activation of mast cells that do not express the A1 receptor. Furthermore, it has been shown recently that genetic removal of the A1 receptor gene from ADA-decient mice resulted in enhanced pulmonary inammation along with increased mucus metaplasia and alveolar destruction, thus indicating that in this experimental model, the activation of A1 receptors serves an anti-inammatory and/or protective role in the regulation of pulmonary disorders triggered by elevated adenosine levels.
A2A Adenosine Receptor

could result in suppression of airway inammation in asthma and COPD. These ndings support the hypothesis that A2A agonists could be potentially useful in controlling the inammatory processes.
A2B Adenosine Receptor

It is now well established that activation of A2A receptors on lymphoid cells leads to inhibition of an inammatory response; this is largely due to its ability to induce accumulation of intracellular cyclic AMP in activated immune cells. Perhaps the strongest evidence for the critical role of A2A receptors in the regulation of inammation in vivo originated from the elegant study of Ohta et al. using mice decient in A2A receptors. In this model, the absence of the A2A receptor resulted in enhanced tissue inammation and damage, thus suggesting a negative regulatory role for the A2A adenosine receptor subtype. In the airways, A2A receptors are present on most of the immunoinammatory cells that have been implicated in asthma. A2A receptors are expressed on mast cells, and their activation results in increases in the intracellular cAMP concentrations, which are known to inhibit the biochemical pathways implicated in the release of histamine and tryptase from human mast cells. Stimulation of A2A receptors on neutrophils inhibits neutrophil adherence to the endothelium, prevents upregulation of integrin expression stimulated with formyl-Met-Leu-Phe and inhibits degranulation of activated neutrophils and monocytes. Activation of T lymphocytes, which plays a key role in the recruitment of leukocytes to the lung in clinical asthma, is also suppressed by A2A receptor activation. Thus, there are a multitude of mechanisms by which activation at A2A receptors

The initial evidence for the role of A2B receptors in asthma and COPD came from selectivity studies of enprofylline, a methylxanthine structurally closely related to theophylline. It was shown that enprofylline is a selective antagonist for the A2B receptors whereas theophylline has similar binding afnities for A1, A2A, and A2B receptors. The therapeutic concentrations of theophylline and enprofylline match their afnities for A2B receptors. Thus, it has been proposed that A2B receptors are possible targets for the long-term clinical benet achieved with relatively low doses of theophylline and enprofylline. On the other hand, because the anti-inammatory effects of endogenous adenosine might be mediated by A1 and A2A receptors, blockade of A1 and A2A receptors could be disadvantageous. It has been proposed that a more selective antagonist of the A2B receptors may be more effective and safer than theophylline. Recently, A2B receptors have been shown to have proinammatory properties in many pulmonary cells. For example, functional human adenosine A2B receptors have been identied in mast cells, endothelial cells, bronchial smooth muscle cells, lung broblasts, and bronchial epithelium. Adenosine, via activation of A2B receptors, increases the release of various inammatory cytokines from human mast cells (HMC-1), from human bronchial smooth muscle cells, human lung broblasts, and human airway epithelial cells. These cytokines, in turn, induce IgE synthesis from human B lymphocytes, and promote differentiation of lung broblasts into myobroblasts. These ndings provide strong support for the hypothesis that adenosine, via activation of A2B receptors, could enhance airway hyperresponsiveness and the inammatory responses associated with asthma. Thus, an A2B antagonist could potentially be benecial in the treatment of asthma and other pulmonary inammatory diseases.
A3 Adenosine Receptor

The functional role of the A3 receptor in the pathogenesis of chronic airway inammatory diseases remains controversial due in large part to differences in the pharmacology of A3 receptors from different species. For instance, in rodents, mast cell degranulation and/or enhancement of mast degranulation in response to allergen appears to be dependent on A3 receptor activation. In humans, a relatively high density of functionally active A3 receptors is expressed in

28

ADENOSINE AND ADENINE NUCLEOTIDES

Adenosine

eosinophils. Transcript levels for the A3 receptor are elevated in lung biopsies of patients with asthma or COPD and appear to be involved in the inhibition of eosinophil chemotaxis when stimulated. Furthermore, inhibition of important proinammatory functions of human eosinophils by the selective A3 receptor agonist, IBMECA, has been reported. Because asthmatic inammation is characterized by extensive inltration of the airways by activated eosinophils, it is possible that the elevated adenosine concentrations associated with asthma could contribute to inhibition of eosinophil activation through stimulation of A3 receptors. In contrast, in their effort of dissecting out specic signaling pathways involved in adenosine-mediated pulmonary inammation and airway remodeling in ADA-decient mice, Young and colleagues have recently demonstrated that mice treated with the selective A3 receptor antagonists MRS 1523 resulted in a marked attenuation of pulmonary inammation, reduced eosinophil inltration into the airways, and decreased airway mucus production.

A2B receptor

Mast cell

Cytokines

Prostaglandins and leukotrienes Histamine

Bronchoconstriction
Figure 3 Mechanism of adenosine-induced bronchoconstriction. Adenosine, possibly via activation of A2B adenosine receptors, enhances the activation of airway mast cells, leading to increases in the release of potent contractile mediators such as histamine, leukotrienes, prostaglandins, and cytokines. These mediators in turn cause bronchoconstriction in human airways.

Adenosine in Respiratory Diseases

Adenosine may play a critical role in the pathogenesis of chronic inammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). Elevated levels of adenosine are present in chronically inamed airways; they have been observed both in the BAL uid and the EBC of patients with asthma. Adenosine levels are also raised after allergen exposure in the plasma and during experimental exacerbation of asthmatic symptoms due to exercise in atopic individuals. The observed adenosine elevations indicate that adenosine signaling may regulate aspects of acute and chronic airway disease. Consistent with the hypothesis of adenosine playing a critical role in the pathogenesis of chronic inammatory disorders, mice decient in ADA develop features of severe pulmonary inammation and airway remodeling in association with elevations of adenosine concentrations in the lung. Features of the pulmonary phenotype noted include the accumulation of eosinophils and activated macrophages in the airways, mast cell degranulation, mucus metaplasia in the bronchial airways, and emphysema-like injury of the lung parenchyma. Although the histology seen in ADA-decient mice fails to accurately resemble that of human asthma, since no epithelial shedding, subepithelial brosis, or muscle/submucosal gland hypertrophy were observed in this model, the ADA-decient mouse is a useful tool to study the pathogenetic role of adenosine in chronic airway inammation. The role of adenosine in the pathogenesis of asthma is not just limited to its biological effects in

airway inammation and remodeling. Adenosine administration by inhalation is known to elicit concentration-related bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. Since these initial observations were made, a considerable effort has been directed at revealing the ne mechanisms of adenosine-induced bronchoconstriction, which appear to involve a selective interaction with activated airway mast cells with subsequent release of preformed and newly formed mediators (Figure 3). An important development from adenosine research is the use of an adenosine (or AMP) inhalation challenge as an innovative diagnostic test for asthma and COPD. Compared with other noninvasive surrogate markers of airway inammation, monitoring of the responsiveness of airways to inhaled adenosine appears to have the selective ability to probe changes in airway inammation and it has been shown to be very useful when evaluating the effectiveness of different treatment regimens with inhaled corticosteroids. Because the airway response to inhaled adenosine is very sensitive to the effect of inhaled corticosteroids and is a good marker of disease activity, bronchoprovocation with adenosine has been proposed as a convenient and accurate biomarker to monitor corticosteroid requirements in asthma and to establish the appropriate dose needed to control airway inammation.

ADHESION, CELLCELL / Vascular 29

Conclusions and Future Direction

Over the course of 20 years, the initial observation of the bronchoconstrictive effect of inhaled adenosine has evolved to provide the basis for a new asthma therapy as well as a diagnostic test. Recognition of the potential role of adenosine receptor signaling in the pathogenesis of chronic airway inammatory diseases advocates the principle that modulating adenosine receptor signaling is likely to constitute a considerable advance in the management of asthma and COPD. The clinical evaluation of selective agonists or antagonists for the adenosine receptors should help elucidate the multiple roles of adenosine and its receptors in the pathophysiology of asthma and COPD.
See also: Asthma: Overview. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview.

Further Reading
Blackburn MR, Lee CG, Young HWJ, et al. (2003) Adenosine mediates IL-13-induced inammation and remodeling in the lung and interacts in an IL-13-adenosine amplication pathway. Journal of Clinical Investigation 112(3): 332344. Blackburn MR, Volmer JB, Thrasher JL, et al. (2000) Metabolic consequences of adenosine deaminase deciency in mice are associated with defects in alveogenesis, pulmonary inammation and airway obstruction. Journal of Experimental Medicine 192(2): 159170. Cronstein BN (1994) Adenosine, an endogenous anti-inammatory agent. Journal of Applied Physiology 76: 513. Cushley MJ, Tatterseld AE, and Holgate ST (1983) Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. British Journal of Clinical Pharmacology 15(2): 161165.

Feoktistov I and Biaggioni I (1995) Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofyllinesensitive mechanism with implications for asthma. Journal of Clinical Investigation 96(4): 19791986. Feoktistov I, Polosa R, Holgate ST, and Biaggioni I (1998) Adenosine A2B receptors: a novel therapeutic target in asthma? Trends in Pharmacological Sciences 19: 148153. Fozard JR (2003) The case for a role for adenosine in asthma: almost convincing? Current Opinion in Pharmacology 3(3): 264269. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, and Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classication of adenosine receptors. Pharmacological Reviews 53: 527552. Ohta A and Sitkovsky M (2001) Role of G protein-coupled adenosine receptors in down-regulation of inammation and protection from tissue damage. Nature 414: 916920. Polosa R, Ng WH, Crimi N, et al. (1995) Release of mast cellderived mediators after endobronchial adenosine challenge in asthma. American Journal of Respiratory and Critical Care Medicine 151: 624629. Rorke S, Jennison S, Jeffs JA, et al. (2002) Role of cysteinyl leukotrienes in adenosine 50 -monophosphate induced bronchoconstriction in asthma. Thorax 57(4): 323327. Ryzhov S, Goldstein AE, Matafonov A, et al. (2004) Adenosineactivated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. Journal of Immunology 172(12): 77267733. Shryock JC and Belardinelli L (1997) Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology and pharmacology. American Journal of Cardiology 79(12A): 210. Spicuzza L, Bonglio C, and Polosa R (2003) Research applications and implications of adenosine in diseased airways. Trends in Pharmacological Sciences 24(8): 409413. Zhong H, Belardinelli L, Maa T, and Zeng D (2005) Synergy between A2B adenosine receptors and hypoxia in activating human lung broblasts. American Journal of Respiratory Cell and Molecular Biology 32(1): 28.

ADHESION, CELLCELL
Contents

Vascular Epithelial

Vascular
H M DeLisser, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Four distinct families of adhesion molecules (cadherins, immunoglobulin superfamily members, selectins, and integrins) mediate

vascular cellcell adhesion. These molecules are required for the formation of the junctional complexes that enable the assembly of endothelial cells into functional vascular networks; they mediate the leukocyteendothelial adhesive interactions involved in the trafcking of leukocytes out of the circulation; and they contribute to contacts between pericytes and the endothelium that are important to the regulation of endothelial cell proliferation. In the lung these vascular cellcell interactions are the bases of the host defense to lung infection or injury, and the regulation of lung vascular permeability, but are disturbed or dysregulated in diseases such as asthma or the acute respiratory distress syndrome.

30

ADHESION, CELLCELL / Vascular

Description
Families of Adhesion Molecules

A diverse group of protein molecules mediate cell cell as well as cellmatrix adhesion. These cell adhesion molecules are grouped into four major families: cadherins, immunoglobulin (Ig) superfamily members, selectins, and integrins (Figure 1).

Cadherins Cadherins were initially identied as a family of single-pass, transmembrane proteins responsible for mediating calcium-dependent, homophilic, cellcell adhesion. These classical cadherins are now recognized to be part of a larger cadherin superfamily that also includes the structural and functionally related desmosomal cadherins as well as other subfamilies with cytoplasmic domains that are unrelated to those of the classical cadherins. (Unless otherwise specied, the term cadherin will refer to the classical protein.)

Proper cadherin expression and function are essential for normal cell segregation during embryonic development and are required for the initiation and maintenance of normal tissue architecture. The extracellular domain has a variable number of homologous repeats of B110 amino acids with putative Ca2 binding sequences (cadherin-repeats), while the cytoplasmic tail forms complexes with cytoplasmic proteins of the catenin family (a-catenin, b-catenin, g-catenin/plakoglobin, and p120) that mediate association with the actin cytoskeleton. Endothelial cells principally express two cadherins: vascular endothelial-cadherin (VE-cadherin), a molecule expressed almost exclusively by endothelial cells, as well as the more widely distributed N-cadherin. They appear to be functionally different, with VE-cadherin mediating endothelial cellcell adhesion, while N-cadherin may promote endothelial cell adhesion to pericytes and smooth cells. Other nonclassical cadherins, such as T-cadherin and VE-cadherin-2 (protocadherin 12), have been described on

Cadherins VE-cadherin

Ig superfamily

Selectins P-selectin

Integrins

PECAM-1 MAdCAM-1

E-selectin

L-selectin JAM-A C C

C C C C C C

C C C C C C C C C

Extracellular repeating elements Ca2+ binding sites Transmembrane domain Anchoring domains Cytoplasmic domain

lg-like domain Mucin-like domain Transmembrane and cytoplasmic domains PDZ binding motif C

Lectin-like domain EGF-like domain Short consensus repeat Transmembrane and cytoplasmic domains

Integrin Integrin

subunit subunit

Transmembrane and cytoplasmic domains

Figure 1 Families of cell adhesion molecules. Ilustrated are structural features that distinguish the members of each of the four major families of cell adhesion molecules. The extracellular domains of cadherins are composed of a variable number of extracellular repeats with putative Ca2 binding sites, and conserved sequences in the cytoplasmic domain. Immunoglobulin superfamily members share a common structure in which a variable number of Ig-like homology domains are present in the extracellular domains of the molecule. The presence of other structural motifs such as mucin-like domains contribute to the structural diversity of these receptors. The selectins are composed of an NH2-terminal lectin-like binding domain, followed by an EGF-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Integrins are heterodimeric proteins composed of two noncovalently linked a and b subunits, each with large extracellular domains and smaller but functionally important cytoplasmic tails.

ADHESION, CELLCELL / Vascular 31

the endothelium, but their functions are currently not known. Immunoglobulin superfamily The Ig superfamily represents a very diverse group of receptors that function in a variety of cell types and in a number of biological processes. Members of this family are dened by the presence of a variable number of Iglike homology domains in the extracellular domains of these molecules. The Ig-like motif represents a sandwich-like structure of two b-sheets each consisting of antiparallel b strands containing 510 amino acids. Appropriately positioned cysteine residues mediate the formation of intrachain disulde bonds and stabilization of each Ig-like domain. The presence of additional structural domains such as mucinlike regions (e.g., mucosal adressin cell adhesion molecule-1 (MAdCAM-1)) contribute to the structural diversity of this family. Several Ig superfamily members, including intercellular cell adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), MAdCAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the junctional adhesion molecules (JAMs), are expressed on the endothelium and play important roles in leukocyte endothelial adhesion. Ligand binding for these endothelial Ig superfamily molecules may be homophilic (PECAM-1 and the JAMs) or involve interactions with b2 (ICAM-1, JAM-A, and JAM-C) or a4 integrins (MAdCAM-1, JAM-B, and VCAM-1). Selectins The selectins are a small family of receptors found only on vascular-related cells that mediate the initial adhesive interactions of circulating leukocytes with the endothelium at sites of inammation or in lymphoid tissues. Selectin structure includes an N2-terminal lectin-like binding domain, followed by an epidermal growth factor (EGF)-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Three selectins (P-selectin, E-selectin, and L-selectin) have been identied, each differing in their patterns of expression and regulation. P-selectin is stored in cytoplasmic granules in platelets and endothelial cells, but is rapidly mobilized to the cell surface, where it is transiently expressed (minutes), in response to agonists such as histamine or thrombin. E-selectin is not present under resting conditions, but is synthesized and expressed briey (hours) on vascular endothelial cells following cytokine stimulation. In contrast, L-selectin is constitutitively expressed on most leukocytes but is proteolytically cleaved from the surface following cellular activation. Selectins recognize sialylated and fucosylated oligosaccharides (e.g., sialyl Lewis x (sLex) and related

tetrasaccharides), as well as sulfated molecules that lack sialic acid or fucose, such as sulfatides and heparin glycosaminoglycans. Thus, in vitro, selectins are able to bind to a variety of glycoproteins on leukocytes and endothelial cells bearing these structural motifs. Several of these are mucin-like glycoproteins with many serine or threonine residues that are potential sites for attachment of O-linked glycans. In vivo, however, P-selectin glycoprotein ligand-1 (PSGL-1) is the dominant ligand for P- and L-selectin in inammatory settings and may also be physiologically relevant for E-selectin. Recent reports also suggest that for neutrophils, CD44 may be also be a physiological E-selectin ligand. Additional L-selectin ligands have been identied on high endothelial venules (HEV) of lymphatic tissues and on some activated endothelial cells. These include CD34, glycosylated cell adhesion molecule (GlyCAM-1), and podocalyxin. Integrins The integrins are a family of heterodimeric membrane glycoproteins composed of noncovalently associated a and b subunits. To date 18 a and 8 b subunits have been identied and more than 24 a/b pairs have been described in vertebrate tissue. The combination of a and b subunits determines the ligand specicity. Although most integrins bind ligands that are components of the extracellular matrix, certain integrins (b2 and a4 integrins) bind receptors of the immunoglobulin superfamily (e.g., ICAM-1, MAdCAM-1, VCAM-1, and the JAMs). Significant redundancy, however, exists in that most integrins are capable of binding several different matrix or cell surface proteins and many of these ligands bind to multiple integrins. Through their cytoplasmic domains integrins are able to bind to a complex of cytoplasmic proteins, including a-actinin, vinculin, talin, and paxillin, linked to actin laments. These integrincytoskeletal assemblies in turn form the foundation for intracellular signaling cascades.
Endothelial CellCell Adhesion

Endothelial cells adhere to one another through two morphologically distinct junctional structures, adherens junctions (AJs) and tight junctions (TJs), each composed of characteristic molecules (Figure 2). Although very similar structures are present in epithelial cells, their distribution within intercellular junction differs in the two cell types. In epithelial junctions TJs are located toward the apical region of the intercellular contact with the AJs positioned below the TJs, while AJs and TJs are intermingled with each other along the endothelial intercellular junction. Gap junctions represent a third type of junctional complex formed by endothelial cells.

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ADHESION, CELLCELL / Vascular

Apical/luminal surface Junctional complexes Associated transmembrane proteins Occludin, claudins, JAMs

Tight junctions

Adherens junctions

Cadherins

Gap junctions

Connexins

Nonjunctional intercellular proteins

PECAM-1, endoglin

Basal lamina
Figure 2 Adhesion structures found in epithelial and endothelial cells. Shown are intercellular junctional complexes (TJs and AJs) and their associated transmembrane proteins. Gap junctions mediate intercellular communication and other proteins such as PECAM-1 and endoglin mediate cell adhesion but are not present in specic junctional complexes.

However, unlike AJs and TJs, these structures mediate cellcell communication rather than cellcell adhesion. Epithelial cells also express another type of adhesive complex, the desmosone, but these structures are absent from endothelial cells. Adherens junctions These junctions are formed by clusters of dimerized cadherin molecules that bind to cadherin dimers on adjacent cells. Cadherins localize at AJs only when cells contact one another. The short cytoplasmic domain of cadherins interacts through a C-terminal domain with b-catenin and plakoglobin (g-catenin). b-catenin and plakoglobin associate with a-catenin which in turn binds to actin microlaments. This association with the catenins is required for the adhesive function of cadherins and thus is essential to the assembly of AJs. An additional binding partner is p120, an src substrate that is homologous to b-catenin and plakoglobin. In contrast to b-catenin and plakoglobin, p120 binds loosely to a membrane proximal region of the cadherin cytoplasmic tail, associating with a-catenin or actin cytoskeleton. It appears that p120 is involved in the regulation of the cadherin turnover and stability. The formation of AJs provides the cellcell attachments that are required for the assembly of the endothelium into patent tubular networks. AJs also contribute to maintenance of endothelial barrier function, in addition to the regulation provided by TJs (see below). Anti-VE-cadherin antibodies, both in vitro and in vivo are found to increase vascular permeability by disrupting VE-cadherin clustering at the junctions while leaving other junctional structures

intact. Further, AJs are required for the organization and/or maintenance of TJs and gap junctions and thus are critical to the integrity of the entire intercellular junctional complex. In addition, at least two other nonadhesive functions have been ascribed to AJs. First, b-catenin, plakoglobin, and p120 are all able to translocate to the nucleus, where in conjunction with other transcription factors they are able to modulate gene expression. Consequently, the association of these catenins with VE-cadherin in AJs may maintain them at the membrane and thus prevent their nuclear translocation. Second, VE-cadherin engagement promotes endothelial cell quiescence by modulating vascular endothelial growth factor (VEGF) receptor-mediated signaling inhibiting the receptor-dependent proliferative responses, while activating receptor-mediated survival signals. Tight junctions These junctions were originally identied in epithelial cells by electron microscopy as dense regions in which membrane leaflets between the adjacent cells are closely opposed such that the membrane bilayers at the junctions are indistinguishable. In freeze-fracture preparations, TJs have the appearance of brillar strands within the membrane. They form a continuous seal around the apical region of the lateral membranes of adjoining cells, subdividing it into apical and basolateral domains. This is somewhat less so for endothelial cells, where the position of TJs relative to other intercellular junctional complexes can vary. Further, the level and extent of TJs depend on the vessel type and location. Endothelial cell TJs are well developed in arteries

ADHESION, CELLCELL / Vascular 33

and arterioles, but significantly less so in veins and postcapillary venules, the principal site for the extravasation of uid and inammatory leukocytes. TJs are also well developed in the vessels of the brain where they contribute to the bloodbrain barrier, but are much less organized in the vasculature of other organs. Like other junctional structures, TJs are composed of both transmembrane and intracellular molecules. Three types of TJ-associated integral membrane proteins have been identied. These are occludin, the claudins, and several Ig superfamily members, including the JAMs, endothelial cell-selective adhesion molecule (ESAM), and coxsackie- and adenovirus receptor (CAR). Occludin and the claudins (of which more than 20 have been identied) dene the presence of this junction and constitute the molecular basis of the TJ strands, while JAM-A may serve accessory functions such as the recruitment of TJ-associated proteins. The intracellular domains of occludin, the claudins, and the JAMs interact with zonula occludens-1 (ZO-1), a member of a family of membrane-associated, PDZ, and guanylate kinase domain-containing proteins. ZO-1 can bind to actin laments and thus anchor TJs to the cells cytoskeleton. In addition to ZO-1, other PDZ-containing and/or actin binding proteins are recruited that contribute to the formation and function of TJs. Tight junctions restrict both the diffusion of solutes through intercellular spaces (barrier function) and the movement of molecules between the apical and basolateral domains of the plasma membrane (fence function). As such, TJs are the major regulator of cell permeability via the paracellular pathway (i.e., the extracellular space between the lateral membranes of neighboring cells) and are important to the maintenance of cell polarity. TJs are also likely involved in leukocyte transendothelial migration, possibly through interactions mediated by the JAMs. Gap junctions Gap junctions constitute another junctional complex that bridges the intercellular space between adjacent endothelial cells. However, unlike the other junctional structures described above, which mediate cellcell adhesion, gap junctions promote intercellular communication. Metabolites, ions, and second messengers, including Ca2 , cAMP, and inositol triphosphate, are able to pass through gap junction channels from one cell to another, enabling the coordination of multicellular responses. Gap junction channels are dodecameric structures made up of the connexin family of proteins. Six individual connexin proteins oligomerize in the plasma membrane of one cell to form a hemichannel or connexon, and the docking of two connexons, one from each

opposing cell, results in a complete gap junction channel. Other intercellular junction proteins Endothelial cells express several other adhesive proteins that are concentrated in intercellular junctions but are not specifically located in either AJs or TJs. These include (1) PECAM-1, the loss of which compromises vascular barrier function and angiogenesis; (2) S-dndo 1, an Ig superfamily member and a mediator of homophilic adhesion; and (3) endoglin, a regulator of transforming growth factor beta (TGF-b) signaling, the absence of which leads to a phenotype reminiscent of VE-cadherin-null mice.
Endothelial CellLeukocyte Adhesion

The recruitment of leukocytes to sites of infection or injury begins with a well-dened set of sequential adhesive interactions between the circulating leukocyte and the activated endothelium (Figure 3). A similar cascade of events is believed to also mediate the trafcking of lymphocytes across high endothelial venules in lymphoid tissues. Endothelial activation Endothelial activation is characterized by the enhanced expression of endothelial selections (P- and E-selectin) and members of the Ig superfamiy (ICAM-1, MAdCAM-1, and VCAM-1). This typically involves de novo synthesis, with peak expression occurring over several hours: 46 h for E-selectin and 1224 h for VCAM-1 and ICAM-1. For P-selectin, however, endothelial surface expression occurs within minutes following stimulation with noncytokine mediators such as thrombin and histamine as preformed P-selectin is mobilized from WeibelPalade bodies. The expression of other molecules such as PECAM-1 and the JAMs that have been implicated in leukocyte trafcking do not appear to be affected by inammatory mediators. Leukocyte rolling The initial leukocyteendothelial adhesive event is one in which the circulating leukocyte rolls along the surface of the inamed endothelium (and on already adherent leukocytes). This process involves an initial capture and fast rolling of the leukocyte, followed by a period of slow rolling and deceleration prior to leukocyte arrest. This step has long been recognized as principally mediated by selectins, with each L- and P-selectin mediating the capture and fast rolling, while E-selectin is preferentially involved in the slow rolling. Interestingly, L-selectin-mediated capture and rolling requires a critical threshold of shear stress to occur, while rolling adhesion mediated P- and E-selectin

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ADHESION, CELLCELL / Vascular

Leukocyte rolling

Firm adhesion

Diapedesis

Capture

Fast rolling

Slow rolling

L-selectin P-selectin E-selectin 2 integrins/ICAM-1/2 4 1/IVCAM-1 or 4 7/MAdCAM-1 PECAM-1 JAMs CD99

Figure 3 Leukocyteendothelial interactions. The sequence of adhesive interactions involved in the emigration of leukocytes from the circulation across the endothelium to extravascular sites is illustrated. The steps of these interactions, in order, are leukocyte rolling, rm adhesion, and diapedesis (transendothelial migration). Each step is mediated, depending on the cell type and context, by the interactions of specic cell adhesion molecules and their ligands.

demonstrates weak or no dependence on a shear threshold. The basis for this appears to reside in the fact that L-selectin engages its ligands through exceptionally labile adhesive bonds that are only significantly stabilized above a critical shear threshold. Although originally thought to mediate only rm adhesion and transendothelial migration (see below), b2 integrins are now recognized to cooperate with Eselectin in the deceleration of the rolling leukocyte. Depending on the cell type, there is also evidence that a4b1, a4b7, and CD44 are able to respectively promote rolling on VCAM-1, MAdCAM-1, and hyaluronate-covered surfaces. Activationrm adhesion As the leukocyte rolls, interactions between selectins and their ligands and between chemokines and chemoattractants associated with the endothelium and G-protein-coupled receptors on the leukocyte surface, activate b2 (and probably a4) integrins on the leukocyte surface. This activation, which involves upregulation of integrin expression and adhesive activity and the shedding of L-selectin, enables the arrest and rm adhesion of the leukocyte to the endothelium. This phase of leukocyte recruitment is mediated by the binding of endothelial Ig superfamily members to (activated)

leukocyte integrins (ICAM-1/aMb2, ICAM-1, -2/a1, b2, VCAM-1/a4b1, and MAdCAM-1/a4b7). Transendothelial migration (diapedesis) The adherent leukocyte then crawls over the luminal surface, a process that involves the cyclic modulation of integrin receptor avidity. Once a junction has been located, the emigrating leukocyte squeezes between the closely opposed endothelial cells, crossing the basement membrane to enter the extravascular tissue. Significantly, this occurs without disrupting the integrity of the endothelium. This process of leukocyte transendothelial migration or diapedesis is mediated by at least ve cellcell adhesion molecules: Ig superfamily members PECAM-1 and JAM-A, -B and -C, as well as CD99, a molecule with a unique structure. With the exception of JAM-B, all of these molecules are expressed on both leukocytes and endothelial cells and thus are capable of homophilic adhesion. The JAMs also bind leukocyte integrins, heterophilic interactions that have also been implicated in their activity in leukocyte trafcking. PECAM-1 appears to be required for the initial entrance or penetration of the junction, while CD99 appears to be involved more distally in the passage through the intercellular junction. While traversing the junction, homophilic

ADHESION, CELLCELL / Vascular 35

interactions of leukocyte and endothelial PECAM-1 also upregulate a6b1 on transmigrating leukocytes, an integrin that is required for the passage of the leukocyte through the matrix of the basement membrane. Although animal studies have implicated the JAMs in leukocyte diapedesis, their specic roles remain to be determined.
PericyteEndothelial Cell Adhesion

Pericytes are perivascular cells imbedded within the basement membrane of the endothelium of capillaries and postcapillary venules. Contact between the endothelial cell and the pericyte is made by cytoplasmic processes of the pericyte indenting the endothelial cell, and vice versa. This results in the so-called peg and socket contact. The normally intervening basal lamina is absent at these pericyteendothelial cell interdigitations and growth factors (e.g., epidermal growth) factor may concentrate at these contacts. In some tissues, TJs between pericytes and endothelial cells have been reported and adhesion plaques have been described in the pericyte membrane. An inverse correlation exists between endothelial cell proliferation and the extent of pericyte coverage, with tissues demonstrating the slowest rate of endothelial cell turnover having the greatest pericyte coverage. This suggests that pericytes are a major negative modulator of capillary growth and hence a promoter of vessel quiescence. This may be mediated by the pericyte-derived TGF-b, an inhibitor of endothelial cell growth and migration, the activation of which is dependent on pericyteendothelial cell contact. Pericytes are also contractile, and thus points of cellcell contact may permit contractions of the pericyte to be transmitted to the endothelial cell to reduce the caliber of the vessel or alter vascular permeability.

the systemic vascular bed, including neutrophil recruitment mediated Streptococcus pneumoniae, HCl, and C5a, these stimuli mediate neutrophil extravasation from the pulmonary circulation that is independent of the b2 integrins. An even more complex picture is suggested by studies of mice decient in both P-selectin and E-selectin or P-selectin and ICAM-1. In these mutant animals, neutrophil emigration into the peritoneal cavity was completely inhibited during S. pneumoniae-induced peritonitis. In contrast, neutrophil extravasation into the alveolar space during S. pneumoniae-induced pneumonia was intact. Together these data suggest that leukocyte recruitment from the pulmonary circulation can occur through pathways that do not require selectins, b2 integrins, or ICAM-1. These observations may be due in part to the fact that primary site for leukocyte extravasation is in the pulmonary capillaries and not in postcapillary venules as occurs in the systemic vasculature. This is significant because the average diameter of the leukocyte (particularly the neutrophil) approaches or exceeds that of the capillaries. As a result, intravascular leukocytes must deform in order to transit through the pulmonary microvasculature and thus are in close apposition with the pulmonary endothelium. Consequently, processes that change the physical properties of the leukocyte or compromise their ability to deform may be sufcient to slow and arrest the leukocyte in the microcirculation of the lung. Thus, it should not be surprising that in the lung there may be less stringent requirements for molecular-meditated processes to capture and attach the intravascular leukocyte to inamed pulmonary endothelium.
Lung Permeability

Vascular CellCell Adhesion in Normal Lung Function

Lung Defenses

The recruitment of leukocytes is an essential component of the lungs defense against infection or inammatory insults. Although data certainly indicate that the leukocyteendothelial interactions described above may operate in the lung to regulate the emigration of circulating leukocytes, there is also evidence that leukocyte recruitment in the pulmonary circulation may differ, depending on the stimulus, from that which occurs in postcapillary venules at extrapulmonary sites. Although the b2 integrins have been shown to be required for neutrophil emigration in various models of acute inammation involving

In the lung, as in other vascular beds, endothelial cell permeability depends largely on the regulation of paracellular uid and solute transport through intercellular junctions. The degree of openness of this pathway is governed by the balance of centrally directed contractile forces and opposing forces generated by cellcell and cellmatrix adhesive complexes that tether endothelial cells to each other and to the basement membrane. Of the various junctional complexes, TJs are recognized as the primary determinants of the endothelial barrier function. However, disruption of VE-cadherin function results in interstitial edema in the lung and there is evidence that in the setting of hyperosmolarity, E-cadherin expression may be upregulated on the endothelium of the lung microvasculature where it acts to promote barrier function. These data suggest that cadherin-dependent

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ADHESION, CELLCELL / Vascular

activity mediated through AJs may also contribute to the regulation of vascular permeability in the lung. Barrier integrity varies with vessel type, as the endothelium of the pulmonary microcirculation is much more restrictive than that of the pulmonary arteries or veins. This suggests that there may well be site-specic differences in junctional architecture and/or cytoskeletal machinery in the pulmonary vasculature.

Vascular CellCell Adhesion in Respiratory Diseases

Asthma

Asthma is an inammatory disease characterized by increased inltration of various inammatory cells in the bronchial mucosa and airways. In allergic asthma, antigen exposure results in the activation of mast cells and TH2 lymphocytes and their subsequent release of a number of cytokines and lymphocyte or eosinophil chemoattractants. These inammatory mediators alter the expression and activity of cell adhesion molecules on these asthma-associated leukocytes and on the bronchial microvasculature in specic patterns that target their recruitment out of the circulation into the bronchial tissues. Analyses of bronchial tissue from allergic and other asthmatics, as well as studies of murine models of asthma indicate that interactions between E-selectin and its ligands, 1CAM-1 and aLb2 and VCAM-1 and a4b1, are involved in the recruitment of eosinophils in the allergic airway response. It should be noted however, that there are reports in which acute antibody inhibition of a4b1, b2 integrin, or ICAM-1 may reduce airway hyperresponsiveness without reducing eosinophil or lymphocyte accumulation. This suggests that these molecules may mediate processes other than leukocyteendothelial interactions, such as leukocyte activation or antigen presentation, that contribute to the development of the asthmatic phenotype.
Acute Lung Injury

as well as cytokines (TNF-a, IL-1, and g-IFN) increase transendothelial permeability, at least in part, by inducing endothelial cell contraction and the resultant formation of interendothelial gaps. These alterations in endothelial morphology require the recruitment and activation of calcium-dependent cytoskeletal proteins (e.g., actin and myosin) that alter endothelial cell contour and reorganize intercellular junctional complexes. The alterations in endothelial permeability induced by inammatory mediators may involve changes in the concentration of VE-cadherin and PECAM-1 in endothelial intercellular junctions as cytokines have been shown to redistribute these molecules out of endothelial cellcell contacts with a concomitant increase in permeability. An important contributor to the pathogenesis of endothelial dysfunction in ARDS appears to be activated neutrophils sequestered in and adherent to the pulmonary capillaries. These trapped leukocytes release reactive oxygen species and granular constituents that undermine the normal barrier function of the endothelium. These products may also contribute the inammatory cascade by activating monocytes and macrophages leading to release of additional proinammatory mediators. Inhibition of b2 integrins, ICAM-1, and selectins blocks neutrophil recruitment and permeability injury in animal models of acute lung injury or infection although there do appear to be alternative pathways as noted above. This has made these molecules attractive therapeutic targets for the treatment of ARDS. However, the potential for inhibition of host defenses and reparative responses continues currently to be an obstacle to the widespread application of this approach.

Acknowledgments
This work was supported by the Department of Defense (PR043482), National Institutes of Health (HL079090), and the Philadelphia Veterans Medical Center.
See also: Acute Respiratory Distress Syndrome. Adhesion, CellCell: Epithelial. Adhesion, Cell Matrix: Integrins. Asthma: Overview. Endothelial Cells and Endothelium. Leukocytes: Neutrophils; Monocytes; T Cells.

A large and diverse group of microbial and inammatory insults may acutely injure the lung culminating in the acute respiratory distress syndrome (ARDS) and respiratory failure. A key feature of this syndrome is endothelial cell injury and subsequent dysfunction manifested by impaired barrier function and increased vascular permeability. This dysfunction must necessarily involve destabilization of intercellular junctions, but the exact molecular basis is still being dened. A variety of inammatory agents including histamine, lipopolysaccharide, leukotriene B4, platelet activating factor, nitric oxide thrombin,

Further Reading
Allt G and Lawrenson JG (2001) Pericytes: cell biology and pathology. Cells Tissues Organs 169: 111. Angst BD, Marcozzi C, and Magee AI (2001) The cadherin superfamily. Journal of Cell Science 114: 629641.

ADHESION, CELLCELL / Epithelial 37

Bazzoni G and Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiological Reviews 84: 869901. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cellcell interactions in regulating lung function. American Journal Physiology (Lung Cell Molecular Physiology) 287: L455L459. Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 14871500. Hogg JC and Doerschuk CM (1995) Leukocyte trafc in the lung. Annual Review of Physiology 57: 97114. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673687. Juliano RL (2002) Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annual Review of Pharmacology and Toxicology 42: 283323. Ley K (2003) The role of selectins in inammation and disease. Trends in Molecular Medicine 9: 263268. Mandell KJ and Parkos CA (2005) The JAM family of proteins. Advanced Drug Delivery Reviews 57(6): 857867. Muller WA (2003) Leukocyteendothelial-cell interactions in leukocyte transmigration and the inammatory response. Trends in Immunology 24: 327334. Sohl G and Willecke K (2004) Gap junctions and the connexin protein family. Cardiovascular Research 62: 228232. Vincent PA, Xiao K, Buckley KM, and Kowalczyk AP (2004) VEcadherin: adhesion at arms length. American Journal Physiology Cell Physiology 286: C987C997. Vorbrodt AW and Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopists view. Brain Research Reviews 42: 221242. are critical in cell motility and differentiation and their loss may be associated with epithelial carcinogenesis. However, little is known about how specic changes in cellcell adhesion contribute to respiratory disease, and this remains an area of intense study.

Description
From the upper airway to distal alveoli, an essentially continuous epithelial sheet comprising of several specialized cell types lines the respiratory tract to create a semipermeable barrier to the outside world and provide protection from invading microorganisms. Essential to these functions are the complex structures that produce cellcell adhesion between individual epithelial cells. Far more than just cellular glue holding epithelial cells together, cellcell adhesion complexes organize development and differentiation of the lung and respiratory tract, promote and maintain structural and functional integrity of the epithelial surface, and regulate cellular responses to injury and disease. Integral to the consideration of cellcell adhesion in respiratory epithelia are the specic structural and functional characteristics of these cells. Respiratory epithelial cells are polarized, with distinct apical and basolateral domains, with the apical domain facing the external environment or lumen and the basolateral domain forming contacts with the substratum or neighboring cells. In addition to regulating cell shape, polarization contributes to the asymmetric distribution of organelles and molecules within epithelial cells and to the arrangement of cytoskeletal networks. Cellcell adhesion complexes are key determinants of epithelial cell polarity and are essential in major respiratory epithelial functions such as maintenance of the barrier to the external environment and other critical epithelial functions including secretion and repair. A high degree of similarity exists across different species in the structure of cellcell junctions and as in most vertebrate epithelia, three major types of junctions mediate intercellular adhesion in respiratory epithelial cells: tight junctions, adherens junctions, and desmosomes. Additionally, all epithelia studied thus far have an adhesive belt called the zonula adherens that encircles the cell just below the apical surface and that is characterized by an electron-dense cytoplasmic actin plaque (Figure 1). Along with the tight junction, which is just apical to the zonula adherens, this adhesive belt delineates the apical and basolateral epithelial compartments and is an important contributor to regulation of epithelial permeability and barrier function. Although the various epithelial cellcell junctions are composed of unique proteins and serve distinct functions, in general, they

Epithelial
J K McGuire, Childrens Hospital and Regional Medical Center, Seattle, WA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
A continuous epithelial sheet lines the respiratory tract to provide a barrier to the outside world and protect against invasion by microorganisms. Central to these functions are the complex junctions that regulate adhesion between adjacent epithelial cells. These junctions create epithelial polarity, regulate paracellular transfer of molecules across the epithelial barrier, and maintain the integrity of the epithelial layer. Far more than just cellular glue, the major junctional complexes that regulate cellcell adhesion, i.e., tight junctions, adherens junctions, and desmosomes, are critical in tissue patterning and differentiation during development, epithelial cell sensing of the pericellular environment, and modulation of intercellular signaling. Dozens of different proteins have been associated with cellcell adhesion functions in mammalian epithelia and only recently have the structure and function of specic proteins expressed at cellcell junctions in the lung and airways been identied. For example, the claudins are major transmembrane proteins regulating extracellular interaction at tight junctions, and the E-cadherin/b-catenin complexes that form adherens junctions

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ADHESION, CELLCELL / Epithelial

Tight junction

Adherens junction

Desmosome

Figure 1 Cellcell junctions in respiratory epithelia. Schematic diagram of the three major types of junctions mediating cell adhesion in epithelial cells. Tight junction transmembrane components occludin (in red above), claudin, and JAM (blue) interact in the extracellular space and cytosolic plaque proteins (green) interact with the actin cytoskeleton (pink). Adherens junctions are formed by transmembrane cadherins (orange) that bind the actin cytoskeleton (pink) via the catenins (yellow and blue). Desmosomes are formed by the desmosomal cadherins (blue and pink) and cytosolic plaque proteins (aqua) that interact with intermediate laments (green). Electron micrograph of mouse tracheal epithelium shows ultrastructure of zonula adherens near the apical/luminal epithelial surface.

Table 1 Major cellcell junction proteins Junction Tight junction Transmembrane proteins Occludin Claudins Junctional adhesion molecule (JAM) Cadherins Desmosomal cadherins Cytosolic proteins Plaque proteins Cytoskeletal linker proteins PDZ domain proteins (zonula occludens, PAR)

Adherens junction Desmosome

b-Catenin Plakoglobin, plakophilins, p0071

a-Catenin Plakins

share common structural features; principal among these are transmembrane receptors, usually glycoproteins that bind proteins on the extracellular surface and determine the specicity of intercellular interactions. The cytosolic domains of these receptors associate with a variety of cytoplasmic proteins that link them structurally to the cytoskeleton and regulate interaction with intracellular signaling pathways (Table 1).

Epithelial CellCell Adhesion in Normal Lung Function

Tight Junctions

Early electron microscopic assessment of the contacts between epithelial and endothelial cells identied a

series of apparent fusions between the outer plasma membranes of adjacent cells where the intercellular space disappeared. These initial ultrastructural studies suggested and subsequent studies have conrmed that tight junctions are a central component of the barrier to the paracellular passage of solutes, molecules, and inammatory cells. Thus, an understanding of tight junction structure is important in considering the regulation of lung barrier function. Over 40 different proteins have been localized to the tight junction in epithelial, endothelial, and myelinated cells that can be further classied as tight junction integral transmembrane proteins and cytoplasmic plaque proteins. The transmembrane proteins include the tetraspan protein occludin and members of the claudin family, which contain four transmembrane domains, two extracellular domains,

ADHESION, CELLCELL / Epithelial 39

and are oriented with both the N- and C-terminal ends towards the cytoplasm, and the single transmembrane Ig-domain-containing, junctional adhesion molecule (JAM). All of these interact directly with cytoplasmic PDZ domain-containing plaque proteins such as the zonula occludens (ZO) and PAR proteins, which function as adapters to recruit cytoskeletal or signaling molecules. The tetraspan extracellular domains interact in the paracellular space with extracellular domains of tight junction proteins on neighboring cells and occludin, the rst integral membrane protein to be found in tight junctions of many cell types, also directly binds F-actin via the last 150 amino acids of its carboxyl tail. The tightness of tight junctions appears to be regulated by changes in the combination and expression ratios of different claudin family members, which can engage in homotypic or heterotypic interactions between several of the 24 distinct claudin gene products thus far identied in humans. One dened role of claudins in regulating permeability is the formation of ionselective pores, and different types of claudins appear to determine the specicity of the pores for individual ions such as Na , Cl , or Ca2 . Whereas tight junctions can form in the absence of occludin, claudins appear to be essential for tight junction formation. It is likely that JAM has an important role in tight junction assembly and resealing after disassembly in epithelia by engaging in homophilic interactions in the paracellular space. Tight junction plaque proteins have important roles in coordinating intracellular signaling by recruiting cytosolic proteins to the tight junction during assembly, and recent reports of ZO proteins localizing to the nucleus, in addition to their tetraspanbinding at tight junction plaques, suggest that they transduce signals from the cell membrane to the nucleus where they may regulate gene expression. Recruitment of plaque proteins to the tight junction may also have a role in regulating cell motility and proliferation. Consequently, although tight junctions perform key functions in regulating epithelial permeability, it is becoming increasingly clear that tight junctions are multifunctional complexes involved in many vital epithelial cell functions. Only recently have specic patterns of tight junction protein expression in the respiratory tract been characterized, and much work remains to further dene how tight junctions are regulated in normal and abnormal lung function.
Adherens Junctions

Adherens junctions are ancient structures present across eukaryotes and even in related structures in

single-celled organisms such as yeast, and as such, adherens junctions have critical functions in coordinating cell polarity, cytoskeletal dynamics, cell sorting, and differentiation. In addition to regulating organization within epithelia, adherens junctions are also important in transmitting information from the environment to the interior of cells. At the core of adherens junctions are the calcium-dependent transmembrane glycoprotein cadherins, and the major epithelial cadherin is E-cadherin, the best characterized member of the cadherin family and the primary cadherin expressed in respiratory epithelium. The extracellular domain of E-cadherin, like other classical cadherins, is comprised of ve calcium-binding cadherin repeats that participate in homotypic interactions with neighboring cells. There is a single-pass transmembrane domain and the cadherin cytosolic domain is linked to cytoskeletal structures via the catenins. Cadherin adhesive activity is regulated in several ways including the level of cadherin gene expression, which correlates with the strength of adhesion, and the type of cadherin expressed, which affects the specicity of cell interaction and plays a role in embryo patterning and determination of cell fate. Posttranscriptional mechanisms regulating cadherin adhesion include modulation of cadherin clustering at the cell surface and changes in cadherin interaction with the catenins. Proteolysis of cadherin domains is now recognized as an important means of posttranslational cadherin modication with extracellular domains being cleaved by metalloproteinases, the transmembrane domains targeted by g-secretases, and intracellular domains degraded by caspases. Cadherins are key mediators of morphogenesis, epithelial sheet integrity, growth control, and maintenance of the terminally differentiated phenotype. Differential expression of different cadherin classes is likely to be important in cell sorting and embryo patterning in development and specic cadherins may be associated with the determination of cell fate. For example, E-cadherin is the primary cadherin expressed in lung epithelia, whereas N-cadherin is expressed at cell cell junctions in pleural mesothelial cells. The cytoplasmic E-cadherin domain binds the armadillo family member protein b-catenin, and this binding induces a tertiary structure that in turn binds a-catenin, which nucleates the assembly of a multimeric complex that links E-cadherin/b-catenin complexes to the actin cytoskeleton. This process both stabilizes the intercellular junctions and allows for coordination of cytoskeletal dynamics with changes in cellcell adhesion. These protein interactions are regulated by phosphorylation events of both the E-cadherin cytoplasmic tail (increase interaction) and

40

ADHESION, CELLCELL / Epithelial

b-catenin (decrease interaction) and by the recruitment of various other potential adherens junctionassociated proteins. Therefore, this complexity allows for many levels of regulation of adherens junctionbased cell adhesion. In addition to its structural role in adherens junctions, b-catenin also functions as a noncadherin-dependent signaling factor. In concert with activation of the Wnt signaling pathway, free cytoplasmic b-catenin translocates to the nucleus and binds to transcription factors of the lymphocyte enhancer-binding factor-1/T-cell factor (LEF-TCF) pathway to regulate expression of downstream target genes associated with cell migration and proliferation including matrilysin (MMP-7), c-myc, WISP, and cyclin D1. A comprehensive consideration of b-catenin in cell signaling is beyond the focus of this discussion of cell adhesion, but recent studies have suggested a role for b-catenin in terminal differentiation of alveolar epithelium. Adherens junctions at the cell surface associate with several other types of intercellular junctions and membrane receptors. Ultrastructural studies of epithelial sheets reveal closely apposed membranes at sites of cellcell contact where adherens junctions alternate with desmosomes, and formation of adherens junctions has been shown to be necessary for desmosome and tight junction formation. Adherens junction-specic cadherins also associate with other cell surface receptors that participate in signaling such as connexins, Notch, receptor tyrosine kinases, and phosphatases, suggesting that adherens junctions can be regulated by extracellular cues.
Desmosomes

function in directing epithelial differentiation. The cytoplasmic cadherin tails directly bind an armadillo protein, usually plakoglobin (g-catenin), and this complex in turn associates with a cytoskeletal linking protein, e.g., desmoplakin, that mediates attachment to the intermediate laments. The result is a series of connections between adjacent cells that maintains tissue tensile strength. Similarities exist between the regulation of adherens junction and desmosomal adhesion; as for adherens junctions, desmosomal adhesion during development is regulated by the timing of and type of desmosomal cadherin or armadillo protein expressed, and as discussed above, desmosome assembly appears to be downstream of and dependent on adherens junction assembly. Posttranslational mechanisms regulating desmosomal adhesion include proteolytic processing of intracellular cadherin domains by caspases and cleavage of extracellular domains by metalloproteinases. Although desmosomes are present throughout the airway and alveolar epithelium, little is known about the specic regulation of desmosome-based cell adhesion in the respiratory tract.

CellCell Adhesion in Respiratory Diseases

Although the net permeability of the lung and airways is jointly regulated by the epithelium and endothelium, disruption of paracellular permeability and leakage of uid and proteins across the airway or alveolar epithelium are hallmarks of a variety of respiratory diseases including asthma, allergic rhinitis, acute lung injury, and pneumonia among others. Furthermore, pathogens may directly target junctional proteins to facilitate entry into the host. However, the specic mechanisms by which changes in tight and adherens junctions and the proteins that regulate these junctions in diseases of the airways and lung remain largely undened. It has been known for a few years that some types of lung cancer, like other carcinomas, show altered staining patterns for several adherens junction proteins, and an increasing amount of evidence suggests that inactivation of E-cadherincatenin complexes plays a significant role in carcinogenesis. Additionally, altered b-catenin signaling has been implicated in idiopathic pulmonary brosis. Thus, these pathways may be attractive targets for therapy in these historically difcult to treat diseases. The available knowledge on the structure, assembly, and function of intercellular junctions in normal and abnormal processes is rapidly increasing. These complexes clearly have more than a structural role and perform critical functions in tissue development,

Desmosomes are specialized junctions that anchor stress-bearing intermediate laments at sites of strong intercellular adhesion. This provides a scaffold that gives integrity to tissues such as skin, heart, and respiratory epithelium that is subject to mechanical stress. At the core of desmosomes are proteins of the cadherin (the desmogleins and desmocollins) and armadillo (plakoglobin, the plakophilins, and p0071) families, which form membrane-associated complexes that are tethered to intermediate laments via the plakins. Analogous to adherens junction cadherins, the single-pass transmembrane desmosomal cadherin extracellular domains interact in the extracellular space; however, in contrast to adherens junction cadherins, desmosomal cadherins interact in a heterotypic manner as each desmosome must contain at least one desmoglein and one desmocollin. Desmocollins and desmogleins are expressed in a tissueand differentiation-specic manner, and thus it has been proposed that desmosomal cadherins may

ADHESION, CELLMATRIX / Focal Contacts and Signaling 41

homeostasis, and response to injury. However, our understanding of the regulation of epithelial cellcell adhesion in respiratory tract development and maintenance of normal lung function, and in injury and disease remains limited. Thus, this remains an area of intense investigation.
See also: Adhesion, CellCell: Vascular. Asthma: Overview. Basal Cells. Bronchiectasis. Bronchiolitis. Connexins, Tissue Expression. Contractile Proteins. Defense Systems. Epithelial Cells: Type I Cells; Type II Cells. Gene Regulation. Lung Development: Overview. Matrix Metalloproteinases. Panbronchiolitis. Signal Transduction. Transcription Factors: Overview. Tumors, Malignant: Overview; Metastases from Lung Cancer.

Further Reading
Braga VMM (2002) Cellcell adhesion and signalling. Current Opinion in Cell Biology 14: 546556.

Bremnes RM, Veve R, Hirsch FR, and Franklin WA (2002) The E-cadherin cellcell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 36: 115124. Godfrey RWA (1997) Human airway epithelial tight junctions. Microscopy Research and Technique 38: 488499. Gonzalez-Mariscal L, Betanzos A, Nava P, and Jaramillo BE (2003) Tight junction proteins. Progress in Biophysics and Molecular Biology 81: 144. Perez-Moreno M, Jamora C, and Fuchs E (2003) Sticky business: orchestrating cellular signals at adherens junctions. Cell 112: 535548. Schneeberger EE and Lynch RD (2004) The tight junction: a multifunctional complex. American Journal of Physiology. Cell Physiology 286: C1213C1228. Wheelock MJ and Johnson KR (2003) Cadherins as modulators of cellular phenotype. Annual Review of Cell and Developmental Biology 19: 207235. Yin T and Green KJ (2004) Regulation of desmosome assembly and adhesion. Seminars in Cell and Developmental Biology 15: 665677. Zhurinsky J, Shtutman M, and Ben-Zeev A (2000) Plakoglobin and b-catenin: protein interactions, regulation and biological roles. Journal of Cell Science 113: 31273139.

ADHESION, CELLMATRIX
Contents

Focal Contacts and Signaling Integrins

Focal Contacts and Signaling

G D Rosen and D S Dube, Stanford University Medical Center, Stanford, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

as lung cancer, acute respiratory distress syndrome, and pulmonary brosis.

Introduction
This article explores the physiology of focal contacts, and cell signaling in normal and pathological states. Focal adhesions (FAs) are specialized multimolecular structures that exist at the points of contact between the cell membrane and the extracellular matrix. Our current understanding of the physiological roles of FAs suggests that in addition to mediating cell adhesion, they also function as a physiological signaling link between the cytoplasm and the extracellular milieu by promoting the bidirectional transmission of biochemical signals. Integrins are the core components of FAs whose orderly function underlies the diverse roles associated with FAs. Integrins are a family of a/b heterodimeric glycoprotein receptors that span the cell membrane. Morphologically, these receptors exhibit an intracellular domain, a transmembrane domain, and an ectodomain that is in association with the cell matrix or other cells. The

Abstract
Focal adhesions (FAs) are contact points for the cell with the extracellular matrix. These complex structures regulate communication of the cell with the surrounding extracellular environment and signaling through these FAs regulates diverse cellular processes, including proliferation, migration, apoptosis, spreading, and differentiation. The principal components of the FAs are integrins, which are ab heterodimers that regulate cell matrix and cellcell interactions. Focal adhesion kinase (FAK) is a kinase that localizes to FAs and regulates signaling in FAs. FAK communicates signals between integrins and intracellular proteins, which regulate diverse cellular processes such as cell polarity, migration, and invasion. FAs play a central role in regulating normal developmental processes such as blood vessel morphogenesis but increased activity of FA can incite aberrant events such as tumor cell invasion and pulmonary brosis. A further exploration of the role of FA in health and disease will provide greater insight into the regulation of normal developmental processes and into the pathogenesis of lung diseases such

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ADHESION, CELLMATRIX / Focal Contacts and Signaling

intracytoplasmic domains of integrins can associate with actin, adaptor proteins, and enzymatic components of signaling cascades and their substrates. This complex association of the cell matrix, integrins, and cytosolic proteins forms the molecular conglomerates that are termed FAs and mediates the bidirectional ow of signals between the cell matrix and the cell. The binding of ligands to integrins in either the extracellular or intracellular domains induces conformational changes within the integrins that affect the binding of ligands on the reciprocal end of the integrin molecule. The activation of FAs by paracrine and endocrine stimuli culminates in the activation of specic intracytoplasmic signaling cascades, including tyrosine phosphorylation, focal adhesion kinase (FAK)-mediated signaling, mitogen-activated protein-kinase (MAPK), and the generation of phosphatidylinositol-4, 5-bisphosphate. These intracellular signals result in a multitude of cytoplasmic responses that regulate, for example, cell proliferation, motility, and differentiation. Based on the multiplicity of functions attributed to FAs, the strict regulation of their function forms the basis of orderly organ structure and tissue function. A better understanding of the mechanisms that are involved in the regulation of FAs has led to the development of potent antithrombosis and anti-inammatory therapies. Moreover, the multiple roles of FAs further promise to aid the elucidation of diverse pathological processes associated with FAs. Some of these processes include tumor growth, pathological organ remodeling, cell barrier defects, and embryogenesis as well as many other pathological entities that are regulated by cellmatrix and cellcell contact.

rearrangements within integrin molecules using diverse tools such as negative stain microscopy, nuclear magnetic resonance, and spectroscopy have further aided the understanding of the function of focal adhesion molecules. These studies have allowed the evaluation of conformational changes within integrins using ligand analogs that bind to ligand-induced binding sites (LIBSs). Site-directed mutagenesis has also aided the characterization of the roles of submolecular components of protein components of FAs, while in vivo studies of these mutagenesis studies have led to the discovery of unique phenotypes, thus further allowing the determination of the functional roles of individual components of FAs. Most recently, the use of uorescent green protein (FGP) tagged proteins has allowed the determination of stoichiometric relationships of the various components of FAs.

Components of Focal Adhesions: Integrins and Extracellular Matrix

Integrins are a large family of heterodimeric transmembrane glycoprotein receptors uniquely found among metazoans whose quaternary structure is characterized by a noncovalent union between a- and b-subunits. There are 18 a-subunits (MW 120 180 kDa) and 8 b-subunits (MW 90110 kDa) that assemble in various permutations to generate 24 distinct integrin heterodimers. These various subunit combinations expand the repertoire of intracellular and extracellular ligands for integrins. In turn, the expanded receptor capability created by the many combinations of a- and b-subunits accounts for the diverse functional roles of integrins. Integrins are central to the structure of FAs. The significance of integrins in metazoan cell structure and tissue organization is emphasized by the observation that the number of integrins parallels the evolutionary complexity of an organism. It has been proposed that the evolution of metazoans as multicellular organisms hinged upon the parallel evolution of integrins as anchor apparatus to the cell matrix and intercellular molecular adhesives. Figure 1 shows the subclassication of the integrin receptor family. Integrins have a large extracellular domain, a transmembrane domain, and an intracytoplasmic domain. The ectodomains of integrins exist in three major conformational states. The rst of these states is a bent conformation that is similar to the crystal structure of avb3. This conformation is considered to be a low-afnity state. The second conformation has been referred to as a closed head piece conformation and is considered to represent an intermediate afnity conformational state. Finally, there is the

Focal Contacts: Structure and Function

Interference reection microscopy rst identied FAs as dark areas at the interface between the ventral cell surface and the extracellular matrix. Diverse types of focal adhesions have since been described. Subsequently, advances in gene cloning have led to the determination of the primary structures of many of the constituents of FAs. Early analysis of FA structure identied integrins as core glycoproteins intimately associated with the structure of FAs. The elucidation of the primary structure of integrins revealed three domains: an extracellular domain, a transmembrane domain, and an intracellular domain as well as sites for tyrosine phosphorylation. In parallel with the elucidation of the primary structure, the solution of the crystal structures of the components of FAs further advanced testable hypotheses on structure function relationships of the individual protein components of FAs. Studies of the conformational

ADHESION, CELLMATRIX / Focal Contacts and Signaling 43

Collagen receptors 3 5 5 V 6 8 8 3 6 7 1 4 9 1 2 10 11

Leukocyte-specific receptors 2 L M X D

7 E

RGD receptors

Laminin receptors 4

Figure 1 The integrin receptor family. Integrins are ab heterodimers that span the cell membrane. This gure shows the mammalian subunits and their ab associations. Asterisks denote alternatively spliced cytoplasmic domains. Reproduced from Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673687, with permission from Elsevier.

high-afnity open head piece conformation, which is induced by ligand binding. The high-afnity conformation is thought to be acquired by the extension of the angle between the b- and hybrid domains. A detailed discussion of the crystal structures of the various integrins and their interacting partners is outside the scope of this article. The evidence so far supports a model whereby the binding of a ligand to the ectodomain mediates the extension and simultaneous separation of the a- and b-subunits of the integrin heterodimers within the transmembrane and intracytoplasmic domains. This model also suggests that the binding of a ligand in the intracellular domain of integrins by adaptor proteins such as talin also mediates counter conformational alterations that cause the exposure of LIBS in the ectodomain. It has further been proposed that these conformational changes are dynamic and dependent on regulatory factors such as growth factors.

constitutively activated integrin receptors. Similarly, deletions of conserved intracytoplasmic b3 sequences also culminate in constitutively activated integrin receptors. These observations have led to the conclusion that the interaction between these two cytoplasmic domains negatively controls the activation of integrins. This interaction seems to occur through a salt bridge between the R995 of the aIIb and the D723 in the b3. In support of this idea, the experimental mutation of either amino acid to the charge neutral alanine leads to a constitutively active integrin. Nuclear magnetic resonance (NMR) studies have demonstrated that the intracytoplasmic portions of the a- and b-domains interact with each other. Furthermore, it has been shown that point mutations in F992A or R995D impair the interaction of the a- and b-domains.
The Role of Talin in Integrin Activation

Regulation of Integrins
Intracytoplasmic Association between the a- and b-Chains

A considerable body of experimental data reveals that the interaction between the a- and b-subunits in the cytoplasm regulates the activation states of the integrin receptors. Studies utilizing the platelet integrin aIIbb3, for example, have revealed that the aIIb short intracytoplasmic domain negatively regulates the activation of the integrin receptors. The experimental deletion of the intracytoplasmic aIIb domain or the conserved GFFKR sequence leads to

Studies support the conclusion that talin, a cytoskeletal actin-binding protein that co-localizes with active forms of integrins, is a crucial element in the activation of integrins. Talin is a homodimer consisting of two antiparallel subunits, each approximately 270 kDa. Each of the talin subunits is comprised of a 220 kDa C-terminal and a 70 kDa globular head, which is thought to contain the binding sites for integrins. Talin has been demonstrated to bind the b1D, b2, b3, b5, and b7 intracytoplasmic integrins at their N-terminus. Structural analysis of the PTB-like 96 amino acid subdomain of the talin FERM (4.1, ezrin, radixin, moesin) domain shows a major integrin-binding site. Besides the ubiquitous interaction

44

ADHESION, CELLMATRIX / Focal Contacts and Signaling

of talin with diverse integrins, the overexpression of a talin fragment containing the head domain leads to the activation of integrin molecules. The role of talin in integrin activation is further supported by the observation that talin knockdown abrogates aIIb3 activation by endogenous agonists. This observation suggests that talin is a critical common downstream target in the activation of at least some integrins. Molecular mechanisms of the role of talin in the regulation of integrins are complex and still being explored. Vonogradova and colleagues have demonstrated that the talin head domain alters the NMR spectrum for the interaction between the b3 and aIIb domains. Other investigators have shown that the talin head domain attenuates the uorescence resonance energy transfer (FRET) between uorophoretagged a- and b-integrins within viable cells. These observations support a model whereby the binding of the talin phosphotyrosine-binding (PTB) domain to integrin disrupts the inhibitory interaction between a- and b-membrane proximal domains.

Regulation of TalinIntegrin Interactions

The preceding section considers that the interaction of talin with intracytoplasmic domains of integrin is pivotal to the regulation of integrin function and hence FAs. A variety of mechanisms have been advanced to account for how these interactions are regulated. The talin head has a sixfold higher afnity in comparison to the intact talin molecule. This observation has led to the hypothesis that in the whole talin molecule, integrin binding sites are obscured. Therefore, a potential mechanism whereby talin regulates integrin activation is through the modulation of its conformations by physiological factors, which then reveal or mask integrin binding sites. Evidence supporting this possibility accrues from observations made from the function of the ERM (ezrin, radixin, moesin) family of proteins. The major integrin-binding site in talin lies within the talin FERM domain, and binding occurs via a variant of the classical PTB domainNPxY interaction. In the ERM family, the intramolecular interaction of the N-terminal FERM domain with the C-terminal obscures ligandbinding sites. Intriguingly, ERM phosphorylation, PIP2 (phosphatidyl inositol 4,5-bis-phosphate), and rho-dependent pathways abrogate the native inhibition in the unstimulated ERM molecules presumably by unmasking the interaction of the FERM domain. Although the role of phosphorylation in the activation of integrins is still debated, it is notable that the binding of PIP2 to talin has been shown to mediate a conformational change within the talin molecule that culminates in the exposure of integrin-binding

sites in the FERM domain. The role of PIP in the regulation of talin is complicated by the observation that talin directly stimulates a splice variant of PIP2producing enzyme PIPKIg-90 (phosphatidylinositol phosphate kinase type Ig-90) thereby increasing PIP2 production. Interestingly, PIPKIg-90 and integrin b tails have overlapping binding sites on the talin F3 subdomain. This suggests a novel control strategy whereby PIPKIg-90 and talin may displace each other from integrin-binding sites following activation. Another regulatory mechanism for talinintegrin interaction is thought to involve calpain-mediated proteolysis of integrin b tails at sites straddling the NPXY/NXXY motifs that are necessary for the anchorage of integrins to the cytoskeleton and FAs. Calpains are calcium-dependent thiol proteases that have a diverse substrate spectrum that includes constituents of the cytoskeletal apparatus and cell-signaling proteins. Structurally, they are composed of an 80 kDa catalytic subunit and a 30 kDa regulatory subunit. Although calpain was historically considered to be involved in the detachment of migrating cells, accumulating evidence suggests that calpain also functions by regulating actin remodeling during cell migration. Supportive data have been obtained through experimental overexpression of calpain in aortic endothelial cells. These experiments reveal that overexpression of calpain leads to formation of focal adhesions. In contrast, when calpain is inhibited in broblasts and endothelial cells, adherence is maintained while cell dispersion is attenuated. Additional evidence points to the fact that the regulation of actin lament formation in endothelial cells is dependent on calpain-mediated proteolysis. The capacity of calpain to cleave integrins with diverse molecular structures suggests that calpains recognize higherorder protein structure rather than primary protein sequence. Calpains also cleave tyrosine kinases (PTK) such as focal adhesion kinase (FAK) and src and phosphatases (PTP) PTP-1B, Shp-1, and PTP-MEG. Other investigators have reported that calpain cleaves the N-terminal 47 kDa of talin, which is known to be hyperphosphorylated by protein kinase A.

Focal Adhesion Structure and Signaling

FAs exhibit dynamic spatialtemporal variations in function. A large group of proteins are core to the structural behavior of focal adhesions. This is in keeping with the diversity of roles that are associated with FAs. The structural integrity of FA molecules relies on adaptor molecules that perform core scaffolding functions. Multiple other molecules also interact with the FAs and perform both structural and enzymatic chores of FAs. The overall function

ADHESION, CELLMATRIX / Focal Contacts and Signaling 45

and net effect of these scaffolds is to juxtapose kinases with their substrates and thereby facilitate FA-mediated signaling. The full repertoire of proteins that have been observed to interact with FAs is large and continuously expanding. In general, the relative expression or activation of different scaffolding proteins depends on the tissue of origin and cellular stimulus. Therefore, scaffolding proteins not only confer on FA structural integrity but are also responsible for the functional diversity associated with FAs. A core component of the FA complex is the p130Cas protein. The structure of p130Cas includes an SH3 domain, a proline-rich motif, and a very complex substrate-binding region that has the Src-SH2 binding region. The p130Cas SH3 domain interacts with the FAK possibly through interactions with the YDYV motif. This same YDYV motif also binds c-Src. It is thought that c-Src mediates the phosphorylation of the substrate domain of p130Cas. It is intriguing to also note that FAK has the capability to phosphorylate an Src binding site within p130Cas. The phosphorylation of the substrate-binding site within p130Cas achieves two major roles. First it allows the localization of p130Cas to the FA and second it allows p130Cas to interact with other proteins containing the SH2 domain such as protein tyrosine phosphatase (PTP)-PEST, NcK, and CrK. Crk/CrkL is an adaptor protein that interacts with phosphorylated p130Cas via an SH2 domain. The interaction of Crk with P130Cas leads to the formation of a complex that activates Dock 180, which is a novel exchange factor. Ultimately, the binding of Dock 180 to Crk and ELMO culminates in the activation of Rac, which augments the activation of p130Cas phosphorylation. In general, the association of p130Cas with Crk activates GTPases that are involved in the maintenance and reorganization of the actin cytoskeleton, which are important for cell motility and are crucial to establishing cellular structure. Paxillin is another adaptor molecule that interacts with a diverse range of proteins such as FAK, PKL, PTP-PEST, and b1 and a4 integrin intracytoplasmic tails. Similar to P130Cas, paxillin also complexes with a wide range of distinct exchange factors in a phosphorylation-dependent and tissue-specic manner. In summary, scaffold proteins bring into close proximity protein moieties important for the function of the FAs. The functional diversity conferred by the combinations afforded through these proteins results in tissue-specic function.
Kinases

generation of focal adhesion-mediated cell signaling. Tyrosine phosphorylation creates sites for binding of Src-homology domain (SH2-containing proteins). Moreover, tyrosine phosphorylation directly regulates a variety of kinases and phosphatases. The principal kinases that regulate focal adhesions are FAK and Src. Multiple other kinases (ser/thr kinases, Abl, PYK2,Csk) also probably play a role but their precise role in regulation of focal adhesions has not been fully elucidated.

Tyrosine phosphorylation of focal adhesion components is considered to be the core event in the

Focal adhesion kinase FAK is a 125 kDa protein kinase that was initially shown to localize to FAs. Moreover, it was observed to be phosphorylated in relation to Src-mediated transformation. FAK is an FERM domain protein and is known to bind to many different signaling proteins. In general, it is considered that phosphorylation of FAK is the sentinel event in FA-mediated cell signaling. In response to integrin association with a ligand, FAK is activated by autophosphorylation at tyrosine Y397. One model proposes that subsequent to this autophosphorylation, Y397 acts as a docking site for Src that in turn mediates the phosphorylation of FAK at Y576 and Y577 and potentially at other sites. The phosphorylation of these tyrosines creates molecular contact points for proteins containing SH2 domains. As a result of this, the Ras-regulated MAPK pathway is activated (Figure 2). In support of this model, it has been observed that phosphorylation of Y926 in FAK creates an interaction site (pYNQV) for the SH2 domain of Grb2, an adapter protein that regulates growth factor signaling through Ras/SOS through binding of its SH2 and SH3 domains. This event is considered to be critical for the activation of the MAPK pathway. Grb2 can be phosphorylated at the Y926 position only when FAK is dissociated from the focal adhesion complex. This observation suggests that the phosphorylation of FAK causes it to dissociate from the FA complex and then phosphorylates its target proteins and in turn initiates signaling cascades. The interaction of FAK with FAs occurs through the FA-targeting domain (FAT) of FAK. Indeed, when the FAT domain in FAK is mutated, the FAK molecules are incapable of autophosphorylation and cannot phosphorylate known endogenous substrates. Although the role of FAK as a regulatory kinase is widely accepted, it is intriguing that FAK molecules lacking the kinase domain are still functional in some cell-signaling pathways. This has led to the proposal that FAK may predominantly perform a structural role by acting as a scaffold for diverse constituents of FAs. However, these same data have been interpreted

46

ADHESION, CELLMATRIX / Focal Contacts and Signaling

Integrin

Graf SH3

Paxillin

COOH Pro Y397 371374 P SH3-SH2 Src/Fyn Paxillin Grb2 Spreading Sow/Ras Erk (MAPK) p130Cas Crk/Nek? Akt? SH2 PI3 kinase Pro Pro Y925 712715 878881 P SH2 PI3 kinase SH2 GRB-2

Proliferation Migration

Apoptosis

Figure 2 Signaling through focal adhesion kinase (FAK). Association of FAK with integrins activates signaling pathways that regulate cell proliferation, apoptosis, spreading, and migration. Reproduced from Laboratory of Molecular Biophysics http://biop.ox.ac.uk, with permission.

to imply that other kinases have the ability to duplicate the kinase functions of FAK. Src tyrosine kinase The c-Src tyrosine kinase performs a pivotal role in the regulation of the function of FAs and the cytoskeleton. In transformed cells, vSrc is localized at three distinct locations: perinuclear, the actin cytoskeleton, and focal adhesions. Studies utilizing vSrc mutants in LA 29 rat broblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat broblasts, transfected with a ts mutant of v-fps, have demonstrated that at restrictive temperatures vSrc is inactive and localizes to the perinuclear regions where it is intimately associated with the microtubular cytoskeleton. In contrast, at more permissive temperatures v-Src localizes to focal adhesions at the cell periphery where it interacts with RhoA-induced actin bers. An interesting observation is that targeting of v-Src is independent of its own tyrosine kinase function; rather it depends on its SH3 domain. However, this localization is dependent on the binding of the p85 regulatory subunit of phosphatidylinositol kinase (P13 K) to the v-Src SH3 domain as well as on P13 K kinase function. Although not completely analogous to v-Src, overexpressed c-Src in broblasts localizes to a perinuclear location and associates with microtubules and organelles such as endosomes and the trans-Golgi network. The localization of c-Src is dependent on

GTPse activity, which directs c-Src to diverse cellular locations. The extrapolation of the function of c-Src from the observations of v-Src is limited by the observation that c-Src does not associate with p85 of p13 K. Thus, the role of c-Src still needs to be fully dened.

Focal Adhesion Molecules in Respiratory Disease

A characterization of the role of focal adhesion kinases in normal cellular function and in disease is an area of active investigation. Focal adhesion molecules such as integrins and FAK play key roles in the regulation of cellcell communications, structure, and remodeling during development of the lung and other tissues. Perturbations of the tightly controlled functions of focal adhesions have been shown to affect tumorigenesis and metastasis but the role of focal adhesions in diseases of the lung and other organs is less well dened. However, studies have begun to reveal a role for focal adhesions in cellular function and disease states. For example, focal adhesions have been shown to be important in lymphocyte homing to sites of inammation as b7 integrins have been shown to be crucial in the localization of lymphocytes to sites of inammation in the gastrointestinal tract. Focal adhesions are also important

ADHESION, CELLMATRIX / Integrins 47

in hemostasis as GII3b integrins are crucial in platelet aggregation and their blockade has given rise to a potent class of antagonists that are used to prevent platelet aggregation following interventional procedures in cardiology. Also, FAK is required for blood vessel morphogenesis and migration of vascular smooth muscle cells. FAK has been shown to be required for microtubule organization, nuclear movement, and neuronal migration during brain development and to be a negative regulator of axonal branching and synapse formation. In the acute respiratory distress syndrome, perturbation of the cell surface barrier is thought to be mediated through impaired focal adhesion function thus leading to increased permeability. Additionally, TGF-b-induced activation of FAK has been shown to mediate the TGF-b-mediated suppression of apoptosis in lung broblasts, which likely plays a role in the pathogenesis of idiopathic pulmonary brosis. We among others have shown that FAK is cleaved during apoptosis and a recent study revealed that FAK interacts with p53 and inhibits p53-mediated apoptosis. Further studies will elucidate the precise role for focal adhesions in normal cellular function and in disease.
See also: Adhesion, CellCell: Vascular ; Epithelial. Adhesion, CellMatrix: Integrins. Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Cytoskeletal Proteins. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Platelets.

Mould AP and Humphries MJ (2004) Cell biology: adhesion articulated. Nature 432(7013): 2728. Petit V and Thiery JP (2000) Focal adhesions: structure and dynamics. Biology of the Cell 92(7): 477494. Schlaepfer DD, Mitra SK, and Ilic D (2004) Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochimica et Biophysica Acta 1692(23): 77102. Schoenwaelder SM and Burridge K (1999) Bidirectional signaling between the cytoskeleton and integrins. Current Opinion in Cell Biology 11(2): 274286. Takagi J, Petre BM, Walz T, and Springer TA (2002) Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110(5): 599611. Vinogradova O, Velyvis A, Velyviene A, et al. (2002) A structural mechanism of integrin alpha(IIb)beta(3) inside-out activation as regulated by its cytoplasmic face. Cell 110(5): 587597. Wen LP, Fahrni JA, Troie S, et al. (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. Journal of Biological Chemistry 272(41): 2605626061. Wozniak MA, Modzelewska K, Kwong L, and Keely PJ (2004) Focal adhesion regulation of cell behavior. Biochimica et Biophysica Acta 1692(23): 103119.

Integrins
L M Schnapp, University of Washington, Seattle, WA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Integrins are a large family of cell adhesion receptors that mediate cellcell and cellextracellular matrix adhesion. Integrin members are expressed on virtually every cell and provide essential links between the extracellular environment and intracellular signaling pathways. In doing so, integrins play a role in most essential cell behaviors, including cell survival, apoptosis, differentiation, and transcriptional regulation. Phenotypes of integrin knockout mice illustrate important roles of integrins in lung development, pulmonary brosis, and emphysema. Integrins contribute to immune response in the lung by mediating trafcking of leukocytes to areas of inammation. In addition, they function as receptors for a variety of pulmonary pathogens. Because of their central role in different disease processes, therapeutic agents are being developed to target integrinligand interactions.

Further Reading
Calderwood DA (2004) Integrin activation. Journal of Cell Science 117(5): 657666. Calderwood DA (2004) Talin controls integrin activation. Biochemical Society Transactions 32(3): 434437. (Review: PMID: 15157154: PubMed indexed for MEDLINE.) Calvete JJ (2004) Structures of integrin domains and concerted conformational changes in the bidirectional signaling mechanism of alphaIIbbeta3. Experimental Biology and Medicine (Maywood) 229(8): 732744. Diagne I, Hall SM, Kogaki S, Kielty CM, and Haworth SG (2003) Paxillin-associated focal adhesion involvement in perinatal pulmonary arterial remodelling. Matrix Biology 22(2): 193205. Frame MC, Fincham VJ, Carragher NO, and Wyke JA (2002) vSrcs hold over actin and cell adhesions. Nature Reviews: Molecular Cell Biology 3(4): 233245. Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673687. Kanda S, Miyata Y, and Kanetake H (2004) Role of focal adhesion formation in migration and morphogenesis of endothelial cells. Cellular Signalling 16(11): 12731281.

Integrins are a major family of cell adhesion receptors. The term integrin was rst used in the 1980s to describe cell surface receptors that integrated the cytoskeleton of one cell with that of another cell or extracellular matrix protein. Since the rst description, it is apparent that integrins are essential for many important biological processes including, but not limited to, development, cell proliferation, cell survival, migration, immune function, and wound healing. Integrins are found in all metazoa, vertebrates, and invertebrates. No homologs have been

48

ADHESION, CELLMATRIX / Integrins

1 Leukocyte integrins L M X D 2 7 E 2 3 4 5 6 7 4 8 9 10 11 1

Platelet integrin IIb

5 6

Figure 1 Integrin family. a/b associations of mammalian subunits are illustrated. Subunits in red recognize the RGD motif, subunits in blue are collagen receptors, and subunits in purple are laminin receptors. Some integrins have restricted cell type expression (i.e., leukocyte integrins and platelet integrin).

Table 1 Nomenclature of b2 leukocyte integrins Leukocyte integrin aLb2 aMb2 aXb2 aDb2 Names Lymphocyte function-associated antigen (LFA)-1, CD11a/CD18 Macrophage adhesion molecule (Mac)-1, CD11b/CD18, CR3 p150,95, CD11c/CD18 CD11d/CD18

b2-containing integrins, which are found exclusively on leukocytes, and the integrin aIIbb3 (GPIIb/IIIa), found exclusively on megokaryocytes and platelets.

Structure
Integrins are heterodimeric glycoproteins that are composed of two noncovalently associated subunits, a and b (Figure 2). Each a and b subunit contains a large extracellular domain, a single pass transmembrane domain, and a short cytoplasmic domain (2060 amino acids, except for the b4 subunit cytoplasmic domain, which contains 1000 amino acids). The cytoplasmic domain interacts with the cytoskeleton and/or cytoplasmic-signaling molecules. A subset of a subunits contains an inserted (I) domain that is homologous to the A domain of von Willebrand factor and is important in ligand binding. The extracellular domains of both subunits form the ligandbinding site. Divalent cations such as Ca2 or Mg2 are required for functional activity. Divalent cations can promote or suppress ligand binding, change ligand specicity, or stabilize integrin structure. In general, Mg2 promotes cell adhesion, Ca2 decreases it, and Mn2 increases ligand afnity. Insight into the structure of the extracellular domain was obtained from X-ray crystal structure analysis. The extracellular domain resembles a globular head atop two legs, one from each subunit (Figure 2). The globular heads form the main contact between the subunits and the ligand. The head of the a subunit is composed of a seven-bladed b-propeller

identied to date in prokaryotes, plants, or fungi. The number of integrin members roughly correlates with the complexity of the organisms. Currently, there are 18 known a subunits and 8 known b subunits, which combine to form 24 different heterodimers in mammals (Figure 1). Different a/b combinations alter the ligand specicity of the receptor. Additional complexity arises due to alternatively spliced variants of several integrin subunits. Integrins can be roughly divided into subfamilies according to their shared b subunit, although a subunits can sometimes bind to more than one b subunit, thus blurring the original subfamilies. The receptors are generally named according to the subunit composition (i.e., a8b1). However, older nomenclature still exists (Table 1). For example, the b1 subfamily of integrins was originally termed VLA (very late activation) antigens because they were originally detected on T cells very late after mitogen stimulation (i.e., a4b1 is also referred to as VLA-4). All mammalian cells express some member of the integrin family. In addition, some integrins have cell-restricted expression, most notably the

ADHESION, CELLMATRIX / Integrins 49

A domain -propeller Hybrid domain PSI domain Thigh domain

Calf domain

EGF repeats

Calf domain Transmembrane domains

domain, followed by an immunoglobulin (Ig)-like thigh domain and two calf domains. The b subunit head has a bA domain (similar to the I domain of a subunits) followed by an Ig-like hybrid domain, PSI domain (plexin, semaphorin, and integrin), and four EGF-like domains. Recently, the crystal structure of the extracellular domain of avb3 bound to a protypical ArgGlyAsp (RGD) ligand was determined. The RGD peptide is inserted in a crevice between the heads of the a subunit and the b subunit. Amino acid residues previously predicted to be important in ligand binding by mutational analysis were conrmed to participate in ligand binding. Furthermore, occupation by ligand resulted in a conformational change of the extracellular domain that is likely to be transmitted to the cytoplasmic domain and contribute to outside-in signaling.

Regulation of Activity
Integrins can exist in different conformational states, and many integrins exist at rest in a low-afnity state (Figure 3). However, in response to agonists, signaling pathways are activated that cause a conformational change in the integrins to allow high-afnity binding (afnity modulation). This inside-out signaling refers to the rapid reversible change in integrin afnity in response to external agonists. The classic

Cytoplasmic domains
Figure 2 Schematic representation of integrin based on structure of avb3. Ligand-binding pocket is formed by the b-propeller and bA domain of a and b subunits, respectively, and is indicated by the arrow.

ECM

Extracellular

Intracellular

Actin cytoskeleton Conformational change/ high affinity

Inactive low affinity

Clustering/ increased avidity

Figure 3 Schematic representation of integrin afnity and avidity modulation in response to inside-out signaling. In the resting (inactive) state, interaction of cytoplasmic tails via salt bridge (black bar) inhibits interaction with cytoskeletal and signaling partners. After agonist-induced signaling, the salt bridge is broken by cytoplasmic proteins such as talin. This results in a conformational change in the extracellular domain, allowing high-afnity binding to ligand. Diffusion of integrin within the plasma membrane leads to clustering and increased avidity.

50

ADHESION, CELLMATRIX / Integrins

example of inside-out signaling occurs in platelets. On a resting platelet, the integrin aIIbb3 exists in a low-afnity state and is unable to bind soluble brinogen. After platelet activation (by agonists such as thrombin, collagen, ADP, or epinephrine), aIIbb3 undergoes a conformational change to a high-afnity state and binds soluble brinogen, causing platelet aggregation. Regulation of integrin activation is also critical for leukocyte trafcking. Leukocytes circulate in a nonadhesive state and require activation (i.e., by cytokines or chemokines) to allow targeted integrinmediated adhesion to the vascular endothelial cells and subsequent transmigration. Activation of b2 integrins is also critical for phagocytosis, antigen presentation, and T-cell killer function. Integrin cytoplasmic domains regulate the activation state of the integrin. Truncation of the a or b integrin cytoplasmic domain results in a constitutively active receptor. In the resting (inactive) state, the cytoplasmic tails of a and b subunits are thought to be in close proximity to each other, connected by a salt bridge formed between several highly conserved residues within the cytoplasmic domain. When the salt bridge is broken, the integrin becomes activated. Several cytoplasmic proteins can bind to cytoplasmic tails and activate integrins, including talin, calcium and integrin binding protein (CIB), and b3 endonexin. Ligand binding can also be strengthened by increased lateral mobility of the integrin within the plasma membrane. This results in clustering of the integrin and increased ligand binding, referred to as avidity modulation.

as mechanotransducers. Integrins also coordinate signaling with many receptor tyrosine kinases and therefore determine cellular responses to soluble growth factors and cytokines. In addition, integrins can associate with other transmembrane or membrane-associated proteins (e.g., PDGF-bR, uPAR, and TM4SF) to inuence cell signaling.

Integrin Ligands: More Than Just Glue

Originally described as receptors that mediated adhesion to extracellular matrix proteins such as bronectin, collagen, and laminin, it is increasingly evident the integrins play a broader role in homeostasis, and their ligand repertoire is much larger than originally appreciated. Many integrins recognize the tripeptide sequence RGD found in many matrix proteins, including bronectin, vitronectin, and brinogen, and other extracellular proteins, including the latency-associated peptide (LAP)-TGF-b1. Integrins bind to a variety of counterreceptors (i.e., VCAM, ICAM, and E-cadherin), illustrating the importance of integrins in cellcell adhesion. An increasing number of different substrates have been identied as biologically relevant ligands for integrins, including VEGF, matrix metalloproteinase (MMP)-2, sperm fertilin, and the endogenous angiogenesis inhibitors angiostatin, endostatin, and tumstatin. Identication of novel ligands has expanded the spectrum of biological processes that integrins regulate.

Integrins in Respiratory Diseases Biological Function

Outside-in signaling occurs after ligand binding to integrins. The cytoplasmic tails do not contain any intrinsic catalytic activity but act as organizing centers and scaffolding for other signaling components and cytoskeletal proteins. Most integrins activate focal adhesion kinase, which subsequently activates a number of classic signaling pathways, such as mitogen-activated protein kinase, PI3-kinase, protein kinase C, and c-JUN kinase. These pathways regulate diverse cell behaviors, including survival, proliferation, migration, and transcriptional activity. Integrins are important links to the actin cytoskeleton through binding of the cytoplasmic tails to actinbinding proteins such as talin, lamin, integrin-linked kinase, and a-actinin. Integrin-mediated adhesion can activate the Rho family of small GTPases that are involved in actin cytoskeleton rearrangement. Integrin activation of RhoA, Rac-1, or CDC42 results in the formation of stress bers, lamellopodia, and lopodia, respectively. Thus, integrins are situated to act The nonredundant, important function of the different integrins is illustrated by the distinct phenotypes of the knockouts. In addition, several human genetic diseases illustrate the clinical importance of the integrin family. Leukocyte adhesion deciency (LAD1) is a rare autosomal dominant disorder in which patients suffer from recurrent life-threatening bacterial infections, despite normal numbers of circulating leukocytes. LAD-1 is caused by lack of functional b2 integrins on leukocytes, which results in the inability of leukocytes to migrate to tissue sites of inammation and infection. Glanzmanns thrombasthenia is an autosomal recessive disorder in which patients suffer from severe mucocutaneous bleeding due to a lack of functional aIIbb3 on platelets. Mutations in a4b6, normally expressed on the basal surface of keratinocytes, cause some cases of the skin blistering disease epidermolysis bullosa. Mutation of the a7 integrin subunit, expressed primarily on skeletal and cardiac muscle, is responsible for some cases of congenital myopathy.

ADHESION, CELLMATRIX / Integrins 51

Because of their diverse roles in many processes, integrins are attractive targets in many diseases. Drugs aimed at directly blocking receptorligand interactions have been developed and include monoclonal antibodies and small molecule ligand mimetics (based on RGD). The platelet integrin aIIbb3 antibody, abciximab, was the rst anti-integrin drug to be approved for clinical use. Several agents targeting integrins are being tested for use in asthma, cancer, and inammatory diseases.
Integrins in Lung Development

Early studies using in vitro models of lung branching showed that administration of RGD peptides decreased branching morphogenesis. These studies suggested a critical role of cellmatrix interactions, mediated through integrins in lung development. Further studies on knockout mice have conrmed the importance of several integrins in lung development. Mice lacking the a3 integrin subunit exhibit decreased branching morphogenesis and immature bronchiolar epithelium, in addition to abnormal kidney development. The underlying epithelial basement membrane was severely disrupted, demonstrating a critical role of a3 in basement membrane assembly. Mice lacking the a9 subunit die soon after birth due to bilateral chylothorax, suggesting abnormalities in lymphatic development. Recently, VEGF-C and VEGF-D, key effector proteins in lymphatic development, were identied as novel ligands for a9b1. a9b1VEGF interactions mediated endothelial cell adhesion and migration, suggesting a mechanism for phenotype found in a9-decient mice. The absence of lung phenotype in other integrin knockouts does not necessarily imply the absence of a role in lung development. In mice, lung development begins at approximately E12.5 and continues until approximately 4 weeks after birth. Knockouts of several potentially important integrins for lung development were embryonic lethal before lung development was completed (i.e., b1, a5); thus, determining their contribution to lung development is difcult. In addition, upregulation of other integrins or related proteins may compensate for the loss of an integrin subunit during development.
Integrins and Fibrosis

was due to a defect in the activation of TGF-b1, an important mediator of pulmonary injury and brosis. TGF-b1 is secreted in a latent form, complexed with LAP, and is unable to signal through its receptor. LAP contains an RGD site that binds to avb6 and results in activation of TGF-b1. The activation of TGF-b1 by avb6 requires contact between avb6-expressing cells (i.e., epithelial cells) and TGF-b receptor-expressing cells (i.e., macrophages); it also requires an intact integrin cytoplasmic tail. Like avb6, avb8 also binds to the RGD sequence in LAPTGF-b1 and activates TGF-b1. However, in contrast to avb6, avb8 activation requires metalloproteinase activity and results in free active TGF-b. Thus, integrin-mediated regulation of TGF-b activity can occur by several distinct mechanisms. Another interesting nding in the b6-null mice is the development of emphysema over time. Backcrossing the b6-null mice onto a MMP-12-null background eliminates the development of emphysema, suggesting that the development of emphysema is due to increased expression of MMP-12, a macrophage elastase. The ndings suggest a model wherein active TGF-b1 normally suppresses MMP-12 expression by macrophages. In the absence of avb6-mediated TGF-b activation, MMP-12 expression is no longer suppressed and ultimately results in a matrixdegrading phenotype in the lung.
Integrins and Asthma

The integrin avb6 is normally expressed at low levels on airway epithelium. Mice that lack the integrin subunit b6 develop exaggerated inammation in the lung at baseline. Interestingly, following bleomycin administration, which typically results in lung injury and brosis, the b6-null mice were protected from development of pulmonary edema and brosis. This

In asthma, integrins, including aLb2 and a4b1, contribute to trafcking of eosinophils and other inammatory cells to the lung during an asthma exacerbation. Therefore, integrins are attractive targets for novel anti-inammatory therapies in asthma. Efalizumab, a humanized anti-aL monoclonal antibody, inhibits leukocyte migration to areas of inammation and is approved for use in moderate to severe psoriasis. Efalizumab was tested in patients with mild asthma. Despite a significant decrease in activated eosinophils in the lung, there was no significant difference in pulmonary function after allergen challenge. There has been much interest in targeting the a4 integrin subunit in asthma and other chronic inammatory diseases, such as Crohns disease, rheumatoid arthritis, and multiple sclerosis. In multiple sclerosis, treatment with the monoclonal a4 antibody natalizumab resulted in a significant reduction in relapses, leading to Food and Drug Administration approval of natalizumab. However, since approval, several unexpected cases of progressive multifocal leukoencelphalopathy (PML) have been reported in patients receiving natalizumab. PML is a rare, fatal demyelinating disease caused

52

ADHESION, CELLMATRIX / Integrins

by reactivation of human polyoma virus JC. The contribution of natalizumab to the development of PML is not entirely clear, but it likely relates to preventing migration of immune cells that normally repress latent virus into the central nervous system. These ndings have raised serious concerns regarding the safety of a4 antagonists and put into question any future work targeting the a4 subunit.
Integrins and Acute Lung Injury

The general paradigm for leukocyte trafcking occurs in several steps: 1. selectin-mediated rolling of leukocytes on endothelium, 2. activation of leukocyte b2 integrins by chemoattractants, 3. integrin-mediated rm adhesion of leukocytes to endothelium, and 4. integrin-mediated transmigration of leukocytes into the interstitium. In the lung, leukocyte trafcking differs from the general model in several aspects: the classical selectinmediated rolling does not occur; transmigration of leukocytes occurs at pulmonary capillaries, not postcapillary venules; and transmigration of leukocytes into interstitium and alveoli may follow a b2-dependent or b2-independent pathway. In the lung, involvement of b2 integrins for leukocyte migration depends on the stimulus. For example, leukocyte trafcking in response to Escherichia coli, Pseudomonas aeruginosa, lipopolysaccharide (LPS), and IL-1 is dependent on b2 integrins, whereas leukocyte trafcking in response to Streptococcus pneumoniae, group B streptococcus, Staphylococcus aureus, and hydrochloric acid is independent of b2 integrins.
Integrins and Pathogens

causes severe pulmonary disease and thrombocytopenia and infects pulmonary epithelial cells and platelets. Pathogenic strains of hantaviruses attach to cells via b3-containing integrins. Another pathogen, foot and mouth disease virus (FMDV), causes a devastating disease of cattle. The primary route of infection by FMDV is through the epithelial cells and associated lymphoid tissues in the upper respiratory tract, followed by dissemination to other epithelial tissues. The outer capsid of FMDV contains a highly conserved RGD tripeptide motif on an exposed loop that interacts with av-containing integrins and facilitates viral attachment and infection of cells. Adenovirus is important both for the diseases it causes in humans, including respiratory infections, and because replicationdefective variants of adenovirus are being used as vectors for gene therapy. Adenovirus contains a conserved RGD sequence within a highly variable region in the penton base protein. In contrast to the viruses just mentioned, adenoviruses do not use integrins for initial attachment. Instead, adenovirus attaches to cells through a common coxsackievirus adenovirus receptor and then the RGD site interacts with av-containing integrins to facilitate internalization. Likewise, the causative virus of Kaposis sarcoma, human herpes virus 8 (HHV-8), interacts with a3b1 on target cells via RGD sequence on an outer envelope to facilitate viral entry and uptake. Thus, viruses and other pathogens have exploited integrins for their advantage.
Integrins and Malignancy

Many pathogens utilize host integrins to facilitate cell attachment and infection (Table 2). Many viruses have RGD motifs in capsid proteins, which facilitates attachment and internalization of organisms. Hantavirus
Table 2 Virusintegrin interactions Virus Hantavirus Echovirus Rotavirus Papillomavirus Coxsackievirus Foot and mouth disease virus Adenovirus HHV-8 (KSHV) Integrin receptor avb3, aIIbb3 a2b1 a2b1, axb2, a4b1 a6b1 avb3, avb6 avb3, avb6 avb3, avb5, avb1 a3b1

Most adherent cells are anchorage dependent; that is, they require integrin-mediated signaling to stimulate cell-cycle progression, promote cell survival, and respond to soluble growth factors such as EGF and PDGF. Loss of integrin-mediated signaling causes a subtype of apoptosis, termed anoikoisis from the Greek word meaning homelessness. Following malignant transformation, the requirement of integrin ligation is often bypassed by mutations in oncogenes or tumor suppressor genes, thus allowing anchorage-independent growth. However, integrin signaling still contributes to tumorigenesis and tumor progression. Depending on the integrin, cell type, and signaling pathway, activated integrins may facilitate tumor growth, metastasis, and attachment at distant sites or suppress tumor activity. In general, tumor cells, either through direct signaling mechanisms or through selection process, increase expression of integrins that promote cell proliferation, survival, and migration and decrease expression of integrins that promote a quiescent, differentiated state. Although it is difcult to make generalizations, a2b1 and a3b1 expression is associated with a tumor

ADRENERGIC RECEPTORS 53

suppressor phenotype, whereas avb3, avb6, and a6b4 expression is associated with tumor progression. The development of blood supply is essential for tumor survival and growth, and it is a potential target of antitumor agents. The endothelial integrin avb3 is upregulated in tumor neovasculature. In vitro and animal studies showed that avb3 antagonists (antibodies or peptides) caused tumor regression, inhibited neovascularization, and induced apoptosis of endothelial cells. Several anti-avb3 agents show promise as antiangiogenic therapy for cancer treatment.
See also: Adhesion, CellCell: Vascular; Epithelial. Adhesion, CellMatrix: Focal Contacts and Signaling. Asthma: Overview. CD11/18. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblasts. Matrix Metalloproteinases.

Further Reading
Calderwood DA (2004) Integrin activation. Journal of Cell Science 117: 657. Doerschuk CM (2000) Leukocyte trafcking in alveoli and airway passages. Respiration Research 1: 136. Guo W and Giancotti FG (2004) Integrin signalling during tumour progression. Nature Reviews: Molecular Cell Biology 5: 816. Humphries MJ, McEwan PA, Barton SJ, et al. (2003) Integrin structure: heady advances in ligand binding, but activation still makes the knees wobble. Trends in Biochemical Sciences 28: 313. Hynes R (2002) Integrins. Bidirectional, allosteric signaling machines. Cell 110: 673. Plow EF, Haas TA, Zhang L, Loftus J, and Smith JW (2000) Ligand binding to integrins. Journal of Biological Chemistry 275: 21785. Schwartz MA (2001) Integrin signaling revisited. Trends in Cell Biology 11: 466. Sheppard D (2000) In vivo functions of integrins: lessons from null mutations in mice. Matrix Biology 19: 203. Sheppard D (2003) Functions of pulmonary epithelial integrins: from development to disease. Physiological Reviews 83: 673. Sheppard D (2004) Roles of alphav integrins in vascular biology and pulmonary pathology. Current Opinion in Cell Biology 16: 552.

ADRENERGIC RECEPTORS
A E Tatterseld, Nottingham University, Nottingham, UK
& 2006 Elsevier Ltd. All rights reserved.

b-Adrenoceptors
b1 and b2 receptors were initially identied on the basis of tissue selectivity to a range of agonists. Both b-adrenoceptors were subsequently characterized and cloned, as was a third (b3) receptor and there are putative claims for a b4 receptor in the heart. There is 54% homology between b1 and b2 receptors.
Structure

Abstract
Adrenergic receptors, which includes a, b and dopamine receptors, belong to the large family of G-protein-coupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung, with both b1 and b2 receptors being widely distributed. b2Adrenoceptors are an important therapeutic target and their polymorphisms may inuence the response to b2 agonist treatment. Their numbers and functions are regulated by b-agonist stimulation and by drugs, such as corticosteroids, and cytokines. a-Adrenoceptors are found on vascular smooth muscle, presynaptic nerve endings, airways, and submucus glands, and they may help to condition inspired air. There is evidence for D1 dopamine expression on alveolar cells, where they help to clear lung edema, and for D2 receptors on sensory nerves in the lung, where they may modulate neurogenic inammation and reexmediated symptoms.

Introduction
Adrenergic receptors include a, b, and dopamine receptors; they are part of the large family of G-proteincoupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung; the role of a-adrenoceptors and dopamine receptors is less well established.

The b2-adrenoceptor contains 413 amino acids with seven transmembrane-spanning domains, three intraand three extracellular loops, an intracellular C-terminus, and an extracellular N-terminus (Figure 1). The third intracellular loop is important for linking to G-proteins. Site-directed mutagenesis studies show that b-agonists bind to residues on the hydrophobic region within the cell membrane between the third and sixth transmembrane domains as shown in Figure 1. The salmeterol molecule anchors to an exosite on the fourth transmembrane domain, allowing the molecule to stimulate the receptor repetitively. The intracellular third loop and tail of the b-adrenoceptor contains phosphorylation sites that are involved in receptor downregulation.
Polymorphisms

Nine single-base mutations or polymorphisms in the coding region of the b2-adrenoceptor gene on

54

ADRENERGIC RECEPTORS

NH2

2-Agonist

Cell membrane I II III IV V VI VII P

s

cAMP PKA PKC PTK P + arrestin Inhibits receptor function

P = Phosphorylation sites P P

H COO

ARK (GRK)

Figure 1 Schematic representation of the human beta 2-adrenoceptor showing the 7 trans membrane domains and the site on the third, fth, and sixth domains that are required for beta-agonist binding. P marks the phosphorylation sites concerned with receptor ancapling. The blue circles indicate the amino acids that are essential for b-agonist binding. Adapted from b-adrenergic receptors and their regulation by PJ Barnes.

chromosome 5Q have been identied, though only four of these cause amino acid substitutions at codons 16, 27, 34, and 167. The two most common polymorphisms are at codon 16, where glycine is substituted for arginine, and codon 27 where glutamic acid is substituted for glutamine. Both polymorphisms are common, with 37% and 23% of subjects being homozygous for the gly 16 and glu 27 polymorphisms, respectively, in a large general population study. There is linkage disequilibrium between polymorphisms at codons 16 and 27, that is, they are more likely to occur together, and this can cause difculties when trying to determine the functional effects of a specic polymorphism.
b-Adrenoceptor Activation

Protein kinase A is also able to phosphorylate and activate a transcription factor, CREB or cyclic AMP response element binding protein. By binding to the cyclic AMP response element (CRE) on the promoter region of target genes, CREB is able to cause transcription of various genes including the b-receptor gene. b2-Receptor expression may therefore be increased transiently by b-agonist stimulation. CREB also interacts with other transcription factors within the nucleus including some proinammatory cytokines such as activating protein (AP-1) and nuclear factor kappa B (NF-kB).
b-Adrenoceptor Regulation and Activity

When a b-agonist binds to the b-adrenoceptor/Gs complex guanosine diphosphate (GDP) is released, allowing guanosine triphosphate (GTP) to bind and activate the a-subunit of Gs, which then dissociates from the b-receptor and is free to stimulate adenylate cyclase. Adenylate cyclase catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (AMP), which in turn activates protein kinase A. Cyclic AMP has a range of actions that promote smooth muscle relaxation in airways and other activities such as inhibition of mediator release in inammatory cells (see Figure 2 and Table 1). Most of the effects of b-agonists are mediated via cyclic AMP and activation of protein kinase A, but some, such as the opening of maxi K channels, may, in part at least, be due to direct activation by the Gs a-subunit.

Until recently, it was thought that b-adrenoceptors existed in high- or low-afnity states, the high-afnity state being increased in the presence of b-agonists and associated with an increased binding afnity for b-agonists. The afnity for b-adrenoceptor antagonists did not differ between high- and low-afnity states. b-Adrenoceptor activation with this model only occurred when an agonist was present. However, recent work suggests that receptors may have constitutive activity, that is, activity when no agonist is present. Some b-adrenoceptor antagonists are now thought to act by stabilizing this constitutive activity rather than as a competitive antagonist and hence they can be described as inverse agonists. The number of b-adrenoceptors in the airways and other tissues depends on the rates at which they are synthesized and degraded; their function also depends on the extent to which they are coupled to the catalytic unit. Both receptor numbers and functions

ADRENERGIC RECEPTORS 55

K+ Gs -Agonist -Adrenoceptor Gs AC cAMP Inactive PKA Active PKA ATP Phosphorylation of myosin light chain kinase Inhibition of PI hydrolysis Ca2+ extrusion and sequestration Stimulation of Na+/K+ ATPase Membrane hyperpolarization Activation of Ca2+ gated potassium channels
Figure 2 Schematic representation of the signaling pathway following stimulation of the b-receptor; this causes coupling of the receptor to the Gs protein and stimulation of adenylate cyclase (AC). This in turn causes increased accumulation of cyclic-AMP (cAMP) and protein kinase A (PKA) and a fall in intracellular calcium. The most important cellular effects for asthma are smooth muscle relaxation and inhibition of mediator release from inammatory cells.

Cell membrane

Smooth muscle relaxation

Inhibition of mediator release

Table 1 Main actions of b-adrenergic agonists at different receptor subtypes Tissue Airways Receptor b2 mainly Response Bronchodilatation, reduction in mediator release from mast cells, increased mucus production, increased ciliary activity Tachycardia, inotropic action Dilatation, fall in blood pressure, compensatory reex increase in heart rate Relaxation Increase in glucose, insulin, lactate, pyruvate, nonesteried fatty acids, glycerol, and ketone bodies; decrease in potassium, phosphate, calcium, and magnesium Tremor

underlie this development: 1. Receptor uncoupling. Exposure of a b-receptor to a b-agonist causes phosphorylation of sites on the 3rd intracellular loop and tail of the b-adrenoceptor, causing the receptor to uncouple from its Gs protein and catalytic unit. Receptor uncoupling occurs within minutes and is rapidly reversible. At least two kinases are involved: the cyclic AMP-dependent protein kinase A and the G-protein-dependent b-adrenergic receptor kinase (bARK); protein kinase G may also contribute. Protein kinase A is activated by fairly low concentrations of b-agonist, as seen with therapeutic doses, whereas activation of bARK requires higher agonist concentrations and the cofactor b-arrestin. bARK causes homologous b-receptor desensitization, that is, to b-agonists only, whereas protein kinase A causes heterologous desensitization, so that the response to other agonists that use the same catalytic unit, for example, forskolin, is reduced. 2. Sequestration of receptors. Following exposure to a b-agonist, b-adrenoceptors may be internalized within the cell, and sequestrated into vesicles. This can also occur within minutes and is again reversible, the receptors being recycled to the cell surface. 3. Downregulation. With longer term exposure to bagonists, however, b-receptors may be internalized and degraded and hence are not available for recycling. Polymorphisms at codon 16 and 27 have been shown to affect b-adrenoceptor downregulation

Heart Blood vessels

b1/b2 b2

Uterus Metabolic

b2 b2/b3

Muscle

b2

are modied by b-agonists and by other factors, of which cytokines and corticosteroids are particularly relevant to asthma.

Regulation by b-agonists With repeated or continuous exposure to a b-agonist there is a reduction in the response, a phenomenon known as desensitization or tolerance. Several mechanisms have been shown to

56

ADRENERGIC RECEPTORS

in vitro. Receptors with the gly 16 polymorphism have shown increased downregulation whilst those with the glu 27 polymorphism were protected against downregulation. 4. Reduced synthesis. b-Adrenoreceptor numbers may also be lower as a result of a reduction in receptor mRNA, following continuous exposure. Regulation by other factors 1. Corticosteroids. Corticosteroids have a number of effects in vitro that may increase or decrease the response to a b-agonist. They are able to increase b-receptor numbers by inhibiting downregulation of b-receptors, and by increasing b-receptor gene transcription. However, the activity of CREB can be inhibited by glucocorticoid transcription factors, thus providing a possible mechanism whereby glucocorticoids might reduce the efcacy of a b-agonist. 2. Cytokines. The proinammatory cytokine interleukin 1b has been shown to attenuate b-receptor responsiveness by uncoupling the receptor from its catalytic unit; this appears to be due to induction of the inducible form of cyclooxygenase (COX 2) and release of inhibitory prostanoids.
Distribution of b-Adrenoceptors

patients with asthma who are homozygous for arginine at codon 16.

a-Adrenoceptors
Classifying a-adrenoceptors has been less straightforward than classifying b-adrenoceptors. The two main classes are the a1-adrenoceptors, classic postsynaptic excitatory receptors, and a2-adrenoceptors, which are usually, though not invariably, presynaptic, where they inhibit neuronal mediator release. There are three fully dened a1-adrenoceptor subtypes (a1A, a1B, a1D, and a putative a1L) and four subtypes of the a2-adrenoceptor (a2A, a2B, a2C, and a2D). Most of the subtypes have now been cloned.
a-Adrenoceptor Activation

The distribution and type of b-receptor within the lung has been determined by receptor-binding studies; airway epithelium and alveoli have the highest receptor density. Some 70% of the b-adrenoceptors in the lung are of the b2-receptor subtype, the remainder being b1-receptors. Only b2-receptors are found on airway and vascular smooth muscle whereas both b1- and b2-receptors are found on submucosal glands and alveoli. b-Receptors are distributed widely throughout the body and some of the more important sites are shown in Table 1.
b-Receptor Numbers and Function in Disease

The postsynaptic a1-adrenoceptor is coupled to a Gq protein and produces physiological effects via activation of phospholipase C, which mobilizes intracellular calcium by increasing inositol 1, 4, 5-trisphosphate concentrations, and diacylglycerol, which activates protein kinase C. a1-Adrenoceptors also activate other pathways such as the mitogen-activated protein kinase pathway. A single base polymorphism has been described in a1A but no effect on receptor function has been identied as yet. Activation of a2 receptors usually causes inhibition of adenylate cyclase through coupling with membrane-linked pertussis toxin-sensitive Gi/o protein.
Distribution

Alpha-adrenoceptors are widely distributed on neuronal and non-neuronal tissues. Within the lung, aadrenoceptors have been located predominantly in smaller airways, on serous cells in submucus glands, and on pulmonary blood vessels. a1-Adrenoceptors are thought to be present on bronchial blood vessels and a2-adrenoceptors on airway ganglia and presynaptic cholinergic nerve endings.
Function

Positron emission tomography studies indicate that there are normal numbers of b-receptors in the lungs of people with mild asthma, and they may even increase in patients dying from asthma. The function of b-receptors in the lungs of patients with asthma also appears to be normal, although it may be affected by previous b-agonist exposure and receptor polymorphisms. There is no evidence to suggest that the presence of asthma is associated with b2-receptor polymorphisms or haplotypes. However, there is some evidence that the response to b2-agonists is reduced in

The role of a-adrenoceptors in the lung is not entirely clear and work is hampered by lack of specicity of many of the ligands used to investigate a-adrenoceptor subtypes. a1-Adrenoceptors classically cause smooth muscle contraction and could therefore help control bronchial blood ow and conditioning of inspired air. Other possible roles include inhibition of histamine release from mast cells and acetylcholine release from cholinergic nerve endings, and modulating the content of airway secretions through serous cell stimulation.

ADRENERGIC RECEPTORS 57 Function in Disease

The possibility that a-adrenergic mechanisms might contribute to asthma was explored several years ago, particularly when the density of a-receptors was found to be higher in patients with asthma. a-Adrenoceptor agonists caused bronchoconstriction in some but not all studies although whether this is a specic or non-specic effect is not clear. Similarly nonselective a-adrenoceptor antagonists such as indoramin caused some degree of bronchodilatation or protected against induced bronchoconstriction in some studies, but all the drugs studied had additional pharmacological effects that could have been responsible. None of the a-adrenoceptor antagonists available caused worthwhile bronchodilatation in asthma when given regularly or by inhalation. These data and the fact that norepinephrine when infused causes bronchodilatation rather than bronchoconstriction suggest that a-adrenergic mechanisms are not making an important contribution to the bronchoconstriction seen in asthma.

breathlessness in patients with chronic obstructive pulmonary disease. Dopamine in low doses stimulates dopamine receptors but as the dose is increased it stimulates b1adrenoceptors followed by a1- and a1-adrenoceptors.
See also: Asthma: Overview. Bronchodilators: Beta Agonists. G-Protein-Coupled Receptors.

Further Reading
Barnard ML, Ridge KM, Saldias F, et al. (1999) Stimulation of the dopamine 1 receptor increases lung edema clearance. American Journal of Respiratory and Critical Care Medicine 160: 982 986. Barnes PJ (1986) Neural control of human airways in health and disease. American Review of Respiratory Diseases 134: 1289 1314. Barnes PJ (1995) Beta-adrenergic receptors and their regulation. American Journal of Respiratory and Critical Care Medicine 152: 838860. Birrell MA, Crispino N, Hele DJ, et al. (2002) Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD. British Journal of Pharmacology 136: 620628. Carswell H and Nahorski SR (1983) b-Adrenoceptor heterogeneity in guinea pig airways: comparison of functional and receptor labelling studies. British Journal of Pharmacology 79: 965971. Goidie RG, Papidimitriou SM, Paterson SW, et al. (1986) Autoradiographic localisation of b-adrenoceptors in pig lung using [125I]-iodocyanopindolol. British Journal of Pharmacology 88: 621628. Green SA, Spasoff AP, Coleman RA, Johnson M, and Liggett SB (1996) Sustained activation of a G protein coupled receptor via anchored agonist binding. Molecular localization of the salmeterol exosite within the b-adrenergic receptor. Journal of Biological Chemistry 39: 2402924035. Green SA, Turki J, Innis M, and Liggett SB (1994) Amino-terminal polymorphisms of the human b-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry 33: 94149419. Hall IP and Tatterseld AE (1998) b-Adrenoceptor agonists. In: Barnes PJ, Rodger IW, and Thomson NC (eds.) Asthma. Basic Mechanisms and Clinical Management, 3rd edn, pp. 651676. London: Academic Press. Koshimizu T, Tanoue A, Hirasawa A, Yamauchi J, and Tsujimoto G (2003) Recent advances in a1-adrenoceptor pharmacology. Pharmacology and Therapeutics 98: 235244. Lands AM, Arnold A, McAuliff JP, et al. (1967) Differentiation of receptor systems activated by sympathomimetic amines. Nature 214: 597598. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the b2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334339. Schwartz J, Carlsson A, Caron M, et al. (1998) The IUPHAR compendium of receptor characterisation and classication: dopamine receptors. IUPHAR Media (London) 142151. Strader CD, Sigal IS, and Dixon AF (1989) Mapping the functional domains of the b-adrenergic receptor. American Journal of Respiratory Cell and Molecular Biology 1: 8186.

Dopamine Receptors
Of the ve subtypes of dopamine receptors identied (D1D5), D1 and D5 receptors have similar homology and both stimulate adenylate cyclase. D2, D3, and D4 receptors are also homologous but they inhibit adenylate cyclase. Most work relates to the D1 and D2 subtypes.
Distribution and Function

Dopamine receptors are found in the central and peripheral nervous system. They have been studied in the brain predominantly and in the context of the lung there is interest in the role that central dopaminergic pathways might play in the development of nicotine addiction. Within the lung there is evidence for D1 receptors on alveoli in the rat, which, when stimulated, increase the clearance of lung edema. D2 receptor mRNA and protein are expressed in sensory ganglia in the airways and dopamine receptor activation has been shown to inhibit depolarization of the vagus in animals and man, and neuropeptide release from nerve endings. D2 dopamine receptors may therefore have a role in modulating neurogenic inammation and reex-mediated symptoms such as cough. D2 receptor agonists have reduced cough, mucus production, and tachypnea in animal models and there is interest in whether they might reduce symptoms such as cough and

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AEROSOLS
S P Newman, Nottingham, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The inhaled route is used to deliver drugs as aerosols for the maintenance therapy of asthma, chronic obstructive pulmonary disease, and other conditions. The deposition of aerosol particles in the respiratory tract is an important prerequisite to obtaining a good clinical effect. Generally, inhaler devices should deliver particles smaller than approximately 5 mm in diameter in order to enter the lungs. A variety of inhaler devices are available for inhalation therapy. Pressurized metered dose inhalers (pMDIs) have been widely used for 50 years, but many patients have problems using them correctly. They are currently being reformulated with ozone-friendly propellants. Breath-actuated inhalers and spacer attachments may be useful supplements to pMDIs for some patients. Dry powder inhalers (DPIs) are easier to use correctly than pMDIs, and they do not require propellants. Many pharmaceutical companies seem to be prioritizing DPIs above pMDI reformulation, and they are also preferred by many patients. Nebulizers continue to be used widely, but the limitations of jet and ultrasonic nebulizers have led to the development of novel systems, sometimes involving vibrating meshes. Finally, a new class of inhalers (soft mist inhalers) is emerging, composed of multidose devices containing liquid formulations, some of which could challenge pMDIs and DPIs in the portable inhaler market.

Inhaled Drug Delivery

The pulmonary route may be used to deliver drugs for the maintenance therapy of some lung diseases, most notably asthma and chronic obstructive pulmonary disease (COPD). Drugs are also given by inhalation to treat other chest problems, including respiratory tract infections in cystic brosis. In addition, it is hoped that inhaled drugs intended to have a systemic action in the body (e.g., insulin) will soon be marketed. The potential benets of the inhaled route have long been recognized, but the importance of good quality inhaler devices that deliver drugs reliably to the lungs has only been appreciated during the past 25 years.

Aerosol Properties
An understanding of aerosol properties and aerosol deposition is an important prerequisite for optimizing inhalation therapy. Drugs are given by inhalation as aerosols of solid particles or liquid droplets, but for simplicity the term particle may be used to describe both solid and liquid dispersions. The most important property of an aerosol particle is its

size, and this is best expressed as the aerodynamic diameter, which also takes into account particle density and shape. For spherical particles, aerodynamic diameter (Da) and physical diameter (Dp) are related by the formula Da DpOr, where r is the specic gravity of the material from which the particles are made. In practice, aerosol particles are seldom spherical; for instance, micronized drug particles are often highly irregular in shape. Aerosol systems found in medicine are usually heterodisperse, indicating that the particles in a particular spray or cloud have a wide range of sizes. Monodisperse aerosols, in which all the particles have approximately the same size, are not normally found in pharmaceutical products, although they can be made using specialized equipment. It is preferable to describe the mass or volume distribution of an aerosol rather than the distribution of particles by number since many small particles may contain much less drug than a few large particles. In practice, particle size spectra from inhaler devices often approximate to log-normal distributions. The mass median aerodynamic diameter (MMAD) may be used to express the average aerosol size. This diameter is such that half the aerosol mass is contained in larger particles and half in smaller particles. The spread of particle sizes may be expressed as a geometric standard deviation (GSD), a dimensionless quantity. A perfectly monodisperse aerosol has a GSD of 1. A typical pharmaceutical aerosol may contain particles ranging in size from o0.5 to 410 mm, with an MMAD of 34 mm and a GSD of 2.02.5. As explained later, deposition of aerosols depends critically on particle size. The fraction of the aerosol mass contained in particles o5 mm in diameter is usually termed the respirable fraction or ne particle fraction (FPF). These are the particles with the greatest likelihood of reaching the lungs in adults, although even smaller particles may be needed for drug therapies in small children. In adults, particles o3 mm in diameter are needed in order to deliver drugs to the alveolated regions for instance, to deliver inhaled a1 antitrypsin to the alveoli of patients with emphysema. Particle size distributions of aerosols intended for pulmonary delivery may be quantied by several methods. The approach favored within the pharmaceutical industry is the cascade impactor, through which the aerosol is drawn by a vacuum pump, and particles of different sizes are collected on a series of stages. Each stage can be washed out with a solvent

AEROSOLS 59

so that the amount of drug associated with different size bands may be quantied by an analytical technique. Supplementary particle size data may be provided by optical methods, the best known of which is laser diffraction. This involves passing the aerosol cloud through a laser beam, and the angle of diffraction of the laser light is inversely proportional to particle size. It is important to remember that these in vitro measurements are undertaken primarily for purposes of quality control and product release, and they may not predict accurately drug delivery to the lungs in vivo.

Deposition of Pharmaceutical Aerosols

Several mechanisms cause aerosol particles to deposit in the respiratory tract, but the two most important ones relating to pharmaceutical aerosols are inertial impaction and gravitational sedimentation. Inertial impaction takes place mainly in the oropharynx and at the bifurcations between major airways, when the aerosol particle has too much inertia to follow the air stream as it changes direction. The probability of inertial impaction occurring is proportional to D2 a Q, where Q is the inhaled ow rate. Deposition in central lung regions may be enhanced by the effects of air turbulence, especially at fast inhaled ow rates. Gravitational sedimentation takes place mainly in smaller conducting airways and in the alveoli, when particles settle onto the airway surface under gravity either during slow steady breathing or during breath-holding. The probability of gravitational sedimentation occurring is proportional to D2 a T, where T is the residence time of the particle in the airways. A third deposition mechanism (Brownian diffusion) is also important for aerosol particles o1 mm in diameter, which may be pushed in a random direction toward airway walls by collisions with gas molecules. Some particles (especially those o1 mm in diameter) are not deposited, and after inhalation they are simply exhaled. In addition to particle size, the patients inhalation also plays a major part in determining the site of aerosol deposition. The inhaled ow rate is particularly important, with slow inhalation usually being recommended in order to reduce impaction losses in the oropharynx. Deep inhalation and a period of breathholding help to increase gravitational sedimentation in the peripheral parts of the lungs. For most pharmaceutical aerosols, lung deposition is enhanced by a combination of aerosol particles o5 mm in diameter and a slow inhaled ow rate (2030 l min 1). As will be explained later, there is an exception to this rule for dry powder inhalers, where faster inhalation

may preferable. Particles are ltered efciently from the inhaled air by the nasal passages, so wherever practicable it is better to deliver an inhaled aerosol via a mouthpiece (with mouth breathing) than via a face mask (with nose breathing). The airways of the patient who inhales the aerosol particles also determine the site and extent of deposition in two major ways. First, random variations in airway geometry between different individuals will lead to random variations in the deposition pattern. Hence, for aerosols delivered from any inhaler device, considerable intersubject variability of deposition is to be expected. Second, in patients with asthma, COPD, and other obstructive conditions, the airways may be narrowed by bronchospasm, inammation, and mucus hypersecretion so that aerosol particles may deposit preferentially in the larger airways of the lungs, with less deposition in the peripheral airways. Both electrostatic charge and humidity affect aerosol deposition in a variety of ways. The most striking effect of humidity is that dry particles composed of water-soluble materials are likely to absorb water when they enter the respiratory tract and, hence, to increase in size. The deposition of pharmaceutical aerosols may be quantied by radionuclide imaging (gamma scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography (PET)). SPECT and PET are three-dimensional imaging methods and provide information about the distribution pattern within the lungs. However, PET is relatively complex and is probably not practical for use on a regular basis. Certain pharmacokinetic methods are also useful for assessing delivery of some drugs to the lungs. For instance, the plasma or urinary concentrations of albuterol in the rst 30 min after inhalation are considered to result solely from pulmonary absorption.

Pressurized Metered Dose Inhalers

The pressurized metered dose inhaler (pMDI) has been the backbone of inhalation therapy for asthma for approximately 50 years, since its introduction by 3 M Riker Laboratories in 1956. Patients and physicians recognized the convenience of the pMDI, which contains 100200 doses in a small portable device that is immediately ready for use (Figure 1). The pMDI consists of an aluminum can mounted in a plastic actuator. Individual doses (25100 ml) are delivered as a spray via a sophisticated metering valve. The drug is usually a micronized suspension of drug particles but may be a solution dissolved in propellants, ethanol, or another excipient as a co-solvent.

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Patient presses canister while breathing in Canister Actuator

Drug formulation in propellants

Metering valve Actuator nozzle

Spray plume

Figure 1 Design and operation of a typical pressurized metered dose inhaler.

The best known pMDI therapies include the b-agonists albuterol, terbutaline, and salmeterol and the glucocorticosteroids beclomethasone dipropionate, budesonide, and uticasone propionate. Successful pMDI therapy is highly dependent on the patients inhalation technique, and patient education about their use is essential. In most pMDI products, it is necessary for the patient to press the pMDI at the same time as inhaling. Failure to do this is sometimes described as poor coordination or handlung dyscoordination, and it is probably the most important problem patients have with pMDIs. A second major problem using pMDIs is the so-called cold Freon effect, where the patient stops inhaling when the cold propellant spray is felt on the back of the throat. Freon is one of the trade names of chlorouorocarbon (CFC) propellants. In order to optimize lung deposition from pMDIs, patients also need to inhale slowly and deeply and to hold the breath for several seconds. Even with perfect inhalation technique, no more than 1020% of the dose from a CFC pMDI is deposited in the lungs, with the majority of the dose being deposited in the oropharynx. However, the lung dose will vary from product to product according to the nature of the formulation and the diameter of the actuator orice. Until recently, all pMDIs were formulated in CFC propellants, giving the pMDI an internal pressure of approximately 300 kPa (3 atm) and a spray velocity at the nozzle exceeding 30 m s 1. However, it is possible to reduce the spray velocity by modications to the actuator design, for instance, in the Spacehaler device (formerly known as Gentlehaler). During the past few years, the pharmaceutical industry has been forced to start reformulating pMDIs in non-CFC propellants, consisting of one of two hydrouoroalkanes (HFA-134a or HFA-227). This challenge arose

following the discovery that the degradation of CFCs damages stratospheric ozone and has proved to be a major stimulus to the development of novel inhaler technologies. The switch to HFA-powered pMDIs is in progress and will take several more years to complete. In the meantime, CFCs have been granted an essential-use exemption in pMDIs under the Montreal Protocol of 1987, reecting their importance to the well-being of society. HFAs are greenhouse gases, and despite the fact that their contribution to global warming is small, this issue could restrict their future use. The development of novel HFA pMDI formulations has not been a simple manner, owing to a range of technical factors and the need to demonstrate clinical efcacy and safety for the reformulated products. Individual companies have adopted one of two strategies. One strategy involves making a product that is bioequivalent with the CFC pMDI that is to be replaced so that the HFA pMDI can be used in exactly the same doses as the CFC pMDI. The alternative strategy is to make a product that deposits drug in the lungs more efciently than a CFC pMDI. This usually involves formulating a corticosteroid product as a solution, enabling a very small particle size to be achieved as the propellant evaporates. With such a product, it is also possible to reduce the spray velocity and to deposit up to half the dose in the patients lung, with greatly reduced oropharyngeal deposition, so that asthma control may be achieved using only a fraction of the CFC pMDI dose. A formulation of beclomethasone dipropionate (Qvar) was the rst of these products to reach the market, and several similar products are either already marketed or in development. Breath-actuated pMDIs may be helpful in patients with poor coordination, who cannot actuate the pMDI at the same time as inhaling. These devices contain triggering mechanisms that are operated by the patients inhalation via the mouthpiece. However, it is unlikely that breath-actuated pMDIs confer any additional benet on patients who can use a conventional pMDI successfully.

pMDIs with Spacer Devices

Spacer devices are widely used with pMDIs. These vary greatly in size and shape, with volumes of commercially available models ranging from 50 to 750 ml. The concept of a spacer is to place some distance between the point at which the aerosol is generated and the patients mouth, allowing the propellant to evaporate and the rapidly moving aerosol cloud to slow down before it is inhaled (Figure 2). The most successful spacers have a one-way valve in

AEROSOLS 61

Formulation: ordered mixture of drug and carrier

DPI device

Powder de-aggregated by patients inhalation

Figure 3 Principle of operation of a dry powder inhaler (DPI). The formulation most frequently consists of an ordered mixture of micronized drug and carrier lactose, which is de-aggregated by the patients inhalation through the device.

Figure 2 pMDI connected to a large volume spacer device.

the mouthpiece, which allows the pMDI to be actuated into the spacer, with a brief pause before the patient inhales so that it is not necessary to actuate and inhale simultaneously. Some spacers function effectively if the patient takes a series of relaxed tidal breaths from the device immediately after actuating a dose. Spacers reduce oropharyngeal deposition of drug and may increase lung deposition, but the majority of the dose is often deposited on the walls of the spacer. This may allow the reduction of the total body burden of inhaled corticosteroids compared with a standard pMDI. Large volume spacers, such as the Volumatic and Nebuhaler, have a well-accepted role in hospital emergency rooms for treating acute asthmatic attacks. Specially designed spacers with a volume of 200300 ml are available for treating young children. Most spacer devices are made of plastic, which may acquire a static charge during handling. This results in a suspended aerosol cloud being attracted to the spacer walls, with a marked reduction in the dose available for inhalation. Specic handling and washing techniques are usually recommended, and at least one lightweight metal spacer is available that is not susceptible to the effects of static charge. With correct use, including control over electrostatic charge effects, large volume (4500 ml) spacer devices may deposit more than 30% of the dose from a CFC pMDI in the patients lungs.

Dry Powder Inhalers

Dry powder inhalers (DPIs) have been available commercially since approximately 1970, although the earliest prototypes were described several decades earlier. DPIs contain a powder formulation, which most frequently consists of an ordered mixture of micronized drug (o5 mm in diameter) and larger

carrier lactose particles that are required to improve powder ow properties. The patients inhalation through the device is used to disperse the powder and to ensure that some of the dose is carried into the lungs (Figure 3). An alternative type of formulation used in some DPIs consists either of micronized drug particles alone loosely aggregated into small spherules or of cospheronized drug and lactose. DPIs are basically of three types: (1) unit-dose devices, in which an individual dose in a gelatin capsule or blister is loaded by the patient immediately before use; (2) multiple unit-dose devices, which contain a series of blisters or capsules; and (3) reservoir devices, in which powder is metered from a storage unit by the patient before inhalation. Unit-dose devices, including Spinhaler and Rotahaler, were the only DPIs available until the mid-1980s. Patients generally nd multiple unit-dose devices, such as the Diskus (Accuhaler), and reservoir DPIs, such as the Turbuhaler, to be more convenient than unit-dose DPIs since they provide several weeks treatment. DPIs tend to deposit a greater fraction of the dose in the lungs compared with CFC pMDIs, but in practice lung deposition varies widely between devices (Figure 4). Powder formulations are susceptible to the effects of moisture, and protecting the formulation against these effects is an important part of DPI design. By the end of 2004, at least 16 DPIs had been marketed in different areas of the world for asthma and COPD therapy, involving a range of unit-dose, multiple unit-dose, and reservoir systems. A further 2030 DPIs were also known to be in development. The anticipated expansion of the generics market for inhaled asthma and COPD drugs is likely to result in a number of these novel DPIs reaching the market. It is interesting to note that the major pharmaceutical companies with an interest in inhaled asthma and COPD drugs appear to be prioritizing the DPI over reformulated HFA pMDIs products. In particular,

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100 90 80 Percentage of dose 70 60 50 40 30 20 10 0
D is kh U ale ltr ah r al Pu er lv Sp inal in ha Ae ler ro liz e Ai r rm M AG ax Tu hal rb er uh Ea aler sy ha C lic ler kh al e N ov r ol iz e Ta r ifu Fl n ow ca p Ec s lip se

Figure 4 Mean percentage of the dose deposited in the lungs from 14 dry powder inhalers (DPIs), obtained in scintigraphic studies. The high lung deposition from the Flowcaps and Eclipse DPIs probably reects the properties of the formulation as much as the DPI.

combination products in DPIs containing a longacting b-agonist and a corticosteroid (e.g., Advair Diskus and Symbicort Turbuhaler) have been very successful. However, DPIs tend to be more expensive than pMDIs, and this may limit their use, especially in developing countries. DPIs have two major advantages over pMDIs. First, they do not contain propellants. Second, all currently marketed models are breath-actuated, and patients nd them easier to use correctly than pMDIs. However, this second advantage is closely linked to a disadvantage. In order to disperse the powder as efciently as possible, and hence to maximize lung deposition, it may be necessary for patients to inhale as forcefully as possible via the DPI, and some patients may be either unable or unwilling to do this. All DPIs exhibit some degree of inhaled ow rate dependence, with forceful (fast) inhalation tending to give higher lung deposition than more gentle (slow) inhalation. For instance, in the Turbuhaler DPI, a reduction in peak inhaled ow rate from 60 to 30 l min 1 was shown to result in a reduction in lung deposition from 27% to 14% of the dose. In this respect, DPIs present a paradox since fast inhalation per se is generally associated with enhanced deposition in the oropharynx, as described previously. Low inspiratory effort through a DPI may result in a reduced emitted dose and poor particle deaggregation. The actual magnitude of the peak inhaled ow rate associated with forceful inhalation will vary between devices from o30 to 4100 l min 1, according to the resistance to airow of each device. Not only the peak inhaled ow rate achieved through the DPI but

also the time taken to reach the peak ow will determine how efciently particles are deaggregated. In practice, it seems that almost all patients with stable asthma or COPD can inhale sufciently well via DPIs to benet from them. Several so-called active DPIs have been developed, in which the powder is dispersed by some mechanism other than the patients inhalation for instance, by an internal source of compressed air or by a fan driven by an electric motor. These active DPIs are generally more complex than breath-actuated DPIs and may come to be used primarily for therapies that require very efcient and reproducible targeting of drugs to specic lung regions, such as inhaled peptides for systemic therapy. Sophisticated formulations for use in DPIs are also in development. These include drug/lactose blends, in which the surface of the lactose particles has been smoothed in order to aid dispersion, or particles made by processes other than micronization. For instance, a spray-dried formulation of the antibiotic tobramycin is under development for the treatment and prevention of respiratory tract infections in patients with cystic brosis, consisting of low-density spherical particles that disperse efciently with minimal inspiratory effort. An advantage of these sophisticated formulations is that often they can be delivered efciently to the lungs using very simple and inexpensive DPI devices.

Nebulizers
Drugs may often be formulated as solutions in water or ethanol, and they may be delivered by nebulizers

AEROSOLS 63
Signal from piezoelectric crystal Mouthpiece Baffle Venturi Drug formulation in nebulizer cup Mesh

Drug formulation in reservoir

Compressed air
Figure 5 Design and operation of a typical jet nebulizer.

To mouthpiece
Figure 6 Principle of operation of a mesh-based nebulizer system. A mesh or grid is vibrated by a piezoelectric crystal, and a dispersion of micron-sized liquid droplets is formed.

that convert the solution into a spray. A variety of devices may be used to form the spray, and the three most common are jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. An important advantage of nebulizers is that they can be used with relaxed tidal breathing. This makes them attractive for delivering inhaled drugs to children, the elderly, and those undergoing acute asthmatic attacks, who may not be able to use pMDIs or DPIs successfully. Currently, nebulizers represent the most practical way to deliver very large drug doses (4100 mg) that are occasionally needed for some inhaled antibiotics. Jet nebulizers are operated by compressed air passing through a narrow constriction (a venturi). A single dose contained in a volume of typically 24 ml in a cup within the nebulizer is drawn up a feed tube and is fragmented into droplets (Figure 5). Only the smallest droplets are delivered directly to the patient; larger droplets impact on bafe structures situated close to the nozzle and are returned to the cup to be nebulized again. Several minutes are required to nebulize the entire dose, and even at completion of treatment the majority of the dose remains within the device as large droplets on internal walls. There are major differences in performance between different commercially available nebulizers, with lung deposition ranging from o2% to 20% of the dose. Jet nebulizers can also be used to aerosolize micronized suspensions of corticosteroids. Recent developments in technology have included breath-enhanced nebulizers, in which passage of inhaled air through the device is used to increase aerosol output, and adaptive aerosol delivery systems, in which aerosol generation is synchronized to coincide with the rst part of the patients inhalation. Adaptive aerosol delivery systems seem to be

able to reduce the intersubject variability of aerosol delivery. Ultrasonic nebulizers have many properties similar to jet nebulizers, but the aerosol is formed in a different way. A piezoelectric crystal is located beneath the cup, and a fountain of droplets is generated. Ultrasonic nebulizers are less popular now than a few years ago, possibly for several reasons. They may not handle either suspensions or viscous solutions well, and there is evidence that they damage some drug molecules, probably by heat generated during the nebulization process. Jet and ultrasonic nebulizers cannot compete with pMDIs and DPIs in the portable inhaler market, partly because they are singledose devices and partly because they generally need either a compressor or a power source in order to function. Several novel nebulizers are available in which the spray is formed by the passage of drug solution through a vibrating mesh or grid of micron-sized holes (Figure 6). The mesh is usually vibrated by a piezoelectric crystal, but unlike ultrasonic nebulizers, there is no evidence that this process damages drug molecules. Mesh-based systems deliver a higher proportion of the dose, and achieve higher lung deposition, compared to jet or ultrasonic nebulizers. A smaller percentage of the dose is retained in the device at the end of treatment, and this can result in less wastage for expensive drug substances. Nebulization time is short compared to that of jet and ultrasonic nebulizers, which should improve patient compliance. Some vibrating mesh nebulizers are small, compact, and battery operated, giving them practical advantages over jet and ultrasonic nebulizers. Careful cleaning of all nebulizers is essential in order to avoid bacterial contamination and to ensure that the working parts (particularly narrow nozzles) function correctly.

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Soft Mist Inhalers

A recent development in inhaler technology has been the development of low-velocity sprays known as soft mist inhalers. These devices represent a new class of multidose inhaler devices and contain liquid formulations similar to those in nebulizers. A variety of principles are utilized, including forcing liquid under pressure through a nozzle array, ultrasonics, vibrating meshes, and several novel approaches, such as condensation of vapors to form particle dispersions. Many of these devices are able to achieve extremely high lung deposition (450% of the dose), and they are capable of delivering drugs to the deepest parts of the lungs. This may allow them to play a major future role in inhalation therapy, particularly in situations in which precise aerosol targeting is needed. In 2004, one soft mist inhaler (Respimat) was launched in Europe for asthma and COPD therapy as a direct replacement for the same drugs given either in a CFC pMDI or in a DPI. The spray is formed by passing a metered dose (typically 15 ml) via a sophisticated nozzle system under pressure. The velocity of the spray is only a fraction of that found in a CFC pMDI. This device deposits a greater percentage of the drug in the lungs compared to a CFC pMDI (Figure 7), and it is clinically effective

using smaller doses. It is probable that other soft mist inhalers will be marketed in the relatively near future, and some could mount a significant challenge to pMDIs and DPIs in the portable inhaler market.
See also: Asthma: Overview. Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive Pulmonary Disease: Overview: Emphysema, Alpha-1Antitrypsin Deciency. Corticosteroids: Therapy. Cystic Fibrosis: Overview. Particle Deposition in the Lung.

Further Reading
Adjei AL and Gupta PK (1997) Inhalation Delivery of Therapeutic Peptides and Proteins. New York: Dekker. Bisgaard H, OCallaghan C, and Smaldone GC (eds.) (2003) Drug Delivery to the Lung. New York: Dekker. Dalby RN, Byron PR, Peart J, and Farr SJ (eds.) (2002) Respiratory Drug Delivery VIII. Raleigh, NC: Davis Horwood. Dalby RN, Byron PR, Peart J, Suman JD, and Farr SJ (eds.) (2004) Respiratory Drug Delivery IX. River Grove, IL: Davis Healthcare. Dolovich M, MacIntyre NR, Dhand R, et al. (2000) Consensus conference on aerosols and delivery devices. Respiratory Care 45: 588776. Hickey AJ (ed.) 2003. Aerosol delivery and asthma therapy (theme issue). Advanced Drug Delivery Reviews 55, 777928. Mitchell JP and Nagel MW (2003) Cascade impactors for size characterization of aerosols from medical inhalers: their uses and limitations. Journal of Aerosol Medicine 16: 341377. More n F, Dolovich MB, Newhouse MT, and Newman SP (eds.) (1993) Aerosols in Medicine: Principles, Diagnosis and Therapy. Amsterdam: Elsevier. Newman SP, et al. (1998) Lung deposition of fenoterol and unisolide delivered using a novel device for inhaled medicines. Chest 113: 957963. Newman SP and Newhouse MT (1996) Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects. Journal of Aerosol Medicine 9: 5570. OCallaghan C and Barry PW (1997) The science of nebulised drug delivery. Thorax 52(supplement 2): S31S44. Pauwels R, Newman SP, and Borgstro m L (1997) Airway deposition and airway effects of antiasthma drugs delivered from metered dose inhalers. European Respiratory Journal 10: 2127 2138. Smith IJ and Parry-Billings M (2003) The inhalers of the future? A review of dry powder devices on the market today. Pulmonary Pharmacology and Therapeutics 16: 7995.

100% 80% 60% 40% 20% 0% CFC pMDI Respimat

Exhaled Device Oropharynx Lungs

Figure 7 Fractionation of the dose from a novel Respimat soft mist inhaler compared to that from a pMDI formulated with chlorouorocarbon (CFC) propellants. Data from Newman SP et al. (1998) Lung deposition of fenoterol and unisolide delivered using a novel device for inhaled medicines. Chest 113: 957963.

Allergic Bronchopulmonary Aspergillosis see Asthma: Allergic Bronchopulmonary Aspergillosis.

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ALLERGY
Contents

Overview Allergic Reactions Allergic Rhinitis

Overview
J A Grant and C C Horner, University of Texas Medical Branch, Galveston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Allergy is the term used for a collection of diseases mediated by immunologic mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis. The prevalence of allergic diseases has been increasing in recent times. They are a major cause of morbidity and decreased quality of life. The development of allergic diseases is inuenced by heritable and environmental factors. Diagnosis often involves documenting responses to allergen such as in skin testing, radioallergosorbant allergen testing, or bronchial provocation testing. Allergic disorders share the common pathology of inammation of affected tissues. Allergy requires sensitization to an allergen and response on reexposure to that same allergen. Pathogenesis involves production and release of cytokines, chemokines, and lipid mediators which cause tissue damage and recruit inammatory cells. Current therapies include allergen avoidance, antihistamines, leukotriene modiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy.

Introduction
Allergy encompasses a wide variety of disorders that share immunologic mechanisms. For centuries, patients had allergic symptoms but the causative agent for sensitivity to allergens was unknown. Allergic symptoms were rst described by Leonardo Bottallo in sixteenth century Europe. Wyman identied ragweed as the trigger of the autumnal catarrh and Blackely identied hay fever as being initiated by grass pollen in the 1870s. Twenty years later Behring described the adverse skin reactions to tubercle bacillus as hypersensitivity. Portier and Richet attempted to confer immunity to sea anemone toxin by injecting dogs with subsequent doses of toxin. They employed the term anaphylaxis (denoting antiprotection) in 1902 to describe a lethal reaction after the dogs received the second dose of the toxin. Von Pirquet used the term allergy in 1906 to describe the skin reaction to cowpox vaccine at 24 h post-vaccination. His definition of allergy described an organisms alteration

by contact with an organic agent. Four years later, Noon observed reactions to pollen extracts on abraded skin and began immunotherapy with these extracts. Dale and Laidlaw produced respiratory distress and anaphylaxis in animals by histamine infusion in 1919. Another major discovery was the identication of cytokines. This class includes chemokines that are important in the recruitment of inammatory cells. In 1921, Prausnitz and Kustner demonstrated that a factor could be transferred to a nonsensitized persons skin and confer sensitivity. This factor was called reagin by Coca and Cooke. In 1966, Ishizakas discovery of IgE established a scientic basis for the specic reactions. Concurrently, S G O Johannson independently identied a protein in multiple myeloma that was also elevated in allergic patients. Subsequently, the WHO determined that both proteins were IgE. In 1968, Gell and Coomb described four classes of immunologic reactions. These classes are IgE-mediated immediate hypersensitivity (e.g., anaphylactic shock), IgG- or IgM-mediated cytotoxic reactions (e.g., immune hemolytic anemia), immune complexmediated reactions (e.g., serum sickness), and delayed hypersensitivity (e.g., contact dermatitis). Allergic diseases can be mediated by any of these mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis.

Etiology
Atopy refers to the tendency of patients to be sensitive to allergens. Atopic diseases include eczema, allergic rhinitis, and asthma. Over the past century, genetics has been thought to affect allergic disease occurrence. The incidence of allergic disease in patients with an allergic family history is higher than in those without one. Atopy is thought to be autosomally transmitted and multigenic. It is probably a complex trait inuenced by both heritable and environmental factors. Environmental exposures likely have effects on gene expression and the development of allergic disease. The hygiene hypothesis proposes

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that in Western countries the developing immune system is deprived of environmental microbial antigens that stimulate Th1 cells. This lack of stimulation increases the presence of atopic disease. Other factors such as diet and exposure to high pollen counts at birth may also increase allergic disease. Linkage studies for allergy and asthma have produced 420 distinct chromosomal regions with linkage to asthma or related traits. The lack of distinct phenotypes, the inexact definitions of allergic diseases, and the presumed inuence of numerous genes have made positional cloning with linkage studies problematic. The linkages reproduced most frequently are on 6p, 5q, 12q, and 13q. Linkage on 14q and 7p was identied in founder populations in Iceland and Finland. Chromosome 2q142q32, which includes the IL-1 gene, has been linked to asthma. Candidate gene analysis is a promising technique. Candidate genes encode biochemical markers that affect allergic diseases. Many of these genes control IgE and cytokine production. Candidate genes include one loci on 5q near the gene cluster for IL-4, IL-5, IL-9, and IL-13. Polymorphisms of the IL-9 gene are associated with human asthma. 11q13, which encodes the b chain of the high-afnity IgE receptor has been linked to asthma. Polymorphisms of the IL-4 receptor a chain are associated with atopic asthma. PHF11, present in the locus for total IgE on 13q14, is expressed in many immune-related tissues. It is associated with total IgE and has been linked with asthma in multiple genome screens. The ADAM33 gene on 20p13 encodes a disintegrin and metalloprotease. This gene was mapped in 2002 as an asthma and airway hyperresponsiveness gene by Van Eerdewegh and co-workers. A cluster of single nucleotide polymorphisms (SNPs) was identied in the ADAM33 gene and demonstrated significant associations with asthma. Howard and co-workers reproduced the association seen by Van Eerdeweghs group and lent support to the potential role of ADAM33 in asthma susceptibility (see Genetics: Gene Association Studies).

by binding endothelial cell adhesion molecules. Major basic protein, lipid mediators, and cytokines enable the eosinophil proinammatory effects. Eosinophils may also repair damage to mucosal surfaces with brogenic growth factor and matrix metalloprotease. This repair mechanism may result in remodeling of airway tissue as seen in asthma. Mast cells are present in tissues throughout the body. Features of an allergic reaction vary with the anatomic site. The site of allergen contact determines the tissue or organ involved. The concentration of mast cells at the site determines the severity. The wheal and are is the typical cutaneous allergic response. When allergen binds to specic IgE on mast cells, mast cell mediators are released and cause local blood vessel dilation. These vessels leak uid and macromolecules and produce redness and swelling (known as the wheal). Dilation of vessels on the edge of the swelling causes redness (known as the are).

Clinical Features
Patients with allergic rhinitis typically experience rhinorrhea, congestion, sneezing, and itchy nose. Patients also may report postnasal drip and associated eye, ear, and throat symptoms. Patients commonly exhibit reactions to dust mite, animal dander, molds, and pollens. Small molecular weight chemicals can also react with self-proteins and become allergens. Reactivity to allergens can be determined by immediate hypersensitivity skin testing. Drops of allergen are placed into the dermis by various methods. Most skin testing employs a prick device composed of metal or plastic to introduce allergen into the skin. Allergen binds IgE and the mast cells in the skin release histamine to produce the wheal and are response. The skin response can be compared to controls of saline (negative control) and histamine (positive control). Radioallergosorbant allergen testing (RAST) quantitates allergenspecic IgE in a patients serum. RAST is usually reserved for patients who have a contraindication to skin testing or are taking medications that either interfere with testing (antihistamines) or interfere with treatment should a reaction occur (beta blockers or ace inhibitors). Nasal cytology can reveal the presence or absence of inammatory cells, especially eosinophils. Adjunct tests such as rhinoscopy and rhinomanometry can provide further characterization of the nasal airway (see Allergy: Allergic Rhinitis). Patients with asthma report symptoms of wheezing, shortness of breath, cough, and chest tightness. These patients airways show hyperresponsiveness

Pathology
Allergic inammation is present in all allergic diseases. Most tissues exhibit vasodilation and increased vascular permeability. Eosinophils, neutrophils, CD4 T cells, and basophils eventually inltrate the site of allergy. Asthma is a disorder which also involves an increase in mucous glands, mucus hypersecretion, smooth muscle hypertrophy, and airway remodeling. Recruitment of eosinophils is a prominent feature of allergic diseases (see Leukocytes: Eosinophils). Eosinophils migrate across blood vessels into tissues

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with reversibility. Patients may experience asthmatic symptoms in response to allergens, infections, exercise, nonsteroidal anti-inammatory drugs, gastroesophageal reux disease, stress, and irritants. Lung function can be assessed by spirometry before and after treatment with bronchodilators. Bronchial hyperresponsiveness can be investigated with bronchial provocation testing such as with methacholine or exercise challenge testing (see Asthma: Overview). Urticaria is localized edema in skin or mucous membranes. Patients have pale to pink wheals that are extremely pruritic. These lesions are transient and usually resolve within 24 h. Angioedema involves local edema in deeper areas of skin or mucous membranes. These lesions are often painful. Urticaria/angioedema can be triggered by temperature, sun, direct pressure, medication, infections, foods, or systemic diseases. Atopic eczema is a skin disorder with pruritis, erythematous macules or papules, and xerosis. Lesions may become excoriated with crust and exudates. Skin tends to be dry and more permeable to allergens and bacteria. Chronic irritation may cause lichenication of the skin and scaling patches or papules. Young children typically have lesions on the face, scalp, and extensor surfaces. Older children and adults have exor surface involvement. Food allergy is most frequent in young children. Symptoms may include urticaria, angioedema, rash, ushing, rhinitis, wheezing, or anaphylaxis after ingestion of the allergenic food. Some patients experience the oral allergy syndrome which is usually conned to the oropharynx. Symptoms may consist of pruritis and angioedema of the tongue, lips, palate, and throat. Patients with birch pollen-induced rhinitis may develop oral allergy symptoms after eating raw potato, carrot, apple, celery, or hazelnut. Patients with ragweed pollen-induced rhinitis may develop symptoms after eating melons or bananas. Major food allergens in children are milk, egg, peanuts, soybeans, wheat, sh, and tree nuts. Major food allergens in adults are peanuts, tree nuts, sh, and shellsh. Conformational and sequential food epitopes are responsible for food allergy. Patients with IgE to sequential epitopes react to all forms of a food and tend to have persistent allergy, whereas those with IgE to conformational epitopes tolerate small amounts of food after heating or partial hydrolysis because these conformational epitopes are destroyed. These patients tend to have clinical tolerance. Prick skin testing for foods can be performed as described previously. Negative prick skin tests have a high negative predictive value. Positive skin tests are not conclusive. RAST may also be performed to aid in diagnosis.

Drug allergy reactions most commonly consist of urticaria, morbilliform rash, or anaphylaxis. Skin testing for drug allergy is not standardized and the predictive value of this technique is unclear. Anaphylaxis is an immediate generalized systemic allergic reaction in response to an allergen. Symptoms may include ushing, pruritis, urticaria, angioedema, wheezing, shortness of breath, chest tightness, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, and hypotension. Serum tryptase can be measured within 6 h of a reaction to determine if mast cell degranulation has occurred during the reaction. Serum histamine and urinary LTE4 may be quantitated to provide evidence of anaphylaxis.

Pathogenesis
Immunology plays a large role in allergic diseases. T lymphocytes are the major effector cells of these immune responses (see Leukocytes: T cells). CD4 lymphocytes are present in two predominant types, Th1 and Th2 cells. Th1 are helper cells that produce IL-2 and interferon gamma (IFN-g) and promote cellmediated reactions. Th2 are helper cells that produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are involved in humoral immunity and allergic inammation. Th2 cytokines augment antibody production (especially IgE), enhance eosinophil production, and are associated with allergic and antibody-driven responses (Figure 1). Th1 and Th2 cells suppress each other. Patients with an allergic phenotype generate responses to allergens that favor Th2 responses. Another type of T cell is the regulatory T cell (T reg). Thymus-derived CD4 CD25 regulatory cells are termed naturally occurring T regs. Adaptive T regs are T-cell populations induced by in vitro or in vivo manipulation. Active T-cell suppression by T regs promotes immunologic tolerance, but the mechanism of this suppression is controversial. There is a potential role for T regs in the control or prevention of Th2 responses. CD4 CD25 T cells and IL-10producing T regs have been shown in humans to prevent T-cell activation by allergens and are possibly decient in atopic patients. T regs may also secrete the immunosuppressive cytokines IL-10 and TGF-b. IL-10 inhibits macrophage activation and antagonizes IFN-g while TGF-b inhibits T- and B-cell proliferation. IgE-mediated allergic responses occur through numerous steps. First, a patient is exposed to an allergen. Then antigen-presenting cells internalize the allergen which is then processed and presented to Th2 cells by class II MHC molecules. T-cell receptors bind the presented allergen thus activating the Th2

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ALLERGY / Overview
APC Th2 cell

Allergen IL-4

Re-exposure to allergen B cell Arachidonic acid Mast cell Preformed mediators (histamine, TNF- )

Specific IgE

Prostaglandins, leukotrienes Early response

Vascular leak, bronchoconstriction, inflammation, tissue damage, late response

Figure 1 Allergen is processed by antigen presenting cells (APCs) and presented to Th2 cells. Stimulated by IL-4, B cells produce allergen-specic IgE. Upon re-exposure to allergen, allergen cross-links specic IgE on mast cells and activates allergic responses.

cells. The activated cells produce IL-4. Atopic patients have more allergen-specic IL-4 secretory T cells in circulation than nonatopics and produce more IL-4 per cell. B cells, in turn, are stimulated by IL-4 to class-switch to produce IgE, specic to the allergen. Approximately 20% of an exposed population will generate specic IgE to an allergen. This IgE binds to receptors on mast cells. When the patient is subsequently exposed to the allergen, the allergen binds the IgE already present on the mast cell surfaces and cross-links the antibodies. Antibody cross-linking of mast cells activates a variety of responses. The early phase of immediate hypersensitivity occurs within minutes and involves the release of preformed mediators from granules. These mediators include biogenic amines (histamine), enzymes (tryptase, chymase), carboxypeptidase, cathepsin G, acid hydrolases, and tumor necrosis factor alpha (TNF-a) (see Leukocytes: Mast Cells and Basophils). Histamine causes endothelial cells to contract and allow plasma to leak into tissue. Endothelial cells also produce prostacyclin and nitric oxide which promote vascular smooth muscle relaxation and lead to vasodilation. Bronchial smooth muscle constriction is also caused by histamine. Antibody cross-linking also triggers the initiation of several pathways which lead to cytokine, prostaglandin, and leukotriene production. Arachadonic acid is converted to prostaglandin D2 and the cysteinyl leukotrienes (LTC4, LTD4, LTE4) (see Lipid Mediators: Leukotrienes). These mediators are responsible for vascular leak, bronchoconstriction, inammation, and tissue damage. These substances lead to inammatory changes hours after exposure, referred to as the late-phase response. The late phase involves recruitment and inltration of the mucosa

with neutrophils, eosinophils, basophils, and Th2 cells. TNF-a activates endothelial expression of adhesion molecules E-selectin and ICAM-1 and promotes cell inltration. IL-3 promotes mast cell proliferation. IL-5 stimulates eosinophil production and activation (see Interleukins: IL-5). The chemokines eotaxin and monocyte chemotactic protein-5 from epithelial cells recruit eosinophils. IL-4 and IL13 induce Th2 differentiation. While mast cells are responsible for the majority of leukotriene production in the early response, basophils and eosinophils produce most of the leukotrienes in the late-phase response.

Animal Models
Mouse models have been used in the study of allergy because mice are readily available, have a well-characterized immune system, and strains are genetically characterized. Knockout mice can be used to evaluate the role of a cell type or mediator. Many allergists propose that allergic rhinitis and asthma may represent a continuum of inammation and should be considered as a united allergy airway disorder. Mouse models have been used to investigate this concept. Mice were systemically sensitized with allergen (most often ovalbumin) and then challenged with airway allergen. The inammatory response in the mouse nose resembles human allergic rhinitis. Nasal mucosal thickening can be seen on imaging. Experimental asthma in mice also mimics human asthma. Bronchial hyperresponsiveness can be documented by plethysmography. Most of the inammation in mice is seen in the lower airways where the minority of allergen is deposited. This may indicate increased sensitivity in the lower airways. Inhaled

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allergen causes an increase in allergen-specic IgE and eosinophils in the blood and increases bone marrow eosinopoiesis. Currently, it is unclear why sensitization causes symptoms in the nose, the lower airway, or both. There may be a genetic cause so studying different strains of mice may be useful. Mouse models have also been used in the study of atopic eczema. Mice have been sensitized epicutaneously with ovalbumin on shaved skin. Elevated serum total and specic IgE and IgG1 and increased dermal IL-4, IL-5, and IFN-g were observed. Deciencies of these cytokines decreased the eczematous phenotype. For example, IL-4-decient mice showed Th1biased skin inammation with decreased eosinophils and eotaxin mRNA in the dermal inltrate. IL-5decient mice similarly had less pronounced epidermal and dermal thickening and the dermal inltrate lacked eosinophils. IFN-g-decient mice showed only slight dermal thickening. The necessity of certain lymphocytes in eczema was demonstrated. ab T cells are essential since T-cell receptor a-chain-decient mice did not develop a dermal inltrate or induction of IL-4 or IgE. Mice that lack gd T cells showed no change in inltrate. Likewise, mice that lack B cells still develop an inltrate and elevated IL-4. Numerous animal models have been utilized in food allergy. Rats and mice have been used to assess foodinduced anaphylaxis. Animals ingested ovalbumin by gavage or in drinking water and were subsequently challenged intraperitoneally. The route, dose, and age of the animal were shown to inuence sensitization. Rats and swine sensitized to allergens and subsequently challenged orally, demonstrated alterations in small intestinal pathology. Tolerance is dose-dependent for specic antigens. Mice fed ovalbumin or peanuts required a 50-fold higher dose of peanuts to develop tolerance; low doses of peanuts were more likely to induce sensitization. The importance of an intact mucosal barrier was shown when mice, fed a novel dietary protein while their gastrointestinal tracts were inamed, developed sensitization and high serum IgE. Allergic conjunctivitis has been studied in guinea pigs, rats, and mice. Mice exposed to ragweed by topical contact with conjunctival and nasal mucosa developed signs of allergic conjunctivitis and ragweedspecic IgE. Regulators of vascular permeability are important in allergic conjunctivitis. Substance P has been shown to be a mediator of allergic conjunctivitis and acts through NK1 receptors on blood vessels to produce conjunctival hyperpermeability. Nitric oxide has been shown to play a major role in regulating vascular permeability and stimulating prostaglandin E2 production. T-cell adhesion molecules are integral in allergic conjunctivitis. Guinea pig models with ovalbumin have shown that the integrin very

late activation antigen-4 (VLA-4) plays a critical role in eosinophil inltration. Other mice studies showed that antibodies against the integrin intercellular adhesion molecule-1 (ICAM-1) and its ligand leukocyte function-associated antigen-1 (LFA-1) inhibited clinical and histological signs of conjunctivitis. The significance of IL-1 for inammatory changes in conjunctivitis was demonstrated using an IL-1 receptor antagonist in mice exposed to cat dander antigens. Studies in rats using ovalbumin showed that IFN-g suppresses the development of allergic conjunctivitis during the induction phase.

Management and Current Therapy

One of the cornerstones of allergy treatment is avoidance. Avoiding or reducing allergen exposure prevents or minimizes the bodys response to allergen. Environmental control measures help one decrease exposure. For example, patients with house dust-mite allergy can encase their mattresses and pillows in special covers to minimize exposure to dust mites while asleep. One study showed that in inner-city children with atopic asthma, a comprehensive environmental intervention decreased exposure to indoor allergens and reduced asthma-associated morbidity. Avoiding allergic foods or drugs can prevent reactions (Figure 2). Depending on ones sensitivities, allergen avoidance may range from simple to extremely difcult. Other therapies available to treat allergic disorders include antihistamines, leukotriene modiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy. H1-antihistamines have been used for decades for the relief or prevention of allergic symptoms. Antihistamines have recently received the designation of inverse agonists because they stabilize the inactive form of the H1 histamine receptor. First-generation antihistamines have marked sedation; second-generation antihistamines that are relatively nonsedating
Therapy algorithm Allergy diagnosis Avoidance of allergen Pharmacotherapy Pharmacotherapy Immunotherapy Immunotherapy
Figure 2 Therapy for both allergic rhinitis and asthma starts with avoidance of allergens. Pharmacotherapy can be added. Some patients with allergic rhinitis and asthma benet considerably from immunotherapy.

Asthma diagnosis Avoidance of triggers, allergens

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ALLERGY / Overview

have also been identied. Antiallergic activities include inhibiting the release of mast cell mediators probably through direct inhibition of Ca channels. Anti-inammatory effects include inhibiting cell adhesion molecule expression and inhibiting inammatory cell chemotaxis (e.g., eosinophil chemotaxis). These inhibitions probably involve the downregulation of NF-kB, a transcription factor that regulates adhesion proteins and proinammatory cytokines. Antihistamines have established roles in the treatment of allergic rhinitis and urticaria. A potential role exists for the treatment of anaphylaxis or asthma. The cysteinyl leukotrienes and LTB4 are products of arachadonic acid metabolism by 5-lipoxygenase. Leukotrienes cause airway inammation and obstruction by affecting mucus production, smooth muscle contraction, and vascular permeability. They may also affect airway remodeling. The effects of leukotrienes are most likely mediated through the CysLT1 receptor. The CysLT1 receptor antagonists montelukast, pranlukast, and zarlukast block the actions of LTC4, LTD4, and LTE4. A nonselective antagonist of the CysLT1 and CysLT2 receptors is not yet clinically available. Zileuton is a 5-lipoxygenase inhibitor and decreases the production of leukotrienes. Leukotriene modiers are useful in allergic rhinitis and asthma. Leukotriene modiers inhibit an important part of the inammatory cascade and decrease eosinophil survival, goblet cell hyperplasia, mucus release, collagen deposition, and airway smooth muscle proliferation. Corticosteroids block the production of inammatory cytokines (see Corticosteroids: Therapy). They may be given topically at the site of inammation (nose, lungs, or skin) or delivered systemically. Glucocorticoids are the most potent therapy for treating all allergic disorders. They are liposoluble

hormones that enter the cell and bind a cytoplasmic glucocorticoid receptor (see Corticosteroids: Glucocorticoid Receptors). This receptor translocates to the nucleus and binds a glucocorticoid-response element in the promoter region of target genes. The glucocorticoid receptor can also bind transcription factors like NF-kB and AP-1 and prevent these factors from binding their DNA-response elements. Glucocorticoids control airway inammation by inhibiting transcriptional activity of genes encoding proinammatory molecules such as cytokines, chemokines, adhesion molecules, and mediator-synthesizing enzymes. They may suppress histone acetylation and stimulate histone deacetylation. They may also interfere with signal transduction pathways, such as MAP kinase enzymatic cascades involved in the regulation of transcription factors. Theophylline is a nonselective phosphodiesterase inhibitor with a narrow therapeutic ratio and significant drug interactions and has been used exclusively in asthma (see Bronchodilators: Theophylline). Inhibitors of phosphodiesterase type 4 have recently been developed. These inhibitors increase the intracellular concentration of CAMP and exhibit a broad range of anti-inammatory effects on effector cells. Blocking the PDE4B receptor subtype appears responsible for anti-inammatory properties of these agents. Cilomast and roumilast are PDE4 inhibitors that are in late phase III clinical trials. Roumilast has demonstrated more selectivity and a superior therapeutic ratio (Figure 3). The humanized monoclonal anti-IgE omalizumab is a new therapy which decreases the amount of IgE available for reactions and downregulates the number of IgE receptors on mast cells. This therapy is currently available for the treatment of severe asthma. Its use in food allergy is being investigated.

Clinical features Allergic rhinitis Asthma Urticaria Angioedema Atopic eczema Food allergy Oral allergy syndrome Drug allergy Anaphylaxis Rhinorrhea, congestion, sneezing, itchy nose Wheezing, shortness of breath, cough, chest tightness Pruritic wheals Deep local swelling of skin or mucus membranes Pruritis, erythematous maculesor papules, xerosis, lichenification Urticaria/angioedema, rash, flushing, rhinitis, wheezing, anaphylaxis Pruritis/angioedema of tongue, lips, palate, throat Uticaria, morbilliform rash, anaphylaxis Flushing, pruritis, urticaria/angioedema, wheezing, shortness of breath, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, hypotension

Figure 3 Clinical features associated with various allergic disorders.

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Diagnostic workup Allergic rhinitis History and physical examination Asthma History and physical examination

Immediate hypersensitivity skin testing or RAST

Spirometry

Medication trial Nasal cytology Bronchial provocation testing

Rhinoscopy or rhinomanometry

Figure 4 Diagnostic workups start with the history and physical exam. Initial testing for allergic rhinitis consists of skin testing or RAST. Adjunct tests include nasal cytology, rhinoscopy, and rhinomanometry. Lung function in asthma is assessed by spirometry and a medication trial is begun. Bronchial provocation testing can assess bronchial hyperresponsiveness if needed.

bacterial DNA and induce a Th1 type response in humans. Synthetic oligodeoxynucleotides mimic the bacterial DNA immunostimulatory sequences. These synthetic nucleotides can be conjugated to allergen to produce an allergen vaccine that is more immunogenic but less allergenic than allergen alone. These vaccines are currently under clinical trials. Recently, there has been an interest in pharmacogenetics. This eld recognizes that medications may work more efcaciously in certain patients because of their genetic makeup. Single nucleotide polymorphisms (SNPs) may signal a change in proteins or amino acids in an individual. These changes may alter a drugs target, uptake, metabolism, or excretion. A polymorphism in Gly 16 promotes bronchodilator resistance while the Arg 16 polymorphism potentiates a greater response to bronchodilators. An alternatively spliced form of glucocorticoid receptor b is present with higher frequency in corticosteroid-resistant patients. A polymorphism in the 5-lipoxygenase promoter decreases the response to the 5-lipoxygenase inhibitor zileuton. Pharmacogenetics is an exciting area that may shape the future of treatment for allergic diseases.
See also: Allergy: Allergic Reactions; Allergic Rhinitis. Asthma: Overview. Bronchodilators: Theophylline. Chemokines. Corticosteroids: Glucocorticoid Receptors; Therapy. Genetics: Gene Association Studies. Immunoglobulins. Interleukins: IL-5. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids. Matrix Metalloproteinases.

Patients with allergic rhinitis and/or asthma who are (Figure 4) poorly controlled on medications may nd immunotherapy (allergy shots) a feasible alternative. Immunotherapy involves administering injections of allergens to which a patient is sensitive. Increasing doses of allergen are given weekly until the patient is at a maintenance dose. This maintenance dose is continued monthly for 3 to 5 years. Patients receive relief from nasal allergy symptoms and their asthma may improve. The mechanisms of immunotherapy are not well dened. Immunotherapy may induce specic T-cell tolerance or a shift from a Th2 to a Th1 phenotype. Possibly, the Th2 response is inhibited, Th1 response is upregulated, or both. Immunotherapy also increases the production of IL-10 and TGF-b by T cells including T regs. IgG is also produced in response to antigen instead of the typical IgE. Allergen-specic IgG or blocking antibodies may compete with IgE for allergen and inhibit IgE activation of mast cells. IgG can also bind epitopes on allergen that are not recognized by IgE. This IgG binding may prevent cross-linking of IgE. Allergen-IgG complexes on antigen-presenting cells might impair antigen processing or the co-stimulation of T cells and render patients anergic. Immunotherapy is a type of desensitization, where small amounts of allergen are given until the patient tolerates the allergen. The same theory is used in treatment of drug allergies by giving increasing doses of drug until a therapeutic dose is tolerated. Unmethylated CG dinucleotides, or CpG motifs, are responsible for the immunostimulatory effect of

Further Reading
Abbas AK and Lichtman AH (2003) Cellular and Molecular Immunology, 5th edn. Philadelphia: Saunders. Adelman DC, Casale TB, and Corren J (2002) Manual of Allergy and Immunology, 4th edn. Philadelphia: Lippincott Williams & Wilkins. Broide DH (2001) Molecular and cellular mechanisms of allergic disease. Journal of Allergy and Clinical Immunology 108: S65S71. Cakebread JA, et al. (2004) The role of ADAM33 in the pathogenesis of asthma. Springer Seminars in Immunopathology 25: 361375. Creticos PS, Chen Y, and Schroeder JT (2004) New approaches in immunotherapy: allergen vaccination with immunostimulatory DNA. Immunology and Allergy Clinics North America 24: 569581. Groneberg DA, et al. (2003) Animal models of allergic and inammatory conjunctivitis. Allergy 58: 11011113. Gutermuth J, et al. (2004) Mouse models of atopic eczema critically evaluated. International Archives of Allergy and Immunology 135: 262276. Hallstrand TS and Henderson WR (2002) Leukotriene modiers. Medical Clinics of North America 86: 10091033. Hellings PW and Ceuppens JL (2004) Mouse models of global airway allergy: what have we learned and what should we do next? Allergy 59: 914919. Helm RM and Burks AW (2002) Animal models of food allergy. Current Opinion in Allergy and Clinical Immunology 2: 541546.

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Systemic allergic reactions vary in severity from mild (such as generalized urticaria) to severe and life-threatening reactions with cardiovascular collapse and death. Anaphylaxis is the clinical syndrome that represents the life-threatening systemic allergic reaction. It results from the immunologically induced release of mast cell and basophil mediators after exposure to a specic antigen in previously sensitized individuals. Clinically indistinguishable reactions, caused by non-IgE-mediated immune mechanisms, are termed anaphylactoid reactions. Common causes of anaphylaxis are foods, drugs, insect stings, latex, and allergen extracts used for immunotherapy. The symptoms of anaphylaxis occur within a few minutes of exposure and are generally related to the skin, gastrointestinal tract, respiratory tract, and cardiovascular systems. Common manifestations include generalized urticaria/angioedema, nausea, vomiting, stridor, wheezing, hypotension, and syncope. Anaphylaxis requires immediate treatment with epinephrine, given intramuscularly. The dosage for adults is 0.30.5 ml, and for children is 0.01 ml kg 1, of a 1:1000 solution. The dose can be repeated at 515 min intervals. Supportive treatment includes cardiopulmonary resuscitation (if required), intravenous uids, oxygen, antihistamine, and corticosteroids. Following the rst episode, an assessment by an allergist is essential to establish the cause and for appropriate advice on preventive measures including avoidance of the offending agent and self-injectable epinephrine.

Holgate ST (2004) Pharmacogenetics: the new science of personalizing treatment. Current Opinion in Allergy and Clinical Immunology 4: 3738. Joad J and Casale TB (1988) Histamine and airway caliber. Annals of Allergy 61: 17. Kay AB (2001) Allergy and allergic diseases: rst of two parts. New England Journal of Medicine 344(1): 3037. Kim DS and Drake-Lee AB (2003) Allergen immunotherapy in ENT: historical perspective. Journal of Laryngology & Otology 117: 940945. Lipworth BJ (2005) Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet 365: 167175. Morgan WJ, et al. (2004) Results of a home-based environmental intervention among urban children with asthma. New England Journal of Medicine 351: 10681080. Pelaia G, et al. (2002) Molecular mechanisms of corticosteroid action in chronic inammatory airway diseases. Life Sciences 72: 15491561. Robinson DS, Larche M, and Durham SR (2004) Tregs and allergic disease. Journal of Clinical Investigation 114: 13891397. Sampson HA (2004) Update on food allergy. Journal of Allergy and Clinical Immunology 113(5): 805819. Shearer WT and Li JT (2003) Primer on allergic and immunologic diseases. Journal of Allergy and Clinical Immunology 111(2): S441S778. Simons FER (2004) Advances in H1-antihistamines. New England Journal of Medicine 351: 22032217. Till SJ, et al. (2004) Mechanisms of immunotherapy. Journal of Allergy and Clinical Immunology 113(6): 10251034. Weiss ST and Raby BA (2004) Asthma genetics 2003. Human Molecular Genetics 13: R83R89.

Immune Responses
Atopy is dened as the genetic predisposition to form immunoglobulin (IgE) antibodies on exposure to allergens. The production of IgE is central to the induction of allergic diseases. Allergens are proteins with the capability to react to the immune system through their antigenic determinants. Initial exposure to the antigen results in sensitization. Antigens enter the body through the respiratory and gastrointestinal mucosa and the skin. Allergenic proteins are engulfed by antigen-presenting cells (APCs) such as monocytes, macrophages, and dendritic cells inducing primary immune response. The antigen is broken down to reveal the specic part of the molecule called antigenic determinant or epitope. Once processed in this way, the antigen is bound to the MHC class II molecules on the surface of these cells and the complex is presented to the T lymphocyte cell receptor. Bacterial antigens favor the production of Th1 cells with the secretion of its prole of cytokines, particularly interferon gamma (IFN-g). In atopic individuals, and in the presence of co-stimulatory signals, na ve T cells are converted to activated CD4 T-helper-2 (Th2) cells. T lymphocytes play a central role in orchestrating the allergic reaction. Th2 cells produce cytokines, such as interleukin-4 (IL-4) and IL-13. These cytokines cause proliferation and switching of B cells to IgE producing B and plasma cells, specic to the antigen (Figure 1). Some of these cells have a long life and are called memory cells. IL-4 is the most

Allergic Reactions
S H Arshad, University Hospital of North Staffordshire, Stoke-on-Trent, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Allergy is a harmful response to an otherwise innocuous substance. Allergic reactions are immunologically mediated reactions to external substances, usually proteins. These are often, but not always, mediated by immunoglobulin E (IgE) antibody. Initial exposure to an allergen results in sensitization with the production of allergen-specic IgE antibodies. These antibodies circulate in the blood but are largely bound to high-afnity receptors on the surface of basophils and mast cells and lowafnity receptors on eosinophils, macrophages, and platelets. On further exposure, the allergen reacts with the IgE bound to mast cells and basophils, causing degranulation and release of preformed mediators such as histamine and tryptase. These mediators cause the immediate phase of the type I reaction, which occurs within a few minutes (immediate hypersensitivity). Other mediators and cytokines are released and eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 46 h after exposure. Immediate hypersensitivity reaction occurs in asthma, rhinitis, and anaphylaxis. In a sensitized asthmatic, early and late phase asthmatic reaction can be observed following bronchial allergen challenge. Repeated or continued exposure to allergen results in chronic airway inammation, which is characteristic of asthma.

ALLERGY / Allergic Reactions 73

Antigen

IgE Naive T cell

Antigen-presenting cell

Th2 cell

B cell

Antigen

Release of mediators such as histamine and tryptase Mast cell

Figure 1 A schematic representation of immediate (IgE-mediated) hypersensitivity reaction.

important pro-allergy cytokine. Apart from switching of B cells to IgE production, it also stimulates T cells and macrophages, and enhances the expression of low-afnity IgE receptor (FceR2) on B cells and adhesion molecules on endothelial cells. This latter action promotes the movement of cells out of the blood vessels. It also inhibits other types of immune reaction, such as antibody-dependent cell-mediated cytotoxicity. IL-13 has similar but weaker biological activity though it lasts longer than IL-4. IFN-g inhibits allergic responses and its effects are opposite to IL-4 and IL-13. Therefore, it is crucial in the regulation of IgE production. Other cytokines, which inhibit allergic responses, are IL10, IL-12, TGF-b, and IL-8. The direction of immune response on exposure to allergen depends on the balance of Th1 and Th2 reactivity and ensuing cytokine milieu. In atopic individuals the balance is tilted towards the production of Th2 type cytokines (IL-4 and IL-13) as opposed to Th1 type, such as IFN-g. The Th2 differentiation and production of IgE is also suppressed by regulatory T cells (CD4 CD25 ). Thus, an inappropriately weak T regulatory mechanism would facilitate Th2 dominance and production of IgE and allergic disease. It is likely that allergen exposure in early childhood results in a lifelong T cell memory pool. In atopic individuals, the immunological memory is dominated by Th2 type cells leading to allergic reactivity, whereas in nonatopic subjects, Th1 cells dominate the memory pool. In addition to genetic predisposition, environmental factors (infections, diet, etc.) may also inuence the outcome of these initial responses by altering the cellular and cytokine milieu within the lymph nodes. In atopic subjects with their Th2 skew, IgE is formed specic to the antigenic protein following initial exposure (sensitization). The IgE circulates in the blood in small quantities but is mostly present in the tissues bound to high-afnity receptors (FceR1) on

4.5 4 3.5 3 2.5 2 1.5 1 0 1 2 3 4 5 6 7 8 9 10 11 12 Hours (postallergen exposure)

Figure 2 Early and late-phase asthmatic reaction following allergen inhalation challenge. FEV1, forced expiratory volume.

the surface of mast cells and basophils and FceR2 on eosinophils, macrophages, and platelets. This IgE can be detected in the serum by immune assays or in the skin by allergy skin tests. On further exposure, multivalent antigens bind and cross-link IgE bound to FceR1 on cell surface leading to the signaling cascade that causes rapid release of preformed mediators such as histamine, tryptase, and heparin (Figure 1). These mediators cause the immediate phase of the type I hypersensitivity reaction, which occurs within a few minutes (hence immediate hypersensitivity). There is also induction of rapid synthesis of arachidonic acid metabolites such as prostaglandins and leukotrienes and expression of cytokines (IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and tumor necrosis factor alpha (TNF-a)) and chemokines. Eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 4 6 h after exposure (Figure 2). Immediate hypersensitivity is central to all IgEmediated allergic reactions and occurs in asthma, rhinitis, and anaphylaxis. During acute allergic reactions, the process is acute with the release of huge quantities of histamine causing typical symptoms and signs, such as acute bronchospasm or anaphylaxis. However, the process may be subacute or chronic and localized to one site such as lung or nose.

FEV1

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ALLERGY / Allergic Reactions

Repeated exposure to allergens leads to the induction of a more chronic inammatory process with the inux of inammatory cells including T lymphocytes and eosinophils. Cytokines, produced by a variety of inammatory cells, including T cells, regulate the inammatory process. Proliferation of Th2 subsets producing predominantly IL-4 and IL-5 results in differentiation and isotype switching of the na ve B cells to IgE-producing plasma cells as well as activation and inux of inammatory effector cells such as eosinophils. Eosinophils are potentially tissue damaging, particularly after priming with IL-5. Various cytokines upregulate adhesion molecules on endothelial and epithelial cells, thereby enhancing migration of eosinophils into the mucosa.

P, which further increase vascular permeability and cause stimulation of parasympathetic reexes augmenting mucous secretion and bronchoconstriction. These changes manifest clinically in cough, wheeze, and dyspnea.
Late Asthmatic Response

Allergic Reaction in the Lung

Allergic reaction in the lung results in airway inammation. Exposure to allergen is recognized as important in initiating and maintaining allergic airway inammation in atopic asthmatics. In the appropriate setting of repeated allergen exposure and Th2 type immune responses, a cytokine milieu is created with upregulation of adhesion molecules and continuous recruitment and activation of inammatory cells from the bloodstream towards the bronchial mucosa. The release of cytokines and inammatory mediators by activated cells causes amplication and persistence of the inammatory process. However, structural cells such as epithelial cells and smooth muscle cells are not merely passive recipients of immune-related tissue damage but are active participants of the complex inammatory cascade, which may well have initiated at the epithelial/ mesenchymal level within the airways. Nonatopics show similar inammation in their airways and thus IgE-mediated allergy in not a prerequisite for airway inammation in asthma or rhinitis.
Early Asthmatic Reaction

Clinically, the effect of early asthmatic reaction diminishes after 30 min (Figure 2). This is followed by a relatively asymptomatic period during which a plethora of cytokines and mediators draw leukocytes to the tissues. Events initiated during the early response result in vascular dilatation and increased permeability, edema formation, and the accumulation of cells. IL-5, secreted from mast cells, lymphocytes, and eosinophils is the most important cytokine for eosinophils. Besides attracting them to the site of inammation, it also causes their proliferation, activation, and increased survival. Other eosinophilic cytokines are IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines. Upon activation, eosinophils release mediators such as eosinophilic cationic proteins, major basic proteins, leukotrienes, and prostaglandins. These and other mediators enhance inammation and cause epithelial damage. This results in bronchoconstriction clinically 412 h later and prolonged bronchial hyperresponsiveness, mucus secretion, and edema formation. Further secretion of a host of cytokines including IL-3, IL-4 and IL-5, contribute to an ongoing inammation.
Chronic Inammation

The effect of allergen exposure can be observed and studied in a controlled fashion during bronchial allergen challenge. In an atopic asthmatic, inhalation of allergen to which the patient is sensitized, results in an immediate hypersensitivity reaction with the release of mast cell mediators in the bronchial mucosa. These mediators enhance vascular dilatation, increase permeability of the venule, and increase mucus secretion, resulting in edema and congestion, typical of an acute phase reaction. Histamine and leukotrienes are potent bronchoconstrictors. Histamine stimulates local type c neurones leading to the release of several neuropeptides, including substance

With continued or repeated exposure to allergen, a state of chronic inammation develops with increased numbers of activated Th2 cells, expressing mRNA for the secretion of IL-3, IL-4, IL-5, and GMCSF. These cytokines are important in the continuation of inammation and the attraction of mast cells and eosinophils. These cells cause further increase in histamine, prostaglandins, and eosinophilic toxic products, causing epithelial damage. There is upregulation of intercellular adhesion molecules in the blood vessels promoting stickiness of the endothelium to leukocytes and facilitating their passage across, into the tissues. Increased permeability and cellular inltration causes mucosal edema. Even in patients with mild, intermittent asthma, a state of low-grade inammation persists, in the absence of symptoms. It is hypothesized that almost continuous exposure to very small amounts of allergens, such as house dust-mite, or pollen during summer, contributes to this ongoing allergic reaction without causing symptoms. Under the inuence of IL-4 from mast cells, more B cells are switched to the

ALLERGY / Allergic Reactions 75

production of IgE antibodies, thus maintaining allergic reaction. Bronchoscopy studies reveal increased numbers of activated inammatory cells and cytokines in the respiratory mucosa and secretions.
Clinical Effects

The clinical features of asthma are due to the airway narrowing causing obstruction to airow. This airway obstruction has three elements: 1. Excessive bronchial smooth muscle contraction. Inammatory mediators such as histamine, bradykinin, prostaglandins, and leukotrienes act directly on their specic receptors to cause bronchoconstriction. In asthma, the smooth muscles contract easily and excessively following exposure to inammatory mediators perhaps due to heightened sensitivity of their receptors. On the other hand, b2-receptors may have a diminished response. This feature is called bronchial hyperresponsiveness. 2. Thickening of bronchial wall. Bronchial wall thickening is due to inammatory and brotic changes. Increased capillary permeability allows plasma exudation into the mucosa causing edema and cellular inltration. Proliferation of broblast and myobroblast leads to thickening of the basement membrane with deposition of collagen and hypertrophy of bronchial smooth muscles (airway remodeling). This leads to irreversible airway obstruction in chronic asthma. 3. Excessive luminal secretions and cellular debris. There is excess mucus secretion due to glandular hyperplasia. The epithelium is fragile and damaged epithelial cells are found in the sputum. Impaired ciliary function encourages retention of thick mucus in the lumen. During severe exacerbation, the lumen of the airway is blocked by thick mucus, plasma proteins, and cell debris. Allergic reactions in the nose follow a similar process with inammation of the nasal mucosa, resulting in rhinorrhea, sneezing, and nasal blockage.

bronchospasm of mild to moderate severity, may be called severe allergic reactions. Traditionally, the term anaphylaxis is used for IgE-mediated reactions. Systemic reactions that clinically resemble anaphylaxis but are caused by non-IgE-mediated mediator release from mast cells and basophils are referred to as anaphylactoid reactions. Anaphylaxis occurs in 30/100 000 population/year with a mortality of 12%. Offending agents include foods, drugs, insect stings, and exercise but in 20% of cases no cause can be found (idiopathic).
Pathogenesis

Systemic allergic reaction occurs as a result of degranulation of mast cells and basophils. Mast cells in respiratory and gastrointestinal tract, skin, and perivascular tissues are involved in both IgE and non-IgEmediated allergic reaction. IgE-mediated release is caused by antigen-specic cross-linking of IgE molecules on the surface of tissue mast cells and peripheral blood basophils. Non-IgE-mediated release may be due to direct stimulation of mast cells. Mast cells produce both histamine and tryptase while basophils secrete histamine but not tryptase. Histamine is a primary mediator of anaphylaxis and signs and symptoms of anaphylaxis can be reproduced by histamine infusion.
Clinical Features

Systemic Allergic Reactions and Anaphylaxis

Allergic reactions vary widely in severity from mild pruritis and urticaria to circulatory collapse and death. An acute systemic allergic reaction with one or more life-threatening features, such as stridor or hypotension, is termed anaphylaxis. Allergic reactions with troublesome but not life-threatening reactions, such as generalized urticaria/angioedema and

Anaphylaxis is a rapidly developing generalized reaction that involves respiratory, cardiovascular, cutaneous, and gastrointestinal systems. Clinical manifestations vary depending on the cause of anaphylaxis, route of entry, host factors (such as degree of sensitization, associated factors such as exercise, comorbidity, etc.) and the amount of allergen exposure. The initial symptoms, such as nasal congestion or pruritis, can quickly progress to collapse or death. Laryngeal edema, cardiovascular collapse, and severe bronchospasm are life-threatening features. In one large series of fatal anaphylactic reactions, 70% of the deaths were from respiratory causes, and 24% were from cardiovascular causes. In 1020% of cases, skin may not be involved. Anaphylaxis can be confused with septic or other forms of shock, asthma, airway foreign body, and panic attacks (Table 1). Symptoms commonly occur within a few seconds or minutes of exposure and death may occur within minutes. Speed of onset of symptoms is indicative of the severity. Occasionally, the onset of symptoms may be delayed for 2 h or more. In general, the later the symptoms begin after exposure to a causative agent, the less severe the reaction. Food allergens may have slower onset or slow progression and gastrointestinal

76

ALLERGY / Allergic Reactions

Table 2 Clinical features of anaphylaxis Symptoms Cardiovascular Faintness, syncope, palpitations Throat tightness, hoarseness, inspiratory stridor, difculty in swallowing Nasal congestion, rhinorrhea, sneezing Pruritis, lacrimation Generalized warmth, tingling, pruritis, rash Chest tightness, wheezing, cough, shortness of breath Nausea, vomiting, abdominal pain, bloating, cramps, diarrhea Dizziness, a sense of impending doom, lightheadedness Physical examination Hypotension, tachycardia, arrhythmias Laryngeal edema

Table 1 Differential diagnosis of anaphylaxis Acute cardiac event Vasovagal syncope Acute angioedema Acute severe asthma Pulmonary embolism Foreign body inhalation Carcinoid syndromes Pheochromocytoma Seizure Systemic mastocytosis Panic attack Vocal cord dysfunction

Throat

symptoms are more common. Onset of anaphylaxis to insect stings or allergen injections is usually rapid: 70% begin in o20 min and 90% in o40 min.
Management

Upper respiratory tract Ocular Skin

Chest

Conjunctival injection Flushing, urticaria, swelling of the lips, tongue or uvula Wheezing, tachypnea, cyanosis, respiratory arrest

Gastrointestinal

A quick initial assessment should determine the nature and progression of the clinical event (Table 2). Continuous monitoring is essential as progression from a mild to a severe episode may occur rapidly. Epinephrine injected intramuscularly into the thigh provides the most efcient absorption (Figure 3). If there is no response to several doses of intramuscular epinephrine, intravenous administration may be needed, by using a formulation of 1:10 000 (0.1 mg ml 1) at 1 mg min 1, which can be increased to 210 mg min 1. If the response is still inadequate, transfer the patient to an intensive care unit for close monitoring and endotracheal intubation, if required. Specic treatment for coexisting medical conditions (e.g., coronary artery disease) may be necessary. There may be complete resolution of the reaction. However, if there are concerns, continued monitoring for remaining or recurring symptoms is essential. A short course of corticosteroids may reduce the risk of recurring or protracted symptoms of a biphasic reaction but this is not proven. Patients receiving badrenergic blocking agents may not respond adequately to epinephrine. They require continued uid replacement and may respond to glucagon. Patients receiving angiotensin-converting enzyme inhibitors may also be at increased risk of anaphylaxis and be more refractory to treatment with epinephrine. If there is any doubt regarding the diagnosis, blood should be taken for plasma histamine or serum tryptase levels within the rst 4 h after the onset of symptoms. Elevated serum levels of b-tryptase indicate mast cell activation and degranulation in both IgE-mediated (anaphylaxis) and non-IgE-mediated (anaphylactoid) reactions. b-tryptase is useful in differentiating anaphylaxis from other events having

Neurologic

Loss of consciousness, seizures

similar clinical manifestations, particularly if hypotension is present. Blood for plasma histamine needs to be processed immediately to avoid detecting articially high levels due to spontaneous basophil histamine release. If this is not possible, urinary histamine (or metabolites) levels can be checked for up to 24 h. After initial treatment of acute anaphylaxis, the patient should be followed-up closely for the possibility of recurrent episodes. For mild to moderate episodes and good response to treatment, further monitoring can be done at home. However, following a severe episode, in-patient monitoring may be required for late-phase reactions.
Subsequent Assessment

All individuals who have had a known or suspected anaphylactic episode require a careful allergy evaluation. The aims are to review the diagnosis of anaphylaxis and prevent or minimize the risk of future anaphylactic episodes by identifying the cause, educate the patient and/or family members regarding avoidance of the offending agent, education and training to deal with future inadvertent exposures, and consideration of desensitization, if appropriate. The level of condence in the diagnosis of the original episode should be reviewed with details of the

ALLERGY / Allergic Reactions 77

Evaluate: breathing status, pulse, blood pressure, and level of consciousness Recognize life-threatening features such as stridor, shock, arrhythmia, seizure, and loss of consciousness

Institute CPR if there is loss of circulation or respiration Maintain airway with an airway device or tracheotomy Oxygen, if there is circulatory or respiratory compromise

Epinephrine (1:1000) 0.30.5 ml i.m. (children: 0.15 ml), repeat every 1015 min, as needed Chlorpheniramine 10 mg i.v. (then 6 hourly) Hydrocortisone 200 mg i.v.

Improve

Problems persist

Late-phase symptoms

Monitor

Discharge home

Hypotension: intravenous fluids (colloids) and, if needed, vasopressor agents (e.g., dopamine) Bronchospasm: nebulized 2-agonist, consider use of i.v. aminophylline. Mechanical ventilation may be required Urticaria/angioedema: oral/i.v. antihistamines and steroids
Figure 3 Emergency management of anaphylactic and anaphylactoid reactions.

events before and during the episode. Results of any laboratory tests (e.g., serum tryptase or urine histamine) may be helpful in supporting the diagnosis of anaphylaxis and differentiating it from other entities. However, a careful and comprehensive history is the most useful part of the assessment. Information on any previous similar episodes, known food or drug allergy, and medication record should be sought. The history might suggest a specic cause such as insect sting or peanut consumption just before the episode. However, the cause may not be obvious from history and sometimes no cause can be found despite thorough searching (idiopathic anaphylaxis).
Diagnostic Tests

specic cause of anaphylaxis in cases of food, insect, and some cases of drug (penicillin, insulin) allergy. An SPT is more sensitive than in vitro testing and is the diagnostic procedure of choice. When possible, standardized extracts should be used. If skin tests or in vitro tests do not provide a cause, then challenge to the suspected agent/agents should be considered. Challenge procedures are helpful in IgE-mediated allergic reaction where standardized test material is not available and in non-IgE anaphylactic reactions (such as to aspirin).
Prevention

Skin prick tests (SPTs) or determination of specic IgE in serum (in vitro test) is helpful in identifying a

Once the offending agent has been identied (e.g., food, medication, or insect sting), patients should be educated regarding the specic exposures and be counseled on avoidance measures (Table 3). All those

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ALLERGY / Allergic Reactions

Table 3 Specic measures to reduce allergic reactions to common provoking agents Provoking agents Foods Specic preventive methods Patients should be taught to read and interpret food labels They should be encouraged to ask about ingredients in restaurant meals They should be provided with a list of alternative foods School personnel should be fully informed of the pupils allergy history Patients should carry a medical identication bracelet or card Use alternatives, when possible, but avoid crossreactive antibiotics If penicillin is essential, desensitization could be performed Remove any hives or nests in the garden Wear long shoes and full trousers when walking in the elds Be alert to the presence of insects when outdoors Discontinue exercise at the earliest symptom Avoid any exacerbating factors such as aspirin or NSAIDs Avoid exercise for 46 h after eating if there is a history of anaphylaxis after food ingestion Patients should carry a medical identication bracelet or card All procedures should be conducted in a latex-free environment Food with known crossreactivity to latex should be avoided Consider an alternative procedure that does not require RCM Use low osmolar RCM Consider pretreatment with steroids, antihistamine, and ephedrine Allergen immunotherapy should only be administered under the supervision of a trained allergist Consider alternative therapy in those who are at higher risk (Table 4) Care should be taken to avoid dosing errors Observe patients for at least 20 min after the injection Consider reducing the dose, if interval between injections had been longer than planned or on opening of new vials Adjust dose if large local reaction occurs Reduce dose in highly sensitive patients or if there is concomitant high allergen exposure

Penicillin

Insect sting

Exercise

Latex

Radiocontrast material (RCM) Allergen extracts

with a risk of future anaphylaxis outside the medical settings should carry and be educated in the use of self-injectable epinephrine and antihistamines. Self-injectable epinephrine is available in two different strengths (for adults, 0.3 ml of 1:1000 solution and for children, 0.3 ml of 1:2000 solution) in readyto-use syringes. Antihistamine (such as chlorpheniramine 48 mg orally) may be sufcient for a mild episode but epinephrine should not be held back if symptoms are severe from the outset or response to antihistamine is inadequate. Humanized, monoclonal anti-IgE antibody has shown protection against peanut-induced anaphylaxis.

suspected food, either by skin prick test or in vitro test, in the presence of a suggestive history, is sufcient to make a denitive diagnosis. However, food challenges (single or double blind) may be required. Strict avoidance of the offending food is essential. Patients should also carry, and be trained, in the use of epinephrine in an emergency following inadvertent exposure.
Penicillin

Allergic Reactions
Foods

Food allergic reactions are common in children and presents clinically with systemic involvement (as described above), although oral (itching, numbness and tingling of lips and mouth) and gastrointestinal symptoms may be more prominent. Although any food can cause a reaction, commonly implicated foods are milk, egg, peanuts, tree nuts, sh, and shellsh. Symptoms often occur within minutes of ingestion and certainly within 2 h. Assessment of specic IgE to

Allergic reaction to penicillin is the most common cause of anaphylaxis. Severe reactions are usually attributable to parenteral administration. Atopy and family history of penicillin allergy does not increase the risk of a reaction. A history of penicillin allergy is not reliable as nearly 80% of these individuals tolerate penicillin without ill effects. However, these subjects should have a skin test to major and minor determinants before penicillin is administered. The risk of a reaction following a negative test is less than 2%. Skin tests do not resensitize a patient to penicillin. A positive skin test in the presence of a history of reaction to penicillin indicates a more than 50% risk of an allergic reaction and penicillin should be avoided, or if penicillin is mandatory, desensitization should be considered. Cross-reactivity exists between

ALLERGY / Allergic Reactions 79

cephalosporins and penicillins due to the common b-lactam ring structure.

Aspirin and Nonsteroidal Anti-Inammatory Drugs

Aspirin and nonsteroidal anti-inammatory drugs (NSAIDs) can induce life-threatening systemic, nonIgE-mediated reaction causing rhinoconjunctivitis, bronchospasm, urticaria, and laryngeal edema. In vitro or skin tests are not available and oral challenges are required for conrmation. If the diagnosis is conrmed, aspirin and NSAIDs should be strictly avoided. Desensitization may be performed if these drugs are considered essential.
Insect Sting

administered during general anesthesia. Plasma tryptase is useful in differentiating allergic reaction (due to mast cell release of mediators) from pharmacologic or other causes. Skin testing is used for diagnosis where IgE-mediated mechanism is suspected. Otherwise, graded challenge may be required.
Latex

Stinging insects belong to the order Hymenoptera. Common stinging insects include honeybees, wasps, yellow jackets, hornets, and re ants. The self-reported prevalence of insect sting allergy is approximately 1%. Insect venoms contain several well-characterized allergens that can trigger anaphylactic reactions. Localized reaction may occur at the site of the sting and a large local reaction may involve, for example, the whole limb. However, these do not predispose to systemic allergic reaction. Urticaria and angioedema are key features of systemic allergic reactions caused by insect sting. Skin test is the preferred method of conrming allergy and identifying the responsible insect species. However, careful interpretation is needed, as falsepositive reactions are common. Following a systemic allergic reaction, only about half of the patients will react to a future sting. Patients should carry self-injectable epinephrine for early treatment of a sting, and allergen immunotherapy should be considered, where risk of future sting is substantial. The immunotherapy is 490% effective but optimal duration is not known.
Intraoperative Drugs

IgE-mediated allergic reactions to natural rubber latex became common during the 1990s due to a sudden increase in the use of rubber gloves. Risk factors for latex allergy include atopy and previous repeated exposure to latex (e.g., multiple surgical procedures, healthcare workers). Skin tests are indicated for investigation of latex allergy in those who have a history of possible latex allergic reaction and for screening those who are at high risk. However, in the last few years, the use of latex-free gloves and other products have been effective in reducing the occurrence of latex allergic reactions.
Blood Transfusions

Common agents responsible for intraoperative anaphylaxis are neuromuscular blocking agents, latex, antibiotics, anesthesia induction agents, radiocontrast material, and opioids. Neuromuscular blocking agents and thiopental are responsible for most anaphylactic reactions during general anesthesia. Both IgE-mediated (muscle relaxants, latex) and direct stimulation of mast cell (opioids, radiocontrast material) occurs. Clinical manifestations of intraoperative reactions differ from anaphylactic reactions due to other causes as cardiovascular collapse, airway obstruction, and ushing is prominent. It may be difcult to differentiate allergic reaction from the pharmacologic effects of a variety of medications

Allergic reactions may complicate 13% of blood transfusions. Most reactions are mild, are associated with cutaneous manifestations such as pruritis, maculopapular, or urticarial rash and ushing, and require no specic treatment except discontinuation of transfusion and perhaps antihistamine. However, severe or life-threatening reactions with hypotension and bronchoconstriction may occur occasionally. Most of these reactions are due to IgE or IgM antibodies to serum proteins (e.g., albumin, complement components, IgG, and IgA). Other mechanisms include transfusion of allergen, IgE antibodies, bacterial components or inammatory substances such as cytokines, histamine, or bradykinin. Blood components containing large amounts of plasma, such as fresh frozen plasma, may be associated with more severe allergic reactions. Serum tryptase, measured soon after the reaction, may differentiate allergic from transfusion-related reaction.
Allergen Extract

Allergen extracts are injected during skin test and allergen-specic immunotherapy. The risk of allergic reaction is extremely low with skin prick test but not insignificant when these extracts are injected for treatment. In a survey between 1985 and 1989 in the US, there were 17 deaths from allergen immunotherapy but none from skin testing. Mild, localized reactions are relatively common and respond to antihistamine. Factors that increase the risk of an allergic reaction should be kept in mind by those involved

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ALLERGY / Allergic Rhinitis

Lieberman P (2002) Anaphylactic reactions during surgical and medical procedures. Journal of Allergy and Clinical Immunology 110(supplement 2): S64S69. Moftt JE (2003) Allergic reactions to insect stings and bites. Southern Medical Journal 96(11): 10731079. Moneret-Vautrin DA and Kanny G (2002) Anaphylaxis to muscle relaxants: rational for skin tests. Allergic Immunology (Paris) 34(7): 233240. Noone MC and Osguthorpe JD (2003) Anaphylaxis. Otolaryngologic Clinics of North America 36(5): 10091020. Sampson HA (2003) Anaphylaxis and emergency treatment. Pediatrics 111(6): 16011608. Sicherer SH and Leung D (2004) Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insect stings. Journal of Allergy and Clinical Immunology 114(1): 118124. Tang AW (2003) A practical guide to anaphylaxis. American Family Physician 68(7): 13251332.

Table 4 Risk factors for systemic reactions during allergen immunotherapy Severe asthma Highly sensitive patient Too rapid increase in the dose of allergenic extract Starting a new vial of extract A history of previous systemic reactions to allergen immunotherapy Asthmatic symptoms present immediately before receiving an injection Administration of pollen extracts during high environmental pollen exposure Concomitant treatment with b-adrenergic blocking agents Fever or an upper respiratory tract infection at the time of administration of allergenic extracts

in administration of allergen-specic immunotherapy (Table 4).

Exercise

Allergic Rhinitis
P van Cauwenberge, J-B Watelet, T Van Zele, and H Van Hoecke, Ghent University Hospital, Ghent, Belgium
& 2006 Elsevier Ltd. All rights reserved.

Exercise-induced anaphylaxis is a physical form of allergy that may occur in isolation or in combination with ingestion of food or drug, such as aspirin or NSAIDs. The episode resembles typical anaphylaxis and emergency treatment is the same as for anaphylaxis due to other causes. Patients should carry self-injectable epinephrine. Exercise may also cause urticaria or bronchospasm without anaphylaxis.
See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: IL-8. Immunoglobulins. Interferons. Interleukins: IL-4; IL-10; IL-12; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a ).

Abstract
Over the last decades, the prevalence of allergic rhinitis has risen to epidemic proportions. Nasal symptoms involve sneezing, nasal itch, rhinorrhea, and nasal congestion. These symptoms result from an immunologically mediated (usually IgE-mediated) inammation of the nasal mucosa, following allergen exposure in sensitized patients. Although allergic rhinitis is often trivialized, it has become clear that the disease can cause serious morbidity beyond the nasal manifestations, that it has a significant impact on quality of life and substantial socioeconomic consequences, and that it is associated with multiple comorbidities, including asthma, conjunctivitis, sinusitis, and otitis media. Recently, the Allergic Rhinitis and its Impact on Asthma (ARIA) Working Group proposed a new classication for allergic rhinitis, based on the duration of symptoms, rather than on the type of exposure. The severity of the disease is categorized based on the impact of symptoms on quality of life parameters. Early and correct diagnosis is the basis for the management of allergic rhinitis and starts with a thorough clinical history and physical examination. To conrm the allergic origin of rhinitis symptoms, allergy tests are performed. The test of rst choice is the skin prick test, which has a good sensitivity and specicity. Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of allergic rhinitis. Nowadays, many effective pharmacological agents are available and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To facilitate and standardize the management of allergic rhinitis and to improve the patient care, satisfaction, and compliance, several clinical practice guidelines have been developed. Among those, the ARIA guidelines provide stepwise treatment recommendations, based on the best available evidence from research.

Further Reading
Cockcroft DW (1998) Airway responses to inhaled allergens. Canadian Respiratory Journal 5(supplement A): 14A17A. El Biaze M, Boniface S, Koscher V, et al. (2003) T cell activation, from atopy to asthma: more a paradox than a paradigm. Allergy 58(9): 844853. Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE and the basis of allergic disease. Annual Review of Immunology 21: 579633. Holgate ST, Davies DE, Puddicombe S, et al. (2003) Mechanisms of airway epithelial damage: epithelialmesenchymal interactions in the pathogenesis of asthma. European Respiratory Journal 44(supplement): 24s29s. Joint Task Force on Practice Parameters, American Academy of Allergy, Asthma and Immunology, American College of Allergy, Asthma and Immunology, and the Joint Council of Allergy, Asthma and Immunology (1998) The diagnosis and management of anaphylaxis. Journal of Allergy and Clinical Immunology 101(6 Pt 2): S465S528. Kemp SF and Lockey RF (2002) Anaphylaxis: a review of causes and mechanisms. Journal of Allergy and Clinical Immunology 110(3): 341348.

ALLERGY / Allergic Rhinitis 81

Introduction
Allergic rhinitis (AR) is dened as a nasal disease with the presence of immunologically mediated hypersensitivity symptoms of the nose, for example, itching, sneezing, increased secretion, and blockage. The great majority of cases are immunoglobulin E (IgE) antibody-mediated. In the recent Allergic Rhinitis and its Impact on Asthma (ARIA) report, AR is considered as a major chronic respiratory disease because of its high prevalence in all countries, its significant impact on quality of life or work performance, its considerable economic burden, and its association with multiple comorbidities, asthma in particular. Several important epidemiological surveys (e.g., European Community Respiratory Health Survey, International Study of Asthma and Allergies in Childhood (ISAAC), and Swiss Study on Air Pollution and Lung Diseases in Adults) have recently improved our knowledge about the prevalence of rhinitis. The majority of monocentric studies have reported a prevalence of seasonal AR ranging from 1% to 40% and a prevalence of perennial rhinitis ranging from 1% to 18%. Furthermore, the ISAAC study phase 1 conrmed this large variation in the prevalence of rhinitis symptoms throughout the world. Over the last 40 years the prevalence of AR, like other allergic disorders, has risen to truly epidemic proportions. Despite the increasing insight into the pathophysiology of allergy and the availability of more effective treatment options, this upward trend in the incidence of AR continues, being most prominent in countries with a Western lifestyle, especially among children and adolescents. As a consequence, great attention is being paid to the identication of the factors responsible for this disease. It is well established that allergic diseases tend to occur within families and have a genetic basis. However, numerous generations are needed before changes in the gene pool occur. Therefore, the recent increase in prevalence of AR and allergy in general cannot be explained by genetic factors and is largely attributed to alterations in the environment. Diverse environmental and lifestyle factors (prenatal maternal inuences, allergen exposure, active and passive smoking, viral and other respiratory infections, early life microbial exposure, indoor air quality/house dampness, outdoor air pollution, urban vs farming living environment, socioeconomic status, dietary factors, etc.) have been identied as possible risk factors or as protective factors in the pathogenesis of allergy, although the evidence supporting their involvement varies widely. In order to allow the introduction of individualized primary and secondary

prevention strategies and to meet the challenge of the growing impact of allergy, further assessment of the complex interactions within and between genetic and environmental determinants is required.

Etiology
Allergens are antigens that induce and react through an IgE-mediated inammation. The number of identied allergens has expanded enormously. They originate from animals, insects, plants, or fungi. In AR, airborne allergens are the most common provoking agents and the increase in time spent indoors in recent years is thought to be responsible for the rise in incidence of allergic diseases. The most common indoor allergens are derived from mites, domestic animals, insects, or plants. Mites, such as Dermatophagoides pteronyssinus or D. farinae, usually induce asthma or perennial AR. They feed on human skin and multiply under hot and humid conditions. Other types of mite, such as Tyrophagus putrecentiae and Acarus siro, are present in our. The dander and secretions of many animals contain powerful allergens capable of inducing severe reactions. They can remain airborne for prolonged periods of time. Specic allergens have been identied in cat, dog, horse, cattle, rabbit, and other rodents. Outdoor allergens include pollen and molds. Pollen can be categorized into two groups based on their mode of transport: anemophilous pollen are carried by the wind and can be transported over long distances, while entemophilous pollen are carried by insects. The nature and number of pollen vary with geography, temperature, and climate. Fungi, molds, and yeasts can liberate large quantities of allergenic spores into the atmosphere. Their development and growth are faster in hot and humid conditions. The main atmospheric molds are Cladosporium and Alternaria. Finally, it has been noted that inhalation of insect waste or some bacteria seems to be able to induce an IgE-mediated reaction. AR can also be triggered by food and occupational allergens. Many allergens have enzymatic activity. Simultaneous exposure to both allergenic and proteolytic activity probably allows greater access to cells of the immune system and enhances sensitization and allergic inammatory reactions.

Pathology
Classication of Allergic Rhinitis

Before the publication of the ARIA report, AR was subdivided into seasonal AR, perennial AR, and by extension occupational AR, based on the time of

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exposure to the offending allergen(s). Seasonal AR is related to a wide variety of outdoor allergens such as pollens and molds. Perennial AR is most frequently caused by indoor allergens such as house dust mite, molds, cockroaches, and animal dander. Occupational AR occurs in response to airborne allegens in the workplace. Common causes are laboratory animals, wood dust (particularly hard woods), chemicals, and solvents. The distinction between seasonal and perennial AR, however, is not applicable in all patients and in all countries because
*

symptoms of perennial rhinitis may not be present all year round; pollen and molds are perennial allergens in some parts of the world; many patients are sensitized to multiple allergens and present with symptoms during a number of periods in the year or even throughout the year; and symptoms of seasonal AR do not always occur strictly within the dened allergen season, due to a priming effect and the concept of minimal persistent inammation.

Pollen-induced rhinitis is the most characteristic IgE-mediated allergic disease and is triggered by the interaction of mediators released by cells that are implicated in both allergic inammation and nonspecic hyperreactivity. AR is characterized by a huge inammatory reaction. The resulting inammatory cell inltrate is made up of different cells. The cellular response begins with chemotaxis, selective recruitment, and transendothelial migration of cells. These cells can be localized within the different compartments of the nasal mucosa and represent an activation state. Their survival is prolonged and they release a large amount of inammatory mediators. They also participate in the regulation of IgE synthesis and communicate with the immune system. Biopsies from allergic nasal tissue demonstrate a thicker basement membrane and a greater number of intraepithelial monocytes, subepithelial eosinophils, and neutrophils (Figure 1).

Clinical Features
Symptoms of rhinitis include rhinorrhea, nasal obstruction, nasal itch, and sneezing; often, patients with rhinitis are subdivided into sneezers and runners and blockers, based on the main symptom(s).

Therefore, this classication was considered to be inaccurate and the ARIA Working Group proposed a major change in the classication of AR. The ARIA classication for AR uses the terms intermittent and persistent to describe the duration of symptoms. Based on the impact of symptoms on quality-of-life parameters, the severity of the disease is classied as mild or moderatesevere (Table 1).
Table 1 ARIA classication for allergic rhinitis Intermittent rhinitis Symptoms are present: p4 days a week or p4 consecutive weeks Mild rhinitis None of following items are present: sleep disturbance impairment of daily activities, leisure, and/or sport impairment of work or school work troublesome symptoms In untreated patients Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147S334. Persistent rhinitis Symptoms are present: 44 days a week and 44 consecutive weeks Moderate/severe rhinitis X1 of following items are present:

Figure 1 Hematoxylin-eosin staining (magnication 40) of normal nasal mucosa demonstrating epithelial cells, seromucous glands, and subepithelial lymphoid layer.

ALLERGY / Allergic Rhinitis 83

These rhinitis symptoms, however, do not necessarily have an allergic origin. In the differential diagnosis, AR must be differentiated from several types of nonallergic rhinitis and other nasal inammatory conditions (Tables 2 and 3). The clinical history remains the most essential step for establishment of the diagnosis. Apart from the classical rhinitis symptoms, the patient must be questioned about the presence of other symptoms commonly associated with rhinitis, such as loss of smell, snoring, sleep disturbance, postnasal drip, cough, sedation, conjunctivitis, and lower respiratory symptoms. The history should also contain an evaluation of the severity and duration of the problem, the impact on daily life, and the response to treatment, and should document potential allergic and nonallergic triggers; it must also include a family and occupational history.

Clinical examination starts with a general inspection of the nose, ears, and throat. A complete and systematic nasal examination is required, especially in patients with persistent rhinitis. However, anterior rhinoscopy gives only limited information. Nasal endoscopy is therefore an essential complementary investigation, not to conrm AR, but to exclude other conditions, such as polyps, foreign bodies, tumors, and septal deformations. During allergen exposure, the sinonasal mucosa of patients with AR can demonstrate a bilateral, but not always symmetrical, swelling. Often, mucosal changes in color are seen, from a purplish to a more common pale coloration. An increase in vascularity is also commonly noticed. In the absence of allergen exposure, the nasal mucosa may appear completely normal, but in patients who have suffered from rhinitis for several years, irreversible mucosal hyperplasia and/or viscous secretions may also occur (Figures 26).
Diagnostic Evaluation

Table 2 Classication of rhinitis Allergic rhinitis Infectious rhinitis: viruses, bacteria, fungi Occupational rhinitis: allergic and nonallergic Drug-induced rhinitis, e.g., aspirin Hormonal rhinitis: puberty, pregnancy, menstruation, endocrine disorders Emotional rhinitis Atrophic rhinitis Irritant-induced rhinitis Food-induced rhinitis, e.g., red pepper NARES: nonallergic rhinitis with eosinophilic syndrome Rhinitis associated with gastroesophagel reux Idiopathic rhinitis Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49 (supplement 9): 534.

To conrm the allergic origin of rhinitis symptoms, the ARIA Working Group states that allergy tests should be performed. In vivo and in vitro tests for the diagnosis of allergic diseases are directed towards the detection of free or cell-bound IgE. Immediate hypersensitivity skin tests are a major diagnostic tool to demonstrate IgE-mediated allergic reactions. If properly performed, skin tests are the best available method for detecting the presence of allergen-specic IgE. The rst choice of test is the skin prick test, which has a good sensitivity and specicity (Figures 7 and 8). It is very important that skin tests are performed carefully and interpreted correctly. Therefore,

Table 3 Differential diagnosis of rhinitis Mechanical factors Deviated septum Adenoidal hypertrophy Hypertrophic turbinates Foreign bodies Choanal atresia Tumors Benign Malignant Granulomas Wegeners granulomatosis Sarcoid Infectious (tuberculosis, leprosy) Malignant midline destructive granuloma Ciliary defects Cerebrospinal rhinorrhea Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 534. Figure 2 Profuse watery anterior rhinorrhea in a child with allergic rhinitis.

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Figure 3 The nasal salute refers to habitual rubbing of the nose due to constant irritation. It usually signies an underlying allergic phenomenon of producing a profuse rhinorrhea requiring frequent wiping. It is most marked in children.

Figure 4 Habitual rubbing of the nose usually produces a horizontal nasal crease below the bridge of the nose.

Before allergen challenge

After allergen challenge

Figure 5 Schematic presentation of sinonasal mucosa swelling before and after allergen challenge.

it is recommended that trained healthcare professionals carry them out. The skin reaction can be affected by the quality of the allergen extract, the patients age, the use of some pharmacological agents (e.g., oral antihistamines and topical skin corticosteroids), and can also demonstrate seasonal variations. In addition, the possibility of false-positive and false-negative results must be considered (Table 4). Measurement of total serum IgE lacks specicity and is of little predictive value in allergy screening in rhinitis. On the contrary, serum-specic IgE is as valuable as skin testing. Skin tests, however, are less expensive, have a greater sensitivity, allow a wide allergen selection, and give results in less than half an hour. Serum-specic IgE is indicated in young children, in patients with dermographism or widespread dermatitis, in patients who are noncompliant for skin testing, or in those who did not continue with antihistamine treatment (short-acting antihistamines for 3648 h, long-acting antihistamines for 46 weeks), as this can result in false-negative skin test results. Serum-specic IgE measurement is also a safer option in patients who are very allergic and where an anaphylactic reaction to skin testing is a possible risk. It is important to remember that positive in vivo or in vitro tests for allergen-specic IgE must always be interpreted in relation to the entire clinical presentation. The presence of allergen-specic IgE antibodies is not sufcient for the diagnosis of allergic disease, as patients can be sensitized in the absence of (or prior to) the development of any symptoms. Nasal challenge tests are used in particular for research purposes and are important in the diagnosis of occupational rhinitis. The International Committee on Objective Assessment of Nasal Airways has set up guidelines concerning the indications, techniques, and evaluation of nasal challenge tests. In addition to allergen provocations, nasal challenge tests with aspirin, non-specic agents (histamine, metacholine), and occupational agents can be performed. Imaging (sinus plain radiographs, computed tomography, and magnetic resonance imaging) is not indicated for the diagnosis of AR, but may be necessary to exclude other conditions or complications. Other diagnostic tests that are employed to assess the nasal airways include nasal peak ow, rhinomanometry, and acoustic rhinometry, but these are rarely used in the diagnosis of AR. Objective testing of a patients ability to smell can be performed by olfactory testing. Mucociliary function can be measured by nasomucociliary clearance, ciliary beat frequency, or electron microscopy, but these tests are of little relevance in the diagnosis of AR.

ALLERGY / Allergic Rhinitis 85

Figure 6 Nasoendoscopic visualization (right nasal cavity: Hopkins 301) of nasal mucosa swelling in an allergic patient before (a) and (b) after allergen challenge. Table 4 Causes of false-positive and false-negative skin tests Causes of false-positive skin tests: * Dermographism * Irritant reactions * Non-specic enhancement of a nearby strong reaction * Improper technique/material Causes of false-negative skin tests: * Poor initial potency or loss of potency of extracts * Use of drugs modulating allergic reaction * Diseases attenuating skin response * Decreased activity of the skin (infants and elderly patients) * Improper technique/material Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147S334.

Figure 7 Kit with standardized allergen extracts and positive and negative control solutions used for allergy skin tests.

Comorbidities

Figure 8 The technique used for skin prick testing involves introducing a drop of diluted allergen followed by puncturing the skin with a calibrated lancet (1 mm) at an angle of 451. The drops should be placed 2 cm apart. All patients undergoing skin prick testing should also have a positive (histamine) and negative diluent (saline) control test included. Skin tests should be read at the peak of their reaction by measuring (in mm) the wheal and the are around 15 min after pricking. The relevance of skin prick testing should always be interpreted in the context of the patients history. Positive results can occur in people without symptoms and, similarly, false-negative results may also occur.

It must be emphasized that AR should be evaluated as a global systemic disease. Allergic inammation does not necessarily limit itself to the nasal airway, but the treating physician must also be aware of the possible comorbidities of AR, including asthma, conjunctivitis, sinusitis, and otitis media. The link between asthma and rhinitis in particular has gained much interest. Epidemiological studies have shown that up to 80% of patients with asthma demonstrate symptoms of rhinitis, while approximately 2040% of patients with AR have clinical asthma. There is growing evidence that rhinitis is a risk factor for the development of asthma, independent of atopy. Additionally, the airway mucosa of nose and bronchi have many similarities and the clinical and pathophysiological changes in asthma and AR are often very comparable. Although there are still some differences that should be highlighted, the strong relationship between rhinitis and asthma has introduced the concept of the united airway disease.

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Based on these ndings the ARIA guidelines recommend that patients with persistent AR should be evaluated for asthma by history, chest examination, and, if possible and where necessary, by the assessment of airow obstruction before and after using a bronchodilator, whereas patients with asthma should be evaluated for rhinitis by history and physical examination. Another frequent comorbidity of AR is conjunctivitis. It is estimated that 42% of patients with AR experience symptoms of allergic conjunctivitis and that 3356% of the cases of allergic conjunctivitis occur in association with AR. This coexistence, referred to as rhinoconjunctivitis, seems to be a typical feature in patients with seasonal pollen allergy. As eye symptoms substantially contribute to the burden of allergic rhinoconjunctivitis, adequate assessment and treatment of conjunctivitis should be part of the overall management. AR is also considered as a contributing factor in acute and chronic rhinosinusitis. Up to 54% of adults with chronic rhinosinusitis have symptoms of AR. Similarly, a high concordance (between 25% and 75%) of these disorders is found in children. Conversely, there is a high prevalence of sinus disease in patients with AR: abnormal sinus radiographs occur in over 50% of adults and children with perennial AR and acute rhinosinusitis occurs often during the allergy season. There is still some controversy regarding the etiological role of AR in otitis media with effusion. Most epidemiological data suggest an association between these two diseases. However, the available evidence is compromised by a possible referral bias and by the lack of prospective, controlled studies. It is still not clear whether AR predisposes to the development of otitis or whether nasal dysfunction worsens otitis. It can be concluded that although the exact pathophysiological links between AR and its several comorbidities still need to be elucidated, AR is not an isolated disorder but is part of a systemic disease process. Hence, the ARIA Working Group recommends a coordinated diagnostic and therapeutic approach instead of a fragmented, organ-based management.

by B lymphocyte, and the clinical disease phase during which symptoms in response to subsequent antigen exposure become manifest. The clinical disease phase, in turn, can be subdivided into two distinct phases: an early phase largely mediated through mast cells; and a late phase, which involves cellular inltration and mediator release.
Sensitization Phase

The development of sensitivity to an allergen requires IgE antibody production directed at the epitope. After allergen exposure, antigen-presenting cells present the allergens to CD4 cells. A subset of these CD4 cells, the T-helper type 2 (Th2) lymphocytes, generate Th2 cytokines, including IL-4 and IL-13, which stimulate IgE synthesis in combination with B-cellT-cell ligandreceptor interactions that are pivotal in the B-cell isotype switching toward IgE synthesis. These B-cellT-cell interactions include a major histocompatibility complex class II and T cell receptor/CD3 interaction and a binding of the CD40L on T lymphocytes and CD40 expressed on B cells. As a result, allergen-specic IgE antibodies are produced and these sensitize mast cells and other IgE receptor-bearing cells. There is now increasing evidence that IgE is produced locally in the nasal mucosa since nasal B cells, in the presence of IL-4 and CD40L positive mast cells, are able to produce IgE locally.
Clinical Disease Phase

Pathogenesis
Symptoms of AR develop upon inhalation of allergens in individuals previously exposed to such allergens and against which they have made IgE antibodies. The pathophysiological process of AR can be subdivided in two phases: the initial sensitization phase during which allergen presentation results in primary allergen-specic IgE antibody formation

The clinical disease phase consists of two phases: an early phase and a late phase. The early phase is largely mediated through mast cells. In sensitized patients, allergen re-exposure mediates cross-linkage, of adjacent IgE molecules bound to mast cell surfaces. If a mast cell is activated by IgE cross-linking, it releases granule products containing histamine, tryptase, chymase, and cytokines such as interleukin-4 (IL-4), IL-5, IL-8, IL-13, and tumor necrosis factor alpha (TNF-a) into the extracellular environment. Second upon activation mast cells generate arachidonic acid products including cysteinyl-leukotrienes (LTC4, LTD4, LTE4) and prostaglandin D2 (PGD2) from the phospholipid cell membrane. Nasal challenge with allergens shows the local release within 10 15 min of histamine, tryptase, PGD2, LTB4, and LTC4. These mediators cause the characteristic watery rhinorrhea by stimulating gland and globet cell secretion, vasodilation, and blood vessel leakage, which is characteristic for the early phase. Histamine is the most important mediator in AR and induces symptoms of nasal itching, sneezing, discharge, and transient nasal blockage whereas leukotrienes appear to be relatively more important than histamine in

ALLERGY / Allergic Rhinitis 87

inducing nasal blockage. In addition, the mast cell is thought to contribute to other features in rhinitis, namely the eosinophilic mucosal inammation. Eosinophils are present in nasal mucosal biopsies within the submucosa and epithelium in active rhinitis. They generate vasoactive mediators and have the capacity to produce cytotoxic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein. Although the eosinophils feature heavily in the late-phase allergic response, their role in the early-phase allergic response is not clear. The late phase starts 48 h after allergen exposure. Clinically, it can be similar to the early phase but, in general, nasal congestion is more prominent. The late response involves a process of cellular accumulation. The expression of adhesion molecules and the presence of cytokines IL-3, IL-4, IL-5, IL-8, granulocytemacrophage colony-stimulating factor, and TNF-a enhances cell activation, accumulation of neutrophils, eosinophils, and T lymphocytes, and prolongs the survival of eosinophils within the nasal mucosal tissue. There is evidence showing that, in persistent rhinitis, there is an upregulation of the adhesion mechanisms with increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. During this late phase, activated basophils are responsible for histamine release. All these events amplify the allergic inammatory response, leading to a real cascade of reactions (Figures 9 and 10).

Although the inammatory reaction in AR is triggered by allergen exposure, it has been demonstrated that even in cases of subliminal exposure to the allergen(s), in the absence of symptoms, a certain degree of inammatory inltration at the mucosal level persists. This is called the minimal persistent inammation.
The Priming Effect

The priming effect refers to the phenomenon where the amount of allergens necessary to evoke an immediate response decreases with repeated allergen challenges or exposures. Nasal challenge induces an immediate clinical response in allergic subjects and a concomitant appearance of an inammatory inltrate. The mucosal inammation may persist 4872 h after allergen exposure. If the subjects are rechallenged within this period the response is more pronounced: the so-called priming effect. This effect is hypothesized to be a result of the inux and subsiding activity of inammatory cells during the latephase allergic response. Clinically, this explains the observation that decreasing allergen quantities are required to elicit symptoms as the pollen season progresses. In patients allergic to tree and grass pollen, the tree pollen season has a priming effect on the subsequent grass pollen season and these patients can often develop symptoms early in the grass pollen season when pollen counts are still very low.

Histamine Leukotrienes Concentration Tachykinins

Early phase

Late phase

Time

Blockage Rhinorrhea Sneezing Itching

Figure 9 Nasal allergen provocation results in a significant increase in mediators (histamine, leukotrienes, and tachykinins) and immediate nasal symptoms (blockage, rhinorrhea, sneezing, and itching) in allergic rhinitis patients. Between 2 and 24 h after allergen provocation, late-phase nasal symptoms, especially blockage, are observed in allergic rhinitis patients.

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IL-4 Allergen IL-13 IgE Mast cell IL-4 IL-6 Th0 APC IL-12 IL-18
Figure 10 Allergic inammation is characterized by a preponderance of T-helper type 2 (Th2) lymphocytes over T-helper type 1 (Th1) cells. APC, antigen-presenting cell; IL, interleukin; IFN-g, interferon gamma; IgE, immunoglobulin e; Th0, precursor T cell.

IL-5 Th2

IL-4

Eosinophil

Th1

IFNIL-2

Systemic Component in the Allergic Rhinitis Response

In addition to the local events in the nose, there is a systemic element to the inammatory process of AR. A variety of mechanisms have been proposed to explain the pathophysiological link between the upper and lower airways, including the loss of nasal protective function, altered breathing pattern, postnasal drip causing pulmonary aspiration of nasal contents, the presence of a nasalbronchial reex, and the progression of systemic inammation. Recent data suggests that bidirectional systemic inammation involving the bone marrow is likely to be important. Local allergen provocation (in the nose or bronchi) leads to upregulation and release from the bone marrow of hemopoietic eosinophil/basophil progenitor cells, which migrate to both nose and lungs where they can undergo differentiation and activation in situ.

Animal Models
Besides clinical and in vitro studies, animal models of allergic airway disease may provide useful information concerning upper airway inammation and serve as a model for relatively invasive experimental procedures. Although AR in animals is rare, chronic rhinitis associated with Aspergillus infection has been reported in rodents, poultry, dogs, and horses. In the absence of a common naturally occurring model of AR, sensitivity to allergens must be induced in healthy animals. A number of research groups are currently applying different experimental protocols to

induce experimental airway allergy. In guinea pigs, ovalbumin (OVA) is the most common sensitizing agent. To induce sensitization OVA is repeatedly injected into the peritoneum over the course of several weeks; this is followed by exposure to aerosolized OVA, which leads to allergen-specic IgE production. In addition to systemic sensitization, intranasal applications of OVA may induce allergen-specic IgE production but this exclusive local sensitization has been described in only a small number of studies. The upper airways of mice are less attractive for research compared to the lower airways, and this is for several reasons. First, mice are obligatory nose breathers, resulting in a significant baseline inammation. Second, eosinophilic inammation present in the nose of mice with experimental airway inammation is rather limited compared to the inammatory response in the lower airways. Several animal models of AR have shed light on the cellular and cytokine proles associated with airway inammation. They demonstrate that the relative importance of mast cells, eosinophils, IgE, and the cytokines IL-4 and IL-5 in the development of allergic inammation varies with the sensitization protocol used and with the animal strain.

Management and Current Therapy

Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of AR. In select cases, nasal surgery may be recommended as an adjunctive intervention.

ALLERGY / Allergic Rhinitis 89 Prevention

The treatment of patients with AR starts with the identication of possible allergens and subsequent prevention. If possible, allergen avoidance is the rst step in the management of AR. The most common indoor allergens are dust mites and dog and cat dander. The use of simple measures to avoid the allergens can relieve the symptoms but there is still a paucity of data relating to the effectiveness of avoidance. Clinical improvement can be expected within weeks. However, some allergens require a longer period before they are effectively removed.
Pharmacological Agents

Cromoglycate and nedocromil Sodium cromoglycate and nedocromil sodium are both drugs that have been demonstrated to inuence mast cell degranulation in in vitro studies. However, no inhibitory effect on histamine release has been demonstrated in mast cells recovered from nasal tissue. These drugs also inhibit the intermediate conductance pathways of mast cells, eosinophils, epithelial cells, broblasts, and sensory neurons. Cromoglycate blocks symptoms of the immediate and late phase and is effective even when used shortly before allergen inhalation. Cromoglycate is more effective than placebo; however, most studies show it to be less effective than nasal steroids. Both drugs do not induce any serious side effects. Ipratropium Ipratropium bromide blocks the activity of atropine and therefore reduces rhinorrhea when used intranasally. It does not block sneezing, pruritus, or nasal obstruction and is thus of greatest use in nonallergic rhinitis with predominant rhinorrhea. Vasoconstrictors Local vasoconstrictors reduce nasal blockage and are therefore indicated to relieve the symptoms of AR. However, they create a tachyphylaxis with rebound rhinitis as a symptom that develops after 37 days. Excessive consumption of nasal decongestants can cause rhinitis medicamentosa. Therefore, nasal decongestants are not recommended in the treatment of AR and should preferably be used for a short period when symptomatic vascular decongestion is indicated for the alleviation of nasal obstruction. Oral pseudoephedrine as a sustained release preparation has been shown to decrease both the nasal congestion and nasal airway resistance in patients with hay fever but only at doses where there is a high risk of systemic side effects. Leukotriene modiers While sneezing and nasal itching correlate best with histamine levels in experimental AR, nasal congestion correlates with LTC4 levels. Leukotriene receptor antagonists provide some benets in patients with AR. However, as demonstrated by some recent studies, leukotriene modiers may be less effective than intranasal steroids. In children, leukotriene receptor antagonists have been shown to reduce exercise-induced asthma especially in combination with inhaled steroids. Allergen-specic immunotherapy Allergen-specic immunotherapy is the practise of administering gradually increasing doses of therapeutic vaccines of standardized allergen extracts until reaching an arbitrary dose that is maintained for several years. Traditionally, allergen-specic immunotherapy

Intranasal steroids Intranasal steroids are a potent and highly effective treatment for patients with AR. Their effect is based on localized repression of the inammatory response. They reduce the number of eosinophils, the amount of eosinophilic cationic protein present, and the number of mast cell progenitors. Their multiple sites of action may account for their extreme potency. Clinically their efcacy exceeds that of antihistamines, decongestants, and cromoglycin. Compared to antihistamines they are more effective in reducing nasal blockage. However, they have a limited effect on associated eye symptoms and a relatively slow onset of action. There is some evidence that intranasal steroids have a benecial effect on asthma symptoms. At the recommended doses, intranasal steroids cause few side effects. Systemic side effects have not been observed, and the risk of systemic absorption and possible effect on growth in children has been extensively studied but has shown no effect at the recommended doses. Despite these ndings, nasal steroids should be used at the lowest possible dose in children. To date, there is no convincing evidence that doses greater than the recommended maximum increase efcacy. Antihistamines Antihistamines typically reduce itching, sneezing, and rhinorrhea but have no or little effect on nasal congestion associated with the late-phase reaction. The currently used antihistamines are clearly less effective than topical nasal steroids. They are subdivided into rst- and second-generation histamines. First- and second-generation antihistamines differ in their side effects. First-generation antihistamines produce sedation and other central nervous system symptoms in X20% of patients and may cause drying of the mouth and urinary hesitancy. Secondgeneration antihistamines also have varying degrees of anticholinergic, antimuscarinic, and antiadrenergic effects, but to a lesser extent than rst-generation antihistamines.

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is administered subcutaneously. There is now good evidence that immunotherapy with seasonal and perennial allergens is clinically effective for the treatment of AR, but it should only be considered in patients with severe symptoms of AR, when allergen avoidance and pharmacotherapy have failed to reduce symptoms or when pharmacotherapy has been associated with unacceptable side effects. In addition, specic immunotherapy for AR, when administered early in the disease process, has been demonstrated to modify the long-term progress of the allergic inammation and disease, by preventing the development of new sensitizations and by preventing the development of asthma. The exact mechanisms at the basis of the benecial effects of allergen-specic immunotherapy are complex and are still not completely understood. Recent evidence suggests both a shift away from a Th2-type response as well as the generation of regulatory T cells. As immunotherapy is not free of risk and may provoke systemic reactions (severe asthma attacks and anaphylaxis in particular), it can only be carried out by or under the supervision of trained specialists, with direct access to the necessary rescue medication. Because of these risks, patients must be closely observed for 2030 min after injection. More recently, other administration routes for allergen-specic immunotherapy have been investigated, including nasal, sublingual-swallow, and oral immunotherapy. Sublingual administration has shown to be effective. Novel treatments Increasing insights into the pathophysiology of AR, the roles of diverse cells and their cytokine products, and receptors and mediators involved in allergic inammation has provided new (potential) targets for pharmacotherapy. Modulation of allergic response through antimediators and antireceptors has gained much interest (e.g., anti-IgE, anti-IL-5, anti-CCR3).
Role of Surgery in the Management of Allergic Rhinitis

diagnostic tests have been developed, many effective pharmacological agents are currently available, and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To provide and disseminate this knowledge from research into practice, to facilitate and standardize the management of AR, and to improve the patient care and, consequently, the patient satisfaction and compliance several clinical guidelines have been developed. Before 1998, European and American guidelines for the management of AR were developed, based on expert opinion. The European guidelines are in many respects very similar to the American guidelines, but a
Table 5 Classication schemes of statements of evidence Category of evidence Ia: Evidence from meta-analysis of randomized controlled trials Ib: Evidence from at least one randomized controlled trial IIa: Evidence from at least one controlled study without randomization IIb: Evidence from at least one other type of quasi-experimental study III: Evidence from nonexperimental descriptive studies, such as comparative studies, correlation studies, and casecontrol studies IV: Evidence from expert committee reports or opinions or clinical experience of respected authorities or both Strength of evidence of recommendations A: Directly based on category I evidence B: Directly based on category II evidence or extrapolated recommendation from category I evidence C: Directly based on category III evidence or extrapolated recommendation from category I or II evidence D: Directly based on category IV evidence or extrapolated recommendation from category I, II, or III evidence Adapted form Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593 596.

Table 6 Strength of evidence for the treatment of rhinitis Intervention Seasonal AR Adults Oral anti-H1 Intranasal anti-H1 Cromones Antileukotrienes Subcutaneous SIT Sublingual/nasal SIT Allergen avoidance A A A A A A D Children A A A A A A D Perennial AR Adults A A A A A D Children A A A A A

Surgery does not relieve allergic inammation and should only be used in case of turbinate hypertrophy or cartilaginous or bony obstruction, contributing to or aggravating rhinitis symptoms, especially nasal obstruction. In these cases, a conchotomy and/or septo(rhino)plasty is recommended. In cases of secondary and independent sinus disease, functional endoscopic sinus surgery can be performed.
Clinical Guidelines for the Management of Allergic Rhinitis

Our insight into the pathophysiology of AR has increased in recent years. Highly sensitive and specic

SIT, allergen-specic immunotherapy. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147S334.

ALLERGY / Allergic Rhinitis 91

prominent feature of the former is the stepwise approach recommended for the treatment of rhinitis, which is similar to the Global Initiative for Asthma guidelines for the treatment of asthma. In 1999, the ARIA Working Group was founded, under the initiative of the World Health Organization. Unlike the European and American guidelines,

the ARIA guidelines are formulated on evidencebased medicine, categorized by Shekelle. Based on these categories of evidence, the strength of evidence for a certain recommendation is graded from A to D (Table 5). The evidence for the recommended pharmacotherapy and immunotherapy for AR treatment is particularly strong (category A evidence strength).

Diagnosis of allergic rhinitis Allergen avoidance

Intermittent symptoms

Persistent symptoms

Mild

Moderate/ severe

Mild

Moderate/severe Intranasal corticosteroids

Not in preferred order: oral H1-blocker intranasal H1-blocker and/or decongestant

Review patient after 2 4 weeks Not in preferred order: oral H1-blocker intranasal H1-blocker and/or decongestant intranasal corticosteroid (cromones)

improved

failure

In persistent rhinitis: review the patient after 2 4 weeks

Step-down and continue treatment for 1 month

Review diagnosis, compliance, query infections or other infections, or other causes

If failure: step-up If improved: continue for 1 month

Intranasal CS dose

Itch/sneeze: +H1 blocker

Rhinorrhea: + ipratropium Blockage: + decongestant or oral CS (short term)

Failure Surgical referral If conjunctivitis add : Oral H1-blocker or intraocular H1-blocker or intraocular cromones (or saline) Consider specific immunotherapy
Figure 11 Stepwise treatment algorithm for allergic rhinitis in adolescents and adults, as recommended by ARIA. CS, corticosteroids. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147S334.

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The evidence for allergen avoidance, on the other hand, is limited (category D evidence strength) (Table 6). Similar to the European guidelines, a stepwise approach for the treatment of AR is recommended by ARIA with the following rst-line treatment approaches (Figure 11): oral or intranasal H1-antihistamines, with limited use of decongestants, for mild intermittent rhinitis; oral or intranasal H1-antihistamines or intranasal corticosteroids, with limited use of decongestants and cromones, for moderate to severe intermittent and mild persistent rhinitis; and intranasal corticosteroids, with step-down and step-up options, in conjunction with H1-antihistamines, decongestants, ipratropium, and eventually oral corticosteroids, for moderate to severe persistent rhinitis. Additionally, specic immunotherapy should be considered in persistent disease and when the symptoms are moderate to severe and do not respond to conventional treatment. When conjunctivitis is present, oral or intraocular H1-antihistamines should be used. Whereas the European guidelines did not take into account the costs and availability of the treatment strategies in different countries, the ARIA guidelines are developed for the whole world and recognize that the outcome of disease management largely depends on compliance with the suggested treatment, which in turn is inuenced by the availability and affordability of the specic interventions. Therefore, a exible stepwise approach is recommended, based on the four cornerstones of patient education, allergen avoidance, pharmacotherapy, and immunotherapy, but modiable in low-income countries.
See also: Allergy: Overview; Allergic Reactions. Asthma: Overview. Chemokines. Chymase and Tryptase. Histamine. Immunoglobulins. Interleukins: IL-4; IL-5; IL-10; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids.

Further Reading
Aalberse RC (2000) Molecular mechanisms in allergy and clinical immunology. Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228238. Barnes PJ (2003) Pathophysiology of allergic inammation. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middletons Allergy Principles and Practice, pp. 483499. Pennsylvania: Mosby. Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147S334. Howarth PH (2003) Allergic and nonallergic rhinitis. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middletons Allergy Principles and Practice, pp. 13911410. Pennsylvania: Mosby. International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 534. Johansson SG, Bieber T, Dahl R, et al. (2004) A revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization. Journal of Allergy and Clinical Immunology 113: 823826. Malm L, Gerth van Wijk R, and Bachert C (1999) Guidelines for nasal provocations with aspects on nasal patency, airow and airow resistance. Rhinology 37: 133135. Salib RJ and Howarth PH (2003) Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clinical and Experimental Allergy 33: 16291633. Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593596. Van Cauwenberge P, Bachert C, Passalacqua G, et al. (2000) Consensus statement on the treatment of allergic rhinitis. Allergy 55: 116134. Wheatley LM and Platts-Mills TAE (1999) Allergens. In: Naclerio RM, Durham SR, and Mygind N (eds.) Rhinitis, pp. 4558. New York: Marcel Dekker, Inc. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema (1998) ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 351: 12251232.

Relevant Website
http://www.ginasthma.com Global Initiative for Asthma. Global strategy for asthma management and prevention (2003).

ALVEOLAR HEMORRHAGE
O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved. life-threatening condition. There are many causes of DAH, including vasculitides, such as Wegeners granulomatosis, microscopic polyangiitis, Good pastures syndrome, connective tissue disorders, and other conditions. Pathologically, the syndrome is due to pulmonary vasculitis, to a bland alveolar hemorrhage, or it represents the nondominant pathology, as seen in diffuse alveolar damage from acute respiratory distress syndrome (ARDS). Most patients present with dyspnea, cough, hemoptysis (the latter in only 66% of the cases), anemia, and

Abstract
Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH) and represents a potential

ALVEOLAR HEMORRHAGE 93
new pulmonary inltrates. Urgent bronchoscopy and bronchoalveolar lavage is generally required to conrm the diagnosis, with a superior yield when it is performed in the rst 48 h. In patients with evidence of DAH and renal involvement (pulmonaryrenal syndrome), kidney biopsy may be considered to identify the etiology and direct the therapy. This chapter will describe the mean features of the DAH syndrome and review in short the main causes of alveolar bleeding. Table 1 Causes of diffuse alveolar hemorrhage (DAH) DAH associated with vasculitis Wegeners granulomatosis Microscopic polyangiitis Goodpastures syndrome Isolated pauci-immune pulmonary capillaritis Connective tissue disorders Antiphospholipid antibody syndrome Mixed cryoglobulinemia nlein purpura, IgA nephropathy HenochScho Pauci-immune or immune complex-associated glomerulonephritis Behcets syndrome Acute lung graft rejection Thrombotic thrombocytopenic purpura and idiopathic thrombocytopenic purpura Bland DAH Mitral stenosis and mitral regurgitation Anticoagulants, antiplatelet agents, or thrombolytics; disseminated intravascular coagulation Pulmonary venoocclusive disease Infections: human immunodeciency virus (HIV) and infective endocarditis Toxins: trimellitic anhydride, isocyanates, crack cocaine Drugs: propylthiouracil, diphenylhydantoin, amiodarone, mitomycin, penicillamine, sirolimus, methotrexate, nitrofurantoin, gold, all-trans retinoic acid (ATRA), bleomycin (especially with high-ow O2), montelukast, zarlukast, iniximab Idiopathic pulmonary hemosiderosis DAH as nondominant pathology Diffuse alveolar damage (DAD) Malignant conditions (e.g., pulmonary angiosarcoma) Lymphangioleiomyomatosis/tuberous sclerosis Pulmonary capillary hemangiomatosis Lymphangiography

Introduction
Hemoptysis is most commonly due to disruption of the bronchial circulation of various (endo)bronchial conditions such as bronchitis, bronchiectasis, neoplastic disorders, or (rarely) disorders of the pulmonary circulation. Hemorrhage from a bronchial source can be rapidly fatal, since a brisk bleeding can lead to a large amount of blood to occupy anatomic and functional dead space of the respiratory tract; in this setting the alveoli can also be ooded quickly, mimicking the true alveolar hemorrhage. Anatomical injury at the alveolarcapillary basement membrane level of the pulmonary microcirculation may cause hemoptysis from arteriolar, venular, or capillary source. Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH). Although alveolar hemorrhage may be focal, generally there are multiple areas affected, hence the term DAH. Diffuse alveolar hemorrhage should always be considered a potentially life-threatening condition; it requires early recognition, care in stabilizing the patient (airway protection, sometimes selective intubation, mechanical ventilation, etc.) and specic treatment once the etiology is established. Synonyms with DAH that can be found in the literature are: (intra)pulmonary hemorrhage, pulmonary alveolar hemorrhage, pulmonary capillary hemorrhage, alveolar bleeding, and microvascular pulmonary hemorrhage. The DAH syndrome is relatively rare, although no studies addressed its specic epidemiology. Currently, our understanding of the etiopathogenesis and the appropriate management relies mainly on case reports or case series of specic disorders leading to DAH. Unfortunately, the therapeutic approach is rather non-specic, with a few notable exceptions (Wegeners granulomatosis (WG), Goodpastures syndrome, systemic lupus erythematosus (SLE), etc.)

accounted for one-third of cases, followed by Goodpastures syndrome (13%), idiopathic pulmonary hemosiderosis (IPH) (13%), collagen vascular disease (13%), and microscopic polyangiitis (9%). A review of 29 cases of DAH associated with capillaritis found that the most common cause was isolated pulmonary capillaritis. Other conditions associated with DAH are briey discussed below. Overall, three different histologic patterns may be seen.
DAH Associated with Vasculitis

Etiology
Various diseases can lead to DAH syndrome (Table 1). To date, no prospective studies of DAH have estimated the relative frequency of various etiologies. A wellknown retrospective review of 34 cases of DAH suggested that the most common cause was WG, which

This pathologic pattern is characterized by neutrophilic inltration of the interalveolar and peribronchiolar septal vessels (pulmonary interstitium). This sequentially leads to anatomic disruption of the capillaries, and red blood cell extravasation into the alveoli and interstitium. This leads to neutrophil fragmentation and apoptosis, with subsequent release of the intracellular proteolytic enzymes and reactive oxygen species, which begets more inammation,

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intra-alveolar neutrophilic nuclear dust, brin and inammatory exudate, and brinoid necrosis of the interstitium.
DAH Associated with Bland Pulmonary Hemorrhage

This pattern is characterized by intra-alveolar red cell extravasation without any evidence of inammation or destruction of the alveolar capillaries, venules, and arterioles; the epithelial lesions are usually discrete.
DAH Associated with Other Conditions (Nondominant Pathology)

The presence of Kerley B lines points toward mitral valve disease or pulmonary venoocclusive disease. Computed tomographic imaging studies may show areas of consolidation alternating with areas of ground-glass attenuation and preserved, normal areas. Gallium scan, rarely ordered today for this purpose, used to be performed in the past to reveal areas of active vasculitis or inammatory activity (rather non-specic and with unclear sensitivity, too). Other nuclear studies, geared to reveal breakdown of the microcirculatory integrity and extravasation of red blood cell out of the vessels, have not been conrmed by the ultimate test of time.
Pulmonary Function Tests

Here, the DAH is secondary to diffuse alveolar damage (DAD), lymphangioleiomyomatosis (LAM), drug-induced lung injury, metastatic neoplasia to the lungs, mitral stenosis, etc. DAD is the main underlying lesion of the acute respiratory distress syndrome (ARDS), and is characterized by intra-alveolar hyaline membranes, interstitial edema with minimal inammation, and at times by secondary DAH.

Clinical Presentation
The syndrome of DAH may present with a constellation of symptoms, signs, and laboratory results that may suggest the underlying etiology (e.g., WG, Goodpastures syndrome, drug-related vasculitis, etc.) or only establish the diagnosis of the syndrome without a specic etiology.
Symptoms

The onset of DAH is most often acute or subacute (less than 1 week). Dyspnea, cough, and fever are the common initial symptoms. Some patients, however, present with ARDS requiring mechanical ventilation. Hemoptysis may be absent at the time of presentation in up to one-third of patients with DAH syndrome. When present, it ranges from fulminant and life-threatening to mild and intermittent in nature.
Physical Examination

The DAH leads to various degrees of oxygen transfer impairment and hypoxemia, sometimes severe enough to require ventilatory support. A sensitive marker for DAH is a sequential increase in diffusing lung capacity for carbon monoxide (DLCO). In this setting, due to an increased availability of intraalveolar hemoglobin, capable of binding with highafnity carbon monoxide, the DLCO is generally normal or elevated. Unfortunately, the severe condition of these patients and the associated hemoptysis generally preclude any DLCO measurements. An obstructive lung disease associated with recurrent DAH may be due to lymphangioleiomatosis, histiocytosis X, or pulmonary capillaritis in the setting of microscopic polyangiitis or WG. Recurrent episodes of DAH generally lead to interstitial brosis and ventilatory restrictive defects, as seen in idiopathic pulmonary hemosiderosis.
Laboratory Abnormalities

The lung examination is usually non-specic, unless there are physical signs of an underlying systemic vasculitis or collagen vascular disorder (rashes, purpura, eye lesions, hepatosplenomegaly, clubbing, etc.).
Imaging Studies

The blood work generally reveals acute and/or chronic anemia, stress leukocytosis, elevated erythrocyte sedimentation rate, and C-reactive protein (particularly in the cases of DAH caused by systemic diseases or associated with a pattern of vasculitis). Since several causes of DAH may present as pulmonaryrenal syndromes (i.e., association of pulmonary hemorrhage with different types of glomerulonephritis), blood urea nitrogen (BUN) and creatinine concentration elevations, and abnormal urine sediments (red blood cells/casts, white blood cells/casts, proteinuria of glomerular origin) can be seen.

The radiographic ndings are generally non-specic and show new and/or old, patchy or diffuse alveolar opacities. Recurrent episodes of DAH lead to reticular interstitial opacities due to pulmonary brosis, usually with minimal honeycombing (if any).

Diagnosis
DAH represents a medical emergency, since it represents a potentially fatal condition. Despite recent advances and renements of the diagnostic and therapeutic tools in DAH, it remains a highly morbid

ALVEOLAR HEMORRHAGE 95

condition, with substantial fatality. One must have a low threshold to entertain the diagnosis, to conrm it and to thoroughly look for the underlying etiology. Methodically, two separate steps are to be followed.
Establish the Diagnosis of DAH Syndrome

The triad of elevated DLCO, hypoxemia, and new pulmonary inltrates in the setting of hemoptysis and dyspnea suggests the diagnosis of DAH. Patients who present with hemoptysis need to be screened for focal sources of pulmonary hemorrhage (i.e., bronchitis, bronchiectasis, infection, neoplastic processes, etc.); upper airway and gastrointestinal sources must also be excluded carefully. It is important to remember that most disorders manifested with severe hemoptysis can cause alveolar inltrates. In patients without hemoptysis, the clinical evaluation needs also to screen for evidence of congestive heart failure, pneumonia, inhalational or drug-related lung injury, and other causes of bleeding. Most patients suspected of having DAH need a bronchoscopic examination, which serves two purposes: documentation of alveolar hemorrhage by visual inspection and bronchoalveolar lavage (BAL; especially if there is no hemoptysis) and exclusion of an associated infection. This procedure has a higher yield if it is performed early (within 48 h) rather than later. An increasing hematocrit or progressively bloodier aspect of three sequential BAL aliquots from an affected area is diagnostic of DAH. In subacute or recurrent episodes of DAH, counting the hemosiderin-laden macrophages (siderophages) as demonstrated by Prussian Blue staining on a pooled BAL specimen centrifugate may be useful for diagnosis (generally more than 30% siderophages). BAL specimens should be sent for routine bacterial, mycobacterial, fungal, viral, and Pneumocystis carinii microscopic studies and cultures. The use of transbronchial biopsy in patients with suspected DAH is of unclear value due to the small size of the specimens and possible sampling bias. Unless another clinical condition is suspected, transbronchial biopsy is generally not useful clinically.
Identify the Specic Etiology

represent the basic panel to be checked in DAH patients (see Autoantibodies). If the diagnosis of DAH is still not clear or the underlying etiology is still not known after a thorough clinical evaluation, imaging studies, serologies, and bronchoscopy, surgical biopsy should be entertained. Besides, the results of a surgical biopsy may become available faster than the serologic tests. The ideal site to biopsy is dependent on the pretest probability of the underlying disorder: for WG a nasal biopsy may sufce, while a kidney biopsy (with immunouorescent studies) may be less invasive than others in diagnosing Goodpastures syndrome, microscopic polyangiitis, or systemic lupus erythematosus. In isolated pulmonary disease or pauci-immune pulmonary capillaritis a lung biopsy is a mandatory test. Immunouorescence reveals linear deposition of immunoglobulins and immune complexes along the basement membrane in Goodpastures syndrome and granular deposits in SLE, whereas the systemic vasculitides appears pauci-immune. In cases of pulmonaryrenal syndrome, the kidney biopsy shows focal segmental necrotizing glomerulonephritis. Additionally, skin biopsy of a lesion can demonstrate leukocytoclastic vasculitis or HenochScho nlein purpura (in the latter case, immunoglobulin A (IgA) deposits are suggestive of the diagnostic).

Current Therapy
Corticosteroids are currently the backbone of DAH syndrome therapy, especially if associated with systemic or pulmonary vasculitis, Goodpastures syndrome, and connective tissue disorders. Most authors recommend intravenous methylpredisolone (up to 500 mg every 6 h, although lower doses seem to have similar efcacy) for approximately 45 days, followed by a gradual taper to maintain doses of oral steroids. In the setting of pulmonaryrenal syndrome, the therapy needs to be initiated as soon as possible, since the potential of reversibility is lost exponentially in the rst week of disease activity. Other immunosuppressive drugs can be used in DAH (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, etanercept, etc.) depending on the disease severity, failures to respond to corticosteroids and the underlying disease (e.g., WG, Goodpastures syndrome). Intravenously administered cyclophosphamide (2 mg kg 1 day 1, adjustable to renal function) is generally the preferred adjunctive immunosuppressive drug in the initiation phase of the treatment, and continued several weeks until blood marrow suppression, infections, or other limiting effects require discontinuation and thereafter switch to consolidative

Identifying the specic etiology is generally equivalent to the process of differential diagnosis. Serologic studies need to be performed early during the course of the disease, although the results are generally not available in a timely manner for immediate management of the disease. Complement fractions C3 and C4 and anti-dsDNA, anti-glomerular basement membrane (GBM), antineutrophil cytoplasmic antibodies (ANCA), and antiphospholipid antibodies

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and/or maintenance therapy with methotrexate or other agents. Plasmapheresis is indicated in the treatment of DAH associated with Goodpastures syndrome or other vasculitis processes when the titers of pathogenetic immunoglobulins and immune complexes are very high (e.g., plasmapheresis in the setting of ANCA-associated vasculitis with overwhelming endothelial injury and/or hypercoagulable state). However, its utility in DAH syndromes other than Goodpastures syndrome has not been evaluated in prospective studies. If intravenous immunoglobulin (IVIG) therapy adds anything to the treatment of DAH due to vasculitis or other connective tissue disease is yet unclear. Besides the treatment of vasculitis and underlying disorder, stabilization of the patients with moderate and severe hemoptysis entails supplemental oxygen, bronchodilators, reversal of any coagulopathy, red blood cell transfusion, intubation with bronchial tamponade/protective strategies for the less involved lung, mechanical ventilation, etc. Several case reports showed success in treating alveolar hemorrhage due to allogeneic hemaopoietic stem cell transplantation, ANCA-associated vasculitis, systemic lupus erythematosus, and antiphospholipid syndrome with recombinant-activated human factor VIIa, which may become a new tool in the (otherwise poor) armamentarium available for this condition.

Prognosis
Recurrent episodes of DAH may lead to various degrees of interstitial brosis, especially in patients with underlying WG, mitral stenosis, long-standing and severe mitral regurgitation, and idiopathic pulmonary hemosiderosis. A post-DAH syndrome has also been described, particularly in microscopic polyangiitis, and idiopathic pulmonary hemosiderosis and consists of progressive obstructive ventilatory defects and anatomic emphysema.
Mortality

etc.) have allowed the indirect study of DAH syndrome and its close interrelation with iron metabolism. Iron is of crucial importance in the inammatory and anti-infectious defense of the respiratory tract and lung parenchymal homeostasis. Local macrophages have a major contribution to iron recycling from red blood cells, through the phagocytosis of the intra-alveolar erythrocytes and eventual return of the electrolyte to the bone marrow erythron, where iron is incorporated in the structure of heme in maturing red blood cells. The understanding of alveolar pathology in DAH has been enriched lately by a literature explosion in the eld of ferroproteins and genetics of the regulatory factors involved in the iron metabolism. It is beyond the scope of this section to review exhaustively the respiratory iron metabolism, hence mention will only be made of the main facts. The free irons presence in the extracellular milieu together with local hypoxia induces reactive oxygen species and other free radical production, with subsequent peroxidation of the cellular membranes, cell apoptosis, and preinammatory cytokine release. The phagocytosis capacity of the alveolar macrophages seems to be easily exhaustible, becoming hemosiderin-laden macrophages (siderophages), which in turn leads to more free iron and heme available in alveolar spaces and pulmonary interstitium. Recently, J774 macrophages have been used among other experimental models to investigate the presence of different ferroproteins and their role. A recently discovered peptide, hepcidin, seems to have a major ferrostat role in local and general iron overload syndromes; its role (if any) is yet unclear in the DAH syndromes. Another interesting protein, ferroportin, seems to be involved in macrophage iron recycling from engulfed erythrocytes. It is possible that macrophages iron retentive mechanism is abnormal, similar to what is seen in human hereditary hemochromatosis type 4, or ferroportin disease.

Specic Causes of DAH

Wegeners Granulomatosis

The disease fatality varies with the underlying cause of DAH; patients with systemic lupus erythematosus, anti-GBM antibody disease and several forms of ANCA-associated vasculitis can have a high mortality (up to 50%) due to the disease activity, infections, and cardiovascular morbidity (e.g., ChurgStrauss syndrome).

Experimental Models
Experimental models of different conditions (e.g., microscopic polyangiitis, anti-GBM antibody disease,

WG is a systemic disease characterized by necrotizing granulomatous inammation of the small vessels, involving predominantly the respiratory tract and kidneys. The organ involvements can be recalled using the mnemotechnic JERKS: (1) Joint disease % (arthralgias and arthritis); (2) ENT (rhinosinusitis) % (both upper and and eye disease; (3) Respiratory % about 7095% of cases); lower respiratory tracts, (4) Kidney involvement (lesions of focal segmental % necrotizing glomerulonephritis); (5) Skin and other % of the lung organs (systemic). The gross appearance

ALVEOLAR HEMORRHAGE 97

ANCA-associated small vessel vasculitis who were treated with plasma exchange in conjunction with induction immunosuppressive regimens had a remission of the DAH. Other therapies, such as trimethoprim sulfamethoxazole, tumor necrosis factor inhibitors, and anti-CD20 monoclonal antibodies have been tried with unproven efcacy or are under scrutiny. Mortality is considerable and most often caused by infections, respiratory failure, and renal failure.
Microscopic Polyangiitis

Figure 1 Diffuse alveolar hemorrhage associated with, necrotizing pulmonary vasculitis (shown) in a patient with Wegeners granulomatosis.

in WG with DAH is dark red, sometimes associated with multiple nodules and cavitary lesions (exceptionally solitary pulmonary nodules), and much more frequently with areas of consolidations and focal or geographic necrosis (Figure 1). The chest radiograms show uffy alveolar and/or interstitial inltrates reecting the diffuse microvasculature disorder. The diagnosis of WG is established based on clinical presentation and serologic conrmation, that is, positive ANCA antibodies (c- or p-ANCA). In organlimited WG there is ANCA positivity in 60% of the cases, while in generalized disease in 9095% of cases. c-ANCA (directed against proteinase-3) is present in 8590% of generalized forms of the disease. If diagnosis is still in doubt, surgical biopsy of affected organs (kidneys, lungs, nasal mucosa) may be useful for diagnosis. In WG, the DAH due to pulmonary capillaritis may mark the disease onset or occur later during the course, in a subclinical and/or recurrent fashion. The progressively bloodier BAL aliquots with large numbers of red blood cells and hemosiderin-laden macrophages in the absence of an infectious etiology conrm the diagnosis of DAH. The standard inductive therapy of DAH is high-dose corticosteroids (e.g., 1 g methylprednisolone daily for 3 days) and daily intravenous cyclophosphamide. Azathioprine generally substitutes cyclophosphamide in about 6 months or after remission in an inductivemaintenance approach and/or after a cumulative high dose of cyclophosphamide is reached. IVIG or therapeutic plasma exchange may be used for persistent disease. In one study, 20 out of 20 patients with

Microscopic polyangiitis (MP), a small-vessel variant of polyarteritis nodosa, is sometimes difcult to differentiate from WG due to similar clinical presentations, serologic and pathologic ndings. The typical pathologic feature in microscopic pauci-immune neutrophilic polyangiitis (found in virtually 100% of cases) is a focal and segmental necrotizing glomerulonephritis, renal lesion also seen in WG, Goodpastures syndrome, and other connective tissue disorders. The lack of granulomatous inammation differentiates pathologically between WG and MP. A positive serum perinuclear ANCA (p-ANCA), directed against myeloperoxidases epitopes, strongly supports the diagnosis, but it can be also seen in 2030% of WG cases. Microscopic polyangiitis causes relatively frequently a syndrome of severe DAH. Treatment with corticosteroids and cyclophosphamide followed by azathioprine is similar to WG therapy. Plasmapheresis and IVIG may be also useful in difcult-to-treat cases. As in other hemorrhagic conditions, factor VIIa has also been used with various success. The short-term and long-term (5-year) mortality rates from DAH in MP are approximately 25% and 40%, respectively.
Goodpastures Syndrome

Goodpastures syndrome is a form of anti-basement membrane antibody disease and is characterized by a combination of DAH and glomerulonephritis. Anti-GBM antibody, the pathognomonic immunoglobulin, is directed against alpha-3 (IV) collagen from GBM and is found in the serum of more than 90% of patients and part of the linear immunouorescent deposits in the glomerular membrane noted in the disease. The syndrome typically involves DAH in a smoker (usually a young male, although older patients, women, and nonsmokers can also have it). Isolated, renal-sparing DAH, a rare occurrence, may have anti-GBM both in the serum and in the glomerular membrane in a typical linear antibody deposition. The treatment of Goodpastures syndrome includes urgent plasmapheresis and corticosteroids,

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cyclophosphamide, and/or azathioprine. The isolated DAH seems to respond well to corticosteroids alone. Alveolar bleeding secondary to Goodpastures syndrome has a 2-year survival rate close to 50%, while patients presenting with renal insufciency have a worse outcome.
ChurgStrauss Syndrome

ChurgStrauss syndrome (allergic and granulomatous angiitis) is a systemic disorder characterized by asthma, peripheral blood eosinophilia, and systemic vasculitis of small-caliber vessels with extravascular necrotizing granulomas. It involves mainly the upper respiratory tract, the lungs, and the peripheral nerves. Tissue eosinophilia may involve the lungs or the gastrointestinal tract. Pulmonary hemorrhage is rare in this condition, while radiographic abnormal ndings are described in 5090% of the cases. p-ANCA positivity occurs in approximately 3540%. Histologically, it is distinguished by small- and medium-vessel involvement and eosinophil-rich inltrates (sometimes presenting with an aspect of chronic eosinophilic pneumonia). Of note, a ChurgStrauss-like syndrome can occur due to chronic ingestion of carbamazepine, quinine, or macrolides, while potential risk of aggravation of the disorder can occur on cysteinil leukotriene receptor blockers.
Isolated Pauci-Immune Pulmonary Capillaritis

(although it is the most frequent disorder in this category presenting with alveolar bleeding); DAH has generally a poor prognostic connotation (50% fatality rate) and is due to a process of pulmonary capillaritis. When DAH occurs in SLE, contrary to WG, glomerulonephritis is generally absent. Immunouorescent studies show granular deposits of IgG and complement (C3) in the pulmonary interstitium, alveolar blood vessels, and the GBM. Of note, the syndrome of DAH is clinically and pathologically distinct from acute lupus pneumonitis, which presents similarly and may be the inaugural presentation of SLE. Therapy consists of corticosteroids and cyclophosphamide and/or azathioprine. Plasmapheresis has no proven benet. In rheumatoid arthritis, the syndrome of DAH secondary to pulmonary vasculitis generally occurs late, in burnout disease.
Hematologic Conditions

DAD seems to be the dominant lesion in the lungs of the patients who undergo chemotherapy for leukemia or stem cell transplantation; this lesion can be accompanied by DAH, sometimes fulminant and fatal, even in the absence of hemoptoic sputa. The offending factors seem to be the chemotherapeutic agents associated with actinic lesions, thrombocytopenia, and superimposed infections. The therapy includes platelet transfusions, reversal of coagulation abnormalities, and high-dose corticosteroids.
DAH of the Immunocompromised Patient

An interesting case of a DAH syndrome is represented by pauci-immune pulmonary alveolar capillaritis in the absence of any other systemic involvement or abnormalities. Several cases have been shown to have elevated serum titers of p-ANCA, but this nding may reveal in fact a fruste form of MP. Isolated, pauci-immune pulmonary capillaritis has been also seen in association with the drug called all-trans retinoic acid (ATRA). Available literature on pauciimmune pulmonary alveolar capillaritis shows that respiratory failure necessitating mechanical ventilation is frequent in this condition and the response to corticosteroids and immunosuppressive agents is favorable.
Connective Tissue Disorders

Diffuse alveolar bleeding has been described in collagen vascular diseases such as SLE, scleroderma, rheumatoid arthritis, polymyositis/dermatomyositis, HenochScho nlein syndrome, Behcet disease, and mixed connective tissue disorder. In SLE, pulmonary complications occur in more than 50% of cases, while DAH can occur in up to 5% of the patients

Alveolar bleeding can occur in immunocompromised hosts due to a myriad of factors (infectious, chemotherapeutic, and immunosuppressive drugs, radiation therapy, thrombocytopenia, pulmonary edema, lung malignancy, other lung comorbidities, etc.) that have a common denominator: the endothelial injury. The exact frequency of the syndrome in the immunocompromised patients is currently unknown, partly because the hemorrhage can be subclinical, and because empirical therapy is instituted early, since the risk of invasive evaluation outweighs the benets. While subclinical DAH in this setting may have minimal impact on survival, the alveolar hemorrhage associated with serious causes like Kaposis sarcoma, invasive fungal infections (Aspergillus, Pseudoallescheria boydii, etc.), Mycobacterium, Legionella, or other invasive bacterial species may have catastrophic consequences. In human immunodeciency virus (HIV) infection or acquired immunodeciency syndrome (AIDS) patients, the cytomegalovirus (CMV) and Kaposis sarcoma represent the main risk factors for developing DAH. In a study of

ALVEOLAR HEMORRHAGE 99

HIV-infected patients with radiographic inltrates, in up to 44% of them more than 20% of the BAL cells were hemosiderin-laden macrophages.
Drug-Induced DAH

Many drugs have been associated with DAH, including anticoagulants (warfarin, heparin, etc.), thrombolytic agents, and platelet antiaggregant agents; as a rule, in order to produce DAH, a second hit (infectious, inammatory, inhalatory, etc.) is required. Several drugs can produce lung injury and DAD, with secondary DAH (e.g., sirolimus, methotrexate, nitrofurantoin, etc.). Other drugs causing DAH trigger a process of pulmonary capillaritis (e.g., propylthyouracil, phenytoin, mitomycin, and ATRA). Penicillamine can cause pulmonary capillaritis associated with glomerulonephritis with granular immunouorescent deposits (pulmonary renal syndrome). Interestingly, these agents are also responsible for p-ANCA generation. The standard therapy is discontinuation of the presumed offending drug and (rarely) plasma exchange.
Other Causes of DAH

Figure 2 Diffuse alveolar hemorrhage without any evidence of pulmonary vasculitis (bland alveolar hemorrhage) in a patient with idiopathic pulmonary hemosiderosis.

Other conditions associated with alveolar bleeding are: different coagulopathies, toxic exposures (isocyantes, trimellitic anhydride, crack cocaine, etc.) primary antiphospholipid antibody syndrome, mixed cryoglobulinemia, Behcets syndrome, Henoch Scho nlein purpura, lung transplant acute rejection, mitral stenosis and regurgitation, pulmonary venoocclusive disease, pulmonary capillary hemangiomatosis, LAM, and tuberous sclerosis Bourneville. In patients with primary antiphospholipid syndrome (APS), the thromboembolic complications occur in up to 15% of cases, pulmonary hypertension in about 2% of the cases, interstitial lung disease in up to 1% of patients, with DAH in less than 1% of cases.
Idiopathic Pulmonary Hemosiderosis

further scrutiny. Corticosteroids, hydroxychloquine, azathyoprine, and other immunosuppressive agents have been used with favorable effects. Lung transplantation has been reported as rather unsuccessful in a couple of patients with progressive disease and significant pulmonary brosis, due to recurrent pulmonary bleeding. Survival with IPH varies widely, although recent data has suggested better outcomes, most likely due to more aggressive immunosuppressive therapies.

Summary of Therapeutic Options

DAH syndromes represent a serious condition with possible catastrophic consequences, caused by a myriad of conditions, associated or not with pulmonary capillaritis. Dyspnea, cough, hemoptysis and new alveolar, uffy inltrates in conjunction with bronchoscopic ndings of bloody BAL, with numerous erythrocytes and siderophages make the syndrome diagnosis evident. Rarely, a surgical biopsy from the lung or another organ involved by the underlying condition may be necessary. The advent of ANCA has revolutionarized the diagnosis of WG, microscopic polyangiitis, ChurgStrauss syndrome, and other ANCA-associated conditions. The therapy in DAH targets both the autoimmune destruction of the alveolar capillary membrane and the underlying condition; corticosteroids and immunosuppressive agents are still the gold standard of therapy in the majority of cases. Factor VIIa seems to be a promising new therapy for DAH, although further evaluation is needed.

Idiopathic pulmonary hemosiderosis (IPH) is a diagnosis of exclusion (see Idiopathic Pulmonary Hemosiderosis); it is a rare disease characterized by recurrent episodes of DAH and bland alveolar hemorrhage (Figure 2). IPH occurs most frequently in children (80% of cases), but adult cases with onset up to the eighth decade of life have been reported (20% of cases). The pathogenesis of IPH is largely unknown. Some cases have been linked to fungi (Stachybotrys atra), others to environmental insecticides; it seems that several cases have initially been called IPH, when in fact coagulopathies such as van Willebrands disease have been found upon

100 ALVEOLAR HEMORRHAGE See also: Acute Respiratory Distress Syndrome. Autoantibodies. Bronchoalveolar Lavage. Granulomatosis: Wegeners Disease. Idiopathic Pulmonary Hemosiderosis. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpastures Syndrome.
isolated, pauciimmune pulmonary capillaritis. American Journal of Respiratory and Critical Care Medicine 155: 1101 1109. Klemmer PJ, Chalermskulrat W, Reif MS, et al. (2003) Plasmapheresis therapy for diffuse alveolar hemorrhage in patients with small-vessel vasculitis. American Journal of Kidney Diseases 42: 11491153. Lai RS, Lin SL, Lai NS, and Lee PC (1998) ChurgStrauss syndrome presenting with pulmonary capillaritis and diffuse alveolar hemorrhage. Scandinavian Journal of Rheumatology 27: 230232. Lauque D, Cadranel J, Lazor R, et al. (2000) Microscopic polyangiitis with alveolar hemorrhage: a study of 29 cases and review of the literature-Groupe dEtudes et de Recherche sur les Maladies Orphelines Pulmonaires (GERM O P). Medicine (Baltimore) 79: 222233. Leatherman JW (1988) The lung in systemic vasculitis. Seminars in Respiratory Infections 3: 274288. Murray RJ, Albin RJ, Mergner W, and Criner GJ (1988) Diffuse alveolar hemorrhage temporally related to cocaine smoking. Chest 93: 427429. Nadrous HF, Yu AC, Specks U, and Ryu JH (2004) Pulmonary involvement in HenochScho nlein purpura. Mayo Clinic Proceedings 79: 11511157. Pastores SM, Papadopoulos E, Voigt L, and Halpern NA (2003) Diffuse alveolar hemorrhage after allogeneic hematopoietic stem-cell transplantation: treatment with recombinant factor VIIa. Chest 124: 24002403. Schwarz MI and Brown KK (2000) Small vessel vasculitis of the lung. Thorax 55: 502510. Schwarz MI and Fontenot AP (2004) Drug-induced diffuse alveolar hemorrhage syndromes and vasculitis. Clinics in Chest Medicine 25: 133140. Schwarz MI, Mortenson RL, Colby TV, et al. (1993) Pulmonary capillaritis: the association with progressive irreversible airow limitation and hyperination. American Review of Respiratory Diseases 148: 507511. Schwarz MI, Zamora MR, Hodges TN, et al. (1998) Isolated pulmonary capillaritis and diffuse alveolar hemorrhage in rheumatoid arthritis and mixed connective tissue disease. Chest 113: 16091615. Segal SL, Lenchner GS, Cichelli AV, et al. (1988) Angiosarcoma presenting as diffuse alveolar hemorrhage. Chest 94: 214216. Specks U (2001) Diffuse alveolar hemorrhage syndromes. Current Opinion in Rheumatology 13: 1217. Travis WD, Colby TV, Lombard C, and Carpenter HA (1990) A clinicopathologic study of 34 cases of diffuse pulmonary hemorrhage with lung biopsy conrmation. American Journal of Surgical Pathology 14: 11121125. Zamora MR, Warner ML, Tuder R, and Schwarz MI (1997) Diffuse alveolar hemorrhage and systemic lupus erythematosus: clinical presentation, histology, survival, and outcome. Medicine (Baltimore) 76: 192202.

Further Reading
Afessa B, Cowart RG, and Koenig SM (1997) Alveolar hemorrhage in IgA nephropathy treated with plasmapheresis. Southern Medical Journal 90: 237239. Afessa B, Tefferi A, Litzow MR, and Peters SG (2002) Outcome of diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. American Journal of Respiratory and Critical Care Medicine 166: 13641368. Agusti C, Ramirez J, Picado C, et al. (1995) Diffuse alveolar hemorrhage in allogeneic bone marrow transplantation: a postmortem study. American Journal of Respiratory and Critical Care Medicine 151: 10061010. Bar J, Ehrenfeld M, Rozenman J, et al. (2001) Pulmonaryrenal syndrome in systemic sclerosis. Seminars in Arthritis and Rheumatism 30: 403410. Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583592. vii. Dhillon SS, Singh D, Doe N, et al. (1999) Diffuse alveolar hemorrhage and pulmonary capillaritis due to propylthiouracil. Chest 116: 14851488. Dweik RA, Arroliga AC, and Cash JM (1997) Alveolar hemorrhage in patients with rheumatic disease. Rheumatic Diseases Clinics of North America 23: 395410. Dweik RA and Stoller JK (1999) Role of bronchoscopy in massive hemoptysis. Clinics in Chest Medicine 20: 89105. Franks TJ and Koss MN (2000) Pulmonary capillaritis. Current Opinion Pulmonary Medicine 6: 430435. Gertner E (1999) Diffuse alveolar hemorrhage in the antiphospholipid syndrome: spectrum of disease and treatment. Journal of Rheumatology 26: 805807. Green RJ, Ruoss SJ, Kraft SA, et al. (1996) Pulmonary capillaritis and alveolar hemorrhage: update on diagnosis and management. Chest 110: 13051316. Henke D, Falk RJ, and Gabriel DA (2004) Successful treatment of diffuse alveolar hemorrhage with activated factor VII. Annals of Internal Medicine 140: 493494. Ioachimescu O (2003) Idiopathic pulmonary hemosiderosis in adults. Pneumologia 52: 3843. Ioachimescu OC, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12: 1625. Ioachimescu OC, Sieber S, and Kotch A (2004) Idiopathic pulmonary hemosiderosis revisited. European Respiratory Journal 24: 162170. Jennings CA, King TE Jr, Tuder R, Cherniack RM, and Schwarz MI (1997) Diffuse alveolar hemorrhage with underlying

Alveolar Proteinosis

see Interstitial Lung Disease: Alveolar Proteinosis.

ALVEOLAR SURFACE MECHANICS 101

ALVEOLAR SURFACE MECHANICS

S B Hall and S Rugonyi, Oregon Health & Science University, Portland, OR, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The major component of the recoil forces that tend to deate the lungs is the surface tension of a thin liquid layer that lines the alveoli. This surface tension is well below the value for a clean air/water interface, indicating the presence of a surfactant. The surface tensions in situ specify that the surfactant lms must have certain characteristics. Following large expansions of the air/water interface during deep inhalations, the surfactant must adsorb rapidly to form the interfacial lm. When compressed by the shrinking surface area during exhalation, the lms must be sufciently rigid to resist the tendency to collapse from the interface. Materials washed from the lungs show that pulmonary surfactant is a mixture containing mostly lipids with some proteins that is synthesized and secreted by the type II pneumocyte. The disorder in which an abnormality of pulmonary surfactant most clearly plays a role is the respiratory distress syndrome of premature babies, although altered surfactant function may also contribute to the acute respiratory distress syndrome that occurs in patients of all ages.

Description
The importance of alveolar surface mechanics is readily evident from the pressurevolume (PV) characteristics of the lungs. Pressures required to maintain any given volume are substantially higher for lungs inated with air than with saline. The fundamental difference between the two procedures is that saline eliminates an air/water interface, the surface tension of which contributes to the inward recoil forces for lungs inated with air. The presence of

surface tension implies that the curved surfaces of the pulmonary airspaces are lined by a layer of liquid. Electron microscopy has demonstrated that such a layer coats the alveoli, and that it is thin, with an average thickness of 0.2 mm, and continuous. The surface tension resulting from the air/water interface of this alveolar lining represents the major component of contractile forces in the lungs. Surface tension results from an imbalance of forces on molecules close to an interface between two separated phases. For molecules deep within a substance, attractive forces towards neighboring constituents are equal in all directions (Figure 1). Within a few molecular diameters of the interface, however, components experience a reduced attraction towards constituents in the adjoining phase, resulting in a net inward pull and a force per unit length, or surface tension, that tends to contract the interfacial area. The force along a curved surface, such as in the alveoli, translates directly into a difference in pressures across the interface. For a spherical interface with radius R, mechanical equilibrium between surface tension, s, and the difference in pressures, p p0, occurs (Figure 2) when pR2 p p0 2pRs which leads directly to the law of Young and Laplace for a sphere: Dp 2s=R Ination of lungs with air therefore requires higher pressure to overcome interfacial forces that are absent during ination with saline.

Air Water

Figure 1 Origin of surface tension: molecules in the bulk water experience an attractive force towards neighboring molecules that is equal in all directions, resulting in no net force. Within a few molecular diameters of the interface, however, the absence of neighbors towards the surface results in a net inward pull that tends to shrink the interfacial area. A lm of surfactant at the interface tends to spread, resulting in an opposing force that lowers surface tension.

102 ALVEOLAR SURFACE MECHANICS

p0 p R

Figure 2 Relationship of surface tension to hydrostatic pressure across a spherical surface. Mechanical equilibrium occurs when the inward recoil force of surface tension (s) and the opposing force of hydrostatic pressure (p p0) are equal. At the midsection of a spherical bubble with radius R, surface tension will produce a force (length s) 2pRs, and the force from hydrostatic pressure will be [area (p p0)] pR2(p p0). The equality leads directly to the law of Young and Laplace, Dp 2s/R.

Several methods have been used to determine surface tension in the lungs. The difference in PV curves between air- and saline-lled lungs can provide surface tensions, either with simple assumptions concerning alveolar geometries and tissue forces, or with an energetic analysis that makes those assumptions unnecessary. Rinsing the lungs with a series of detergents or liquids provides an air/liquid interface with constant known surface tensions, and the intersection of PV curves from these and normal lungs indicates points of common surface tension. Fluorocarbon droplets deposited on the interfacial lm in peripheral alveoli spread according to the relative surface tensions of the uorocarbon and lm, and the shape of droplets containing different materials has provided the most direct estimates of surface tensions in situ. These different approaches have produced remarkably consistent results. During excursions through large volumes, surface tension shows a major hysteresis between ination and deation that explains the hysteresis of the PV mechanics. Deation from total lung capacity to functional residual capacity lowers surface tension from B30 mN m 1 to o5 mN m 1. Over tidal volumes, surface tension varies between 10 mN m 1 and values as low as 1 mN m 1. These surface tensions are well below the values for a clean air/water interface, which would be 70 mN m 1, and indicate the presence of a surfactant. Surfactants are the general class of compounds that have higher concentrations at the surface than in the aqueous medium. They are amphipathic, with distinct hydrophilic and hydrophobic regions of the molecule, and best satisfy the energetics of both portions by orientation across an interface. Once trapped within the two-dimensional surface, surfactants tend to spread, resulting in a force that expands the interface and opposes surface tension. Surfactant lms with higher densities produce larger reductions in surface

tension. The very low surface tensions in the lungs indicate lms with particularly high densities. Surfactants can lower surface tension by adsorbing to an interface only to a limited extent. Adsorbed surfactants reach a maximum density, above which further constituents form a three-dimensional bulk phase at the interface rather than adding to the twodimensional lm. Surface tensions in the lungs reach values well below this minimum equilibrium value, and they also vary with volume. These observations indicate that the low surface tensions and high densities of the lms result not from insertion of more constituents, but from a decrease in surface area during deation. The low surface tensions reached during deation are impressively stable in static lungs for prolonged periods. The low magnitude and particularly the stability of the surface tensions indicate that the compressed lms have specic characteristics. When compressed above the maximum equilibrium density, lms that can ow from the surface will collapse to form the three-dimensional bulk phase, thereby reestablishing equilibrium surface tensions. Only lms that are solid, dened by their inability to ow, can resist the tendency to collapse and demonstrate the behavior observed in the lungs. When compressed sufciently, even solid lms collapse. An interface without surface tension has no basis for existence, and at sufciently high densities, solid lms must also rupture. The hysteresis of surface tension, and of hydrostatic pressures, between deation and ination over large volumes reects at least partially material lost from the interface during collapse. Lavaging the lungs produces the increase in recoil pressures expected from removal of a surfactant and a resulting increase in surface tension. Material recovered from the lungs can form lms with characteristics indicated by the in situ measurements. Material obtained from alveolar foam, when suspended in saline, can form small bubbles that persist for prolonged periods, indicating that the pressuredifference across their surface, which would tend to dissolve the gas in the surrounding liquid, must be low, and that according to the law of Young and Laplace, surface tension must also be small. When compressed in vitro by changing their surface area, lms formed from lavaged material can reach and sustain surface tension below the minimum equilibrium value. Material puried from lavaged material can also restore the PV mechanics of the original lungs. These observations provide the basis for the identication of the surfactant in the lungs. Material recovered by lavage contains two sets of phospholipid vesicles that differ in size. The larger

ALVEOLAR SURFACE MECHANICS 103

Air Molecular view Lamellar body Tubular myelin Surfactant film

Liquid layer

Type I cell

Small vesicles

Type II cell

Figure 3 Schematic of pulmonary surfactant in the alveolus. Constituents of pulmonary surfactant are synthesized in type II pneumocytes and assembled into lamellar bodies, which are secreted into a thin liquid layer that lines the alveolus. The vesicles unravel and rst form tubular myelin, which appears to represent the immediate precursor of a lm at the air/water interface. Lavage of the lungs recovers both the large multilamellar vesicles, which contain mostly phospholipids with small amounts of cholesterol and proteins, and small unilamellar vesicles, which contain only the lipids.

form has the same morphological appearance as a subcellular organelle, the lamellar body, of the type II pneumocytes (Figure 3). Organic extracts of these larger particles can restore the PV mechanics of lavaged lungs, which perhaps best denes these vesicles as pulmonary surfactant. The smaller particles may represent material excluded from the interface during compression to very low surface tensions. Vesicles of both sizes contain the same set of phospholipids with small amounts of cholesterol. The larger particles are much more capable of lowering surface tension in vitro than the smaller vesicles. Four proteins copurify with the larger particles but are absent from the smaller forms. The organic extracts of the large particles, which function well in lavaged lungs, lack surfactant proteins SP-A and SP-D, and based on a variety of assays, their primary function appears related to processes other than the lowering of surface tension. SP-B and SP-C, which are sufciently hydrophobic to extract with the lipids into organic solvents, determine the difference in surface activity between the large and small particles.

performance of lms in the lungs has been difcult, and the mechanisms by which pulmonary surfactant functions in the alveoli remain the subject of active investigation.
Adsorption

Normal Physiological Processes

To achieve the surface tensions observed in the lungs, pulmonary surfactant must satisfy conicting constraints. Vesicles must rst adsorb rapidly to form the interfacial lm. Pulmonary mechanics become normal during the rst few breaths following the initial air-ination of uid-lled lungs, suggesting that adsorption to the newly created air/water interface forms a lm having the equilibrium density within seconds. The low surface tensions reached during deation indicate that the compressed lm avoids the reverse process of desorption to re-establish the equilibrium density. Replicating in vitro the full

The adsorption of pulmonary surfactant is fundamentally different from the process for other more common surfactants, which insert into the interface as individual molecules. Surfactants share the general characteristic that they exist in solution as individual monomers only up to a certain concentration, above which they aggregate into structures such as micelles and bilayers (Figure 4), the nature of which depends on the effective shape of the particular molecule. The critical micelle concentration at which phospholipids aggregate is less than 10 9 M. Constituents of pulmonary surfactant therefore exist in the alveolar lining exclusively as vesicles, which insert as collective units into the interface. Because the vesicles themselves are stable in aqueous medium, an energy barrier limits adsorption, which occurs quite slowly for lipid vesicles without the hydrophobic proteins. In the alveolar lining, lamellar bodies secreted by the type II pneumocytes unravel to form a distinct intermediate structure known as tubular myelin that apparently represents the direct precursor of the interfacial lm (Figure 3). SP-A is required in vitro for the reconstruction of tubular myelin, which is absent from extracted surfactants and transgenic animals that lack SP-A. Extracted surfactants, however, function well in surfactant-decient lungs, and mice without SP-A have normal pulmonary mechanics. The functional significance of tubular myelin for adsorption is therefore unclear. The absence of SP-B, whether in transgenic animals or in patients with

104 ALVEOLAR SURFACE MECHANICS

Figure 4 Aggregation of surfactants. Above a certain concentration, the hydrophobic portion of surfactants makes them insoluble as individual molecules, and causes their aggregation into structures that expose their hydrophilic components to the aqueous medium while sequestering the hydrophobic segments. The particular form of the aggregate depends on the effective shape of the specic compounds. Single chain surfactants tend to form micelles. Biological phospholipids form bilayers that may stack into the concentric layers of a multilamellar vesicle.

genetic abnormalities, does produce abnormal pulmonary function, consistent with the crucial role suggested by in vitro studies of this protein for adsorption.
Stability of Compressed Film

Under equilibrium conditions, attempts to increase the density of surfactant monolayers above a maximum density, either by adding more constituents or by decreasing interfacial area, instead forms a threedimensional bulk phase that coexists with the twodimensional lm. The bulk phase of phospholipids is a liquid-crystal, in which layers of material stack in the regularly repeating manner characteristic of a crystal, but with each layer having the disordered structure that is characteristic of liquids. Slowly compressed monolayers of pulmonary surfactant in vitro ow into these stacked structures (Figure 5), and their ability to ow indicates that the lms are uid. In the lungs, the prolonged low surface tensions, well below the minimum equilibrium values, indicate lms that resist ow, and that therefore have the dening characteristic of a solid. The structure of two-dimensional solid lms could be either highly ordered, analogous to a three-dimensional crystal, or amorphous, like a glass. At physiological temperatures, a single constituent of pulmonary surfactant, dipalmitoyl phosphatidylcholine (DPPC), can form highly ordered lms that approach the structure of a two-dimensional crystal. DPPC has the unusual characteristic relative to other biological phospholipids that both acyl chains are fully saturated and that it constitutes an unusually large amount (3050%) of pulmonary

surfactant. A widely held view contends that the functional lm in the lung consists of essentially pure DPPC. The difference in composition between the secreted vesicles and the hypothetical functional lm of DPPC could result either from selective adsorption of DPPC or from selective collapse of other constituents. Both processes are difcult to reconcile with current understandings of how adsorption and collapse occur. One prediction of the model is that PV curves should change abruptly over a narrow range of temperatures. Films of DPPC, like three-dimensional crystals, melt from solid to uid structures at specic temperatures. At surface tensions below the minimum equilibrium value, rates of collapse increase when solid lms melt, resulting in increased surface tensions that would produce higher recoil pressures. Measurements of the temperature dependence for PV curves have yielded conicting results, and the presence of a highly ordered lm remains unconrmed. Although lms containing only DPPC would explain the stability of low surface tensions in the lungs, experimental evidence to support that possibility is limited. The solid lms that sustain low surface tensions in situ could also have a structure that resembles a two-dimensional glass. Three-dimensional liquids, if cooled fast enough and far enough below their freezing temperatures, retain their disordered structure but become frozen in place, forming amorphous solids, or glasses. Two-dimensional uid lms, dened by their ability to ow into collapsed structures, similarly become jammed into a form that resists collapse if supercompressed to sufciently high

ALVEOLAR SURFACE MECHANICS 105

Figure 5 Liquid-crystalline collapse. Under equilibrium conditions, surfactant lms reach a minimum surface tension at which they undergo a phase transition to form a three-dimensional bulk phase. Phospholipids form bulk smectic liquid crystals, and compression of uid phospholipid lms at the minimum equilibrium surface tension can cause the lm to ow into stacked structures. Solid lms, which can resist ow and remain at the interface below the minimum equilibrium surface tension, eventually also collapse from the interface at very low surface tensions, although probably by a process more like fracture.

densities and low surface tensions. These supercompressed lms retain their solid behavior and slow rates of collapse when expanded, even when returned to the surface tensions at which they originally collapsed. If they reach low surface tensions in the lungs during a single exhalation, the lms would be transformed, and their ability to avoid collapse could persist through multiple cycles of tidal breathing. To achieve low surface tensions, however, the initially uid lms must be compressed faster than they can collapse. The required rates may occur during normal breathing, but they are faster than rates in quasi-static experiments with excised lungs. The supercompressed lms, which would require no compositional change, could explain surface tensions observed in the lungs, but like the lms of pure DPPC, the process by which they would form remains unclear. Original views concerning surface tension in the lungs considered an interfacial lm with the thickness of one molecule. Although perhaps difcult to explain how they might form, monolayers that have the characteristics of lms in the lungs are well described, and more complicated structures were unnecessary to explain the observed behavior. Electron microscopy, however, has demonstrated that in situ, at least parts of the interface are occupied by lms that are multilayered. Whether these structures are formed during adsorption or collapse, and the extent to which the additional material might affect the mechanical characteristics of the lm, are both unknown.

Physiological Processes in Respiratory Diseases

The disorder in which an abnormality of pulmonary surfactant most clearly represents a major pathogenic factor is the respiratory distress syndrome (RDS) that occurs in premature babies. Ventilation of immature lungs that lack adequate amounts of surfactant injures the alveolocapillary barrier, resulting in pulmonary edema and respiratory failure. Two

mechanisms, both involving shear stresses, could explain how a deciency of pulmonary surfactant would produce the injury. First, elevated surface tension would produce an increased tendency for small alveoli to collapse, and the shear stresses involved in reopening the closed airspaces could produce the injury. Second, the meniscus of any uid column in the small airways would have an increased surface tension, and the greater pressure-difference across the interface could rupture the epithelial cells over which it passes. Elevated surface tensions would also lower interstitial and pericapillary pressures, resulting in a greater transmural pressure-difference that would increase the ow of uid across the alveolocapillary membrane. The most direct evidence that decient surfactant causes RDS comes from manipulation of surfactant levels. Subsequent to removing surfactant by lavage, ventilation of animals produces an injury that replicates RDS. Conversely, giving exogenous surfactant to premature babies at risk for RDS prevents or reverses the disorder. The acute respiratory distress syndrome (ARDS) was originally called adult RDS to point out the clinical similarities between adults with injured lungs and the infants with RDS, and to suggest that the common presentation resulting from a variety of insults might reect an abnormality of surfactant acting as the nal common pathway. Although the primary defect in ARDS is an inammatory process, abnormal surfactant might perpetuate the initial injury by the same processes that result from an elevated surface tension in RDS. Surfactant function could be altered in ARDS by either deciency or inhibition. Injured lungs have reduced levels of the large active surfactant vesicles, suggesting a deciency. Pulmonary surfactant could also be inhibited by the large number of extraneous compounds, such as plasma proteins and membrane lipids, that reach the alveolus in injured lungs and that can act as surfactants. Direct evidence for elevated surface tensions in ARDS, however, is limited. The presence of edema complicates the interpretation

106 ALVEOLAR WALL MICROMECHANICS

of PV mechanics, and the distinction of small lungs, caused by uid-lled airspaces that occur with any pulmonary edema, from stiff lungs, caused by increased surface tension, has been difcult. Evidence that exogenous surfactants can mitigate ARDS has also been lacking. The larger doses required to treat adults with ARDS relative to premature babies with RDS has limited the therapeutic agents that can be used. Initial trials with surfactants that lack SP-B have produced no improvement, just as early attempts to treat babies with RDS using aerosolized DPPC had no benet. The role of therapeutic surfactants in ARDS, and of abnormal surfactant in its pathogenesis, therefore remains unresolved.
See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Alveolar Wall Micromechanics. Breathing: Breathing in the Newborn; Fetal Lung Liquid; First Breath. Bronchoalveolar Lavage. Drug-Induced Pulmonary Disease. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Imaging. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D).

Further Reading
Bastacky J, Lee CY, Goerke J, et al. (1995) Alveolar lining layer is thin and continuous: low-temperature scanning electron

microscopy of rat lung. Journal of Applied Physiology 79: 16151628. Goerke J and Clements JA (1985) Alveolar surface tension and lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of Physiology The Respiratory System, vol. III, part 1, pp. 247 261. Washington, DC: American Physiological Society. Hoppin J, Frederic G, Joseph C, et al. (1986) Lung recoil: elastic and rheological properties. In: Fishman AE (ed.) Handbook of Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, pp. 195215. Bethesda, MD: American Physiological Society. Keough KMW (1992) Physical chemistry of pulmonary surfactant in the terminal air spaces. In: Robertson B, van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant: from Molecular Biology to Clinical Practice, pp. 109164. Amsterdam, New York: Elsevier. Lewis JF and Jobe AH (1993) Surfactant and the adult respiratory distress syndrome. American Review of Respiratory Diseases 147: 218233. Piknova B, Schram V, and Hall SB (2002) Pulmonary surfactant: phase behavior and function. Current Opinion in Structural Biology 12: 487494. Robertson B (1984) Pathology and pathophysiology of neonatal surfactant deciency (respiratory distress syndrome, hyaline membrane disease). In: Robertson B, Van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant, pp. 383418. Amsterdam: Elsevier. Schu rch S, Green FH, and Bachofen H (1998) Formation and structure of surface lms: captive bubble surfactometry. Biochimica et Biophysica Acta 1408: 180202. Stamenovic D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 11171134. Wilson TA (1981) Mechanics of the pressure-volume curve of the lung. Annals of Biomedical Engineering 9: 439449.

ALVEOLAR WALL MICROMECHANICS

F G Hoppin Jr, Brown University, Providence, RI, USA
& 2006 Elsevier Ltd. All rights reserved.

Introduction
The alveolar wall is the site of gas exchange by passive diffusion between inspired gas and the pulmonary capillary blood. Its tiny scale supports passive diffusion by providing an immense net-diffusing area and a short path length. Because it is elastically extensible, the gas-exchanging lung units can readily expand and contract during breathing. How does this diaphanous structure meet these functional needs and at the same time avoid structural weakness, congurational instability, and uid accumulation?

Abstract
The site of gas exchange in the lung is the alveolar wall. Its structure supports passive diffusion of the respiratory gases by spreading the pulmonary capillary bed over an immense surface area and by having an extremely thin air/blood barrier. It enables the alveolar gas spaces to inate and deate by its great extensibility. Yet it remains stiff enough to withstand the severe collapsing forces of surface tension acting in tiny connes and to transmit elastic tensions throughout the network of alveolar walls, maintaining by those tensions the ne and gross conguration of the lung and a reasonable distribution of ventilation within the lung. It can modulate local ventilation by activation of its smooth muscle. The mechanical properties that meet these exacting requirements can be understood in terms of the behavior of elastic networks and the elastic properties of the walls solid components and of pulmonary surfactant. Dysfunction at the level of the alveolar wall lies at the core of most disorders of the lung by impairing diffusion, ventilation, and perfusion.

Normal Mechanics
The lung, like a parachute or a spinnaker, is a tensed structure (Figure 1). Patency of the airspaces and a relatively even distribution of expansion and contraction during breathing depend on satisfactory

ALVEOLAR WALL MICROMECHANICS 107

50 m E J B

Figure 1 Photomicrograph of inated lung, showing the network of alveolar walls, partitioning the lung into alveolar gas spaces. The predominant termination of the prole of the alveolar wall is a triple junction with two other walls (J). Where alveolar gas spaces open into an alveolar duct (right top), the proles either appear bent (B) or simply end (E), and these edges of the wall are invariably reinforced with connective tissue cables, often accompanied by smooth muscle. Reproduced from Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal bend structures in lung parenchyma. Journal of Applied Physiology 81: 18001806, used with permission of The American Physiological Society.

transmission of tensions (lung elastic recoil) throughout. The evidence for the network of alveolar walls being the major tension-bearing structure in the lung include the following:
*

It contains the majority of the lungs tension-bearing material. Its tension-bearing structures are continuous throughout the lung. Distortions of the inated lung whether by gravity, local indentation of the surface, or local internal expansions or contractions behave as expected for a diffuse, isotropic elastic network. Estimates of lung elastic recoil, based on stereological data and elastic properties of the tensionbearing components of the alveolar wall are close to observed lung-distending pressure differences over a range of ination volumes.

The other tension-bearing structures, acting mechanically in parallel, include the lungs outer rind (pleura), which has been thought to account for only B20% of the work of ination, and the brous connective tissue systems of the airway and bronchovascular trees, which have been estimated to bear B10%. The network of alveolar walls is a cable/membrane structure. The membrane portion of the alveolar wall is tensed at its edges either by other walls or by an embedded cable of relatively heavy connective tissue. It is thin, polygonal, and pseudoplanar. It has three

main tension-bearing components: the ne, isotropic brous network; the basement membranes of the epithelium and capillary endothelium; and the air-liquid interfaces on either side (Figure 2). Along most of its perimeter (B66%), the wall joins two others (J in Figure 1). At this junction, there is no cable; the ne brous network, basement membranes, and air-liquid interfaces are directly continuous from one wall to the adjacent walls. The tensions of the walls at such junctions are resolved by the balance of the three walls pulling in three directions. Another mechanism is needed where the alveolar spaces open into the alveolar duct. At these locations, the wall either meets one other wall (B24% of its perimeter, B in Figure 1) or simply ends (B9% of the perimeter, E in Figure 1) and it requires some ancillary support. This is provided by relatively heavy connective tissue cables that are curved so as to oppose the walls tensions. The ne brous network of the walls connects directly with these cables. The cables of adjoining walls form a lattice that frames the alveolar duct. Centrally, their connective tissues merge with the still heavier connective tissues of the terminal bronchioles. The difference between the E and B congurations, incidentally, probably does not reect a functional difference beyond the geometric complexity of space packing of alveoli and of the branching airway system. The cables and membranes, being continuous structures throughout the network, are capable of transmitting tensions from membrane to membrane or cable to cable across the lung in any direction. However, there is also a distinct serial connection between the cables and membranes at the level of the alveolar duct and its surrounding alveoli, where the membranes pull radially against the cables. This is dramatically apparent as dilation of the ducts of lungs that have been washed with detergent to increase the walls interfacial tension. Normally, however, the elastic properties of these very different systems are sufciently matched so that expansion and contraction of the duct and its surrounding alveoli are relatively symmetrical during breathing. Lying alongside most of the cables are bundles of smooth muscle. Its function relates to its characteristic stiffening when it is activated and passively length-cycled at typical breathing frequencies. As a result of the series relationship of the alveolar duct and its surrounding alveoli (above), stiffening of the cables reduces the compliance of the respiratory unit. Indeed, the lung is substantially stiffened in response to low levels of carbon dioxide (hypocapnia). This suggests a homeostatic mechanism. Relatively under ) lung units are characteristically =Q perfused (high V hypocapnic. A local response that stiffens the lung

108 ALVEOLAR WALL MICROMECHANICS

Alveolar lining liquid Epithelial cell Basement membrane Interstitium Basement membrane Endothelial cell

Epithelial cell nucleus Fine fiber network Endothelial cell nucleus Red blood cell Plasma

Endothelial cell Fused basement membrane Epithelial cell Alveolar lining liquid
Figure 2 Schematic depiction of the structure of an alveolar wall. It is pseudoplanar and has dimensions on the order of 100 100 10 mm. In the middle is the interstitium, containing the supporting ne ber network, the marsh-like pulmonary capillary bed, and small amounts of interstitial uid. Outside the interstitium on both sides of the wall is a basement membrane supporting the single layer of thin alveolar epithelial cells and, outside that, a uid alveolar lining. The capillary itself is a single thin endothelial cell and its supporting basement membrane, fused on one side of the capillary with the basement membrane of the epithelium.

locally reduces the ventilation of such units. This hypocapnic pneumoconstriction improves the overall efciency of gas exchange, much as hypoxic pulmonary vasoconstriction reduces the perfusion of un ) units. =Q derventilated (low V Tensions in adjoining walls are quite uniform. This conclusion is drawn from a vector analysis of the tensions at J and B junctions, the assumption that the walls are free to rotate relative to each other, and data for the conguration of xed inated specimens. At J junctions, the distribution of dihedral angles between walls, extremely narrow around 1201, is consistent with B2% variation of wall tensions. At B junctions, the inferred distribution is somewhat wider, but has a notably sharp cutoff below 1201. Open soap lms supported on complex wire frames show these several features at their J and B junctions, where they are well understood to reect (1) locally uniform lm tensions and (2) a mechanism for adopting the least energy conguration, that is, minimal surface area. This nding suggests, intriguingly, that lung structure during growth and development (perhaps even during remodeling of the adult lung?) responded to these same physical principles. Local uniformity of wall

tensions, of course, does not imply global uniformity, which varies systematically over large distances in the lung, for example, vertically in the gravity eld. The wall is highly extensible, undergoing up to about twofold change in linear dimensions for the deepest breaths, that is, about eightfold change of volume. (Birds followed a different evolutionary route by having unidirectional air ow through a relatively stiff gas exchange apparatus.) The risks inherent in such extensibility include instability, collapse, and gross unevenness of ventilation among the lungs different air compartments. These risks are limited by the walls positive compliance and by the effects of network geometry. A given distortion of the elastic network stretches the walls on the side away from the displacement and realigns them closer to the direction of the displacement. As the walls are positively compliant, the effect is to increase tension on that side, and the effect of that increase is enhanced by the realignment of the walls to better oppose the displacement. The converse occurs on the side towards the displacement. This mechanism acts to stabilize conguration at the ne (alveolus) and gross (regional) levels.

ALVEOLAR WALL MICROMECHANICS 109

The walls positive compliance is conferred independently by both the airliquid interface and the solid components (ne brous network and basement membranes). Interfacial tension modulated by pulmonary surfactant varies monotonically from near 0 to B30 dynes cm 1 over a fourfold change of surface area. The ne brous connective tissues are collagen bers (stiff as steel in isolated bers) and elastin (the prototypical rubber). They are substantially entangled and cross-linked, and the compressibility of a proteoglycan matrix modies the extent to which collagen bers fold and stretch. The resulting solid components are capable of more than the about twofold linear extension, that is, of maintaining tension over the full operating range of lung volume. The tensions in the interface interact with those of the solid elements of the wall through curvature. There is no reason to postulate shear forces between them. The interface tenses the cables at E and B junctions by curvature that is convex to the airspace, that is by draping over the cable region like washing on a clothes line. At the inner corners of the alveolus (J junctions and the inner side of the B junctions), by contrast, the curvature of the interface is sharply concave to the airspace, and therefore interfacial tension reduces the pressure in (i.e., sucks out on) the junctional tissues, transmitting tension to the cable and/or other septae at that junction. In lungs prepared in protocols that permit collapse, the freedom to shear permits the solid elements of the wall to pleat or fold beneath a smoothly curved interface in the corners of alveoli. This is probably not the conguration in vivo. Transiently low distending pressures (o23 cmH2O) permit folding. High distending pressures (416 20 cmH2O) are required to resolve the folds. With the appropriate protocols, the walls ne ber network remains quite planar right into the corners of the alveoli even down to 23 cmH2O. This is consistent with the capability of the ne ber network to bear tension over the full feasible range of lung ination, and with its roles of supporting the capillary network and of maintaining the conguration of the alveolus, particularly at low ination volumes. The pulmonary capillaries are notably affected by the mechanical state of the wall. The majority of the capillary bed lies within the at portions of the wall. There, the capillary distending pressure (the difference between intracapillary and alveolar gas space pressures) is in equilibrium with the combined opposing forces of intervening structures, namely the basement membranes of the capillary and alveolar epithelium, the ne ber network, and the airliquid interface. The effects are evident in the prominent bulging of the pulmonary capillaries into the alveolar space when hydrostatic capillary pressure increases

or interfacial tension is reduced (e.g., in the salinelled lung). Conversely, the planar forces of the walls solid structures and interfacial tension atten the capillaries when the wall is stretched at high lung volumes. By contrast, the vessels in the corners, for example, within the J junctions, where the curvatures of the solid structures and interface are concave to the alveolar space, are distended at high lung volumes, and may remain open even when the vessels within the at portions are compressed. Under those conditions, bloodow in such regions is not completely eliminated and there is gas exchange evidence compatible with very low perfusion units. Fluid balance is also impacted by mechanical factors, again with different mechanisms in the at portions of the wall and the corners. Throughout the body, the volume of uid in the interstitial spaces is controlled by the balance between hydrostatic and osmotic factors across the capillary walls (Starling equilibrium). The lung, however, is a special case because of the powerful forces that develop when interfacial tension acts on very tight curvatures. In particular, the tissue pressure in the alveolar corners (J junctions) is lowered. This may be benecial by helping to draw interstitial uid from the at portions of the alveolar wall into the corners, whence it may be cleared by the lymphatics. But it is also counterproductive insofar as it opposes the uid-absorptive forces across the microvascular walls and inhibits lymphatic drainage. Of greatest concern, it can lead to uid accumulation within the alveolus. Ordinarily, a simple mechanical negative-feedback loop limits the accumulation of uid any increase of alveolar uid initially decreases the curvature of the interface, reducing the pressure-lowering effect of interfacial tension and thereby favoring the return of uid to the capillaries and lymph. If, however, the uid gathers to the point that the corners of the alveolar space are lled, the feedback loop reverses; any further gathering of uid increases the curvature, leading to further alveolar lling or collapse. This problem is substantially mitigated by the interfacial tension-lowering effect of pulmonary surfactant, which has enabled evolution of alveolar walls at a much smaller scale than would otherwise have been possible, providing an immense surface area for gas exchange within a reasonably sized chest.

The Mechanical Role of the Alveolar Wall in Respiratory Diseases

Mechanical dysfunction of the alveolar wall is at the core of most major acute and chronic lung diseases, as readily predictable from the above discussion and seen in the following examples.

110 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

The essence of emphysema is loss of alveolar walls. This impairs gas exchange by reducing surface area of the pulmonary capillary bed and increasing the path lengths for passive diffusion. It also relaxes the network of alveolar walls. This has the effect of reducing the dilating effects of the network on the embedded and extrapulmonary airways and blood vessels. Narrowing of the airways increases their airow resistance and forces the subject to breathe at disadvantageously high chest wall volumes in order to mount sufcient elastic tensions to hold the airways open. Inhomogeneity of damage to the network of alveolar walls and its effects on airway and blood vessel calibers cause substantial maldistribution of ventilation and perfusion, itself a major cause of inefciency of gas exchange. A range of mechanical dysfunctions at the level of the alveolar wall permits blood to shunt through the lung without exchanging gas. Fluid can gather (pulmonary edema) secondary to the high hydrostatic pressures of heart failure, or even in normal individuals at very high altitudes. Toxic, infectious, or immunological entities can allow excessive uid or protein leakage from damaged capillaries. Airspaces can collapse (atelectasis) from local underination and gas absorption, from dysfunctional surfactant, from trauma, or from chest wall dysfunction. High distending forces (e.g., deep breaths) are required for reopening. Repeated opening and closing of airspaces in a patient breathing with ventilator support can have its own direct physical and inammatory consequences. Pulmonary brosis due to a wide range of causes can thicken and stiffen the alveolar wall, impairing

gas exchange and placing a mechanical load on breathing.

See also: Alveolar Surface Mechanics. Atelectasis. Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General. Diffusion of Gases. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matrix Proteoglycans. Fluid Balance in the Lung. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Lung Development: Overview. Myobroblasts. Occupational Diseases: Overview. Pulmonary Fibrosis. Smooth Muscle Cells: Airway. Stress Distribution in the Lung. Surfactant: Overview. Ventilation: Uneven.

Further Reading
Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal bend structures in lung parenchyma. Journal of Applied Physiology 81: 18001806. Greaves IA, Hildebrandt J, Hoppin FG Jr (1986) Micromechanics of the lung. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 217231. Bethesda: American Physiological Society. Hoppin FG Jr, Stothert JC Jr, Greaves IA, Lai Y-L, Hildebrandt J (1986) Lung recoil: elastic and rheological properties. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 195215. Bethesda: American Physiological Society. Mead J (1961) Mechanical properties of lungs. Physiological Reviews 41: 281330. Stamenovic D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 11171134. West JB (2003) Thoughts on the pulmonary bloodgas barrier. American Journal of Physiology. Lung Cellular and Molecular Physiology 285: L501L513.

Amyloidosis

see Interstitial Lung Disease: Amyloidosis.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

P A DAmore, Harvard Medical School, Boston, MA, USA M K Sakurai, Boston Childrens Hospital, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved. gas exchange across a thin blood gas barrier. This network of blood and air conduits begins to form in early gestation and continues through early childhood, requiring intricate epithelialmesenchymal interactions that are regulated by various angiogenic and growth factors. Disruptions in this complex system lead to inadequate gas exchange and diseases of respiratory distress. At this time, the precise roles of these growth factors and interactions between the epithelial and mesenchymal cells are poorly understood. A better understanding of these processes may lead to improved therapies for various respiratory diseases.

Abstract
Angiogenesis is crucial in pulmonary development to establish the groundwork structure for the lungs main function of

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 111

Introduction
The basic function of the lung is gas exchange, and efcient gas exchange requires a thin airblood interface. Hence, epithelialmesenchymal interactions are crucial to the formation of a blood gas. Lung development, which begins in early gestation and continues into early childhood, requires complex interactions that are regulated by a number of angiogenic and growth factors. Although it is not whether mesenchymal development drives epithelial growth or vice versa, several factors have been identied as important for the development of a functional blood gas barrier. Any disruption to this intricate and highly orchestrated process can cause ineffective gas exchange with resulting respiratory distress. Further knowledge of the factors inuencing pulmonary development may lead to therapeutic interventions for various respiratory diseases.

General Embryology
Lung development is divided into ve stages, starting in early gestation and ending postnatally in early childhood. Basic organogenesis occurs during the initial embryonic phase. The next three phases pseudoglandular, canalicular, and saccular describe the morphogenesis of primitive lung parenchyma during those stages. The nal alveolar stage extends into childhood. Concurrent with mesenchymal development, the lung vasculature develops from mesodermal cells by a combination of vasculogenesis and angiogenesis. Vasculogenesis is the process of de novo blood vessel development from mesenchymal precursor cells, whereas angiogenesis involves new vessels sprouting from existing vasculature. Initially, the blood lakes, formed by vasculogenesis, create a capillary plexus and connect to drain into the pulmonary veins. Eventually, the pulmonary arteries bud off the aortic arches by angiogenesis and grow into the mesenchyme where they anastamose with this capillary plexus. This vascular plexus surrounds the developing airways. The time frame of these phases overlaps, given the independent development of each lobe. During the embryonic stage, the initial lung buds originate from ventral diverticuli of the primitive foregut and proliferate into the surrounding mesenchyme. These buds undergo branching morphogenesis to create a complex network of airways that are lined with columnar epithelium. Simultaneously, vascular lakes of hematopoietic cells that have formed by vasculogenesis appear in the mesenchyme. Neither the pulmonary artery nor veins have formed at this time. At rst the capillaries only drain into

systemic veins but eventually connect to the pulmonary veins that arise from the primitive cardiac atrium during the pseudoglandular phase. Bronchial tree formation and acinar structure development at approximately 517 weeks into gestation mark the pseudoglandular stage. Epithelial mesenchymal interactions determine and regulate the branching patterns. Transplantation experiments have demonstrated that the mesenchyme directs the growth and branching of epithelial tubules forming the conductive airways. In vitro experiments have shown that removing the mesenchyme from the tip of a budding epithelial tube prevents further branching and that transplanted mesenchyme can induce new budding. During the pseudoglandular stage, differentiation of the cells comprising the airway walls occurs proximal to distal. These cells become ciliated, nonciliated, goblet, and basal epithelial cells. At the distal ends, cuboidal epithelial cells line the forming acini, the mesenchyme begins to thin, and a network of capillaries develops. During this stage, the venous system connects to the heart. Later in the pseudoglandular phase, the pulmonary artery buds from the aorta and primitive arterial branches begin to develop alongside the airways. At this point, the basic airway pattern has been established. The canalicular stage at gestational weeks 1626 encompasses acini and pulmonary vasculature development, as well as the initiation of surfactant synthesis. At the crucial gas-exchange region, acini development involves widening of the peripheral tubules, further thinning of the mesenchyme and increased vascularization. The cuboidal epithelium thins to form the blood gas barrier and differentiates into type I and type II pneumocytes. The type II pneumocytes initiate surfactant production. During the saccular stage, which occurs during 2438 weeks of gestation, continued expansion of the air spaces occurs and is accompanied by increased vasculature and decreased interstitial tissue as the lung prepares for its main function of gas exchange. At this phase, the airways end in thin-walled terminal sacs, hence the name saccular stage. The vessels continue to grow and elongate. With the lessening of interstitial tissue, the airspaces begin to approach one another and a capillary bilayer is formed in the intersaccular septa. The surfactant system continues to mature in preparation for the function of gas exchange. The alveolar stage starts at the end of gestation and continues for 12 years postnatally. Less than 10% of alveoli are present at birth and a majority of alveolar formation occurs via the process of septation after birth. The walls of the airspaces are called primary septa and contain a distinct double capillary

112 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

network. Secondary septa form to partition the saccules and form alveoli. This process increases the alveolar surface area and thus the gas-exchange area. These thick septa transform into a thin epithelial endothelial cell lining via a process that is not well understood. The exact mechanisms that underlie the ve stages of lung development have yet to be elucidated; however, various angiogenic growth factors and developmental factors have been demonstrated to play important roles in this process.

Angiogenic Growth Factors, Developmental Factors, and Their Roles

Epithelialmesenchymal interactions are crucial in the formation of a thin blood gas interface during lung development. Multiple angiogenic and growth factors interact throughout the ve stages of pulmonary development. A variety of in vivo and in vitro techniques have been used to elucidate the role of particular growth factors in this process. These studies point to the importance of maintaining a delicate balance of growth factors during the process of pulmonary development.
Vascular Endothelial Growth Factor

The tight regulation of VEGF presumably contributes to the formation and maintenance of a normal blood gas barrier. Experiments involving transgenic mice have revealed the importance of precise regulation of VEGF expression. Targeted disruption of VEGF expression results in embryonic mortality, and inactivation of just a single VEGF allele causes abnormal blood vessel formation, leading to early embryonic lethality. Furthermore, overexpression of VEGF by the respiratory epithelium produces abnormal vascular formation, which also leads to disruption of lung morphology and embryonic lethality. Hypoxia increases VEGF production by lung epithelial cells, whereas hyperoxia depresses the expression of VEGF as well as its receptors.
Platelet-Derived Growth Factors

Vascular endothelial growth factor (VEGF) is a wellestablished angiogenic factor. Differential splicing of a single gene generates three distinct VEGF isoforms in the mouse and at least ve in humans. VEGF acts to stimulate the proliferation and migration of vascular endothelium as well as to increase vascular permeability. In the lung, VEGF is expressed by type II pulmonary epithelial cells and localizes to the basement membrane. An analysis of the various VEGF isoforms in the mouse revealed that the lung produces primarily VEGF188. This isoform includes two domains, encoded by exons 6 and 7, which are highly charged and bind with high afnity to the heparan sulfate, thus accounting for the basement membrane localization. This specic localization is postulated to be important for inducing capillary development. VEGF interacts with tyrosine kinase receptors to exert its cellular functions. These receptors (VEGFR1 or Flt-1; VEGFR2 or Flt-2 and VEGFR3) are localized on endothelial cells. Recent studies have revealed that they are also expressed by a variety of nonendothelial cell types. VEGF expressed by the pulmonary epithelium induces vascular development via the VEGFR2 on the endothelial precursor cells. Alterations in VEGF expression lead not only to abnormal vasculature but also to abnormal lung morphology. Additional evidence indicates that VEGF is important for maintenance of newly formed blood vessels as well.

Platelet derived growth factor (PDGF) is composed of dimers of A and/or B chains and acts via two tyrosine kinase receptors PDGFR-a and -b. Early in gestation, PDGF protein localizes to airway epithelial cells as well as to mesenchymal cells. PDGF is produced by the epithelium and interacts with PDGF receptors (PDGFRs) on interstitial mesenchymal cells. Expression of PDGF appears to change throughout gestation, increasing during the pseudoglandular phase with minimal production by the saccular stage. The localization of PDGF as well as the gestation-dependent expression pattern indicates an important role during lung development. PDGF is also involved in the formation of the vessel wall. PDGF B produced by immature endothelial cells acts as a mitogen and chemoattractant for undifferentiated mesenchymal, recruiting them to the nascent vessel. Upon contact with the endothelium, latent transforming growth factor beta (TGF-b) is activated. The activated TGF-b induces the differentiation of the mesenchymal to become smooth muscle cells/pericytes. It also acts upon the endothelial cells to inhibit their proliferation and migration, and it induces the production of basement membrane. Taken together, these factors lead to the formation of a stable, mature vasculature. PDGF appears to regulate the generation of myobroblasts. The development and analysis of PDGF A and PDGF B knockout mice has revealed a role for PDGF in lung development. Homozygous PDGF A null mutants do not develop alveolar myobroblasts and therefore have reduced elastin deposition, resulting in abnormal alveolarization. Failure of alveolar septation leads to emphysema in the surviving mutant mice. PDGF B-decient mice are embryonic lethal, due to generalized hemorrhage and edema, most likely secondary to defective blood wall formation.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 113

Precise regulation of PDGF levels during each phase of development is also important. Overexpression of PDGF A by the respiratory epithelium causes overgrowth of the mesenchyme and results in death from abnormal lung architecture.
Transforming Growth Factor Beta

The TGF-b family of cytokines regulates cell proliferation, differentiation, recognition, and death via serine/threonine kinase activity receptors. The TGF-b1, TGF-b2, and TGF-b3 have been identied in the developing lung where they have been shown to stimulate extracellular matrix deposition by lung broblasts and inhibit epithelial cell proliferation. Spatial and temporal patterns of expression during lung development suggest a role for TGF-b in branch morphogenesis. Both mesenchymal and epithelial cells of the primitive lung express TGF-b1. TGF-b2 expression localizes to the tips of developing bronchioles, while TGF-b3 has been demonstrated in the proximal respiratory tract epithelium as well as the terminal growing buds. Signaling via the TGF-b type I receptor (TGFbRI) directs the formation of branch points while activation of the TGF-b type II receptor (TGFbRII) exhibits an inhibitory effect. The inhibitory effect of TGF-b on lung branching is mediated by the stimulation of the production and deposition of extracellular matrix proteins and inhibition of production of collagenase and proteolytic enzymes. Control over these molecules allows TGF-b to modulate lung branching. In culture, TGF-b1 and TGF-b2 decrease lung explant size and inhibit branching. TGF-b3 null mice, which die at birth, have delayed lung development and exhibit cleft palate as well as pulmonary epithelial, mesenchymal, and vascular dysplasia. In contrast to TGF-b3-decient mice, TGF-b1 knockout mice have normal lung development but die by 1 month of age from fulminant pulmonary inammatory inltrates. The normal lung development has been attributed either to functional redundancy provided by the other TGF-b isoforms or to rescue via transplancental transfer of maternal TGF-b. Overexpression of TGF-b1 results in neonatal lethality and hypoplastic lungs, with decreased saccular formation and epithelial differentiation. Conversely, inhibition of TGFbRIIs stimulates lung development.
Insulin-Like Growth Factors

IGF-IR and IGF-IIR. IGF-IR is a tyrosine kinase receptor, whereas IGF-IIR is a cation-independent mannose 6-phosphate receptor. In addition, IGF binding proteins (IGFBPs), which modulate IGF function, have been identied. IGF-I, IGF-II, and IGF-IR have been localized during early gestation to endothelial cells lining the primary vascular plexus, suggesting a role for these factors in pulmonary vascular development. Studies suggest that IGFs may function as survival factors during pulmonary vascular development. In vitro experiments involving the treatment of fetal lung explants with IGF-IR inhibitors exhibit not only a reduction in endothelial cell number but also an increase in mesenchymal cell apoptosis. In transgenic mice, disruption of the IGF-IR gene is lethal postnatally from respiratory failure. Lungs in these mutants appear hypoplastic although no gross defect in lung architecture has been observed. Other studies have suggested that VEGF may act downstream of IGF-1 in vascular development. IGF is important in pulmonary development as a survival factor perhaps via signaling through other growth factors.
Epidermal Growth Factor and Transforming Growth Factor Alpha

Insulin-like growth factor-I ( IGF-I) and Insulin-like growth factor-II (IGF-II) are peptides similar in structure to proinsulin. These growth factors modulate cell proliferation and differentiation via two receptors,

Epidermal growth factor (EGF) and TGF-a are members of the EGF family that act through the EGF receptor (EGFR). EGF and TGF-a are produced by the mesenchyme and appear to act on the EGFR located on pulmonary epithelial cells as well as in neighboring mesenchyme. The source of these factors and the localization of EGFR indicate an important role for EGF and TGF-a in epithelial mesenchymal interactions. EGF is also expressed by alveolar epithelial cells and regulates type 2 cell proliferation in an autocrine manner. Both EGF and TGF-a affect growth and branching of the pulmonary tree. In vitro experiments demonstrate that administration of exogenous EGF increases cellular proliferation as well as branching, whereas inhibition of EGF leads to decreased branching in explants. Targeted disruption of TGF-a demonstrates no adverse effects, most likely due to the functional redundancy provided by EGF. However, disruption of EGFR results in respiratory distress, leading to neonatal mortality. The lungs of these mutants exhibit septal thickening, decreased branching, decient alveolarization, and reduced epithelial differentiation. Overexpression in the lung of TGF-a disrupts alveolar development and causes brotic lesions. Clearly the IGFs have an important role in epithelialmesenchymal interactions during lung development.

114 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS Fibroblast Growth Factors

The broblast growth factors (FGFs) are a family of proteins that mediate a variety of processes, including cell proliferation, differentiation, cell angiogenesis, and development. These growth factors interact with specic tyrosine kinase receptors to mediate their effects. Binding to heparin sulfate proteoglycans on cell surfaces and within the extracellular matrix potentiates the effects of some FGFs. Specic FGFs and FGF receptors (FGFRs) regulated temporally and spatially during lung development implicate them in epithelialmesenchymal interactions. Abnormal FGF expression and signaling during pulmonary development lead to anomalous epithelial branching and differentiation. FGF-10 and keratinocyte growth factor (KGF or FGF-7) induce pulmonary epithelial proliferation and branching. FGF-10 regulates patterning by chemotaxis, whereas KGF inuences patterning by promoting epithelial growth. FGF-10 null mice display perinatal mortality from aberrant lung development. Disruption of FGFR2 expression results in pulmonary atresia distal to the main stem bronchi. Interruption of FGFR3 and FGFR4 signaling leads to excess elastin deposition, aberrant alveolization, and postnatal lethality. Transgenic overexpression of KGF results in pulmonary malformations that resemble cystic adenomatoid malformations. The function of KGF appears to overlap with other factors since KGF knockout mice have no gross pulmonary abnormalities. FGF signaling is critical not only for lung development but for postnatal repair following lung injury as well.

growth due to the loss of opposition to the lungs inherent tendency to collapse. Intrusion of the abdominal viscera into the thoracic cavity is a secondary event that may further hinder lung growth and development. In addition to being small in size, the lungs in CDH are also immature. Despite timely repair of the diaphragmatic defect, many infants suffer postnatally from respiratory compromise due to underdeveloped lungs. Survival and long-term outcome is dependent on the severity of pulmonary hypoplasia, the extent of bronchopulmonary dysplasia from ventilatory injury, and other associated anomalies. Lungs from patients with pulmonary hypoplasia associated with CDH exhibit significantly decreased number of airways, decreased vessel density, and more muscularized arteries. A murine model phenotypically similar to CDH has been developed using nitrofen exposure. Lungs from nitrofen-treated mice have decreased levels of VEGF, which may contribute to their immature state. Nitric oxide, important in the regulation of smooth muscle cell proliferation, and nitric oxide synthase are also reduced. The decreased production and diffusion of nitric oxide may contribute to the muscularized arteries, which in turn lead to pulmonary hypertension. The possibility of growth-factor-induced pulmonary growth is currently under investigation in hopes of a therapeutic intervention for CDH.
Bronchopulmonary Dysplasia

Diseases and Therapeutic Considerations

Pulmonary development is a complicated system involving multiple growth factors and extensive epithelialmesenchymal signaling. Any disruptions in this system can lead to inadequate gas exchange and diseases of respiratory distress. Both the vascular and airway development are affected in diseases with abnormal lung formation such as pulmonary hypoplasia. A better understanding of these developmental processes may elucidate the mechanisms underlying various respiratory diseases and thus lead to improved therapies.
Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a condition that affects approximately 1 in every 30005000 infants and is one of the most common causes of fetal hypoplastic lungs. In CDH, improper diaphragmatic development leads to abdominal content herniation into the thoracic cavity. This results in impaired lung

Bronchopulmonary dysplasia (BPD), also known as neonatal chronic lung disease, is an important cause of respiratory illness, especially in preterm infants. In BPD, acute injury of the lung may be caused by multiple factors, including pulmonary oxygen toxicity, barotrauma from mechanical ventilation and cellular immaturity. BPD is more common among premature infants because of higher susceptibility of the immature lungs to injury. The acute injury causes an acute inammatory reaction, which leads to interstitial brosis and emphysema with associated histologic features including airway mucosal metaplasia, smooth muscle hyperplasia, and atelectasis. Long-term histologic changes include decreased alveolar numbers and surface area. Airway lavage samples from patients with BPD contain increased levels of TGF-b, which are associated with an adverse prognosis. As described above, in vivo experimental models have shown that excessive expression of TGF-b inhibits lung morphogenesis and induces pulmonary brosis; EGF and PDGF are also thought to be involved in the development of pulmonary brosis in BPD. Decreased levels of VEGF in the presence of alveolar damage are also

ANTICOAGULANTS 115

likely to contribute to the abnormal pulmonary vasculature associated with lung injury. Manipulation of TGF-b may lead to therapeutic interventions to prevent the development of chronic pulmonary brosis associated with BPD.

Acknowledgments
The authors were supported by CA45548 (PDA) and EY05318 (PDA).
See also: Bronchopulmonary Dysplasia. Epidermal Growth Factors. Fibroblast Growth Factors. Insulin-Like Growth Factors. Keratinocyte Growth Factor. Platelet-Derived Growth Factor. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading
Alescio T and Cassini A (1962) Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium. Journal of Experimental Zoology 150: b83b94. Burri PH (1984) Fetal and postnatal development of the lung. Annual Review of Physiology 46: 617628.

Carmeliet P, Ferreira V, et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380: 435439. Chinoy MR (2003) Lung growth and development. Frontiers in Bioscience 8: 392415. deMello DE, Sawyer D, et al. (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568581. Gaultier C, Bourbon J, and Post M (eds.) (1999) Lung Development. New York: Oxford University Press. Han RN, Post M, et al. (2003) Insulin-like growth factor-I receptor-mediated vasculogenesis/angiogenesis in human lung development. American Journal of Respiratory Cell and Molecular Biology 28: 159169. Jankov RP and Keith A (2004) Tanswell. Growth factors, postnatal lung growth and bronchopulmonary dysplasia. Paediatric Respiratory Reviews 5(supplement A): S265S275. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464480. Shannon JM (1997) Epithelialmesenchymal interactions in lung development. In: McDonald JA (ed.) Lung Growth and Development, pp. 81118. New York: Dekker. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Paediatric Respiratory Reviews 5(supplement A): S283S287. Warburton D, Schwarz M, et al. (2000) The molecular basis of lung morphogenesis. Mechanisms of Development 92: 5581.

ANTICOAGULANTS
nther and C Ruppert, University of Giessen A Gu Lung center (UGLC), Giessen, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Anticoagulants are widely used for the prevention and treatment of venous and/or arterial thrombosis. Anticoagulants comprise a chemically heterogeneous group of drugs acting at different steps within the coagulation cascade. Heparin and heparin-based anticoagulants are indirect anticoagulants that bind to antithrombin and enhance the inhibitory capacity of this natural anticoagulant. Coumarin derivatives (e.g., warfarin) interfere with the hepatic synthesis of coagulation factors (vitamin K antagonists). A third class comprises direct inhibitors of enzymes of the clotting cascade, primarily thrombin. Classic anticoagulants such as unfractionated heparin and coumarins have some clinical drawbacks. Heparin requires parenteral application, has serious adverse effects, and is difcult to dose and monitor due to variable and unpredictable pharmacokinetics. The orally active coumarin derivatives have a narrow therapeutic window and multiple interactions with food and drugs, necessitating individualized dosing and monitoring. During the past 10 years, the search for safer and more effective antithrombotic agents resulted in the development or discovery of new molecules, including fondaparinux and idraparinux (both selective inhibitors of factor Xa), thrombin inhibitors for parenteral use such as recombinant hirudin and hirulogs, and the orally active ximelagatran.

The rst effective agent for therapy of thromboembolism was unfractionated (UF) heparin. It has become the anticoagulant of choice for the treatment and prevention of arterial and venous thrombotic diseases, including pulmonary embolism. Due to the problems accompanying heparin therapy (unpredictable pharmacokinetics, low bioavailability at low doses, unpredictable dose response, need for careful laboratory monitoring, bleeding, and thrombocytopenia), the search for improved anticoagulants resulted in the introduction of low-molecular-weight (LMW) heparins (Table 1). These heterogeneous compounds have been shown to produce a more predictable anticoagulant response and fewer adverse effects than UF heparin, reecting the improved pharmacokinetic properties (greater bioavailability after subcutaneous injection (B30%), longer half-life, and dose-independent clearance). The expectation, however, that these new compounds might eliminate the risk of bleeding was not conrmed. Another newer anticoagulant is danaparoid sodium, a heparinoid that is often used for treatment of heparin-induced thrombocytopenia.

116 ANTICOAGULANTS
Table 1 Heparins and heparinoids INN name Brand name Manufacturer Method of preparation Mean molecular weight Anti-Fxa: anti-FIIa ratio 1.0

Unfractionated heparin Heparin-sodium Generic (e.g., Liquemin) Low-molecular-weight heparins Ardeparin Normio

Extraction from porcine mucosa

14 000 (range: 5 00040 000)

WyethAyerst

Certoparin

Mono-Embolex

Novartis

Dalteparin Enoxaparin

Fragmin Clexane

Pharmacia Aventis-Pharma

Nadroparin Reviparin

Fraxiparine Clivarine

Sano Synthelabo Abbott

Tinzaparin

Innohep

LeoLabs

Peroxidative depolymerization of UF heparin Isoamylnitrite depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Benzylation and alkaline depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Nitrous acid depolymerization of UF heparin and size exclusion chromatography Haparinase digestion of UF heparin

5 300

2.0

5 200

2.2

6 000 4 500

1.93.2 3.35.3

4 300 3 900

2.54.0 3.66.1

6 500

1.52.5

Heparinoids Danaparoid Pentosanpolysulfate

Orgaran Elmiron Hemoclar Fibrezym

Celltech Pharma Ortho-McNeal Sano Aventis Bene Arzneimittel

Extraction from porine mucosa Sulfation of pentosan derived from beech tree bark

6 500 6 000

Anti-FXa 4 anti-FIIa n.a.

Oral anticoagulants (4-hydroxycoumarin derivatives), the second major class of traditional anticoagulants, were developed in parallel to heparin, based on a serendipitous discovery. Today, several coumarins are used as agents of choice for longterm anticoagulant therapy (Table 2). For historical and marketing reasons, some countries use more warfarin (e.g., United States, Canada, and United Kingdom), others phenprocoumon (e.g., Germany), and yet others acenocoumarol. Another class of drugs, called indaniones, are similar to coumarins in terms of pharmacodynamics and are mostly used in France. The traditional anticoagulants UF heparin and coumarin derivatives are highly effective agents and relatively safe when administered at appropriate dosages and carefully monitored. LMW heparins improved and simplied anticoagulant therapy by obviating the need for routine coagulation monitoring. Newer agents, including fondaparinux and

idraparinux, represent the rst agents of a new class of FXa inhibitors. Both are fully synthetic analogs of the unique antithrombin-binding pentasaccharide sequence found in UF and LMW heparin. Advances in molecular biology and biotechnology have led to the recombinant production of hirudin, the most potent naturally occurring direct thrombin inhibitor. Hirudin also serves as a standard for the development of LMW inhibitors of thrombin. Argatroban was the rst synthetic thrombin inhibitor approved for prophylaxis and treatment of various thrombotic disorders mainly in patients with heparin-induced thrombocytopenia. The major drawback of argatroban and hirudins is their requirement for parenteral administration. Ximelagatran and its active metabolite, melagatran, are members of a class of newly developed oral direct thrombin inhibitors. These agents have been approved for anticoagulant treatment very recently and expand the treatment options for thrombotic disorders. Whether these new agents

ANTICOAGULANTS 117
Table 2 Coumarin and indandione derivatives INN Brand name Manufacturer Pharmacokinetics t1 2 (h) Acenocoumarol (Nicoumalone) Ethylbiscoumacetate Dicumarol Phenprocoumon Tioclomarol Warfarin Sintrom Tromexane Generic Marcumar Apegmone Coumadin Warlone Miradon Previscan Dindevan Pindione Novartis Ciba-Geigy Generic Roche Merck Lipha Bristol-Myers Squibb Merck Frosst Schering Procter & Gamble Goldshield Merck Lipha 9 2.5 n.a. 150 24 3545 Duration of action (days) 24 12 n.a. 710 23 45

Anisindione Fluindione Phenindione

9 6 6

34 23 23

will be more effective and safer than traditional anticoagulants remains to be determined.

Chemical Structure
Heparin and Heparinoids

Heparin is a naturally occurring, highly sulfated glycosaminoglycan that has been found in mast cells in a large number of mammalian and nonmammalian vertebrates. Material for clinical use is derived from bovine lungs or pig intestinal mucosa and is prepared either as UF heparin or depolymerized LMW heparin. Heparin is a heterogeneous mixture of unbranched polysaccharide chains composed of 15100 alternating 1-4-linked mucosaccharide units of D-glucosamine and L-iduronic acid or D-glucuronic acid (Figure 1(a)). Heparin is the most negatively charged molecule in the human body. The molecular weight of UF heparin ranges from 5 to 40 kDa with an average of B14 kDa. LMW heparins are much smaller (mean MW, B5 kDa) and are produced from UF heparin by chemical or enzymatic depolymerization. The chemical composition is similar but not identical. The anticoagulant effect is mediated by a unique pentasaccharide unit (Figure 1(b)) that binds antithrombin with high afnity and activates the inhibitor by approximately 1000-fold. This pentasaccharide sequence is found in B30% of the chains of UF heparin (known as high-afnity heparin) but in only 1525% of the chains of LMW heparin. Danaparoid sodium is an LMW heparinoid isolated from porcine mucosa with a mean MW of 6 kDa (range, 412 kDa). It consists of a mixture of 84% heparan sulfate (of which 4% has high antithrombin activity), 12% dermatan sulfate, and 4% chondroitin sulfate. The characteristic disaccharide

repeat units of these glycosaminoglycans are depicted in Figure 1(c). Pentosanpolysulfate (PPS) is a highly sulfated, semisynthetic polysaccharide derived from beech tree bark with a mean molecular weight of 6 kDa (range, 29 kDa). The degree of sulfation is higher than in heparin, resulting in a higher negative charge density. Pentosan consists of b-1-4-linked D-xylose units branched with 4-O-methyl-D-glucuronic acid in a ratio of 1 uronic to 9 xylose units (Figure 1(d)). In contrast to heparin and other glycosaminoglycans, PPS is orally bioavailable.
FXa Inhibitors

Fondaparinux is a fully synthetic analog of the unique pentasaccharide sequence found in heparins. Idraparinux represents the second generation of this class. The O-methylation and O-sulfation result in a higher afnity for antithrombin and a higher negative charge, which in turn accounts for the tight binding of antithrombin and the prolonged half-life of idraparinux (80 h vs. 17 h for fondaparinux) (Figure 2).
Vitamin K Antagonists

The clinically used vitamin K antagonists are derivatives of either 4-hydroxycoumarin or indan-1,3dione (Figure 3). The substituent at the 3 position of the coumarin backbone or side-chain variations inuence pharmacokinetic and pharmacodynamic properties (Table 2). Pharmaceutical preparations of warfarin, acenocoumarol, and phenprocoumon contain a racemic mixture of R- and S-enantiomers. The stereoisomers exhibit different pharmacodynamics and pharmacokinetics since they undergo stereoselective biotransformation in the liver.

118 ANTICOAGULANTS
k

COOk 4 OH

O 1
k

CH2OSO3 O O 4 OH 1

O NHSO3k

OSO3

(a)
CH2OSO3k O O OH O NHCOCH3 OH OH COOk O O

Heparin
CH2OSO3k O OSO3
k

COOk O OH

O O

CH2OSO3k O

OH OSO3k

O NHSO3k

NHSO3k

(b)

Heparin pentasaccharide

CH2OSO3k O 4 OH 1 O 4

COOk O O 1 OH OH 4 OH

COO

O 1

CH2OH O O 1 3

O3SO 4 O

OH

NHCOCH3

Heparan sulfate
COOk O 4 OH O OH 1 CH2OR2 O O 1 3 NHCOCH3

Dermatan sulfate

Chondroitin sulfate A: R1 = SO3k R2 = H C: R1 = H R2 = SO3k n

R1O 4

(c)

O O OSOk 3 OSO3k O

O OSOk 3 OSO3k COOk O O

O OSOk 3 O

O OSOk 3 OSO3k O

O OSOk 3 OSO3k O

H3CO OSO3 OSO3k

(d)

Pentosanpolysulfate

Figure 1 Chemical structures of heparin and other members of the glycosaminoglycan family. (a) The major repeat unit of heparin is depicted, which is found in up to 90% in heparin from beef lung and up to 70% in heparin from pig mucosa. The disaccharide consists of 1-4 linked sulfated L-iduronic acid and sulfated D-glucosamine. (b) The specic pentasaccharide sequence that is responsible for highafnity binding to antithrombin is depicted. (c) The major repeat units of heparan sulfate, dermatan sulfate, and chondroitin sulfate A are depicted. (d) A typical sequence of pentosanpolysulfate is shown.

Hirudin and Hirulogs

Hirudin is a naturally occurring inhibitor of thrombin that is a single-chain polypeptide of 65 amino acid residues with an apparent molecular weight of 7 kDa. The structure is stabilized by three intramolecular disulde bridges. In 1986, recombinant hirudin (r-hirudin) became available. Desirudin

([Val1, Val2]-63-desulfohirudin) and lepirudin ([Leu12]-63-desulfohirudin) are therapeutically used recombinant hirudins produced in yeast (Table 3). They are not sulfated and differ only in the rst two amino acids of the N terminus. Both natural and recombinant hirudins show almost identical pharmacodynamic and pharmacokinetic properties.

ANTICOAGULANTS 119
CH2OSO3k O COOk O O CH2OSO3k O CH2OSO3k O O O OH OSO3k OH OCH3 NHSO3k

O COOk

HO OH

O NHSO3k

OH OH

OSO3k

NHSO3k Fondaparinux

CH2OSO3k O

COOk O O

CH2OSO3k O OSO3k OSO3k Idraparinux

O COOk O O OCH3 OCH3k

CH2OSO3k O

H3CO OCH3 OCH3

OCH3 OCH3

OSO3

OCH3 OSO3k

Figure 2 Chemical structure of FXa inhibitors. The structures of fondaparinux and idraparinux are given.

OH 4 12 O

O 3 O 1 2 3 O Indan-1, 3-dione

4-Hydroxycoumarin

O OH O

O OH OH

O Phenindione

Warfarin O OH

O O Dicumarol

O F

O Acenocoumarol

NO2

OH

OH

O Fluindione

O OH

O O

O O

Ethylbiscoumacetate O O

OCH3 O Anisindione

Phenprocoumon

Figure 3 Chemical structures of coumarin and indandione derivatives. The lead structures and derivatives are given. Clinically used coumarin derivatives are substituted at the 3-position, and indandiones are substituted at the 2-position. Asymmetric carbons are indicated by asterisks.

120 ANTICOAGULANTS
Table 3 Direct thrombin inhibitors INN name Desirudin Lepirudin Bivalirudin Argatroban Melagatran Ximelagatran Brand name Revasc Refludan Angiomax Novastan Melagatran Exanta Manufacturer Aventis/Novartis Schering, Berlex The Medicine Company Texas Biotechnology AstraZeneca AstraZeneca Route of administration Parenteral (i.v., s.c.) Parenteral (i.v.) Parenteral (i.v.) Parenteral Parenteral Oral Half-life 23 h 1.3 h 25 min 45 min 35 h 35 h

1 H2N 20 Val Val Tyr Thr Asp Cys Thr Glu Ser Gly 10 Val Asn Ser Gly Glu Cys Leu Cys Leu Asn Gln Cys Gly Gln Gly Asn Lys Cys IIe Leu 30

40 Thr Val Cys Gly Glu Gly

Gln Asn Lys Glu Gly Asp Ser Gly

65 60 50 Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln SO3

COOH

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln Hirudin

DPhe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Bivalirudin
Figure 4 Structure of hirudin and hirulogs. The amino acid sequences of natural hirudin and the hirulog bivalirudin are given. The C-terminal sequences blocking the anion-binding exosite (amino acids 5465 of hirudin) are depicted in blue, and the sequences binding to the active site of thrombin (amino acids 4548 of hirudin) are depicted in light blue.

The discovery of the bifunctional inhibition mechanism of hirudin resulted in the development of a new type of bivalent oligopeptide inhibitors, termed hirulogs. Bivalirudin, also termed hirulog-1, is a semisynthetic 20-amino acid peptide consisting of the N-terminal and the C-terminal active peptide domains from hirudin joined by a glycine linker (Figure 4).
Synthetic Active Site Inhibitors of Thrombin

mimicking the cleavage site in brinogen. The arginine-derived structures are shown in Figure 5. The pharmaceutical preparation consists of a mixture of R and S stereoisomers at a ratio of approximately 65:35. Ximelagatran has a molecular weight of 473.6 Da and is a pro-drug without intrinsic anticoagulant activity. Following resorption, it is rapidly converted into its active metabolite, melagatran (MW, 429.5 Da).

Argatroban and the orally active ximelagatran are LMW active site inhibitors of thrombin. They are the result of structure-based drug design and belong to the peptidomimetic (arginominetic) group of inhibitors

Mode of Action
The classical and new anticoagulants interact with different clotting factors at different levels of blood

ANTICOAGULANTS 121
O H2N OH

NH H2N NH

Arginine

H3C

NH O O S O NH O CH3 NH H2N NH CH3 Ximelagatran Argatroban O O HO N NH2 O N HN OH N O NH

Hydrolysis

Reduction

NH N O HN O
OH

HN

NH2

Melagatran
Figure 5 Chemical structures of synthetic thrombin inhibitors. The chemical structures of the arginomimetics argatroban, ximelagatran, and melagatran are shown. Bioconversion of ximelagatran into its active metabolite, melagatran, involves hydrolysis of the carboxyl ester and reduction of the hydroxyamidino group.

coagulation. A summary of their targets within the coagulation cascade is shown in Figure 6.
Heparin and Heparinoids

Heparin acts as an accelerator of antithrombin (formerly called antithrombin III). The pentasaccharide sequence of heparin binds to the lysine site in antithrombin inducing a conformational change of

the antithrombin molecule, which facilitates binding to specic clotting factors and accelerates the rate at which antithrombin inhibits these factors by approximately 1000 times. UF heparin inhibits factors Xa, IIa (thrombin), IXa, XIa, and XIIa, with FXa and thrombin representing the most responsive and most critical factors within the clotting cascade. Heparin, antithrombin, and thrombin form a ternary complex in which thrombin initially binds to the

122 ANTICOAGULANTS
Extrinsic pathway Coumarins, indandiones Vascular injury Tissue factor (TF) FVII FVII TF FVIIa

TFPI, FVIIai Coumarins, indandiones

Intrinsic pathway

HMW kininogen Prekallikrein FXIIa FXI

Surface

FIX

Ca2+

FXIa

FX

UF heparin, LMW heparin Danaparoid, fondaparinux

FIXa
Ca2+, PL PL, Ca2+ Tenase complex

Antithrombin FXa Hirudin, hirulogs Argatroban, melagatran Thrombin FXIII

FVIII APC

FIXa, thrombin

FVIIIa
PL, Ca Prothrombinase complex
2+

Prothrombin FV

Coumarins, indandiones APC Danaparoid Dermatan sulfate

2+ Ca , PL, FXa

FVa

FXIIIa Heparin cofactor II Fibrinogen Fibrin Ca2+ Crosslinked fibrin

Figure 6 Overview of the coagulation cascade and targets of anticoagulants. The targets of anticoagulants within the clotting cascade are given.

heparinantithrombin complex in a non-specic manner to any site of the heparin molecule and then slides along the surface until it binds to the inhibitor. The afnity of heparin for the antithrombinthrombin complex is much lower than that for unreacted antithrombin. Thus, heparin can dissociate from the complex and bind to additional unreacted antithrombin molecules, resulting in a continuing anticoagulant effect. The complex, however, is not effective in inhibiting brin-bound thrombin. UF heparin also blocks the thrombin-induced feedback activation of factors V and VIII. The sliding mechanism requires heparin chain lengths consisting of at least 18 saccharide units. The lower content of glycosaminoglycan chains 418 saccharide units in LMW heparin accounts for the greater relative activity against factor Xa. This difference is described in an activity ratio (anti-FXa:anti-FIIa ratio), which is 1:1 for UF heparin and 1.5:1 to 6:1 for LMW heparin (see Table 1). The anticoagulant activity of UF heparin is expressed in relation to the fourth international standard: Pharmaceutical preparations have specic activities of 150190 U mg 1. The

anticoagulant activity of LMW heparin is expressed relative to the rst international standard for LMW heparin; the specic activity ranges between 80 and 120 anti-FXa U mg 1 and between 35 and 45 antithrombin U mg 1. In addition to the interaction with antithrombin, both UF and LMW heparin enhance the release of tissue factor pathway inhibitor (TFPI), which forms a complex and inactivates FXa and subsequently FVIIa. UF and LMW heparins also bind to platelet factor 4, which is the predicate for heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy that is associated with paradoxic clotting, including deep vein thrombosis, pulmonary embolism, or occasional arterial thromboses. In HIT, antibodies form against the heparinplatelet factor 4 complex, leading to platelet activation and aggregation. LMW heparins bind less extensively to platelet factor 4 as well as to plasma proteins, endothelial cells, and macrophages. However, LMW heparins should not be used in patients with established HIT since HIT antibodies have an almost 100% cross-reactivity with LMW heparin.

ANTICOAGULANTS 123

The heparinoid danaparoid sodium exerts its pharmacological effects in a similar manner via binding and activation of the serpin inhibitor heparin cofactor II. Danaparoid has little effect on platelet function and exhibits low cross-reactivity with heparin-induced antibodies, making it an option for the treatment of HIT. Similar to LMW heparins, danaparoid also exerts a higher inhibitory activity against FXa. The overall antithrombotic activity is much lower compared to that of UF heparin (anti-FXa activity B10% and antithrombin activity B1% of that of UF heparin). PPS may act via an antithrombin or heparin cofactor II-independent pathway, but the exact mechanism of action is not known. It has been used since the 1960s as an anticoagulant but does not seem to play a major role in todays clinical medicine with regard to anticoagulation. In the United States, PPS is approved for pain relief in the management of interstitial cystitis.
Fondaparinux and Idraparinux

the response of different thromboplastin reagents. The therapeutic INR range is 2.03.0. An INR o2.0 may be ineffective in preventing thrombotic events, and an INR 43.0 is associated with increased bleeding. The manifold interaction of coumarins with food and drugs has an important impact on the anticoagulatory response by either increasing or decreasing anticoagulant activity.
Direct Thrombin Inhibitors (Hirudin, Hirulogs, and Synthetic Inhibitors)

Fonaparinux and idraparinux are selective indirect inhibitors of FXa. Their activity also requires the presence of antithrombin, but inhibition is achieved solely through binding of the pentasaccharide sequence to antithrombin and induction of a conformational change.
Vitamin K Antagonists

Coumarin and indandione derivatives act as vitamin K antagonists and exert their anticoagulatory effect by interfering with the hepatic synthesis of vitamin K-dependent clotting factors. Synthesis of factors II (prothrombin), VII, IX, and X involves carboxylation of glutamate residues to g-carboxyglutamates by a specic carboxylase, which is required for their biological activity. These g-carboxyglutamate residues promote binding of the clotting factors to phospholipids, thereby accelerating coagulation. Coumarin and indandione derivatives interfere with the vitamin K cycle by inhibiting vitamin K epoxide reductase. By this mechanism, coumarins decrease the synthesis of coagulation factors by 3050%. The anticoagulant response to coumarins is delayed by 2 or 3 days since previously formed coagulation factors circulate with a long half-life (46 h for FVII, 2050 h for factors IX and X, and 5070 h for thrombin). A major disadvantage of vitamin K antagonists is the narrow therapeutic index and the need for intensive monitoring. The anticoagulatory effect is assessed by the international normalized ratio (INR), which is dened as the ratio of the patient prothrombin time (PT) compared to the mean PT of normal donors normalized to the international sensitivity index, a correction factor for

Hirudin is a highly potent (Ki 22 fM) inhibitor specic for the serine protease thrombin. It forms a stable noncovalent 1:1 stoichiometric complex with the B-chain of a-thrombin, thereby abolishing its ability to cleave brinogen (Figure 7). Hirudin inhibits both free and brin-bound thrombin. During inhibition, hirudin displaces brin from thrombin. Bivalirudin is a bivalent inhibitor (Ki 1:9 nM) blocking both the active site and the substrate recognition site (anion-binding exosite). Argatroban (Ki 39 nM) and melagatran (Ki 2 nM) are peptidomimetics of the sequence of brinopeptide A that interact with the active site of thrombin. They are reversible, noncovalent, competitive inhibitors blocking the enzymes interaction with its substrate. The pro-drug ximelagatran has no intrinsic anticoagulant activity but is rapidly converted into melagatran following gastrointestinal resorption. The direct thrombin inhibitors are recommended agents for prevention and treatment of HIT. Lepirudin and argatroban are approved for the treatment of HIT in the United States. Bivalirudin, which is approved for anticoagulation during percutaneous coronary intervention, appears to be promising in patients with HIT. Although displaying pharmacologic advantages, its use for this indication is only weakly recommended due to the limited data available.
Novel Anticoagulants

In addition to the previously described anticoagulants, novel agents that directly target specic steps within the coagulation cascade have been developed and tested in clinical phase II or phase III studies or are currently undergoing preclinical and clinical testing. They include LMW inhibitors of FXa and recombinant forms of naturally occurring anticoagulants. Recombinant tissue factor pathway inhibitor (tifacogin) Tifacogin is a recombinant form of the endogenous extrinsic pathway inhibitor TFPI, also referred to as anticonvertin or lipoprotein-associated coagulation inhibitor. TFPI is a 276-amino acid residue, high-afnity Kunitz-type serine protease inhibitor.

124 ANTICOAGULANTS

Fibrin

Hirudin

Bivalirudin

Heparin recognition site = anion-binding exosite II

Thrombin + + + + + + Substrate recognition site = anion-binding exosite I

Binding of heparin, prothrombin 2 fragment (F2) platelet gp Ib Specificity pocket Active site

Binding of fibrin(ogen), PAR-1, heparin cofactor III thrombomodulin

Synthetic inhibitors (Argatroban, melagatran)

Figure 7 Mechanism of action of thrombin inhibitors. The interaction of hirudin, hirologs, and synthetic thrombin inhibitors with thrombin is shown.

Inhibition of the tissue factor-mediated extrinsic coagulation pathway occurs in a two-step manner: TFPI directly binds to FXa at or near its active site via the second Kunitz domain and produces a FXadependent feedback inhibition of the TFFVIIa catalytic complex by formation of a quaternary complex (FXaTFPITF/FVIIa), in which the second Kunitz domain binds to FXa and the rst Kunitz domain binds FVIIa (Figure 8). Despite showing promising results in animal thrombosis models, a phase III trial failed to demonstrate significant benet in outcome in sepsis patients. Recombinant nematode anticoagulant protein c2 Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent 85-amino acid residue anticoagulant protein originally identied in the hookworm Ancylostoma caninum. Like TFPI, rNAPc2 inhibits initiation of the extrinsic coagulation pathway by binding to a noncatalytic exosite on FX or

FXa prior to the formation of the quaternary inhibitory complex with tissue factorFVIIa. In a phase II study on patients who underwent knee replacement surgery, a low dose of rNAPc2 decreased the rate of deep vein thrombosis and major bleeding.

Active site inhibited factor VII Active site inhibited factor VII (FVIIai; ASIS) is a recombinant variant of activated factor VII in which the catalytic function is irreversibly blocked. The resulting molecule retains its TF binding capacity but is enzymatically inactive. FVIIai exerts its effects by competing with plasma FVII(a) for tissue factor binding on cell surfaces. The formation of an inactive binary FVIIaiTF complex attenuates the initiation of coagulation. However, although recombinant FVIIai was shown to inhibit TF-mediated injury in animal models, it failed to elicit a benecial response in coronary patients in a phase II trial.

Fibrin

+ + +

+ + +

Fibrin

+ + +

+ + +

+ + +

+ + +

ANTICOAGULANTS 125
3 TFPI 1 2 FXa

Ca2+

TFPI

FXa

Ca2+

TFPI

FVIIa Ca2+ Ca2+

FVIIa

FXa

TF

TF Ca2+

Figure 8 Mechanism of action of tissue factor pathway inhibitor (TFPI). TFPI binds to FXa in solution forming a binary complex. Subsequent binding of FXaTFPI to the TFFVIIa complex results in the nal quaternary complex inhibiting initiation of coagulation.

Direct FXa inhibitors DX-9065a (Daiichi) is a synthetic, LMW, nonpeptidic, direct reversible and parenteral inhibitor of factor Xa. BAY 59-7939 (Bayer), razaxaban (Bristol-Myers Squibb), LY 517717 (Eli Lilly), and YM-60828 (Yamanouchi Pharmaceutical) are orally active synthetic FXa inhibitors. Activated protein C (drotrecogin alpha (activated)) The protein C system also regulates coagulation by modulation of the activity of the two clotting factors, FVa and FVIIIa. Protein C, the key component of this system, is a vitamin K-dependent zymogen of an anticoagulant protease, which is activated on the surface of intact endothelial cells by thrombin that has bound to the integral membrane protein thrombomodulin. Activated protein C (APC) can cleave the phospholipid membrane-bound (activated) clotting factors FVa and FVIIIa, which blocks the function of the prothrombinase and tenase

complexes and thus inhibits the propagation of coagulation (Figure 9). Activation of protein C is augmented by the endothelial cell protein C receptor, and the anticoagulant activity of APC is supported by protein S, a vitamin K-dependent cofactor. The protein C system also has anti-inammatory and antiapoptotic properties. Drotrecogin alpha (activated) is a recombinant version of APC. Drotrecogin alpha was approved for treatment of severe sepsis in adults based on a phase III trial in which 28-day mortality was reduced. Thrombomodulin Thrombomodulin is a complex multifunctional endothelial cell surface glycoprotein receptor. High-afnity binding to thrombin involving EGF domains 46 converts thrombin from a procoagulant into an anticoagulant state that can activate protein C. Thrombomodulin not only regulates hemostasis but also plays an important role in the

126 ANTICOAGULANTS
Protein C APC TM Th EPCR

Protein S

FVIIIa A P C

Protein S

FVIIIi

FVi FVa A P C

FV

Figure 9 Protein C pathway: mechanism of action of protein C and thrombomodulin. (Top) Thrombin (Th) generated in the vicinity of intact endothelial cells binds to thrombomodulin (TM) and activates protein C. This process is enhanced by the endothelial cell protein C receptor (EPCR). (Bottom) Activated protein C (APC) and protein S form a complex on the membrane of endothelial cells that cleaves and inactivates FVIIIa (-FVIIIi) and FVa (-FVi) and thus inhibits coagulation. In the case of FVIIIa, this process is further enhanced by FV, which in this context functions as an anticoagulant cofactor protein.

modulation of inammation. The EGF 36 domains are involved in activation of thrombin-activatable brinolysis inhibitor, a natural anti-inammatory molecule that inhibits vasoactive peptides such as the complement anaphalotoxin C5a. Soluble thrombomodulin is a recombinant variant that is undergoing clinical testing.

Role of Anticoagulants in Respiratory Medicine

Thromboembolic events, including deep vein thrombosis and pulmonary embolism, are commonly encountered in clinical practice. In addition to the occlusion of a vessel, periembolic processes, such as activation of circulating platelets and inammatory cells at the surface of thrombi and the liberation of vasoactive mediators (thromboxan, serotonin, and leukotriene B4) and brin(ogen)-derived split products, markedly contribute to cardiopulmonary dysfunction, with increased pulmonary vascular resistance, elevated right heart pressure, and gas exchange disturbances. Anticoagulants are used for prevention and treatment of a variety of indications, including prevention of deep vein thrombosis and pulmonary embolism postoperatively after general or orthopedic surgery (e.g., hip replacement/fracture), in patients with acute spinal cord injuries, multiple trauma, ischemic stroke, after coronary angioplasty, heart valve replacement, and in many other medical conditions as well as for treatment of established deep vein thrombosis, unstable angina, and ischemic stroke. In a meta-analysis,

subcutaneous LMW heparin was shown to be as effective and safe as intravenous UF heparin for the initial treatment of nonmassive pulmonary embolism. Intravascular clot formation due to predominance of a procoagulant and suppression of brinolytic factors is a key event in a variety of inammatory and infectious diseases, such as septic organ failure. Microthrombosis/microembolism is commonly found in patients with acute respiratory distress syndrome (ARDS), of which sepsis is the major underlying cause, and in patients with chronic pulmonary hypertension. The lung microvascular compartment represents the largest surface of the pulmonary vascular bed and may be of crucial importance in the dissolution of microthrombi. Under physiological conditions, patency of capillaries is provided by a high concentration of antithrombin that is activated by glycosaminoglycans (e.g., heparan sulfate) and thrombomodulin on the surface of endothelial cells. In addition, clearance of thrombi is achieved through the production and secretion of tPA and uPA by microvascular endothelial cells. Under inammatory or infectious conditions (e.g., sepsis and ARDS), this balance between anticoagulation and brinolysis regulation is shifted toward coagulation with a decrement in brinolysis, mainly due to activation of the extrinsic tissue factor/FVII-dependent coagulation pathway and secretion of plasminogen activator inhibitor-1 (PAI-1). In experimental models of acute lung injury (ALI), APC prevented intravascular coagulation, edema formation, and inammatory cell recruitment in the lung. Although administration of APC has been shown to decrease mortality in patients

ANTICOAGULANTS 127

with severe sepsis, clinical studies demonstrating its efcacy in ARDS are lacking. In ALI/ARDS, brin deposition occurs in both intravascular and extravascular locations. In patients with ALI/ARDS, the alveolar hemostatic balance is shifted toward predominance of procoagulant activity, which is mainly attributable to the extrinsic pathway enzymes tissue factor and FVII. In contrast, the brinolytic capacity of the alveolar space is markedly reduced. In lavage uid from patients with ARDS, concentrations of urokinase, representing the predominant plasminogen activator in this compartment, were markedly decreased, whereas elevated activities of PAI-1 and a2-antiplasmin were consistently encountered. Under these conditions, brinogen leaking into the alveolar space due to an impaired barrier function is rapidly converted into brin. The function of brin formation in the alveolar space is largely unsettled. It may well exert benecial effects by preventing pulmonary hemorrhage and serve as a primary matrix of wound repair. On the other hand, alveolar brin is a potent inhibitor of surfactant function, thus promoting atelectasis formation and contributing to the severe impairment of gas exchange properties. In vitro studies have demonstrated an incorporation of hydrophobic surfactant compounds into nascent brin strands, resulting in an almost complete loss of the surface tension-lowering properties. Alveolar/interstitial brin deposition and aberrant local brin turnover have likewise been shown in patients with chronic interstitial lung disease, including idiopathic pulmonary brosis, sarcoidosis, and hypersensitivity pneumonitis. Alveolar brin appears to be a key factor in triggering the broproliferative process in the lung. Delayed clearance of brin from the lung may promote broblast activation, proliferation, and tissue remodeling (i.e., replacement of the primary brin matrix by a secondary collagenous matrix associated with scarring and honeycombing). Thrombin also acts as a chemoattractant for inammatory cells and broblasts and stimulates the release of proinammatory cytokines. Moreover, it induces the release of growth factors and brogenic mediators, thereby stimulating broblast proliferation and connective tissue synthesis. Although the use of anticoagulants is not part of the standard clinical practice for these conditions, blockade of the extrinsic coagulation pathway and prevention of extravascular brin formation may be a promising approach for acute inammatory and chronic interstitial lung disease. Experimental studies addressing anticoagulant intervention (e.g., by administration of APC, heparin, and direct thrombin inhibitor) in animal models of ALI and pulmonary brosis have been shown to

protect the lung and to reduce the broproliferative response. In addition, different anticoagulants are being tested in ongoing experimental and clinical studies. A phase II study of recombinant TFPI in patients with severe sepsis showed improvement in lung function and a trend toward improved survival in the subset of patients with ARDS. A follow-up phase III study, however, failed to demonstrate improved survival. Similarly, the benecial effects of recombinant antithrombin observed in animal models could not be conrmed in humans. Heparin is being evaluated in humans with idiopathic pulmonary brosis. In a similar vein, heparin is considered an alternate agent in the treatment of allergic asthma. Glycosaminoglycans such as heparan sulfate are expressed as part of a proteoglycan on cell surfaces and are thought to play a role in the regulation of the inammatory response (e.g., by binding chemokines). Heparin, which is synthesized by and stored exclusively in mast cells, has been found to exert anti-inammatory effects in animal models and in human disease. Heparin binds and inhibits a variety of cytotoxic and inammatory mediators, including eosinophilic cation protein and peroxidase. Furthermore, heparin has been associated with the inhibition of lymphocyte activation, neutrophil chemotaxis, smooth muscle growth, and vascular tone. The rst clinical trial with inhaled heparin was performed in 1969, but further human studies using inhaled heparin were not published until the early 1990s. Three smaller studies of patients with exercise-induced asthma showed that inhaled heparin preserved specic airway conductance better than inhaled cromolyn or placebo following exercise. Studies investigating the effects of inhaled heparin on bronchial reactivity following inhaled allergen challenge have yielded mixed results.

Side Effects and Contraindications

The most devastating complication of anticoagulant therapy is bleeding. The occurrence of bleeding, primarily in the gastrointestinal tract and the central nervous system, is a major cause of morbidity and mortality. Consequently, anticoagulants are contraindicated in any case in which the risk of hemorrhage may be greater than the potential clinical benet of anticoagulation. Risk factors for bleeding include advanced age, serious illness (cerebral, cardiac, kidney, or liver disease), cerebrovascular or peripheral vascular disease, and an unstable anticoagulant effect. Whereas the action of unfractionated heparin may be antagonized by protamine sulfate, no specic antidote is available for LMW heparins, danaparoid, fondaparinux, or the direct thrombin inhibitors

128 ANTICOAGULANTS

hirudin and ximelagatran. The anticoagulant effect of coumarin derivatives may be antagonized by low doses of vitamin K and substitution with fresh frozen plasma. Other adverse effects of heparins are heparin-induced thrombocytopenia and osteoporosis, which can occur after long-term treatment and increase the risk for fractures. An absolute contraindication for coumarins is pregnancy because they cause fetal bone abnormalities by interfering with g-carboxylation of proteins synthesized in the bone. Heparins do not cross the placental barrier and do not cause these problems. Long-term use of ximelagatran carries the risk of severe liver injury. Administration in patients with hepatic disease is not recommended. As outlined previously, HIT is an antibody-mediated adverse effect of heparin associated with an increased thrombotic risk. According to the seventh American College of Clinical Pharmacology consensus conference guidelines, alternative, nonheparin anticoagulants should be used in patients with strongly suspected or conrmed HIT. Recommendations include direct thrombin inhibitors as well as danaparoid. Although withdrawn from the US market, danaparoid remains approved and available for prevention and treatment of HIT in Canada, Europe, Australia, and Japan. Because fondaparinux does not cross-react with HIT antibodies, it may also be useful for this indication. A general recommendation, however, cannot be made due to the minimal data available. Antihirudin antibodies are commonly generated during treatment with lepirudin. Although they are usually not clinically significant, the European Agency for the Evaluation of Medical Products recommended the use of nonhirudin anticoagulants in patients who have previously been exposed to lepirudin. Coumarins should not be used in patients with conrmed or strongly suspected HIT because they can contribute to skin necrosis and venous limb gangrene. For patients receiving coumarins at the time of diagnosis of HIT, the use of vitamin K for reversal of coumarin-induced anticoagulation is recommended.
See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Overview; Antithrombin III;

Factor X; Protein C and Protein S; Thrombin. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading
Ansell J, Hirsh J, Poller L, et al. (2004) The pharmacology and management of the vitamin K antagonists: the seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 204S233S. Bussey H, Francis JL, and the Heparin Consensus Group (2004) Heparin overview and issues. Pharmacotherapy 24: 103S107S. Desai UR (2004) New antithrombin-based anticoagulants. Medical Research Reviews 24: 151181. Ginsberg JS, Greer I, and Hirsh J (2001) Use of antithrombotic agents during pregnancy. Chest 119: 122S131S. Happel KI, Nelson S, and Summer W (2004) The lung in sepsis: Fueling the re. American Journal of the Medical Sciences 328: 230237. Hirsh J and Raschke R (2004) Heparin and low-molecular-weight heparin: The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 188S203S. Hirsh J, Fuster V, Ansell J, and Halperin JL (2003) American Heart Association/American College of Cardiology Foundation guide to warfarin therapy. Circulation 107: 16921711. Idell S (2001) Anticoagulants for acute respiratory distress syndrome. Can they work? American Journal of Respiratory and Critical Care Medicine 164: 517520. Idell S (2002) Adult respiratory distress syndrome: Do selective anticoagulants help? American Journal of Respiratory Medicine 1: 383391. Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31(supplement): S329S336. Nowak G (2002) Pharmacology of recombinant hirudin. Seminars in Thrombosis and Hemostasis 28: 415423. Srivastava S, Goswami LN, and Dikshit DK (2005) Progress in the design of low molecular weight thrombin inhibitors. Medical Research Reviews 25: 6692. Warkentin TE and Greinacher A (2004) Heparin-induced thrombocytopenia: Recognition, treatment, and prevention. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 311337. Weitz JI (1997) Low-molecular weight heparins. New England Journal of Medicine 337: 688698. Weitz JI, Hirsh J, and Samama MM (2004) New anticoagulant drugs. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 265S286S. Wittkowsky AK (2003) Warfarin and other coumarin derivatives: Pharmacokinetics, pharmacodynamics, and drug interactions. Seminars in Vascular Medicine 3: 221230.

Antioxidants

see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic.

ANTIVIRAL AGENTS 129

ANTIVIRAL AGENTS
M A Parniak, E N Vergis, and M E Abram, University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Most respiratory tract infections (RTIs) are viral in origin; numerous different viruses cause RTIs, many of which can be quite serious. However, only ve drugs are approved for their treatment, and these drugs are directed at only two viruses. Four drugs amantadine, rimantadine, zanamivir, and oseltamivir are used for the prophylaxis and treatment of inuenza virus infection. These drugs target two different stages of inuenza replication. Orally administered amantadine and rimantadine act only against inuenza A and block the ion channel formed by the viral M2 protein, thereby preventing viral ribonucleoprotein release. Zanamivir and oseltamivir are active against inuenza A and B, and they are competitive inhibitors of the viral neuraminidase. These drugs block nascent viral release from the infected cell and thus viral spread by preventing neuraminidase-catalyzed removal of sialic acid residues from membrane glycoproteins. Oseltamivir is administered orally, whereas zanamivir must be delivered topically by inhalation of the dry powder. The fth approved drug, the ribonucleoside analog ribavirin, is administered by nebulization and is approved for the treatment of pediatric respiratory syncytial virus infection. Its mechanism of action is uncertain, but it may involve alteration of cellular nucleotide pools and inhibition of viral RNA synthesis.

annually, with up to 500 000 deaths. Inuenza viruses that cause human disease are classied into groups AC. Inuenza A is the most serious pathogen since it leads to large recurrent epidemics with significant morbidity and mortality. Four drugs (Figure 1) the adamantane derivatives amantadine and rimantadine and the neuraminidase inhibitors zanamivir and oseltamivir are used for the treatment or prophylaxis of inuenza virus infection. These drugs target different stages of inuenza virus replication (Figure 2). Amantadine and rimantadine inhibit an early stage involved in the uncoating of the viral ribonucleoprotein, whereas zanamivir and oseltamivir inhibit a viral enzyme needed to allow nascent virus release from the infected cell.
Amantadine and Rimantadine

Introduction
Viruses are the cause of most respiratory tract infections (RTIs); numerous different viruses can infect the respiratory tract. Many of these infections can be quite serious, especially in the young, the elderly, chronically ill, and immunocompromised individuals. However, there are only ve drugs approved for the treatment of viral RTIs, and these drugs are directed at only two viruses. Four of the approved drugs are used for the treatment or prophylaxis of inuenza, and one is used for the treatment of pediatric respiratory syncytial virus infection.

Drugs for the Treatment of Inuenza

Inuenza virus mainly infects the upper respiratory tract, and most individuals recover within approximately 1 week without medical treatment. Nevertheless, RTI due to inuenza is a major cause of morbidity and mortality worldwide. The World Health Organization estimates that inuenza can account for up to 15% of worldwide upper RTIs

The inhibitory activity of amantadine (adamantan-1ylamine hydrochloride) against inuenza A was rst identied in 1964, and this drug was rst marketed in 1966. The close analog, rimantadine (1-adamantan-1-yl-ethylamine hydrochloride), was developed subsequently due to the adverse neurological side effects associated with the use of amantadine. These structurally similar compounds are used primarily for prophylaxis of inuenza A infection, particularly for individuals with high risk of exposure such as healthcare workers, and prevent inuenza-like illness in up to 80% of treated individuals. Both drugs can also reduce the duration of symptoms of established inuenza A infection if therapy is initiated within 48 h of discernible symptoms. The adamantanes are effective only against inuenza A. However, since inuenza A is the source of most serious cases of inuenza-related RTI, the adamantanes are valuable drugs for the treatment and especially prophylaxis of inuenza infections. Amantadine and rimantadine are administered orally, once or twice daily with adult dosages of 100200 mg per day. For prophylaxis, the drugs are administered for up to 7 days during high-risk periods of an epidemic. The two drugs have very different pharmacokinetic proles despite their similarity in structure. Amantadine is rapidly absorbed after oral delivery, has a half-life of approximately 15 h, is not metabolized, and is excreted almost entirely in the urine. In contrast, rimantadine is more slowly absorbed, has a half-life twice that of amantadine, and undergoes extensive hepatic metabolism. Although peak plasma levels of rimantadine are less than those

130 ANTIVIRAL AGENTS

H3C NH2 HCl NH2 HCl

Amantadine

Rimantadine

OH

OH

O H HO N H HN OH OH O

NH H NH2 O HN NH2

Zanamivir

Oseltamivir

Figure 1 Drugs used for prophylaxis or treatment of inuenza virus infection. The drug oseltamivir is shown as the active caboxylic acid form, although it is administered as the ethyl ester pro-drug.

of amantadine, the former is more highly concentrated in respiratory secretions than amantadine, thereby providing better access to the viral target. Mechanism of action Amantadine and rimantadine target the inuenza A virus M2 membrane protein. The inuenza virus enters the cell via endocytosis, and fusion of the virus membrane envelope with the endosome membrane provides a mechanism for the release of the viral ribonucleoprotein (RNP) into the cytoplasm and thus to the nucleus, where replication of the viral genomic RNA can take place. The RNP interacts with the viral M1 protein that underlies the viral envelope. Furthermore, M1 masks a nuclear localization signal on the RNP. Unless the M1RNP interactions are disrupted, the RNP can neither be released into the cytoplasm nor imported into the nucleus. Disassembly of the complex may also be important for activation of the viral RNA polymerase. The inuenza M2 membrane protein is critical in enabling disruption of the M1RNP complex. The M2 protein is a homotetramer that is considered to form an ion channel in the viral membrane envelope (Figure 3). During acidication of the viruscontaining endosome, the M2 protein enables protons to enter the virus particle. Acidication of

the interior of the virus particle results in dissociation of the RNP from the M1 protein. This allows release of the RNP into the cytoplasm and subsequent import into the nucleus. Amantadine and rimantadine bind to the viral M2 membrane protein, blocking the channel and preventing proton transport into the viral particle. The M2 protein is not found in inuenza B virus, which accounts for the lack of antiviral activity of amantadine and rimantadine against inuenza B. Contraindications Central nervous system side effects are noted in significant numbers (up to 10%) of amantadine-treated individuals. Symptoms include dizziness, anxiety, difculty in concentrating, and insomnia, all of which are problematic for healthcare workers, a major target group for prophylactic treatment during inuenza epidemics. Central nervous system side effects reverse once treatment is stopped. Severe toxic reactions or death can occur in patients with renal insufciency and include serious neurotoxicity (mental status changes, hallucinations, tremor, myoclonus, seizures, and coma) or cardiac arrhythmias. Dosages of the drugs, especially amantadine, must be lowered and carefully monitored in such patients. Rimantadine is significantly better tolerated than amantadine.

ANTIVIRAL AGENTS 131

Zanamivir, oseltamivir

Amantadine, rimantadine

Figure 2 Inuenza virus replication cycle showing stages inhibited by approved therapeutic drugs. Following attachment, endocytosis, and acidication of the virion-containing endosome, the virion core is also acidied in a process mediated by the viral M2 membrane proton channel protein. This enables a pH-dependent release of viral ribonucleoprotein (RNP). Adamantane drugs target the virus M2 protein, preventing virion acidication and release of RNP. Nascent virus release from an infected cell requires cleavage of terminal sialic acid residues from cell surface and virion envelope proteins. Neuraminidase inhibitors target this stage of replication, preventing spread of virus to uninfected cells.

Neuraminidase Inhibitors

The viral neuraminidase inhibitors zanamivir (5-acetylamino-4-gunadino-6-(1,2,3-trihydroxypropyl)-5,6dihydro-4H-pyran-2-carboxylic acid) and oseltamivir (4-acetylamino-5-amino-3-(1-ethylpropoxy)-cyclohex1-ene carboxylic acid) were approved for therapeutic use in 1999. Both drugs are reversible competitive inhibitors of inuenza virus neuraminidase. The drugs are effective against both inuenza A and inuenza B and also against strains of inuenza A virus resistant to

the adamantane drugs. Zanamivir and oseltamivir have virtually no activity against human cell neuraminidases at levels that provide potent antiviral activity. Viral resistance to zanamivir or oseltamivir is uncommon. Both drugs are approved for treatment of inuenza in adults and children (1 year or older for oseltamivir and 7 years or older for zanamivir) within 48 h of onset of discernible symptoms. Oseltamivir can also be used for prophylaxis in adults. Zanamivir has low oral bioavailability and is administered topically by dry powder inhalation, whereas oseltamivir can be

132 ANTIVIRAL AGENTS

H+

NH2HCl V A S H G H H V A S G

Viral membrane envelope

M2 protein subunits
Figure 3 Schematic of the mechanism of adamantane drug inhibition. The drugs interact with specic residues of the inuenza A virus M2 transmembrane protein. This protein forms a channel for the inux of protons into the virion. Only two of the four M2 subunits that form the channel are shown. Binding of amantadine or rimantadine blocks the channel, preventing intravirion acidication.

administered orally (bioavailability 460%, with a plasma half-life of approximately 8 h). Oseltamivir is not metabolized and is excreted almost entirely in the urine. Oseltamivir is administered as the ethyl ester pro-drug that lacks antiviral activity. This is hydrolyzed to the active oseltamivir carboxylate by esterases in the intestinal mucosal epithelia and in the liver. Mechanism of action Zanamivir and oseltamivir target the viral enzyme neuraminidase. Neuraminidase, also called sialidase, is one of the two major inuenza viral proteins (the other is hemagglutinin, which is important for binding and fusion of the virus envelope with the host cell). Neuraminidase is essential for release of newly formed virus from infected cells and for virus spread throughout the respiratory tract of the infected host. Nascent virions bud off from the infected cell encased in an envelope composed of a lipid bilayer derived from the cell plasma membrane with associated viral envelope proteins. Many cell surface glycoproteins of mammalian cells possess sialic acid as the terminal sugar, a residue added during glycosylation events in subcellular Golgi organelles. Inuenza virus envelope proteins are formed in the same subcellular compartment and are therefore also sialylated. However, the cell surface receptor for inuenza virus attachment is sialic acid. Removal of the cell surface and viral envelope protein sialic acid residues is essential to prevent reattachment of the nascent virions

to the same cell and to prevent aggregation of the virus particles. Neuraminidase cleaves terminal sialic acid residues from cellular and viral membrane glycoproteins, thus destroying the cellular receptors recognized by the viral hemagglutinin. Inuenza neuraminidase is a tetramer of identical subunits (Figure 4). The active site in each subunit is a deep groove on the protein surface that comprises residues that are apparently identical in all strains of inuenza A and B. The inhibitors zanamivir and oseltamivir result from rational drug design based on the neuraminidase sialic acid-binding site determined by crystallography. Both drugs bind with high afnity to the active site of viral neuraminidase (Figure 4), thus preventing binding of the normal sialic acid glycoprotein substrate. However, the inhibition mechanisms of the two drugs differ. Zanamivir is an analog of sialic acid and acts as a classical competitive inhibitor to prevent substrate binding to the unliganded enzyme. Oseltamivir is a transition-state analog of sialic acid cleavage and thus interferes with neuraminidase conformational changes needed to allow substrate binding. Nonetheless, the result is the same, and both drugs have similar antiviral potency. Contraindications Few serious clinical toxicities have been reported with zanamivir or oseltamivir. Inhaled zanamivir is generally well tolerated, although exacerbations of asthma may occur. Orally administered oseltamivir may cause nausea and

ANTIVIRAL AGENTS 133

Figure 4 Structure of inuenza B virus neuraminidase bound with the inhibitor zanamivir (in red). The structure was based on pdb1A4G. Adapted from Taylor NR, Cleasby A, Singh O et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of inuenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from inuenza virus types A and B. Journal of Medicinal Chemistry 41: 798807.

gastrointestinal discomfort, although these are transient and less likely if the drug is administered with food.

NH2 O N

Drug for the Treatment of Respiratory Syncytial Virus Infection

Ribavirin

N N O HO

The synthetic nucleoside ribavirin (1-(3,4-dihydroxy5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide) is a guanosine analog that is approved for the treatment of pediatric respiratory syncytial virus (RSV) infection (Figure 5). RSV is the most common cause of severe lower respiratory tract disease in infants, causing up to 90% of bronchiolitis and 40% of bronchopneumonia cases. Disease symptoms are much milder in older children and adults. Although orally administered ribavirin is rapidly absorbed with good bioavailability (approximately 45%), treatment of pediatric RSV infection requires the drug to be administered by continuous aerosol (nebulizer) for 12 18 h daily for 36 days. This therapy is expensive and requires specialized equipment and monitoring. Its use is therefore generally reserved for treatment of RSV infection in high-risk infants (e.g., premature infants, immunocompromised children, or infants with congenital heart disease). Mechanism of action Ribavirin is in fact a broadspectrum antiviral agent that inhibits a wide range of RNA and DNA viruses. The mechanism for the

HO

OH

Figure 5 Structure of ribavirin, which is used for the treatment of high-risk pediatric respiratory syncytial virus infection.

antiviral activity of ribavirin is unclear and may differ for different types of viruses. However, it is generally thought that ribavirin therapy alters cellular nucleotide pools, thereby affecting viral mRNA synthesis. It is likely that ribavirin must be phosphorylated by cellular kinases to provide antiviral activity. Ribavirin-50 -monophosphate inhibits the cellular enzyme inosine-50 -phosphate dehydrogenase, blocking the conversion of inosine-50 -monophosphate to xanthosine-50 -monophosphate. This decreases levels of guanosine triphosphate (GTP), which impacts on both RNA and DNA metabolism. Ribavirin-50 triphosphate may also be a competitive inhibitor of GTP-dependent 50 -capping of viral mRNA. Contraindications Aerosolized ribavirin may cause bronchospasm or conjunctival irritation. It requires close supervision, especially with mechanical

134 APOPTOSIS

ventilation, because precipitation of the drug may occur. Healthcare workers involved in the administration and monitoring of aerosol ribavirin treatment may occasionally experience irritation of the eyes and respiratory tract. Ribavirin is mutagenic, teratogenic, and embryotoxic; thus, aerosolized ribavirin is a risk to pregnant healthcare workers.
See also: Aerosols. Bronchiolitis. Pneumonia: Viral. Upper Respiratory Tract Infection. Vaccinations: Viral. Viruses of the Lung.

Further Reading
Abdel-Magid AF, Maryanoff CA, and Mehrman SJ (2001) Synthesis of inuenza neuraminidase inhibitors. Current Opinion in Drug Discovery and Development 4: 776791. Broughton S and Greenough A (2004) Drugs for the management of respiratory virus infection. Current Opinion in Investigative Drugs 5: 862865. De Clercq E (2004) Antiviral drugs in current clinical use. Journal of Clinical Virology 30: 115133. Dreitlein WB, Maratos J, and Brocavich J (2001) Zanamivir and oseltamivir: two new options for the treatment and prevention of inuenza. Clinical Therapeutics 23: 327355.

Garman E and Laver G (2004) Controlling inuenza by inhibiting the viruss neuraminidase. Current Drug Targets 5: 119136. Johnston SL (2002) Anti-inuenza therapies. Virus Research 82: 147152. Kandel R and Hartshorn KL (2001) Prophylaxis and treatment of inuenza virus infection. BioDrugs 15: 303323. Lew W, Chen X, and Kim CU (2000) Discovery and development of GS4104 (oseltamivir): an orally active inuenza neuraminidase inhibitor. Current Medical Chemistry 7: 663672. Oxford JS, Bossuyt S, Balasingam S, et al. (2003) Treatment of epidemic and pandemic inuenza with neuraminidase and M2 proton channel inhibitors. Clinical Microbiology and Infection 9: 114. Snell NJC (2001) New treatments for viral respiratory tract infections opportunities and problems. Journal of Antimicrobial Chemotherapy 47: 251259. Tam RC, Lau JY, and Hong Z (2001) Mechanisms of action of ribavirin in antiviral therapies. Antiviral Chemistry and Chemotherapy 12: 261272. Taylor NR, Cleasby A, Singh O, et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of inuenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran6-carboxamides and sialidase from inuenza virus types A and B. Journal of Medicinal Chemistry 41: 798807. Torrence PF and Powell LD (2002) The quest for an efcacious antiviral for respiratory syncytial virus. Antiviral Chemistry and Chemotherapy 13: 325344.

APOPTOSIS
A M K Choi and X Wang, University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The phenomenon of programmed cell-death, which is more commonly known as apoptosis, was rst described by C Vogt in 1842. Apoptosis, the term was proposed by Kerr and colleagues in 1972, is an active process of cellular self-destruction with distinctive morphological and biochemical features . It is indispensable in the development and maintenance of homeostasis within all multicellular organisms. The molecular machinery of apoptosis is also important for the regulation of homeostasis during adulthood, which is important for the control of neoplasia and autoimmunity. Understanding how apoptosis occurs in different situations will help to understand the pathogenesis of a number of human diseases and therefore provide clues to the treatment.

Apoptotic Pathways
Caspases are involved in various programmed celldeath pathways reported in the literature. They are a

family of cysteine proteases, and many of them are implicated as important initiators or effectors of the apoptosis process. To date, at least 14 members of this family have been identied, but only a subset of them are partially or fully characterized. Like many enzymes, caspases are synthesized in the cell as inactive precursors and can be cleaved to active form comprised of two associated heterodimers. They are activated or inactivated through a series of intracellular steps, or pathways, in response to death or survival signals, which are subject to multiple regulations. Clearly, the discovery of diverse apoptosis pathways involving signals primarily via the death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) using caspases as effector molecules has dominated the eld. Lately, a more sophisticated view of signaling pathways has arisen which leads to the observation that cell death can occur even in the absence of caspases. The death receptor pathway. The extrinsic pathway is initiated at the cell surface through the

APOPTOSIS 135

Fas/TNF-R1 family protein. Ligation of Fas either by its ligand, FasL, or by its agonistic antibodies triggers the homotrimeric association of the receptors. The clustering of the death domains (DDs) in the intracellular portion of the receptors recruits the adapter molecule, Fas-associated DD containing protein (FADD), which then recruits procaspase-8. Activation of procaspase-8 through self-cleavage leads to a series of downstream events, including activation of procaspase 3, cleavage of multiple caspase substrates, and induction of mitochondrial damages. Fas (CD95/APO-1) is the best-characterized member of the tumor necrosis factor (TNF) superfamily of receptors. Its main and best-known function in signaling is the induction of apoptosis. Fas receptors are expressed on the surface of cells as preassociated homotrimers. Fas is a prototype death receptor characterized by the presence of an 80 amino acid DD in its cytoplasm tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-Fas antibodies or the cognate Fas ligand that initiate apoptosis. The complex of proteins that form upon triggering of Fas is called the death-inducing signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. In the DISC, the adaptor molecule FADD is bound to Fas. FADD has been shown to interact with several proteins through its death effector domain (DED), including caspase-8. These two molecules, FADD

and caspase-8 are the key components of the Fas DISC. Once caspase-8 associates with FADD, the high local concentration of caspase-8 is believed to lead to its autoproteolytic cleavage to p43/41 and p20, and activation. Studies of knockout mice and mutant cell lines established that the DISC is essential for Fas apoptosis signaling. DISC assembly differs between cell types in ways that inuence the efciency of Fas signaling. In type I cell lines and re-stimulated primary T cells, the DISC forms efciently, and apoptosis can be induced with bivalent anti-Fas stimuli without additional cross-linking. The activated caspase-8 directly cleaves caspase-3 in its downstream. However, in type II cell lines and recently activated primary T cells, the DISC is formed inefciently, hypercrosslinking of Fas is necessary to induce apoptosis, and caspase-8 does not activate caspase-3 and cleaves Bid directly instead. The truncated Bid (tBid) translocates to mitochondria (Figure 1). TNF receptor type I (TNFR1) is another important member in the death receptor family. TNF is a highly pleiotropic cytokine that elicits diverse cellular responses ranging from proliferation and differentiation to activation of apoptosis. The different biological activities are mediated by two distinct cell surface receptors: TNFR1 and TNFR2. TNFR1 appears to be the key mediator of TNF signaling. Upon binding of its ligand, TNFR1 recruits the adaptor protein TRADD directly to its cytoplasmic DD. In turn, TRADD serves as an assembly platform to diverge TNFR1 signaling from the DD: interaction of

FasL Fas Membrane FADD

Caspase-8 Activated caspase-8 tBid

DISC

Bid

Bax

Activated-Bax Mitochondria

Cytochrome c Caspase-9 Caspase-3 Cell death

Figure 1 A schematic diagram outlining the cell death pathways.

136 APOPTOSIS

TRADD with RIP-1 and TRAF-2 leads to nuclear factor kappa B (NF-kB) activation. Alternatively, TRADD can recruit FADD and procaspase-8, which is subsequently activated to initiate apoptosis. Under native conditions, a controversy has been reported about the formation of a TNFR1-associated DISC at the plasma membrane. It was reported that TNFR1-induced apoptosis involves two sequential signaling complexes. The initial plasma membrane bound complex (complex I) consists of TNFR1, the adaptor TRADD, the kinase RIP1, and TRAF2 and rapidly signals activation of NF-kB. In a second step, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II) in human brosarcoma cells, HT1080. Recently, it has been reported that the DISC forms at the plasma membrane in several cell lines. We agree with this report in that the difference between the results of Micheau and co-workers and Harper and co-workers may be based on the different compositions of the lysis buffers used and the fact that different labeling and precipitation protocols are employed; we also discovered that the DISC formed at the plasma membrane in primary cultured hepatocytes (our unpublished results), which is consistent with the results of Schneider-Brachert and co-workers. The mitochondrial pathway. Many death stimuli do not seem to depend on the death receptor pathway. Instead, the death signals are transmitted to mitochondria through unique intracellular signaling pathways, where release of cytochrome c is induced. Cytochrome c activates Apaf-1, in the presence of dATP, which in turn activates procaspase-9. Activated caspase-9 can then cleave downstream effector caspases. Mitochondria apoptosis pathway is involved in many types of cell death induced by stress signals, such as irradiation, DNA-damaging drugs, hormone, or growth factor withdrawal. It has to be pointed out that cytochrome c release and caspase activation may not be the only effects caused by the various insults on mitochondria. Others include mitochondrial depolarization and free-radical generation. Death stimuli transmitted through the Fas/TNFR1 death receptor family are mainly mediated directly by caspase cascades in cytosol. However, in certain types of cells, such as hepatocytes, the effector caspases may not be efciently activated by caspase-8 and thus the mitochondria pathway mediated by Bid becomes critical. As mentioned above, Bid is cleaved by caspase-8 and translocated to mitochondria to induce cytochrome c release. The mitochondria pathway is subject to regulation by Bcl-2 family proteins. This family of proteins consists of both death antagonists (Bcl-2, Bcl-XL, Bcl-W, B-1, and Mcf-1) and death agonists (Bax, Bak, Bid,

Bim, Bnip3, Bnip3L (Nix), Bad, Bik, and Bok). They share structural homology in Bcl-2 homology (BH)1, 2, 3, and 4 domains, although not all members have all domains. The BH1 and BH2 domains of the antagonists are required to heterodimerize with the death agonists and repress cell death. Conversely, the BH3 domain of the death agonists is required for them to heterodimerize with the death antagonists and to promote cell death. Bcl-2 family proteins can regulate caspase activation through the regulation of cytochrome c release from mitochondria, which is inhibited by the death antagonists (Bcl-2 or Bcl-XL), and promoted by the death agonists (Bax or Bid). However, the exact mechanisms by which Bcl-2 proteins modulate apoptosis are still subject to much debate and controversy. One hypothesis is that both proapoptotic and antiapoptotic Bcl-2 proteins bind directly to components of the mitochondrial pore, leading to either its opening or closure, respectively. A second hypothesis is that upon activation, proapoptotic members such as Bax and Bak insert into the outer mitochondrial membrane where they oligomerize to form a protein-permeant pore of their own. Regulation of Bcl-2 proteins can occur at multiple levels. For example, Bid is cleaved by caspase-8 to form tBid, which then translocates to the mitochondrion and induces permeability transition, suggesting a linkage between the extrinsic and intrinsic pathways. In the context of signal transduction, phosphorylation also plays a critical role in regulating Bcl-2 proteins. In general, serine phosphorylation of Bad by multiple kinases causes its sequestration and hence inactivation by 14-3-3 proteins, and phosphorylation of Bim can target it for proteosomal degradation (13). The other apoptosis is the endoplasmic reticulum (ER)-involved pathway. As mentioned above, two major apoptotic cascades are triggered by specic initiator caspases: the death receptor pathway and the mitochondrial pathway. These are activated by caspase-8 and caspase-9, respectively. The key caspase in the ER pathway is caspase-12. Proper folding of polypeptide into a three-dimensional structure is essential for cellular function, and protein malfolding can threaten cell survival. Various conditions can perturb the protein folding in the ER, which is collectively called ER stress. In order to adapt ER stress conditions, the cells respond in three distinct ways such as transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation. After ER functions are severely impaired, the cell is eliminated by apoptosis via transcriptional induction of CHOP/GADD153, the activation of c-Jun NH2-terminal kinase, and/or the activation of caspase-12. This type of cellular stress is receiving increased attention because it is considered a cause of

APOPTOSIS 137

pathologically relevant apoptosis and is especially implicated in neurodegenerative disorders. ER stress activates caspase-12 on the surface of the ER, and caspase-12-decient cells are resistant to ER stress inducers, indicating that caspase-12 is significant in ER stress-induced apoptosis. However, the mechanism responsible for caspase-12 activation is largely unknown, unlike in the case of both caspase-8 and caspase-9 whose activation mechanisms have been revealed at the molecular level.

Organelle Dysfunction
Mitochondria. As the main energy source for all cellular processes, the proper function of the mitochondrion is important to the survival of the cell. The dysfunction of this organelle can usually be attributed to depolarization of the membrane, resulting in the loss of its property of selective mitochondrial membrane permeability (MMP), usually resulting in cell death. Many proapoptotic signals and antiapoptotic defenses converge in the mitochondrion. The congregation of proteins like Bax on the outer membrane cause alteration in the membrane permeability and compromise the integrity of the mitochondrial membrane, where MMP ensue. On the other hand, the mitochondrion can mount a defense through the antideath members of the Bcl-2 family, namely Bcl-2 and Bcl-XL. For example, these proteins usually reside at the mitochondrial membrane and can inhibit the function of Bax and consequently prevent the onset of MMP. It has been reported that caspase-8 resides in mitochondria. In the human breast carcinoma cell line MCF7, caspase-8 predominantly colocalizes with and binds to mitochondria. After induction of apoptosis through Fas or TNFRI, active caspase-8 translocate to plectin, a major cross-linking protein of the three main cytoplasmic lament systems, whereas the caspase-8 prodomain remain bound to mitochondria. Golgi apparatus. To date, there is no direct evidence suggesting that the Golgi apparatus is directly involved in the initiation of any apoptotic pathway. However, it has been reported that many apoptosissignaling proteins are enriched at the Golgi membrane. Among them are caspase-2, TNFR, Fas, and TNF-related apoptosis-inducing ligand receptor 1. Recently, we have found that the DISC (Fas/FADD/ caspase-8) forms rst in Golgi complex, and then translocates to plasma membrane. The blockage of the DISC transport dramatically decreases the DISC level in plasma membrane. Plasma membrane. The plasma membrane is the rst line of defense for the cell against extracellular

stimuli. With the right type of stimulus, the corresponding receptor will instigate a signal cascade that results in cell death. To date, three different types of death receptors have been identied: the TNFR, the Fas, and the APO-3. There was a report that Fas in type I lymphocytes associates with glycosphingolipid-enriched detergent-resistant membrane microdomains termed lipid rafts. Although there is a controversy if the DISC formation is involved in lipid raft, we have found that the DISC formation localizes in the lipid raft of lung endothelial cells exposed to hypoxia/reoxygenation. The lipid-based machinery may be involved in the formation of carriers trafcking from the Golgi complex to the cell surface.

Bcl-XL
It is well known that Bcl-XL protects cells from apoptosis mediated by a variety of stimuli, but the mechanisms are not fully understood. Overexpression of Bcl-XL reportedly confers protection upon mitochondria, making it more difcult for numerous stimuli to induce permeability transition pore opening; or Bcl-XL tends to form small channels that assume a mostly closed conformation, preferring cations; Bcl-XL sequesters BH3 domain-only molecules in stable mitochondria complexes, preventing the activation of Bax. Bcl-XL was reported to block Bax translocation. Studies by others reported that Bcl-XL could not block activation of Fas by FasLinduced apoptosis, especially caspase-8 activation. A role for caspase-8 mediating Bid cleavage accompanied by cytochrome c release was observed in focal cerebral ischemia endothelial cells and focal cerebral ischemia in rats. It has been established that Bcl-XL blocks Bid cleavage and functions downstream of caspase-8 to inhibit Fas-induced apoptosis of MCF7 breast carcinoma cells. Recently, we have found that Bcl-XL inactivates caspase-8 by disrupting DISC formation in the plasma membrane, Golgi complex, and nucleus. Bcl-XL retains the DISC in mitochondria where caspase-8 is inactivated. Bcl-XL breaks the physical association of Fas and caspase-8 with GRASP65, a Golgi-apparatus-related protein. This indicates that Bcl-XL downregulates the transfer of DISC to the plasma membrane by the Golgi component, at the same time diverting the DISC formation to the mitochondria. FLIP (FLICE (Fas-associated death-domain-like IL-1beta-converting enzyme)-inhibitory protein)) was identied as an inhibitor of Fas signals. At least four splice variants have been identied (3134). The largest variant FLIP long (FLIPL), a protein of 440 amino acids, is highly homologous to caspase-8. In fact, FLIPL contains a

138 APOPTOSIS

caspase-like domain at the carboxy-terminus but, as indicated by the name, was originally characterized as a molecule with inhibitory activity on caspase-8, in which FLIPL inhibits the nal cleavage between the prodomain and the p20 subunit of the p43/41 intermediate. In addition to inhibition of receptor-mediated apoptosis, more recently, it has been reported that FLIP also promotes activation of NF-kB and Erk signaling pathway. Therefore, FLIP is not simply an inhibitor of death-receptor-induced apoptosis but also mediates the activation of NF-kB. The death effector domain (DED) of FLIP binds to Fas/FADD complexes and inhibits the recruitment and activation of procaspase-8 and therefore acts as antiapoptotic molecule. We have reported that, in addition to inhibiting the recruitment of caspase-8 into the DISC by decreasing DISC formation in the plasma membrane, FLIP blocks the transfer of the DISC formed in the Golgi to the plasma membrane. FLIP expression also inhibits Bax activation and Bax-induced apoptotic cell death by promoting the association of the inactive form of PKC to Bax, which inactivates Bax.

The Effect of Tyrosine Kinases on Apoptosis

Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes such as growth, differentiation, metabolism, and apoptosis in response to external and internal stimuli. Tyrosine kinases are a family of enzymes, which catalyze phosphorylation of select tyrosine residues in target proteins, using ATP. Tyrosine kinases are primarily classied as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and c-Met, and nonreceptor tyrosine kinases, such as SRC, ABL, FAK, and Janus kinase. The receptor tyrosine kinases are not only cell surface transmembrane receptors, but also enzymes having kinase activity. The structural organization of the receptor tyrosine kinase exhibits a multidomain extracellular ligand for conveying ligand specicity, a single pass transmembrane hydrophobic helix, and a cytoplasmic portion containing a tyrosine kinase domain. The kinase domain has regulatory sequence both on the N- and C-terminal end. Nonreceptor tyrosine kinases are cytoplasmic proteins, exhibiting considerable structural variability. The nonreceptor tyrosine kinase has a kinase domain and often possesses several additional signaling or proteinprotein interacting domains such as Src homology (SH)2, SH3, and the phosphotyrosine homology (PH)

domain. Generally, receptor tyrosine kinases activate many signaling pathways to inhibit apoptosis by regulating Bcl-2 family member protein expression and their phosphorylation. For example, receptor tyrosine kinases activate the serine/threonine kinase Akt, which inhibits apoptosis through the Bad phosphorylation. Here, we focus on recent novel ndings that tyrosine kinases are involved in apoptotic regulation. The initiation of Fas-mediated apoptotic pathway is to stimulate Fas activities through Fas phosphorylation. The phosphorylation at the Fas tyrosine is thought to be prerequisite for Fas membrane trafcking and DISC formation in hepatocytes exposed to hyperosmolarity and Fas ligand in endothelial cells of the lung exposed to hypoxia/reoxygenation (our unpublished results). EGFR is reported to associate with Fas and induce Fas phosphorylation at the tyrosine site through EGFR tyrosine kinase activity. Hepatocyte growth factor (HGF) receptor, c-Met, can inhibit Fas-mediated apoptosis by physically binding to Fas, which may block the Fas conformation change required for trafcking and DISC formation in hepatocytes. HGF can upregulate the c-Met/Fas association through c-Met-signaling pathway in lung epithelial and endothelial cells (our unpublished results). HGF also inhibits Bax and Bid activation by activating p38 MAPK that induces Bax and Bid phosphorylation, which decreases their conformation change and activation. The Src kinase family comprises three ubiquitously expressed members (Src, Fyn, Yes), which share functional domains such as an amino-terminal myristoylation sequence for membrane targeting, SH2 and SH3 domains, a family member-specic unique region, and a kinase domain and a carboxy-terminal noncatalytic domain. Recent studies on vascular smooth muscle cells and endothelial cells indicated an involvement of c-Src, but not of Fyn, in the c-Jun NH2-terminal kinase (JNK) activation in response to oxidative stress. Also, oxidative stress-induced activation of Erk-5 in broblasts was shown to be Src-dependent, but not Fyn-dependent, whereas Fyn, but not Src, was required for activation of p90 ribosomal S6 kinase by reactive oxygen species. It has been reported that hydrophobic bile acids rapidly activate Yes but not Fyn, Src, or Lck. Activated Yes associates with the EGFR in a protein kinase A-sensitive way and acts as an EGFR-activating kinase, thereby triggering a decisive event in bile acid-induced apoptosis. Phosphorylation of the p53 tumor suppressor protein is a critical event in the upregulation and activation of p53 during cellular stress. It was demonstrated that the signaling pathway linking oxidative stress to p53 through PDGFb receptor. Hydrogen peroxide-induced phosphorylation of the

APOPTOSIS 139

PDGFb receptor and p53 activation was inhibited by kinase-inactive forms of the PDGFb receptor.

Concluding Remarks
Apoptosis plays an important role in cell growth, differentiation, and homeostasis. It is obvious that our current knowledge in terms of the mechanism of apoptosis and its regulation is far from complete. For example, we do not have conclusive answers to questions such as: how does the DISC formation transport from Golgi complex to plasma membrane? What is the importance/significance of the DISC formed rst in Golgi complex? How does FLIP affect the Bax/mitochondria apoptotic pathway? What is the mechanism of the dual function (antiapoptosis and proapoptosis) of tyrosine kinases? Much more effort is required to dissect and document these pathways. Studies of the apoptotic regulation will help understand the mechanisms of cell death or cancer development. We must better characterize the novel pathways of cell death and further our understanding of the pathologies underlying a variety of human health problems.
See also: CD14. Cysteine Proteases, Cathepsins. DNA: Repair. Extracellular Matrix: Collagens. Hepatocyte Growth (Scatter) Factor. Ion Transport: Overview. Myobroblasts. NADPH and NADPH Oxidase. Oncogenes and Proto-Oncogenes: jun Oncogenes. Transcription Factors: AP-1; NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Anto RJ, Mukhopadhyay A, Denning K, and Aggarwal BB (2002) Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 23: 143150. Bin L, Li X, Xu LG, and Shu HB (2002) The short splice form of casperc-FLIP is a major cellular inhibitor of TRAIL-induced apoptosis. FEBS Letters 510: 3740. Cao XX, Mohuiddin I, Chada S, et al. (2002) Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Molecular Medicine 8: 869876. Chen K, Albano A, Ho A, and Keaney JF Jr (2003) Activation of p53 by oxidative stress involves platelet-derived growth factorbeta receptor-mediated ataxia telangiectasia mutated (ATM) kinase activation. Journal of Biological Chemistry 278: 39527 39533. Cheng EH, Wei MC, Weiler S, et al. (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAXand BAK-mediated mitochondrial apoptosis. Molecular Cell 8: 705711. Creagh EM, Conroy H, and Martin SJ (2003) Caspase-activation pathways in apoptosis and immunity. Immunological Reviews 193: 1021.

Eramo A, Sargiacomo M, Ricci-Vitiani L, et al. (2004) CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. European Journal of Immunology 34: 19301940. Gniadecki R (2004) Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. Biochemical and Biophysical Research Communications 320: 165169. Harper N, Hughes M, MacFarlane M, and Cohen GM (2003) Fasassociated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. Journal of Biological Chemistry 278: 2553425541. Hsu H, Shu HB, Pan MG, and Goeddel DV (1996) TRADDTRAF2 and TRADD-FADD interactions dene two distinct TNF receptor 1 signal transduction pathways. Cell 84: 299308. Huang DC, Hahne M, Schroeter M, et al. (1999) Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x (L). Proceedings of the National Academy of Sciences, USA 96: 1487114876. Kataoka T, Budd RC, Holler N, et al. (2000) The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Current Biology 10: 640648. Kerr JF, Wyllie AH, and Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239257. Kroemer G and Reed JC (2000) Mitochondrial control of cell death. Nature Medicine 6: 513519. Krueger A, Schmitz I, Baumann S, Krammer PH, and Kirchhoff S (2001) Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. Journal of Biological Chemistry 276: 2063320640. Liou AK, Clark RS, Henshall DC, Yin XM, and Chen J (2003) To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways. Progress in Neurobiology 69: 103142. Los M, Wesselborg S, and Schulze-Osthoff K (1999) The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10: 629639. Micheau O and Tschopp J (2003) Induction of TNF receptor Imediated apoptosis via two sequential signaling complexes. Cell 114: 181190. Momoi T (2004) Caspases involved in ER stress-mediated cell death. Journal of Chemical Neuroanatomy 28: 101105. Muppidi JR and Siegel RM (2004) Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nature Immunology 5: 182189. Paul MK and Mukhopadhyay AK (2004) Tyrosine kinase role and significance in Cancer. International Journal of Medical Science 1: 101115. Peter ME and Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death and Differentiation 10: 2635. Plesnila N, Zinkel S, Amin-Hanjani S, Qiu J, Korsmeyer SJ, and Moskowitz MA (2002) Function of BID a molecule of the bcl2 family in ischemic cell death in the brain. European Surgical Research 34: 3741. Reed JC (1998) The Bcl-XL family proteins. Oncogene 17: 3225 3226. Reinehr R, Schliess F, and Haussinger D (2003) Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafcking and DISC formation. FASEB Journal 17: 731733. Scafdi C, Fulda S, Srinivasan A, et al. (1998) Two CD95 (APO-1/ Fas) signaling pathways. The EMBO Journal 17: 16751687.

140 AQUAPORINS
Scafdi C, Schmitz I, Krammer PH, and Peter ME (1999) The role of c-FLIP in modulation of CD95-induced apoptosis. Journal of Biological Chemistry 274: 15411548. Schneider-Brachert W, Tchikov V, Neumeyer J, et al. (2004) Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21: 415428. Srinivasan A, Li F, Wong A, et al. (1998) Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells. Journal of Biological Chemistry 273: 45234529. Stegh AH, Barnhart BC, Volkland J, et al. (2002) Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein. Journal of Biological Chemistry 277: 43514360. Stegh AH, Herrmann H, Lampel S, et al. (2000) Identication of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis. Molecular and Cellular Biology 20: 56655679. Szegezdi E, Fitzgerald U, and Samali A (2003) Caspase-12 and ERstress-mediated apoptosis: the story so far. Annals of the New York Academy of Sciences 1010: 186194. Tschopp J, Irmler M, and Thome M (1998) Inhibition of fas death signals by FLIPs. Current Opinion in Immunology 10: 552558. Wajant H, Pzenmaier K, and Scheurich P (2003) Tumor necrosis factor signaling. Cell Death and Differentiation 10: 4565. Wang X, DeFrances MC, Dai Y, et al. (2002) A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Molecular Cell 9: 411421. Wang X, Wang Y, Zhang J, et al. (2005) FLIP protects against hypoxia/reoxygenation-induced endothelial cell apoptosis by inhibiting Bax activation. Molecular and Cellular Biology 25: 47424751. Wang X, Zhang J, Kim HP, et al. (2004) Bcl-XL disrupts deathinducing signal complex formation in plasma membrane induced by hypoxia/reoxygenation. FASEB Journal 18: 18261833. Wang X, Zhou Y, Kim HP, et al. (2004) Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. Journal of Biological Chemistry 279: 5237 5243. Xiao C, Yang BF, Asadi N, Beguinot F, and Hao C (2002) Tumor necrosis factor-related apoptosis-inducing ligand-induced deathinducing signaling complex and its modulation by c-FLIP and PEDPEA-15 in glioma cells. Journal of Biological Chemistry 277: 2502025025. Yin XM (2000) Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Research 10: 161167. Yin XM and Ding WX (2003) Death receptor activation-induced hepatocyte apoptosis and liver injury. Current Molecular Medicine 3: 491508.

AQUAPORINS
A S Verkman and Y Song, University of California, San Francisco, CA, USA
& 2006 Elsevier Ltd. All rights reserved. uid transport in airways, pleura, and lung compared to renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress/injury not yet tested or in functions unrelated to transepithelial uid transport.

Abstract
Aquaporins (AQP) are a family of water transporting proteins expressed in many epithelial, endothelial, and other tissues. AQP1 is expressed in microvascular endothelia throughout the lung/airways, AQP3 in basal cells in large airways, AQP4 at the basolateral membrane of epithelia throughout the airways, and AQP5 at the apical membrane of type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these aquaporins increases near the time of birth and appears to be regulated by growth factors, inammation, and osmotic stress, suggesting a role of aquaporins in lung physiology. However, studies in transgenic mouse models of AQP deciency have provided evidence against an important physiological role for aquaporins in many lung functions. Although AQP1 and AQP5 provide the principal route for osmotically driven alveolar water transport, alveolar uid clearance in the neonatal and adult lung is not affected by AQP deletion, nor is lung CO2 transport or uid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, airway surface liquid layer volume and composition, and airway uid absorption are not impaired by AQP3/AQP4 deletion. In airway submucosal glands, AQP5 deletion significantly reduced the rate and increased the protein content of uid secretions. Thus, although AQPs have many important extrapulmonary physiological functions, in the lung/ airways AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of

Description
Fluid movement between the distal airspace and interstitial and vascular compartments of the lung/airways is important in the maintenance of airspace hydration, the absorption of airspace uid near the time of birth and in pulmonary edema, and the secretion of uid onto the airway surface by submucosal glands. Aquaporins (AQPs), a family of small (B30 kDa monomer) integral membrane water channel proteins, are expressed in many cell types involved in uid transport. There are more than 10 aquaporins in mammals, at least four of which are expressed in the respiratory system (Figure 1). AQP1 is expressed in microvascular endothelia near airways and alveoli, as well as in microvessels and mesothelial cells of visceral and parietal pleura. AQP3 is expressed in the basolateral membrane of basal epithelial cells lining the trachea and large airways, and at the basal membrane in human (but not rodent) alveoli. AQP4 is expressed in the basolateral membrane

AQUAPORINS 141
AQP3 AQP4 AQP5 Nasopharnyx (upper airways) Submucosal gland Microvessels

Airway surface liquid

AQP1 Trachea Pleura Lung AQP1 Alveolus Type I Type II Microvessels Distal airway AQP4 AQP3

AQP5

AQP1

Figure 1 Aquaporin expression in lung and airways. AQP1 is expressed in microvascular endothelial and pleural membranes; AQP3 in large airway epithelia; AQP4 at the basolateral membrane in epithelia throughout the airways; and AQP5 at the apical membrane of alveolar type I cells and of serous acinar cells in submucosal glands.

High osmolality

Low osmolality

Water

Figure 2 Tetrameric structure of AQPs. Each AQP tetramer consists of four B30 kDa monomers, each of which contains a narrow water-transporting pore. Water moves across AQP pores in response to osmotic gradients.

of ciliated columnar cells in bronchial, tracheal, and nasopharyngeal epithelia. AQP5 is expressed in the apical membrane of type I alveolar epithelial cells and of acinar cells in submucosal glands. AQP5 has also been reported in human lung to be expressed in bronchial and nasopharyngeal acinar and ciliated duct columnar cells. This expression pattern in uid

transporting cells provides indirect evidence for a role of aquaporins in lung/airway physiology. AQPs 1, 4, and 5 are water selective, and consist of tetramers of four monomeric B30 kDa subunits, each of which contains an independent narrow pore pathway for water transport (Figure 2), whereas AQP3 (an aquaglyceroporin) transports both water and glycerol.

142 AQUAPORINS

Normal Physiological Processes

Developmental Regulation of Aquaporin Expression

Fluid Transport in Distal Lung

Rodent studies have shown developmental regulation of lung aquaporin expression with distinct patterns for each aquaporin. AQP1 is detectable just before birth in rodents, increasing several-fold perinatally and into adulthood. Functional measurements in rabbit showed significantly increased lung water permeability in the perinatal period in parallel to increasing AQP1 expression. AQP1 expression is also upregulated by treatment with corticosteroids. In contrast, little AQP5 is expressed at birth and gradually increases until adulthood, whereas AQP4 expression strongly increases just after birth and is upregulated by -agonists and glucocorticoids.
Regulation of Aquaporin Expression in Adult Lung

Regulated aquaporin expression is also seen in the adult lung. As in prenatal lung, AQP1 expression can be upregulated by corticosteroids. AQP1 and AQP5 expression are reduced in rodent lung following adenoviral infection, and AQP5 expression is increased after bleomycin exposure. Reduced AQP5 expression was found after exposure of a mouse lung epithelial cell line (MLE-12) to tumor necrosis factor alpha (TNF-a), suggesting a possible mechanism for its downregulation in viral infection in vivo. AQP5 expression was increased in MLE-15 and alveolar epithelial cells exposed to hypertonicity. Although potentially interesting, the physiological relevance of many of these observations is unclear, as the airway/ lung is probably not exposed to significant hypertonicity, and regulated AQP expression is a general phenomenon not specic to the lung/airways.
Role of Aquaporins from Functional Studies in Knockout Mice

Transgenic mice lacking each of the lung aquaporins (AQP1, AQP3, AQP4, and AQP5) were generated in our laboratory both individually and in combinations. Comparative studies were done in wild-type and AQPdecient mice to investigate the physiological role of aquaporins in the lung/airways. These mice have been quite informative in elucidating important roles of various aquaporins outside the lung. For example, mice lacking AQPs 14 are defective in their ability to concentrate urine, mice lacking AQP1 have impaired angiogenesis and cell migration, mice lacking AQP3 have reduced epidermal hydration, mice lacking AQP4 have altered brain water balance and impaired neural signal transduction, mice lacking AQP5 have impaired saliva secretion, and mice lacking AQP7 manifest progressive fat accumulation and adipocyte hypertrophy.

The proposed aquaporin functions in distal lung include alveolar uid absorption at the time of birth and in the adult lung, gas (CO2, O2) exchange, and formation/resolution of lung edema in response to acute and subacute lung injury. AQP5 is expressed in type I alveolar epithelial cells and AQP1 in microvascular endothelial cells. Osmotic water permeability between alveolus and capillary was B10-fold reduced in lungs of AQP1 and AQP5 null mice compared to wild-type mice, and 30-fold reduced by AQP1/AQP5 codeletion (in double knockout mice). Interestingly, AQP1 deletion mildly reduced hydrostatic lung edema in an isolated perfused lung preparation, and computerized tomographic analysis of two humans lacking AQP1 showed a blunted increase in airway wall thickness following saline infusion compared to control subjects. Reduced hydrostatic lung edema in AQP1 deciency may be related to an abnormality in microvasculature deciency, since on theoretical grounds hydrostatic driving forces should be unable to produce significant net uid movement across a water-only pathway. Alveolar uid clearance is an important function of the alveolar epithelium. Fluid absorption is the result of sodium absorption through the epithelial sodium channel (ENaC) in response to the electrochemical driving force created by the basolateral membrane Na-K-ATPase of type II alveolar epithelial cells. The consequent osmotic imbalance drives water absorption primarily through the type I cells. Alveolar uid clearance is measured in uid-lled lung models from the kinetics of increasing concentration of an airspace volume marker such as radiolabeled albumin. Remarkably, even with maximal stimulation of alveolar uid absorption with betaagonists and pretreatment with keratinocyte growth factor (to increase the number of type II cells), AQP1 or AQP5 deletion did not reduce alveolar uid clearance. Further, the rapid absorption of uid from the airspace just after birth was not impaired by aquaporin deletion, nor was lung edema following acidinduced epithelial cell injury, thiourea-induced endothelial cell injury, or hyperoxic subacute lung injury. The much slower rate of maximal alveolar uid absorption (0.016 ml min 1cm 2) compared to uid absorption in kidney proximal tubule or saliva secretion in salivary gland (410 ml min 1 cm 2) may account for the lack of effect of AQP1 and AQP5 deletion on alveolar uid clearance. Because rates of uid transport are relatively low in lung, the low intrinsic (aquaporin independent) water permeability of the alveolar epithelial and capillary membranes appears to be adequate to allow uid

AQUAPORINS 143

transport to occur without impairment under normal physiological conditions and in response to clinically relevant stresses.
Fluid Transport in the Pleura

Fluid is continuously secreted into and reabsorbed from the pleural space. Little uid is present in the pleural space (0.2 ml kg 1) despite its large surface area (4000 cm2 in man, 10 cm2 in mouse). Fluid entry into the pleural space involves ltration across microvascular endothelia near the pleural surface, and movement across a mesothelial barrier lining the pleural space, whereas uid clearance is thought to occur primarily by lymphatic drainage. Pleural uid can accumulate in pathological conditions such as congestive heart failure, lung infection, lung tumor, and the acute respiratory distress syndrome. AQP1 is expressed in microvascular endothelia near the visceral and parietal pleura and in mesothelial cells in visceral pleura. Osmotic water permeability across the pleural barrier, measured from the kinetics of pleural uid osmolality changes after instillation of hypertonic or hypotonic uid into the pleural space, was rapid in wild-type mice (50% osmotic equilibration in 2 min), and slowed by fourfold in AQP1 knockout mice. However, the clearance of saline instilled in the pleural space was not affected by AQP1 deletion, nor was the accumulation of pleural uid in a uid overload model produced by intraperitoneal saline administration or in a thiourea model of acute endothelial injury. Thus, although rapid osmotic equilibration across the pleural surface is facilitated by AQP1, as found in distal lung, AQP1 does not appear to play a major role in physiologically important mechanisms of pleural uid accumulation or clearance.
Fluid Transport in the Airways

air through a tracheotomy, or of upper airways, as measured from the moisture content of dry air passed through the upper airways in mice breathing through a tracheotomy. Also, the depth and salt concentration of the ASL in the trachea, as measured in vivo using uorescent probes and confocal microscopy, was not altered by AQP3/AQP4 deciency. Finally, active isosmolar uid absorption, measured in nasopharyngeal airways (using a volume marker as done for alveolar uid clearance) was not impaired by aquaporin deletion. Thus, although AQP3/AQP4 facilitate osmotic water transport in the airways, they play at most a minor role in airway humidication, ASL hydration, and isosmolar uid absorption. Interestingly, one study showed increased airway reactivity in response to bronchoconstricting agents in AQP5 null mice. The mechanism of this phenotype was not established, but may be related to indirect effects of AQP5 deletion on agonist-induced uid secretion from submucosal glands as described below.
Fluid Secretion by Airway Submucosal Glands

Potential functions of aquaporins in the airways include humidication of inspired air, regulation of airway surface liquid (ASL) volume and composition, and absorption of uid from the airways. Evaporative water loss in the airways is thought to drive water inux from capillaries and interstitium into the ASL by the generation of an osmotic gradient. The depth and ionic composition of the ASL should depend theoretically on the ion transporting properties of the airway epithelium and the rate of evaporative water loss, as well as the water permeability of the airway-capillary barrier. Osmotic water permeability in upper airways, measured by dilution of an airway volume marker in response to an osmotic gradient, was reduced in mice lacking AQP3 and/or AQP4. However, there was little effect of AQP3/AQP4 deletion on humidication of lower airways, as measured from the moisture content of expired air during mechanical ventilation with dry

Submucosal glands in mammalian airways secrete a mixture of water, ions, and macromolecules onto the airway surface. Glandular secretions are important in establishing ASL uid composition and volume, and in antimicrobial defense mechanisms. Abnormally viscous gland secretions in cystic brosis have been proposed to promote bacterial adhesion and inhibit bacterial clearance. Submucosal glands contain serous tubules, where active salt secretion into the gland lumen creates a small osmotic gradient driving water transport across a water-permeable epithelium, as well as mucous cells and tubules, where viscous glycoproteins are secreted. AQP5 is expressed at the luminal membrane of the serous epithelial cells. Pilocarpinestimulated uid secretion was found to be reduced by twofold in AQP5 null mice, as determined by nasopharyngeal uid collections and video imaging of uid droplets (covered with mineral oil) secreted by individual submucosal glands. Analysis of secreted uid showed a twofold increase of total protein concentration in AQP5 null mice, suggesting intact protein and salt secretion across a relatively water-impermeable epithelial barrier. There was no significant difference of submucosal gland morphology or density in wildtype versus AQP5 knockout mice. AQP5 thus facilitates uid secretion in submucosal glands, indicating that the luminal membrane of serous epithelial cells is the rate-limiting barrier to water movement.
CO2 Transport by Aquaporins in Lung

AQP1-dependent CO2 transport has been proposed based on measurements in AQP1-overexpressing

144 ARTERIAL BLOOD GASES

Xenopus oocytes; however, subsequent studies showed unimpaired CO2 permeability in AQP1-decient erythrocytes, where rapid CO2 transport occurs by a passive membrane solubility-diffusion mechanism. Measurements of CO2 movement in the perfused and in vivo mouse lung showed no effect of AQP1 deletion, providing direct evidence against the role of AQP1 in lung CO2 transport.

expression/function to reduce secretion/ASL viscosity in cystic brosis.

See also: Basal Cells. Fluid Balance in the Lung.

Further Reading
Agre P, King LS, Yasui M, et al. (2002) Aquaporin water channels from atomic structure to clinical medicine. Journal of Physiology 542: 316. Bai C, Fukuda N, Song Y, et al. (1999) Lung uid transport in aquaporin-1 and aquaporin-4 knockout mice. Journal of Clinical Investigation 103: 555561. Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in uid transport in lung and airways. Journal of Applied Physiology 93: 21992206. Dobbs L, Gonzalez R, Matthay MA, et al. (1998) Highly waterpermeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung. Proceedings of the National Academy of Sciences, USA 95: 29912996. Folkesson H, Matthay MA, Frigeri A, and Verkman AS (1996) High transepithelial water permeability in microperfused distal airways: evidence for channel-mediated water transport. Journal of Clinical Investigation 97: 664671. King LS, Nielsen S, and Agre P (1996) Aquaporin-1 water channel protein in lung-ontogeny, steroid-induced expression, and distribution in rat. Journal of Clinical Investigation 97: 2183 2191. King LS, Nielsen S, Agre P, and Brown RH (2002) Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proceedings of the National Academy of Sciences USA 99: 10591063. Krane CM, Fortner CN, Hand AR, et al. (2001) Aquaporin-5 decient mouse lungs are hyperresponsive to cholinergic stimulation. Proceedings of the National Academy of Sciences, USA 98: 1411414119. Ma T, Fukuda N, Song Y, Matthay MA, and Verkman AS (2000) Lung uid transport in aquaporin-5 knockout mice. Journal of Clinical Investigation 105: 93100. Saadoun S, Papadopoulos M, Hara-Chikuma M, and Verkman AS (2005) Targeted AQP1 gene deletion impairs angiogenesis and cell migration. Nature 434: 786792. Song Y and Verkman AS (2001) Aquaporin-5 dependent uid secretion in airway submucosal glands. Journal of Biological Chemistry 276: 4128841292. Verkman AS (2002) Physiological importance of aquaporin water channels. Annals of Medicine 34: 192200.

Aquaporins and Respiratory Disease

A small number of AQP1-decient humans have been identied. Although initially reported to have no phenotype, subsequent studies showed that they manifest a urine concentrating defect that is qualitatively similar to that found in AQP1-decient mice. As mentioned above, AQP1-decient subjects have also been found to have a small reduction in the increase in bronchiolar wall thickness following intravenous volume overload compared to normal controls, though other lung phenotypes have not been reported. The significance of this observation is unclear. Together with a considerable body of data in transgenic mice, the functional studies suggest that aquaporins play at most a minor role in normal lung physiology and clinically relevant states of lung injury, with the possible exception of AQP5 in submucosal uid secretion. It remains unresolved why aquaporins are expressed at multiple sites of uid movement in the lung/airways, and why the expression of lung aquaporins appears to be altered in states of stress. When available, nontoxic aquaporin-selective inhibitors will be useful to examine effects of acute aquaporin inhibition, which may reveal lung/ airway phenotypes that might not be manifest in mice or humans with chronic aquaporin deciency. If significant aquaporin-dependent phenotypes are found, then pharmacological modulation of aquaporin function may have clinical applications, such as AQP5 inhibition in reducing glandular uid secretions in allergic and infectious rhinitis, or increasing AQP5

ARTERIAL BLOOD GASES

J W Severinghaus, UCSF, San Francisco, CA, USA
& 2006 Elsevier Ltd. All rights reserved. following inventions by R Stow (CO2) and L Clark (PO2) both dating from 1954. From these outputs, internal computers calculate O2 saturation, base excess, bicarbonate, and other derived variables such as the compensation by the body for acidbase abnormalities. Arterial PO2 and PCO2 can be approximated using heated skin surface transcutaneous electrodes, which are commonly used in premature infants and nurseries. Hemoglobin oxygen saturation, SO2%, is also directly measured by

Abstract
Blood gas analyzers consist of three electrodes measuring pH, PCO2 , and PO2 at 371C. They were introduced in about 1960

ARTERIAL BLOOD GASES 145

multiwavelength blood oximeters. Arterial SO2 is approximated by pulse oximeters, which detect the arterial pulsatile variations in red and infrared light penetrating a nger, ear, or other tissue, a method invented by T Aoyagi in Tokyo in 1973 that became commercially available in 1983. Interpretation of blood gases and acidbase balance is briey discussed. Figures include schema of the three electrodes, a pulse oximeter probe, an acidbase compensation diagram, and photographs of the rst three-function blood gas analyzer, a combined PO2 PCO2 transcutaneous electrode in use on a child, and a pulse oximeter probe on a nger.

HendersonHasselbalch (HH) equation: pH pK0 logHCO 3 =SPCO2 In plasma, pK0 6.1, the effective dissociation constant of H2CO3 (carbonic acid), calculated as S PCO2 . S is CO2 solubility, 0.031 mM l 1 Torr 1. HCO3 is plasma bicarbonate content calculated as plasma [CO2 content SPCO2 ].

Introduction
Arterial blood gas analyzers directly measure pH, PCO2 and PO2 , (mmHg or Torr) and calculate stand ard base excess (SBE), bicarbonate (HCO3 ), oxygen saturation (SO2), and other variables useful in diagnosis and clinical management of patients in emergencies, anesthesia, surgery, recovery, and intensive care. These tests were rarely done until the 1950s when electrodes were invented and developed. Understanding of acidbase and blood gas theory depended on discoveries in physical chemistry.
Ionic Theory

Electrodes
pH Electrode

Electrochemistry and physical chemistry of solutions of acids, alkalis, metals, and salts were transformed from empiricism to theory by the 1884 thesis of Svante Arrhenius in Uppsala, Sweden. Wilhelm Ostwald then used a platinum electrode to measure hydrogen ion strength electrically. In 1893, Ostwalds student Walther Nernst applied the longestablished laws of gases to ions in solution to calculate the electrical potential of batteries or cells.
Buffers

In 1905, Max Cremer noted that hydrogen ions permeated some kinds of very thin glass, developing electrical potential gradients across the glass. Fritz Haber and Z Klemensiewicz made the rst glass pH electrode in 1909. Its potential was a linear function of pH, not H ion concentration. In 1925, the rst glass cup-shaped blood pH electrode was produced by Phyllis T Courage. By 1933, capillary blood pH electrodes were being made commercially. Blood pH was corrected to 371C with the Rosenthal factor ( 0.0147 1C 1) until thermostatted blood pH electrodes became available in the 1950s. A pH and reference electrode is schematically shown in Figure 1. Details Special glass compositions permit hydrogen ions to diffuse through imperfectly annealed submicroscopic cracks, probably exchanging loci with loosely bound alkali cations, especially lithium. Some pH electrodes are sensitive to very high Na concentrations. At 371C, the electromotive force (EMF) across the glass measured with reference electrodes (usually silversilver chloride) is 61.5 mV per pH unit change (a 10-fold change in H ion concentration).
Sample

Shortly after 1900, Lawrence J Henderson at Harvard adapted the mass action law to relate [H ] to PCO2 and HCO3 : K H HCO 3 =H2 CO3 He demonstrated how respiration could buffer metabolic acids by reducing PCO2 . The kidneys can also change blood and extracellular uid buffer base to partially normalize pH in respiratory acidosis or alkalosis.
Origin of pH

Glass body AgCl internal reference H+ permeable glass Liquid junction Saturated KCl AgCl reference

In order to dene H ion concentrations such as 0.000 000 04 moles liter 1 (e.g., in blood) more elegantly, in 1907, S P L Srensen suggested dening pH as the negative log of hydrogen ion concentration (or activity). In 1915, K A Hasselbalch converted Hendersons equation to log form, later dubbed the

Sample
Figure 1 Schema of a blood pH electrode with a liquid junction to a reference electrode in a thermostatted cuvette.

146 ARTERIAL BLOOD GASES

For use in blood, the reference electrode usually contacts blood via a liquid junction containing saturated KCl. Rapid diffusion of K into red cells causes an often-ignored error of 0.01 pH at the liquid junction when calibration is done with aqueous buffers.
PCO2 Measurement

Until the worldwide polio epidemics of 195052, PCO2 was measured by the HH equation. This required measuring plasma CO2 content by acidication and extraction in a manometric Van Slyke apparatus and measuring pH and correcting it to 371C. In Copenhagens communicable disease hospital in 195051, sometimes up to 100 patients at a time were manually ventilated by volunteers using a bag and mask with O2. The laboratory director Poul Astrup, needing a faster analytic method than the HH equation, devised a new simple method. He measured pH before and after equilibrating the sample with known PCO2 . He then computed patient PCO2 by extrapolation. His method became the standard for the rest of the decade.
PCO2 Electrode

Details An internal, nearly at, glass pH electrode is separated from the sample by a membrane permeable to CO2 but not ions (e.g., Teon or silastic). Under the membrane, a spacer (e.g., lens-cleaning tissue paper) holds a lm of electrolyte containing about 10 mM NaHCO3 and usually 0.1 M salt or KCl. Thermostatted to 37oC, the output signal is a log function of PCO2 , about 30 mV per decade in distilled water (Stows design), doubling to 61 mV per decade with bicarbonate electrolyte.
Oxygen Electrode

At Ohio State University in 1954, while also trying to resolve the polio problem, Richard Stow invented a PCO2 electrode. He covered a glass bulb-shaped pH electrode with a rubber glove (through which CO2 can diffuse but H cannot), over a lm of distilled water. However, Stows electrode drifted because various cations in pH glass altered the distilled water pH. This electrode was stabilized by John Severinghaus at the National Institute of Health (NIH) by adding bicarbonate and salt, and was manufactured by many rms from 1959 onwards. A blood PCO2 electrode is illustrated in Figure 2.

In 1950, Leland Clark at Antioch College, Ohio used perfused isolated liver to study steroid metabolism. He needed oxygenated blood so he built a bubble oxygenator and discovered how to defoam the blood using silicone oil on glass wool. The journal Science rejected his paper on the basis that he hadnt measured the PO2 in the oxygenated blood; to this end, Clark invented a polarographic oxygen electrode. He covered a platinum disc cathode sealed in glass with cellophane to keep blood protein from poisoning the cathode. It served his purpose but was not accurate, requiring very high ow past the sensor due to the depletion of oxygen at the membrane surface due to its consumption by the cathode. In October 1954, a sudden inspiration led Clark to substitute a less O2-permeable and electrically insulating polyethylene membrane for cellophane, by mounting a reference electrode and cathode in electrolyte in a sealed probe (Figure 3). His invention was presented at a meeting of the FASEB in April 1956. Details In a polarographic oxygen electrode, a negatively biased platinum cathode donates electrons to

StowSeveringhaus PCO2 electrode Sample

AgCl external reference electrode AgCl internal reference H+ permeable glass 0.1M KCl + 0.01M KHCO3 Electrolyte in paper spacer Sample
Figure 2 Schema of a blood PCO2 electrode with Teon CO2 permeable membrane and spacer to contain electrolyte for pH measurement.

Glass body

membrane

Teflon or silastic

ARTERIAL BLOOD GASES 147

Clark-type oxygen electrode 25 m polypropylene membrane Sample

0.1 m KCl electrolyte 10 m Platinum wire

Solid glass AgCl reference Sample

Figure 3 Schema of Clarks polarographic PO2 electrode, after cathode size was greatly reduced to avoid stirring effect.

dissolved oxygen: O2 4e 2H2 O ) 4OH The only major functional change since Clarks original invention has been to reduce the cathode diameter from 2 mm to about 10 mm, requiring far more sensitive current analysis, which was not available 50 years ago. This almost eliminated the need for the sample to be rapidly stirred. The electrolyte usually contains KCl, and may have added agents for viscosity. No separator is needed between cathode and membrane. The cathode is biased to about 0.65 V at which all oxygen molecules reaching the cathode are reduced. Cathode current is a linear function of the membrane surface PO2 .
Blood Gas Analyzers

Figure 4 The rst blood gas analyzer containing three electrodes in a water bath at 371C with tonometer for preparing blood for calibration of PO2 electrode. Reproduced from Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253256, with permission from Lippincott Williams & Wilkins.

In 1958, Severinghaus and Bradley created the rst three-function blood gas analyzer by mounting a Clark PO2 electrode with a tiny stirring paddle, a StowSeveringhaus PCO2 electrode, and a commercial pH electrode in a water bath at 371C (Figure 4). A small tonometer was included in which blood could be equilibrated with air or a known gas to calibrate the Clark electrode. Modern blood gas analyzers compute many variables from the three measured values.
Transcutaneous PO2

Figure 5 Transcutaneous combined PO2 and PCO2 electrode monitoring a patient recovering from anesthesia.

temperature or skin metabolism are thus needed. Introduced in the mid-1970s, these devices are widely used on premature and term infants to help control oxygen therapy and prevent blindness following retinal vascular growth interference (Figure 5).
Transcutaneous PCO2

PaO2 can be estimated transcutaneously using a at Clark type PO2 electrode, typically internally heated to about 431C. Heating causes sufcient dermal vasodilation to raise skin capillary PO2 to nearly equal arterial PO2 . Heating also raises blood PO2 by about 7% per degree, while skin oxygen consumption reduces surface PO2 , these two factors approximately canceling each other out. No correction factors for

Arterial PCO2 can also be estimated transcutaneously using at CO2 electrodes heated (e.g., to 431C) to increase skin capillary blood ow. The signal must be corrected 4.7% per degree to 371C, and reduced about 4 Torr to compensate for skin metabolism and electrode surface cooling.

148 ARTERIAL BLOOD GASES Hemoglobin Oxygen Saturation

Prior to about 1970, this was measured by vacuum extraction of oxygen from blood, before and after equilibrating a sample with air.
Multiwavelength Oximetry

Compared with oxygenated blood, desaturated blood strongly absorbs red light (at about 660 nM wavelength). At the isobestic point, 805 nM (near infrared), absorption is unaffected by oxygenation. Multiwavelength oximeters use the ratio of optical density of a thin lm of hemolyzed blood at red and infrared wavelengths to calculate saturation and hemoglobin concentration, with small corrections for other pigments such as bilirubin, detected at other wavelengths. A typical laboratory bench oximeter using at least ve wavelengths can be precise to at least 0.1% saturation. Some oximeters use lters to select wavelengths while others use more stable and precisely dened diffusion-grating monochromators, avoiding the need for user calibration. Conrmatory testing with dyes is recommended. Some blood gas analyzers include a multiwavelength oximeter (often termed CO-oximeter because it also measures the fraction of hemoglobin bound to carbon monoxide).
Pulse Oximetry

Figure 6 Pulse oximeter probe taped on ngertip with red and infrared LEDs on nail side and a photo diode on the dorsal side.

Photodiode

Finger

Red and infrared LEDs

Figure 7 Schema of pulse oximeter probe on a nger.

Light passing through a nger or ear is partly absorbed by the blood in its path. Arteries expand with each pulse, absorbing a bit more light. Pulse oximeters measure the amplitude of the pulsatile variation of light as a fraction of total transmitted red (e.g., 660 nM) and infrared (e.g., 900950 nM) light (Figures 6 and 7). The ratio of these two ratios was shown by T Aoyagi in 1973 (Nihon Kohden Co, Tokyo) to be a unique function of arterial oxygen saturation, theoretically independent of venous or capillary saturation, or skin color, tissue thickness, or other pigments. In the late 1940s, Earl Wood (Mayo Clinic) modied G Millikans 1942 original ear oximeter by adding a pneumatic pressure capsule. Wood showed that when blood was readmitted to a pressure-blanched ear, the ratios of the decreases in red and infrared light passing through the ear were unique functions of oxygen saturation.

be the most useful and important diagnostic procedure available. Its indications are global and will not be listed here.

Common Patterns of Results and Interpretation

Diagnostic Terminology

Indications
Blood gas analysis has become so commonplace that its use is nearly universal in diagnosis during admission, in emergencies, trauma, intensive care, anesthesia, and surgery. It is considered by physicians to

A pH of less than 7.35 is called acidemia, while that over 7.45 is termed alkalemia. A PCO2 over 45 Torr indicates respiratory acidosis or hypercapnia, while values under 35 (males) or 30 (females) indicate respiratory alkalosis or hypocapnia. A standard base excess (SBE) more negative than 5 mM is metabolic acidosis and over 5 mM is metabolic alkalosis. Presence of compensatory responses to chronic acid base respiratory or metabolic imbalances can be predicted and used in diagnosis (Figure 8). Normal PO2 at sea level in young adults is 90 100 mmHg. It falls with age to 6070 mmHg at age 80. There is no consensus on what PO2 level is dened as hypoxia. Arterial oxyhemoglobin functional saturation (100 x HbO2/[HbO2 HHb]) is normally 9798% (i.e., 23% deoxyhemoglobin,

ARTERIAL BLOOD GASES 149

30 Metabolic SBE (mM) 20 10 0 10 20 30 10
CR

7.7

7.6

7.5

7.4

CR
AR

AR

7.3 Metabolic alkalosis 7.2

Metabolic acidosis 7.1

7.0 pH

Respiratory alkalosis

Respiratory acidosis

20

30 40 50 60 70 Respiratory P aCO2 (Torr)

80

90

difference (SID) can identify the causes of many metabolic abnormalities in addition to obtaining an approximation of the degree of plasma metabolic acid base abnormality, provided that all anions and cations are measured. This unfortunately is not a measure of the whole body extracellular uid acidbase condition. For that one needs the SBE, which is computed from directly measured arterial pH, PCO2 , and PO2 without separate ion measurements. SBE is thus a quantitative analysis of imbalance while SID is an approximation of imbalance with additional causative suggestions.
Hydrogen Ion Concentration or pH?

Figure 8 Acidbase compensation diagram predicting in vivo compensation for respiratory and metabolic acidbase imbalance. AR and CR, acute and chronic respiratory, respectively; M, metabolic. Reproduced from Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human P aCO2 and standard base excess compensation of blood gas apparatus. Critical Care Medicine 26: 11731179, with permission from Lippincott Williams & Wilkins.

HHb). Carboxyhemoglobin or methemoglobin or other abnormal forms, if present, reduces fractional saturation (or % oxyhemoglobin) computed as 100 x HbO2/total Hb but is not counted in functional saturation. The terms hypoxia or hypoxemia generally imply that SaO2 is at least 5% lower than expected (at that age and altitude).
Temperature Correction

pH is used widely both for convenience and because chemical activities and potentials are log functions of concentrations. All animals no matter what their normal body temperature maintain their pH 0.6 unit above neutrality, i.e., a ratio of [OH ]/[H ] of about 16 whereas [H ] varies by a factor of more than 4 between 01C (sh) and 401C (hummingbirds). Buffering is a linear function of pH, an exponential function of [H ] making straight pH lines on acid base compensation plots. Some clinicians prefer to interpret acidbase abnormalities using an approximation of Hendersens equation: H 24PCO2 =HCO 3 using nM l 1 for H , mmHg for PCO2 and mM l 1 for HCO3 . This requires a constant 24 combining the dissociation constant K, CO2 solubility, equating carbonic acid with dissolved CO2, and a factor for the three different units.

Clinicians often ask whether they should instruct the laboratory to correct blood gas values to patient temperature. In general, this is not necessary. The appropriate pH and PCO2 for optimal physiologic function is the same function of temperature as are the in vitro temperature correction factors. At 301C in a hypothermic patient, the appropriate pH is 7.4 measured at 371C, or 7.55 corrected to 301C. Animals with a normal body temperature of 301C have a pH of 7.55. Fish in Antarctic waters at 01C have a pH of 8.0, as does normal human blood cooled in vitro to 01C. Blood at 90% SaO2 with PO2 60 Torr will have PO2 30 Torr at 251C, but delivers oxygen at least as effectively to tissues in hypothermia (as shown by decreased tissue lactate). However, physiologists who wish to study pulmonary gas transport and compare alveolar and arterial blood gas tension gradients must correct blood values from the laboratory (at 371C) to the body temperature under study.
SID or SBE?

Oxygen Dissociation Curve Computations and Corrections

Blood gas analytic apparatus commonly provides a computed value of SO2 (oxygen saturation). The observed PO2 is rst corrected from observed pH to pH 7.4 by
obs :4 obs logP7 O2 logPO2 0:487:4 pH

At pH 7.40, 371C, the relationship of SO2 to PO2 is most simply and accurately expressed by
1 SO2 % 10023; 400P3 11 O2 150PO2

The interpretation of acidbase balance divides clinicians into two schools of thought. Strong ion

No temperature correction (e.g., to patient temperature) is needed, all measurements being at 371C.

150 ARTERIES AND VEINS

SO2 in a blood sample does not vary with sample temperature.

Further Reading
Geha DG (1990) Blood gas monitoring. In: Blitt CDK (ed.) Monitoring in Anesthesia and Critical Care Medicine. New York: Churchill-Livingston. Nunn JF (1993) Nunns Applied Respiratory Physiology, 4th edn. Oxford: Butterworth-Heinemann. Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human PaCO2 and standard base excess compensation for acid-base imbalance. Critical Care Medicine 26: 11731179. Severinghaus JW (1979) Simple, accurate equations for human blood O2 dissociation computations. Journal of Applied Physiology 46: 599602. Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253256. Severinghaus JW and Astrup PB (1987) History of Blood Gas Analysis. International Anesthesiology Clinics, vol. 25(4). Boston: Little Brown. Severinghaus JW, Astrup P, and Murray J (1998) Blood gas analysis and critical care medicine. American Journal of Respiratory Critical Care Medicine 157: S114S122. Severinghaus JW and Bradley AF Jr (1958) Electrodes for blood PO2 and PCO2 determination. Journal of Applied Physiology 13: 515520.

State of the Art

Electrodes versus Optical Sensors

Optodes can measure pH, PCO2 and PO2 using dyes, uorescence, quenching, and other optically responsive material, permitting the use of extremely small sensors on optical ber tips in blood at catheter tips or inside tissue cells. They rival electrodes in accuracy and cost, in particular providing portable and disposable sensors (e.g., for cardiac bypass oxygenator control, bedside and eld blood gas analysis).
See also: AcidBase Balance. Diffusion of Gases. Diving. Erythrocytes. High Altitude, Physiology and Diseases. OxygenHemoglobin Dissociation Curve. Permeability of the BloodGas Barrier. Ventilation: Control.

ARTERIES AND VEINS

D E deMello, Saint Louis University Health Sciences Center, St Louis, MO, USA
& 2006 Elsevier Ltd. All rights reserved. Blood vessel assembly begins in primitive mesenchymal cells that undergo a complex series of steps before the mature vessel structure and function is attained. These stages in vessel development are under the control of a large number of transcriptional and growth factors. The timing and dose of some of these angiogenic factors is critical to normal embryonic and fetal development; absence or even reduced expression of some factors is lethal for the developing embryo.

Abstract
The lungs vasculature is different from that of most other organs because it has a double arterial supply and a double venous drainage system. The pulmonary artery which carries deoxygenated blood to the lungs branches alongside the airways into conventional and supernumerary arteries before it empties into the vast capillary network in the alveolar walls where gas exchange occurs across airblood barriers. Conventional and supernumerary pulmonary veins drain oxygenated blood to the left atrium of the heart. The smaller (intra-acinar) vessels develop at the same time as do the acini of the lung and a major component of acinar lung development occurs postnatally. Alveoli increase from about 20 million at birth to about 300 million by the time lung growth is complete in adolescence. Consequently, a large component of intra-acinar vessels develop postnatally and during this critical growth phase are susceptible to local inuences such as increased blood ow from intracardiac left to right shunts which can curtail vessel growth. Arterial wall structure is composed of endothelial cells that line the lumen, smooth muscle cells that make up the media, and broblasts that contribute the adventitial brous sheath. The wall structure changes from proximal to distal vessels in the lung, and alterations in the normal structure may occur during intrauterine life or postnatally in response to a variety of stimuli. Altered wall structure results in functional changes reected in pulmonary artery pressure and resistance.

Anatomy, Histology, and Structure

Unlike most other organs, the lung, because of its gas exchange function, has a double arterial supply and double venous drainage (Figure 1). One of the arterial systems, the pulmonary arterial tree, serves as a conduit for deoxygenated blood from the body to the alveoli where it drains into a vast capillary network within which gas exchange and oxygenation occur. From the alveoli, oxygenated blood is transported to the left atrium of the heart via the pulmonary veins. The other arterial system is the bronchial arterial tree which serves a nutrient function to the airways and perihilar structures. The arterial supply to the pleura, except at the hilar region, is from the pulmonary artery. For the intrapulmonary structures there are no bronchial veins, so all intrapulmonary structures drain to the pulmonary vein, resulting in a small amount of venous admixture in the left atrium. The hilar structures, however, drain to true bronchial

ARTERIES AND VEINS 151

Alveoli Pulmonary vein Respiratory bronchiolus

Pulmonary artery

Bronchioli Bronchial artery Bronchi

To left atrium

Precapillary shunts Capillary bed

To right atrium

Azygos vein

Figure 1 The lungs vasculature consists of a double arterial and a double venous system. The pulmonary artery supplies the alveoli whereas the bronchial artery supplies the airways, the pulmonary artery, and the gas exchange region. Both systems drain via the pulmonary veins to the left atrium, except for the true bronchial veins at the hilum which drain via the azygos veins to the right atrium. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221263. New York: Churchill Livingstone, with permission from Elsevier.

veins and then to the azygos system and the right atrium.
Conventional and Supernumerary Arteries

Segmental hilum

Lateral pathway

The main pulmonary artery that forms the outow path of the right ventricle divides into the right and left pulmonary arteries which enter the lung at the hilum. Within the lung the pulmonary arteries travel alongside the airway branches within a connective tissue sheath. The arterial branching pattern is similar to that of the airway it accompanies, but dissection has revealed many more branches from the pulmonary artery than from its accompanying airway. Two types of arterial branches exist. Those that branch dichotomously next to the airways are conventional branches, the longest of which are the axial pathways running the longest possible course from hilum to distal pleura. The lateral branches of the conventional pulmonary arteries supply the alveoli near the axial pathways (Figure 2). The other type of branch is the supernumerary artery which is short, arises at right angles to the axis of the pulmonary artery, and supplies the alveoli in the immediate vicinity of the artery (Figure 3). Supernumerary arteries are more numerous toward the periphery of any arterial pathway. Over the preacinar length of an axial artery, the ratio of the number of supernumerary to conventional

Axial pathways

Pleura

Figure 2 The path of axial and lateral airways in the human lung. The pulmonary arteries are alongside the airways within the bronchoarterial sheath and follow the same path. Reproduced from Davies P, deMello DE, and Reid LM (1990) Structural methods in the study of development of the lung. In Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 409472. New York: Dekker, with permission.

branches is of the order of 3 : 1 and the lumen crosssection area of the supernumerary side branches is about one-third of the total cross-sectional area of all side branches (Figure 4). Supernumerary arteries have

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19-week fetus Hilum 1 1 2 2 4 5 4 6 5 6

Conventional artery Supernumerary Airway

7 8 7 8

Airways Conventional arteries Supernumerary arteries

Figure 3 The anatomy of the pulmonary arterial tree. Conventional arteries divide alongside the airways at acute angles to the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short, arise at right angles to the main axis, and supply the airspaces adjacent to the axial vessel. Modied from deMello DE and Reid L (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature and its Regulation, vol. 2, pp. 211237. Boston: Birkhauser, with permission.
11 11

9 10 10 12 12 15 15 16

13 14 14

18 19 21.23 21 23 25

a special sphincter at their point of origin which probably serves a functional purpose in recruitment. This valve is responsive to vasoconstrictive agents suggesting that it may be responsible for regulation of blood ow into the supernumerary artery. Because the diameter of the supernumerary vessel is smaller than that of the conventional artery from which it arises, it has a higher critical opening pressure, providing a built-in mechanism for recruitment as pulmonary artery pressure rises. It is noteworthy that the two types of pulmonary artery have different pharmacological proles; for example, supernumerary arteries are 30 times more sensitive in their response to a vasoconstrictor such as 5-hydroxytryptamine (5-HT) compared to conventional arteries, and endogenous nitric oxide selectively attenuates the vasoconstrictor response to 5-HT in the supernumerary but not in the conventional artery. In pathological conditions, these short vessels provide an alternate route for blood in the conventional artery obstructed by thromboemboli. The capillary bed between an artery proximal to the block can open to conduct blood to an arterial bed distal to the block. Blood ow can produce remodeling of a vessel whether artery or vein, and injection techniques have demonstrated that such compensatory or collateral ow produces arcades between axial pulmonary arteries in a number of pathological states (Figure 5).

17 17 20 20 22 24 Terminal bronchiolus

Figure 4 Posterior basal lung segment in a 19-week fetus. Airway and accompanying conventional artery branches are numbered. The more numerous supernumerary arteries (58) are also shown. Reproduced from Hislop A and Reid L (1972) Intrapulmonary arterial development in fetal life: branching pattern and structure. Journal of Anatomy 113: 3548, with permission from Blackwell Publishing Ltd.

Conventional and Supernumerary Veins

The venous drainage resembles the arterial pattern in that there are more venous tributaries than airway branches. The conventional veins arise from the points of division of an airway and pass to the periphery of the unit, combining as tributaries to form progressively larger venous conduits. The supernumerary veins are short and drain small regions around the conventional veins. Additional tributaries to the pulmonary veins arise from pleura and connective tissue septa within the lung. Lung development is unique in that there is a set timetable for the growth and maturation of its major tissue components, airways, alveoli, and blood vessels, and that a large portion of its development continues postnatally until about 12 years of age.
Laws of Lung Development

The template for the growth pattern of the lung is reected in the three laws of lung development.

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Airway Hilum

Artery

Vein

m Preacinar Resistance artery TB Intraacinar RB AD A Precapillary unit nm Capillary

Figure 7 The arteriovenous loop between the right and left side of the heart. The acinus is a unit of lung and includes that portion of lung that is subtended by the terminal bronchiole, i.e., respiratory bronchioles, alveolar ducts, and alveoli. The pulmonary artery that accompanies the airways is shown, including the resistance segment that is immediately precapillary in the newborn child when the acinus is 1 mm in diameter. In the adult the acinus is 1 cm in diameter and the resistance vessels are further downstream at the level of the alveolar ducts and alveoli. m, muscular; pm, partially muscular; nm, nonmuscular; TB, terminal bronchiole; RB, respiratory bronchiole; AD, alveolar duct; A, alveolus. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, pp. 767777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

pm

Postcapillary unit

Figure 5 Lung arteriogram from a premature infant who died at 9 months of age with pulmonary hypertension and vascular hypo plasia. Note dilated, tortuous vessels and interpulmonary artery arcades (arrows). Reproduced from Rendas A et al. (1980) American Review of Respiratory Disease 121: 873880, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Figure 6 Human lung arteriograms. Top, newborn; bottom left, 18 months; right, adult. Increase in radiodensity with age reects progressive postnatal increase in number of intra-acinar arteries. Reproduced from Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531546, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Law II Alveoli. At birth the alveolar spaces are more primitive than in the adult. They have been described as primitive saccules. Of these saccules about 20 106 are present at birth. After birth the alveoli multiply, so that by the age of 8 the number is about 300 106 and within the adult range. Law III Vascular. The vascular law reects the rst two. The preacinar branches of the pulmonary artery (i.e., those that accompany bronchi or bronchioli), as well as the preacinar venous tributaries, appear at virtually the same time as do the accompanying airways. The intra-acinar vessels appear as alveoli grow (Figure 6).
Preacinar Arteries: Structure and Size

These generalizations are also predictive of the effect of perturbation at different times during development and they offer a framework against which to assess vascular growth patterns and to interpret anomalous development. Law I Airways. The airways (i.e., bronchi and bronchioli) are present by the 16th week of intrauterine life.

The cellular structure of any vessel is composed of three cell types that make up the three coats of a vessel: the intima is constituted by endothelial cells, the media is composed of smooth muscle cells or their precursors, intermediate cells or pericytes, and the adventitia is made up of broblasts. Intercellular products also contribute to the composition of each coat. Regional and organ specicity dictate functional differences. A number of mediators, cytokines, growth factors, or hormones determine and modulate these differences.

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Conventional branches Supernumerary branches Diameter of axial artery and side branches (m) 1000

External diameter of axial pathway 500

Acinus Lobule
Figure 8 The relative sizes and numbers of conventional and supernumerary pulmonary arteries. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, pp. 767777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

Muscular

Partially muscular

Non muscular

Capillary (a)

I P E Artery Lumen

(b)

(c)
Figure 9 (a) Diagram depicting the light microscopic appearance of the structure of a pulmonary artery. In its path from the hilum to the periphery, the muscle coat gradually disappears so that the most peripheral segment still precapillary, is nonmuscular. (b) Diagrammatic illustration of the ultrastructural features of the pulmonary artery. From the hilum toward the periphery, muscle cells (M) are replaced by intermediate cells (I), and in the immediate precapillary segment, pericytes (P) are present. (c) Same as (a), in cross-section. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, pp. 767777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

ARTERIES AND VEINS 155

Fetus 100 60 20 Percentage arterial population

Child 100 60 NM 20 PM

Adult 100 60 20 100 200 Arterial size (ED, m)

Figure 10 The structure of vessels of different sizes at different ages in the human. The distribution of muscular (M), partially muscular (PM), and nonmuscular (NM) arteries is similar in the fetus and adult; however, in the child, larger arteries are nonmuscular and partially muscular. ED, external diameter. Reproduced with permission from the BMJ Publishing Group Davies G and Reid L (1970) Thorax 25: 669681.

300

400

In the lung, the vessels proximal to the capillary bed are arteries and those distal to the capillary bed are veins. The immediate pre- and postcapillary vascular segments are functionally specialized and serve to protect the capillary bed (Figure 7). They are the key reactive site for remodeling in disease. Preacinar artery structure is established early in fetal life as elastic, transitional, or muscular and the program for structure is a regional or topographic one. In the adult along an axial pathway, the rst seven generations which are vessels down to a diameter of 3000 mm (distended) are elastic. Vessels from 3000 to 2000 mm in diameter representing generations seven to nine have a transitional structure and smaller vessels down to a diameter of 30 mm have a muscular structure. The largest partially muscular and nonmuscular arteries are found in diameters up to 150 and 130 mm respectively (Figure 8). Along any arterial pathway, a complete muscular coat gives way to an incomplete coat which at rst consists of a spiral before disappearing from vessels, still larger than capillaries, to produce a nonmuscular artery (Figure 9). On the arterial side of the capillary bed, nonmuscular, partially muscular, and muscular small arteries are identied. On the venous side a similar arrangement occurs. By

electron microscopy intermediate cells and pericytes, each a precursor of the smooth muscle cell, are identied: the intermediate cell in the nonmuscular part of the partially muscular artery and the pericyte in the nonmuscular wall. Whereas the pericyte lies within the basement membrane of the endothelial cell, the intermediate cell, like a smooth muscle cell, has its own basement membrane, but, unlike the latter does not have dense bodies. Characteristics of an artery include size, structure, and the thickness of the media as well as its position within the branching pattern. Because vessel remodeling occurs with growth and in disease, it is best to characterize a pulmonary artery by its position in the branching pattern dened by the structure of the accompanying airway, a landmark that remains relatively constant from fetal life. In arteries, the relative circumferences of the internal and external elastic laminae appear to inuence the size and morphology of the endothelial cell and its microenvironment. For example, in vessels larger than 200 mm, the external lamina is shorter than the internal suggesting that it is the former that restricts distension and that the internal lamina may never be smoothed out. In the smaller arteries the situation is

156 ARTERIES AND VEINS

reversed suggesting that the internal lamina is the limiting structure. The balance between these is changed in disease. The functional significance of this, in pulsatile ow and propagation of the pulse wave, has not been established. Postnatally, the length and diameter of the entire pulmonary vascular tree changes, but structural changes occur mainly at the sites of alveolarization in the acinar region, that is, in vessels distal to the level of the terminal bronchiole. In the newborn lung the resistance arteries, that is, vessels with a smooth muscle wall, are upstream from the alveolar wall. Normally before birth pulmonary arterial muscularization stops at the beginning of the acinus, so that the peripheral vessels are muscularized later (Figures 10 and 11). In conditions such as idiopathic pulmonary hypertension of the newborn, congenital heart lesions associated with increased pulmonary artery ow or pressure before birth, and pulmonary hypoplasia, a well-developed muscle coat is found in more peripheral and smaller arteries than is normal. The functional correlate of this structural change is an elevation in pulmonary artery pressure and resistance.
Alv AD

Species Variation

Species differences in structure and rate of lung growth occur and need to be considered when interpreting experimental results. In general, animals like the sheep that can walk or run within hours of birth have a prenatal burst in lung development, preparing the animal as it were for considerable, immediate postnatal activity. In mammals like the rat, mouse, and pig a similar burst in lung growth occurs postnatally, and in the human, postnatal lung growth continues for several years until adolescence.
Postacinar Veins: Structure and Size

The veins are present at the periphery of an acinus within their own connective tissue sheath. As additional tributaries are added in their course toward the hilum, they increase progressively in size. Conventional axial veins enter the larger veins at an acute angle and are the main pathways from periphery to hilum. Their numbers are equivalent to the airway generations and to conventional arteries. The supernumerary tributaries are shorter, drain the lung immediately around the axial vein, and connect with
RB TB

Pleura

Normal Fetus 3 days 10 months 3 years 10 years 19 years Cases PPHN Mec Asp (fatal) HLHS IDTAPVR
Figure 11 Top: diagram of an acinus of the lung which includes the terminal bronchiole (TB), respiratory bronchioles (RB), alveolar ducts (AD), and alveoli (alv). Below: the open bars indicate the level beyond which at different ages the arteries are nonmuscular. The blue bars indicate the extent of muscularization (precocious muscularization) in different disease processes: PPHN, persistent pulmonary hypertension of the newborn; Mec Asp (fatal), fatal cases of PPHN with meconium aspiration; HLHS, hypoplastic left heart syndrome; IDTAPVR, infradiaphragmatic total anomalous venous return. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive stages. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221263. New York: Churchill Livingstone, with permission from Elsevier.

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Pathway 1

1200

800

External diameter of axial pathway

400 Diameter of axial vein and tributaries (m)

Hilum Pathway 2 1200

800 + + +

400

Hilum Pathway 3 Conventional veins 400 Supernumerary veins

Figure 12 Diagrammatic reconstruction of conventional and supernumerary venous tributaries in a 20 week fetus. Pathway 2 is 9.31 mm in length. *Point where pathway 2 joins pathway 1. Point where pathway 1 joins pathway 2. z Point where pathway 3 joins pathway 2. There is a higher density of venous tributaries at the periphery. Reproduced from Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University, with permission from A A Hislop.

the conventional veins at right angles (Figure 12). The preacinar tributaries are developed by 20 weeks gestation, and subsequent tributaries arise within the acinus. Supernumerary tributaries exceed conventional, their ratio being 3.5 : 1, and more vessels drain from the capillary bed than enter. Postcapillary veins have an endothelial lining, but no muscle coat. Larger veins contain an internal elastic lamina with only occasional medial smooth muscle bers. The medial thickness increases in yet larger veins, but even the largest veins do not have a well-dened external elastic lamina. Elastin and

collagen are present between the smooth muscle cells. The largest nonmuscular veins are approximately 150 mm in diameter and as with the arteries, overlap in size groups occurring between muscular, partially muscular, and nonmuscular veins. At 20 weeks gestation, the vein structure consists of endothelial cells resting on collagen mixed with an occasional elastic ber. Even the largest veins have no muscle in their wall. Scattered muscle bers appear by 28 weeks, but a continuous layer is only developed at term, and even then, no elastic lamina is present. Postnatally, the thickness of the muscle

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increases but not as much as in arteries. At birth the smallest muscular vein measures about 105 mm in diameter, at 3 years the smallest is 130 mm, and at 10 years 70 mm. No significant structural change occurs after this time.
Intra-Acinar Arteries and Veins: Structure and Size

celiac axis. A portion of the lung bud may remain attached and manifests later as a lung sequestration.

Arteries and Veins in Normal Lung Function

Blood Vessel Assembly

By 5 years of age, axial pathways have nished development and future growth occurs in the more peripheral and smaller arteries that appear as alveoli are formed, but in these vessels, muscularization is slower, so that during childhood the transition from muscular to partially to nonmuscular occurs in vessels of a larger size than in the fetus or adult (Figure 10). Therefore, in the child, the structure of intra-acinar arteries cannot be predicted by their size.
Bronchial Arteries

Early in gestation, primitive bronchial arteries arise from the dorsal aorta in the neck region near the celiac axis and are distributed with the early branches of the airways (Table 1). When lobar or segmental airway branches are present at about the 6th week of embryonic life, the central bronchial artery branches disappear. Between the 9th and 12th weeks, denitive bronchial arteries arise from the aorta, pass along the superior surface of the airways, and communicate with the pre-existing capillary bed in the distal airways. Sometimes the primitive bronchial arteries persist and migrate with their point of origin, to a site below the diaphragm, still near the
Table 1 The development of the bronchial arteries Week of gestation 4th Airway Main Blood vessels Primitive ventral aorta Pulmonary vein links to heart 6th arch supplies lung Paired systemic arteries from dorsal aorta Only blood from right ventricle to pulmonary artery Systemic arteries disappear Bronchial arteries enter peribronchial plexus

5th 6th 9th to 12th

Lobar Segmental

The primitive paired bronchial arteries arise from the dorsal aorta near the celiac axis in the neck. These have usually disappeared before the 5th week; if they persist, they then migrate with the celiac axis to below the diaphragm as seen in certain abnormal conditions (e.g., sequestered segment). Reproduced from deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, 2nd edn., pp. 11171127. Philadelphia: Lippincott-Raven Press, with permission from Lippincott Williams & Wilkins.

Two processes are involved in the development of blood vessels: (1) angiogenesis, the branching of new vessels from pre-existing ones, and (2) vasculogenesis, the formation of blood lakes that undergo transformation into vessels. In the lung, blood vessel development must be coordinated temporally and spatially with airway and alveolar development. Angioblasts are the precursor cells from which blood vessels are derived and they are of mesodermal origin. In the development of the pulmonary vasculature, angioblasts must interact with epithelial and mesenchymal components of the lung. Central events determine for a given vessel, the direction of blood ow, its distribution or drainage and its central connections, and local or peripheral events serve as modiers. These determine the ne structure of the vessels as they supply the ultimate functioning unit of the lung. Vessel branching occurs at the end of a pathway and lengthening or widening results from cell multiplication. Chimeric experiments have shown that angioblasts migrate widely and to considerable distances from their sites of origin. Biologic differences in vessel cells, for example, endothelial cells from different sites in the vascular tree, exist, reecting the inuence of the local milieu. The transcription factor TAL1/SCL is a marker of angioblasts. In the presence of extracellular matrix, endothelial cell precursors form protrusions that result in the formation of vascular cords and then vessels. This process requires integrin-mediated adhesions. Vascular endothelial growth factor (VEGF) plays a role as a mitogen and as a vascular morphogen in vasculogenesis. Whereas vascular smooth muscle cells arise through progenitors within the mesoderm, they are also derived from endothelial cells. In the fetal mouse, transmission electron microscopy shows that between 9 and 10 days, primitive angioblast precursors within the mesenchyme surrounding the lung bud, form vascular lakes that have hematopoietic cells in their lumen (Figures 13(a) 13(c)). Mercox pulmonary vascular injections combined with scanning electron microscopy of the vascular casts indicate that conventional and supernumerary branches from the main pulmonary artery are derived by angiogenesis and that the more distal vessels, those of the future alveolar region, arise by vasculogenesis, that is, from a lumen that appears

ARTERIES AND VEINS 159

Figure 13 Transmission electron micrographs of fetal mouse thoraces. (a) 9 day fetus: a space between densely packed mesenchymal cells contains membrane-bound vesicles. Magnication 8750. (b) 10 day fetus: mesenchymal cells around intercellular spaces appear thin and endothelial-like. Magnication 5250. (c) 10 day fetus: some intercellular spaces contain hematopoietic precursor cells. Magnication 5250. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568581, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

locally within the mesenchyme: at 12 days, there are four generations of central arterial branches but no luminal connection is seen between these vessels and the lakes in the peripheral lung mesenchyme, where already a dense collection of lakes containing hematopoietic cells is present. By 14 days, ve to seven generations of central artery branches, supernumerary and conventional, are present. There is now a connection between the central vessels and the peripheral system so that casts of the peripheral vessels are obtained by central injection (Figures 14(a) 14(c)). Between 15 days and term, there is increasing complexity of the peripheral vascular casts reecting an increase in the connections between the central and peripheral systems (Figure 14(d)). Whereas the processes of angiogenesis and vasculogenesis occur separately but concurrently, a third process, fusion, between these two systems is necessary for the circulation to be established.

In the human, examination of serial sections of embryos and fetuses of different ages in the Carnegie Collection of Human Embryos housed in the Carnegie Institute of Washington, DC (now located in the Museum of Human Development in the Armed Forces Institute of Pathology in Washington, DC) revealed that the blood lakes are the rst to form and are present in the primitive mesenchyme around the lung bud in the neck between stages 14 and 18 (32 44 days of gestation). As gestation progresses, abundant lakes appear in the subpleural mesenchyme. At stage 23 (56.5 days), pulmonary artery branches accompany the airways but lag behind the airway by two to three generations. The thick-walled arteries end blindly in a solid cord of cells. Between 12 and 16 weeks, an extensive capillary network is present in the subpleural mesenchyme surrounding the most distal airway buds but separated from them by mesenchyme. By 22 to 23 weeks, the capillary network

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PA

PA L

PA

PA

Aorta Aorta (a) 12 days (b)

13 days

PA

R PV

L PA L R PV

CL

15 days (c)

16 days (d)

Figure 14 Photomicrographs of pulmonary arterial mercox casts of mouse fetuses. (a) 12 day fetus: up to four generations of arterial branches are present. (b) 13 day fetus: isolated patches of peripheral vessels are lled with mercox. (c) 15 day fetus: mercox lling of more peripheral vessels results in visualization of several generations of arterial branches with an increase in diameter. (d) 16 day fetus: mercox lling of the expanded peripheral vascular network results in a markedly dense cast that obscures the proximal vessels. S, systemic; R, right; L, left; PA, pulmonary artery; PV, pulmonary vein; CL, cardiac lobe. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568581, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

approaches the airway epithelium and bulges into the air space indicating that blood barriers for future gas exchange have formed. At this time, the pulmonary artery accompanies even the most distal airway branch just beneath the pleura. So from this study, it appears that in the human also, the three processes of vascular development, that is, angiogenesis, vasculogenesis, and fusion, contribute to the establishment of the pulmonary circulation. Other studies using markers for endothelial cell precursors in

mouse embryos and three-dimensional reconstruction of serial sections of human embryos suggest that the intrapulmonary vascular tree is predominantly developed by the process of vasculogenesis.
Controlling Mechanisms: Genes and Factors Involved in Vessel Assembly

The path from angioblast precursor within primitive mesenchyme to mature blood vessel is complex and

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Endothelial cell commitment ('blood island') Proteases Smooth muscle recruitment and differentiation Mature blood vessel

Primitive mesenchyme

Hemangioblasts

Migration and tube formation

SCL/tal-1

Ets-1 Fra1 Vezf1

ARNT ELF-1 EPAS Fli-1 GATA2 GATA3 HIF-1 HOXD3 NERF-2

Ets-1

AML-1 COUP-TFII HESR1 HOXB3 PPAR-

dHAND MEF2C SMAD5 SmLIM

LKLF

Transcription factors Growth factors and receptors bFGF Flk-1 Flt-1 integrin P/GH TGFTIE2 VEGF Angiopoietin-1 TIE2
V 3

Figure 15 The multiple steps involved in vessel assembly from primitive mesenchyme requires the sequential action of numerous factors. Reproduced from Pediatric and Developmental Pathology vol. 7, 2004, pp. 422424, A matter of life and breath: context article, deMello DE, gure 1, with kind permission of Springer Science and Business Media.

involves a number of steps including commitment to differentiate into an endothelial cell, the action of proteases, migration and tube formation, recruitment and differentiation of smooth muscle cells, and acquisition of mature vessel structure and function. This complex process is under the coordinated control and inuence of a large number of genes and growth factors (Figure 15), which have been identied by experiments involving overexpression or knockout of growth factors or genes. A ne-tuned and delicate balance in the temporal and spatial expression of a variety of genes and factors is essential for both normal intrauterine vessel development and postnatal vessel maintenance. For example, knockout of the VEGF gene or even its reduced expression in heterozygosity results in lethal defects in vessel formation in the mouse embryo, whereas overexpression of VEGF in the lung results in the formation of oversized vessels and disordered airway morphogenesis. Even the relative ratios of VEGF isoforms is critical for normal vessel and airway development. This was demonstrated in the VEGF 120 mouse in which the other VEGF isoforms, VEGF 164 and VEGF 188, are not expressed. Homozygous VEGF 120 mice have a severe reduction in the number of intra-acinar arteries and airblood barriers and a

delay in lung development resulting in hypoplastic lungs (Figures 16 and 17). Knockout of the Flt-1 (VEGF receptor) gene produces a lethal defect in angiogenesis and knockout of the Flk-1 (VEGF receptor) gene results in a lethal failure of vasculogenesis. Transforming growth factor beta (TGF-b) is important for the eventual wall structure of the developing vasculature and inuences growth, migration, and differentiation of endothelial, smooth muscle, and mesenchymal cells and pericytes. The receptor tyrosine kinase gene (tie-1) and receptor (Tie -2) are involved in the regulation of vasculogenesis and angiogenesis.
Transitional Circulation and Perinatal Adaptation in Humans

At birth, as the lung expands with air, structural changes occur within the resistance segment of the pulmonary arteries producing an increase in compliance, and drop in resistance and pulmonary artery pressure as a result of an increase in external diameter and decrease in wall thickness. By 4 months the resistance arteries lose their fetal wall thickness, but in vessels less than 200 mm in diameter, wall thickness gradually increases.

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1 mm

E13.5

1 mm

E15.5

1 mm

1 mm

1 mm

E18.5

E18.5

E18.5

Figure 16 Photographs of fetal mouse lungs from three gestational ages: top, E13.5; middle, E15.5; bottom, E18.5. Lungs of wild-type (VEGF / , left) and heterozygous (VEGF 120/ , middle) fetuses do not differ in size. At all gestational ages, the lungs of homozygous fetuses (VEGF 120/120, right) are smaller than the lungs of wild-type and heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194203, with permission.

Structure

The distribution of preacinar elastic and transitional arteries is unchanged during childhood, although the size range varies with age. Intra-acinar arteries which develop as new alveoli are formed postnatally, are nonmuscular initially, and acquire muscle coats gradually with time. By 10 years of age, even alveolar wall level arteries have muscle coats.
Veins

Arteries and Veins in Respiratory Diseases

Abnormalities of pulmonary arteries or veins can result from disorders of vessel assembly or from postnatal events that interfere with postnatal vascular growth or cause structural remodeling of the existing vasculature.

Disorders of Vessel Assembly

During childhood, the veins increase in size and peripheral veins increase in number. Within an acinus, the number of veins exceeds that of the arteries presumably to facilitate blood ow through the capillary bed.

These disorders are the consequence of aberrations in the processes involved in vessel assembly, that is, angiogenesis or vasculogenesis and result in failure of growth, overgrowth, or structural/functional abnormalities.

ARTERIES AND VEINS 163

1 mm (a) (b)

1 mm (c)

1 mm

1 mm (a) (b)

1 mm (c)

1 mm

Figure 17 Lung mercox vascular casts of VEGF 120/120 fetal mouse littermates. Genotypes: (a), wild-type; (b), heterozygous; (c), homozygous. Gestational ages: top, E17; bottom, E18. The homozygous fetal casts are smaller, and fewer peripheral vessels make the casts less dense than those of the wild type or heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194203, with permission.

Aberrant angiogenesis Absence of the main pulmonary artery or its branches The main pulmonary artery or one of its branches fails to grow and the lung is supplied instead by collateral vessels from the systemic circulation. Usually the pattern of intrapulmonary artery branching is unaffected so it can be presumed that central sprouting or angiogenesis fails, but that peripheral vasculogenesis is normal and the systemic collaterals assume the role of the intrapulmonary arterial tree. Misalignment of blood vessels This disorder often accompanies another serious condition, alveolar capillary dysplasia (Figure 18). The central pulmonary arteries and veins are misaligned so that the veins are displaced from their normal location at the periphery of a lung lobule and instead share the bronchoarterial sheath. When present in all lobes of

the lung and in association with alveolar capillary dysplasia, the condition is fatal. Hypoplastic vascular tree: dwarfism and congenital diaphragmatic hernia The small thoracic cage in most forms of dwarfism or skeletal dysplasia is associated with small lungs. The main structural components of the lung, airways, alveoli, and vessels are hypoplastic but additional abnormalities suggest a direct metabolic effect as well. In congenital diaphragmatic hernia, the restricted space available for fetal lung growth produces hypoplastic lungs with a reduced number of airways and alveoli. Because the pulmonary arteries travel and branch with the airways, their number is also reduced and the size is small but appropriate for the smaller lung volume. Arterial structure is also altered so that the walls are thicker and muscle extends into smaller, more peripheral vessels.

164 ARTERIES AND VEINS

(a)

(b)

(c)
Figure 18 (a) Post-mortem lung barium angiograms. (left) Preterm neonatal lung reveals lling of preacinar and intra-acinar arteries. (right) Lung of a term infant with alveolar capillary dysplasia; the angiogram has the look of a pruned tree because of a reduced number of intra-acinar arteries. (b) In the normal lung (left), the pulmonary artery (long arrow) is present within the bronchovascular sheath and the vein (short arrow) lies within the pulmonary septum at the periphery of the lung lobule. In misalignment of the pulmonary vessels (right), the pulmonary arteries (long arrows) and veins (short arrows) lie within the same bronchovascular sheath. The pulmonary arteries are lled with barium (post-mortem injection) and the veins are empty. Movat pentachrome stain. Magnication 100. (c) The normal term lung (left) has numerous airblood barriers (arrows) within alveolar walls. In alveolar capillary dysplasia (right) airblood barriers are absent and vessels larger than normal capillaries are present in the middle of thickened alveolar septa (arrows). Movat pentachrome stain. Magnication 200. Reproduced from deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311 329, with permission.

Disordered vasculogenesis Alveolar capillary dysplasia In this rare and fatal disorder, airblood barriers which are critical for gas exchange fail to form within alveolar walls. When the entire lung is affected, the condition is incompatible with independent air-breathing existence (Figure 18). Sometimes, however, a single lobe is

affected suggesting failure of a local inductive mechanism for triggering capillary growth. Fusion Intrapulmonary arteriovenous malformations point to aberrant connections between the central and peripheral pulmonary vasculature during fetal development. This is usually a circumscribed

ARTERIES AND VEINS 165

lesion suggesting that while overall angiogenesis and vasculogenesis has occurred normally, the mechanism that regulates fusion (? via chemotaxis) is faulty and results in communications between arteries and veins. Rarely, the process may involve an entire lobe. Postnatal Disorders Idiopathic (persistent) pulmonary hypertension of the newborn (PPHN), with or without meconium aspiration (also known as meconium aspiration syndrome) In both instances, the overall pattern of arterial growth is normal, but significant functional abnormalities at birth result from structural alterations in preacinar and intra-acinar arteries. The vessels are often small and have thick muscle walls and adventitial coats. There is precocious muscularization of intra-acinar arteries so muscle coats are present within smaller, normally nonmuscularized arteries. Congenital heart disease with left-to-right shunts In many types of congenital heart disease, the pulmonary circulation develops normally in utero, but adaptational changes occur after birth. An increase in pulmonary blood ow from left to right shunts interferes with postnatal intra-acinar artery growth and if left uncorrected will result in a reduction in intra-acinar artery number, increased pulmonary vascular resistance, and pulmonary hypertension. Systemic arteriovenous anastomoses in which high pulmonary blood ow occurs before birth will result in an abnormal vascular structure at birth.
See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Bronchial Circulation. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Coagulation Cascade: Factor VII. Diffusion of Gases. Epithelial Cells: Type I Cells; Type II Cells. Hypoxia and Hypoxemia. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Lymphatic System. Neonatal Circulation. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Pediatric Pulmonary Diseases. Permeability of the BloodGas Barrier. Pulmonary Circulation. Pulmonary Edema. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Vascular. Surgery: Transplantation. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpastures Syndrome. Vascular Disease. Vascular Endothelial Growth Factor. Vasculitis: Overview; Microscopic Polyangiitis.

Further Reading
Davies G and Reid L (1970) Thorax 25: 669681. Davies P, deMello DE, and Reid LM (1990) Methods in experimental pathology of pulmonary vasculature. In: Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 843904. New York: Dekker. deMello DE (1999) Structural elements of human fetal and neonatal lung vascular development. In: Control Mechanisms in the Fetal and Neonatal Pulmonary Circulation, Proceedings of the Ninth Pulmonary Circulation Conference, Sedalia, CO, Oct 1999, pp. 3764. deMello DE (2004) A matter of life and breath: context article. Pediatric and Developmental Pathology 7: 422424. deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311329. deMello DE and Reid L (2000) Embryonic and early fetal development of human lung vasculature and its functional implications. Pediatric and Developmental Pathology 3: 439449. deMello DE and Reid L (2004) Pre- and post natal development of the pulmonary circulation. In: Haddad GG, Abman SH, and Chernick V (eds.) Basic Mechanisms of Pediatric Respiratory Disease, pp. 77101. Ontario: BC Decker. deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, pp. 767777. New York: Raven Press. deMello DE and Reid LM (1991) Pre and post-natal development of the pulmonary circulation. In: Chernick V and Mellins RB (eds.) Basic Mechanisms of Pediatric Respiratory Disease: Cellular and Integrative, pp. 3654. Philadelphia: BC Decker. deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientic Foundations, 2nd edn., pp. 11171127. Philadelphia: Lippincott-Raven. deMello DE and Reid LM (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature, vol. 2, pp. 211237. Boston: Birkhauser. deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568581. Galambos G, Ng YS, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194203. Hall SM, Hislop AA, Pierce CM, and Haworth SG (2000) Prenatal origins of human intrapulmonary arteries. American Journal of Respiratory Cell and Molecular Biology 23: 194203. Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University. Hislop A and Reid L (1972) Intra-pulmonary arterial development during fetal life: branching pattern and structure. Journal of Anatomy 113: 3548. Hislop A and Reid L (1973) Fetal and childhood development of the intrapulmonary veins in man: branching pattern and structure. Thorax 28: 313319. Hislop A and Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28: 129135. Oettgen P (2001) Transcriptional regulation of vascular development. Circulation Research 89: 380388.

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Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531546. Reid L, Fried R, Geggle R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, Vol. 4, pp. 221263. New York: Churchill Livingstone. Rendas A, et al. (1980) American Review of Respiratory Disease 121: 873880. Schachtner SK, Wang YQ, and Baldwin SH (2000) Quantitative analysis of embryonic pulmonary vessel formation. American Journal of Respiratory Cell and Molecular Biology 22: 157165.

Asbestos

see Occupational Diseases: Asbestos-Related Lung Disease.

ASTHMA
Contents

Overview Allergic Bronchopulmonary Aspergillosis Aspirin-Intolerant Occupational Asthma (Including Byssinosis) Acute Exacerbations Exercise-Induced Extrinsic/Intrinsic

Overview
pital Arnaud de Villeneuve, Montpellier, P Chanez, Ho France
& 2006 Elsevier Ltd. All rights reserved.

Asthma is one of the most frequent chronic diseases. It is responsible for absenteeism from school and work, thereby handicapping daily life. Its management is based on drug therapy, control of the environment, therapeutic education, and management of triggering factors.

Abstract
Asthma is one of the most prevalent chronic diseases in most of the countries in the world. Its continuous increase is clearly described in most places, conrming the potential importance of the environment. These environmental factors interact in a susceptible individual with some complex polygenic background, to reveal the asthma phenotype. Several triggers including inhaled allergens, viruses and some indoor and outdoor pollutants have been pointed as potential culprits to induce, perpetuate, or exacerbate asthma. Inammation and structural changes are hallmarks of asthmatic airways occurring from the nose to the distal part of the lung. The relationships between those structural changes and clinical and functional abnormalities clearly deserve further investigations. Those ndings led to the reinforcement of the use of inhaled corticosteroids as the pivotal treatment for asthma. The adjunction of long-acting b2 agonists has been shown to be the next logical step of pharmacology, in case of poor control when using inhaled steroids alone. The long-term management should include some tailor-made environment control and educational measures leading to a better partnership with the patients. In some occasions, the addition of leukotriene receptor antagonists, specic immunotherapy, and more recently, subcutaneous anti-IgE may offer better control for a subset of patients. A better understanding of the mechanisms, especially in the most severe forms of the disease, is paramount to develop better preventive strategies and innovative therapies.

Physiopathology
Asthma is an inammatory disorder accompanied by remodeling of the airways. This inammation is secondary to polymorphic inammatory inltration, rich in mast cells, and eosinophils. In genetically predisposed subjects, this inammation may cause symptoms which, in general, are related to diffuse variable bronchial obstruction that is spontaneously reversible or subsides under the inuence of treatment. This inammation is also associated with bronchial hyperresponsiveness to a wide variety of stimuli. This definition of asthma is supported by the physiopathological and clinical ndings on the disease. The variability and reversibility of the airow impairment, under the inuence of bronchodilators and glucocorticoids, distinguish asthma from other bronchial disorders. Chronic rhinosinusitis is very often associated with asthma (approximately 80% of cases) and must be investigated, not only by questioning, but also by careful nasal examination.

ASTHMA / Overview 167

Several arguments support a link between persistent rhinosinusitis and asthma: the common characteristics of inammation and tissue reorganization, similar epidemiology and chronicity, higher risk of asthma in cases of rhinitis, higher risk of bronchial hyperreactivity in patients suffering from rhinitis, occurrence of bronchial inammation after nasal challenge with an allergen, and the same aggravating and triggering factors. The efcacy of local corticoid therapy constitutes another link. On the other hand, there is no solid evidence in favor of the bronchial efcacy of nasal treatment. Systemic treatments also require additional evaluation of their joint efcacy in rhinosinusitis and asthma.

Table 1 Differential diagnosis of asthma in the child and adult In the child Obliterating bronchiolitis Cystic brosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction Upper airway abnormalities Immunodeciency (IgA, IgG2, IgG4) Ciliary dyskinesia Abnormal aortic arch In adults Bronchiectasis Cystic brosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction COPD Heart failure Bronchial amyloidosis Abnormal aortic arch Tracheobronchial cancer Bronchiolitis

Diagnosis
Positive Diagnosis

Alternative Diagnosis

This must be based on the definition of asthma. The two characteristic elements are clinical (chronicity, variability, and reversibility of symptoms), and functional. Tests of respiratory function clearly conrm the diagnosis provided that: 1. spirometry shows a reversible obstructive ventilatory disorder from 12% to 15% in comparison with theoretical values, or at least 180 ml in absolute value, with short-acting b2-mimetics; 20% or 250 ml during a 10-day test with glucocorticoids; 2. peak expiratory ow (PEF) has a variability X20%; and 3. bronchial hyperreactivity is another feature of the diagnosis. There have been few studies on the sensitivity and specicity of the functional signs of asthma and their predictive values seem insufcient. Those maintained in the international recommendations are cough, dyspnea, sibilant rhonchi (wheezing), chest tightness, and expectoration. There may be one or several symptoms. Symptoms may be absent at the time of the examination. Definition of reversibility or its significance is only obtained from expert opinions or by consensus. The same is true for the usefulness of the corticosteroid test to identify asthmatic patients; a short course of oral corticosteroid is used generally. The dose and duration of steroid treatment given in the literature are highly variable. However, these criteria are generally considered to be sufcient to propose the diagnosis of asthma and to potentially qualify nonresponding asthmatic patients as steroidresistant.

The main differential diagnoses are summarized in Table 1.

Clinical Definitions
From the point of view of the terminology, three essential terms must be used and seem to be operational in clinical medicine and for the follow-up of asthmatics the seriousness of the asthma which refers to the current state of the patient (serious acute asthma), the control of the asthma which refers to recent events (symptoms of brief duration and exacerbations), and the severity of the asthma which is mainly evaluated over the past year.
Exacerbations

The definition of an exacerbation is variable and it is based on the unanticipated use of drugs, the persistent symptoms (repetition of short-lasting symptoms generally on two consecutive days), the increased bronchial obstruction, and the requirement for substantial change in treatment with oral corticoids being the most frequent. Exacerbations form part of poor control of the asthma, but differ from it. The permanence of shortlasting symptoms with return to the baseline state between these symptoms denes simple poor control, whereas an exacerbation is characterized by a persistent worsening without a return to the baseline state.
Control

Several asthma evaluation control questionnaires have been developed and concern the events occurring during the 14 weeks before the visit. Some even cover a period of 3 months, or even the time since the previous visit. There are no data in the literature pointing to one or the other of these periods.

168 ASTHMA / Overview

Juniper showed that her questionnaire (ACQ) was more discriminating than a daily diary. The authors clearly specied that this comparison concerned clinical trials and no conclusion could be deduced for daily clinical practice. These indexes are often correlated with generic and specic quality of life questionnaires: AQLQ, SF 36, Saint Georges questionnaire. The definition of optimal (or excellent) and suboptimal (or acceptable) control is based on experts agreement and their clinical experience.
Severity

Table 2 Factors aggravating controlled asthma Interruption of anti-inammatory treatment Viral infections Administration of aspirin or a betablocker Hormonal factors with premenstrual recurrence in woman Contact with allergen Atmospheric pollution Domestic pollution Meteorological factors Stress

The notion of severity was initially based on the same parameters as control, though these were assessed retrospectively over a longer period, often including the number of successful anti-inammatory treatments and the best level of respiratory function. At present, the assessment of asthma severity must be made more objectively, according to the control and the quantity of drugs required to obtain and maintain it.
Difcult Asthma

Table 3 Examples of candidate genes implicated in the development of asthma Chromosome 5q 6q 11q Candidate gene Th2 related cytokines TNF-a Clara cell protein CC10 FceRIb: IgE highafnity receptor Interferon g T-lymphocytes receptor IL-4 receptor a ADAM 33 Potential relation with asthma Airway inammation Airway inammation, treatment Airway inammation, treatment

12q 14q 16q 20q

Difcult asthma may only be dened after answering questions about its diagnostic reality, patient adherence to treatment, and the existence of major comorbidity which may interfere with management, making it impossible to obtain an acceptable control. Positive answers to these questions aid in diagnosing severe asthma. Severe asthma is characterized by frequent and serious exacerbations (stays in intensive care units), the persistence of airway obstruction, resorting to high doses of inhaled corticosteroids or even oral steroid dependence, and in some cases, by lack of steroid sensitivity. It should be pointed out that patients, nursing staff (nurses, educators in asthma schools etc.) , general practitioners, and even respiratory physicians are not always familiar with these notions of seriousness, control, exacerbations, and severity. They, therefore, often differ in their assessment of the same clinical situation. Efforts to provide information, education, and coherence are therefore required more than ever.
Precipitating Factors

Airway inammation Airway inammation, treatment Airway remodeling

Certain authors note that an attack leading to a change in primary therapy must be considered to be potentially serious.

For the doctor, it is always a potentially fatal medical emergency. Table 3 presents the clinical signs of serious acute asthma (SAA). Various items have been recognized as essential to management of SAA: 1. Value of evaluation protocols. 2. Requirement to measure respiratory function (PEF) and blood gases. 3. Efcacy of treatment with inhaled short-acting b2mimetics, systemic glucocorticoids, inhaled anticholinergics (48 h mainly in the child), and oxygen therapy. 4. Value of second-line therapy with intravenous b2mimetics, nebulization using helium oxygen as vector (heliox), intravenous magnesium. 5. Requirement for regular clinical re-evaluation. 6. The criteria for hospitalization are not validated, though they are widely used and consist of PEF o60% of the predicted value or o100 l min 1, no clinical improvement, severe underlying asthma,

The risk factors of loss of control of asthma are summarized in Table 2.

Serious Acute Asthma

From the patients point of view, the seriousness of an attack may be dened by the following characters:
* *

it is unusual and a doctor must be called; it leads to the discontinuation of current activity (work, school, or play); and

ASTHMA / Overview 169

Table 4 Clinical signs of serious acute asthma Threatening syndrome Worsening in a few days Increase in the frequency of attacks Increase of severity of attacks Resistance to treatment Increase in drug consumption Disease-free intervals less and less frequent Progressive decrease in PEF Signs of immediate seriousness Unusual and/or progressive dyspnea Difculty in speaking or coughing Agitation Sweats and/or cyanosis SCM muscle permanently tensed RR 430 min 1 HR 4120 min 1 Paradoxical pulse 420 mmHg PEF o150 l min 1 Gain in PEF under treatment o60 l min 1 PaCO2 440 mmHg Signs of distress Consciousness disorders Collapsus Pause in breathing Respiratory silence PEF, peak expiratory ow; HR, heart rate; RR, respiratory ow; SCM, sternocleidomastoid. Table 6 Why new therapies are needed in asthma? Therapy Intermittent mild persistent None High Low Moderate persistent Few potential Variable Variable Severe persistent Highest frequency Low Usually better

Side effects Efciency Compliance

Table 7 Goals for asthma treatment Minimize symptoms Achieve best lung function Prevent asthma exacerbations Obtain treatment with the best therapeutic balance Educate patients for best partnership Avoid decline in lung function

Table 8 ICS side effects usually not clinically significant if low doses are used Frequent and mild Oral candidosis Hoarseness Potential and mild Cough Skin bruising Rare and potentially severe Surrenal insufciency Biologically proven but long-term clinical significance unknown Osteoporosis Impaired growth velocity Cataract

Table 5 Risk factors of mortality in serious acute asthma Severe asthma (bronchial obstruction during intercritical period or frequent exacerbations) Patients presenting major rapid variations in bronchial obstruction (variation in PEF 430%) Major reversibility under bronchodilators Psychosocial instability Use of three drugs (or more) for asthma Frequent resort to ER for SAA, with hospitalization and/or intensive care Poor perception of symptoms Elderly patients Patients with hypereosinophilia Poor compliance and denial of disease

inhaled corticoids were given as primary therapy. These data validate a fundamental aspect of the longterm therapeutic management of asthma (Tables 6, 7, and 8).
Asthma Associations

and psychosocial problems. The same is true for criteria concerning the return home (PEF 4 60% or 300 l min 1). After an episode of SAA, oral glucocorticoid therapy must be prescribed for from 7 to 14 days, combined with inhaled glucocorticoid therapy and follow-up with therapeutic education. The risk factors for SAA mortality are given in Tables 4 and 5. Suissa showed that the death rate due to asthma decreased by 21% per additional vial of inhaled corticoids received during the previous year. The death rate due to SAA therefore decreased if low doses of

Bronchopulmonary allergic aspergillosis This entity is dened by the following major symptoms: 1. recurrent lung inltrates with eosinophils, sensitive to corticoids, 2. highly characteristic proximal bronchiectasis, 3. sometimes high blood eosinophilia (41000), 4. increased total IgE and specic IgE (measured by RAST) and IgG (precipitins) immune reaction against Aspergillus fumigatus, and 5. Aspergillus can sometimes be present in sputum.

170 ASTHMA / Overview

These point to severe asthma, usually justifying long-term oral corticoid therapy and, in certain circumstances, antifungal treatment. ChurgStrauss syndrome This is a granulomatous and necrosing vasculitis. It is a rare form of asthma characterized by the severity of the respiratory symptoms, the levels of blood eosinophilia (generally above 1500 mm3), and the existence of extrarespiratory signs (neurological and cutaneous). It is often oral steroid-dependent and sometimes requires immunosuppressive agents.

Arthropods. They are insects such as locusts or cockroaches, which can cause asthma. Atmospheric molds and yeasts. They are an important source of allergens. Allergens present in insalubrious habitats are associated with more severe asthma. Food allergens Food and drinks may cause respiratory reactions after allergic sensitization, but nonallergic reactions or toxic reactions may also occur due to non-specic histamine release. Drugs Very often, drugs behave as haptens. Atopic subjects and asthmatics do not have any predisposition to hypersensitivity to drug products but when these occur, they are more violent. Aspirin intolerance This sometimes involves severe asthma, often with a late-onset with pan-rhino sinusitis with polyposis and systemic reactions, often with anaphylactic shock after ingestion of aspirin or nonsteroidal anti-inammatory drugs. Occupational sensitizers They may be allergenic, but they may also act by toxic, irritative, or pharmacological mechanisms. The most important directly allergic allergen is wheat our, responsible for bakers asthma. Another classical example of occupational asthma is due to work in contact with isocyanates. Demonstration that the asthma is caused by an occupational agent requires the use of a coherent, clinical, and functional diagnostic procedure, sometimes including bronchial challenge tests in the laboratory. Other factors Importance of viruses This is variable as a function of age in the triggering of asthma exacerbations. In the infant, wheezing bronchitis is usually caused by viruses though the possibility of asthma must not be over- or underestimated. Certain studies show that more than 60% of asthma exacerbations are related to a viral respiratory infection. Viral infections can be involved at various levels:
* * * *

Triggering and Aggravating Factors

Asthma is a multifactorial syndrome. It is more appropriate to speak of triggering factors rather than etiological factors. The diagnostic procedure should take place in two stages rst, by investigating for the presence of such a factor in a patient and second, by evaluating its role in the global evaluation of the asthma (seriousness, control, and severity).
Genetic Component

The genetic component is indisputable. It is well known that asthma runs in families. Conventionally, this hereditary constituent seems to be associated with atopy. However, recent work has demonstrated that even asthmatics with no demonstrable personal or family allergic factor (intrinsic asthma) had asthmatics in their family. Asthma is almost certainly polygenic in origin. Association with numerous polymorphisms have been reported at different loci in relation with or not with the known physiopathological data for the disease. The nding that different patients have different responses to antiasthmatic treatments has made it possible to develop potentially promising strategies for pharmacogenetic studies. For instance, data concerning the polymorphism of b2-mimetic receptor and the potential severity of the disease represent an interesting approach. Some examples of candidate genes implicated in the development of asthma are reported in Table 3.
Environmental Component

Inhaled allergens These are allergens present in the ambient air. When inhaled in small quantities, they are capable of sensitizing subjects and triggering symptoms when they reach the level of the respiratory mucosa. Dermatophagoides pteronissimus. These form one of the major allergens of household dust. Animal proteins. Proteins produced by pets, laboratory experiments, or leisure activities.

occurrence of exacerbations, prevention of the occurrence of asthma, induction of asthma, and persistence and development of chronic asthma due to chronic infections.

Bacterial infection This plays a secondary role in the physiopathology of asthma and as a triggering factor in exacerbations. The presence of endotoxins in the environment may be involved in the triggering

ASTHMA / Overview 171

of exacerbations in asthmatic patients, though it may also potentially protect an individual from the development of asthma symptoms (hygiene theory). Pollution SO2, NO2, and ozone act in synergy to trigger an attack. These different pollutants are responsible for exacerbations, sometimes serious or even very serious, especially in a child. The main sources of pollution are boiler plants (using fuel-oil and coal), household and industrial refuse incinerators, and motor vehicles (diesel engine). Particulate pollution has been shown to be associated in industrialized areas with an increase in asthma exacerbations. Diesel exhaust particles worsen allergic symptoms by amplifying the release of inammatory mediators. Indoor pollution should not be forgotten and in particular that produced by home boilers. There are several evidences indicating that indoor pollution indicating a potential role for endotoxins, cockroaches, and parents smoking are major contributors for asthma exacerbations and lack of control in children with asthma. There is no denitive argument proving the involvement of atmospheric pollution in the acquisition of the asthma phenotype of a given subject. Gastroesophageal reux Gastroesophageal reux (GER) is more frequent in asthmatics than in a normal population. The acidity of the lower esophagus induces reex bronchoconstriction. Contamination of the bronchi may also occur following incompetence of the cardia. Controlled studies have not shown the value of systematic treatment of GER to improve the asthma, even in the most severely affected patients. Psychological and/or psychiatric factors These are certainly involved, both in the expression of symptoms and beliefs on treatment, and therefore therapeutic compliance. They are, however, always difcult to evaluate. Inuence of endocrine factors This is probable and the role of sex hormones is always mentioned rst (effect of puberty, episodes of genital life in woman). Premenstrual exacerbations are rare events which can contribute to the severity of the disease. It has been shown recently that severe asthma is more frequent in women than in men. Menopause is an important period for asthma revival, poor control, or late-onset, but the exact sustaining mechanisms are yet to be understood. Sexual female hormonal replacement has been shown in one epidemiological study to be associated with an increase in emergency room attendance for asthma.

Exercise Induced Asthma

This is characterized by the occurrence of bronchial obstruction at the end of exercise. In certain cases, the asthma occurs during exercise though it is then possible to run through the asthma if the exercise is continued. This is practically always present in children and adolescents and it may constitute a marker of bronchial hyperreactivity and inammation. It is essential to control this form of asthma to allow normal physical activity and a harmonious physical and psychological development. Intercritical bronchial obstruction may be responsible for exertional dyspnea though this is not exertional asthma in the strict sense of the term.
Smoking

Tobacco smoke (active and passive) by itself induces inammation of the airways with hypersecretion, paralysis, and ciliary destruction, and inltration of the distal aerial spaces by neutrophils. Active smoking is associated with a more severe asthma which responds less well to corticosteroids. It is not always easy to distinguish between asthma in the smoker and chronic obstructive pulmonary disease (COPD) with a certain degree of variability of the respiratory function. Maternal smoking may induce more asthma or atopy-associated diseases in children. Passive smoking by children is clearly responsible for uncontrollable asthma and this factor must be always looked for in the child. Many asthmatic adolescents are smokers and they have a high risk of loss of control and SAA. Adapted educational strategies must always be proposed.

Treatment
Asthma treatment is based on three main axes: drug therapy, control of aggravating and triggering factors, and education of patients and health professionals. The objectives must be discussed with the patient, though the main aim is to obtain an acceptable control of the disease.
Pharmacology

Corticocorticosteroids The efcacy of treatment with inhaled corticocorticosteroids is well established. It decreases functional signs, exacerbations, deaths, and costs, and improves the level of respiratory function and bronchial reactivity. All these effects have been demonstrated in comparison with placebo. Inhaled corticoids considerably improve the riskbenet ratio of long-term treatment (Figure 1). They are indicated in persistent, mild, moderate, or

172 ASTHMA / Overview

severe asthma. The local side effects of inhaled corticoids (candidiasis, hoarse voice) are effectively prevented by rinsing the mouth after inhalation or by the use of a spacer. At recommended dosages, in persistent mild to moderate asthma (and up to 1500 mg day 1 of beclomethasone equivalents), the risks of systematic adverse effects (adrenal insufciency, osteoporosis) are low. The main adverse effects are susceptibility to bruises and skin atrophy. In the child, the mean dosage of 400 mg day 1 of budesonide does not modify growth. However, questions still remain about inhaled corticosteroids their efcacy in modifying the severity of asthma, their interaction with the natural history of the disease, or the long-term systemic side effects are still a topic of debate.

100

75

50 Efficacy

25

0 Mild Moderate Severe

Figure 1 Riskbenet ratio potential for drugs in the treatment of persistent asthma.

Bronchodilators b2-mimetics are very potent bronchodilators. They stimulate bronchial smooth muscle b2-receptors. Short-acting b2-agonists (from 4 to 6 h) may be distinguished from long-acting b2-agonists (12 h and more). This class of drug has no or very few anti-inammatory effects, and causes little tachyphylaxis. Side effects are minor with mainly tremors, which disappear during treatment, and usually nonserious tachycardia-type palpitations. In persistent asthma, on-demand treatment with short-acting b2-agonists must be combined with continuous anti-inammatory treatment. In case of suboptimal or unacceptable control in patients requiring daily doses of these short-acting b2-agonists and/or if symptoms occur at night, a long-acting b2-agonist may be prescribed. The use of shortacting b2-agonists is then reserved for the treatment of episodes of dyspnea occurring in spite of wellconducted treatment. A high consumption is then associated with loss of control. It is better to give them on demand, rather than systematically, and they are not associated with a risk of an increased death rate. The value of long-acting b2-agonists is demonstrated by a high level of evidence. These agents improve the control of asthma when inhaled corticoid therapy is insufcient alone. Their combination with inhaled corticoid therapy is better than a double dose of the latter drug. This strategy improves the control of the asthma, decreases the incidence and seriousness of exacerbations, and improves patient quality of life. At present, LABAs should never be considered as the sole treatment for asthma (Figures 2 and 3).

FEV1

Side effects

BHR

Hours

Days

Weeks

Months

Years

Symptoms

% DEP

Exacerbations

Figure 2 Improvement of control components after rst treatment initiation in asthma.

ASTHMA / Overview 173

+ High-dose ICS OCS Theophylline AntiIGE Steroid sparing Ipratropium Leukotriene antagonists

+ LA 2 agonists + Moderate-dose ICS Leukotriene antagonists

+ Low-dose ICS

SA 2 agonists as required

Mild controlled

Severe uncontrolled

Education Environment control specific immunotherapy Comorbidities treatment

Figure 3 Treatment of asthma according to control.

Figure 4 Pathology of asthma.

Inhaled corticoidlong-acting b2-agonist combination Combination in a same inhaler of a corticoid and a long-acting b2-agonist follows recommendations for the treatment of persistent, moderate to severe asthma, with facilitated administration, and the objective of improving compliance, notably of corticoid therapy. Two combinations have been developed: uticasonesalmeterol and formoterol budesonide. The benet of these xed combinations in comparison with separate combination of longacting b2-agonists and inhaled corticocorticosteroids

is not completely established and nor is the value of a xed combination administered systematically or modulated on demand (Figure 4). Cysteinylleukotriene receptor antagonists The cysteinylleukotriene receptor antagonists used at present are montelukast and zarlukast. They are used orally and potentially can treat asthma and rhinosinusitis. These agents have been shown to be useful in exercise induced-asthma, as well as a singleagent therapy of mild or moderate asthma. They lead

174 ASTHMA / Overview

to bronchodilation and have been able to improve asthma control in long-term placebo-controlled studies. Their equivalence with low-dose inhaled corticoid therapy (400 mg day 1 of beclomethasone) is suspected but not fully demonstrated. The equivalence of a combination of an antileukotriene in comparison with a long-acting b2-agonist in noncontrolled patients treated with inhaled corticoid therapy alone is not proven. Their role in severe asthma is uncertain; it should be interesting, though, to follow their benet in aspirin-induced asthma, which was recently demonstrated in some clinical studies. Theophylline This is a less potent bronchodilator than the b2-mimetics. It also has numerous other actions and in particular an anti-inammatory activity. Its effects are strictly dependent on its serum concentration, which must be maintained in a relatively narrow range between 10 and 20 mg l 1. Side effects are also dose-dependent. At present it is not used much for primary treatment of asthma, except in the most severely affected patients. Anticholinergics Ipratropium and oxitropium are antagonists of muscarinic receptors. They are bronchodilators but are less potent and slower to act than b2-agonists. They are indicated in the treatment of SAA and exacerbations of chronic asthma as a complement to short-acting b2-agonists. Their efcacy beyond the rst 48 h of the treatment is unknown. Proof of their efcacy exists especially for treatment in children. There are few adverse effects. Their efcacy in the management of chronic asthma has not been evaluated. Tioptropium (Spiriva) is a long acting anticholinergic drug, which has been developed for the treatment of COPD. Its potential interest in asthma and specifically severe asthma deserves further investigations.
Control of the Environment

asthma with a maximum of evidence may force a patient to change jobs. It is more difcult to adopt outdoor pollution-control measures which mainly concern the authorities.
Immunotherapy

Specic immunotherapy is of some help in allergic asthmatic patients if simple rules are applied. Patients with persistent severe rhinitis and persistent mild asthma benet most from this treatment. It is contraindicated in uncontrolled and severe asthma. On the other hand, the development of anti-IgE strategies may give hope to better control in severely allergic patients.
Education

Education of patients is indicated in all asthmatics, though it should be adapted to the severity, importance of triggering factors, and specic personality of each patient. It must be based on a precise educational diagnosis. Objectives must be dened in partnership with the patient. The educational methods used depend on these objectives. This therapeutic education may be individual or may be provided in institutions such as asthma schools.

Conclusion
Asthma is a bronchial inammatory disease that alters the structure of the airways including the upper airways, which may sometimes have systemic repercussions. It is chronic, variable, and reversible and also a multifactorial, polygenic, and environmental disease. Management must be based on a long-term strategy. The major objective is to obtain satisfactory control to allow an optimal quality of life. Treatment involves use of a potent and well-tolerated pharmacopoeia, but may only be implemented in partnership with the patient.
See also: Asthma: Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchodilators: Anticholinergic Agents. Capsaicin. Carbon Monoxide. CD14. Chemokines. Chymase and Tryptase. Cilia and Mucociliary Clearance. Cyclic Nucleotide Phosphodiesterases. DNA: Repair. Dust Mite. Environmental Pollutants: Oxidant Gases. Eotaxins. Epidermal Growth Factors. Extracellular Matrix: Surface Proteoglycans. Fibroblasts. Gastroesophageal Reux. G-Protein-Coupled Receptors. Histamine. Immunoglobulins. Interferons. Kinins and Neuropeptides: Vasoactive Intestinal Peptide; Other Important Neuropeptides. Leukocytes: Eosinophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. Oxidants and Antioxidants: Oxidants. Pediatric Pulmonary

Although environmental control seems useful, there is no complete evidence of its efcacy. It comprises the reduction of exposure to allergens and non-specic irritants and the prevention of active and passive smoking. Allergy is one of the major factors associated with asthma and the most important for children of school age. Prevention of exposure to allergens is logically the rst measure to be taken. This obviously concerns pets though this is not necessarily well accepted by patients or close relatives and friends. It is possible to try to eradicate house-dust mites though this is rarely complete. Diagnosis of occupational

ASTHMA / Overview 175 Diseases. Proteinase-Activated Receptors. Signs of Respiratory Disease: Lung Sounds. Symptoms of Respiratory Disease: Dyspnea. Tumor Necrosis Factor Alpha (TNF-a ).
review in adults with asthma. Cochrane Database of Systematic Reviews 3: CD001279. Gibson PG, Henry RL, and Coughlan JL (2003) Gastro-oesophageal reux treatment for asthma in adults and children. Cochrane Database of System Review 2: CD001496. Holgate ST (1999) Genetic and environmental interaction in allergy and asthma. Journal of Allergy and Clinical Immunology 104: 11391146. Juniper EF, OByrne PM, Ferrie PJ, King DR, and Roberts JN (2000) Measuring asthma control. Clinic questionnaire or daily diary? American Journal of Respiratory and Critical Care Medicine 162: 13301334. Juniper EF, OByrne PM, Guyatt GH, Ferrie PJ, and King DR (1999) Development and validation of a questionnaire to measure asthma control. European Respiratory Journal 14: 902907. Kips JC, OConnor BJ, Inman MD, et al. (2000) A long-term study of the anti-inammatory effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma. American Journal of Respiratory and Critical Care Medicine 161: 996 1001. Kips JC, Peleman RA, and Pauwels RA (1999) The role of theophylline in asthma management. Current Opinion in Pulmonary Medicine 5: 8892. Lemiere C, Bai T, Balter M, et al. (2004) Adult asthma consensus guidelines update 2003. Canadian Respiratory Journal 11(supplement A): A9A18. Meslier N, Racineux JL, Six P, and Lockhart A (1989) Diagnostic value of reversibility of chronic airway obstruction to separate asthma from chronic bronchitis: a statistical approach. European Respiratory Journal 2: 497505. OHollaren MT, Yunginger JW, Offord KP, et al. (1991) Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. New England Journal of Medicine 324: 359363. Parker JM and Guerrero ML (2004) Airway function in women: bronchial hyperesponsiveness, cough and vocal cord dysfunction. Clinics in Chest Medicine 25: 321330. Pauwels RA, Lofdahl CG, Postma DS, et al. (1997) Effect of inhaled formoterol and budesonide on exacerbations of asthma. New England Journal of Medicine 337: 14051411. Reddel H, Ware S, Marks G, et al. (1999) Differences between asthma exacerbations and poor asthma control. Lancet 353: 364369. Rosenstreich DL, Eggleston P, Kattan M, et al. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. New England Journal of Medicine 336: 13561363. Salmeron S, Liard R, Elkharrat D, et al. (2001) Asthma severity and adequacy of management in accident and emergency departments in France: a prospective study. Lancet 358: 629635. Shrewsbury S, Pyke S, and Britton M (2000) Meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (MIASMA). British Medical Journal 320: 13681373. Sistek D, Tschopp JM, Schindler C, et al. (2001) Clinical diagnosis of current asthma: predictive value of respiratory symptoms in the SAPALDIA study. Swiss study on air pollution and lung diseases in adults. European Respiratory Journal 17: 214219. Suissa S, Ernst P, Benayoun S, Baltzan M, and Cai B (2000) Lowdose inhaled corticocorticosteroids and the prevention of death from asthma. New England Journal of Medicine 343: 332336. ten Brinke A, Ouwerkerk ME, Zwinderman AH, Spinhoven P, and Bel EH (2001) Psychopathology in patients with severe asthma is associated with increased health care utilization. American Journal of Respiratory and Critical Care Medicine 163: 10931096.

Further Reading
Abramson MJ, Puy RM, and Weiner JM (2003) Allergen immunotherapy for asthma. Cochrane Database of Systematic Reviews 4: CD001186. Agertoft L and Pedersen S (2000) Effect of long-term treatment with inhaled budesonide on adult height in children with asthma. New England Journal of Medicine 343: 10641069. American Thoracic Society (1995) Standardization of spirometry, 1994 update. American Journal of Respiratory and Critical Care Medicine 152: 11071136. ANAES (2004) Recommandations pour le suivi me dical des patients asthmatiques adultes et adolescents. Paris 169pp (with an english translation on site www.anaes.fr). Anderson SD and Holzer K (2000) Exercise-induced asthma: is it the right diagnosis in elite athletes? Journal of Allergy and Clinical Immunology 106: 419428. Barnes PJ (1995) Inhaled glucocorticoids for asthma. New England Journal of Medicine 332: 868875. Barnes PJ (2004) Corticoresistance in airway disease. Proceedings of the American Thoracic Society 1: 264269. Bateman ED, Boushey HA, Bousquet J, et al. (2004) GOAL Investigators Group. Can guideline-dened asthma control be achieved? The gaining optimal asthma control study. American Journal of Respiratory and Critical Care Medicine 170: 836844. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58(supplement 1): S1S83. Chung KF, Godard P, Adelroth E, et al. (1999) Difcult/therapyresistant asthma: the need for an integrated approach to dene clinical phenotypes, evaluate risk factors, understand pathophysiology and nd novel therapies. European Respiratory Journal 13: 11981208. Cookson W (1999) The alliance of genes and environment in asthma and allergy. Nature 402(supplement 6760): B5B11. Cottin V and Cordier JF (1999) ChurgStrauss syndrome. Allergy 54: 535551. DAmato G, Liccardi G, DAmato M, and Cazzola M (2002) Outdoor air pollution, climatic changes and allergic bronchial asthma. European Respiratory Monograph 21: 3051. De Blay F, Casel S, Pauli G, and Bessot JC (2000) Respiratory allergies and household allergenic environment. Revue des Maladies Respiratories 17: 167176. Ducharme FM (2003) Inhaled glucocorticoids versus leukotriene receptor antagonists as single agent asthma treatment: systematic review of current evidence. British Medical Journal 326: 621. Eggleston PA, Bush RK, and American Academy of Asthma, Allergy and Immunology (2001) Environmental allergen avoidance: an overview. Journal of Allergy and Clinical Immunology 107(supplement 3): S403S405. Everard ML, Bara A, Kurian M, Elliott TM, and Ducharme F (2002) Anticholinergic drugs for wheeze in children under the age of two years. Cochrane Database of Systematic Reviews 1: CD001279. Gern JE and Busse WW (2000) The role of viral infections in the natural history of asthma. Journal of Allergy and Clinical Immunology 106: 201212. Gibson PG, Coughlan J, and Wilson AJ (1998) The effects of self management and asthma education and regular practioner

176 ASTHMA / Allergic Bronchopulmonary Aspergillosis

Thomas NS, Wilkinson J, Lio P, et al. (2000) Genetic factors involved in asthma and atopy. Studies in British families. Revue des Maladies Respiratories 17: 177182. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 734739. Venables KM and Chan-Yeung M (1997) Occupational asthma. Lancet 349: 14651469. Vignola AM, Chanez P, Godard P, and Bousquet J (1998) Relationships between rhinitis and asthma. Allergy 53: 833839. Vollmer WM, Markson LE, OConnor E, et al. (1999) Association of asthma control with health care utilization and quality of life. American Journal of Respiratory and Critical Care Medicine 160: 16471652. Wark PA and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 17. WHO/NHLBI Workshop Report (1995) Global Strategy for Asthma Management and Prevention. National Institutes of Health, National Heart, Lung and Blood Institute, Publication Number 95-3659 (revised 2002). Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews 3: CD001281. Woolcock AJ and Peat JK (1997) Evidence for the increase in asthma worldwide. Ciba Foundation Symposium 206: 122134.

formation of aspergilloma in those with pre-existing pulmonary cavities; a form of extrinsic allergic alveolitis; and hypersensitivity diseases. Subjects with asthma or cystic brosis (CF) may become sensitized to Af following inhalation of spores; once sensitized this results in a type I, IgE-mediated reaction and a spectrum of clinical responses from acute exacerbations of asthma to a more sustained and intense inammatory response that shows features of both type I and type III hypersensitivity and leads to ABPA. ABPA is unique in these disorders as there is evidence of persistence of the organism within the airways resulting in an intense local and systemic immune response that is associated with mucus impaction and may lead to bronchiectasis and pulmonary brosis.

Etiology
Fungi belonging to the genus Aspergillus are ubiquitous spore-forming lamentous fungi and while any member of the group can cause allergic sensitization most disease is attributable to Af. The organism is present in outdoor environments where it readily grows in decomposing organic matter, such as decaying vegetation in soil, mulches, wood chips, freshly mown grass, and sewage treatment debris. Indoors, Af can be found in damp areas especially on walls or ceilings where water damage has occurred; in addition, spores can be found in very large quantities in bird excreta. Acute inhalation of spores is known to trigger acute asthma in sensitized asthmatics, while in subjects with ABPA inhalation of Af antigens leads to a dramatic worsening of the acute and late-phase response with falls in FEV1 of 5079%. Exacerbations of clinical disease have been reported to occur coincident with high outdoor spore counts but a direct temporal relationship to inhalation and an acute exacerbation of ABPA remains unclear. A genetic predisposition to ABPA is also possible, with reports of familial occurrence. Two small studies have shown a higher carriage rate of at least one mutation of the cystic brosis transmembrane regulator (CFTR) gene in subjects with ABPA, compared to subjects who are skin-test positive to Af and those with asthma alone. The alleles HLA-DR2 and -DR5 have also been linked to severity of disease in ABPA. Most recently, a polymorphism of surfactant proteins has been shown to be associated with increased serum IgE and blood eosinophilia in patients with ABPA. These ndings suggest there may be a link between ABPA and an inability to effectively clear the organism in a group whose immune response is likely to be one associated with atopy and a T-helper (TH)-2 response.

Allergic Bronchopulmonary Aspergillosis

P Wark, Southampton University, Southampton, UK
& 2006 Elsevier Ltd. All rights reserved.

Introduction
Allergic bronchopulmonary aspergillosis (ABPA) is a complex condition that results from hypersensitivity to the fungus Aspergillus fumigatus (Af). ABPA was rst described in the UK in 1952, and has been estimated to occur in 12% of chronic asthmatics and up to 15% of patients with cystic brosis. It is unclear whether the prevalence of ABPA has declined with the widespread use of inhaled corticosteroids to treat asthma. There is an excessively high prevalence of ABPA in cystic brosis where evidence suggests that ABPA may cause a faster decline in lung function. In chronic asthma, ABPA follows a more variable course with recurrent exacerbations and, at least in some patients, it leads to proximal bronchiectasis and irreversible brotic lung disease. Exposure to Af can cause a wide range of pulmonary diseases, which are summarized in Figure 1, including: severe life-threatening pneumonia in the immunosuppressed; subacute infections such as the

ASTHMA / Allergic Bronchopulmonary Aspergillosis 177

No previous lung disease

Asthma

Cystic fibrosis

Cavitatary lung disease

Immunocompromised

Inhale Af spores

Acute asthma
(those sensitized up to 30%)

No immune response

Colonization with Af

Mucoid impaction extrinsic allergic alveolitis (all extremely rare)

ABPA (1 2%)

ABPA (10 15%)

Aspergilloma

Invasive pulmonary aspergillosis

Figure 1 A summary of the wide range of pulmonary diseases caused by exposure to Aspergillus fumigatus. Aspergillus fumigatus is a ubiquitous environmental fungus and spores are regularly inhaled. For most individuals with no previous lung disease, this appears to cause no immune response and no symptoms. High-dose exposure or exposure of susceptible individuals may trigger extrinsic allergic alveolitis or, more rarely, colonization leading to an intense immune response as in ABPA with areas of mucoid impaction and inammation. Up to 30% of subjects with asthma are sensitized to Af. In those individuals exposure to Af may initiate an acute asthma response. A proportion of asthmatics seem to be particularly prone to the effects of Af and are unable to clear the organism. It persists in the lower airways and initiates the intense immune response resulting in ABPA, which occurs in 12% of asthmatics. In CF, particularly in those with atopy or a history of asthma-like symptoms, sensitization to Af occurs. In at least 1015% of CF individuals the organism is not cleared and colonization ensues. This initiates an immune response like that seen in asthma but is associated also with the endobronchial inammation and chronic infection already present in CF. An aspergilloma (fungal ball) consists of masses of fungal mycelia, inammatory cells, brin, mucus, and tissue debris, these usually developing in a preformed lung cavity. In a study of 549 subjects with pulmonary cavities due to old tuberculosis, aspergillomas were present in 11%. Subjects with immunosuppression subsequent to chemotherapy or the use of immunosuppressives or with disorders such as chronic granulomatous disease are unable to clear Af. In these cases the immune response is so ineffective that direct fungal invasion occurs, leading to a severe acute pneumonia, sepsis, and often death.

Pathology
In the early stages of ABPA, pulmonary lesions consist of bronchial mucoid impaction, and areas of eosinophilic pneumonia. In mucoid impaction, bronchi are dilated and lled with mucus that contains necrotic eosinophils, Curschmans spirals, Charcot-Leyden crystals, and occasionally fungal hyphae. Eosinophilic pneumonia is synonymous with radiographic inltrates, composed of focal areas of alveoli lled with eosinophils and macrophages. As the disease progresses, endobronchial inammation increases, which begins as a mixed eosinophilic/neutrophilic obliterative bronchiolitis. This then progresses to the complete replacement of bronchial structures by inammatory cells (histiocytes, lymphocytes, and plasma cells),

known as bronchocentric granulomatosis. At the center of these areas are necrotic inammatory cells and Af hyphae. Destruction of the bronchial wall matrix and subsequent scarring and repair are thought to account for the development of bronchiectasis and brosis. The intensity of the inammatory inltrate present in the airways of subjects with ABPA certainly correlates with the severity of bronchiectasis present. Pathological features of ABPA are demonstrated in Figure 2.

Clinical Features
Diagnosis and Disease Staging

The criteria for diagnosis were standardized in 1977 by Patterson and co-workers and are summarized in

178 ASTHMA / Allergic Bronchopulmonary Aspergillosis

Table 1 Diagnostic criteria for ABPA Essential criteria Asthma Immediate skin-prick test (SPT) positive to Af Total serum IgE 4 1000 ng ml 1 (400 IU ml 1) Serum specic IgG and IgE antibodies to Af (or positive precipitins) Presence of bronchiectasis Yes, dene as ABPA-CB No, dene as ABPA-S Nonessential criteria Transient pulmonary inltrates on chest radiograph Blood eosinophilia Precipitating antibodies to Af Expectoration of mucus plugs

(a)

(b)

(c)
Figure 2 (a) Hyphae of Aspergillus fumigatus grown in vitro; (b) mucoid impaction. The dilated bronchus is lled with mucus that contains eosinophils, cell debris, and the products of eosinophil degranulation (Charcot-Leyden crystals). The bronchial epithelium is heavily inltrated by inammatory cells. (c) Bronchocentric granulomatosis: the epithelium of the bronchus becomes completely replaced by a granulomatous inltrate of histiocytes, lymphocytes, plasma cells, and eosinophils. In addition, there are necrotic inammatory cells and mucus within the dilated airway and marked generalized bronchial wall thickening. Slides Courtesy of Dr B Addis, Southampton General Hospital.

Table 1. They require the patient to have a pre-existing diagnosis of asthma (or cystic brosis), immediate-type skin reactivity to Af, and, at least during exacerbations or in the absence of treatment,

peripheral blood eosinophilia, precipitating antibodies to Af antigen, elevated serum IgE, and IgG antibodies against Af. Radiological evidence of proximal bronchiectasis is frequently found with ABPA, but is no longer felt to be a prerequisite for diagnosis. However, the presence of bronchiectasis along with skin-test reactivity and eosinophilia is quite specic for ABPA. ABPA has been subdivided into ve stages. Stage I is the initial acute presentation, with eosinophilia, immediate-type skin reactivity to Af, total serum IgE greater than 2500 ng ml 1, and pulmonary inltrates on a chest radiograph. Stage II is the disease in remission, where there is persistent immediate-type skin reactivity and precipitating antibodies to Af antigens. In stage III there is an exacerbation of symptoms with all the characteristics of stage I but with a twofold rise in serum IgE and new pulmonary inltrates. Stage IV patients have asthma where control of symptoms is dependent on chronic use of highdose corticosteroids. In stage V, chronic disease has progressed to predominately xed airow obstruction with extensive bronchiectasis and brosis. Skin-prick testing is a useful screening test to identify potential patients with ABPA. It is highly sensitive but not specic. Consequently, a negative skin-prick test for Af can rule out ABPA whereas a positive test warrants further investigation, particularly in the case of an asthmatic with frequent exacerbations or parenteral corticosteroid dependence. Patients with a positive skin test should be evaluated with serology including measurement of total serum IgE, specic IgG, and IgE antibodies to Af and precipitins. Patients with an IgE 4 1000 ng ml 1 (400 IU ml 1) and positive IgE Af and IgG Af are likely to have ABPA and warrant further investigation with a high-resolution computed tomography (CT) scan of the chest to determine the presence of bronchiectasis. Patients with central bronchiectasis are classed as

ASTHMA / Allergic Bronchopulmonary Aspergillosis 179

ABPA-CB (ABPA with central bronchiectasis) and those without are classed as ABPA-S (ABPA serology). In milder disease, a high index of suspicion is required, with the criteria fullled only during an exacerbation or when off parenteral corticosteroids.
Radiology

During acute disease ares chest radiographs may demonstrate transient pulmonary inltrates that occur as a result of eosinophilic pneumonitis and areas of mucoid impaction that appear as areas of atelectasis with toothpaste shadows and the nger-in-glove appearance of bronchi lled with inspissated mucus. Permanent radiographic changes are a feature of more advanced disease with the development of central bronchiectasis and in some areas of parenchymal brosis. While bronchial wall thickening and minimal bronchiectasis (limited to 12 segments) has been described in asthma, the presence of widespread central bronchiectasis, which is best seen on high-resolution CT scans, is characteristic of ABPA. In addition, areas of bronchial wall thickening, mucoid impaction, circular opacities, and areas of atelectasis are common.
ABPA and Cystic Fibrosis

Diagnosis of ABPA in CF is more difcult though the criteria have recently been reviewed and standardized by a consensus conference; these criteria are summarized in Table 2. The diagnosis in CF is complicated by the pre-existing presence of bronchiectasis and chronic endobronchial infection. A multiple regression analysis of over 14 000 individuals with CF determined that wheezing, a diagnosis of bronchial asthma, and colonization with Pseudomonas aeruginosa were independent risk factors for ABPA. The development of ABPA in someone with cystic brosis heralds a more complicated clinical course and is associated with more bacterial colonization, lower lung function, and an increase in pulmonary complications.

Pathogenesis
Af is an effective pathogen in humans as intrinsic qualities of the organism enhance its ability to infect
Table 2 Diagnostic criteria for ABPA in cystic brosis An acute or subacute deterioration in clinical symptoms (cough, wheeze, exercise capacity, change in pulmonary function, or increase in sputum) not attributable to another cause Immediate skin test reactivity to Af A total serum IgE 4 400 IU ml 1 Along with one of the following: precipitins to Af or specic IgG to Af new or recent inltrates, mucus plugging, or proximal bronchiectasis on either chest radiograph or CT scan

the lungs and cause disease. The spores of Af are 2 5 mm in size with a hydrophobic coat that allows them to be inspired into the lungs. Once inspired, the conidia of Af are able to bind to surfactant molecules in the distal airway lumen, as well as complement (C3) and brinogen. The conidia germinate within the airways and form focal areas of mycelia. The mature organisms are then capable of releasing allergens, virulence factors, and proteases. These factors contribute to: (1) impaired mucociliary clearance; (2) impaired action of fungicidal proteins and complement in the airway lining uid; and (3) inhibition of phagocytosis and the killing capacity of phagocytic cells (macrophages, neutrophils). As the organism is ubiquitous in the environment, sensitized individuals are likely to regularly inhale spores and this will always represent a fresh source of antigenic stimulus. However, Af is unique as an aeroallergen and inhalation alone is insufcient to lead to ABPA. Persistence of viable Af within the airways appears to be an important factor in determining the development of ABPA. Viable Af has been found growing on and between bronchial epithelial cells, despite an intense inammatory cell inltrate while Af proteases lead to the release of proinammatory mediators from epithelial cells. These proteases also have the ability to detach epithelial cells from their basement membrane and are particularly potent. This loss of epithelial integrity may lead to exposure of the underlying matrix, to which Af can also adhere allowing direct damage of the airways and the development of bronchiectasis; in addition, the disruption of mucosal integrity enhances antigen uptake and activation of T cells. In addition to its direct effect on the airway epithelium, Af appears to be able to elicit a powerful TH2 immune response. The presence of Af infection in mice induces a TH2 lymphocyte response, while the extracts from Af when co-cultured with B cells elicit the release of IgE and subjects with ABPA develop specic TH2 CD4 cells in response to exposure. Epithelial cell activation and a TH2 immune response favors recruitment and activation of eosinophils. Eosinophil activation and release of toxic granular proteins are likely to contribute to damage to both the epithelium and airway matrix. In asthma TH2 cells also release IL-4 and IL-13, which induces the release of TGF-b from epithelial cells, leading to a repair response with myobroblast activation, brosis, and airway wall remodeling. This intense recruitment of inammatory cells to the airway lumen and the development of progressive airway wall damage is in keeping with airway inammation found in patients with ABPA and the development of radiographic bronchiectasis.

180 ASTHMA / Allergic Bronchopulmonary Aspergillosis

Af conidia

Mucoid impaction Alveolar macrophage 2 3 Proteases

Tenacious mucus

Epithelial detachment

Mycelia

Fibroblast 8
Inflammato ry cytokines/chemokines

Allergens 4 5

6 Eosinophil

IgE IL-5 TH2 lymphocyte IL-4 & IL-13

B cell

Figure 3 A model for the pathogenesis of ABPA. 1: The conidia of Af are just 25 mm in diameter and are easily respired into the distal airways. 2: The conidia are trapped within the tenacious mucus bilayer, binding to surfactants, complement, and brinogen. The conidia attach to bronchial epithelial cells. Here they germinate and mature, forming mycelia or small fungal balls. 3: Mature Af produces proteases and allergens. Aspergillus proteases are extremely potent. They activate epithelial cells, releasing proinammatory cytokines and chemokines, leading to the hypersecretion of mucus, thus damaging epithelial cells, which results in widespread epithelial detachment and the loss of mucosal integrity. This allows the proteases to damage the airway matrix also. In addition, proteases can block the ability of alveolar macrophages to ingest Af, the net effect being impaired mucociliary clearance and facilitated colonization. 4: Activated epithelial cells release proinammatory mediators, such as interleukin-8 (IL-8), IL-6, RANTES, and MCP-1, activating and attracting lymphocytes, eosinophils, and neutrophils to the airways. 5: Aspergillus allergens are potent activators of TH2 type immune response from TH cells. These cells release IL-5, which recruits and activates eosinophils. 6: Eosinophils migrate to the airways and inltrate. Once activated, they release toxic granular proteins that further damage the epithelium and airway matrix. 7: Activated TH2 lymphocytes produce IL-4 and IL-13, which induce isotype switching of bells to produce IgE antibodies. In addition, IL-4 and IL-13 have been shown to induce epithelial cells to secrete the anti-inammatory but pro-brotic mediator TGF-b. 8: Epithelial and airway matrix damage elicits a wound-repair response in the airways. Release of TGF-b has been shown to induce broblasts to release growth factors and collagen. This is thought, at least in part, to induce airway wall remodeling in asthma.

A model for the pathogenesis of ABPA is proposed in Figure 3. ABPA appears to develop in susceptible individuals who are sensitized to Af; they are unable to clear the organism, which then adheres to the bronchial epithelium releasing allergens and proteases. This activates an intense TH2 immune response and leads to progressive airway destruction and remodeling.

Animal Models
A number of investigators have employed animal models to determine the role of Af antigens in triggering the immune response and in determining the

constitutive components responsible for this response. Most investigators have used crude Af extract to sensitize animals, often followed by ongoing exposure to viable conidia or Af spores. Uniquely, sensitization with Af extract does not require an adjunct; it is thought that Af proteases effectively disrupt the mucosal barrier enhancing antigen presentation. Recently, recombinant antigen components of Af extract have been used; identifying Asp f 1, 3, and 4 as independent inducers of airway inammation and bronchial hyperresponsiveness (BHR). The most widely employed animal model has been murine. As in humans, sensitization to Af leads to a

ASTHMA / Aspirin-Intolerant
Table 3 Components of the immune response to Aspergillus elucidated by murine models of ABPA Immune mediator Block IL-13 Response observed in the model Reduced pulmonary eosinophilia and goblet cell hyperplasia No change to subepithelial brosis Reduced IgE and pulmonary eosinophila More pronounced TH1 response and consequent pulmonary inammation Inhibit all aspects of the TH2 inammatory response to Af Reduced pulmonary eosinophils Airway inammation occurs with monocyte inltrate and BHR still increased (a) Treated at time of Af sensitization, increased airway inammation, and BHR (b) Treated at 1430 postsensitization, reduced airway inammation, and BHR Increased TH2-mediated inammation Increased IgE production Reduced TH1 response with lower levels of interferon-g and reduced pulmonary neutrophils Reduced clearance of Af

181

Block IL-4

Addition of IL-10 Block IL-5

Inhibit MCP-1 or inactivate receptor CCR2 CCR2 knockout mice

potent response with production of IgE, IgG1, pulmonary eosinophilia, and TH2 activation and their recruitment to the airways. In addition, such models have identied Af as potent inducers of BHR, airway remodeling, goblet cell hyperplasia, and mucus hypersecretion. The ability to use knockout animals and block specic cytokines, chemokines, and their receptors in murine models has allowed a greater understanding of the specic components of the immune response to Af; the most important of these are summarized in Table 3. The profound TH2 immune response to Af has been well demonstrated in these models along with the suggestion that such a response is relatively ineffective in clearing the organism.
See also: Chemokines, CXC: IL-8. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Interleukins: IL-4; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Grunig G and Kurup VP (2003) Animal models of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e157e171. Kauffman HF (2003) Immunopathogenesis of allergic bronchopulmonary aspergillosis and airway remodelling. Frontiers in Bioscience 8: e190e196. Kauffman HF and Tomee JF (1999) Inammatory cells and airway defense against Aspergillus fumigatus. Immunology and Allergy Clinics of North America 18: 619640. Kurup VP and Banerjee B (1996) Allergic aspergillosis: antigens and immunodiagnosis. Advances in Medical Mycology 2: 133154. Patterson R, Greenberger P, et al. (1982) Allergic bronchopulmonary aspergillosis: staging as an aid to management. Annals of Internal Medicine 96: 286291. Soubani AO and Chandrasekar PH (2002) The clinical spectrum of pulmonary aspergillosis. Chest 121: 19881999. Stevens D, Moss RB, et al. (2003) Allergic bronchopulmonary aspergillosis in cystic brosis state of the art: Cystic Fibrosis Foundation Consensus Conference. Clinical Infectious Diseases 37(supplement 3): S225S264. Varkey B (1998) Allergic bronchopulmonary aspergillosis: clinical perspectives. Immunology and Allergy Clinics of North America 18(3): 479501. Wark PAB and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 17. Wark PAB, Gibson PG, et al. (2003) Azoles for allergic bronchopulmonary aspergillosis associated with asthma. Cochrane Database System Review 4(CD001108).

Aspirin-Intolerant
A P Sampson, University of Southampton School of Medicine, Southampton, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Aspirin-intolerant asthma (AIA) is a phenotype experienced by 1020% of persistent asthmatics, in whom acute bronchoconstriction is induced by ingestion of aspirin and other nonsteroidal anti-inammatory drugs (NSAIDs). These drugs share the ability to inhibit synthesis of prostanoids by blockade of cyclooxygenase (COX). Acute reactions to NSAIDs can be life threatening and may be associated with rhinoconjunctival and dermal symptoms. Drugs that selectively inhibit COX-2 appear to be better tolerated than nonselective inhibitors of COX-1 and COX-2. Patients with AIA usually have persistent underlying asthma, often associated with nasal polyposis. Pathologically, the bronchial and nasal airways of AIA subjects show chronic eosinophilia, with evidence of activation of eosinophils and mast cells during acute reactions. The etiology of AIA is unclear, but the proposed mechanism focuses on the inhibition by NSAIDs of the synthesis of a prostanoid, putatively prostaglandin E2, that would normally suppress local inammatory reactions. The consequent synthesis of cysteinyl-leukotrienes and other leukocyte-derived mediators contributes to bronchoconstriction and other acute features. Treatment of AIA involves avoidance of NSAIDs combined with conventional management of underlying asthma, with 75% of AIA patients requiring corticosteroids. Controlled desensitization with regular doses of an NSAID can provide protection against acute reactions.

Further Reading
Bateman ED (1994) A new look at the natural history of aspergillus hypersensitivity in asthmatics. Respiratory Medicine 88: 325. Bosken CH, Myers JL, et al. (1988) Pathological features of allergic bronchopulmonary aspergillosis. American Journal of Surgical Pathology 12(3): 216222. Chetty A (2003) Pathology of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e110e114. Greenberger P, Miller T, et al. (1993) Allergic bronchopulmonary aspergillosis in patients with and without evidence of bronchiectasis. Annals of Allergy 70: 333338.

182 ASTHMA / Aspirin-Intolerant

Introduction
The classical aspirin-intolerant asthma (AIA) syndrome was described by Samter and Beers in 1968 as a triad of rhinosinusitis (often with nasal polyps), asthma, and aspirin sensitivity. Patients with fullblown AIA are about twice as likely to be female as male, but no more likely to be atopic than the general population. Patients typically present in early middle age with nasal congestion, anosmia, and rhinorrhea, and many develop nasal polyps, often with secondary infection of the paranasal sinuses. Bronchoconstriction and airway inammation usually emerge later, and this becomes severe, perennial asthma, with about 75% of patients needing oral or inhaled corticosteroids to maintain control of their symptoms. Acute respiratory reactions to nonsteroidal anti-inammatory drugs (NSAIDs) may be accompanied by rhinoconjunctival symptoms and by dermal symptoms such as facial ushing and exacerbation of preexisting urticaria. Aspirin challenges reveal that the tendency to bronchoconstrict in response to therapeutic doses of NSAIDs is more prevalent than the full-blown aspirin triad recognized clinically. Based on patient history alone, the prevalence of NSAID intolerance in adult asthmatics is 35%, but it rises to 19% when consecutive asthmatic patients are challenged with oral aspirin. Prevalence in asthmatic children is less than 2% based on history alone, but 1316% of postpubertal asthmatic children respond adversely when aspirin challenged. NSAID intolerance is overrepresented in the severe asthmatic population. Among patients who have experienced a near-fatal asthma exacerbation requiring treatment in the intensive care unit, 24% are aspirin-sensitive based on history alone. Most life-threatening acute exacerbations in AIA patients are directly due to inadvertent use of NSAIDs, and in a few cases the precipitating NSAID was prescribed by the patients own physician. However, over 40% of life-threatening asthma exacerbations in AIA patients cannot be attributed to NSAID ingestion, illustrating the severity of the underlying chronic asthma in these patients.

Table 1 Chemical classes of some nonsteroidal anti-inammatory drugs (NSAIDs) Acetic acids Indomethacin Sulindac Diclofenac Propionic acids Ibuprofen Benoxaprofen Fenamates Mefenamic acid Oxicams Piroxicam

Pyrazoles Phenylbutazone Oxyphenbutazone

Salicylates Aspirin (acetylsalicylic acid) Sodium salicylate

In 1919, Cooke recognized that in some patients with asthma, aspirin may also precipitate life-threatening acute exacerbations. Such reactions have now been recognized in response to a wide variety of NSAIDs of diverse chemical classes (Table 1). A family history of AIA is reported by only 6% of AIA patients, suggesting that any genetic inuences are subtle and expressed phenotypically only in the presence of relevant environmental factors. Leukotriene C4 synthase is the terminal enzyme for the synthesis of the bronchoconstrictor cysteinylleukotrienes postulated to contribute to airway narrowing in AIA. A biallelic polymorphism in the promoter region of the LTC4 synthase gene has been reported, involving an A to C transversion at a position 444 bp upstream of the transcription start site. The variant 444C allele may lead to enhanced transcription of the LTC4 synthase gene in relevant cells. The prevalence of the variant allele has been reported to be significantly elevated in AIA patients from a Polish population, but this has not been replicated in US Caucasian or Japanese populations.

Pathology
A classication system for allergic and pseudoallergic reactions to NSAIDs was proposed by Stevenson, Sanchez-Borges, and Szczeklik in 2001. AIA is classied as a type 1 pseudoallergic reaction in which asthmatic patients with a high frequency of sinusitis and nasal polyps experience lower respiratory tract reactions and/or rhinoconjunctival symptoms after exposure to therapeutic doses of aspirin and other NSAIDs. Reactions are dose-dependent and may be life threatening. The type 2 category describes urticarial reactions or angioedema induced by NSAIDs in patients with pre-existing chronic urticaria, and types 3 and 4 describe urticarial or sporadic reactions in patients who are otherwise normal. In contrast to types 14 above, in which patients show extensive cross-reactivity to many NSAIDs, patients classied in types 58 react adversely only to a single

Etiology
Aspirin (acetylsalicylic acid) and other members of the family of NSAIDs inhibit formation of the prostanoid family of lipid mediators by blocking isozymes of cyclooxygenase (COX). They are used therapeutically for their mild analgesic, antipyretic, and anti-inammatory actions. NSAIDs are variably associated with adverse reactions including prolonged bleeding, nephritis, and gastric ulceration.

ASTHMA / Aspirin-Intolerant

183

drug. These reactions include urticaria/angioedema, anaphylaxis, aseptic meningitis, and hypersensitivity pneumonitis. A consistent, although not diagnostic, pathological nding in AIA patients is chronic eosinophilia in the blood, nasal polyps, and bronchoalveolar lavage (BAL) uid. Immunohistochemical studies in bronchial biopsies have conrmed a marked bronchial mucosal eosinophilia in AIA patients, with eosinophil counts three- to fourfold higher than in aspirintolerant asthmatics and 10- to 15-fold higher than in normal subjects. After NSAID challenge, further increases in eosinophils, their activation marker ECP (eosinophil cationic protein), and histamine have been described in the nasal airways, BAL uid, and plasma of AIA patients, suggesting activation of eosinophils and mast cells. In the nasal airway, aspirin challenge of AIA patients is associated with increments in nasal tryptase and histamine, strongly suggesting mast cell activation. Tryptase and histamine levels also rise in the serum of patients experiencing systemic reactions to oral aspirin, but not in those with localized respiratory reactions. Release of tryptase is a recognized marker of mast cell activation in the pulmonary airway, and occurs within 5 min of allergen challenge in allergic asthmatics. However, tryptase did not rise in the BAL uid of AIA patients challenged with inhaled or endobronchial lysine-aspirin. In contrast, a rise in urinary levels of the PGD2 metabolite 9a, 11b-PGF2 following endobronchial lysine-aspirin challenge has been interpreted as evidence for mast cell activation in AIA. Together, the evidence suggests that inammatory mediators released both from mast cells and from eosinophils contribute to the pulmonary and rhinoconjunctival symptoms following NSAID exposure in AIA patients.

50 min after oral aspirin ingestion, ranging from 20 to 120 min. A shorter protocol involves the inhalation of incremental aerosolized doses of lysine-aspirin, a soluble and nonirritant form (this is not available in the US). Respiratory reactions to inhaled lysineaspirin often occur within 1 min, so shorter dosing intervals of 3060 min allow the entire challenge to be completed within 1 day. Inhaled lysine-aspirin challenges are safer than oral challenges as reactions are localized to the airways and are easily reversible with inhaled b2-agonists. The sensitivity of inhaled lysine-aspirin challenge is similar to oral aspirin challenge.

Pathogenesis
The Cyclooxygenase Theory

The most successful model to explain acute respiratory reactions to NSAIDs is the cyclooxygenase theory promulgated by Szczeklik in 1975. This postulates that reactions are related directly to the pharmacological activity of NSAIDs in inhibiting isozymes of COX, the key enzymes in the synthesis of the prostanoid family of lipid-inammatory mediators. Intolerance to an individual NSAID in vivo was shown to be predictable by its potency in inhibiting COX in vitro, with strong inhibitors (including aspirin, indomethacin, mefenamic acid, ibuprofen, and piroxicam) being common precipitants of adverse reactions, while weak inhibitors (such as sodium salicylate) precipitated reactions rarely or only at high doses. The concept that inhibition of prostanoid synthesis is the trigger for NSAID-induced reactions is recognized as a key advance.
The Role of Cysteinyl-Leukotrienes: The Shunting Hypothesis and the PGE2 Brake Hypothesis

Clinical Features
As there are no acceptable in vitro tests for AIA, conrmation of aspirin intolerance can only be obtained by NSAID challenge under controlled conditions. Lung function is monitored while patients ingest incremental oral doses of aspirin or inhaled doses of a lysine-aspirin conjugate or sulpyrine. Concomitant use of b2-adrenergic agonists, cromones, and inhaled corticosteroids may mask responses to NSAID challenge, leading to a high rate of false-negative results. In a 3-day oral challenge protocol, incremental doses of aspirin are given at 3-hourly intervals up to a maximum of 650 mg. The challenge is terminated when forced expiratory volume in 1 s (FEV1) falls by at least 20%. Reactions begin around

The second key advance was the elucidation in 1979 of the structure of slow-reacting substance of anaphylaxis (SRS-A) as a mixture of potent bronchoconstrictor products of a related family of lipid mediators, the cysteinyl-leukotrienes (cysteinyl-LTs). The cysteinyl-LTs LTC4, LTD4, and LTE4 are now recognized to have important bronchoconstrictor and proinammatory roles in many phenotypes of asthma. Their particular relevance to AIA emerged from the recognition that their biosynthetic pathway, the 5lipoxygenase (5-LO) pathway, shares arachidonic acid as a common precursor with the prostanoid pathway (Figure 1). The leukotriene pathway is not inhibited by NSAIDs. Specic blockade of the prostanoid pathway by NSAIDs was therefore proposed to shunt arachidonate away from conversion into

184 ASTHMA / Aspirin-Intolerant

Membrane phospholipids Phospholipase A2 Arachidonic acid

NSAIDs Cyclooxygenase-1 Cyclooxygenase-2

PGG2 PGH2

Zileuton

5-lipoxygenase FLAP LTA4 LTC4 synthase

Prostanoid synthases & isomerases

LTC4 LTD4 LTE4 (Cysteinyl -leukotrienes)

PGE2

TXA2

PGD2 PGF2

Montelukast Pranlukast Zafirlukast

CysLT1 receptors Bronchoconstriction Vascular permeability Edema Mucus secretion Eosinophil migration

Prostanoid receptors (EP1 4, TP, DP, CRTH2, FP)

Figure 1 The 5-lipoxygenase and cyclooxygenase pathways of eicosanoid metabolism.

prostanoids, which have relatively little bronchoconstrictor activity, towards the formation of cysteinylLTs, which are highly potent bronchoconstrictors. This shunting hypothesis is supercially attractive but measurements of lipid mediators in isolated leukocytes treated with NSAIDs argues against such a simple mechanism. It has therefore been superseded by an alternative notion, the PGE2 brake hypothesis. This proposes that NSAIDs block the formation of an anti-inammatory prostanoid, PGE2, which is otherwise known to suppress leukotriene synthesis by leukocytes. Exposure to NSAIDs may thus liberate the 5-LO pathway from suppression by endogenous PGE2 in vivo, at least in susceptible individuals (Figure 2). There are three main lines of experimental evidence supporting this model: 1. The triggering effect of NSAIDs on LT synthesis can be mimicked in vitro in a number of inammatory leukocyte subtypes. Endogenous PGE2 suppresses, and NSAIDs consequently enhance, leukotriene synthesis in eosinophils, neutrophils, basophils, and macrophages, but apparently not in human lung mast cells. Eosinophils themselves generate sufcient PGE2 to suppress their own LTC4 synthesis by about 90%. Treatment of eosinophils with indomethacin inhibits endogenous PGE2 synthesis and stimulates LTC4 release. Replacement experiments showed that

exogenous PGE2 restores the braking effect, returning LTC4 synthesis to the levels seen before indomethacin treatment. PGE2 may act in an autocrine or paracrine manner at EP2 receptors on the cell surface, followed by an increase in intracellular cAMP and activation of protein kinase A, but the subsequent steps by which 5-LO activity is inhibited are unknown. 2. Eicosanoids can be measured in biological uids, including bronchoalveolar lavage (BAL) uid, and urine. Endoscopic challenge with lysine-aspirin reduces PGE2 and thromboxane A2 levels in the BAL uid of AIA patients and aspirin-tolerant asthmatics, but a dramatic rise in BAL uid cysteinyl-LTs is seen only in the AIA group. Following challenge with oral aspirin or inhaled lysine-aspirin, urinary LTE4 levels, used as a marker of whole-body cysteinyl-LT production, rise three to sevenfold in AIA patients, but not in aspirin-tolerant asthmatics; this response is not seen after methacholine-induced bronchoconstriction or placebo challenge. At the same time, there is a fall in urinary markers of prostanoid synthesis, such as 11-dehydro-thromboxane A2. In AIA patients, preinhalation of PGE2 before challenge with inhaled lysine-aspirin completely ablates the rise in urinary LTE4 and prevents the consequent bronchoconstriction, providing strong evidence that cysteinyl-LT synthesis has a functional role in the NSAID-induced airway narrowing. The

ASTHMA / Aspirin-Intolerant
Mechanism of acute NSAID reactions Mast cell / eosinophil COX-1 FLAP 5-LO NSAID

185

LTC4 synthase

cAMP

EP2

PGE2

LTC4

ECP

Histamine Tryptase IL-5

Bronchoconstriction Eosinophillia Vasodilation / edema Remodeling / BHR

Figure 2 In this model, PGE2 derived from constitutive cyclooxygenase-1 (COX-1) normally acts via cell surface EP2 receptors to suppress the activity of mast cells and eosinophils, including the activity of the 5-lipoxygenase pathway. However, when an NSAID ingested by a susceptible individual inhibits COX-1, the reduced synthesis of PGE2 is insufcient to suppress the leukocyte effectively. The result is a surge in the synthesis and release of LTC4 and other mediators, leading to bronchoconstriction and inflammations, AIA patients may be susceptible to this response because they have exaggerated LTC4 synthase expression, and/or a defect in the PGE2 brake, based perhaps on insufcient COX-1 activity or more likely on impaired signaling from EP2 receptors.

protective effect of inhaled PGE2 does not correlate with its relatively weak bronchodilator activity, conrming that PGE2 preinhalation protects by restoring the suppression of cysteinyl-LT synthesis, not by dilating airways directly. 3. The effector role of cysteinyl-LTs in adverse respiratory and rhinitic reactions to NSAIDs in most AIA patients has been conrmed with placebocontrolled clinical trials of specic leukotriene modier drugs. The 5-LO inhibitors zileuton and ZD-2138 markedly blocked the rise in urinary LTE4 and the fall in FEV1 following oral aspirin challenge of AIA. Rhinoconjunctival and dermal reactions to oral aspirin are also blocked by zileuton. Antagonists of CysLT1 receptors block oral NSAID-induced respiratory reactions in AIA patients.
The PGE2 Brake is Derived from COX-1

The cyclooxygenase theory and the PGE2 brake hypothesis are thus crucial in understanding how NSAIDs trigger LT synthesis and bronchoconstriction, but they do not explain why only some asthmatics are susceptible to these responses. AIA patients tolerate selective COX-2 inhibitors including nimesulide, meloxicam, and rofecoxib, and respond adversely most often to NSAIDs with a greater selectivity for COX-1, such as aspirin and indomethacin. This suggests that cytoprotective/anti-inammatory PGE2 in the lung is produced by constitutive COX-1. COX-1 is expressed in a large number of cell types,

including mast cells, eosinophils, macrophages, vascular endothelial cells, bronchial epithelium, and bronchial smooth muscle. The exact cellular sources of the putative PGE2 brake remain unclear. AIA patients may overproduce cysteinyl-LTs chronically and acutely after NSAID exposure because of a defect in endogenous PGE2 synthesis. However, inhaled lysineaspirin equieffectively inhibits airway PGE2 synthesis both in AIA patients and in aspirin-tolerant asthmatics. Baseline levels of PGE2 and other prostanoids are not consistently different in the BAL uid and urine of AIA patients and control groups. Immunohistochemical studies of AIA bronchial biopsies have found little evidence for a meaningful anomaly in the cellular expression of COX isozymes. One possibility is that a defective PGE2 brake may derive not from a failure of PGE2 synthesis, but of leukocytes to be suppressed by PGE2. An anomaly in EP2 receptor structure, expression, or signaling might be one explanation, and this would also be consistent with the lack of clinical benet shown in AIA patients treated with the stable PGE1 analog misoprostol. Suggestive evidence of an anomaly within the cysteinyl-LT biosynthetic pathway itself is that baseline cysteinyl-LT synthesis appears to be two- to sevenfold higher in AIA patients than in control groups, even in the absence of exposure to NSAIDs, as demonstrated by measurements of cysteinyl-LTs in BAL uid, induced sputum, and urine (Figure 3). The increases seen after NSAID exposure are superimposed upon this chronically elevated baseline. Immunohistochemical analysis of bronchial biopsies from

186 ASTHMA / Aspirin-Intolerant

NSAID AIA

ATA N 0 1 2 3

Time (h)
Figure 3 Schematic diagram showing that cysteinyl-leukotriene production is chronically elevated in patients with aspirin-intolerant asthma (AIA) compared to those with aspirin-tolerant asthma (ATA) and normal subjects (N). A further rise in cysteinyl-LT production after exposure to a nonsteroidal anti-inammatory drug (NSAID) occurs only in the AIA group.

AIA and aspirin-tolerant asthmatic subjects shows that counts of cells immunostaining for LTC4 synthase, the terminal enzyme for cysteinyl-LT synthesis, were vefold higher in AIA biopsies than in aspirintolerant asthma biopsies and 18-fold higher than in normal biopsies. The numbers of LTC4 synthasepositive cells in the bronchial mucosa correlated with elevated levels of cysteinyl-LTs in the BAL uid and with bronchial responsiveness to inhaled lysine-aspirin. Persistent overproduction of cysteinyl-LTs in steady-state AIA may therefore be related to overexpression of LTC4 synthase in the bronchial wall, much of it within eosinophils and mast cells. This may also contribute to the further surge in cysteinylLT synthesis when PGE2 suppression is removed by NSAIDs.

Animal Models
Although the anti-inammatory actions of NSAIDs and their effects of prolonged bleeding, nephritis, and gastric ulceration can be replicated in animal models, there has been relatively little interest in developing animal models of AIA. This may reect, at least in part, the difculty of replicating the diverse immunopathological features of asthma, especially chronic remodeling of structural airway tissues, and also of replicating the typically high degree of disease severity and glucocorticoid dependency observed in AIA patients.

Management and Current Therapy

National and international management guidelines for asthma (e.g., GINA guidelines) are based on disease severity and make no therapeutic distinctions between AIA and other asthma phenotypes. Asthmatics should

avoid using NSAIDs either prescribed inadvertently or purchased over the counter. NSAID-induced acute reactions in susceptible asthmatics can be treated with nebulized b-2 adrenergic agonists, repeated frequently over several hours where necessary. Decongestants and antihistamines (topical or oral) can be used for associated rhinoconjunctival symptoms. In the most severe cases, intubation and mechanical ventilation in the intensive treatment unit may be required. Treatment of chronic AIA focuses on anti-inammatory therapy of the upper and lower airways using topical and inhaled/insufflated corticosteroids. Antibiotics may be required when purulent nasal secretions indicate infection, and many AIA patients require repeated polypectomies. Some AIA patients may require NSAIDs for concomitant inammatory disease such as arthritis, and in such cases, aspirin desensitization may be an option. Acute respiratory reactions to NSAIDs in AIA patients are always followed by a refractory period lasting 25 days during which further reactions to NSAIDs cannot be induced. Sensitivity to NSAIDs re-emerges within a week, but regular low-dose NSAIDs can maintain the refractory state indefinitely. Daily or alternate-day dosing of NSAIDs is therefore used clinically to desensitize AIA patients to inadvertent ingestion of NSAIDs and to allow NSAID therapy of concomitant diseases such as arthritis. Cross-desensitization occurs such that repeated dosing with one NSAID provides protection against adverse reactions to other NSAIDs. Chronic desensitization reduced the numbers of acute exacerbations and hospital admissions in AIA patients compared to a control group of AIA patients who avoided NSAIDs, associated with reductions in corticosteroid use. The mechanism of desensitization is unknown, but is tentatively linked to reductions both in the quantity of cysteinyl-LTs synthesized following NSAID exposure, and with reduced bronchial responsiveness to cysteinyl-LTs. In the nasal airway, desensitization has been linked to a reduction in expression of the CysLT1 receptor on inltrating leukocytes. In small studies in AIA patients, the antiallergic cromone sodium cromoglycate, the antiviral agent acyclovir, the antibiotic roxithromycin, and the longacting b2-agonist salmeterol have all been reported to block not only the bronchial responses to inhaled NSAIDs, but also the associated rise in urinary LTE4 excretion. The mechanism of these drugs is difcult to understand in this context, but may involve prevention of mast cell degranulation. On the basis of the pathophysiological evidence of a central role of cysteinyl-LTs in AIA, clinical trials of 5-LO inhibitors and CysLT1 receptor antagonists have been reported. Zileuton improved lung function and rescue beta-2

Cys-LT production

ASTHMA / Occupational Asthma (Including Byssinosis) 187

agonist use and restored the sense of smell in a 6week study of 40 AIA patients. In an 8-week crossover trial of montelukast in 80 AIA patients, there were significant improvements in lung function, use of rescue therapy, symptom scores, night-time awakenings, and asthma quality-of-life (QOL) scores compared with placebo. In the clinic, treatment response to leukotriene modiers is variable, but a treatment trial is advised in patients with AIA uncontrolled by topical corticosteroids.
See also: Allergy: Overview. Asthma: Overview. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chymase and Tryptase. Genetics: Overview. Histamine. Immunoglobulins. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Pulmonary Function Testing in Infants.

Further Reading
Drazen JM, Israel E, and OByrne PM (1999) Treatment of asthma with drugs modifying the leukotriene pathway. New England Journal of Medicine 340: 197206. Pavord ID and Tatterseld AE (1994) Bronchoprotective role for endogenous prostaglandin E2. Lancet 345: 436438. Samter M and Beers RF (1968) Intolerance to aspirin: clinical studies and consideration of its pathogenesis. Annals of Internal Medicine 68: 975983. Sanak M and Szczeklik A (2000) Genetics of aspirin-induced asthma. Thorax 55(supplement 2): S45S47. Stevenson D, Sanchez-Borges M, and Szczeklik A (2001) A classication of allergic and pseudoallergic reactions to drugs that inhibit cyclooxygenase enzymes. Annals of Allergy, Asthma and Immunology 87: 14. Stevenson DD, Simon RA, and Zuraw BL (2003) Sensitivity to aspirin and nonsteroidal antiinammatory drugs. In: Adkinson NF, Bochner BS, Yunginger JW, et al. (eds.) Middletons Allergy: Principle and Practice, 6th edn., pp. 16951710. St Louis, MO: Mosby. Szczeklik A, Gryglewski RJ, and Czerniawska-Mysik G (1975) Relationship of inhibition of prostaglandin biosynthesis of analogues to asthma attacks in aspirin sensitive patients. British Medical Journal 1: 6669. Szczeklik A and Stevenson DD (2003) Aspirin-induced asthma: advances in pathogenesis, diagnosis, and management. Journal of Allergy and Clinical Immunology 111: 913921.

of occupational asthma (OA) of 25100 per million employed in industrially developed countries. For 510% of cases, toxic mechanisms are responsible, and asthma is the result of accidents that release agents such as chlorine, sulfur dioxide, and acetic acid into occupational environments. For the remaining 9095%, asthma appears to arise through hypersensitivity mechanisms. Many of the several hundred causal asthmagens are reactive chemicals of low molecular weight, though some are naturally occurring allergens of high molecular weight. Some agents (e.g., di-isocyanates, epoxy resins, our) have such sensitizing potency that at current exposure levels in some occupations (e.g., spray painting and baking) the risk of OA exceeds that of asthma arising spontaneously. OA is thus important in an epidemiologic sense, and it serves as a useful model of asthma in general. Asthma of occupational origin is like asthma resulting from any other cause. However, when due to hypersensitivity mechanisms, it has one unusual characteristic. If the diagnosis is recognized within 624 months and exposure ceases, there is a meaningful possibility that active disease will resolve. Therefore, the onus is on physicians to consider the possibility of an occupational cause in every adult presenting with asthma.

Introduction
It was not until the twentieth century that investigatory techniques acquired the sophistication to distinguish airway disorders from those of the lung parenchyma/interstitium. The definition of asthma, the elucidation of its clinical characteristics and pathogenic mechanisms, and the evolution of strategies for its recognition, management, and prevention have consequently been comparatively recent events. Nevertheless, it is likely that asthma was the explanation for many of the respiratory disorders of antiquity and the middle ages that were recognized to afict (and sometimes devastate) workers in certain trades and industries. The realization that asthma may arise as a direct consequence of inhaled occupational agents has been the focus of particular attention over the last three to four decades, and our understanding of occupational asthma (OA) has largely arisen during this period. OA has no recognized differences from asthma in general with respect to its pathology and genetic etiology, and so this article will focus on its features of special interest.
Definitions and Classication

Occupational Asthma (Including Byssinosis)

D J Hendrick, Royal Victoria Inrmary, Newcastle upon Tyne, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Respirable agents in the workplace are responsible for about 10% of asthma arising in adult life, and for an annual incidence

Asthma is a disease of the intrathoracic airways that is characterized, and often dened, by its means of clinical expression (diffuse airway obstruction that varies in degree over time) and its underlying pathogenic basis (a state of enhanced airway responsiveness). OA is caused by exposure to agents, almost exclusively airborne, that are encountered primarily in the workplace. Such exposure in susceptible individuals causes the level of airway responsiveness to

188 ASTHMA / Occupational Asthma (Including Byssinosis)

increase into the asthmatic range. Two distinct mechanisms are recognized. First, hypersensitivity mechanisms appear responsible since there is a latency period of presumed sensitization between exposure onset and symptom onset; this accounts for 9095% of cases. Second, acute toxicity is the initiating event; this is generally a consequence of inhalation of toxic agents during an industrial accident. Asthma following the latter is sometimes identied as the reactive airways dysfunction syndrome (RADS) or, more simply, irritant asthma. Neither term is fully satisfactory. The rst may be interpreted, incorrectly, to indicate a disorder other than asthma, while the second may be mistaken for pre-existing asthma that is exacerbated non-specifically at work (e.g., because of exertion in cold air); this is work-aggravated not occupational asthma, and is not associated with an increase in airway responsiveness. It is currently unclear whether repeated exposures to toxic agents at dose levels insufcient to provoke clinical reactions might nevertheless cause minor increases in airway responsiveness. If so, the cumulative effect might elevate airway responsiveness to a level at which asthma eventually becomes inevitable (low-level RADS). This would simulate the latency period association with presumed hypersensitivity mechanisms, and ongoing exposure might then provoke acute symptoms in a non-specic fashion. This would appear to simulate hypersensitivity mechanisms also, but such exposure should provoke reactions in any asthmatic individual with a sufcient level of airway responsiveness.
Key Clinical Features

the issue, rather, is whether an individuals level of airway responsiveness is sufciently high to make meaningful bronchoconstriction likely when there are appropriate provoking stimuli. Whether the resulting degree of bronchoconstriction is perceived to be distressing (or is perceived at all), is very dependent on psychological factors. This adds an important further level of complexity and variability, since at all degrees of asthmatic severity as dened physiologically, there will be a wide spectrum of perceived disability. This is particularly so in OA because of resentment, even anger, over the possible liability of a third party (the employer), and the possibility of compensation. Longstanding OA, like asthma generally, is commonly associated with airway obstruction that has a reduced capability for reversal. It may come to simulate chronic obstructive pulmonary disease (COPD).
Epidemiology

When hypersensitivity mechanisms are responsible, further exposures to the particular inducing asthmagen may provoke specic asthmatic reactions. Symptoms are dependent on the degree of hypersensitivity, the magnitude of the provoking stimulus, the level of airway responsiveness, and the ease with which the affected individual perceives changing respiratory sensations. Airway responsiveness varies in level from subject to subject over a considerable range, and can be quantied throughout the population at large. Its distribution (like that of most biologic parameters) is unimodal, and it follows the common biologic pattern of a bell-shaped curve. The tail in which responsiveness is most marked gives rise to asthma, but the adjacent segment implies vulnerability, and the opposite tail implies comparative impunity. It is misleading to think of asthmatic and nonasthmatic subjects being fundamentally different because of the presence or absence of airway hyperresponsiveness;

Over recent decades, OA has proved consistently to be the commonest type of newly diagnosed occupational lung disease in industrially developed countries, though the various disorders attributable to asbestos have an equal cumulative incidence. Between them, asthma and asbestos account for 6570% of all incident respiratory disorders of occupational origin. In most outbreaks of OA no more than a few per cent of exposed workers become affected, but there are examples with both pathogenic pathways of prevalences approaching 50%. The reported incidence of OA varies widely at a global level, depending on local employment patterns and diagnostic criteria. Estimates from statutory notication schemes, compensation registers, voluntary surveillance schemes, and general population surveys, suggest that 515% (median 910%) of asthma beginning in adult life is occupational. When asthma arises for the rst time in a working adult, the background odds favoring an occupational cause over a nonoccupational cause are consequently of the order 1 in 10 only. This is consistent with the estimate from SWORD (Surveillance of Work-Related and Occupational Respiratory Disease) data that about 40 cases of OA per million employed workers arise in the UK each year. SWORD has usefully considered the incidence of new cases within specic working groups for which there are particular occupational exposures. For example, among spray painters, who may use asthmagenic di-isocyanate, epoxy resin, and acrylic paints, the average annual incidence of OA from 1992 to 1997 was 1464 per million more than threefold the UK national average for all cases of incident asthma.

ASTHMA / Occupational Asthma (Including Byssinosis) 189

Thus, in the case of spray painters developing asthma in adult life, the background odds favor an occupational cause over a coincidental cause. Other settings associated similarly with greater than even odds include baking, metal treatment, chemical processing, and plastics manufacture. Most national estimates range from 25 to 100 million per year.

Table 1 Commonly reported causes of occupational asthma Animal sources Arthopods/ insects Birds Mammals Marine species Bees, locusts, mites, silkworms, weevils Feather bloom, excreta Rodent urinary protein, pancreatic enzyme supplements Corals, crabs, shmeal, oysters, prawns, sponges

Etiology
Many occupational agents (some 400) have been reported to be definite or probable sensitizers capable of inducing asthma. Some of the most prominent are classied in Table 1, and the most common reports to SWORD over a 9-year period are listed in Table 2. Notable agents reported to cause RADS over recent years have been acetic acid, chlorine/chlorine dioxide/hydrochloric acid, di-isocyanates, dinitrogen tetroxide, endotoxin, re smoke, freons, hydrobromic acid, Iraq/Iran war gases, pentamidine, phosphoric acid, silo and swine connement gases, sulfur dioxide/sulfuric acid, tear gas, and welding fume.
Vegetable sources Beans Castor, coffee, soy Colophony Pine resin solder Enzymes Bromelain (pineapple), papain (papaw) Grain/our Gums Acacia, arabic, karaya Mushrooms Oil mists Tea Tobacco Wood Iroko, latex, redwood, western red cedar Microbial sources Endotoxin Gram-negative organisms Enzymes Bacillus subtilis (alcalase, maxatase, subtilisin) Spores Chemical sources Inorganic Salts/oxides of aluminum, chromium, cobalt, nickel, platinum, vanadium Stainless-steel welding fume Organic Acid anhydrides/phthalates, acrylates, amines, azodicarbonamide, benzalkonium, chloramine, di-isocyanates, uxes, formaldehyde, furfuryl alcohol, glutaraldehyde, isothiazolinones, organophosphate pesticides, plexiglass, polyvinyl pyrolysis fume, reactive dyes, styrene, sulphone chloramides, tannic acid Pharmaceutical sources Intermediates 6-amino penicillanic acid Drugs Cimetidine, cephalosporins, ispaghula (psyllium), penicillins, piperazine, sulphonamides, tetracycline

Clinical Features
Once a diagnosis of asthma is suspected from the clinical history or physical examination and conrmed objectively (by the demonstration of reversible airway obstruction or the measurement of airway responsiveness), the diagnostic issue turns to whether it has arisen occupationally. If the toxicity pathway has provided the mechanism, the diagnosis is clear and needs no further investigation. Asthma becomes evident during the recovery phase from what is usually a combination of conjunctivitis, rhinitis, pharyngolaryngitis, tracheobronchitis, and (possibly) pneumonitis induced by a major exposure to a toxic chemical or organic dust. In survivors, full recovery is the rule. If asthma arises, it is generally the only persisting respiratory problem, though occasionally bronchiectasis or an obliterative bronchiolitis is also detectable. If hypersensitivity has provided the mechanism, the diagnosis is more challenging and may be extremely difcult. This is partly because the latency period during which sensitization occurs (usually 324 months) may be very short (days or weeks) or very prolonged (several years), and partly because in most working environments associated with OA, most cases of incident asthma are coincidental and quite unrelated to occupation. What follows addresses this diagnostically more challenging variety of OA.
Clinical History

Table 2 Causes of occupational asthma reported to SWORD over 9 years Agent Di-isocyanates Laboratory animals/insects Flour/grain Fluxes/glues/resins/solders Aldehydes Wood dust Welding fume Enzymes Latex Others Total 199098 (%) 16 9 8 7 5 5 2 1998 (%) 13 12 7 9 5

The history provides an obvious starting point, but may be importantly distorted. Affected workers

47 100

14 6 34 100

The gures are rounded to the nearest whole number.

190 ASTHMA / Occupational Asthma (Including Byssinosis)

anxious to remain employed may deny or minimize relevant symptoms; they may also exaggerate or falsify critical aspects. An overwhelming belief that occupational exposure (and/or employer negligence) is responsible may, curiously, make a diagnosis of OA the independent variable on which the symptoms depend: if improvement during holidays/vacations is a cardinal diagnostic feature of OA then, yes, this must be so in my case since I know I have OA. For a classical case, there is exposure to a known asthmagen and other exposed workers are affected. For the affected individual, there is recognition that symptoms arise or worsen after a minimum of 12 hours and a maximum of 8 h from the onset of each sufciently strong exposure, and persist for hours or days. Such late asthmatic reactions are characteristically associated with increases in airway responsiveness and so are much more denitive of OA than immediate reactions, which may simply result non-specifically through irritant mechanisms. Mild late reactions may resolve within hours so that there is full recovery by the following day, but more commonly the rise in airway responsiveness is sufcient to worsen asthmatic severity for several days. A weekend away from work may therefore be insufcient to allow full recovery, and the association between occupational exposure and symptoms may not be recognized until there is a 2-week period of vacation (or sick leave). Even then gradual recovery, particularly cessation of disturbed sleep, may not become obvious until the second week, only to be reversed within a matter of days of returning to work.
Serologic Investigations

for periods of several weeks, so that any differences in pattern between work days and rest days can be detected. Statistical software can aid interpretation. The unsupervised recordings may lack reliability and precision, and work-related changes may reect nonspecic irritant reactions rather than specic hypersensitivity responses. There is consequently some difference of opinion over the value of PEF monitoring. In practice, however, it provides the most widely used diagnostic tool for OA. Figure 1 illustrates a typical positive outcome.
Inhalation Challenge Tests

Laboratory investigation for diagnostic IgE antibodies to relevant asthmagenic agents has proved disappointing for two reasons. First, many occupational asthmagens are reactive chemicals of low molecular weight. They are not thought to act as sensitizers until coupled with appropriate haptogenic body proteins, and it has proved difcult to produce suitable complexes for antibody detection. Second, exposed individuals may develop IgE responses without any apparent ill effect. Antibodies appear to correlate more closely with exposure than disease. Nevertheless, when radioallergosorbant allergen testing to relevant asthmagens is available, positive tests increase the probability of OA, even if sensitivity and specicity are limited.
Peak Expiratory Flow

The greatest diagnostic condence comes from laboratory inhalation challenge tests that are monitored by both supervised serial measurements of spirometry and repeated measurements of airway responsiveness. When there is deteriorating ventilatory function and increasing airway responsiveness, and the changes can be evaluated statistically, a diagnosis of OA can be considered conrmed with considerable condence, especially if the outcome is shown to be repeatable and the tests are carried out in a double-blind fashion. Thus, neither test subject nor the immediately supervising physician knows whether the challenge exposure involved the suspected asthmagen or a dummy placebo. These principles, with two methods of statistical evaluation, are illustrated in Figures 2 and 3. In practice, such tests require sophisticated equipment, are very time-consuming, pose potential risks, and are inevitably restricted to a few centers. They involve a fraction of 1% of all cases, but are particularly valuable (arguably indispensable) when hitherto unrecognized occupational asthmagens are rst investigated. Figure 3 illustrates one such case involving a novel asthmagen.
Return-to-Work Studies

More popular and more readily available is peak expiratory ow (PEF) monitoring. Test subjects take measurements on several occasions each day

A useful, and practical, compromise is the return-towork challenge test. For this, the test subject is kept from work (or at least from exposure to the suspected asthmagen) for a period of 23 weeks, during which time any asthmatic medication is reduced to a minimum (ideally discontinued). In true OA, some improvement is likely (or there is no deterioration with treatment reduction), and can be demonstrated by serial measurements of spirometry and airway responsiveness. Hourly spirometric monitoring over the 3 days prior to the return-to-work generates condence limits, and so allows the detection of any statistically significant deterioration subsequently (usually within a few days only if the asthma is occupational). If airway responsiveness too increases significantly, OA in the individual is reasonably conrmed. The method

ASTHMA / Occupational Asthma (Including Byssinosis) 191

Diurnal variation (%) PEF (l min1) 50 20 460 440 420 400 380 360 340 320 300 M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 11 12 Readings 9 9 9 8 9 8 7 9 8 7 9 8 8 7 5 9 9 8 8 8 8 9 8 3 6 8 6 7 8 9 9 7 7 9 8 6 9 7 8 8 7 6 W W W W W Comments 280

Figure 1 Serial PEF measurements in a laboratory technician sensitized to a oor cleaning material. Serial 2-hourly PEF measurements, waking to sleeping, for 6 weeks. Top panel: diurnal variation expressed as % predicted. Middle panel: daily maximum, mean, and minimum PEF. Bottom panel: date and number of PEF readings per day. W, days without waking measurement. Dotted line: PEF 359 represents the predicted PEF. The shaded columns represent work days. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 54. London: Saunders, with permission from Elsevier.

FEV1 (l)

does not conrm the identity of the asthmagenic agent, and is only suitable if the test subject is still employed and the employer cooperates fully. In many cases the possibility of an occupational cause does not arise until after the affected individual has ceased employment or the work environment has changed. Figure 4 illustrates this methodology in a newly appointed factory safety ofcer who found himself working with di-isocyanates.

Animal Models
80 2 Min 4 6 8 10 12 H 24

0 40 0

Figure 2 Inhalation challenge test with nebulized ceftazidime (3.2 mg) in a production worker. Upper plot: mean FEV1 at each hourly measurement point from 3 control days. Middle plot: lower 95% condence band for the hourly control FEV1 derived from the pooled variance. Lower plot: FEV1 following nebulized ceftazidime administered in a double-blind fashion. The lower condence band is breached for well over an hour, indicating a significant decrement consistent with a late asthmatic reaction. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

Several animal species have provided invaluable insight as to how asthma arises following exposure to occupational agents. Sensitization has been achieved by inhaled, dermal, and/or peritoneal routes for many occupational asthmagens (notably acid anhydrides, colophony, di-isocyanates, latex, plicatic acid), and both immediate and late asthmatic reactions have been provoked by subsequent inhalation challenge. The airways are then characterized by inammation, eosinophil inltration, mucus hypersecretion, and hyperresponsiveness. Hypersensitivity mechanisms have been conrmed by transferring lymphocytes or serum from affected to unaffected animals, which then respond to inhaled challenge in a similar fashion

192 ASTHMA / Occupational Asthma (Including Byssinosis)

5.0 4.0 FEV1 (l) 3.0 2.0 1.0 0 40 (a) 9 FEV1 area decrement 212 h (l h) 8 7 6 5 4 3 2 1 0 1 (b) 2 Control days 3 SINOS (32 g) 95% 99% Confidence limits

0 Min

60

2 H

10 12

24

Figure 3 Inhalation challenge test with nebulized SINOS (32 mg) in a research and development technician. (a) The FEV1-time plot following challenge with a newly developed low-temperature bleach-activating agent, SINOS. The shaded zone denes the 212 h area decrement (212 hAD). It provides a summary measure to quantify a late asthmatic reaction. The line demarcating the upper boundary represents the mean of the measurements during the 40 min before challenge. (b) Comparison of the 212 hAD following the SINOS challenge with those following three double-blind control challenges with nebulized solvent alone. It exceeds their 95% and 99% condence limits, conrming that a significant late asthmatic reaction has occurred. The outcome was reproduced when the procedure was repeated. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

to the donor animals. Specic IgE antibodies to the inducing agents (or hapten conjugates) have been evident commonly, but not invariably. Occasionally specic IgG antibodies are reported. There is involvement of both CD4 and CD8 T cells, with a dominant T-helper-2 cells (Th2) response. The mechanisms include deposition of excess extracellular matrix (and increased activity of matrix metalloprotease, MMP) and activation of the vascular endothelium associated with the release of vascular endothelial growth factor (VEGF). Experiments with inhibitors of MMP and VEGF have shown substantial reductions of the markers of asthmatic activity, possibly pointing a way to novel strategies for future management. Animal models have additionally conrmed that airway inammation induced by exposure to toxic levels of certain reactive chemicals may also induce airway hyperresponsiveness. This simulates the RADS pathway. Ozone and chlorine exert their effects

through oxidative stress, which is associated with increased expression of inducible nitric oxide synthase and an increase in airway responsiveness. Ozone has also been shown to suppress Th1 responses (possibly thereby enhancing Th2 responses), and acute exposure to ozone or nitrogen dioxide amplies asthmatic responses to allergenic agents in already-sensitized animals. Animal models have also been used to investigate the exposure threshold levels at which airway inammation and hyperresponsiveness rst develop. The importance of this for occupationally induced asthma in humans is readily evident, though extrapolation from animals is necessarily fraught with uncertainty. The threshold levels of exposure that trigger meaningful responses once sensitization has occurred may differ critically from those that are responsible for initial sensitization, and are likely to differ appreciably from individual to individual.

ASTHMA / Occupational Asthma (Including Byssinosis) 193

3.00

2.00 FEV1 (l s1)

1.00

Test day 3 Mean of three control days Lower boundary for control days Work

0.00 07:30 08:30 09:30 10:30 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30 Time (h)
Figure 4 Return-to-work study of a safety ofcer employed by a factory using di-isocyanates. He developed asthma within months of employment onset. He was kept from work for several weeks during which airway responsiveness improved steadily (PD20.methacholine; 3.2-25.0 mg). An increase in PD20 of twofold or more indicates a significant change. The gure illustrates the third successive day of the return-to-work study. The lowered initial FEV1 measurement reects an equivocal late reaction from the preceding day. On Day 4, following the 3 days at work, the PD20 was significantly decreased to 9.0 mg (i.e., to less than half the pretest value of 25.0 mg).

Management and Current Therapy

Drug and ancillary therapies for OA are those of asthma of any cause, but OA arising by the hypersensitivity route offers an additional and critical means of management. If the relevance of an occupational sensitizer is recognized within 624 months of symptom onset, and if exposure then ceases, there is a meaningful probability of the asthmatic state resolving entirely (i.e., the level of airway responsiveness falls into the nonasthmatic range). This is almost unknown for adults with nonoccupational asthma, unless it is drug induced (beta-blockers, nonsteroidal anti-inammatory drugs (NSAID). There is inevitably much variability from individual to individual, but if exposure ceases within as short a period as 6 months, the probability of complete resolution may exceed 50%. If the period exceeds 24 months, it is more likely that active asthma will continue, even in the absence of ongoing exposure. Most individuals developing OA have unskilled or semiskilled jobs. Their susceptibility to develop asthma with exposure to sensitizing agents will inevitably put them at a disadvantage in the labor market,

and this will be enhanced if active asthma persists. It is unfortunate that most lose their current jobs and never become gainfully employed again. This poses several management challenges. A correct diagnosis is the cornerstone, since mild asthma rarely leads to job loss of itself. A false-positive diagnosis may have devastating but unnecessary economic consequences for both the affected individual and the employer. A dilemma arises when asthma is occupational, but is mild, has been active for several years, and does not improve much after an experimental period away from the workplace. If the affected individual cannot nd (or does not wish for) alternative employment, the risk of continuing exposure needs to be assessed. It will certainly diminish the long-term probability of regaining a nonasthmatic level of airway responsiveness, but this may be minimal anyway. More importantly it may cause a further and permanent increase in airway responsiveness, and hence worsening asthma with the possibility of remodeling and xed airway obstruction. A period of close surveillance, with objective serial measurements of spirometry and airway responsiveness, may help the affected individual and his medical advisors to judge whether the

194 ASTHMA / Occupational Asthma (Including Byssinosis)

obvious benets justify the uncertain risks. There is anecdotal evidence that tolerance develops in some affected individuals and that they have little to lose by continued exposure. Complete cessation of exposure offers the best outcome, especially if this can be achieved by using an alternative, nonsensitizing agent for the particular industrial process. If this is not possible or practical, it may be that exposure levels can be reduced by modications to job plans, task sharing/exchanging, improved industrial hygiene (ventilation/extraction), or the use of respiratory protection equipment.
Prevention

Prevention offers the best means of control. When a risk of OA is identied, surveillance programs may identify emergent cases at a point when exposure cessation is likely to produce cure. This assumes such programs are efcient. Some employers rely primarily on environment measurements, and reassure themselves (inappropriately) that if they have complied with regulatory limits, any emergent cases of asthma must be nonoccupational. When a clear risk of OA is recognized, the onus should lie with disproving the diagnosis when new cases of asthma arise, rather than the converse. It is the very nature of allergy that some affected individuals become sensitized at exposure levels that are harmless to the majority, and that lie within regulatory limits.
Compensation

Compensation is inevitably an issue for affected workers, particularly those who become disabled and unemployed. Equally inevitably, compensation systems differ from country to country. Regretfully, few include provision for retraining and re-placement. The physician managing OA needs to be familiar with local procedures in order to offer proper advice. At a time of increasing litigation, the physician too may nd him- or herself the focus of a legal (negligence) suit if he or she has failed to provide correct advice.

Byssinosis
The respiratory effects of dust from cotton and other biologic bers became prominent in the nineteenth and early/mid-twentieth centuries. They appeared initially to be specic to the cloth manufacturing industry and the appellation, byssinosis, arose to identify the disorder characterized by them. This has had the misleading consequence of segregating cotton dust asthma from the many other types of OA that

were identied later, and of disguising the other respiratory disorders that may result from cotton dust. A very characteristic feature of reported byssinosis has been a prominence of symptoms on the rst work day following periods without exposure. These tend to diminish or even cease on subsequent days, at least in some individuals, despite similar levels of ongoing exposure. While chest tightness and breathlessness may be a consequence of asthma, some affected individuals describe an inuenza-like reaction with fever and malaise (Monday fever). The latter may occur on the very rst employment day, that is, before any possibility of sensitization. Such symptoms have also been generated in experimental conditions when na ve volunteers are exposed. The same febrile reaction may occur in subjects heavily exposed to metal fume or the fume arising from certain polymers. It is closely related to neutrophil inammation and the release into plasma of pyrogenic interleukin6 (IL-6). These non-specic systemic symptoms are features of inhalation fevers generally. They occur widely with exposure to a variety of organic dusts, and in these contexts are now known more commonly by the term organic dust toxic syndrome (ODTS). Products from microbial overgrowth appear to be responsible, and high airborne concentrations of respirable spores and endotoxins are characteristically seen. This is consistent with the recognition that byssinosis is most prevalent among those who harvest, transport, store, sort, process, and clean cotton before its decontaminated bers are woven into cloth. The matter is complex since heavy exposure of na ve subjects to organic dust may also provoke cough, chest tightness, and bronchoconstriction. These symptoms are rarely severe, and rarely last for more than a day or two. Nevertheless, this response simulates RADS, and no clear sensitizer has been incriminated in the pathogenesis of byssinosis. Some individuals affected by other forms of OA may also have more prominent symptoms on Mondays that peter out as the working week progresses. A further characteristic of byssinosis has also proved to be non-specic, namely the development of COPD. Early studies of byssinosis were necessarily cross-sectional, and it was assumed that subjects with COPD had initially experienced the typical acute and reversible features (i.e., asthma and/or inhalation fever), which had progressed to produce chronic byssinosis. It seems more likely now that COPD and asthma (and inhalation fever/ODTS) are independent disorders arising from cotton dust exposure, which result from different levels of susceptibility within exposed workers and different patterns of cellular response. Even so, it is to be expected that COPD might develop

ASTHMA / Acute Exacerbations 195

because of asthma as well as independently of it, and there is convincing evidence to support this from recent longitudinal studies.
See also: Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Occupational Diseases: Asbestos-Related Lung Disease.

Acute Exacerbations
S L Johnston, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Acute exacerbations of asthma are acute worsenings of asthma symptoms accompanied by reductions in lung function, normally provoked by some external event or combination of events. Exacerbations may be relatively mild or severe. The most severe may lead to asthma death. Symptoms include increased breathlessness, wheeze, cough, or chest tightness. Severity is graded on a combination of symptoms, clinical signs, and lung function. The majority of asthma exacerbations, particularly in children, are precipitated by acute respiratory tract viral infections. These may interact with a number of other cofactors such as allergen exposure, air pollution, and exercise. Exacerbations are more likely to occur on a background of poorly controlled rather than well-controlled underlying disease. Pathology involves increased airway inammation with most inammatory cell types implicated in pathogenesis. Most prominent, however, are neutrophils, lymphocytes, and eosinophils. A wide variety of acute inammatory mediators are increased during exacerbations. Severity of virus infection is the major determinant of severity of exacerbation. Asthmatics have increased susceptibility to viral and bacterial infection. Treatment includes optimal control of underlying disease, inhaled/ oral steroids, and bronchodilators. The role of anti-infective therapy is under investigation.

Further Reading
Beach JR, Young CL, Avery AJ, et al. (1993) Measurement of airway responsiveness to methacholine: relative importance of the precision of drug delivery and the method of assessing response. Thorax 48: 239243. Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376384. Burge PS, Pantin CFA, Newton DT, et al. (1999) Development of an expert system for the interpretation of serial peak expiratory ow measurements in the diagnosis of occupational asthma. Occupational and Environmental Medicine 56: 758764. Cote J, Kennedy S, and Chan-Yeung M (1990) Outcome of patients with cedar asthma with continuous exposure. American Review of Respiratory Diseases 141: 373376. Glindmeyer HW, Lefante JJ, Jones RN, et al. (1994) Cotton dust and across-shift change in FEV1 as predictors of annual change in FEV1. American Journal of Respiratory and Critical Care Medicine 149: 584590. Hendrick DJ and Burge PS (2002) Occupational asthma. In: Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) Occupational Disorders of the Lung Recognition, Management, and Prevention, pp. 3376. London: Saunders. Herrick CA, Xu L, Wisnewski AV, et al. (2002) A novel mouse model of diisocyanate-induced asthma showing allergic-type inammation in the lung after inhaled antigen challenge. Journal of Allergy and Clinical Immunology 109: 873878. Lee KS, Jin SM, Kim SS, and Lee YC (2004) Doxycycline reduces airway inammation and hyperresponsiveness in a murine model of toluene diisocyanate-induced asthma. Journal of Allergy and Clinical Immunology 113: 902909. Lee YC, Kwak YG, and Song CH (2002) Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inammation in a murine model of toluene diisocyanate-induced asthma. Journal of Immunology 168: 595600. Meyer JD, Holt DL, Cherry NM, and McDonald JC (1999) SWORD 98: surveillance of work-related and occupational respiratory disease in the UK. Occupational Medicine 47: 485489. Newman Taylor A (2000) Asthma. In: McDonald JC (ed.) Epidemiology of Work Related Diseases, 2nd edn., ch. 8, pp. 149 174. London: BMJ Books. Schilling RSF, Hughes JPW, Dingwall-Fordyce I, et al. (1995) An epidemiological study of byssinosis among Lancashire cotton workers. British Journal of Industrial Medicine 12: 217227. Stenton SC, Avery AJ, Walters EH, and Hendrick DJ (1994) Technical note: statistical approaches to the identication of late asthmatic reactions. European Respiratory Journal 7: 806812. Stenton SC, Dennis JH, Walters EH, and Hendrick DJ (1990) The asthmagenic properties of a newly developed detergent ingredient sodium iso-nonanoyl oxybenzene sulphonate. British Journal of Industrial Medicine 47: 405410. Venables KM, Topping MD, Howe W, et al. (1985) Interaction of smoking and atopy in producing specic IgE antibody against a hapten protein conjugate. British Medical Journal 290: 201204.

Introduction
Asthma is itself a heterogeneous disease. Asthma exacerbations also, therefore, are by definition heterogeneous as exacerbations are dened as a worsening of a pre-existing state. Given that the etiology (see below) of exacerbations is also heterogeneous, heterogeneity of both the underlying cause and of the etiology of the exacerbation makes the exacerbation itself very varied in its presentation. One of the difculties in clinical practice and clinical research is dening exacerbations accurately and differentiating them from poor control of the underlying disease. There is no agreed definition of an exacerbation, but most clinicians and clinical researchers would agree on something like episodes of relatively sudden onset and rapidly progressive increase in symptoms of shortness of breath, cough, wheeze, or chest tightness accompanied by reduction in lung function and normally provoked by some external event or combination of events. Exacerbations of asthma are very important as they are the major cause of morbidity and mortality in asthma and are also responsible for the greater part of healthcare costs associated with asthma treatment. Currently available treatments for asthma exacerbations include supportive care, oxygen if required, bronchodilators, and oral/inhaled corticosteroids.

196 ASTHMA / Acute Exacerbations

This treatment is at best only partially effective. Understanding the causes and mechanisms of asthma exacerbations is therefore of great importance as there is an urgent need for more effective preventive and interventional therapy.

Etiology
In infants and young children the vast majority (approaching 100%) of exacerbations are precipitated by acute respiratory tract virus infections. Respiratory syncytial virus (RSV) is the major cause of acute wheezing illness severe enough to require hospitalization in infants and 12-year-old children. There is, however, increasing evidence that rhinoviruses also play an important role and most recent data suggest RSV is implicated in 7080% of hospitalized wheezing lower respiratory tract infections and rhinoviruses in around 40%. The incidence of dual infection with these two viruses and with a variety of other respiratory viruses is likely to be as high as 2030%. There is much less data available on community lower respiratory wheezing illness but birth cohort studies have now reported that rhinoviruses are the dominant cause of acute attacks of wheezing in this age group, perhaps causing around three times as many episodes of wheezing illness as RSV and other respiratory viruses. In school-age children and teenagers, viruses precipitate at least 80% of acute asthma exacerbations. Rhinoviruses again are by far the most common virus implicated accounting for around two-thirds of viruses detected. In adults, viruses likely cause around two-thirds to three-quarters of asthma exacerbations. In adults in particular where virus loads tend to be much lower than in children, the evidence is less strong than it is in children and further work is required to clarify the role of viruses. In all these age groups, viruses can interact with a number of other factors in provoking asthma exacerbations. There are good data in both adults and children that virus infection and exposure to an allergen to which the patient is sensitized interact in a synergistic manner in increasing the risk of exacerbation. There is also evidence that air pollution interacts with virus infection in increasing the risk of lower respiratory illness when infected and it is possible that a number of other cofactors also play a role. Poor control of the underlying disease is a major risk factor for asthma exacerbation and treatment with prophylactic therapy, principally with inhaled corticosteroids, but also with leukotriene receptor antagonists and long-acting beta-agonists is a major protective factor against exacerbation.

Asthma exacerbations show marked seasonality in all age groups, peaking 12 weeks after school return, most especially in the autumn and most especially in school-age children. This seasonal pattern is important as it emphasizes the times at which prophylactic therapy is most needed. Asthmatics have recently been shown to have increased susceptibility to virus infection through impaired innate immunity and there is a biological rationale that they are also likely to have impaired acquired antiviral immunity. Clinical studies conrm that asthmatics are more susceptible to virus infection and bacterial infection, having a greater risk of invasive pneumococcal disease. To date, very little is known about susceptibility to infection in asthma but this is an important area for future research. Genetic susceptibility is also an underresearched area and very little is known in this regard. Finally, there is increasing evidence that chronic infection with the atypical bacteria Chlamydophila pneumoniae and Mycoplasma pneumoniae may play a role in exacerbations of asthma and a recent study conrms therapeutic benet of treating asthma exacerbations with an antibiotic active against these organisms. Further studies are required to conrm these ndings and to shed further light on the role of chlamydia and mycoplasma.

Pathology
Relatively little is known about the pathology of asthma exacerbations for an obvious reason: sampling the lower respiratory tract during an exacerbation is extremely difcult. In one study asthmatic patients were bronchoscoped during naturally occurring colds, and eosinophilic, neutrophilic, and CD8 T-cell inammatory responses were found. Studies of post-mortem samples from patients dying of asthma exacerbation also reveal CD8 T cells, activated CD8 T cells, increased perforin expression, and impaired ratios of interferon gamma (IFN-g) to interleukin-4 (IL-4), possibly implicating impaired antiviral immunity in asthma death. Noninvasive methods of sampling the lower airway include measurement of exhaled breath condensate or exhaled nitric oxide, and sputum sampling. Studies with sputum again show both eosinophilic and neutrophilic inammation with increased levels of IL-8 and brinogen. Markers of both eosinophil and neutrophil activation are also increased and sputum lactate dehydrogenase (LDH) as a marker of virus-induced cytotoxicity is also markedly elevated. Levels of sputum LDH and eosinophil cationic protein were associated with longer hospital stay, indicating that virus-induced cell damage was the major predictor of

ASTHMA / Acute Exacerbations 197

the severity of asthma exacerbation and that eosinophilia as a consequence of either viral and/or allergen exposure was also an important contributor.

Clinical Features
Asthma exacerbations present with a sudden worsening of wheeze, cough, shortness of breath, and chest tightness with a reduction in lung function. Exacerbations can range from mild to severe, the most severe leading to asthma death. Diagnosis is normally made on the clinical history with the assistance of peak expiratory ow measurement and/or spirometry. Clinical examination will normally reveal a distressed, anxious patient with tachycardia, tachypnea, and audible wheeze on auscultation (though in the most severe exacerbations the chest can be almost silent). Patients will have prolonged expiration, and signs of severe exacerbation include inability to talk in complete sentences, tachycardia above 110 beats min 1, a respiratory rate above 25 breaths min 1, and a peak expiratory ow below 50% of predicted or best. Presence of pulsus paradoxus also indicates a severe attack and life-threatening attacks are suggested by a silent chest, presence of cyanosis, bradycardia, or hypotension, and a peak expiratory ow below 30% of predicted or best. Measurement of blood gases should be carried out. Hypoxia is usual. In milder exacerbations, hypocapnia is common due to hyperventilation while in more severe and life-threatening exacerbations, PCO2 starts to rise. A raised PCO2 or rising PCO2 is an indication for intensive care. Chest X-rays should be performed to exclude pneumonia and pneumothorax. The response to therapy and the progress of the exacerbation are monitored with serial peak expiratory ow or spirometry testing. Routine hematology and biochemistry are indicated but other blood testing is not normally required.

that have been investigated have been found to be elevated in exacerbations. Perhaps the most prominent are interleukin-8, interleukin-1b, tumor necrosis factor alpha (TNF-a), the regulated upon activation normally T-cell expressed and secreted factor (RANTES), and IFN-g. The mechanisms of induction of lower airway inammation in the context of respiratory virus infection are of great interest as these represent potential targets for interventional therapy. Nuclear factor kappa B (NF-kB) is very strongly implicated in virus-induced inammation, as is activating protein 1 (AP-1). However, a number of other transcription factors are also implicated. Mechanisms of induction of mucus secretion are also of great interest complex pathways are involved, but NF-kB, Sp1 transcription factors, and the epidermal growth factor receptor signaling pathway are all implicated. Airway obstruction is a result of acute smooth muscle contraction, chronic smooth muscle hypertrophy, mucus secretion, acute tissue edema, and chronic tissue inammation with airway brosis/remodeling. An important aspect of pathogenesis is host resistance to infection as virus infection is the major precipitant to exacerbation. Recent studies have conrmed that asthmatic bronchial epithelial cells mount defective apoptotic and IFN-b innate immune responses to rhinovirus infection. In consequence, rhinovirus infection is robust while in normal epithelial cells infection is largely abortive. The clinical studies indicating increased susceptibility to virus infection in asthma are consistent with this biological evidence, and more recent evidence that asthmatics are also susceptible to invasive pneumococcal disease possibly indicates a more generalized immune deficit. There is an urgent need to increase our understanding of host immunity to both viral and bacterial infection in asthma.

Experimental Models
There are no small-animal models of rhinovirus infection as rhinoviruses only infect humans and nonhuman primates. There are animal models of other virus infections including inuenza, RSV, and parainuenza, and these models have been combined in some instances with allergen exposure to try to increase our understanding of the pathogenesis of virus-induced asthma exacerbations. These studies indicate that impaired T helper (Th)-1 immune responses increase susceptibility to virus infection and that airway inammation, airway obstruction, and bronchial hyperreactivity are increased when virus infection occurs in the presence of ongoing allergic inammation. Other studies have conrmed an increased risk of developing allergen sensitization if

Pathogenesis
Asthma exacerbations are always mixed in their pathogenesis. They involve a mixture of acute and chronic inammation provoked by virus infection and other stimuli including allergen exposure, air pollution, tobacco smoke, etc. Much of the information we have gained regarding the pathogenesis of asthma exacerbations has come from experimental studies of rhinovirus infection in asthmatic and normal subjects. These studies have implicated neutrophils, CD4 and CD8 lymphocytes, and eosinophils, but in addition mast cells, macrophages, and mediators of acute inammation are also shown to play important roles, including leukotrienes. Most cytokines/chemokines

198 ASTHMA / Acute Exacerbations

allergen exposure occurs in the context of an acute respiratory virus infection. Studies with RSV indicate that the major protective immune responses are production of neutralizing antibody, natural killer (NK) cell, and CD8 T-cell IFN-g responses. A great deal of further work is needed to increase our understanding of the interaction between virus infection and allergen exposure in the context of asthma exacerbations. In the absence of an animal model, investigators have carried out human rhinovirus experimental infections in asthmatic and normal subjects, and have demonstrated bronchial hyperactivity and airway obstruction in asthmatic volunteers. These studies have also implicated Th1 responses in increased susceptibility to virus infection and further work in these models is ongoing to try to increase our understanding of the pathogenesis of virus-induced asthma. In vitro models of host immunity to virus infection and of virus-induced lower airway inammation include infection of airway epithelial cells and macrophages with a variety of respiratory viruses including rhinoviruses, RSV, and inuenza. These studies demonstrate that virus infection induces many proinammatory cytokines and chemokines, dependent upon transcription factor activation such as NF-kB, AP-1, and NF-IL-6. In turn, much of the activation of transcription factors is dependent upon activation of oxidant pathways. Production of nitric oxide appears to exert some degree of protection against virus infection.

severe exacerbations, oral/systemic corticosteroid therapy is indicated. Intravenous magnesium has been shown to be of some benet in more severe exacerbations failing to respond to initial therapy. Oxygen and supportive care should be given to all exacerbations where hypoxia is a feature. Leukotriene modiers have been shown to be more benecial in exacerbations in infants and young children. Their intravenous use in the acute setting has also been shown to produce some benet, though there is not much evidence available as yet. Use of standard antibiotics in two placebo-controlled studies has produced no evidence of benet. However, a recent study with an antibiotic active against atypical bacteria and with anti-inammatory properties has shown clinically significant benet over placebo. The most severe exacerbations require high dependency unit or intensive care monitoring and some require invasive ventilation. Hospitalization with acute exacerbation is a major risk factor for asthma mortality, and preventive therapy including inhaled corticosterioids and self-management plans are strongly indicated in such patients.
See also: Chemokines. Leukocytes: Eosinophils; Neutrophils. Viruses of the Lung.

Further Reading
British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guidelines on the management of asthma. Thorax 58(Supplement 1): 1194. Corne JM, Marshall C, Smith S, et al. (2002) Frequency, severity, and duration of rhinovirus infections in asthmatic and nonasthmatic individuals: a longitudinal cohort study. Lancet 359(9309): 831834. Gern JE (2002) Rhinovirus respiratory infections and asthma. American Journal of Medicine 112(Supplement 6A): 19S27S. Gern JE and Busse WW (2002) Relationship of viral infections to wheezing illnesses and asthma. Nature Reviews: Immunology 2(2): 132138. Gern JE and Lemanske RF Jr. (2003) Infectious triggers of pediatric asthma. Pediatric Clinics of North America 50(3): 555575. Green RM, Custovic A, Sanderson G, et al. (2002) Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study. British Medical Journal 324(7340): 763. (Erratum British Medical Journal 324 (7346): 1131.) Johnston SL and Martin RJ (2005) Chlamydophila pneumoniae and Mycoplasma pneumoniae: a role in asthma pathogenesis? American Journal of Respiratory and Critical Care Medicine, doi: 10.1164/rccm.2004121743pp. Message SD and Johnston SL (2002) Viruses in asthma. British Medical Bulletin 61: 2943. Message SD and Johnston SL (2004) Host defense function of the airway epithelium in health and disease: clinical background. Journal of Leukocyte Biology 75(1): 517. Talbot TR, Hartert TV, Mitchel E, et al. (2005) Asthma as a risk factor for invasive pneumococcal disease. New England Journal of Medicine 352(20): 20822090.

Management and Current Therapy

Perhaps the most important aspect to management of asthma exacerbations is prevention of asthma exacerbations. Optimal control of underlying disease with optimal therapy including inhaled corticosteroids, leukotriene modiers, and long-acting betaagonists has been shown to reduce exacerbation frequency by approximately 50%. Once exacerbations occur, the initial response is to give short-acting bronchodilators, if mild via inhalation through a spacer, and if more severe by nebulization. Short-acting beta-agonists are initial therapy. The addition of anticholinergic bronchodilators has been shown to further improve lung function and to reduce the need for hospitalization. In mild exacerbations, increasing the dose of inhaled corticosteroids can reduce the severity of the exacerbation and speed recovery. However, evidence indicates that doubling the dose is usually not adequate and that quadrupling the dose or giving high-dose therapy may give better responses. In moderate to

ASTHMA / Exercise-Induced 199

Wark PA, Johnston SL, Bucchieri F, et al. (2005) Asthmatic bronchial epithelial cells have a decient innate immune response to infection with rhinovirus. Journal of Experimental Medicine 201(6): 937947. Wark PA, Johnston SL, Moric I, et al. (2002) Neutrophil degranulation and cell lysis is associated with clinical severity in virusinduced asthma. European Respiratory Journal 19(1): 6875.

Epidemiology
EIA is common in known asthmatic subjects and exercise has been recognized as a trigger for asthma since the seventeenth century. At least 90% of asthma sufferers will experience a fall in forced expiratory volume (FEV1) or peak ow during or shortly after an appropriate exercise challenge. Some authorities believe that all asthmatics will have demonstrable bronchospasm following sufcient exercise challenge. In atopic patients who suffer only from allergic rhinitis prevalence drops to 40%. Studies of other groups are complicated by the definition of EIA used. For example, comparing the prevalence of EIB, hypersensitivity to methacholine, and self-reported symptoms of EIA consistently identify different groups within a given population. Most of the prevalence data provided are therefore relatively generalized (Table 1). EIB is a useful model for clinical asthma and it has been used as a screening tool. These cohorts are frequently drawn from populations engaged in regular exercise so may not represent a truly unselected population. Based on these studies the prevalence of EIA in the general adult population is estimated at between 6% and 13% and this varies geographically with higher levels in the UK and Australia and lower levels in developing countries. In cohorts of normal school children challenged with exercise then tested by spirometry up to 12% are shown to have EIA. The identication of exercise-induced bronchial narrowing is often a surprise to both child and adult subjects, who may not have been aware of any such problem. A poor perception of airow restriction is generally recognized in asthma but also casts some doubt on the definition of EIA based on spirometry alone. It is not known if otherwise normal subjects who experience a fall in FEV1 with exercise will benet from asthma therapy or will go on to develop clinical asthma later in life. Failure to recognize potentially reversible EIA may not be a trivial matter. Children who suffer distressing

Exercise-Induced
N C Thomson and M Shepherd, University of Glasgow, Glasgow, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Exercise-induced asthma (EIA) is described in asthmatics, elite athletes, and the general population alike. Difculties reconciling the variety of presentations complicate our understanding of the disease process and prevalence. Exercise-induced bronchospasm (EIB) can be measured during and after exercise in susceptible individuals, but whether this signies EIA is unclear. Exercise can cause an inammatory response in the airway that appears to be dose dependent and exaggerated by breathing cold dry air. Classical understanding of the pathogenesis of EIA suggests that drying of the airway mucosa leading to an osmotic gradient across the epithelium and basement membrane combined with cooling of the bronchial wall triggers bronchospasm in EIA. Modern investigation suggests that airway surface stress can be communicated to the bronchial wall by chemical signals arising from the epithelium. Managing a patient with EIA requires careful investigation to ensure the correct diagnosis and consideration of alternative disease processes should be made. Pharmacological intervention can both treat and limit the extent of EIB, but behavioral modications can also be introduced. Finally, clinicians managing EIA should be aware of the conicts that arise when regular asthma medications are used by patients engaging in competitive sport.

Introduction
Increased airway resistance triggered by vigorous exercise is variously called exercise-induced asthma (EIA) or exercise-induced bronchospasm (EIB). Although a consensus exists on the criteria for making a diagnosis of EIB, the range of circumstances in which it has been recognized mean a precise clinical definition of EIA is difcult to achieve. EIB has been described in asthmatics and those with no history of asthma and subjects ranging from school children to elite athletes. It is not clear whether the disease is the same in all cases. While it may be more accurate to reserve the term EIA for exercise-induced, symptomatic bronchial narrowing occurring in previously diagnosed asthma we will use the term broadly to describe asthmatic symptoms or physiological changes consistent with asthma developing during or immediately after exercise.

Table 1 Estimated prevalence (percent study population) of exercise induced asthma based on a number of studies using different methodology Study population General population Asthma Atopic nonasthmatic Athletes Elilte Recreational Children (o18 years) (%) 9 4580 15 Adults (%) 420 7090 40 4.220 1150

19

EIA prevalence has been reported using physiological based parameters and self-reporting questionnaires.

200 ASTHMA / Exercise-Induced

symptoms will tend to avoid exercise leading to possible psychosocial and neurological development problems. Similarly, the importance of correct identication of EIA is highlighted by studies demonstrating lower self-esteem scores in children who perceive themselves to be socially disadvantaged by asthma. A surprisingly high prevalence of EIA is reported in both recreational and elite athletes (Table 2). Anecdotally, this association has been explained by the focus of some asthmatic children on ventilatory control and on athletic pursuit to achieve this. Whatever the cause, around 23% of recreational athletes report asthma induced by exercise while within the elite ranks prevalence rates of between 16% in summer outdoor events and 50% in winter events such as cross-country skiing or ice skating are reported. While asthmatics frequently state that swimming tends to cause less bronchospasm than other sports, a particularly high prevalence of EIA has been observed in elite swimmers in whom up to 79% have been reported to have demonstrable bronchial hyperresponsiveness to methacholine and 33% have asthma. The prevalence of EIA reported in elite athletes has raised concern among sporting governing bodies regarding the potential for inappropriate use of performance enhancing medications. As a result a considerable body of research has focused on EIA developing in elite athletes. However, elite athletes are subject to a variety of additional possible triggers of EIB including the stress of competition. Thus, whether this research is equally applicable to recreational athletes or nonathletic sufferers from EIA is not known. Therefore, it appears that EIA, dened by a fall in FEV1, occurs in a relatively high proportion of the general population. This prevalence is increased by underlying atopy, asthma, and athletic pursuit,

particularly outdoor winter endurance events. Accurate assessments of prevalence are hampered by difculty in definition stemming from the range of possible criteria for diagnosis and the variety of presenting complaints, which will be outlined later. Further difculties stem from poor perception and variable self-reporting. This difculty in precise definition has also limited understanding of the pathology underlying EIA.

Pathogenesis
Pathophysiology

Table 2 The prevalence of EIA based on sporting pursuit Athletes Olympians Elite/events Season Summer Winter Summer Winter Track/eld Indoor Nordic skiing Skating Nonendurance Swimming Activity Asthmaa (%) 23 22 19 12 60 6.4 1 4450 12 50 35 EIB (%)

The normal respiratory responses to exercise result in an increase in ventilation of up to 200 l min1 . In order to facilitate the increased airow, mild bronchodilation occurs early during an exercise event and this can be measured using spirometry. Classically, EIA was described as taking place after the exercise challenge was completed, but it is clear that airway narrowing can occur during exercise lasting more than 12 min and in the postexercise period. The stopstart nature of some sporting events means that symptoms developing during exercise are entirely consistent with a diagnosis of EIA. A variable period of protection from further exercise-induced bronchial narrowing known as the refractory period is well described. A refractory period may last from 30 min to 2 h and may be due in part to the bronchodilator properties of prostaglandin E2 (PGE2). The cause of EIA and the refractory period are not fully understood; however, considerable evidence points to a cellular response to increased ventilation triggering an inammatory reaction in the airway. EIA may therefore be considered as a subset of chronic asthma in which exercise is the trigger to inammation. While this hypothesis is attractive, a variety of studies have produced conicting data regarding the inammatory character of EIA. The three-stage model proposed by R Gotshall serves as a useful framework to consider the pathogenesis of EIA. In this model an exercise challenge serves as a trigger sensed in the airway that ultimately signals to the cells of the airway controlling caliber. Hyperventilation alone can cause bronchoconstriction in human and canine subjects and has been identied as the key element of exercise that triggers EIA. How this trigger leads to bronchoconstriction is a matter of controversy.
Airway Cooling and Hyperosmolarity

All seasons
a

Definition of asthma varies among reports and includes selfreporting of ever diagnosed asthma and current or ever used asthma medications.

In 1864, H H Slater noted that cold air triggered asthma and offered pulmonary vascular congestion as a possible explanation. This theory has been

ASTHMA / Exercise-Induced 201

developed by McFadden and others. Airway cooling in response to inspired cold air may trigger vasoconstriction of the bronchial vasculature. Subsequent reex vasodilation and hyperemia with extravasated uid leading to mucosal edema could lead to airway narrowing. Although attractive, this theory fails to acknowledge the capacity of the upper airways to warm inspired air such that it is unlikely that cold air is delivered to medium-sized airways, the main site of narrowing in asthma. However, cooling of the airways could take place if evaporation of mucosal water exceeded the replacement capacity of the airway. Exercise is associated with mouth breathing, which bypasses the humidifying effect of the nasal mucosa, and dry air has been shown to increase the capacity of an exercise challenge to trigger EIB. Evaporation of water from the upper airways might take place as a result of a large increase in ventilation of relatively dry air. This would cause an increase in the osmolarity of the airway mucosa. This concept of airway hyperosmolarity has been developed by Anderson and colleagues and has become popular in current literature. Inhaling hyperosmolar mannitol or saline solutions can trigger bronchospasm in the absence of an exercise stimulus. It has been difcult to separate the specic elements of these two processes and it may be that both are partly responsible for the effect of hyperventilation on the airway in EIA. Since cold air holds less moisture than warm air, this may add to the drying of the airway. Alternative mechanisms for transducing the proasthmatic stimulus to a bronchoconstrictive response can be postulated. These include stretching of the airway wall cells and altered pressure dynamics of the airway lumen. Ultimately, a signal to alter the diameter of the airway lumen is generated that results in a fall in FEV1. The nature of this signal is also controversial but biochemical mediators associated with inammation offer a possible explanation.
Inammation in EIA

caused by a variety of inammatory triggers such as allergen exposure or viral infection. While less intuitive it seems possible that EIA may also have an inammatory basis. Table 3 details the variety of inammation markers that have been studied with reference to EIA. Biological markers of inammation A variety of studies have demonstrated elevated inammatory cells in the airway lumen and bronchial wall from athletes engaged in a wide spectrum of athletic pursuits. These cells include eosinophils, T lymphocytes, macrophages, and mast cells all of which are recognized as key cellular elements of the asthmatic inammatory response. Changes in airway inammatory cell number correlate with changes in bronchial reactivity and some groups have demonstrated that the degree of bronchospasm in response to exercise challenge more closely reects the level of airway eosinophilia than the prechallenge methacholine sensitivity. Bronchial biopsies taken from resting cross-country skiers demonstrated increased inammatory cells in the bronchial wall compared to nonathlete controls. Regular exercise may therefore lead to a persistent airway inammation beyond the acute effects of exercise challenge. Cell-based studies appear to support the role of exercise as a trigger for airway inammation. The study of winter athletes described above demonstrated that airway wall inammation occurred even in the absence of symptomatic EIB. This suggests that airway inammation is not sufcient to cause EIA and that exercise per se can induce inammation in normal subjects. Unfortunately, none of these studies offer an explanation for why some athletes are susceptible to the inammatory effects of exercise while others are not. Inammatory cells in the airway are presumed to cause bronchial hyperresponsiveness by the production of chemical mediators of inammation. A variety of such mediators have been studied in asthma and EIA (Table 3). Early studies investigating the role of the mast cell stabilizers such as nedcromil and sodium cromoglycate identied histamine as a likely

It has become clear that an inammatory process in the airway leads to bronchial hyperresponsiveness and chronic asthma. Exacerbations of asthma can be
Table 3 Inammatory mediators in EIA Mediator Histamine Leukotrienes Prostaglandin D2 Role in asthma

Induced by exercise

Efcacy of inhibitor ? Variable

Bronchoconstrictor Bronchoconstriction, smooth muscle proliferation, chemoattractant

Various inammatory mediators have been investigated for their capacity to induce or maintain airway inammation in EIA. Evidence for induction during exercise comes from studies looking at plasma, urine, or sputum levels of the mediator or product. The role of histamine has recently been challenged due to the overriding effect of leukotriene antagonists with no additional effect of antihistamine and a consistent difculty in demonstrating exercise induction.

202 ASTHMA / Exercise-Induced

trigger for EIA although this has been challenged recently. The role of other chemical mediators of inammation including cysteinyl leukotrienes (LTD4 and LTC4) and prostaglandins (PGD2 and PGF2) have been supported by the discovery of increased levels in postexercise plasma and urine and by the effects of specic antagonists. The role of inammatory cells and their products in the development of EIA is supported by studies demonstrating the capacity of exercise to increase their availability to airway cells. However, not all investigators have found increased levels of proasthmatic mediators in response to exercise, and the presence of a particular agent does not imply a causal link. Further evidence is available from interventions aimed at reducing airway inammation.
Do Anti-Inammatory Therapies Prevent EIA?

Inhaled corticosteroids (ICs) are the most widely used airway-delivered anti-inammatory agents. Among well-controlled asthmatic subjects who use regular ICs to abolish normal symptoms 50% will still experience EIB on testing. This suggests that EIA is relatively resistant to standard doses of IC therapy. This hypothesis is supported by early studies demonstrating only a 50% reduction in exercise-induced fall of FEV1 when ICs were introduced. When a short-acting b-adrenergic receptor agonist was added to the IC therapy, this further reduced EIB by 30%, hinting that increased airway resistance in EIA is a complex phenomenon. The introduction of modern anti-inammatories has reinforced the theory that EIA is an inammation-mediated phenomenon. Leukotrienes and in particular LTD4 are regarded as among the most potent proasthmatic inammatory mediators. Leukotriene receptor antagonists (LTRAs) inhibit the effect of these mediators at their specic receptors. The introduction of LTRAs to common practice suggested that they might have efcacy in treating EIA, a suggestion supported by subsequent examination. LTRA therapy provides a dose-dependent protection from EIB and shortens the time to recovery of FEV1. In contrast antihistamine therapy does not appear to add any further protection over LTRA medication. Several lines of evidence support the role of inammatory processes in EIA. Exercise can trigger and maintain airway inammation that is associated with bronchial hyperresponsiveness. Intervention studies show that combating inammation can improve EIA. What is unclear, however, is what predisposes an individual to develop exercise-induced airway inammation and why otherwise potent antiinammatory agents are only partially effective in

preventing EIA despite improved control in underlying asthma. Finally, various chemical triggers of asthma have been investigated for their ability to trigger or enhance bronchoconstriction on exercise challenge. Of these adenosine, the product of ATP breakdown, seems worthy of further investigation. Exercise increases circulating adenosine levels suggesting that any proasthmatic activity of this chemical could then be enhanced by exercise. A correlation between the fall in FEV1 following exercise challenge in asthmatic subjects and plasma adenosine levels has been reported. Adenosine is believed to trigger inammatory cell degranulation, and in the context of asthma this may enhance a mild asthmatic response making it considerably more severe. Such studies may ultimately explain why exercise can be the only trigger for symptomatic EIA in otherwise nonasthmatic subjects.
Integrating the Two Pathogenic Theories

It can be concluded that exercise-induced hyperventilation can trigger physical changes in the airway that are subsequently transduced to an inammatory signal in the bronchial wall, which can be assumed to lead to bronchoconstriction and possibly chronic inammation in susceptible people. Can physical changes at the surface of the airway communicate to the bronchial wall to cause inammation and bronchial hypersensitivity? Evidence for pathways communicating signals between different airway compartments has been accumulating over the last decade, offering possible mechanisms to complete the model. As yet these pathways are untested in the context of EIA but do offer intriguing possibilities. It is becoming clear that airway epithelial stress can cause the release of inammatory mediators that promote airway inammation. Recent research supports the development of an integrated epithelium mesenchyme complex (the epithelial mesenchymal trophic unit) reminiscent of the embryonic lung. Epithelial monolayers in vitro can trigger inammatory signals in broblasts and smooth muscle cells of the airway supporting such a communication. Inammatory cells found supercially in the airway, such as eosinophils, can be activated by osmolar stress and release mediators capable of eliciting an asthmatic response offering a more direct line of communication. Thus, drying and cooling of surface epithelium could in theory at least trigger generalized airway inammation as seen in chronic asthma. The hyperemia following airway rewarming would support an inammatory process by supplying exudates and inammatory cells (Figure 1).

ASTHMA / Exercise-Induced 203

Hyperventilation/exercise

Cooling of the airway & bronchial vasoconstriction

Reactive vasodilitation & mucosal hyperemia

Hyperosmolarity of airway mucous layer

Mucosal congestion & exudate formation

Accumulation of inflammatory cells in the airway lumen

Osmotic stress communicated to superficial cells of the airway

Degranulation Degranulation of inflammatory cells release of inflammatory mediators LTD4 and PGE2

Release of proinflammatory cytokines from epithelial cells

Bronchial wall inflammation, airway smooth muscle contraction, mucosaledema, possible epithelial cell shedding
Figure 1 Summary diagram of the proposed pathogenesis of EIA. A series of pathological events following increased ventilation in exercise are proposed that trigger an asthmatic response in susceptible individuals.

Clinical Presentation
Symptoms associated with exercise can arise from a variety of sources including the lungs, heart, and gastrointestinal tract. A careful history can help to identify the most likely source, but EIA may present with classical symptoms such as dyspnea, wheeze, and cough or with more obscure symptoms making the diagnosis difcult. Complaints such as headache, abdominal pain, chest pain, cramps, and severe fatigue may all respond to treatment for EIA. Symptoms may develop early during an exercise event or following its completion. The association with exercise should

alert the clinician to the possibility of EIA although the complaint may seem unrelated to the lungs. Frequently patients believe they are simply out of condition and even well-controlled asthmatics may not associate their exercise-induced symptoms with asthma. EIA can be the only manifestation of hyperresponsive airways so the absence of a formal diagnosis of asthma does not exclude the possibility of EIA.

Differential Diagnosis
Since the diagnosis of EIA is not always straightforward to make a high index of clinical suspicion, it

204 ASTHMA / Exercise-Induced

Table 4 Differential diagnosis of the patient presenting with exercise-induced breathlessness System Respiratory Disease EIA COPD Pulmonary brosis Pulmonary vascular hypertension Angina Ventricular dysfunction Dysrhythmia Poor tness

Cardiovascular

General

COPD, chronic obstructive pulmonary disease; EIA, exerciseinduced asthma.

should be combined with an open mind regarding the variety of other potential sources of symptoms. In particular, true vocal cord dysfunction can be difcult to distinguish from EIA when symptoms predominantly present with exercise. A list of alternative diagnoses for exercise-induced dyspnea is presented (Table 4). It is not yet clear if patients who have demonstrable exercise-induced bronchoconstriction but no symptoms will benet from pharmacological treatment. Examination of the lungs of a subject with exerciseinduced dyspnea will usually be normal in the context of asthma or lone EIB. The presence of wheeze or hyperination might point to chronic airow obstruction that has gone unrecognized. A full examination might point to an alternative diagnosis as listed. Again quiescent asthma or lone EIB will usually be associated with normal pulmonary function tests while abnormalities may point to alternative pulmonary diagnoses or chronic airow obstruction.

or cromoglycate have been shown to have some preventative efcacy in this setting. b2-adrenergic agonists have been shown to relieve exercise-induced bronchoconstriction but their efcacy is reduced in EIB. Patients should be able to record the effect of treatment on symptoms or on peak ow with a small degree of training. Where it is desirable to direct investigations towards a specic diagnosis of EIA, a variety of alternative procedures, ranging from exercise provocation to bronchial challenge, are available. Various procedures have been described that are capable of triggering airway narrowing in asthma consistent with a diagnosis of EIA. The principal element of all these tests is to raise minute ventilation as occurs with exercise. It is possible to trigger bronchial narrowing by hyperventilation and this has been recommended as a surrogate for more formal exercise challenge. More exercise specic tests have been developed for both eld and laboratory. While testing in the eld has the advantage of replicating the conditions that normally trigger asthma in the subject, the equipment required to make useful measurements has to be portable. This has led to the development of protocols designed to measure pulmonary responses to exercise under laboratory conditions that replicate the essential features of outdoor activity. Guidelines recommending the best practice for performing these tests are available, the essential features of which are summarized below.
Patient Instructions

Investigation
The need for further investigation will be directed by the clinical ndings but may fall into two groups. In the rst a pragmatic trial of pharmacological intervention prior to a predictable exercise trigger of symptoms can be the most useful and easiest test. In some cases, however, detailed pulmonary function in response to exercise is desirable either to monitor treatment, to make a difcult diagnosis, or if the presence of EIA would limit the performance of essential life-saving work (American Thoracic Society (ATS) Guideline). Other uses for detailed exercise testing include the diagnosis of asthma in elite athletes for drug monitoring or performance testing. In a pragmatic trial a patient should be prescribed either a preventative or a reliever inhaled therapy and advised to use this prior to or during exercise that would normally trigger the reported symptoms. Antiinammatory drugs such as inhaled corticosteroids

Subjects should avoid bronchoprotective or bronchoreliever medication for 48 h and have eaten only a light meal. Antihistamines and caffeine should also be avoided. Exercise should be kept to a minimum prior to the test, as around 50% of EIA sufferers will experience a refractory period of up to 4 h after vigorous exercise. Any medical or orthopedic contraindications to exercise should be considered and the ability of the patient to fulll the physiological requirements should be ensured.
Exercise

Exercise can be performed on an electronic treadmill or stationary bicycle as both methods have been validated. The desired level of exercise is based on 8090% of maximal heart rate (based on an HR 220 age) or 5060% of maximal voluntary ventilation. The degree of exercise required to achieve this level of exercise response will vary considerably among subjects and it is advised that a period of rapid progressive increase in workload is used to achieve these targets and then maintained for at least 4 min. The

ASTHMA / Exercise-Induced 205

total exercise time for adults should be around 6 8 min. During the exercise, heart rate and where possible minute ventilation should be measured to ensure that an adequate stimulus to the airway is being achieved.
Climatic Considerations

Outdoor exercises are generally better than indoor exercises at triggering bronchospasm. This is believed to be due to lower humidity and temperature and cool dry air is known to trigger asthma in exercise sensitive subjects better than indoor room air. Maintaining the ambient temperature at 20251C and relative humidity at 50% is satisfactory. Exercise should be performed with a nose clip to prevent nasal humidication of inspired air. Severe responses to exercise have been described in susceptible people and it is advisable to have medical supervision available to monitor patients responses and administer bronchodilator therapies as required.
Measurements

What constitutes a positive exercise test is controversial due to the variety of techniques described for measurement. Most authorities consider a fall of more than 10% of resting FEV1 as abnormal especially as the normal response to exercise is bronchodilation. Some groups require a greater than 15% fall in FEV1 to diagnose EIA and this appears to be the most frequently quoted value. Plotting the FEV1 against time allows an area under the graph calculation to be made that improves the reliability of the test.
Alternative Testing Procedures

FEV1 is the most useful and convenient measurement to make in the laboratory. Where an alternative diagnosis is suspected full ow-volume loops can offer additional information, while peak expiratory ow might be used if eld testing is performed. FEV1 should be measured before exercise and following the exercise challenge. Intervals of 5, 10, 15, 20, and 30 min are recommended by the ATS; however, additional measurements can be taken and should be in the event of severe symptoms of bronchial obstruction. Changes in FEV1 can then be observed by plotting percent resting FEV1 against time (Figure 2).

Other techniques to mimic the airway response to exercise have been described. These are generally thought to be more convenient than full exercise challenge, but are necessarily less specic. Osmotic drying of the airway has been achieved by inhalation of mannitol and this method has received some attention from testers from the eld of elite sport. Eucapnic hyperventilation achieves a ventilation rate that mimics that achieved by exercise and has also been used as an outpatient measure of the airway responses.
Management

20 18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 60 70 Time from start of exercise (min)

FEV1 (% fall)

The efcacy of asthma medications for exerciseinduced symptoms will be dealt with elsewhere. Patients should be encouraged to manage their symptoms rather than stop the exercise activity that triggers it. Occasionally, altering the exercise environment is helpful, for example, changing to indoor from outdoor activity. Some elite athletes have learned to manage their asthma by introducing a targeted warm up to trigger a mild episode of EIA and the resultant refractory period elite may allow the completion of the competitive element of the activity. Sporting authorities restrict the use of most inhaled therapeutic agents for asthma. It is sensible for athletes and their coaches to be aware of these restrictions, which can be accessed easily. A range of websites for agencies involved in regulating drugs in sport is given at the end of this article.

Conclusion
EIA may be a specic disease entity in some circumstances or may be the only manifestation of latent asthma. It is common in asthma sufferers and in athletes of all capabilities. The basis for the development and persistence of exercise triggered airway pathology is not fully understood but probably reects the ability of hyperventilation to trigger airway inammation. Diagnosis can be complicated by the variety of presentations and detailed investigations

Figure 2 A plot of change in FEV1 as percent baseline with time exercised. Exercise begins at time 0 and ceases at the point marked by the arrow. FEV1 is measured and the percent change is calculated. , change in FEV1; , the effect of an agent , the effect of an agent that changes maximum fall in FEV1; that reduces the duration of EIB. As can be seen it is frequently more accurate to represent changes to EIB by describing the area under the curve than any single element of the plot.

206 ASTHMA / Extrinsic/Intrinsic

are sometimes required. Asthma management is not always successful at controlling symptoms and behavioral changes may have to be made to achieve control of the disease.
See also: Exercise Physiology. Lipid Mediators: Leukotrienes.

Further Reading
Anderson SD and Brannan JD (2002) Exercise-induced asthma: is there still a case for histamine? Journal of Allergy and Clinical Immunology 109(5): 771773. Anderson SD and Daviskas E (2000) The mechanism of exerciseinduced asthma is. Journal of Allergy and Clinical Immunology 106(3): 453459. Gotshall RW (2002) Exercise-induced bronchoconstriction. Drugs 62(12): 17251739. Milgrom H (2004) Exercise-induced asthma: ways to wise exercise. Current Opinion in Allergy and Clinical Immunology 4: 147153. Seale JP (2003) Science and physicianly practice: are they compatible? Clinical and Experimental Pharmacology and Physiology 30(11): 833835. Storms WW (2003) Review of exercise-induced asthma. Medicine and Science in Sports and Exercise 35(9): 14641470. Tan RA and Spector SL (2002) Exercise-induced asthma: diagnosis and management. Annals of Allergy, Asthma, and Immunology 89(3): 226235.

which many cells and cellular elements play a role, in particular, eosinophils, mast cells, T lymphocytes, neutrophils, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling, possibly due to ongoing inammation and abnormal repair processes. Susceptible individuals experience recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airow obstruction, which is often reversible, and bronchial hyperresponsiveness to a variety of stimuli. Acute asthma is a common medical emergency and requires prompt assessment and treatment. Advances in the understanding of the genetic and environmental factors that account for asthma and its pathogenesis should lead to improved management strategies.

Introduction
Historical Perspective

The symptoms of asthma were described by Aretaeus over 2000 years ago. However, despite significant progress in our understanding of its pathogenesis and considerable improvements in pharmacological treatment, we have been unable to halt the relentless increase in prevalence that has taken place over the last 30 years.

Definition Relevant Websites

http://www.olympic.org Home page of the International Olympic Movement, offering insight into the role of antidoping authorities in elite sport. It includes useful links to national Olympic committees detailing specic national guidelines for athletes. http://www.wada-ama.org Homepage of the world antidoping authority. Including details of the use of prescribed proscribed therapeutics in sport. http://www.asthma.org.uk Asthma UK link to exercise induced asthma with tips for patients and some general information regarding school and preschool sporting activities for asthma sufferers.

Extrinsic/Intrinsic
N C Thomson and G Vallance, University of Glasgow, Glasgow, UK
& 2006 Elsevier Ltd. All rights reserved.

Several different definitions have been devised that describe the asthma phenotype. In 1997 the National Asthma Education and Prevention Program Expert Panel Report dened asthma as: A chronic inammatory disorder of the airways in which many cells and cellular elements play a role, in particular, mast cells, eosinophils, T lymphocytes, neutrophils, and epithelial cells. In susceptible individuals, this inammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airow obstruction that is often reversible either spontaneously or with treatment. The inammation also causes an associated increase in the existing bronchial hyperresponsiveness to a variety of stimuli.

Epidemiology
Prevalence of Asthma and Atopy

Abstract
Asthma is one of the most common chronic diseases, affecting 300 million people worldwide. There has been a significant increase in prevalence over the last 30 years, particularly in the West. Complex relationships between genetic and environmental factors, such as viral infections, allergens, and occupational agents, inuence the origin and progression of the disease. Asthma is a chronic inammatory disorder of the airway in

Asthma is one of the most common chronic conditions affecting 300 million people worldwide. The Global Initiative for Asthma (GINA) estimates that one in 20 people in the world now have asthma, with a significant increase in the prevalence of disease over the last 30 years. This is in parallel with an increase in other atopic diseases, such as allergic rhinitis and

ASTHMA / Extrinsic/Intrinsic
Scotland Jersey Guernsey Wales Isle of Man England New Zealand Australia Republic of Ireland Canada Peru Trinidad & Tobago Costa Rica Brazil United States of America Fiji Paraguay Uruguay Israel Barbados Panama Kuwait Ukraine Ecuador South Africa Finland Malta Czech Republic Ivory Coast Colombia Turkey Lebanon Kenya Germany France Japan Norway Thailand Sweden Hong Kong United Arab Emirates Philippines Belgium Austria Saudi Arabia Argentina Iran Estonia Nigeria Spain Chile Singapore Malaysia Portugal Uzbekistan FYR Macedonia Italy Oman Pakistan Tunisia Latvia Cape Verde Poland Algeria South Korea Bangladesh Morocco Occupied Territory of Palestine Mexico Ethiopia Denmark India Taiwan Cyprus Switzerland Russia China Greece Georgia Romania Nepal Albania Indonesia Macau

207

Country

10

15

20

25

30

35

40

Prevalence of asthma symptoms (%)

Figure 1 Ranking of the prevalence of current asthma symptoms in childhood by country: written questionnaire. Reproduced with permission from GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6.

atopic dermatitis. These increases have been most noticeable in afuent countries with a mild climate such as the UK, New Zealand, Australia, and North America (Figure 1) and correlate with urbanization and the adoption of a westernized life style. It is more common in children than in adults. The clearest risk factor for the development of asthma is atopy. Atopy is the genetic predisposition for the development of an IgE-mediated response to common aeroallergens. Complex relationships between atopy and

environmental factors such as viral infections, allergens, and occupational agents inuence the origin and progression of the disease.
Morbidity

The morbidity from asthma is considerable. Surveys of patients with asthma indicate that many have poorly controlled symptoms, impaired indices of quality of life, and are often receiving inadequate

208 ASTHMA / Extrinsic/Intrinsic

treatment. Hospital admission rates for asthma, particularly in children, increased from the 1970s until the mid 1980s and have since then remained stable. In the US during 2002 there were 13.9 million outpatient visits, 1.9 million emergency room visits, and 484 000 hospitalizations. Many children and adults with asthma lose time from school and work, respectively. The nancial impact of asthma is considerable; in the US the estimated total cost exceeds $6 billion per annum, with hospitalization and emergency room visits making up 50% of that gure.
Mortality

Classication
Asthma can be classied on the basis of severity and etiology.
Severity

The severity of asthma can be graded by assessing the frequency and severity of symptoms and measurements of lung function before treatment is started or by the level of treatment required to achieve asthma control (Table 1).
Etiology

International mortality gures for asthma are often unreliable due to misclassication of the cause of death. In Western countries, where studies were restricted to the 534 years age group, the mortality from asthma increased steadily from the mid-1970s to the late 1980s. More recent studies suggest a plateau or decline in deaths from asthma. Risk factors for increased morbidity and potential mortality include socioeconomic deprivation, ethnicity, urban dwelling, and comorbid issues such as drug abuse. The vast majority of deaths occur among those with chronic severe asthma; few deaths occur among those with previously mild disease. Deaths are associated with inadequate treatment with inhaled or oral steroid and with poor follow-up and monitoring.
Natural History

In 1947, Rackeman was the rst to subdivide asthma into intrinsic and extrinsic asthma. He noted that extrinsic asthma, now described as allergic asthma, started before the age of 30 years and was associated with atopy. Intrinsic or nonallergic asthma was noted to begin in middle age and was not associated with allergy, but with nasal polyps. It is more common in women.

Etiology
Genetic

The ndings of the Tucson Childrens Respiratory Study suggested three clinical phenotypes of childhood asthma. Transient infant wheezing occurs during infancy, but not after the age of three years. These children have no family history of atopy and have a good prognosis. The second phenotype is the nonatopic wheeze of the toddler and early school years, after an early lower respiratory tract infection. The third phenotype is persistent atopic wheeze, which describes children who continue to wheeze at age 10 and have associated atopy and airway hyperresponsiveness. Many children have a favorable outcome with spontaneous remission in their adolescence. Risk factors for progression into adulthood include early onset with severe symptoms, poor lung function, and airway hyperresponsiveness. It occurs more commonly in girls with associated atopy. Most adults with mild-to-moderate asthma appear to continue to have symptoms of a similar severity. Remission of adult asthma is rare. Irreversible airow obstruction can develop in nonsmokers with asthma particularly in those individuals with severe symptoms and mucus hypersecretion. Smokers with asthma have an accelerated decline in lung function.

Twin and family studies have demonstrated that atopic diseases cluster in families and have a genetic basis. Genome-wide scans have shown that many genes determine the risk of asthma. The region of chromosome 5q31-33 controls the production of interleukin (IL)-4 and IL-13 and has been linked to atopy. Linkage studies have implicated other candidate genes on chromosomes 2, 3, 4, 6, 7, 11, 12, 13, 17, and 19. Classical positional cloning approaches have led to the identication of new genes of potential significance such as ADAM33, IL4RA, and CD14. This may contribute to our understanding of the mechanism of disease, such as the role of ADAM33 in airway hyperresponsiveness. They may also cast light on different individual responses to therapy, as there are polymorphisms of a number of common drug targets. The delineation of the precise relationships between these genetic factors and environmental agents is now required.
Environment

Hygiene hypothesis In 1989, Strachan noted an inverse relationship between family size and hay fever, with children with more siblings having a lower risk of atopy. It has been suggested that childhood exposure to infection may protect against risk of atopy. Low levels of infection in infancy, associated with improvements in public health, may deprive the immune system of the Th1 stimulus that normally balances the Th2 predominance of the neonate. This

ASTHMA / Extrinsic/Intrinsic
Table 1 Classication of asthma severity by daily medication regimen and response to treatment Patient symptoms and lung function on current therapy Current treatment step Step 1: intermittent Level of severity Step 1: intermittent Symptoms less than once a week Brief exacerbations Nocturnal symptoms not more than twice a month Normal lung function between episodes Step 2: mild persistent Symptoms more than once a week but less than once a day Nocturnal symptoms more than twice a month but less than once a week Normal lung function between episodes Step 3: moderate persistent Symptoms daily Exacerbations may affect activity and sleep Nocturnal symptoms at least once a week 60%oFEV1 o80% predicted OR 60%oPEFo80% of personal best Step 4: severe persistent Symptoms daily Frequent exacerbations Frequent nocturnal asthma symptoms FEV1 p60% predicted OR PEFp60% of personal best Intermittent Mild persistent Step 2: mild persistent Step 3: moderate persistent

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Moderate persistent

Mild persistent

Moderate persistent

Severe persistent

Moderate persistent

Severe persistent

Severe persistent

Severe persistent

Severe persistent

Severe persistent

Reproduced with permission from GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figures 57, p. 7.

unrestrained Th2 response is postulated to predispose to allergic disease. European studies showing lower levels of atopic disease amongst children raised in rural communities have suggested that exposure to bacterial endotoxin may play a role in the development of tolerance to common allergens. However, the high levels of asthma associated with cockroach allergy in inner-city areas of the US, and the simultaneous increase in Th1 diseases, such as type 1 diabetes, illustrate that our understanding of the relationship between environmental exposure and disease is not yet complete. Allergen exposure Sensitization to the house dust mite Dermataphagoides pteronnysinus is the most common risk factor for the development of asthma in adults and children. Early exposure to the dust mite antigen Der p1 has been shown to increase the risk of asthma vefold. Peat elegantly demonstrated the doseresponse relationship between dust mite level and severity of asthma symptoms across six different regions

of Australia with increasing humidity levels. Dust mites thrive in a humid environment, and improved westernized building construction techniques, favoring colonization by house dust mites, may have contributed to the increase in asthma. Studies at altitudes where the levels of house dust mite are extremely low have suggested that a low allergen environment does improve symptoms of asthma. However, simple measures such as mattress covers and carpet cleaning have shown little effect on reducing the domestic burden of dust mite and asthma symptoms. Exposure to other indoor allergens from animal dander and cockroach are also important risk factors for asthma. Removal of the pet from the family is advised to reduce exposure to the allergen. However, early exposure to cat allergen has recently been demonstrated to protect against development of asthma and the full intricacies of the relationship remain to be elucidated. Cockroach infestation has become rampant in inner-city areas of the US and sensitization to the

210 ASTHMA / Extrinsic/Intrinsic

German cockroach Blatella germanica has become an important risk factor for asthma. Outdoor air environment Sensitization to the fungus Alternaria is a risk factor for asthma. It has been suggested that outbreaks of asthma after thunderstorms are related to release of fungal spores of Cladosporium. The relationship between air pollution and asthma is complex as there has been considerable reduction in air pollution over the time period when asthma prevalence has increased. Extremely high levels of asthma have been noted in the rural Highlands of Scotland. It has been suggested that particulate pollution may protect against incidence but exacerbate symptoms in those sensitized. Diet Changes in the modern processed diet have been linked with an increased risk of asthma. High levels of omega-3 oils associated with eating fresh sh have been shown to reduce the risk of asthma. Breast-feeding to 3 months has also been associated with lower levels of asthma. Cigarette smoking There is much evidence to suggest that exposure to tobacco smoke in utero and in early childhood increases the risk of allergy and wheeze. Nevertheless, as the total numbers of smokers in the UK have fallen during the last 30 years, it is not the whole answer to the rise in asthma prevalence.

to small airways with mucus and inammatory cells, particularly eosinophils. Histologically, the airways show characteristic changes: an intense inltration by inammatory cells, particularly eosinophils and T lymphocytes, sloughing of the surface epithelium, thickening of the reticular basement, increase in the airway smooth muscle mass, increased numbers of epithelial goblet cells, vasodilatation, and edema (Figure 2). Neutrophils are found also in those who die suddenly from acute asthma.
Chronic Asthma

Information on the pathology of asthma has been obtained from bronchial biopsies obtained from patients with mainly mild asthma. The histological changes are similar although less pronounced than those obtained from cases of fatal asthma. The similarity of the histological changes in allergic or extrinsic and nonallergic or intrinsic asthma suggests a nal common pathogenic mechanism in both types of asthma. Neutrophils are found more commonly in patients with severe asthma.

Clinical Features
Acute Asthma

Pathology
Fatal Asthma

In cases of fatal asthma the lungs are hyperinated due to air trapping caused by plugging of the medium

Acute asthma is a common medical emergency. It is characterized by a progressive increase in dyspnea, cough, or wheeze. The decrease in expiratory airow can be quantied by a fall in peak expiratory ow (PEF) or forced expiratory volume in 1 s (FEV1). Deterioration usually progresses over hours to days, although in some cases it may be more sudden and require rapid treatment within minutes. The severity of exacerbation is highly variable. Respiratory tract

Mucus Epithelial cells and goblet-cell hyperplasia Thickening of sub-basement membrane Cellular infiltrate

Hypertrophy of smooth muscle

Vascular congestion
Figure 2 Autopsy specimen of airway from a subject who died from acute asthma showing characteristic histological changes. Hematoxylin and Eosin, 40. Photograph courtesy of Dr F Roberts, Department of Pathology, Western Inrmary Glasgow.

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211

infections are thought frequently to precipitate attacks of asthma, particularly in children. Infections are mainly viral, especially human rhinoviruses but also respiratory syncytial virus, adenoviruses, parainuenza, and inuenza viruses. The role of infection in provoking asthma attacks in adults is less certain. Clinical assessment Clinical features A short history of the features of an exacerbation should be elicited. The aims are to ascertain duration and severity of symptoms, with the perspective of current medication and prior admissions. Assessment must be rapid and accurate to permit prompt treatment. The history is usually one of increasing breathlessness and wheeze. Patients often have difculty speaking and sleep is disturbed by the severity of these symptoms. There is increasing need for bronchodilator treatment, which becomes less effective. The patient may show signs of exhaustion and reduced conscious level. There is invariably an associated tachycardia, increase in respiratory rate, and auscultation of the chest may reveal severe wheeze or absent breath sounds that indicates very severe airow obstruction. The chest becomes hyperinated and patients may use accessory respiratory muscles. Investigations Measurement of pulse oximetry is necessary in acute asthma to evaluate oxygen saturation. The aim is to maintain SpO2492%. Arterial blood gas analysis is necessary if SpO2o92%. If oxygenation remains inadequate despite supplemental oxygen, additional complications should be considered, particularly pneumonia. The earliest abnormality is respiratory alkalosis and hypocarbia, but normal oxygen tension. As airow obstruction increases there is uneven distribution of inspired air and changes in the normal ventilation-to-perfusion ratio. As severity increases, hypoxemia develops. The presence of normal levels of arterial carbon dioxide tension is ominous as it indicates the patient is becoming exhausted. Monitoring response to treatment should be on the basis of PEF and clinical examination. A chest radiograph may show evidence of hyperination, mucus plugging, and atelectasis in an acute exacerbation, but these ndings may add little to management. A chest radiograph should be performed if pneumothorax is suspected. Levels of severity Asthma guidelines have been developed to ensure prompt, systematic history and examination, and ensure accurate assessment of severity. The British Guideline on the Management of Asthma denes a moderate exacerbation as one presenting with increasing symptoms of wheeze,

dyspnea, or breathlessness and a fall in PEF to 5075% of the best or predicted. However, if the PEF falls to 3355% best or predicted and is accompanied by an inability to speak in complete sentences, tachypnea of 425 breaths per min, or tachycardia of 110 beats per min, the exacerbation is classied as severe. Life-threatening features are a PEF less than one-third of best or predicted or hypoxia demonstrated by arterial oxygen saturations of less than 92% on air or arterial partial pressure of less than 8 kPA. Normal levels of CO2, a silent chest on examination, or feeble respiratory effort, cyanosis, bradycardia, dysrhythmia, hypotension, exhaustion, confusion, or coma are all signs of a near fatal episode.
Chronic Asthma

The diagnosis based on a history of episodic respiratory symptoms especially after exercise or during the night is usually not difcult. Demonstration of reversible airow obstruction gives a simple, reliable, and objective diagnosis of asthma. Evidence of reversibility can be found through the history of symptoms of episodic cough, wheeze, chest tightness, or dyspnea, measurement of PEF, or spirometry, and trials of therapy. Conditions to be considered in the differential diagnosis are listed in Table 2. Clinical assessment Clinical features Asthma may present with wheeze, shortness of breath, cough, or chest tightness. The hallmark of asthma is that these symptoms tend to be variable and intermittent. They are often worse at night and early morning and provoked by triggers such as allergens or exercise (Table 3). Less common factors are rhinitis, bacterial sinusitis, menstruation, gastroesophageal reux, and pregnancy. When cough is the predominant symptom without wheeze, this is
Table 2 Differential diagnosis of asthma Disease Cystic brosis Gastroesophageal reux Bronchiectasis Ciliary dyskinesia Developmental disorder of the airway Inhaled foreign body Chronic obstructive pulmonary disease Left ventricular function Pulmonary thromboembolism Vocal cord dysfunction Upper airway obstruction Pulmonary eosinophilia Bronchial carcinoid O, Diagnosis should be considered. Children O O O O O O O O O O Adults O O O O O O O O O O O

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Table 3 Triggers of asthma Infections, particularly viral Allergens, e.g., house dust mite, pollens, animals Occupational agents, e.g., isocyanate-containing paints, our Environmental pollutants, e.g., cigarette smoke, sulfur dioxide Drugs, e.g., beta-blockers Exercise Cold air Hyperventilation Foods Psychological factors

of exacerbations, number of emergency consultations for asthma, and hospital admissions. Good control is described as the presence of minimal symptoms during day and night, minimal need for reliever medication, no exacerbations, no limitation of physical activity, and normal lung function. It may not be possible to achieve good asthma control in patients with moderate or severe persistent asthma (Table 1). Specic clinical problems Asthma during pregnancy The course of asthma during pregnancy varies, with a similar proportion of women improving, remaining stable, or worsening. The risk of an exacerbation of asthma is high immediately postpartum, but the severity of asthma usually returns to preconception level after delivery. Changes in b2-adrenoceptor responsiveness and changes in airway inammation induced by high levels of circulating progesterone have been proposed as possible explanations for the effects of pregnancy on asthma. Gastroesophageal reux Gastroesophageal reux can trigger attacks of asthma although the incidence is unclear. The mechanism is unknown; possibilities include aspiration or an esophagio-bronchial reux triggered by acid irritation of the esophageal mucosa.

referred to as cough-variant asthma. The physical sign of wheezing (usually expiratory, bilateral, polyphonic, and diffuse) is associated with asthma but has low sensitivity and specicity, and in many patients examination will be normal. Investigation A simple measure of pulmonary function by PEF is helpful, not only for initial assessment, but also to monitor symptoms, alert to deterioration in airow obstruction, and evaluate response to treatment. Twice-daily recording of PEF for two weeks is a simple and cheap method of demonstrating variation in airow obstruction, with a diurnal variation of greater than 15% conrming the diagnosis. However, in patients with mild asthma, the PEF may show normal variability. Spirometry demonstrates an obstructive pattern with reduction of the ratio of FEV1 to forced vital capacity (FVC). The key feature is that this airway obstruction may be reversible. Administration of a bronchodilator typically causes an increase in FEV1 of 1215%. However, failure to demonstrate reversibility does not exclude asthma, or prove irreversible disease. Airway hyperresponsiveness is a characteristic feature of asthma and can be demonstrated by bronchial provocation techniques. The most common methods are provocation by inhalation of methacholine or histamine and exercise challenge. Fall in FEV1 is measured by serial spirometry after inhalation of increasing concentrations of methacholine. Results are expressed as the concentration of the agent that elicits a fall of 20% in FEV1. This concentration denes the degree of bronchial responsiveness and severity of disease. Skin prick testing, measurement of total and specic IgE levels, and blood eosinophilia are difcult to interpret in asthma because they have variable sensitivity and specicity. Routine chest radiographs in asthma may yield no new information and may be normal in chronic asthma. Assessment of control Asthma control can be determined by assessing symptoms, inhaled b2 adrenoceptor agonists use, lung function as well as rate

Pathogenesis
The pathogenesis of asthma involves acute and chronic inammation as well as remodeling (Figure 3). The underlying process is one of inammation involving eosinophils and T lymphocytes, with the release of various mediators and cytokines, although recent evidence indicates a role for other cells including mast cells, neutrophils, macrophages, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling. The mechanisms involved in remodeling remain to be claried but probably consist of ongoing inammation and abnormal repair processes. Remodeling occurs in many asthmatic patients, although the extent varies. It is thought that remodeling may play an important role in causing symptoms and loss of lung function in severe asthma, although this hypothesis remains to be established.
Airway Inammation

There are two distinct responses to inhalation of an allergen. The immediate hypersensitivity reaction, in which wheeze occurs within minutes, is comparable to the wheal-and-are response of skin. Further wheeze is caused by the late phase response, mounted between 6 and 9 h after allergen provocation.

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213

Inducers of asthma

Inflammatory mediators and asthma

Allergen Antigen presenting cell Dendritic cell Lymph node Acute airway inflammation Chronic airway inflammation Airway remodeling Th2 lymphocyte Cytokines (e.g., IL-4, IL-5, IL-13)

Symptoms Exacerbations Disability

Figure 3 In susceptible individuals allergens, occupational agents, and other known and unknown inducers of asthma cause airway inammation. The inammation may be acute and resolved, but in most individuals the inammation is chronic and may be associated with airway remodeling. Airway inammation and remodeling cause asthma symptoms, exacerbations, and disability.

Mast cell (e.g., leukotrienes, histamine, tryptase)

Epithelium (e.g., prostaglandins, IL-6, IL-8)

Eosinophil (e.g., leukotrienes, major basic proteins)

Inflammatory mediators

BronchoMucosal constriction edema

Mucus hypersecretion

Bronchial reactivity

Airway remodeling

The immediate reaction involves the activation of mast cells by allergen cross-linking two IgE antibodies (Figure 4). IgE antibodies are produced by B cells in response to processed antigen, which is presented by airway dendritic cells in the draining lymph node. The IgE antibodies bind mast cells by their high-afnity receptors (FceRI). Cross-linking by allergen of IgE antibodies initiates signal transduction by the FceRI effecting degranulation of the mast cell. This promotes release of mediators such as histamine, tryptase, eicosanoids, and reactive oxygen species. These spasmogens cause secretion of mucus, smooth muscle constriction, and vasodilation. Leakage of plasma protein causes edema of the airway wall, impedes clearance of mucus, and causes formation of plugs; all result in reduced airway conductance. The late phase reaction includes the accumulation of activated eosinophils, lymphocytes, macrophages, neutrophils, and basophils. The ability of cytokines to induce the expression of adhesion molecules provides a mechanism for cell migration from the circulation to the airway. Eosinophils are thought to play a central role in the pathogenesis of chronic asthma. IL-5 controls the production of eosinophils by the bone marrow and their subsequent release into the circulation. They migrate from circulation to the airway under the inuence of chemokines and release toxic granule proteins, including major basic proteins, eosinophil peroxidase and eosinophil cationic protein, Th2

Symptoms of asthma
Figure 4 Possible pathways in the development of airway inammation in asthma following exposure to allergen.

cytokines, and leukotrienes. Major basic proteins causes direct airway damage with epithelial shedding. Leukotrienes increase vascular permeability and constrict smooth muscles. Challenge of the airway with allergen increases the local levels of IL-5, which correlates directly with the degree of airway eosinophilia. Recently, doubts have arisen about the role of eosinophils in causing airway hyperresponsiveness in asthma. Treatment of patients with allergic asthma using anti-interleukin-5 monoclonal antibody does not prevent allergen-induced bronchoconstriction or airway hyperresponsiveness, despite markedly suppressing eosinophil numbers within the airways. T cells are found in abundance in the inamed airways of asthma patients and it is widely held that the Th2 subset is a driving force in allergic inammation. The T helper subsets were characterized by Mosman on the basis of their signature cytokines. Th2 cell cytokines include IL-4, IL-5, and IL-13. IL-5 is involved in eosinophil maturation and activation, whereas IL-4 and IL-13 control synthesis of IgE. However, the Th2 paradigm for allergic asthma is now thought to be too simplistic and an additional role for Th1 cells has been postulated.

214 ASTHMA / Extrinsic/Intrinsic

The mast cell degranulation is crucial to the acute response to allergen, with release of histamine and synthesis of mediators effecting early recruitment, adhesion, and proliferation of cells. It may also contribute to remodeling as it contains proteoglycans with various functions, including support structures for remodeling. The role of other inammatory cells in the pathogenesis of asthma including neutrophils and macrophages is less clearly characterized. There are also complex interactions between neural control of airways and inammation.
Airway Remodeling

inammation and hyperresponsiveness have provided important information on the acute inammatory response to allergen exposure but have been less relevant to the study of mechanisms of chronic asthma and airway remodeling. The interpretation of data from experiments in animal models is inuenced by the protocol used to sensitize and challenge the animal and the strain of animal used.
See also: ADAMs and ADAMTSs. Allergy: Overview. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced. Bronchoalveolar Lavage. Bronchodilators: Anticholinergic Agents; Beta Agonists. Carbon Dioxide. Chymase and Tryptase. Corticosteroids: Therapy. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Environmental Pollutants: Overview. Epidermal Growth Factors. Genetics: Overview; Gene Association Studies. Histamine. Immunoglobulins. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Overview. Matrix Metalloproteinases. Neurophysiology: Neural Control of Airway Smooth Muscle. Oxygen Therapy. Pneumothorax. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signs of Respiratory Disease: Breathing Patterns; General Examination; Lung Sounds. Smooth Muscle Cells: Airway. Symptoms of Respiratory Disease: Cough and Other Symptoms. Tumor Necrosis Factor Alpha (TNF-a ). Upper Airway Obstruction. Upper Respiratory Tract Infection.

There has been considerable evidence that inammation alone may not explain the full pathophysiology of asthma. Two large longitudinal studies of inhaled corticosteroids have shown little effect on the natural history of asthma. Acute airway inammation should be resolved to permit resumption of normal function. In chronic asthma, a state of increased injury and repair is thought to lead to remodeling. It is coordinated by inammatory cells, such as eosinophils, mast cells and T cells, and structural cells such as broblasts. Growth factors are secreted in response to epithelial damage and cause thickening of the smooth muscle and basement membrane, resulting in airway narrowing. It is thought that a primary abnormality of the epithelium predisposes to such damage by toxins. The interaction between the thickened basement membrane and altered submucosa, known as the epithelial-mesenchymal trophic unit, is postulated to be important early in the origins of disease. This hypothesis is supported by the recent identication of ADAM33 as an asthma susceptibility gene expressed abundantly by the smooth muscle and broblasts, but not by inammatory cells.

Further Reading
Barnes P, Drazen J, Rennard S, and Thomson NC (eds.) (2002) Asthma and COPD Basic Mechanisms and Clinical Management. London: Academic Press. Bel E (2004) Clinical phenotypes of asthma. Current Opinion in Pulmonary Medicine 10(1): 4450. Bousquet JP, Jeffery Busse W, Johnson M, and Vignola A (2000) Asthma from bronchoconstriction to airways inammation and remodeling. American Journal of Respiratory and Critical Care Medicine 161: 17201745. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58: i1i94. Busse W (2001) Asthma. New England Journal of Medicine 5: 350362. GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6. GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figure 57, p.7. Kay AB (2001) Allergy and allergic diseases. New England Journal of Medicine 344: 3037. Kips JC, Anderson GP, Fredberg JJ, et al. (2003) Murine models of asthma. European Respiratory Journal 22(2): 374382. Larche M, Robinson R, and Kay AB (2003) The role of T lymphocytes in asthma. Journal of Allergy and Clinical Immunology 111: 450459.

Animal Models
Animal models have been used to study both the pathogenesis and treatment of asthma. Asthma does not occur spontaneously in animals although both horses and the Berenji greyhound develop a respiratory condition that has some features of asthma. Most animal models involve sensitization to an allergen such as ovalbumin or house dust mite allergen challenge. The species commonly used include mice, guinea pigs, sheep, rats, monkeys, and dogs. The mouse is often used in these models, mainly because this species allows for the application in vivo of gene deletion technology as well as the low cost and availability of inbred species with known characteristics. Animal models of allergen-induced airway

ATELECTASIS 215
McFadden E (2003) Acute severe asthma. American Journal of Respiratory and Critical Care Medicine 168: 740759. National Asthma Education and Prevention Program Expert Panel Report: guidelines for the diagnosis and management of asthma, Update on Selected Topics 2002. Journal of Allergy and clinical Immunology 110(5 pt 2): S141S219. NHLBI/WHO (2004) Global initiative for asthma: global strategy for asthma management and prevention. NHLBI/WHO Workshop Report NIH 02-3659. Bethesda: NIH. OByrne PM and Thomson NC (eds.) (2001) Manual of Asthma Management, 2nd edn. London: W B Saunders. Rodrigo G, Rodrigo M, and Jesse B (2004) Acute asthma in adults: a review. Chest 125(3): 10811102. Tatterseld A, Knox A, Britton J, and Hall I (2002) Asthma. Lancet 360: 13131322. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 822833.

ATELECTASIS
P A Kritek, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. The mechanisms that cause atelectasis can be divided into three categories: passive, adhesive, and resorptive. Passive atelectasis results from space-occupying lesions in either the pleural space or the parenchyma compressing adjacent normal lung tissue. Adhesive atelectasis is caused by a decrease in the level or activity of surfactant leading to an increase in surface tension in the alveolus and subsequent collapse. Resorptive atelectasis ensues when there is partial or complete occlusion of ow of gas between alveoli and the trachea. Oxygen, carbon dioxide, and nitrogen will diffuse from the alveolus into the capillary until all gas is removed from the alveolar space. Small areas of atelectasis can be found in normal lungs due to the effects of gravity. Pneumothoraces or large bullae can result in passive atelectasis. Adhesive atelectasis is a feature of respiratory distress syndrome of the neonate as well as acute respiratory distress syndrome in adults. Resorptive atelectasis is found distal to an obstructive lesion, such as a tumor or mucus plug. Atelectasis associated with anesthesia is complex and caused by a combination of these mechanisms.

thereby creating a smaller space in which to maintain the inated lung (Figure 1). As a result, adjacent lung tissue will lose gas and subsequently collapse. This form of atelectasis is referred to as passive because the collapsed lung is not inherently abnormal but is being affected by an adjacent pathologic process. If the inciting cause of the atelectasis is resolved (e.g., a pneumothorax is evacuated), the underlying atelectatic lung should reexpand and return to normal function. It should be noted, however, that after a segment of lung has collapsed, there are local changes in permeability, inammatory markers, alveolar macrophage function, and surfactant associated with all types of atelectasis that may predispose to abnormal function upon reination.
Adhesive Atelectasis

Description
Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. These changes can be small, affecting only subsegmental regions, or more dramatic, leading to collapse of an entire lung. The mechanisms that result in atelectasis can be divided into three categories: passive, adhesive, and resorptive. Each of these is discussed individually. Note that although discussions of radiographic descriptions of atelectasis are included, the discussion is grounded in these pathophysiologic aspects of atelectasis.
Passive Atelectasis

Adhesive atelectasis results from the absence, loss, or decreased activity of surfactant within the alveoli (Figure 2). Surfactant, produced by type II alveolar cells, decreases surface tension as alveolar surface area decreases and balances the retraction forces of the lung in order to avoid end-expiratory alveolar collapse. When surfactant is decreased or inactivated, the balance is upset and atelectasis ensues. In this situation, there is loss of gas volume based on mechanical forces within the alveolus as opposed to external compression. This form of atelectasis can occur in the neonatal infant in the setting of immature type II alveolar cells and decreased surfactant production. Alternatively, certain adult disease states, including ventilator-associated pneumonia and acute respiratory distress syndrome (ARDS), are associated with decreases in absolute surfactant levels or its activity.
Resorptive Atelectasis

Passive atelectasis results from a space-occupying lesion within the pleural space or the parenchyma

Resorptive atelectasis results when there is partial or complete occlusion of ow of gas between alveoli

216 ATELECTASIS

Pneumothorax

Pleural space

Normal alveolus
Figure 1 Normal alveolus and passive atelectasis.

Passive atelectasis

Edema and decreased surfactant

Normal alveolus
Figure 2 Normal alveolus and adhesive atelectasis.

Adhesive atelectasis

and the trachea (Figure 3). As the section of obstructed lung continues to be perfused, the partial pressure of oxygen in the alveolus equilibrates with that in the alveolar capillary. The loss of oxygen in the alveolus leads to increased concentrations of nitrogen and carbon dioxide in the alveolus, and subsequent gradients result in movement of both gases from the alveolus into the capillary. This process will continue until all gas has been removed from the airspace, a phenomenon that takes 1824 h in normal volunteers with a completely occluded lobe. Resorptive atelectasis can result from any cause of obstruction to airow. Common etiologies include

tumor, mucus plug, and foreign body. The process of resorptive atelectasis is thought to occur more quickly in the setting of oxygen-rich gas because the rst step of oxygen absorption is more rapid and there is a larger portion of gas that is absorbed initially.

Atelectasis in Normal Lung Function

By definition, atelectasis is caused by loss of gas from a normally gas-lled section of lung. However, there is a form of passive atelectasis that is commonly seen in normal lungs that is referred to as dependent atelectasis. This is the loss of volume in alveoli and

ATELECTASIS 217

Tumor

Normal alveolus
Figure 3 Normal alveolus and resorptive atelectasis.

Resorptive atelectasis

small airways in the lower lung zones based on gravity-related decreases in transpulmonary pressures. Although these areas of atelectasis may have physiologic consequences in patients with underlying lung disease (e.g., ARDS), the small loss of volume most likely has no physiologic consequence in normal subjects. The increased use of computed tomography (CT) has led to a greater awareness of these changes. Dependent atelectasis will resolve with position change, as demonstrated by repeated tomography with the patient in the prone position.

Atelectasis in Respiratory Diseases

Passive Atelectasis

reserved for collapse of lung associated with pleural thickening, most commonly found in association with asbestos exposure. The etiology of rounded atelectasis is not well understood but is thought to result from pleural brosis contracting and causing adjacent lung to curl upon itself and collapse. The radiographic ndings are best characterized on chest tomography. The lesions are classically peripheral, subpleural, uniform in density, and mass-like in appearance. They often have a curvilinear opacity of bronchi and vessels (termed a comet tail) extending toward the hilum. Care needs to be taken in diagnosing a lung nodule as rounded atelectasis purely on CT ndings because there are many reports of malignancy masquerading as rounded atelectasis.
Adhesive Atelectasis

Passive atelectasis can occur in a generalized or localized manner. In the setting of a large pneumothorax, the majority of the parenchyma of the ipsilateral lung will undergo passive atelectasis. In contrast, lung tissue adjacent to a bleb or a parenchymal cyst can collapse in a more localized form of passive atelectasis. Both examples illustrate a space-occupying phenomenon that causes subsequent loss of gas and collapse. With relief of a large space-occupying lesion, such as a pneumothorax, the underlying lung may develop reexpansion pulmonary edema. This process is thought to result from changes in pulmonary capillary permeability but is not well understood. The pulmonary edema is usually transient and resolves with supportive care. There is a unique form of passive atelectasis termed rounded atelectasis. This description is

The classic example of absorptive atelectasis is that of respiratory distress syndrome (RDS) of the neonate. As discussed previously, type II alveolar cells are often immature and not fully functional in the preterm infant, predisposing the infant to RDS. There have been significant decreases in morbidity and mortality in RDS with the initiation of surfactant (natural or synthetic) replacement in the immediate neonatal period. This improvement is in part due to the marked decrease in atelectasis and resulting improved gas exchange with surfactant therapy. Many studies demonstrate decreased surfactant levels in adults with ARDS. There is also experimental evidence for decreased surfactant activity in the setting of leakage of plasma proteins into the alveolar space. At the same time, studies using CT have

218 ATELECTASIS

demonstrated areas of atelectasis in ARDS. The etiology of this atelectasis is likely multifactorial. There are increased changes in dependent lung zones suggesting a component of passive atelectasis accompanying the adhesive atelectasis, the latter presumably from decreased quantity or activity of surfactant. Some believe that the recurrent opening and closing of small, atelectatic lung units contributes to ventilator-induced lung injury (termed atelectrauma). In response to this, there has been increasing research on how to avoid or overcome the atelectasis associated with ARDS. Efforts to replace surfactant through a variety of modalities have not demonstrated a mortality benet, although some studies have shown improved gas exchange. Lung ventilation strategies aimed at maintaining an open lung, such as recruitment maneuvers, high-frequency jet ventilation, and conventional ventilation with high positive end expiratory pressure (PEEP), have shown transient rises in oxygenation but no improvement in mortality. There is no conclusive evidence supporting any specic treatment of atelectasis associated with ARDS.
Resorptive Atelectasis

Endobronchial tumors are a common cause of resorptive atelectasis, often resulting in segmental or lobar collapse. One retrospective review reported an incidence of atelectasis in one of ve patients with small cell lung cancer. Because these are gradual processes occurring over weeks to months, the atelectatic lung often does not completely reexpand or function normally if the obstruction is relieved. However, because there is a risk for postobstructive pneumonitis and there is evidence for increased bacterial growth in atelectatic lung, clinicians will often attempt to minimize the obstruction. In contrast to the more gradual development of atelectasis with tumor growth, resorptive atelectasis can occur rapidly with acute occlusions of large airways. Mucus plugs, in both patients with asthma and those with altered secretion clearance, have been described as causes of resorptive atelectasis. Resorptive atelectasis has also been reported with malpositioned endotracheal tubes that selectively intubate the right lung. In a short period of time, the entire left lung can undergo atelectasis that will resolve with repositioning of the endotracheal tube.
Atelectasis Associated with General Anesthesia

by CT) and is often clinically significant. Lobar atelectasis can result in parenchymal shunt and be manifest as marked hypoxemia. There is evidence of a positive correlation between the amount of atelectatic lung and the degree of hypoxemia. The atelectasis associated with anesthesia is caused by multiple mechanisms. The rst contributor is dependent atelectasis (a form of passive atelectasis) from prolonged recumbent positioning with exaggerated effects of gravity. There is also evidence of changes in diaphragm position and shape with supine positioning for anesthesia contributing to dependent atelectasis. These effects are more pronounced when neuromuscular blockade is used in conjunction with anesthesia. All these changes are especially pronounced in morbidly obese patients. Some anesthesiologists advocate for intermittent use of large tidal volume breaths to overcome atelectasis intraoperatively, whereas others advocate for elevated levels of PEEP with ventilation. Administration of supplemental oxygen as part of mechanical ventilation contributes to resorptive atelectasis. Results are mixed with regard to whether higher inspired fractions (80100%) during induction and maintenance of anesthesia increase the likelihood of atelectasis compared to lower oxygen/ nitrogen mixtures. There is also evidence of early airway closure during anesthesia contributing to the resorptive mechanism of atelectasis. It has been suggested that repeated episodes of atelectasis, from the previously mentioned means, can also lead to impaired surfactant function resulting in a component of adhesive atelectasis as well. The issues of atelectasis often persist into the postoperative period, resulting in persistent hypoxemia. The resorptive atelectasis associated with higher inspired fractions of oxygen persists after extubation despite attempts at recruitment at the end of the operation. Dependent atelectasis, which in the normal subject will resolve with changes in position and deep breathing, often persists due to decreased mobility, pain, and sedation in the postoperative period. These conditions also commonly lead to impaired clearance of secretions predisposing to obstruction of airways and resorptive atelectasis. Deep breathing, incentive spirometry, vibration beds, chest physiotherapy, noninvasive ventilation, and therapeutic bronchoscopy all seem to have similar results in terms of resolution of postoperative atelectasis.
See also: Alveolar Surface Mechanics. Breathing: Breathing in the Newborn. Diffusion of Gases. Lung Imaging. Mucus. Oxygen Therapy. Peripheral Gas Exchange. Physiotherapy. Signs of Respiratory

Atelectasis associated with general anesthesia deserves special mention because it occurs at rates as high as 90% of anesthetized patients (as detected

AUTOANTIBODIES 219 Disease: Lung Sounds. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.
Kreider ME and Lipson DA (2003) Bronchoscopy for atelectasis in the ICU: a case report and review of the literature. Chest 124(1): 344350. Magnusson L and Spahn DR (2003) New concepts of atelectasis during general anaesthesia. British Journal of Anaesthesia 91(1): 6172. Peroni DG and Boner AL (2000) Atelectasis: mechanisms, diagnosis and management. Paediatric Respiratory Reviews 1(3): 274278. Rouby JJ, et al. (2003) Acute respiratory distress syndrome: lessons from computed tomography of the whole lung. Critical Care Medicine 31(4 supplement): S285S295. Spragg RG, et al. (2004) Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome. New England Journal of Medicine 351(9): 884 892. Vaaler AK, et al. (1997) Obstructive atelectasis in patients with small cell lung cancer. Incidence and response to treatment. Chest 111(1): 115120. Woodring JH and Reed JC (1996) Types and mechanisms of pulmonary atelectasis. Journal of Thoracic Imaging 11(2): 92108.

Further Reading
Brower RG, et al. (2003) Effects of recruitment maneuvers in patients with acute lung injury and acute respiratory distress syndrome ventilated with high positive end-expiratory pressure. Critical Care Medicine 31(11): 25922597. es Diagnosis of Fraser RS and Para e PD (1999) Fraser and Para Diseases of the Chest, 4th edn. Philadelphia: Saunders. Gunther A, et al. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome. Respiration Research 2(6): 353364. Hallman M, Glumoff V, and Ramet M (2001) Surfactant in respiratory distress syndrome and lung injury. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 129(1): 287294. Hedenstierna G and Rothen HU (2000) Atelectasis formation during anesthesia: causes and measures to prevent it. Journal of Clinical Monitoring and Computing 16(56): 329335.

AUTOANTIBODIES
O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Autoimmunity represents an immune response directed against self-antigens, which may be (articially) separated into T-cell and B-cell responses. The importance of the B cells in autoimmunity is correlated to the production of several autoantibodies, which have a role in diagnosis, pathogenesis, guiding therapy, and predicting outcome of the patients with these conditions. Among these autoantibodies, antineutrophil cytoplasm antibodies (ANCAs) have been distinguished as very important in several conditions, such as Wegeners granulomatosis, microscopic polyangiitis, Goodpastures syndrome, drug-related vasculitides, etc. We review in this article the importance of different classes of autoantibodies and their relationship to underlying disorders.

Introduction: Autoimmunity and Lung Disorders

The phenomenon of autoimmunity has been the object of exploration for over a century. Recent technical and methodological advances in the study of cellular and biochemical processes of autoimmunity have led to a publication big bang. Nevertheless, the etiopathogenesis of most autoimmune disorders remains, to date, largely unknown. In this article, we discuss the basic mechanisms leading to autoimmunity, as we understand them today, and then the

relevance of several classes of autoantibodies in various lung conditions. An autoimmune disorder is a pathologic condition caused by an autoimmune response, which is critically dependent upon antigen quality, concentration, and persistence, and the magnitude of the interaction between self and foreign components. The autoimmune response leads to end-organ organ-damage through cellular or humoral mechanisms. The humoral response is represented by antibody-mediated injury, which could be differentiated in direct, cytotoxic antibody damage or immune complex-related damage. Despite their diverse etiology, there are several pathogenetic mechanisms which are common to all autoimmune conditions. With few exceptions, they require the presence of self-reactive CD4 positive lymphocytes. The immune system is naturally endowed with myriad mechanisms responsible for recognition of and defense against foreign assaults. Direct and rapid responses can be mediated by a set of germline-encoded receptors, called Toll-like receptors (TLRs), which recognize specifically the molecular determinants of various pathogens. Activation of the innate immune defense mechanisms leads to a response from different cell types (local dentritic cells, natural killer lymphocytes, neutrophils, monocytes, macrophages, basophils, eosinophils, and mastocytes), ranging from production of chemokines, cytokines, adhesion molecules, antimicrobial, pro- and antiapoptoic factors. This response is reproducible (acts

220 AUTOANTIBODIES

by the same intensity at each antigenic exposure), is not antigen specic, and creates a pool of memory cells for long-term immunity. The adaptive immunity requires contributions from cells whose receptors are generated by VDJ recombination, the T and B lymphocytes. Furthermore, inducible expression of TLRs in B cells may provide a link between the innate and adaptive branches of the immune system. Genetic susceptibility to autoimmune diseases may occur at several levels: the major histocompatibility complex (MHC) haplotype and polymorphisms of genes involved in establishing self-tolerance and immune regulation, for example, the autoimmune regulator (AIRE), the T-cell immunoglobulin and mucin-domain-containing (TIM) family, and cytolytic T-lymphocyte-associated antigen 4 (CTLA-4). There are several major pathways that can initiate or modulate autoimmunity: 1. molecular mimicry of self-proteins by viral agents can activate specic T cells that attack both the specic virus and the host; 2. bacterial infections can cause intense inammation, with secondary polyclonal activation of bystander T cells, which will act in concert in intensifying the local injury; 3. epitope exposure and maintenance in the local milieu as a result of self local damage may perpetuate the immune response; and 4. drug metabolism or infection can cause the formation of proteins which are seen as foreign and thus result in a neo-antigen-specic response to the modied self. There is also a range of possible posttranslational modications (PTMs) of proteins that can allow immune recognition of neo-self epitopes. The most direct way the posttranslational changes can inuence T-cell reactivity is to generate a new peptideMHC (pMHC) complex that can stimulate stronger binding to T lymphocytes through T-cell receptors. In general, the immune recognition can be modied by posttranslational changes of the antigens in a number of ways: 1. Different additions (glycosylation, methylation, phosphorilation, etc.), as in collagen-induced arthritis. 2. Enzymatic or spontaneous conversions (deamidation or citrullination) as in celiac disease or rheumatoid arthritis (RA). In the latter, multiple autoantibodies have been described: antiperinuclear factor, antilaggrin, and antikeratin antibodies. The target antigens are the result of PTM, namely deimination of the natural aminoacid

arginine to the amino acid citrulline by the activity of peptidylarginine deiminase (PAD). This discovery led to the development of a new serologic test for RA, that is, antibody against cyclic citrullinated peptide (CCP), which has a high specicity (497%) for the disease. Anti-CCP binding also seems predictive of progression to RA in recent-onset arthritis, or retrospectively in blood samples positive for rheumatoid factor (RF), donated years before onset of symptoms of the disease. There is also evidence that citrullination may play a role in T-cell autoreactivity in RA by enhancing the strength of the bonds to MHC II molecules. Therefore, expression of PAD4 and citrullinated proteins, presence of anticitrulline antibody-secreting plasma cells in the inamed synovium, the strong MHCpeptide bonds, and the predictive strength of anti-CCP testing, all prove that this autoimmune cascade is involved in the progression of RA. 3. Extracellular modications by proteolysis or antibody binding. The current understanding is that differential PTMs might therefore provide the means of provoking a local autoaggressive immune response as a consequence of an infection. This would be an alternative explanation of the autoimmunity by self-mimicking microbial antigens, for which denitive proof remains elusive in humans as yet.

ANCAs and Associated Lung Disorders

In 1982, antineutrophil cytoplasm antibodies (ANCAs) were rst described in patients with pauci-immune glomerulonephritis. By 1985, ANCA had already been linked to Wegeners granulomatosis (WG); within a few more years, a link with microscopic polyangiitis (MP), and renal-limited vasculitis has been made. As of today, ANCAs have become very important factors in the diagnosis, pathogenesis, and classication of vasculitides. ANCAs can be determined in two different ways: indirect immunouorescence assay (IIA) and enzyme-linked immunosorbent assay (ELISA). While IIA is more sensitive than ELISA, the latter is clearly more specic; hence, in clinical testing for ANCA it is recommended to screen with IIA, and to conrm all positive results with ELISA directed against the specic antigens, if possible in a reference laboratory. Furthermore, and unfortunately, there is a significant subjective component to the interpretation of the immunouorescence tests, so conrmation by other tests is needed before releasing the denitive positive test. The main target antigens for ANCAs are

AUTOANTIBODIES 221

proteinase 3 or PR3 (PR3ANCA) and myeloperoxidase or MPO (MPOANCA). PR3 and MPO are located in the azurophilic granules of neutrophils, and the peroxidase-positive lysosomes of monocytes, respectively. Other azurophilic granule proteins can cause p-ANCA autoantibodies: lactoferrin, elastase, cathepsin G, bactericidal permeability inhibitor, catalase, lysozyme, azurocidin, beta-glucuronidase, etc.
Immunouorescence Assay

specimens, because formalin-xed neutrophils do not cluster charged cellular components around the nucleus. Mixed or atypical pattern, seen mostly in patients with other immune-mediated conditions than systemic vasculitis (e.g., connective tissue disorders, inammatory bowel disease, and autoimmune hepatitis). Such patterns may be confused with p-ANCA patterns.

When the sera of patients with ANCA-associated vasculitis (AAV) or other conditions are incubated with ethanol-xed human neutrophils, three major IIA patterns are observed (Figure 1):
*

Enzyme-Linked Immunosorbent Assay

c-ANCA pattern (cytoplasmic), with diffuse staining of the cytoplasm. In most cases, the responsible antibodies are PR3ANCA in the setting of WG; rarely, MPOANCA can present in the cytolasmic, uniform immunouorescence. p-ANCA pattern (perinuclear), which results from staining around the nucleus, which in fact is xation artifact when ethanol is used. With ethanol xation, positively charged granule constituents cluster around the negatively charged nuclear membrane, leading to perinuclear uorescence. The usual antibody responsible for this pattern is MPO ANCA (rarely PR3ANCA), generally in the setting of MP. A positive p-ANCA immunouorescence staining pattern may also be detected in a wide variety of inammatory illnesses; it has a low specicity for vasculitis. The p-ANCA pattern on IIA can be similar to that caused by antinuclear antibodies (ANAs), the reason why individuals with ANA frequently have false-positive results on ANCA testing by immunouorescence. Rigorous IIA testing procedures for ANCA (especially when a diagnosis of vasculitis is entertained) entails the use of both formalin- and ethanol-xed neutrophil

Specic ELISA kits for antibodies directed against PR3, MPO, and other azurophilic granule components are commercially available. PR3ANCA and MPOANCA should be part of any standardized approach to the testing for ANCA; they are associated with substantially higher specicities and positive predictive values than their corresponding IIA patterns (c- and p-ANCA, respectively). Antineutrophil cytoplasm antibodies are mainly associated with Wegeners granulomatosis, microscopic polyangiitis, ChurgStrauss syndrome (CSS), renal-limited vasculitis (pauci-immune glomerulonephritis without evidence of extrarenal disease), and drug-induced vasculitides. In these conditions, there is either PR3ANCA or MPOANCA, but almost never both. ANCA with different antigen specicities may be detected in various other rheumatologic and gastrointestinal disorders.
Wegeners Granulomatosis

The histopathologic hallmark of WG is necrotizing granulomatous inammation that involves the respiratory tract, kidneys, skin, and/or the joints. Nearly 90% of patients with generalized, active WG are ANCA-positive, while in limited disease (restricted to upper or lower respiratory tract disease and no renal involvement) only 4060% of patients may be ANCA-positive. Thus, the absence of ANCA does

Figure 1 Indirect immunouorescence assay patterns on ethanol-xed human neutrophils. (a) Diffuse, cytoplasmic cANCA; (b) perinuclear pANCA; (c) antinuclear (ANA) illustrated as control.

222 AUTOANTIBODIES

not exclude the diagnosis; among WG patients with ANCA positivity, 8095% have PR3ANCA by ELISA, the rest nearly always have MPOANCA. The diagnostic performance of PR3ANCA for WG (positive and negative predictive values) is related mainly to disease prevalence in the population searched, and the disease activity. Persistently, high or rising titers of ANCAs are often associated with disease relapses. However, this association may not occur in 1030% or more of those with such ANCA proles during one or more years of follow-up.
Microscopic Polyangiitis

Approximately 70% of patients with MP are ANCA positive. Most ANCA-positive MP patients have MPOANCA, with a minority having PR3ANCA. ANCA serologies are useful in distinguishing MP from classic polyarteritis nodosa (PAN), a vasculitis of medium-sized muscular arteries. In general, PAN is associated with neither PR3ANCA, nor MPO ANCA. Since PR3ANCA or MPOANCA may occur in both WG and MP, it is important to know that these diseases cannot be distinguished solely on ANCA tests. However, distinction between MP and WG is not clinically that important, since their treatment and prognosis are similar.
ChurgStrauss Syndrome

positive. One study, for example, found that 38 of 100 sera with anti-GBM antibodies also had ANCA; of these, 25 had MPOANCA, 12 had PR3ANCA and 1 both types of ANCAs. Almost all such patients have both ANCAs and anti-GBM antibodies at the rst serum examination. The clinical signicance of combined ANCA and anti-GBM serologies is unclear. In some, the titers of ANCAs are low and there are no clinical manifestations of vasculitis. In others, however, there are disease features of anti-GBM antibody disease, but quite typical of systemic vasculitis, including purpura, arthralgias, and granulomatous inammation, suggesting the concurrence of two disease processes. The incriminated self-antigen for the anti-GBM antibodies is the same as in patients with anti-GBM antibody disease alone, suggesting that the inciting epitopes are the same. Alternatively, the production of ANCA could precede that of anti-GBM antibodies, with pulmonary or renal damage caused by ANCA leading to secondary anti-GBM antibody formation.
Other Rheumatologic Disorders

Both PR3ANCA and MPOANCA have been detected in patients with CSS; overall, 50% of CSS patients are ANCA positive, whereas in those with active, untreated disease the percentage is even higher. So far, no identication has been made of any consistent clinical differences with therapeutic or prognostic implications between ANCA-positive CSS and ANCA-negative CSS.
Renal-Limited Vasculitis

ANCAs have been reported in virtually all rheumatic diseases, including RA, systemic lupus erythematosus (SLE), Sjogrens syndrome (SS), scleroderma, inammatory myopathies, dermatomyositis, relapsing polychondritis, and the antiphospholipid syndrome. In most cases, the IIA pattern is p-ANCA. Many reports of ANCAs in these diseases preceded the era of reliable ELISA assays for PR3 and MPOANCA, and used only IIA. Various target antigens have been described in these disorders, such as lactoferrin, elastase, lysozyme, cathepsin G, and others. In other cases, the specic target antigens have not been identied yet.
Cystic Fibrosis

Pauci-immune vasculitis limited to the kidney is characterized by focal and segmental glomerular inammation and necrosis with little or no deposition of immunoreactants (IgG, IgA, IgM, and complement fractions). Almost all patients are ANCA positive, and up to 80% of them have MPOANCA. Some consider this disorder as part of the WG/MP spectrum because the renal histologic ndings are indistinguishable, and because some patients with renal-limited vasculitis eventually develop extrarenal manifestations of either WG or MP.
Anti-GBM Antibody Disease (Goodpastures Syndrome)

Non-MPO p-ANCAs are common in patients with cystic brosis (CF), particularly among those with bacterial airway infections. The ANCA is generally directed against BPI (bactericidal/permeability-increasing) protein. In one series of 66 patients with CF, BPI-IgG and BPI-IgA ANCAs were found in 91% and 83%, respectively. Anti-BPI titers were directly related to the severity of airway destruction. It is unclear whether this relationship represents an epiphenomenon or a response to overwhelming infection, with major release of endotoxins.
Others

Up to 40% of patients with antiglomerular basement membrane (anti-GBM), antibody disease are ANCA

ANCA has also been observed in isolated patients with autoimmune hepatitis, Bu rgers disease,

AUTOANTIBODIES 223

(pre)eclampsia, subacute bacterial endocarditis, leprosy, malaria, and chronic graft-versus-host disease. ANCA positivity is seen in 6080% of patients with ulcerative colitis and in primary sclerosing cholangitis. It can be observed in only 1027% of patients with Crohns disease, in whom only low titers are present. The p-ANCA is the predominant appearance, and is directed against a myeloid cell-specic 50 kDa nuclear envelope protein. Other reported antigens include BPI, lactoferrin, cathepsin G, elastase, lysozyme, and PR3. The pathogenetic significance of these antibodies is unclear. The titers of ANCAs do not vary with the activity or severity of the disease and, in ulcerative colitis, do not fall even after colectomy.
Drug-Induced ANCA-Associated Vasculitis

neutrophils and then it binds to MPO altering its structure, which could lead to ANCA production in susceptible individuals. Discontinuation of the offending drug may be the only intervention necessary in mild cases. Some patients, however, require high doses of corticosteroids and even cyclophosphamide, while others require maintenance therapy. ANCA titers usually persist at low levels, even after active vasculitis goes into resolution, which points out that previous ingestion of PTU is an important piece of information in the history of patients with other pathologies and prior PTU-induced, by-stander ANCA.
Hydralazine

Several medications can induce various forms of AAV. Most patients diagnosed with drug-induced AAV have high titers of MPOANCA. In addition, most have also antibodies to elastase or lactoferrin. Relatively few have PR3ANCA positivity. Many cases of drug-induced AAV are associated with constitutional symptoms, arthralgias/arthritis, and cutaneous vasculitis. However, the full range of clinical features associated with ANCAs, including crescentic glomerulonephritis and alveolar hemorrhage, can also occur. Discontinuation of the offending agent may be the only intervention necessary for mild cases of AAV induced by medications; such cases have in general only constitutional symptoms, arthralgias/arthritis, and/or cutaneous vasculitis. Some patients, however, require high doses of corticosteroids and even cyclophosphamide because of more severe manifestations. The most renowned medications capable of causing AAV are propylthiouracil (PTU), carbimazole, thiamazole, hydralazine, procainamide, minocycline, penicillamine, allopurinol, phenytoin, and clozapine. Drug-induced AAV is quite rare; consequently, the above medications should not be incriminated until other etiologies have been thoroughly excluded.
Propylthiouracil

Hydralazine may cause drug-induced lupus and drug-induced AAV. Unlike hydralazine-induced lupus syndrome, hydralazine-induced AAV is frequently associated with a pauci-immune glomerulonephritis, antibodies to double stranded DNA (dsDNA), high titers of MPOANCA, and is a more serious condition.
Minocycline

Minocycline had produced fever, livedo reticularis, arthritis, and ANCA in a seven-patient case series. The p-ANCA IIA pattern associated with this disorder is usually directed against minor antigens such as cathepsin G, elastase, and bactericidal/permeability increasing (BPI) protein, rather than against major antigens, such as MPO. Symptoms typically resolve after minocycline discontinuation, and recur with drug rechallenge. Some patients require treatment with corticosteroids for short periods of time. More serious manifestations of minocyclineinduced AAV are crescentic glomerulonephritis, lupus-like syndrome, and cutaneous classic PAN.
Pathogenesis of ANCA-Associated Disorders

PTU may be the most common offending agent causing drug-induced AAV. Generally, the medication is taken for long periods of time before the complication occurs (months to years). Vasculitis is a rare complication, whereas a much higher percentage develops serological evidence of ANCA. In a cross-sectional study, 27% of patients receiving long-term treatment with PTU developed MPOANCA. The postulated mechanism by which PTU leads to AAV stems from the observation that PTU accumulates in

Substantial evidence in animal models and human observations supports a significant pathogenetic role of ANCA in producing widespread tissue damage. The hypothesis is that antibodies produce a necrotizing vasculitis by inciting a respiratory burst with diffuse endothelial damage, chemotaxis, and degranulation of neutrophils and monocytes. If autoantibody (ANCA) generation is secondary to a cryptic epitope exposure or a primary event, it is still unclear. After a cryptic antigen exposure, the epitope spread may occur, leading to a more generalized, systemic reaction (Figure 2). The number of activated B lymphocytes seems to correlate with the activity score of the disease (e.g., Birminham vasculitis score), which supports the hypothesis that B lymphocytes

224 AUTOANTIBODIES
AAT Insult ANCA PR3 or MPO Macrophage MMP-12 Neutrophil

TNFIL-8 Monocyte

Endothelial cell
Figure 2 A simplied model of ANCA pathogenesis and neutrophil/monocyte priming. Of note, alpha-1 antitrypsin (AAT) is a natural tissue inhibitor of proteinase 3 (PR3), myeloperoxidase (MPO), or other lysosomal enzymes. In addition, binding ANCA to the cell surface is mediated by specic FcgR receptors for immunoglobulins. IL-8, interleukin-8; MMP-12, matrix metalloproteinase 12; TNF-a, tumor necrosis factor alpha. Copyright (2005) from Journal of COPD by Ioachimescu O and Stoller J. Reproduced by permission of Taylor & Francis Group, LLC., http://www.taylorandfrancis.com.

are involved in the disease pathogenesis. Furthermore, while in normal conditions MPO and PR3 mRNA transcripts are found almost exclusively in early promyelocytes, it was noted that in AAV disorders (and not in SLE or other conditions), both MPO and PR3 mRNA are found in high concentrations in the peripheral neutrophils, and this correlates well with neutrophil total number and disease activity, but not with ANCA titer, left shift, or cytokine levels, including tumor necrosis factor alpha (TNF-a). Cell membrane PR3 and MPO expression has been found in the affected glomeruli of the ANCA-associated diseases (e.g., WG, MP), but not in normal glomeruli, suggesting that local factors also play an important role in gauging the organ involvement. In CSS, it is still unclear what the eosinophils contribution to the disease pathogenesis is.

Table 1 The main pathogenic autoantibodies in different lung conditions Antibody ANCA ANA Anti-Sm, Anti-dsDNA Anti-U1-RNP RF CCP Anticentromere Anti-Scl Anti-Jo1 Cryoglobulins Anti-GM-CSF Lung disease WG, MP, CSS, drug-related AAV SLE, SS, scleroderma, RA, MCTD, UCTD SLE MCTD RA, SLE, MCTD, UCTD RA CREST syndrome Scleroderma DM/PM ILD Essential and secondary cryoglobulinemia PAP

Autoantibodies in Connective Tissue Disorders

Laboratory screening is commonly used for evaluation of connective tissue disorders (CTDs), although there are rare tests which are sensitive or specic enough to establish the diagnosis. For example, Westergren sedimentation rate and C-reactive protein are commonly elevated in infections, malignant, or inammatory conditions; therefore, a high value does not add too much towards the diagnosis, while a low titer may make an active CTD seem unlikely. Table 1 illustrates a synopsis of autoantibodies found in different immune conditions.

ANAs are found positive in most of CTD patients, with different frequencies (from 30% in RA, to 95% in SLE and scleroderma). On the other hand, other lung conditions such as idiopathic pulmonary brosis and coal workers pneumoconiosis may have positive ANAs in titers above 1:40 in up to 33% of the cases, as opposed to 23% of the general population. An extractable nuclear antigen (ENA) panel is available in most of the reference laboratories, which will guide the testing against common antigens if ANA is positive. Anti-dsDNA and anti-Smith (Sm) antibodies are relatively specic for SLE: 9597%, and 5099%, respectively. While anti-dsDNA antibodies seem to correlate with the disease activity, anti-Sm antibodies tend to persist after normalization of anti-dsDNA titers. An interstitial lung disease (ILD)

AUTOANTIBODIES 225

identical to usual interstitial pneumonia (UIP) or non-specic interstitial pneumonia (NSIP) can be seen in SLE, RA, scleroderma, or dermatomyositis, while bronchiolitis obliterans with organizing pneumonia (BOOP) can be frequently seen in RA. In SLE, the most frequent pulmonary complications are pleurisy and pleuritis, lupus pneumonitis, shrinking lung syndrome, bacterial pneumonia, diffuse alveolar hemorrhage (DAH), thromboembolic disease, and pulmonary arterial hypertension. Traditionally, RF is found with higher frequency in patients with RA and long-standing disease, with multiple extra-articular manifestations. In other lung conditions, such as hypersensitivity pneumonitis (bird fanciers lung) or idiopathic pulmonary brosis (IPF), RF can be positive in up to half of the cases. Since part of the workup for interstitial lung disorders is exclusion of associated rheumatic conditions, reliance on more specic testing is generally required to diagnose a systemic disease like RA. This can be achieved by anticyclic citrullinated peptide (CCP) antibodies, which have a much higher specicity, of 9198%. In RA, the lung involvement can take the form of ILD (UIP, NSIP, BOOP), DAH, pleuritis and pleural effusion, rheumatoid nodules (follicular, constrictive, or obliterative) bronchiolitis, cricoarythenoid arthritis with upper airway obstruction or drug-induced pneumonitis (methotrexate, gold, cyclophosphamide, rituximab etc.). In the workup of disorders associated with myopathy or myositis, the determination of muscle creatinphosphokinase (or aldolase) is important. Polymyositis (PM) and amyotrophic dermatomyositis (ADM) can both present with lung disease in more than 65% of cases, as one study found at the time of diagnosis; it is important to know that, contrary to rheumatoid lung disease, in PM or ADM, the pulmonary involvement may be inaugural. In DM/PM, ANA is positive in up to 30% of the patients; one in three patients will also have antibodies against an ENA called histidyl tRNA synthetase, or Jo-1 antibodies. Other antiaminoacyl tRNA synthetases have been identied to date: anti-PL7 or anti-treonyl tRNA synthetase, anti-PL12 or anti-alanyl tRNA synthetase, anti-OJ or anti-isoleucyl tRNA synthetase, anti-EJ or anti-glycyl tRNA synthetase, anti-KS or anti-asparaginyl tRNA synthetase, and anti-Wa directed against a 48 kDa protein yet uncharacterized, but known to be bound to acetylated tRNA. It has been noted that patients with anti-synthetase syndrome (arthritis, Raynauds syndrome, mechanics hands, and anti-Jo-1 antibodies) have a much higher incidence of pulmonary disease. Pathologic pictures of NSIP, UIP, or BOOP can be seen in these settings.

The interstitial lung disease in scleroderma can occur with both limited and diffuse disease. ANA is generally positive in both forms of the disease, anticentromere or anti-TO/TH antibodies found in limited disease, while anti-Scl antibodies are more often found in the diffuse variants of scleroderma. The cellular and brotic form of NSIP can together account for 70% of patients with scleroderma, suggesting a high rate of pulmonary involvement. Sjogrens syndrome is an autoimmune exocrinopathy and disorder of the epithelia characterized by lymphocytic inltration of the glandular and nonglandular subepithelial tissue. It presents with xerostomia (dry mouth), xerophtalmia (dry eyes) and keratoconjunctivitis, xerotrachea (dry trachea, presenting as dry cough and frequent infections), and arthritis. The pulmonary involvement in SS manifests as lymphocytic bronchitis, lymphocytic follicular bronchiolitis, bronchial-associated lymphoid tissue lymphoma, lymphocytic interstitial pneumonia (LIP) or cystic lung disease. The serologic markers of this condition (either primary or secondary SS) are represented by SS-A (anti-Ro) and SS-B (anti-La) antibodies. Mixed connective tissue disorder (MCTD) is an overlap syndrome with features of RA, SLE, scleroderma, and DM/PM, which do not meet the criteria for the individual disorders, in the presence of antiribonucleoprotein (anti-RNP) antibodies on ENA testing, directed against U1 RNP, which is rich in uridine. Pulmonary involvement is common in patients with MCTD, and has feature of UIP or NSIP, with significant septal thickening, less ground-glass attenuation and honeycombing on CT scans. Incomplete rheumatic conditions can occur and are usually called undifferentiated connective tissue disorder (UCTD) and may involve the respiratory tract in a fashion similar to MCTD, although without any evidence of anti-RNP antibodies. In cryoglobulinemia, a condition characterized by cryoglobulins in the serum, there is a systemic inammatory response with involvement of small and medium-size vessels, and generated by cryoglobulincontaining immune responses. By definition, cryoprecipitation is a phenomenon of protein precipitation at temperatures lower than 371C, and it can be present when serum proteins or plasma proteins precipitate (cryobrinogens and cryoglobulins respectively). Cryoglobulins are a mixture of immunoglobulins and complement components, which generally precipitate upon refrigeration of the serum. Three types of cryoglobulinemic conditions have been described (Brouets classication): type I, characterized by isolated monoclonal immunoglobulins (Ig) and seen mostly in hematologic conditions; type II with polyclonal Ig seen in hepatitis C, HIV

226 AUTOANTIBODIES

and other viral conditions; and type III, with mixed cryoglobulins, encountered in CTDs. Pulmonary involvement may be commonly seen in type III cryoglobulinemia and, rarely, in type I, and is mostly subclinical. In up to 50% of cases, there may be cough, pleuritic chest pain, and/or dyspnea. Pulmonary function tests usually show evidence of reactive small airway disease, and occasionally a decreased diffusing lung capacity for CO. Pulmonary vasculitis, DAH, or BOOP can also be found (though rarely). Another rare condition is hypocomplementemic urticarial vasculitis syndrome, which presents with urticarial vasculitis, arthritis, glomerulonephritis, and obstructive lung disease; it is thought to be caused by serum IgG against C1q fraction of the complement, which will decrease significantly (similar to SLE, but more commonly, with angioedema and eye inammation).

PAP. To what degree a high end-titer of anti-GM-CSF represents an indication to treat more aggressively, or with a larger dose of GM-CSF, remains to be proven.

Conclusions
During the past century, much has been accomplished in dening, understanding, and treatment of autoimmune conditions, yet more is to be learned. Autoantibodies play an important role in these immune processes, and of particular importance are ANCAs, involved in the pathogenesis of the so-called ANCA-associated vasculitides. The importance of the autoantibodies stems not only from their contribution to the diagnosis of different conditions, but also from their role in pathogenesis and the importance in monitoring the disease progression.
See also: Cryoglobulinemia. Cystic Fibrosis: Overview. Granulomatosis: Wegeners Disease. Interstitial Lung Disease: Overview. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpastures Syndrome. Toll-Like Receptors. Tumors, Malignant: Overview. Vasculitis: Overview.

Autoantibodies in Other Conditions

Lung Cancer

The antibodies responsible for the paraneoplastic syndromes are discussed in articles Tumors, Malignant: Overview; Bronchogenic Carcinoma.
Sarcoidosis

Further Reading
Boneld TL, Russell D, Burgess S, et al. (2002) Autoantibodies against granulocyte macrophage colony-stimulating factor are diagnostic for pulmonary alveolar proteinosis. American Journal of Respiratory Cell and Molecular Biology 27: 481486. Csernok E (2003) Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides. Autoimmunity Reviews 2: 158164. Imbert-Masseau A, Hamidou M, Agard C, Grolleau JY, and Cherin P (2003) Antisynthetase syndrome. Joint, Bone, Spine 70: 161168. Ioachimescu O and Stoller J (2005) A review of alpha-1 antitrypsin deciency. Journal of COPD 2(2): 263275. Mahadeva R, Dunn AC, Westerbeek RC, et al. (1999) Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/ permeability-increasing protein (BPI) and cystic brosis lung disease. Clinical and Experimental Immunology 117: 561567. Miescher PA, Zavota L, Ossandon A, and Lagana B (2003) Autoimmune disorders: a concept of treatment based on mechanisms of disease. Springer Seminars in Immunopathology 25(supplement 1): S5S60. Paran D, Fireman E, and Elkayam O (2004) Pulmonary disease in systemic lupus erythematosus and the antiphospholpid syndrome. Autoimmunity Reviews 3: 7075. Quintana FJ and Cohen IR (2004) The natural autoantibody repertoire and autoimmune disease. Biomedicine & Pharmacotherapy 58: 276281. Schmitt WH (2004) Newer insights into the aetiology and pathogenesis of myeloperoxidase associated autoimmunity. Japanese Journal of Infectious Diseases 57: S7S8. Seo P and Stone JH (2004) The antineutrophil cytoplasmic antibody-associated vasculitides. American Journal of Medicine 117: 3950. Sharma OP (2002) Sarcoidosis and other autoimmune disorders. Current Opinion in Pulmonary Medicine 8: 452456.

A possible relationship between sarcoidosis and autoimmunity was described more than a century ago, although is still not accepted to be an autoimmune condition. Good preliminary results of the CD20 targeting in several autoimmune conditions (sarcoidosis, SLE, RA, type II cryoglobulinemia, neuropathies, WG, Goodpastures syndrome, etc.) have opened important avenues for research, conceivably capable of improving our understanding of the pathogenesis of these conditions and the effectiveness of pathogenesis-directed therapy.
Pulmonary Alveolar Proteinosis

Animal and human studies have conrmed a pivotal role played by granulocyte-macrophage colony-stimulating factor (GM-CSF) in pulmonary alveolar proteinosis (PAP) pathogenesis. A decreased GM-CSF pathway activity seems to be the common pathogenic pathway. It was shown that a neutralizing (or blocking) anti-GM-CSF IgG antibody can be found in bronchoalveolar lavage uid and sera of patients with idiopathic PAP. The sensitivity of the serum anti-GM-CSF assay is close to 100% and the specicity too is close to 100% when using a cutoff titer of 1:400. Furthermore, anti-GM-CSF antibodies are increasingly used as a diagnostic tool in

AUTOANTIBODIES 227
Strange C and Highland KB (2004) Interstitial lung disease in the patient who has connective tissue disease. Clinics in Chest Medicine 25: 549559. Uchida K, Nakata K, Trapnell BC, et al. (2004) High-afnity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 103: 10891098. Wiik A (2003) Autoantibodies in vasculitis. Arthritis Research & Therapy 5: 147152. Wisnieski JJ, Baer AN, Christensen J, et al. (1995) Hypocomplementemic urticarial vasculitis syndrome. Clinical and serologic ndings in 18 patients. Medicine (Baltimore) 74: 2441.

B
BASAL CELLS
M J Evans, University of California, Davis, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Despite differences in basal cell distribution, the unifying feature in all animal species is that the number of basal cells present is related to the height of the columnar epithelium. This relationship is associated

Abstract
Basal cells are an integral part of pulmonary airway epithelium. They exist as a separate layer of cells covering most of the basement membrane zone. In this central position, they can interact with columnar epithelium, neurons, the basement membrane zone, and underlying mesenchymal cells. In addition, they interact with inammatory cells, lymphocytes, and dendritic cells. The interactions with trafcking leukocytes and neurons take place in the lateral intercellular space between basal cells. In this central position basal cells become a very important part of the epithelialmesenchymal trophic unit of larger airways. Structurally, basal cells function in attachment of columnar epithelium with the basement membrane zone. Failure of attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. They also function in regulation of broblast growth factor-2 signaling from the basement membrane zone, neurogenic inammation, the inammatory response, transepithelial water movement, and oxidant defense of the tissue and formation of the lateral intercellular space for airway epithelium. A subpopulation of basal cells (parabasal cells) has the potential to function as progenitor cells. Clinically, basal cells are probably also involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma.

LIS LIS

LIS BC BC PCB

*
BC (a)

BMZ

Introduction
Basal cells are derived from undifferentiated columnar epithelium in the developing airway. They are characterized by their basal position in the columnar epithelium, the presence of hemidesmosomes (characterized by alpha 6 beta 4 integrins), cytokeratins 5 and 14, and the nuclear protein p63 (Figure 1(a)). The distribution of basal cells varies by airway level and animal species. Airways that are larger in diameter have more basal cells than airways with smaller diameters. For example, the largest numbers of basal cells are found in the trachea. As the airway decreases in diameter, the number of basal cells also decreases, and none are present in the terminal bronchioles. As basal cells increase in number, they displace columnar cells on the basement membrane zone (BMZ). In human airways, 9095% of the BMZ is covered by basal cells from the trachea to bronchioles 13 mm in diameter (Figure 1(b)).

(b)
Figure 1 (a) Electron micrograph of tracheal epithelium from a young rhesus monkey demonstrating basal cells (BC), parabasal cells (PCB), the lateral intercellular space (LIS), and the basement membrane zone (BMZ). The lateral intercellular space is the open area between cells (asterisks). The lateral intercellular space is reduced or absent between ciliated cells and ciliated cells next to secretory cells (arrowheads). Magnication 1500. Copyright 2001 from Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401415 by Evans MJ, Van Winkle LS, Fanucchi MV, et al. Reproduced by permission of Taylor & Francis, Inc. (b) Scanning electron micrograph of a sheep tracheal whole mount treated with EDTA. The columnar epithelium has been released leaving a layer of basal cells attached to the BMZ. The basal cells cover between 90% and 95% of the BMZ. Magnication 1100.

230 BASAL CELLS

with the role of basal cells in attachment of columnar epithelium to the BMZ. Functionally, large airway epithelium is stratied, with a layer of basal cells attached to the BMZ and a layer of columnar epithelium attached to the basal cells. Thus, basal cells act as a separate layer of cells in a central position, that can interact with columnar epithelium, neurons, BMZ, and the underlying mesenchymal cells. In addition, they can interact with inammatory cells, lymphocytes, and dendritic cells in the epithelium. These interactions take place in the lateral intercellular space. The lateral intercellular space is a distinct space between basal cells, basal and adjacent secretory cells, and secretory cells (Figure 1(a)). The lateral intercellular space is hydrated with the aid of the proteoglycan hyaluronan, which is bound to CD44 adhesion molecules on the surface of basal cells. In this central position, basal cells become a very important and integral part of the epithelialmesenchymal trophic unit of large airways. The large number of receptors found on basal cells that bind growth-regulating proteins and trafcking leukocytes (Table 1) supports this concept.

Table 1 Cellular and molecular characteristics of basal cells Cell surface characteristics Alkaline phosphatase Aquaporin 3 transmembrane water channels b-adrenergic receptors CD44 transmembrane glycoproteins Epidermal growth factor receptor Fibroblast growth factor receptor-1 Fas receptors and ligand ICAM-1 IgE receptor Integrins (a6b4) Lectins LEEP-CAM MRP transmembrane transporters MUC 1,4,8 Neurokinin-1 receptor Neutral endopeptidase Syndecan-4 4-1 BB receptor Intracellular characteristics Adrenomedullin receptor (mRNA) Autotaxin (mRNA) Annexin II Bcl-2 protein Extracellular SOD (mRNA) Leukemic inhibitory factor P63 nuclear protein Reproduced from Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401415.

Basal Cell Functions in the Normal Lung

Junctional Adhesion

The structural role of basal cells in the airways is for attachment of columnar epithelium to the BMZ. Epithelial cells are attached to the BMZ by hemidesmosomes and cell adhesion molecules. Cytokeratins 5 and 14 link anchoring junctions of basal cells with the cytoskeletons of adjacent cells through desmosomes and to the BMZ with hemidesmosomes. This arrangement of junctional adhesion provides mechanical stability to a group of cells or tissue. In airway epithelium, basal cells are the only cells that form hemidesmosome junctions with the BMZ. Columnar cells are attached to the BMZ via desmosome attachment with basal cells. The significance of basal cells in junctional adhesion can be demonstrated by treating the tissue with ethylenediaminetetraacetic acid (EDTA). Desmosome junctions are dependent on calcium. When the tissue is treated with EDTA, the calcium is removed from the desmosome, and the columnar epithelium is released leaving the basal cells attached to the BMZ (Figure 1(b)). The number of basal cells present at a particular airway level and their morphology is related to their role in junctional adhesion. When the columnar epithelium increases in height, there is an increase in the size and shape of basal cells along with a corresponding increase in desmosome attachment with the columnar epithelium and hemidesmosome attachment with the BMZ. These changes maintain a

constant amount of junctional adhesion between the columnar epithelium and the BMZ. Thus, the relationship between basal cell junctional adhesions and height of the epithelium is constant and not related to airway level or animal species.
Fibroblast Growth Factor-2 Signaling

Fibroblast growth factor-2 is stored in the BMZ of the airways where it binds with perlecan, a heparan sulfate proteoglycan that is an intrinsic constituent of the BMZ. Fibroblast growth factor-2 is released from perlecan in response to various conditions and becomes an important cytokine within the local microenvironment of the epithelialmesenchymal trophic unit. In airway epithelium, basal cells are the only cell type involved with broblast growth factor-2 signaling. Fibroblast growth factor-2 signals by forming a ternary complex with broblast growth factor receptor-1 (FGFR-1) and syndecan-4. When the broblast growth factor-2 ternary complex is formed, it initiates tyrosine kinase signaling associated with cell proliferation, migration, and differentiation. Basal cells express the cell surface receptors FGFR-1 and syndecan-4 whereas columnar cells do not (Figure 2). Presumably, broblast growth factor-2 is stored in airway BMZ as an intact growth factor to aid in rapid cellular responses to changes in local environmental

BASAL CELLS 231

Syndecan-4 FGFR-1

Basal cell FGF-BP FGF-2

Perlecan

Collagen I, III, & V Basement membrane zone

Perlecan

Figure 2 Ternary signaling complex in airway epithelium. In this illustration, BMZ-bound FGF-2 is released, and formation of the FGF-2 ternary complex with basal cells occurs via diffusion or binding with FGF-binding protein (FGF-BP). This is an example of how basal cells may function in growth factor signaling, neurogenic inammation, and the inammatory response through interactions with substances in the lateral intercellular space. Reproduced from Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931L939, with permission from The American Physiological Society.

inammatory cells and lymphocytes. Human basal cells upregulate intercellular adhesion molecule-1. Human basal cells can also upregulate expression of IgE receptors indicating that they may be involved with allergic responses of the airway. An unusual cell adhesion molecule, lymphocyte endothelialepithelial cell adhesion molecule, is expressed in the basal cell layer of human bronchial epithelium. Lymphocyte adhesion to epithelia and endothelia is mediated by lymphocyte endothelialepithelial cell adhesion molecule. Human basal cells also express the receptor 4-1BB. The receptor 4-1BB is a member of the tumor necrosis factor receptor super-family and is associated with T cell activation. Human basal cells express the Fas receptor and its ligand FasL. Ligation of the Fas receptor by migratory inammatory cells can lead to their apoptosis. Expression of these molecules by basal cells may play an important role in regulation of the inammatory response. They interact with inammatory cells when the latter are moving through the lateral intercellular space of airway epithelium.
Transepithelial Water Movement

conditions such as sloughing of damaged columnar epithelium or damage to the BMZ by leukocyte trafcking.
Neurogenic Inammation

Basal cells are involved with regulation of neurogenic inammation. In response to various inhaled foreign materials, axons in the airway epithelium release neuropeptides into the lateral intercellular space, initiating the process of neurogenic inammation (increased vascular permeability, neutrophil adhesion, vasodilatation, gland secretion, ion transport, smooth muscle contraction, increased cholinergic transmission, and cough). Basal cells contain the protein leukemic inhibitory factor. Leukemic inhibitory factor is thought to function in neurogenic inammation by stimulating the release of neuropeptides (tachykinins) from axons and the formation of neurokinin receptors. Neutral endopeptidase is a cell surface enzyme also associated with the process of neurogenic inammation. The enzyme neutral endopeptidase is expressed mainly on the surface of basal cells. The enzyme neutral endopeptidase cleaves neuropeptides in the lateral intercellular space. Cleavage of neuropeptides by neutral endopeptidase modulates the neurogenic inammatory responses in the airways.
Inammation

Basal cells have the aquaporin water channel AQP3, which is not found in the columnar epithelial cells. Instead, columnar epithelial cells have the aquaporin water channel AQP4. Water transfer between cells and the matrix occurs through these water channels. AQP3 is found in a basolateral position in the airways and in other tissues, implying movement of water between the extracellular matrix and epithelium. The presence of AQP3 water channels in the membranes of basal cells of normal airway epithelium demonstrates a unique role for the basal cell in uid modulation of airway surface liquids and also in the lateral intercellular space. The cellular distribution of AQPs 3 and 4 in airway epithelium implies the presence of cell-specic pathways for transcellular water movement between the extracellular matrix and epithelium. The cystic brosis transmembrane conductance regulator protein is a regulator of AQP3 water channels in basal cells suggesting a role in this disease. Downregulation of AQP 3 is thought to play a role in the pathogenesis of bronchiectasis.
Oxidant Defense of the Tissue

Basal cells participate in the inammatory response by upregulating expression of receptors for migratory

Basal cells in normal human airway epithelium express extracellular superoxide dismutase mRNA. Extracellular superoxide dismutase is a secreted protein found in the extracellular matrix responsible for metabolizing superoxide free radicals. The physiologic functions are not fully dened but it is thought to be critical for the protection of extracellular matrix elements against oxidative damage. The multidrug resistance-associated protein transmembrane transporter is

232 BASAL CELLS

found in bronchial epithelium and plays a major role in cell detoxication and defense against oxidant stress via efux of glutathione conjugates into the lateral intercellular space. The pattern of multidrug resistance-associated protein transmembrane transporter expression differs markedly according to cell type. In basal cells it is distributed over the entire circumference of the cell whereas in ciliated cells it is restricted to the basolateral surface. Expression of extracellular superoxide dismutase and multidrug resistance-associated protein transmembrane transporter by basal cells in normal subjects indicates that basal cells participate in defense of the tissue against oxidative stress.
Progenitor Cells

Basal Cells in Respiratory Disease

Asthma

The basal cell has the capacity to be the progenitor of columnar airway epithelium. This has been demonstrated in studies where denuded airways were repopulated with enriched populations of basal cells, in biphasic organotypic cultures, and in vivo following loss of the columnar epithelium. Under these conditions, basal cells dedifferentiate into a highly proliferative cell phenotype from which a mucociliary epithelium redifferentiates. In vivo there are two populations of proliferating basal cells (basal and parabasal cells). Basal cells have their nuclei next to the basement membrane. Parabasal cells are taller, and their nuclei are above the layer of basal cell nuclei. Basal cells make up 31% of the cell population in large airways and the taller parabasal cells make up 7%. However, parabasal cells have a proliferative fraction 4 to 5 times greater than basal cells. The higher proliferative fraction in parabasal cells suggests they may be more active in reparative proliferation following injury when compared with basal cells. Parabasal cells probably are the intermediate cells seen with electron microscopy. Intermediate cells are known to be the primary proliferating cells following injury to the columnar epithelium. During growth of the airway, the basal cell has a high rate of proliferation. The purpose of such proliferation in the growing airway is for the formation of new basal cells. The increase in basal cells per millimeter is related to their role in attaching columnar epithelium to the BMZ. Increased proliferation of parabasal cells during development is probably associated with formation of the columnar epithelium. In the normal adult airway epithelium, the rate of basal cell proliferation is low. Such proliferation may be for replacement of dying basal cells. When basal cells are lost due to injury or apoptosis, proliferation of surviving basal cells occurs, and they are replaced. Proliferation of parabasal cells is most likely associated with normal turnover of the columnar epithelium.

Dening the mechanism of columnar cell attachment to the BMZ was critical to the understanding of asthma and other disease conditions associated with sloughing of epithelium. In conditions where columnar cells are sloughed from the epithelium of larger airways, basal cells remain attached to the basal lamina. Desmosomal attachment between the columnar epithelium and basal cells represents a plane of cleavage between the two cell populations. Failure of desmosomal attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. The mechanism of desmosomal failure between airway cells is not known. However, it may not be failure per se but rather a specic protective function of basal cells. Shortly after the columnar epithelium has been sloughed, basal cells atten out and form a protective barrier. This is considered to be an important protective function of the basal cell along with the ability to quickly release desmosomal attachments to damaged columnar epithelial cells. It has also been speculated that stimulation of the epithelial neural system causes edema of the lateral intercellular space with a subsequent local sloughing of the epithelium. In a similar manner, an inux of inammatory cells could overwhelm the lateral intercellular space and cause local sloughing of the epithelium. However, these scenarios are speculations and the reasons for or mechanisms of epithelial sloughing are not known at this time.
Bronchiogenic Cancer

The p63 nuclear protein is specic for basal cells. Its functions are not clear, however, it has been used as a marker for tumors of basal cell origin in several tissues. In the lung, p63 was shown to be expressed in basal cells of normal tissue, in areas of squamous metaplasia, and in squamous cell carcinomas of the bronchi. In poorly organized carcinomas, p63 is expressed in most of the cells. However, it is only found in the basalar external cells of organized carcinomas. In adenocarcinomas, p63 is present occasionally but is not present in small cell carcinomas. These ndings indicate that basal cells are probably involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma. Basal cells have also been implicated in abnormal epithelium found in idiopathic pulmonary brosis when p63 was used as a marker.
See also: Adhesion, CellCell: Vascular; Epithelial. Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Aquaporins. Cell Cycle and Cell-Cycle

BREATHING / Breathing in the Newborn 233 Checkpoints. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast Growth Factors. Stem Cells.
Evans MJ, Van Winkle LS, Fanucchi MV, and Plopper CG (1999) The attenuated broblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. American Journal of Respiratory Cell and Molecular Biology 21: 655657. Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401415. Ford JR and Terzaghi-Howe M (1992) Basal cells are the progenitors of primary tracheal cultures. Experimental Cell Research 198: 6977. Inayama Y, Hook GE, Brody AR, et al. (1988) The differentiation potential of basal cells. Laboratory Investigation 58: 706717. Johnson NF and Hubbs AF (1990) Epithelial progenitor cells in rat trachea. American Journal of Respiratory Cell and Molecular Biology 3: 579585. Mercer RR, Russell ML, Roggli VL, and Crapo JD (1994) Cell number and distribution in human and rat airways. American Journal of Respiratory Cell and Molecular Biology 10: 613624. Persson CGA and Erjefalt JS (1997) Airway epithelial restitution after shedding and denudation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung, pp. 26112627. Philadelphia: Lippincott-Raven.

Further Reading
Boers JE, Ambergen AW, and Thunnissen FB (1998) Number and proliferation of basal and parabasal cells in normal human airway epithelium. American Journal of Respiratory Critical Care and Medicine 157(6 Pt 1): 20002006. Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931L939. Evans MJ and Moller PC (1991) Biology of airway basal cells. Experimental Lung Research 17: 513531. Evans MJ and Shami SG (1989) Lung cell kinetics. In: Lenfant C and Massaro M (eds.) Lung Cell Biology, vol. I. of the series Lung Biology in Health and Disease, pp. 136. New York: Marcel Dekker, Inc.

Basement Membranes Berylliosis Bradykinin

see Extracellular Matrix: Basement Membranes.

see Occupational Diseases: Hard Metal Diseases Berylliosis and Others.

see Kinins and Neuropeptides: Bradykinin.

BREATHING
Contents

Breathing in the Newborn Fetal Breathing Fetal Lung Liquid First Breath

Breathing in the Newborn

J A Adams, Mount Sinai Medical Center, Miami Beach, FL, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The complex physiology of breathing in the newborn has important developmental and maturational aspects. Early in fetal

life the respiratory system and controllers respond to intrauterine stimuli (PaCO2 , PaO2 , and lung ination). The complex network of control of breathing can be summarized into three basic components: (1) controllers, (2) effectors, and (3) sensors. At birth a multitude of inputs are responsible for initiation of rhythmic ventilation, and adaptation of the pulmonary circulation to extrauterine life. The normal physiology of breathing in the newborn is affected by posture, sleep state, and gestational age. Pathophysiological aspects of breathing in the newborn can be divided into: (1) fetal pathologies that occur during fetal life and ultimately result in abnormal lung development,

234 BREATHING / Breathing in the Newborn

(2) immediate postnatal events that occur primarily as a result of poor or abnormal transition to extrauterine life, and (3) neonatal events occurring in the neonatal period that lead to intrinsic lung disease or deranged control of breathing such as apnea. This article emphasizes the general importance of understanding the inuence of age, posture, sleep state, and maturation on breathing in the newborn, and provides a general overview of these. Paramount to the interpretation of normative and study data is the understanding of the inuence of these factors.

weeks of gestation, but even after birth the continual adaptation to air-breathing is an ongoing process.
The First Breaths

Description
The undertaking of a complex vital physiological function such as breathing in the newborn is a marvelous feat. Initiation of breathing in the newborn requires a multitude of appropriately synchronized events and signaling pathways. In preparation for extrauterine life and in order to assume the responsibility for extrauterine gas exchange, an anatomical, maturational, and functional process must occur. The alveoli are developed by the 25th week of gestation and by the 35th week of gestation, adequate quantities of surfactant (surface active material that keeps alveoli open and maintains alveoli stability) are present. Pulmonary circulation parallels alveolar development, and thus by the 25th week of gestation alveolar gas exchange can take place, but requires assisted ventilation. During fetal life, respiration is present in human fetuses from 10 weeks onward. These fetal breathing movements are not as well synchronized as those occurring in the newborn period. In fetal life, breathing is discontinuous and becomes continuous after birth. The fetus spends nearly 30% of its time engaged in discoordinate form of breathing associated with rapid irregular electrocortical activity, as seen in active sleep state. In addition, since the entire airways are lled with amniotic uid, at term almost 600 ml of amniotic uid is inhaled per day. Maintenance of normal amniotic uid volume, and respiratory activity, are crucial in the development of the airway, and maturation of lung structure and function. The fetus is also capable of modulating breathing movements in response to: (1) PaCO2 (hypercarbia increases fetal breathing), (2) PaO2 (hypoxia abolishes fetal breathing in sleep, and (3) pulmonary reexes (ination reex of HerringBauer lung distension with saline infusion decreases frequency of breathing). The overall framework for the control of breathing can be schematically viewed as: (1) controller (central nervous system and brainstem); (2) effectors (respiratory muscles and airway); and (3) feedback (chemoreceptor, mechanoreceptors, i.e., stretch receptors) (Figure 1). Functional maturity of the controller, effectors, and feedback mechanism allows for extremely premature newborns to survive, albeit requiring ventilatory assistance. The maturational process continues during gestation until term at 3840

During vaginal birth but less so during Cesarean section delivery, the thoracic cage is compressed to as much as 160 cmH2O pressure; this produces an ejection of tracheal uid via the airways. The recoil of the chest wall causes a passive inspiration and establishes an airliquid interface. The rst active breath is made slightly easier by the fact that some fetal lung uid is retained in the alveoli and smaller airway, thus requiring less distending pressure than a totally collapsed lung. In addition, in near-term and term newborns, surfactant produced by type II pneumocytes decreases alveolar surface tension, which prevents the lungs from total collapse at the lower transpulmonary pressures that occur in the subsequent breaths (Figure 2). The stimuli for the rst active inspiration is debatable but is likely to be a multifactorial set of events with which the newborn is confronted including change in temperature, light, noise, gravity, hypercapnea, sudden change in PaO2 , etc. A complete discussion on the control of breathing during the rst breaths is beyond the scope of this article, but sufce it to say that environmental nonrespiratory stimuli will enhance or facilitate the general tone of the respiratory neurons. In addition to the mechanical effects of the rst breath, the pulmonary circulation that parallels lung development and is maintained at high pulmonary vascular resistance during fetal life must also transition from a fetal to a newborn circulation. The latter is characterized by a gradual lowering of the pulmonary vascular resistance and eventual closure and redirection of blood ow via the foramen ovale and ductus arteriosus, and a change from a parallel circulation to one in series (Figure 3). The resultant effect is ultimately to match ventilation to perfusion for the most efcient oxygen extraction and delivery and carbon dioxide removal. Sleep state modulates control of breathing. The sleep and waking cycles begin to develop during fetal life. In the premature newborn at 24 weeks gestation, rapid eye movement (REM) periods account for nearly 80% of the total sleep time. At term, REM sleep is 6065% of sleep time, while in adulthood it accounts for only 2025%. There are major differences in the control of breathing between awake and sleep, which are independent of age. During sleep the brain is controlled more by stimulation related to gas exchange than any other stimuli. When comparing the breathing patterns and pulmonary mechanics

BREATHING / Breathing in the Newborn 235

Controllers Cerebrum Cerebellum Midbrain Effectors Sensors

Brainstem

Upper airway receptors Vagal resp. motor efferents Chemoreceptors Upper airway Lung receptors Lung

Spinal cord Proprioreceptors Resp. muscles

Figure 1 Schematic representation of the control of breathing. Adapted from Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271280, with permission from Elsevier.

among newborns, measurements should always be made in the same sleep state and in the same posture. Noninvasive methods to quantify these breathing patterns over various sleep states can serve as a basis for comparison. Breathing is affected by gestational age. Maturation of ventilatory response to chemical and mechanical stimuli is also inuenced by gestational age. The ventilatory response to hypoxia in newborns is biphasic. It is characterized by an initial phase of hyperventilation followed by hypoventilation below baseline levels. The etiology of such biphasic response is unclear, and may be related to metabolic demands or neurotransmitters (Figure 4). This response is clearly different to that which occurs in older infancy and adults. The ventilatory response to carbon dioxide in premature infants is also different from that in term newborns. In premature infants the ventilatory response to hypercapnea is quantitatively and qualitatively different from that in older term neonates and adults. Term infants and adults increase ventilation through an increase in tidal volume

and frequency; premature infants do not increase frequency in response to hypercarbia, and have a prolonged expiration. This response appears to be centrally mediated at the level of the brainstem and probably involves the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Furthermore, the ventilatory response to hypercapnea is inuenced by sleep state in term newborns. There is a greater response in minute ventilation during quiet state compared to REM (Figure 5). Breathing is affected by posture in newborns. The characteristics of a very compliant chest wall in newborns have a significant mechanical effect on breathing as efciency of ventilation is reduced due to increased chest wall compliance. In full term infants, a change from supine to prone posture increases minute ventilation and respiratory drive, with a concomitant decrease in thoracoabdominal asynchrony. In premature infants (o35 weeks gestation) supine posture is associated with higher respiratory rate, lower arterial oxygen saturation, lower ventilatory response to hypercapnea, and increased thoracoabdominal

236 BREATHING / Breathing in the Newborn

Saline Deflation 150 Volume (ml) Air

200

100

50 Inflation 0 0 (a) 4 8 12 Pressure (cmH2O) 16 20

200

After surfactant

150

Volume (ml)

100

50

Before surfactant 0 4 8 12 16 20

-50 (b) Pressure (cmH2O)

Figure 2 (a) Pressurevolume curves after saline and air expansion of the lung. Lower pressures are required to expand a saline-lled lung compared to air. The deation curve in air is not superimposable on the ination curve (hysteresis) due to mobilization and orientation of surfactant during deation decreasing alveolar surface tension as the alveolar surface contracts. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins. (b) Pressure volume loops of a normal lung (red) and surfactant-decient lung. Note that the slope of the loop is decreased in the surfactant-decient lung. A greater amount of pressure is required for a lower change in lung volume. (Compliance D volume/D pressure.)

synchrony. Thus, posture appears to play a role in pulmonary mechanics, primarily in the efciency of ventilation. Whether these changes occur as a result of conferring greater chest wall stability or diaphragmatic mechanical advantage in the prone posture in both full term and preterm infants remains to be clearly elucidated. In spite of this, conferring a mechanical advantage in the prone posture for full term infants does not justify its use in the care of the full term newborns upon hospital discharge. Sudden infant death rates have clearly decreased in the US as a result of the Back to Sleep Campaign.

Pathophysiology
In order to understand the effects of various pathological states on breathing in the newborn it is useful to describe three distinct time periods for occurrence of pulmonary pathology: (1) fetal, (2) immediate postnatal, and (3) neonatal.

Fetal

To a large extent, lung growth is dependent on amniotic uid production and volume, and the mechanical

BREATHING / Breathing in the Newborn 237

Pulmonary vascular resistance

Pulmonary venous ret

Left atrial pressure

Ventilation

PaO2 (>30) Closed foramen ovale

Closed ductus venosus

Closed ductus arteriosus

LR shunt ductus Right atrial pressure

Umbilical venous ret

Peripheral vascular resistance

Figure 3 Schematic of the transition to neonatal circulation once ventilation is established. LR, left to right; ret, return. Multiple factors play a role in the conversion of this circulation, including prostaglandins, endothelin, and endothelial-derived nitric oxide. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins.

30 % Change in baseline 20 10 0 10 20 30 Time (s)

Figure 4 The biphasic ventilatory response to hypoxia. Note the increase in ventilation, followed by a marked decrease in ventilation below baseline values. Adapted from Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphase ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960964, with permission from Elsevier.

Control

60

180

300

shear it exerts on the primitive airways and alveolar ducts. Thus, a spectrum of pulmonary hypoplasia due to multiple etiologies is the major pathological problem in the fetal period.

Immediate Postnatal

Failure to transition from a uid-lled to a gas-lled pulmonary tree and to reabsorb the fetal lung uid results in a transient condition called transient

238 BREATHING / Breathing in the Newborn

1000 Min vent (ml kg1 min1)

Quiet

750

REM

500

pulmonary hypertension with increased pulmonary vascular resistance and right to left shunting via the patent ductus arteriosus and foramen ovale. This condition has several underlying etiologies including intrauterine hypoxia, pneumonia, and other pulmonary vascular pathologies.
Neonatal

250 45 50 55 PET CO2 60 65

% Increase in min vent from BL

100.0% 80.0% 60.0% 40.0% 20.0% 0.0%

Quiet REM

Figure 5 Ventilatory response to hypercapnea. Note the difference in slope of the line between active state (REM) and quiet state in full term infants. End tidal CO2 PET CO2. Reproduced from Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168174, with permission from The American Physiological Society.

tachypnea of the newborn (TTN or respiratory distress syndrome (RDS) type II). This condition is characterized by the early appearance of tachypnea and a variable degree of respiratory distress in an otherwise healthy appearing newborn. The greater occurrence of TTN in newborns delivered by Cesarean section make this condition rather common. Onset of respiratory distress after the initial newborn transitional period (about 46 h) usually signies intraparenchymal lung pathology due to pneumonia (viral or bacterial). Medications administered during labor and delivery can also have an effect on breathing; these include narcotics or magnesium sulfate, both of which can depress respiratory drive. In the premature infant, failure to transition from fetal to extrauterine life can be due to pulmonary insufciency secondary to RDS type I (surfactant deciency). Surfactant administration has markedly improved mortality and morbidity of this disease process and has radically changed the outcomes for premature newborns. In both premature and term newborns, failure of the pulmonary circulation to transition from intrauterine fetal circulation to that of the newborn results in the well known persistent pulmonary hypertension of the newborn (PPHN) or persistent fetal circulation (PFC) characterized by

In premature infants, particularly those born at less than 34 weeks gestation who have immature lungs or an intrinsic pulmonary pathology, a frequent respiratory problem is apnea. Apnea is typically dened as cessation of breathing for more than 15 s; however, the literature is replete with various definitions for the duration of cessation of breathing. Apnea is most commonly seen in premature infants, and the younger the gestational age the more common its occurrence (Figure 6). Apnea in the premature infant can be caused by intracranial pathology, metabolic derangements, and infection, among others. The diagnosis of apnea of prematurity is one of exclusion and thus an exhaustive undertaking of diagnostic tests should be done prior to categorizing apnea as such (Figure 7). In addition to cessation of breathing or ineffective ventilation, clinically relevant apneas decrease arterial oxygen saturation and cause bradycardia. With the advent of computerized monitoring and noninvasive technologies to measure breathing and arterial oxygen saturation, long-term studies in newborns have become possible in the home environment. It has been found that apneas can occur at any gestational age and that their severity can only be detected if various physiological parameters are simultaneously monitored. Apnea has been typically classied into three types: (1) central (no respiratory efforts and thus no airow), (2) obstructive (respiratory efforts against a partially or totally occluded airway), and (3) mixed (combination of central and obstructive within the same event). With the advent of more rened monitoring techniques, it is evident that purely obstructive apneas in the newborn are rare, and that most apneas in premature infants have a mixed/obstructive characteristic. Cessation of airow is present followed by obstructive breaths or obstructive breaths are followed by cessation of respiratory effort. Perhaps the most clinically relevant and important aspect of apnea relates to the changes in arterial oxygen saturation that it can cause and bradycardia (Figures 8(a)8(c)). Using respiratory inductive plethysmography that measures both ribcage and abdominal excursions and can be calibrated in the newborn to provide a relative estimate of tidal volume, we have been able to study apneas in premature newborns. In

BREATHING / Breathing in the Newborn 239

Apnea of prematurity

Relative incidence of apnea

Prematurity

Term 1 mo 2 mo Term 1 mo 2 mo 6 mo Age

Figure 6 Relative incidence of apnea as a function of maturation. Note the dramatic decrease in apnea after term gestation. Reproduced with permission from NeoReviews, Vol. 3, pages e66e70, Copyright 2002.

Immaturity

Hypercapnic response

Inhibitory reflexes

Hypoxic depression

Apnea

Seizures

Infection IVH Reflux Head & body position

Hypoxemia or anemia

Drugs

addition to apneas, newborns also experience hypopnea (decreased tidal volumes below 25% of the unimpeded baseline value). Hypopnea is a commonly recognized problem in sleep disorder breathing in adults but not commonly appreciated in newborns due to the lack of ability to measure changes in tidal volume over prolonged periods. The end result of both apneas and hypopneas is to cause decreased functional residual capacity (FRC). Since newborns typically have relatively low FRC and oxygen stores, the concomitant decrease in arterial oxygen saturation during apneas or hypopneas is not surprising. Additionally, we have found using noninvasive methods that during periods of apnea in the premature newborn the cardiac output can decrease by as much as 50%. Cerebral blood ow uctuations have also been shown to occur to a greater degree during periodic breathing, apnea, and REM sleep. Whether cardiac output and episodic arterial oxygen desaturation and changes in cerebral blood ow will have untoward long-term neurological effects needs to be determined.

Figure 7 A schematic representation of the etiology of apnea in premature newborns. Examples of cause of apneas are highlighted in yellow. IVH, intraventicular hemorrhage. Adapted with permission from NeoReviews, Vol. 3, page(s) e66e70 and NeoReviews, Vol. 3, page(s) e59e65, Copyright 2002.

Conclusion
Disordered breathing in the premature and term infant can be ascribed to a multitude of causes, which

240 BREATHING / Breathing in the Newborn

30.5 mm s1 400 %Vt

NIMS respi. events

Vt

400 %Vt

RC

400 %Vt

AB

(a) 9.6 mm s1 300 %Vt Vt 300 %Vt RC 300 %Vt AB 180 deg EP Ang 0 deg 5.00 mV ECG 200 B/M HR 0 B/M 0 (b)
Figure 8 Examples of respiratory inductive plethysmographic recordings: (a) normal tidal breathing in the newborn; (b) central apnea of 14 s duration; and (c) mixed/obstructive apnea of 21 s duration (note a decrease in HR to 90 bpm, followed by a decrease in arterial oxygen saturation to a nadir of 80%). Vt, the tidal volume from calibrated respiratory inductive plethysmography derived as a percent of the calibration value. RC, the volume contribution of the ribcage to tidal volume. AB, the abdominal volume contribution to tidal volume. EPANG, the phase angle between the RC and AB, obtained from a plot of RC vs. AB; values of 180o denote complete paradoxical motion of the RC and AB in opposite directions. ECG, electrocardiogram. HR, heart rate derived from the electrocardiogram. OxiP, the arterial pulse obtained from the pulse oximeter. SAT, percent arterial oxygen saturation.

NIMS respi. events

10

15

20

BREATHING / Breathing in the Newborn 241

5.6 mm s1

300 %Vt Vt 300 %Vt RC 300 %Vt AB

NIMS respi. events

Apnea 2.00 mV ECG 200 B/M HR 50 B/M 255 cu OxiP 0 cu 100 % SaO2 50 % 0 (c)
Figure 8 (Continued )

10

15

20

25

30

35

have their basis in pulmonary developmental problems, intrinsic pulmonary disease, or deranged control of breathing. The inuence of sleep state, posture, and gestational age are paramount in the interpretation of both normative and study data.
See also: Breathing: Fetal Breathing; Fetal Lung Liquid; First Breath. Infant Respiratory Distress Syndrome. Sudden Infant Death Syndrome.

Further Reading
Adams JA, Zabaleta IA, and Sackner MA (1994) Comparison of supine and prone noninvasive measurements of breathing patterns in fullterm newborns. Pediatric Pulmonology 18: 812. Adams JA, Zabaleta IA, and Sackner MA (1997) Hypoxemic events in spontaneously breathing premature infants: etiologic basis. Pediatric Research 42: 463471. Adams JA, Zabaleta IA, Stroh D, Johnson P, and Sackner MA (1993) Tidal volume measurements in newborns using respiratory inductive plethysmography. American Review of Respiratory Diseases 148: 585588. Avery GB, Fletcher MA, and McDonald MG (1999) Neonatology Pathophysiology & Management of the Newborn. Philadelphia: Lippincott Williams & Wilkins. Baird TM, Martin RJ, and Abu-Shaweesh JM (2002) Clinical associations, treatment, and outcome of apnea of prematurity. NeoReviews 3(4): e66e70. Campbell AJ, Bolton DP, Taylor BJ, and Sayers RM (1998) Responses to an increasing asphyxia in infants: effects of age and sleep state. Respiration Physiology 112: 5158.

Chernick V (1981) The fetus and the newborn. In: Hornbein T (ed.) Regulation of Breathing, Part II, pp. 11411179. New York: Dekker. Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168174. Curzi-Dascalova L (1992) Physiological correlates of sleep development in premature and full-term neonates. Neurophysiology Clinincs 22: 151166. Fanaroff AA and Martin RJ (2002) NeonatalPerinatal Medicine Disease of the Fetus and Infant. St Louis: Mosby. Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271280. Guilleminault C and Robinson A (1996) Developmental aspects of sleep and breathing. Current Opinion in Pulmonary Medicine 2: 492499. Martin RJ, Abu-Shaweesh JM, and Baird T (2002) Pathophysiologic mechanisms underlying apnea of prematurity. NeoReviews 3: e59e65. Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphasic ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960964. Mortola JP and Saiki C (1996) Ventilatory response to hypoxia in rats: gender differences. Respiration Physiology 106: 2134. Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257278. Philadelphia: Lippincott Williams & Wilkins. Polin RA and Fox WW (1992) Fetal and Neonatal Physiology. Philadelphia: Saunders. Rigatto H (1992) Maturation of breathing. Clinical Perinatology 19: 739756. Stocks J, Sly PD, Teppers RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley-Liss.

242 BREATHING / Fetal Breathing

Fetal Breathing
R Harding and S B Hooper, Monash University, Melbourne, VIC, Australia C A Albuquerque, Santa Clara Valley Medical Center, San Jose, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Fetal breathing movements (FBMs) are breathing-like movements that occur episodically in healthy mammalian fetuses. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. Owing to the airways being lled with liquid, FBMs cause only minor changes in lung volume but typically lower intrathoracic pressure by up to 5 mmHg and alter the shape of the fetal chest. By altering intrathoracic pressure they affect blood ow within the fetus and uid movement within the uid-lled fetal airways. FBMs are characteristically highly variable in frequency and amplitude, but become more organized with increasing gestational age and relate to fetal behavioral states. They are inhibited by fetal hypoxia and hence can be used in the diagnosis of fetal compromise. FBMs are critical for normal in utero lung growth and development as they maintain the fetal lung in an expanded state by opposing lung recoil: in the absence of FBMs the fetal lungs tend to deate leading to lung hypoplasia. At birth, breathing becomes continuous, possibly by removal of inhibitory substances produced by the placenta or fetal brain and by increased carbon dioxide production.

Two other types of inspiratory efforts have been recognized in the fetus; isolated deep inspiratory efforts and asphyxial gasping. Isolated deep inspiratory efforts, often termed hiccups, are commonly observed in healthy fetal humans, pigs, and sheep; typically, these occur in low-frequency bouts. Asphyxial gasps are strong inspiratory efforts initiated by fetal asphyxia and involve intense activation of many respiratory muscles.

Central and Peripheral Control of FBMs

FBMs are an expression of rhythmic activation of neurons in the fetal brainstem. These brainstem neurons generate rhythmic bursts of activity in phrenic motoneurons and in vagal preganglionic bers destined for dilator muscles of the upper respiratory tract. With development, episodes of FBMs become temporally associated with fetal behavioral states involving body movements, rapid eye movement (REM) sleep, or arousal. During the last third of gestation, FBMs are usually infrequent or absent in association with the fetal state resembling quiet, or non-REM, sleep (low activity state). The periods of FBM/REM sleep/activity and the intervening periods of apnea/non-REM sleep/inactivity are of similar duration, both occupying B50% of the time. Compared to postnatal life, respiratory drive in the fetus is relatively low and is regulated mainly by fetal CO2 levels, as the incidence and amplitude of FBMs are increased by elevated fetal PaCO2 (partial pressure of carbon dioxide in arterial blood) levels and decreased by low fetal PaCO2 levels. It is assumed that this CO2 drive is mediated by acidication of the uid surrounding the central chemoreceptors, as altering the pH of fetal blood or cerebrospinal uid (CSF) can have effects similar to those of alterations in PaCO2 levels. FBMs are inhibited by moderate to severe hypoxia (Figure 1), by what is thought to be a central inhibitory mechanism that also inhibits other forms of fetal skeletal muscle activity and alters fetal behavioral state favoring reduced activity. This inhibitory mechanism may override the stimulatory effects of mild hypoxia. Although peripheral chemoreceptors are active in the fetus and do respond to hypoxia, they do not apparently play a role in the hypoxic inhibition of FBMs. Recent studies suggest that central adenosine receptors play a major role in the central inhibition of FBMs and alteration in fetal behavior by hypoxia.

Introduction
Fetal breathing movements (FBMs) are breathinglike movements that occur episodically in healthy fetuses during much of gestation. FBMs have been observed in many mammalian species, including man, as well as in birds and reptiles. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. They play no role in fetal gas exchange, as the fetal airways are lled with liquid; in sheep, they typically lower intrathoracic pressure by up to 5 mmHg. Most of the available information on FBMs has been obtained from two species, humans and sheep. In humans, FBMs can be detected by ultrasonography from about 10 weeks of gestation. They are observed as rhythmic descending movements of the diaphragm and are usually coincident with inward movement of the chest wall and outward movement of the abdominal wall. In chronically catheterized fetal sheep, FBMs can be detected as electrical activity of the diaphragm muscle, rhythmic reductions in intratracheal or intrathoracic pressure, or uid movement within the trachea.

Effects of FBMs on Airway Fluid Movement and Lung Volume

As FBMs alter fetal intrathoracic pressure, it is to be expected that they affect uid movement within the

BREATHING / Fetal Breathing 243

50

40 Incidence of FBMs (min h1)

30

20 24 h normoxia 24 h hypoxia

10 0 4 0

12

16

20

24

28

Period of hypoxia (h)

Figure 1 Effects of 24 h of fetal hypoxia on the incidence of FBMs in a late gestational fetal sheep. Hypoxia causes a profound but transient inhibition of FBMs, with normal values returning after 1418 h of hypoxia. Asterisks show values that differ significantly from control values. Reproduced from Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127135, used with permission from the American Physiological Society.

respiratory tree. Oscillatory uid ows synchronous with diaphragm movements have been detected in the fetal trachea and at the nose in humans. Although there is some controversy as to the volume of uid moved with each fetal breath, it is clear that tidal volume in the fetus is much smaller than after birth, which can be attributed to the much greater viscosity of liquid relative to air. In fetal sheep, for example, tidal volumes measured by ow meters in the trachea are much less than 1 ml, whereas after birth, in the same species, tidal volumes are typically 30 50 ml. Thus, FBMs are essentially isovolumic, causing only very small changes in thoracic volume with each breath. However, in human fetuses, tidal volumes of about 2 ml have been estimated by ultrasound, in the trachea and at the nose. The reason for the larger tidal volume in humans is unknown but may be due to the slower breathing rates of human fetuses compared to fetal sheep. Although FBMs individually may have little effect on volume ow within the airways, it is clear that episodes of FBM can result in the movement of much greater volumes, relative to total lung uid volume. This effect on uid movement can be largely attributed to FBM-related changes in transpulmonary pressure and upper airway resistance. Studies in sheep have shown that net uid movement to and from the fetal lungs is affected by FBM episodes such that most of the efux occurs during these episodes; during episodes of FBM the laryngeal dilator muscles

are rhythmically active (in phase with the diaphragm) and laryngeal adductor muscles are largely quiescent. Hence, the resistance to tracheal uid movement offered by the upper airway is lowered during FBMs, allowing uid that has accumulated within the airways to ow into the pharynx, from where it is either swallowed or ows into the amniotic sac. During periods of fetal apnea, the laryngeal dilator muscles become quiescent and adductor tone is usually present, resulting in raised resistance of the upper airway. Together with a lack of inspiratory muscle activity, this results in low rates of uid efux from the trachea, although increased efux may occur with fetal postural adjustments or other activities, including those resembling straining movements or Valsalva maneuvers that are common in the fetus. The fetal upper airway plays a critical role in regulating the ow of uid to and from the lungs, thereby preserving lung volume and the composition of liquid within the airways. Removal of the inuence of the upper airway by creating a tracheo-amniotic bypass results in a major loss of lung liquid during periods of apnea and a large ingress of amniotic uid during FBM episodes. This loss of upper airway function not only allows near total lung deation during periods of fetal apnea, it also allows large inuxes of amniotic uid during FBM episodes. This brings the alveolar epithelium into contact with undiluted amniotic uid with potentially harmful effects, especially if the amniotic uid contains meconium.

244 BREATHING / Fetal Breathing

Lung volume (% of intact fetus)

Although the net ow of liquid within the fetal trachea is away from the lungs, primarily due to continuous liquid secretion, periods of inux may occur, especially in association with periods of vigorous FBMs. This may explain the entry of substances into the lungs following their deposition in the amniotic uid.

100 80 60 40 20 0

Effects of FBMs on Fetal Blood Flows

As FBMs alter intrathoracic pressure, it is to be expected that they will affect blood ows within the fetus. FBM-related changes in blood ows have been observed in the umbilical vein, fetal vena cava, foramen ovale and, more recently, in the pulmonary artery. In the pulmonary artery, episodes of vigorous FBMs are associated with increased blood ow, likely to be a consequence of reduced resistance in the pulmonary capillaries as a result of an altered transmural pressure.

Intact fetus

No No FBMs Collapsed FRC FBMs & lung newborn no UA

Functional Importance of FBMs

A major function of FBMs is to maintain lung liquid volume, and hence lung expansion, which is known to be essential for normal growth and structural maturation of the fetal lung. This role of FBMs has been demonstrated in experimental models that have eliminated FBMs while preserving the integrity of the diaphragm. The long-term abolition or suppression of normal FBM results in lung hypoplasia and structural immaturity of the lungs, which is probably due to a chronic reduction in lung expansion rather than abolition of the small phasic movements of the chest wall. A chronic reduction in fetal lung expansion leads to a reduction in lung tissue growth and alterations in the structure of the alveolar wall and epithelium; similar changes in lung development are caused by the abolition of FBMs. Abolishing the diaphragmatic movements that cause FBM is thought to reduce the force that normally opposes the inherent elastic recoil of the lungs, thereby allowing the fetal lungs to deate; a further reduction occurs if the resistance offered by the upper airway is removed (Figure 2).

Figure 2 Lung luminal volumes in fetal and neonatal sheep, expressed in relation to values in the intact, late gestation ovine fetus in utero. Lung liquid volume, and hence lung expansion, is reduced by the prolonged absence of FBMs (no FBMs) induced by phrenic nerve blockade or a high section of cervical spinal cord. A further reduction occurs if the fetal upper airway is bypassed, allowing direct continuity between the fetal lungs and the amniotic sac (no FBMs & no UA). Lung luminal volume is reduced further when the lungs are removed from the fetus (collapsed lung) due to unopposed recoil of the uid-lled lung. Functional residual capacity (FRC) in the air-breathing newborn is shown for comparison. Data from Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209224.

Use of FBMs in Clinical Assessment

In the healthy human fetus, FBMs occur at an average frequency of 60 per min and are accompanied by increased body movements and heart rate variability. FBMs are rst detectable at about 1012 weeks of gestation when they are usually irregular and sporadic. From about 28 weeks of gestation onwards, FBMs become more regular and organized

into discrete episodes. Using color Doppler and spectral ultrasonography analyses, the breath-to-breath interval and the inspiratory phase of the respiratory cycle have been shown to increase from 22 to 35 weeks gestation, but then decrease towards term. FBMs are used as one component of the fetal biophysical prole which is widely used to assess fetal health; the occurrence of FBMs is observed, together with assessments of fetal heart rate, amniotic uid volume, fetal body movements, and fetal body dimensions. If FBMs are not detected, the fetus may be hypoxic, may have a neural disorder affecting the brainstem and phrenic motor nerves, or it may have an abnormality of skeletal muscle function. However, it must be recognized that the inhibition of FBMs by chronic fetal hypoxia may be only transient, as has been shown in sheep (Figure 1). FBMs have also proven to be useful for the diagnosis of intrauterine infection in patients with preterm rupture of membranes as they have a high negative predictive value for intra-amniotic infection; a depression in FBM incidence is non-specic but is suggestive of infection. Studies of patients with premature rupture of membranes have shown a decrease in FBM incidence in those patients positive for amniotic uid infection, clinical chorioamnionitis, or neonatal sepsis. This may be related to inammatory cytokines affecting fetal behavioral states.

BREATHING / Fetal Breathing 245

Fetal Breathing, Amniotic Fluid Volume, and Lung Growth

There is conicting evidence on the role of FBMs in the development of pulmonary hypoplasia in human fetuses with preterm rupture of membranes and reduced amniotic uid volume (oligohydramnios). Studies examining the role of FBM in the lung hypoplasia associated with premature rupture of membranes have shown either no change or a reduction in the incidence of FBM. However it is likely that both the oligohydramnios following membrane rupture and a reduced incidence of FBMs may contribute to the associated fetal lung hypoplasia. In humans, it has been shown that a prolonged reduction in FBMs at critical periods of development (i.e., o24 weeks of gestation) can lead to pulmonary hypoplasia; similarly, pulmonary hypoplasia can develop in human fetuses after prolonged (more than 6 days) membrane rupture. It is likely that oligohydramnios leads to lung hypoplasia due to increased exion of the fetal trunk, which compresses the fetal lungs causing their deation and reduced growth rates.

of maternal smoking reduces the oxygen carrying capacity of fetal blood. FBMs are inhibited by maternal tobacco smoking, and the inhibition may continue for up to an hour following a single cigarette. It is thought that this effect is due mostly to fetal hypoxemia, or more specifically to reduced cerebral oxygen delivery. Maternal smoking has recently been shown to increase the resistance in the fetal cerebral artery, and this may also contribute to reduced cerebral oxygen delivery. The inhibition of FBMs by maternal smoking may contribute to the known adverse effects of smoking on fetal lung development. The same may apply to other drugs taken by the mother, such as narcotics, sedatives, or analgesics.

Respiratory Transition at Birth

The physiological mechanisms underlying the transition from discontinuous fetal breathing to continuous postnatal breathing are complex and not fully understood. During parturition and when the umbilical cord is cut at birth, the neonate may become profoundly hypoxemic, hypercapnic, and acidemic. It is also exposed to lower environmental temperatures, leading to increased heat loss, and to a greatly increased degree of external sensory stimuli, thereby changing the behavioral state to one of arousal. It is also possible that the removal of circulating inhibitory or suppressive substances that originate in the placenta (e.g., prostaglandin E2, adenosine, neuroactive progesterone metabolites) may contribute to the onset of continuous breathing. For example, their removal may lead to an increase in metabolic activity (e.g., by stimulating thermogenesis), and hence an increase in the rate of CO2 production, or to an increase in the sensitivity of central chemoreceptors to CO2. It has also been suggested that endocrine changes at birth, such as a large increase in circulating catecholamines, lead to respiratory stimulation, possibly via an increase in fetal metabolism and hence CO2 production. Studies of fetal sheep maintained ex utero by extracorporeal oxygenation also support the notion that CO2 plays a crucial role in the maintenance of continuous breathing after birth. The integrity of the vagus nerves has been shown to be essential for the onset of adequate breathing at birth. Although the critical pathways have not yet been identied, it is likely that volume receptive feedback from the lungs is involved. Studies in neonatal lambs have shown that a reduction in end-expiratory lung volume (functional residual capacity, FRC), which reduces vagal sensory trafc from pulmonary stretch receptors, results in profound hypoventilation, periodic breathing, and active glottic adduction during periods of apnea. This indicates

Effects of Labor on FBMs

At term, the incidence of FBMs decreases. In sheep, the incidence of FBMs decreases 23 days before the onset of labor and remains reduced until delivery. This may be related to increased circulating concentrations of prostaglandin E2, which is known to inhibit FBMs. Similarly, in humans, fetal apnea of 2060 min duration is considered to be a reliable indicator of premature labor with delivery within 4872 h. During spontaneous or induced labor, the incidence of FBMs is decreased to less than 10% of the time during the latent phase and is further decreased during the active phase of labor. In human pregnancy, the rupture of fetal membranes at term does not appear to affect FBMs. However, a study before term showed a significant reduction in FBMs for the rst 2 weeks of membrane rupture, compared to controls, with a return to near normal by the third week. With increased uterine contractility there is a decrease in the incidence of FBMs.

Maternal Smoking and Fetal Breathing

As with most drugs taken by the mother, tobacco smoking has been shown to affect fetal behavior. Nicotine readily crosses the placenta, and is thought to reduce utero-placental and/or umbilico-placental blood ow, contributing to fetal hypoxemia; furthermore, the formation of carboxyhemoglobin as a result

246 BREATHING / Fetal Lung Liquid

that receptive vagal feedback of lung volume at endexpiration, which is normally maintained by an adequate FRC, is essential for continuous breathing in the newborn, and explains, at least in part, the benets of positive end-expiratory pressure (PEEP) in the treatment of infantile apnea.
See also: Breathing: Fetal Lung Liquid. Lung Development: Overview.

stretch stimulus that is essential for normal fetal lung growth. If the distending inuence of fetal lung liquid is absent, the lungs fail to grow, which is the primary mechanism for fetal lung hypoplasia in humans. At birth, the airways are cleared of liquid to allow the entry of air and the onset of air breathing, due to a reversal of the transepithelial ionic gradient that promotes reabsorption into the lung parenchyma; this is stimulated by the release of stressrelated hormones during labor. Increases in transpulmonary pressure, due to changes in fetal posture, are likely to also contribute to the clearance of fetal lung liquid at birth.

Further Reading
Bissonnette JM (2000) Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 278: R1391R1400. Bocking AD (2003) Assessment of fetal heart rate and fetal movements in detecting oxygen deprivation in-utero. European Journal of Obstetrics and Gynecology 110: S108S112. Champagnat J and Fortin G (1997) Primordial respiratory-like rhythm generation in the vertebrate embryo. Trends in Neuroscience 20: 119124. Cosmi EV, Anceschi MM, Cosmi E, et al. (2003) Ultrasonographic patterns of fetal breathing movements in normal pregnancy. International Journal of Gynaecology and Obstetrics 80: 285290. Harding R (1997) Fetal breathing movements. In: Crystal RG, West JB, Weibel ER, et al. (eds.) The Lung, Scientic Foundations, pp. 20932103. Philadelphia: Lippincott-Raven. Harding R and Bocking AD (2001) Fetal Growth and Development. Cambridge: Cambridge University Press. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209224. Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127135. Laudy JA and Wladimiroff JW (2000) The fetal lung. 1: Developmental aspects. Ultrasound in Obstetrics and Gynecology 16: 284290. Rigatto H (1996) Regulation of fetal breathing. Reproduction, Fertility and Development 8: 2333.

Description
Throughout embryonic and fetal development, the future airways of the lung are lled with liquid and the lungs take no part in gas exchange. This liquid (fetal lung liquid) is a unique secretory product of the lung and is quite unlike fetal plasma or amniotic uid in composition; it has a low pH, a high Cl concentration, and low protein content. Fetal lung liquid is secreted across the pulmonary epithelium and leaves the lungs by owing out of the trachea.
Control of Fetal Lung Liquid Secretion

Fetal Lung Liquid

S B Hooper and R Harding, Monash University, Melbourne, VIC, Australia
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Fetal lung liquid is secreted across the pulmonary epithelium and enters the future airspaces due to an osmotic gradient created by the transepithelial ux of ions into the lung lumen. This liquid leaves the lungs by owing out of the trachea, whereby it is either swallowed or contributed to amniotic uid volume. The high resistance to liquid movement through the fetal upper airway promotes the retention of lung liquid within the future airways, which provides a small (1 or 2 mmHg) internal distending pressure on the lungs. This acts as an internal hydrostatic splint that maintains the fetal lungs in a distended state and provides a

Fetal lung liquid is formed by the net movement of Cl and Na across the epithelium into the lumen, which provides an osmotic gradient for the movement of water in the same direction. This mechanism is thought to be driven by Na /K ATPase, which creates an electrochemical gradient for Na to enter epithelial cells via the Na /K /2Cl cotransporter. The resultant Na -linked Cl entry across the basolateral membrane increases intracellular Cl concentrations that passively exit the cell, down its electrochemical gradient, via selective channels located in the apical surface. The net movement of Cl into the lung lumen generates a small transepithelial potential difference (lumen negative) that also promotes the movement of Na . As a result, water moves down an osmotic gradient generated by the net movement of Na and Cl (Figure 1). However, water movement across the epithelium must also depend on the intraluminal hydrostatic pressure existing, which will oppose its movement into the lung lumen. At rest, a small hydrostatic distending pressure (1 or 2 mmHg above ambient pressure) is present within the lung lumen and, therefore, the secretion of lung liquid must normally occur against a small hydrostatic pressure. When intraluminal pressures are increased above 5 or 6 mmHg, lung liquid secretion ceases, indicating that the osmotic pressure driving lung liquid can be counterbalanced by a hydrostatic pressure of 5 or 6 mmHg. On the other hand, when the fetal lungs are partially deated and the intraluminal hydrostatic pressure is reduced, fetal lung liquid secretion rates increase.

BREATHING / Fetal Lung Liquid 247

Interstitial space

Epithelial cell

Lung lumen

H2O K+

Na+

2Cl Cl

2K+ 3Na+ Na+

Figure 1 The proposed mechanism for fetal lung secretion across the pulmonary epithelium. Na /K ATPase, located on the basolateral surface of pulmonary epithelial cells, provides the free energy for Na to enter the cell via the Na /K /2Cl cotransporter, which promotes the entry of Cl against its electrochemical gradient. Cl exits the cell across the apical membrane down its electrochemical gradient, which causes a transepithelial potential difference (lumen negative) that promotes the movement of Na into the lung lumen. The combined net ux of Na and Cl into the lung lumen provides an osmotic gradient for water to ow in the same direction.

Although the mechanisms for fetal lung liquid secretion are relatively clear, the precise cellular origin is less clear. Early in gestation most epithelial cells are likely to contribute, but later in gestation the most likely candidates are the epithelial cells residing in the smaller, terminal airways because they constitute the vast majority (o90%) of the internal surface area of the lung. This is further supported by the nding that fetal bronchial epithelial cells predominantly absorb Na , whereas cultured fetal type II epithelial cells exhibit similar ionic uxes as that described in the intact fetal lung. However, the relative contribution of type I and type II cells to fetal lung liquid secretion is unknown.
Control of Fetal Lung Liquid Volume

transpulmonary pressure gradient, the degree of lung recoil, and the resistance to liquid efux offered by the fetal glottis and upper airway. During apnea, when fetal breathing movements (FBMs) are absent, active adduction of the glottis increases the resistance to lung liquid efux and promotes its retention within the future airways. This promotes lung expansion, and the resultant increase in lung recoil generates the small intraluminal hydrostatic pressure (1 or 2 mmHg above ambient pressure) that characterizes fetal apneic periods. During FBMs, phasic abduction of the glottis greatly reduces the resistance to lung liquid efux via the trachea and, therefore, the liquid leaves the lungs at a greater rate; usually two or three times the rate observed during apnea (Figure 2). Individual FBMs are essentially isovolumetric because the chest wall partially collapses when the diaphragm contracts, giving rise to small changes in thoracic volume with each movement. This is mainly due to the high viscosity of lung liquid, compared with air, which greatly increases the resistance to uid movement through the fetal airways, particularly at high ow rates. Because the fetal chest wall is very compliant, it is unable to sustain the transpulmonary pressure gradients required to move liquid at ow rates equivalent to the postnatal ow of air. As a result, the tidal volume in the fetus is very small (o0.3 ml kg 1) compared to that of the newborn (710 ml kg 1). The transpulmonary pressure gradient is a major determinant of fetal lung liquid volume and is inuenced by a number of factors, particularly fetal posture. For example, when intrauterine space is limiting, which can occur in the absence of amniotic uid (oligohydramnios), the fetus is forced into a exed position that greatly increases the curvature of the thoracoabdominal spine. This chronically increases abdominal pressure, elevates the diaphragm, and increases the transpulmonary pressure gradient leading to a decrease in lung liquid volume. The resultant decrease in lung expansion is considered to be the primary mechanism for the lung hypoplasia induced by oligohydramnios. Other changes in fetal posture (e.g., stretching), resulting in a decrease in spinal curvature, explain the inuxes of lung liquid that are occasionally observed during periods of FBMs. Because fetal body movements most commonly occur during FBMs, reductions in transpulmonary pressure associated with these movements would promote the inux of lung liquid when upper airway resistance is low.
Fetal Lung Liquid Clearance at Birth

Fetal lung liquid exits the lung by owing out of the trachea, whereby it is either swallowed or enters the amniotic sac. The factors regulating liquid movement within the fetal airways, particularly during late gestation, are complex but primarily involve the

Fetal lung liquid must be removed rapidly at birth to allow the onset of air breathing, and failure to do so

248 BREATHING / Fetal Lung Liquid

Glottis adducted High resistance to liquid efflux

Dilated glottis Low resistance to liquid efflux

0 mmHg

Intraluminal distending pressure 12 mmHg

Lung liquid

Intraluminal pressure 0 mmHg

Lung liquid

Figure 2 Control of fetal lung liquid volumes during periods of apnea and FBMs. During apnea, the glottis is actively adducted, which restricts the efux of lung liquid and promotes the accumulation within the future airways, thereby maintaining an intraluminal distending pressure of 1 or 2 mmHg above ambient pressure (amniotic sac pressure). During periods of FBMs, the glottis phasically dilates, which greatly reduces the resistance to lung liquid efux. As a result, the liquid leaves the lungs at a higher rate, causing a reduction in lung liquid volume and the distending pressure (at end-expiration) reduces to ambient pressure.

is a major cause of respiratory morbidity in newborn infants. In particular, it is relatively common in preterm infants and near-term infants delivered by cesarean section in the absence of labor. Thus, the processes responsible for lung liquid clearance at birth mature relatively late in gestation, and labor plays a major role in activating and/or facilitating these processes. One mechanism for lung liquid clearance at birth results from a reversal of the osmotic gradient driving liquid secretion. This process is initiated by a cAMP-mediated activation of amiloride-inhibitable Na channels, located on the apical surface of lung epithelial cells, resulting in an increase in Na entry into the cell. This increases Na/K ATPase activity, located within the basolateral membrane, resulting in increased Na and Na -linked Cl ux across the epithelium, from lung lumen to interstitium. This reverses the osmotic gradient and promotes the uptake of lung liquid into the interstitial tissue, from where it is gradually cleared via the circulation and lymphatics. The increase in intracellular cAMP levels is thought to be due to an epinephrine-mediated increase in b-adrenoceptor activity because epinephrine is a potent stimulator of lung liquid reabsorption in late gestation. The rigors of labor, particularly during delivery of the head, induce a major stress

response within the fetus, leading to a large increase in a number of stress-related hormones, including epinephrine and vasopressin. Because vasopressin can also stimulate lung liquid reabsorption, it is likely that a number of stress-related hormones act through the same pathway. The ability of epinephrine to induce lung liquid reabsorption matures late in gestation and is mediated by the prepartum increase in circulating glucocorticoids (perhaps in synergy with thyroid hormones), which also mature many other aspects of the lung. This explains why preterm infants commonly suffer from liquid retention within the pulmonary airways after birth and explains one of the many benecial effects that antenatal glucocorticoids, administered to women at risk of preterm labor, have on neonatal respiratory outcome in preterm infants. The maturational effect of glucocorticoids on lung liquid reabsorption involves an increase in the response of epithelial cells to epinephrine as well as an increase in the intracellular machinery responsible for lung liquid secretion/ reabsorption; this includes Na/K ATPase activity as well as Na channel expression. Although liquid uptake across the epithelium is a major mechanism for clearing liquid from the terminal airways, it is unlikely to be the only mechanism.

BREATHING / Fetal Lung Liquid 249

The maximum reabsorption rates (810 ml h 1 kg 1) demonstrated experimentally cannot account for the large volume of liquid that must be cleared, particularly because this process does not begin until delivery of the head. Similarly, the measured increase in lung tissue water content at birth cannot account for the total volume of liquid that is cleared from the airways. Although it was once assumed that lung liquid was forced from the airways due to compression of the chest as it passed through the birth canal, this theory has been discounted because the head and shoulders offer the widest obstacle for human delivery. However, because transpulmonary pressure is a major factor regulating the volume of fetal lung liquid, the effects of labor on fetal posture are likely to significantly contribute to liquid loss from the airways. Reduced amniotic uid volumes, particularly following rupture of the membranes, as well as uterine contractions during labor, are known to cause posture-related increases in transpulmonary pressure and lung liquid loss. This may explain the large loss of liquid that occurs after labor has begun but before (by many hours) second-stage labor commences and delivery of the head begins (i.e., when stress-related hormones are released).

The Role of Fetal Lung Liquid in Development and Disease

Fetal lung liquid plays a major role in the growth and development of the fetal lung by acting as an internal splint that maintains the lungs in an expanded state. The luminal volume of the fetal lung is higher than the end-expiratory lung volume (functional residual capacity) of the postnatal air-lled lung. The decrease in resting lung volume at birth (by 515 ml kg 1) is due to the removal of the distending inuence of lung liquid in addition to the entry of air into the airways. The latter causes an air/liquid interface to form, which creates surface tension and, despite the presence of surfactant, increases lung recoil. This causes partial collapse of the lung and the formation of a subatmospheric intrapleural pressure after birth, which does not exist before birth. During fetal life, the distending inuence of the lung liquid provides a stretch stimulus that is essential for lung growth and determines the three-dimensional tissue structure of the lung as well as the differentiated state of alveolar epithelial cells. If the distending inuence of lung liquid is removed, growth and structural development of the lung ceases, particularly that of the terminal air sacs, and alveolar epithelial cells differentiate into the surfactant secreting type II cell phenotype. On the

other hand, overdistension of the fetal lungs induces a rapid increase in lung growth that can result in a near doubling in lung cell number within 7 days. Similarly, structural development accelerates and alveolar epithelial cells differentiate into type I cells following prolonged overdistension, which can reduce the proportion of type II alveolar epithelial cells to o2% of all alveolar epithelial cells. Because lung growth and development are very sensitive to the degree of fetal lung expansion, it is not surprising that most instances of fetal lung hypoplasia in humans are caused by conditions that reduce fetal lung expansion. These conditions include oligohydraminos, whether it is induced by premature rupture of the membranes or urinary tract abnormalities (which reduce or prevent the ow of fetal urine into the amniotic sac), congenital diaphragmatic hernia, or other space-occupying thoracic lesions (e.g., tumors and cysts). As indicated previously, oligohydramnios causes an increase in fetal trunk exion, which increases the transpulmonary pressure gradient and reduces the degree of lung expansion. Herniation of the diaphragm allows abdominal contents to migrate into the fetal thorax, which acts as a space-occupying lesion that prevents lung expansion. In both conditions, the reduction in lung growth can be so severe that, after birth, the resulting respiratory insufciency is fatal. The presence of lung liquid within the fetal airways may also affect the pulmonary circulation. Studies indicate that the distending inuence of lung liquid, and the small hydrostatic pressure it generates, may contribute to the high pulmonary vascular resistance (PVR) in the fetus. As a result of the high PVR, B88% of right ventricular output bypasses the fetal lungs and enters the systemic circulation via the ductus arteriosus, which connects the main pulmonary artery with the descending aorta. With the clearance of lung liquid and the onset of ventilation at birth, PVR markedly decreases, allowing a rapid increase in pulmonary blood ow, to accept the entire output of the right ventricle, and the ductus arteriosus to close. The mechanisms responsible for the high PVR near term and the sudden decrease at birth are multifactorial. However, evidence indicates that the small hydrostatic distending pressure, caused by the retention of liquid within the future airways, may be a major contributing factor by causing compression and closure of perialveolar capillaries. Loss of this distending pressure at birth and the increase in lung recoil may contribute to the rapid decline in PVR when air rst enters the lungs.
See also: Breathing: Breathing in the Newborn; Fetal Breathing; First Breath. Lung Development: Overview.

250 BREATHING / First Breath

Further Reading
Barker PM and Olver RE (2002) Invited review: clearance of lung liquid during the perinatal period. Journal of Applied Physiology 93: 15421548. Bland RD and Nielson DW (1992) Developmental changes in lung epithelial ion transport and liquid movement. Annual Review of Physiology 54: 373394. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209224. Harding R and Hooper SB (2004) Physiologic mechanisms of normal and altered lung growth. In: Polin RA, Fox WW, and Abman SH (eds.) Fetal and Neonatal Physiology, pp. 802811. Philadelphia: Saunders. Hooper SB and Harding R (1995) Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clinical and Experimental Pharmacology and Physiology 22: 235247. Olver RE, Walters DV, and Wilson M (2004) Developmental regulation of lung liquid transport. Annual Review of Physiology 66: 77101.

attempts to separate the rst breath from the breathing-like activities preceding birth. In fact, it is wellknown that the fetus presents intermittent breathing from at least the second trimester of gestation (see Breathing: Fetal Breathing). Breathing-like movements in utero are not for gas exchange, which is provided by the placenta, but are important in controlling the pulmonary uid and, with it, fetal lung growth. In egg-laying animals such as birds, the denition of rst breath is blurry because, even before hatching, breathing contributes to gas exchange, in conjunction with that provided by the chorioallantoic membrane. Small marsupials are another special case. In fact, these newborn mammals do not need to ventilate the lungs at birth because gas exchange occurs by diffusion through their skin. Hence, in these neonates, breathing acts occur sparsely, seemingly randomly, for several days after birth.

Physiological Processes

First Breath
J P Mortola, McGill University, Montreal, QC, Canada
& 2006 Elsevier Ltd. All rights reserved.

Mechanics of the Onset of Breathing

Abstract
In mammals and other vertebrates, intermittent breathing activities originate early during development; however, the term rst breath is used to indicate the rst breathing act of a new independent organism. Usually, this coincides with the establishment of continuous pulmonary ventilation, although this is not always the case. What triggers the onset of regular breathing at birth remains an unsolved question. The general state of stress, created by all the new stimuli and events surrounding the time of birth, and the sudden increase in oxygenation, raises metabolic rate. The gaseous component of metabolism could be the common mechanism for the establishment of a sustained ventilation after birth. The respiratory work of the rst inspiration is high because of the friction of the column of pulmonary uid as it is displaced toward the peripheral airways. The formation of the airliquid interface generates surface pressure, which is the primary determinant of the recoil pressure of the lungs. Lung expansion with air and the rise in oxygenation are the main factors responsible for the changes in pulmonary circulation at birth. The early breathing pattern is irregular, and the expiratory ows are briey interrupted by closure of the vocal folds. This mechanism raises intra-airway pressure and contributes to the clearing of the pulmonary uid.

Description
First breath indicates the rst breathing act of a new independent organism. When applied to mammals, it refers to the rst active muscle effort aiming to pump air into the lungs at birth. The specications independent organism and at birth are semantic

The fetal lung is lled by uid of its own production. At birth, some of the uid is squeezed out of the upper airways during the passage through the pelvic canal. However, this mechanism is not crucial because differences in the mode of delivery (by breech or head, by cesarean section or vaginal) have no major impact on the cardiopulmonary adjustments at birth. Indeed, in many species with large litters, the mode of delivery alternates among pups, and in marine mammals delivery by tail rst is the norm. What is important, though, is the occurrence of labor because the hormonal surge accompanying this phase of delivery is responsible for the switch from the prenatal production of the fetal pulmonary uid to its postnatal absorption. Quantitative analyses of the mechanical events accompanying the rst breath are based on observations and measurements performed in infants and, to a lesser extent, in lambs; very little information is available on other species. The rst inspiration does not accomplish an immediate and homogeneous aeration of the lungs. In fact, although some alveolar areas pop open, many others will be ventilated only several minutes or even hours later. The work that the inspiratory muscles need to produce to expand the lungs with air is quite high, probably 810 times more than the respiratory work required during quiet breathing at older ages or in adults. The distortion of the thorax during inspiration, favored by the high compliance of the chest wall, is an additional factor that increases the total work of the inspiratory muscles.

BREATHING / First Breath 251

Lung volume (ml) P /r A few days 100

P V R

50 First breath

40

30

20

10

10

20

30

40

Pleural pressure (cmH2O)

Figure 1 Schematic representation of the changes in pleural pressure and lung volume during the rst breath and during some of the following breaths (dashed lines) until a few days. The arrows indicate the direction of the loop, and the areas of the loop are an approximate indication of the external work of the respiratory muscles. (Left) The silhouette of the lungs indicates that during the rst breath (bottom) of the total pressure (P ) required to generate ow (V ), the largest fraction is to overcome airow resistance (R ). Later (top), the largest component of the inspiratory pressure is due to surface tension (g ) and the radius of curvature (r ) of the airliquid interface.

The total inspiratory pressure is mainly contributed by the airow resistance and by the surface pressure (Figure 1). This latter is determined by the product of the surface tension at the airliquid interface and the radius of curvature of the interface. In the liquid-lled lung before the rst breath, because there is no interface, the surface pressure is nil. The interface forms with the entrance of air during the rst inspiration, and as the air progresses toward the small peripheral airways, the surface pressure continues to rise because the curvature of the interface continues to decrease. Eventually, after the rst few breaths, the pressure determined by surface forces will be the primary reason for the tendency of the lungs to deate. Surfactants produced by specialized cells of the alveolar epithelium play a fundamental role in lowering the surface tension and the propensity of the lungs to deate. This reduction in lung recoil pressure is important for the progressive establishment of the end expiratory reserve of air, or functional residual capacity (see Surfactant: Overview). Indeed, the rst breath contributes 1020% to what will be the functional residual capacity in an infant a few days old. The airow resistance of the rst inspiration is high because of the viscosity of the column of pulmonary uid. The incoming air displaces this uid toward the lung peripheral airways. Eventually, the uid will pass in the pulmonary interstitium, where it will be cleared by the pulmonary circulation and, to

a lesser extent, the lymphatics. The process of clearing the airways from the uid takes a few hours in human infants and probably a longer period of time in larger species. During this time, air and uid mix with the formation of foam, a process favored by the presence of surfactant. The air trapped in the pulmonary liquid in the form of foam contributes to gas exchange, and this explains why blood oxygenation rises rapidly in the minutes after birth, despite the much longer time required for the clearance of the uid. Once the airways are liquid-free, the pressure to overcome airow resistance is small. This is the main reason for the large decrease in the work of breathing of the following breaths compared to the rst breath. Theoretically, at birth several mechanisms may help the newborns respiratory muscles in generating the large pressures required for the rst breathing acts. For example, the contraction of the upper airway muscles, such as during sucking, or the outward recoil of the chest during recovery after the squeeze through the pelvic passage can produce subatmospheric pressures. Also, the erection of the pulmonary capillaries with the rise in pulmonary blood ow may contribute some negative (and therefore inspiratory) pressure within the airways. In reality, when subjected to experimental scrutiny, none of these mechanisms have demonstrated an appreciable contribution to the pressure required for the rst inspirations.

252 BREATHING / First Breath Changes in Pulmonary Circulation

Birth is accompanied by dramatic vascular changes. The lung shifts from receiving less than 10% of the cardiac output during fetal life to receiving practically all of it after birth. To a great extent, this change is due to the closure of the ductus arteriosus, which in fetal life permits a large fraction of the blood of the pulmonary trunk to bypass the pulmonary circulation, and to the drop in resistance of the pulmonary vessels (Figure 2). Because the ductus arteriosus enters the aortic arch, its postnatal closure reduces blood-ow to the descending aorta, in favor of the upper body and brain. The rst breath and the onset of continuous ventilation play a crucial role in the cardiovascular changes accompanying birth, mostly because of two mechanisms lung expansion and oxygenation. The increase in lung volume, by itself and independent from the rise in oxygen pressure, increases pulmonary blood-ow approximately vefold, according to animal experiments. This may seem astonishing given that lung volume does not differ much before and after the rst breath; in fact, the volume of the fetal liquid-lled lung is not lower, and most probably larger, than that of the air-lled postnatal lung. However, after birth, the surface tension created at the airliquid interface reduces the pressure in the

lung interstitial tissue, which promotes vascular expansion and a decrease in pulmonary vascular resistance. Lung ventilation also promotes the secretion of pulmonary prostaglandins, with vasodilating effects. The importance of the rise in oxygen levels in lowering pulmonary vascular resistance has been appreciated for a long time. Sustained hypoxia delays this process, leading to right ventricular hypertrophy. It also seems that the respiratory alkalosis accompanying the onset and establishment of pulmonary ventilation contributes to the vascular adjustments at birth.
The Early Breathing Pattern

Despite the mechanical constraints, in infants the rst inspiration is approximately 40 ml deep one of the deepest of the rst few days (Figure 1). It is not clear why this is the case. Among the various possibilities is a change in the threshold of the mechanisms normally controlling tidal volume, notably the airway slowly adapting stretch receptors and the central integration of their inputs. Although the activity of these receptors is related to changes in lung volume, their stimulus is transpulmonary pressure. In utero, the lungs are liquid-lled; hence, the transpulmonary pressure is low because of the absence of surface forces. At birth, with the entrance of air,

Fetus

Newborn

Aorta Ductus arteriosus 0.05 Pulmonary trunk Pulmonary 7

Aorta

Right ventricle

Left ventricle Lungs

Right ventricle

Left ventricle Lungs

Left atrium Right atrium Foramen ovale

Left atrium

Descending aorta 0.5

Figure 2 Schematic representations of the main features of cardiopulmonary circulation before birth (left) and after birth (right). Numbers indicate the approximate changes in blood ow occurring at birth as the result of lung expansion and oxygenation.

BREATHING / First Breath 253

surface forces immediately increase transpulmonary pressure. Therefore, intrapulmonary airway receptors may have to reset to the new mechanical condition, and in the process, the control of tidal volume may not be as accurate as it will be in the following breaths. Peripheral chemoreceptors are also known to undergo resetting with the abrupt change in stimulus. In the case of peripheral chemoreceptors, the abrupt change at birth is caused by the rise in arterial oxygenation with the onset of ventilation. Another set of airway receptors, the rapidly adapting ones, may be involved in the deep inspiration of the rst breath. These receptors are normally activated by a sudden lung stretch, and their reex effect (called Heads reex) is that of promoting the activity of the inspiratory muscles. Hence, the increase in transpulmonary pressure with the rst air expansion may provoke this reex, resulting in further activation of the inspiratory muscles. During the minutes following birth, the breathing pattern is very irregular, and short apneas are interspersed with bursts of high-frequency and shallow breathing. Tachypnea, with average rates between 70 and 90 breaths per minute, has been observed after both vaginal and cesarean section deliveries, and it usually subsides within a few hours. In infants who have difculty clearing pulmonary uid, transient tachypnea can last much longer. It seems likely that the high-frequency pattern is the reex response resulting from the stimulation of a group of receptors located in the pulmonary interstitium. These receptors are known to cause rapid and shallow breathing when activated by an increase in pulmonary interstitial pressure, such as during lung congestion. After the rst breath, and for much of the following hours, during expiration the ow is decreased, and often briey interrupted, by the narrowing of the vocal folds. Some widening and narrowing of the glottis with inspiration and expiration, respectively, are normal events during breathing. However, in the neonatal period narrowing is exaggerated, and in the rst minutes after birth interruptions of expiration at various times are common (Figure 3). This expiratory pattern, or grunting, is controlled by neural information from the lungs and serves important purposes. By delaying expiration, it maintains the average air volume within the lungs higher than it would be with unobstructed expiration, and it raises the end expiratory level. Because it increases the intra-airway pressure, grunting contributes to drainage of pulmonary uid from the peripheral airways into the lung interstitium, where it is picked up by the lymphatic circulation. This mechanism can be effective because the rise in intra-airway pressure is

Tidal volume inspiration

Obstructed expirations

Unobstructed expiration

End-expiratory volume

Figure 3 Schematic representation of a breath with unobstructed expiration (blue) and of several types of obstructed expirations (red). In the unobstructed condition, the lung volume decreases during expiration along a quasi-exponential trajectory. In case of obstruction, the expiratory trajectory is distorted, lung volume takes longer to decrease, and the end expiratory volume rises.

small. If airway pressure reached higher values, as can occur during crying or other forced expiratory maneuvers or during assisted positive airway pressure ventilation, it would hinder the pulmonary circulation and aggravate gas exchange.
What Triggers the Onset of Regular Breathing at Birth?

This question is still unanswered. Once outside the shielded intrauterine environment, the newborn is bombarded by many stimuli visual, acoustic, thermal, tactile, and pressure either totally new or of unusual intensity. Also, neural and chemical information present before birth assumes a new dimension after birth. For example, as air enters the lungs, the increase in transpulmonary pressure brings the airway slowly adapting receptors to a new level of activity. The arterial chemoreceptors, sensing a major increase in the partial pressure of oxygen, decrease or cease their prenatal activity; it will take a few days before they reset to the new oxygen level. The prenatal surge of many antioxidant enzymes testies to the uniqueness of birth as a hyperoxic event. The relative contribution of so many stimuli and new conditions to the onset of ventilation and the establishment of a steady pattern is difcult to quantify. Various experimental approaches have emphasized or dismissed one or the other mechanism. It is worth noting that at birth, as a result of the multiple stimuli and of the general state of stress, the newborns metabolic rate rises much higher than the fetal value. Also, the level of the metabolically produced carbon dioxide increases, which may play a fundamental role in the maintenance of pulmonary ventilation. In animal fetuses exteriorized and kept alive with extracorporeal circulation to provide for their gas exchange, continuous breathing did not

254 BREATHING / First Breath

1st breath 2nd 3rd 4th 5th 6th

Birth

Metabolic level

Thermal, mechanical and sensory stimuli, neural and chemical inputs

Figure 4 Many new stimuli are produced during the events surrounding birth. Either individually or by creating a general state of stress, they increase the metabolic level, which may represent the common mechanism for the maintenance of a continuous breathing pattern. Sensory stimuli include tactile, pressure, and pain stimuli. Neural inputs refer to the facilitation originating from the airway rapidly adapting receptors, as well as to neurochemical substances that may be increased within the brainstem as a result of air-breathing. Chemical inputs refer to the putative role of the chemoreceptors and to the state of relative hyperoxia. The spirometric recording at the top is from a newborn infant delivered by cesarean section. Time bars of 1 s are plotted along the zero volume line. Starting from the rst breath, the amount of air exhaled is less than the amount inhaled a surfactant-dependent process that forms the functional residual capacity.

start even after umbilical cord occlusion as long as carbon dioxide was not allowed to rise. Hence, it is possible that the many stimuli thought to be involved in the onset of breathing do so, ultimately, by favoring the increase in metabolic rate (Figure 4). Also, the relative hyperoxic condition accompanying birth may cause increased metabolism since hyperoxia has a hypermetabolic effect in young animals. Metabolic rate plays a key role in controlling the level of pulmonary ventilation in many circumstances, ranging from muscle exercise to responses to cold or hypoxia. Therefore, its crucial involvement in the maintenance of continuous respiration at birth would not be a special case. Contrary to the rather abrupt onset of continuous breathing observed at birth in the majority of mammals, in birds and other egg-laying animals breathing commences gradually. At rst, breathing is intermittent, and it becomes more regular and continuous during the many hours of the hatching process, in parallel with the progressive decline of the gas exchange function of the chorioallantoic membrane. This process is similar to that observed in some small marsupials, in which pulmonary ventilation becomes continuous over a period of several days, whereas gas exchange through the skin gradually regresses. These examples support the view that the establishment of continuous ventilation at birth is not necessarily an event timed with a particular stage of development. Rather, it may be so closely linked to gaseous

metabolism that as long as extrapulmonary organs take care of gas exchange, pulmonary ventilation does not need to become a continuous process.
See also: Breathing: Breathing in the Newborn; Fetal Breathing. Lung Development: Overview. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Pulmonary Circulation. Signs of Respiratory Disease: Breathing Patterns. Surfactant: Overview.

Further Reading
Bland RD (1997) Fetal lung liquid and its removal near birth. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientic Foundation, 2nd edn., pp. 21152127. Philadelphia: Lippincott-Raven. Hodson WA (1997) The rst breath. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientic Foundation, 2nd edn., pp. 21052114. Philadelphia: LippincottRaven. Lagercrantz H and Slotkin TA (1986) The stress of being born. Scientic American 254: 100107. Mortola JP (2001) Respiratory Physiology of Newborn Mammals. A Comparative Perspective. Baltimore: Johns Hopkins University Press. Strang LB (1991) Fetal lung liquid: secretion and reabsorption. Physiology Review 71: 9911016. Tod ML and Cassin S (1997) Fetal and neonatal pulmonary circulation. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientic Foundation, 2nd edn., pp. 2129 2139. Philadelphia: Lippincott-Raven.

BRONCHIAL CIRCULATION 255

BRONCHIAL CIRCULATION
E M Wagner, Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The bronchial circulation is the systemic vascular supply to the lung, and it supplies blood to conducting airways down to the level of the terminal bronchioles as well as nerves, lymph nodes, visceral pleura, and the walls of large pulmonary vessels. Within the airway wall, the circulation is composed of parallel vascular plexuses adjacent to airway smooth muscle. The density of this vascular network predicts its role in the clearance of aerosols delivered to the airway mucosa. As in other systemic vascular beds, inammatory cells are recruited to the airway wall through postcapillary venules. Inammatory proteins are largely vasodilatory in their effect on bronchial vascular smooth muscle tone. Most vasodilatory action is mediated at least partially through endothelial cell-derived nitric oxide. Inammatory proteins play a major role in the loss of bronchial endothelial barrier function causing airway wall edema, as demonstrated in conditions of asthma and allergy. The most prominent pathologic feature of the bronchial circulation is its proliferative capacity. Unlike the pulmonary vasculature, the bronchial circulation is pro-angiogenic in asthma, chronic pulmonary thromboembolism, interstitial pulmonary brosis, cystic brosis, and other inammatory conditions. Although the recruitment of new bronchial vessels to ischemic lung parenchyma may prove benecial, hemoptysis resulting from rupture of abnormal, bronchial vessels can be life-threatening.

Anatomy, Histology, and Structure

The variability in origin and number of systemic arteries perfusing the airways both among and within species has contributed to the difculty in study and the relative paucity of information concerning the physiological function of the bronchial vasculature. Leonardo da Vinci is frequently credited with providing the rst anatomical drawings of the bronchial circulation. Although he understood the need for this circulation as described in his notebooks that nature gave a vein and an artery to the trachea which would be sufcient for its life and nourishment, reproduction of his efforts demonstrated that his drawings were those of pulmonary veins. The rst complete anatomical drawings of the bronchial circulation in 1721 should be credited to the Dutch anatomist Frederich Ruysch. Since that time, the anatomical arrangement of the bronchial circulation has been described for humans, sheep, pigs, dogs, opossum, rabbits, and several small rodents. The bronchial vasculature originates from the aorta, subclavian, or

intercostal arteries (Figure 1). The bronchial artery courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. The bronchial arteries send arterioles throughout the airway adventitia that perfuse capillaries that are prominent in both the adventitia and the mucosa of the airway wall. Thus, the bronchial vasculature forms parallel vascular plexuses, situated on either side of the airway smooth muscle. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Bronchial anastomoses with pulmonary precapillary vessels have also been reported. Additionally, the bronchial artery sends branches to large pulmonary vessels as vasa vasorum, nerves, lymph nodes, and the visceral pleura. Values reported for absolute bronchial blood ow vary according to the method used to measure ow and the species evaluated. However, all reports demonstrate that ow through the bronchial vasculature represents less than 3% of cardiac output. The vasculature of the airway wall, which constitutes the major perfusion pathway, is tortuous and forms a dense network of vessels. Morphometry of postmortem or resected lung specimens from normal human subjects showed that blood vessels of the mucosa comprise o1% of the airway wall area and within the advential area approximately 8% of the wall area. Subjects with inammatory airways disease show a signicant increase in the overall number of airway vessels. However, several studies have suggested that the increase is proportionate to the increase in wall area. Few studies of the embryonic development of the bronchial vasculature exist. In humans, between weeks 9 and 12 of gestation, the bronchial artery arises as an outgrowth from the aorta. This process occurs later than the development of the pulmonary vasculature. One or two vessels extend from the dorsal aorta and form along the cartilaginous plates of the large airways. The vessels develop longitudinally along the airways to the lung periphery as far as the terminal bronchioles. However, the process by which the parallel vascular plexuses interdigitate and proliferate on either side of airway smooth

256 BRONCHIAL CIRCULATION

Aortic arch Carina Bronchial artery

Descending aorta

Figure 1 The bronchial artery originates from the aorta and courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Additionally, the bronchial artery sends branches to the pulmonary artery as vasa vasorum, nerves, lymph nodes, and the visceral pleura.

muscle has not been studied. Whether the ne capillary networks within the airway wall, the vasa vasorum within large pulmonary vessel walls, or vessels anastomosing with pulmonary vessels form as a continual outgrowth of arterial sprouting or as de novo formation from mesoderm is unknown.

Table 1 Methods to measure bronchial blood ow Method Flow probe Microspheres Artery cannulated/perfused Isolated vascular pouch Anastomotic blood collection Inert gas Species Sheep Dog, sheep Sheep Sheep Dog, sheep Human

Bronchial Circulation in Normal Lung Function

Given the separate structures within the lung that are perfused by branches of the bronchial vasculature, it is likely that the bronchial circulation regulates and supports diverse physiologic functions. However, due to the difculty in accessing this vascular bed in most species, its complex anatomy within the lung, and its small proportion of blood ow relative to pulmonary ow, the role of the bronchial circulation in supporting airway and lung function is incompletely described. Most published studies have focused on characterizing regulators of bronchial blood ow, with few directed at understanding function (Tables 1 and 2). However, several specic physiological functions have been dened and supported by experimental evidence, including that of airway cell nutrition, clearance of inhaled substances, recruitment of inammatory cells, and conditioning inspired air. The evidence for each of these functions is presented in the following sections.
Airway Cell Nutrition

Although all vascular beds exist to support the metabolic needs of the tissue perfused, assessment of

BRONCHIAL CIRCULATION 257

Table 2 Bronchial blood ow responses Vasoconstrictors a-Adrenergic agonists Endothelin Glucocorticoids Increased airway pressure Increased left atrial pressure Vasopressin Vasodilators b-Adrenergic agonists Adenosine Antigen Bradykinin Histamine Hypercarbia Hypoxia Nitric oxide Prostanoids

cellular function during acute changes in perfusion can provide suggestive evidence for the importance of maintaining a critical level of blood ow. Within the airway wall, it is questionable whether oxygen delivery by the blood is necessary given the proximity to airway luminal oxygen. In animal models, bronchial vascular smooth muscle demonstrated signicant reduction in tone when exposed to hypoxemia and hypercarbia. Additionally, when the sheep bronchial artery was perfused with an articial perfusate without calcium, a prompt reduction in airway smooth muscle tone was noted. In an effort to determine the importance of bronchial perfusion on mucociliary function in sheep, clearance of an insoluble tracer (99mTc-sulfur colloid) was determined. When bronchial blood ow was substantially reduced from a normal level, mucociliary activity was signicantly slowed. Whether this response was due to changes in mucus gland production, ciliary function, or neural control mechanisms, each could be related to alterations in substrate delivery to specic cells of the airway wall supplied by the bronchial artery. These studies demonstrate the importance of normal bronchial perfusion to maintain diverse cell function within the airway.
Clearance

These studies demonstrate the complexity of the interaction between aerosol uptake and the underlying airway vasculature. Vasomotor and endothelial barrier changes of the airway vasculature can impact aerosol uptake of both therapeutic and toxicological substances. Several studies have shown that the level of blood ow through the bronchial circulation can affect both the magnitude and the time course of agonistinduced airway smooth muscle constriction by contributing to the passive washout of delivered agonist. Furthermore, it is interesting to note that most airway smooth muscle agonists cause bronchial vasodilation, thus suggesting a homeostatic mechanism for preserving normal airway smooth muscle tone. Another aspect of clearance by this vasculature is conrmed by the study of the metabolic capacity of the bronchial endothelium. Angiotensin-converting enzyme (ACE) inhibitor signicantly depressed metabolism of a synthetic peptide substrate for ACE in an in situ perfused bronchial preparation. This study concluded that the bronchial circulation is pharmacokinetically and metabolically active with respect to bradykinin and that the enzymes responsible for this metabolic activity line the vascular lumen. Recent work conrmed the importance of ACE activity in vivo by demonstrating inhibition of bradykinin vasodilation after treatment with an ACE inhibitor. This result further suggests an important regulatory role for bronchial endothelial ACE in the metabolism of kinin peptides known to contribute to airway pathology. Each aspect of clearance, from inhaled substances removed from an active site causing physiologic changes to endothelial metabolism, suggests an important, albeit incompletely characterized, function.
Recruitment of Inammatory Cells

The normal function of any vascular bed is to support the removal of excess metabolites from a tissue. In addition, the bronchial circulation is juxtaposed between the airway epithelial barrier to the external environment of inhaled substances and the internal environment of the bloodstream. This circulation could be important in the systemic uptake of soluble, inhaled substances. Studies have shown that both large increases or decreases in bronchial blood ow decreased the blood uptake of a soluble tracer (99mTc-DTPA) deposited on airway epithelial surface. Subsequent work demonstrated that increased ow accompanied by increased pressures caused a hydrostatic edema that limited soluble tracer uptake.

Recruitment of leukocytes to the airway provides an important defense function due to their antimicrobial, secretory, and phagocytic functions. Leukocyte recruitment in systemic organs involves an orchestrated series of molecular events between rolling leukocytes and postcapillary venular endothelium. Since the tracheal/bronchial circulations are systemic circulatory beds, the same endothelial cell adhesion molecules likely are responsible for the welldocumented leukocyte recruitment of inammatory airways diseases. Although this is generally assumed, only recently have studies using intravital microscopy to document specic molecules and mechanisms of recruitment in vivo been performed in airways. Research suggests that ventilatory stresses imposed on airway endothelium may exert

258 BRONCHIAL CIRCULATION

additional cell stimulation that is not predicted by static endothelial cell culture conditions, thus conrming the molecules and mechanisms responsible for specic leukocyte recruitment in models that are relevant for airways disease appear to be essential.
Conditioning Inspired Air

Typically ascribed to the airway circulation is the function of heating and humidifying inspired air. Both the tracheal and the bronchial circulations have been shown to dilate when healthy human subjects or animals are challenged with dry air hyperventilation. However, whether the airway circulation contributes signicantly to heating and humidication has been debated because the pulmonary circulation provides a huge heat sink. In experimental systems in which bronchial perfusion could be limited, little effect on exhaled temperatures or on airway obstruction was observed. However, measurements of water exchange demonstrated that the bronchial vasculature served an important role in hydrating the airway mucosal surface and interstitial compartments of peribronchial tissues and limited the degree of airway mucosal injury after hyperpnea. Thus, it appears that the airway circulation is important in preserving normal humidication of airway cells.

Bronchial Circulation in Respiratory Diseases

Proliferation

The unique proliferative capacity of the bronchial circulation compared to the pulmonary vasculature in a variety of lung diseases has long been recognized. Chronic inammatory conditions and chronic pulmonary thromboembolism are the two most cited conditions leading to bronchial vascular proliferation. Inammation can contribute signicantly to bronchial vascular remodeling and life-threatening hemoptysis. Hemoptysis in the vast majority of patients originates from the systemic rather than the pulmonary vasculature, and the bronchial vessels are almost universally involved. Techniques for identifying and embolizing bronchial vessels have been well described. However, the etiology of hemoptysis requiring therapeutic embolization is variable, and the pathology of bronchial vessels leading to this acute condition is poorly understood. Several studies have focused on the chronic inammation of asthma and airway vascular remodeling. Li and Wilson demonstrated increased vascular density in biopsy specimens of subjects with mild asthma compared to normal control volunteers. However, others have shown that only biopsy specimens from

asthmatic subjects with concurrent Mycoplasma pneumoniae infections demonstrated signicantly increased vessel numbers. In studies of alterations in airway vascularity in models of inammation, rodent models have shown increased vessel numbers, size, and permeability characteristics. Airway remodeling following Mycoplasma pulmonis infection showed sustained alterations in the tracheal vasculature that could be reversed with corticosteroid treatment. Additionally, tracheal vessels of rats with chronic M. pulmonis infection demonstrated signicantly increased permeability when challenged with substance P. These results suggest abnormal endothelial cell barrier function after inammationinduced proliferation. Perhaps the most extensively studied form of systemic vascular proliferation within the lung is that which occurs after pulmonary artery embolization. In 1847, Virchow recognized that the bronchial circulation could proliferate and sustain lung tissue distal to a pulmonary embolism. Bronchial arteriograms in patients with chronic thromboembolic disease demonstrate the unique capacity of systemic vessels to proliferate and to invade the ischemic lung parenchyma. Both a dilated bronchial artery and a ne meshwork of vessels distal to the pulmonary occlusion can be seen. Neovascularization of the systemic circulation into the lung after pulmonary artery obstruction has been conrmed and studied in humans, sheep, dog, pig, guinea pig, rat, and mouse. Systemic blood ow to the lung has been shown to increase as much as 30% compared to the original pulmonary blood ow after pulmonary artery occlusion. Although precise mechanisms of vascular proliferation are still under investigation, in a model of chronic pulmonary thromboembolism in the mouse, the pro-angiogenic CXC chemokines appear to play a major role in vascular proliferation. This observation further supports a growing body of evidence, reported by Strieter and colleagues, from studies of non-small cell lung cancer and idiopathic pulmonary brosis that the ELR CXC chemokines are important pro-angiogenic factors within the lung.
Loss of Endothelial Barrier Function

Numerous studies on a variety of models, as well as autopsy specimens of human lungs, demonstrate the propensity for the systemic airway circulation to contribute to uid accumulation within and around airways. In particular, inammatory cytokines have been shown to cause the postcapillary venular endothelium to form gaps and allow transudation of plasma and protein into the interstitium. Changes in airway wall geometry due to uid and inammatory

BRONCHIECTASIS 259

cell accumulation and vasodilation of the bronchial vasculature have contributed to a geometric theory of asthma. Whether mechanisms are related to physical encroachment on small airway lumen or the loss of parenchymal tethering, inammationinduced changes involving bronchial vascular uid exudates likely contribute to the pathology of asthma. Another means by which the bronchial circulation can alter barrier function within the lung is through a portal-like response. Several studies have demonstrated that inhalation of injurious chemicals can result in loss of pulmonary endothelial barrier function. Pulmonary vascular leak due to smoke inhalation has been shown to be attenuated when the bronchial vasculature is occluded. Authors of separate studies concluded that inammatory proteins released within the large airways after smoke inhalation are carried by the bronchial vasculature to pulmonary tissue, where they impact pulmonary endothelial barrier function. However, these results are in contrast to those of Pearse and colleagues, who showed that bronchial perfusion attenuated the loss of pulmonary endothelial barrier function in ischemiareperfusion. These authors suggested that bronchial endothelial-derived nitric oxide helps to preserve pulmonary barrier function. However, in each of these models, the bronchial vasculature serves as a conduit to transport substances to the pulmonary circulation where function is altered.

See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Asthma: Exercise-Induced. Bronchodilators: Beta Agonists. Chemokines, CXC: IL-8. Cilia and Mucociliary Clearance. Endothelial Cells and Endothelium. Fluid Balance in the Lung. Leukocytes: Neutrophils. Lung Development: Overview. Neonatal Circulation. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

Further Reading
Butler J (ed.) (1992) The bronchial circulation. In: Lung Biology in Health and Disease. New York: Dekker. Cudkowicz L (1968) The Human Bronchial Circulation in Health and Disease. Baltimore: Williams & Wilkins. Deffebach ME, Charan NB, Lakshminarayan S, and Butler J (1987) The bronchial circulation small, but a vital attribute of the lung. American Review of Respiratory Disease 135: 463481. Wagner EM (1995) The role of the tracheobronchial circulation in aerosol clearance. Journal of Aerosol Medicine 8(1): 15. Wagner EM (1997) Bronchial circulation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientic Foundations, pp. 10931105. New York: LippincottRaven. Wagner EM (1997) Bronchial vessels. In: Barnes PJ, Grunstein MM, Leff AR, and Woolcock AJ (eds.) Asthma. New York: LippincottRaven. Wanner A (1989) Circulation of the airway mucosa. Journal of Applied Physiology 67: 917925. Widdicombe J (ed.) (2003) Archives of Physiology and Biochemistry. Eighth International Meeting of the DaVinci Society, Nimes, France, pp. 287368.

Bronchial Infections

see Bronchiectasis. Bronchiolitis. Panbronchiolitis.

BRONCHIECTASIS
R Wilson, Royal Brompton and Hareeld NHS Trust, London, UK R Boyton, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved. airways are permanently dilated, tortuous, and contain excess secretions. Characteristically, the elastin layer of the wall is decient, and muscle and cartilage show signs of destruction. Increased mucus production and impaired clearance predispose to bacterial infection, which stimulates chronic inammation in which lymphocytes predominate within the wall and neutrophils in the lumen. The inammation causes more damage, which in turn further impairs the lung defenses, leading to more infection and a vicious cycle of events. Treatment involves daily physiotherapy to drain affected areas and appropriate antibiotics to treat infection and thus reduce inammation.

Abstract
Bronchiectasis is abnormal chronic dilatation of one or more bronchi due to loss of structural elements in the bronchial wall. There are a number of known causes and several associated conditions, but the underlying cause is not known in many cases. The prevalence is unknown, but thin-section computed tomography has increased its recognition. Symptoms are of chronic productive cough, breathlessness, recurrent exacerbations usually provoked by infection, and tiredness. There may be crackles over affected areas but not in all cases, and there may be wheezes due to airow obstruction that is largely irreversible. Subsegmental

Introduction
Bronchiectasis is dened as abnormal chronic dilatation of one or more bronchi. This structural abnormality predisposes the bronchi to bacterial

260 BRONCHIECTASIS

infection. The most common symptoms are chronic cough and sputum production. Recurrent bronchial infections occur that may become chronic, causing daily purulent sputum production. Cases of cystic or saccular bronchiectasis, in which severe loss of bronchial wall structure leads to large balloon-like dilatations, are now infrequently seen in developed countries. This type of bronchiectasis often followed severe lung infections, usually in childhood, and was characterized by production of very large volumes of sputum and nger clubbing. Improved socioeconomic conditions, vaccination programs, and ready availability of antibiotics are responsible for the reduced incidence of cystic or saccular bronchiectasis. Much more common today is a cylindrical form in which the damage to the bronchial wall is less severe. This type of bronchiectasis, which is usually bilateral and may be diffuse, although the lower lobes are usually worst affected, has been called modern bronchiectasis. In varicose bronchiectasis, there are localized constrictions caused by scarring superimposed on cylindrical changes. Traction bronchiectasis occurs in brotic lung conditions such as brosing alveolitis, in which the airway walls are pulled apart by the brotic process. Interestingly, in these cases the bronchiectasis is usually asymptomatic, probably because the mucosa is unaffected. The current prevalence of bronchiectasis is unknown. Published data from older studies were based on chest radiographs, which have been shown to be very insensitive for detecting bronchiectasis. The availability of thin-section, high-resolution computed tomography (CT), which is a much easier investigation to perform than the old gold standard of bronchography, has led to increased recognition in patients who might otherwise have been undiagnosed or classied as having chronic bronchitis or asthma. CT has enabled a diagnosis of bronchiectasis to be made in much milder cases, and this must be kept in mind when comparing older studies to those published recently. Indeed, this can cause confusion in terminology because some patients with asthma or smoking-related chronic bronchitis have bronchiectatic airways by CT criteria. A high prevalence of bronchiectasis has been reported in specic ethnic groups (e.g., Native Americans in North America, New Zealand Maoris, and Western Samoans). However, it is unclear whether genetic predisposition, environmental factors, or social practices are responsible. Most patients with bronchiectasis are nonsmokers. This may be because a history of childhood respiratory problems is common, and consequently starting smoking is less likely, but it could also be inuenced by referral practice because a smoker presenting with a chronic

productive cough is more likely to be advised on smoking cessation rather than be referred for investigation.

Etiology
There are a number of known causes of bronchiectasis (Table 1) and several other conditions that have been associated with bronchiectasis (Table 2) without a clear understanding of the pathological link.
Congenital

Several types of congenital bronchiectasis occur because of the absence or deciency of elements of the bronchial wall that are crucial to the retention of its normal architecture. In WilliamsCampbell syndrome, there is no or much reduced cartilage in the bronchi, generally extending beyond the rst division. Congenital tracheobronchomegaly (Munier Kuhn syndrome) is another example of this type, and bronchiectasis can occur in Marfans and Ehlers Danlos syndromes. Patients with intralobar pulmonary sequestration have blind-ending bronchi that
Table 1 Causes of bronchiectasis Congenital (e.g., deciency of structural elements of the bronchial wall) Infective (e.g., tuberculosis, whooping cough, or nontuberculous mycobacteria) Mechanical obstruction within lumen (e.g., inhaled foreign body) or external compression (e.g., tuberculous lymph node) Decient immune response (e.g., common variable immune deciency or human immunodeciency virus) Inammatory pneumonitis (e.g., aspiration of gastric contents or inhalation of toxic gases) Excessive immune response (e.g., allergic bronchopulmonary aspergillosis or lung transplant rejection) Abnormal mucus clearance (e.g., primary ciliary dyskinesia, cystic brosis, or youngs syndrome) Fibrosis (e.g., brosing alveolitis, sarcoidosis, or radiation pneumonitis)

Table 2 Conditions associated with bronchiectasis Infertility (e.g., primary ciliary dyskinesia, cystic brosis, or Youngs syndrome) Inammatory bowel disease (e.g., ulcerative colitis, Crohns disease, or coeliac disease) Connective tissue disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, or Sjogrens disease) Malignancy (e.g., acute or chronic lymphatic leukemia) Diffuse panbronchiolitis, predominantly reported in Japan Yellow nail syndrome I dystrophic and colored (usually yellow) nails, lymphedema, and pleural effusions a1-Antiproteinase deciency, usually causes emphysema Mercury poisoning may cause Youngs syndrome (obstructive azoospermia, sinusitis, and bronchiectasis)

BRONCHIECTASIS 261

trap mucus and are prone to repeated infections that lead to bronchiectasis.
Postinfective

A severe pneumonia of any sort can damage the bronchial wall sufciently to leave residual bronchiectasis. Mycobacterium tuberculosis, Bordetella pertussis, and measles virus are particularly likely to cause damage, but the diagnosis can be difcult to make because the patients history of the illness is vague. Chronic cough and sputum production should follow the severe lung infection, and bronchiectasis should be conned to the affected area. Some patients give a history of childhood pneumonia when they present in adult life with bronchiectasis, but they have diffuse disease on CT and have had an asymptomatic period in between. The importance of the childhood history in such cases is uncertain, but it may be that the earlier event provides a starting point for later problems if a bronchial infection occurs and is not cleared from the damaged area. Bronchiectasis is a common but not universal nding in SawyerJames (Macleods) syndrome. This condition is a form of obliterative bronchiolitis that most commonly follows an infectious insult to the developing lung during the rst 8 years of life. There is unilateral hyperlucent, hypovascular lung with marked air trapping. Nontuberculous mycobacteria, particularly Mycobacterium avium complex, can cause bronchiectasis. This type of infection seems to occur particularly in middle-aged females who present with a long history of dry cough.
Mechanical Obstruction

bronchiectasis before they die. Bronchiectasis is a frequent complication of the rare X-linked hypogammaglobulinemia, in which there is complete absence of the B-lymphocyte system. Common variable hypogammaglobulinemia, in which there is a reduction in the amount of antibody present in one or more of the major classes, is much more commonly found when investigating an adult population with bronchiectasis. These patients will often give a history of acute severe respiratory infections (pneumonia) in addition to their chronic symptoms. Solitary IgA deciency occurs in approximately 0.2% of the population and can be present without any clinical problems, although some patients suffer frequent viral-like illnesses. IgG subclass deciency may be transient and can also occur in healthy subjects. More important is functional antibody deciency, which involves failure to mount an antibody response to polysaccharide antigens (e.g., Streptococcus pneumoniae capsular antigen). This is associated with low IgG-2 levels, low pre-immunization pneumococcal antibody levels, and failure to mount a sustained antibody response to pneumococcal vaccination. An acquired immunodecient state can occur secondary to multiple myeloma, chronic lymphatic leukemia, protein-losing enteropathy, nephrotic syndrome, or lymphoma. Bronchiectasis can also complicate HIV infection, and this may be seen more frequently now that patients are surviving much longer with antiretroviral therapy.
Inammatory Pneumonitis

Localized bronchiectasis can occur as a result of bronchial obstruction from any cause, and the obstruction can either be in the lumen of the airway (e.g., inhaled foreign body) or due to compression from outside (e.g., tuberculous lymph node at the origin of the right middle lobe bronchus). The obstruction leads sequentially to impaired mucus clearance, distal infection, chronic inammation, damage to the bronchial wall, and bronchiectasis. Middle lobe syndrome, which involves recurrent acute infections at this site, has been described in cases without evidence of obstruction. It may be that the anatomy of the right middle lobe, which is long and acutely angulated with a collar of lymph nodes at its origin, is the reason.
Immune Deciency

Inhalation of toxic gases, such as ammonia or smoke by victims caught in res, and aspiration of gastric contents can cause sufcient damage to lead to bronchiectasis. Acid reux in patients with hiatus hernia can cause cough and wheeze, and may aggravate bronchiectasis symptoms, but is unlikely to be the sole cause of the disease.
Excessive Immune Response

Children with severe immune deciencies do not usually survive, although a proportion develop

Bronchiectasis in patients with allergic bronchopulmonary aspergillosis (ABPA) typically affects the proximal bronchial tree and the upper lobes, although more generalized disease is not uncommon. It is caused by an immune reaction involving eosinophils and fungus colonizing the airways. Atelectasis occurs because of obstruction by plugs of inspissated secretions containing fungal hyphae. Acute episodes of fever, wheeze, expectoration of viscid sputum plugs, and pleuritic pain, sometimes associated with consolidation in different areas on the chest radiograph, merge insidiously over time as bronchiectasis develops into chronic purulent sputum production,

262 BRONCHIECTASIS

where exacerbations of ABPA are difcult to distinguish from infective exacerbations of bronchiectasis. Some disorders causing constrictive obliterative bronchiolitis also damage the large airways. This pathology is seen in lung transplant rejection, chronic graft versus host disease, rheumatoid arthritis, and after inhalation of toxic gases.
Abnormal Mucociliary Clearance

Three different forms of impaired mucociliary clearance result in bronchiectasis: primary ciliary dyskinesia (Kartageners syndrome), cystic brosis (CF), and Youngs syndrome. Forms of infertility are associated with all three. Primary ciliary dyskinesia is rare and thought to be an autosomal recessive condition with incomplete penetrance involving numerous genes. There are usually, but not always, ultrastructural abnormalities in the cilia that make them move in a slow, disordered manner or the cilia may be completely static. Most commonly, there is the absence of one or both dynein arms (Figure 1), but abnormalities of the microtubules or radial spokes have been described. In cases with normal ultrastructure, the abnormality may be beyond the resolution of the electron microscope or may involve the orientation of the cilia on the cell surface. Cilia

line the nose, paranasal sinuses, and bronchi as far as the respiratory bronchioles and also form the tails of spermatozoa. Impaired ciliary function occurs at all these sites, and diffuse bronchiectasis is usually associated with chronic sinusitis, middle-ear disease, and often, but not invariably, male infertility. Females may have some reduction in fertility, and an increased risk of ectopic pregnancy, due to abnormal ciliary movement in the fallopian tubes. The condition may present in the neonatal period with pneumonia or segmental collapse due to mucus impaction or in childhood with recurrent infections. Patients cough continuously because this is their only form of mucus clearance. Approximately 50% of cases have dextracardia and a smaller proportion full situs inversus, which is thought to be due to abnormal cellular microtubules that are involved in rotation of organs in the embryo. Kartageners syndrome is bronchiectasis, chronic sinusitis, and dextracardia named after the German pediatrician who rst described it. Youngs syndrome is bronchiectasis, chronic sinusitis, and azoospermia due to a functional blockage of the sperm in the caput epididymis, which is usually enlarged and palpable in the scrotum. The cause is unknown, and it may occur later in life after successful parenthood. The respiratory secretions are viscid, making ciliary clearance inefcient. The
Outer arm Inner arm

Dynein

Normal cilium

Nexin link

Radial spoke

Cell membrane

Figure 1 (Top) Diagram of normal ultrastructure of a cilium and (bottom) electron micrograph of an abnormal cilium from a patient with primary ciliary dyskinesia that has no dynein arms.

BRONCHIECTASIS 263

syndrome has been linked to mercury poisoning in childhood (pink disease), and its incidence has decreased since calomel (mercurous chloride) was removed from teething powders and worm medication. A Youngs syndrome equivalent is recognized in women who had mercury poisoning in childhood. CF is caused by mutation of a gene on the long arm of chromosome 7 that codes for the CF transmembrane conductance regulator (CFTR), a cyclic-AMPdependent chloride channel that has wide ranging effects, including hydration of the airways. Most cases present in childhood and are associated with severe disease. However, late presentation has been described with atypical CF alleles, some of which are associated with milder bronchiectasis, normal pancreatic function, normal sweat electrolytes, and male fertility. Upper lobe bronchiectasis and culture of Staphylococcus aureus are clues in this type of patient, and CF genotyping should be performed in suspected cases.
Fibrosis

Traction bronchiectasis is common in the CT scans of many patients with all forms of pulmonary brosis, but this is rarely symptomatic. When symptomatic bronchiectasis does occur, it is most common in association with rheumatoid arthritis. In these cases, there may be an immunological explanation, or bacterial infection may establish itself within damaged lung, particularly if patients are immunosuppressed or if bronchial architecture is distorted. Infection causes neutrophilic airway inammation and mucus hypersecretion that can become self-perpetuating if the infection is not eradicated.
Associated Conditions

Pulmonary involvement is a frequent extraarticular manifestation of rheumatoid arthritis, and bronchiectasis may occur in the absence of interstitial lung disease. Airow obstruction and constrictive obliterative bronchiolitis may be associated with the bronchiectasis. Other connective tissue diseases, including Sjogrens syndrome and systemic lupus erythematosus, have varied pulmonary manifestations, including bronchiectasis. Bronchiectasis is associated with ulcerative colitis, Crohns disease, and celiac disease. The association of bowel and lung disease may be related to an underlying immune system dysregulation, with cells migrating between both sites and exposure of both epithelia to common environmental antigens and shared epithelial antigens. The association with ulcerative colitis is best established and two presentations are recognized. First, patients with a history

of severe colitis eventually undergo colectomy and then develop abrupt onset of cough and sputum production soon afterwards. Second, patients with one condition develop the other several years later. In the latter patients, are ups of their bowel disease may or may not be associated with exacerbations in their lungs. Bronchiectasis associated with ulcerative colitis is varied in severity, but in some cases it is associated with orid diffuse neutrophilic airway inammation and very large volumes of purulent sputum, which is often sterile. Another feature is that bronchiectasis regresses with anti-inammatory medication, which contradicts the concept that bronchiectasis is always an irreversible condition. Onset of chronic cough and sputum production has also been linked to bowel resection in Crohns disease. Diffuse panbronchiolitis is a chronic airway condition rst described in Japan, where it seems to be much more common. Patients present in middle age with a productive cough and breathlessness. The condition has subsequently been found in Korea and China, and occasional Caucasian patients with a similar syndrome have been described. There is inammatory hypertrophy of the walls of the respiratory bronchioles, which involves inltration with plasma cells, lymphocytes, and foamy cells. If the condition is untreated, it progresses to bronchiectasis. There is a remarkable response to treatment with long-term low-dose erythromycin, which has led to the investigation of macrolide antibiotic treatment for other forms of bronchiectasis. Alpha-1-antiproteinase deciency predisposes to emphysema, particularly in cigarette smokers. Sometimes, bronchiectasis is also present, and occasionally it is the predominant pathology. There is a proteinaseantiproteinase imbalance in the airways of all patients with bronchiectasis, and possibly these patients were predisposed to airway as well as alveolar problems following an infection that caused initial airway damage. Yellow nail syndrome is a rare condition involving yellow discoloration of dystrophic nails, primary lymphedema, and pleural effusions. Chronic rhinosinusitis is common, and a signicant proportion of patients have bronchiectasis.
Idiopathic Bronchiectasis

Even at centers that perform a full set of investigations into possible causes of bronchiectasis, approximately half of patients remain idiopathic. There is a predominance of females in this group and also in many cases a history of wheezy bronchitis in childhood. This is followed by a period of good health before the onset of chronic cough and sputum

264 BRONCHIECTASIS

production in adult life, often in their 20s or 30s. Patients may describe the onset of their problems as a severe viral-like illness that went into their chest and did not resolve. Patients usually have chronic rhinosinusitis, which suggests that whatever abnormality is predisposing to the illness is present throughout the respiratory tract. These patients suffer from profound tiredness and difculty concentrating, much more so than patients with bronchiectasis due to a postinfective cause or primary ciliary dyskinesia. This condition may involve a dysregulated innate and/or adaptive immune response.

Table 3 Bacteria isolated from sputum of patients with bronchiectasis Common Hemophilus inuenzae Hemophilus parainuenzae Pseudomonas aeruginosa Less common Streptococcus pneumoniae Moraxella catarrhalis Stenotrophomonas maltophilia Staphylococcus aureus Mycobacterium species Other Gram-negative bacilli

Pathology
In parts of the lung with bronchiectasis, subsegmental airways are permanently dilated, tortuous, and partially or totally obstructed by copious amounts of secretions. Side branches of the airways are frequently obliterated. Structural proteins are lost from the bronchial wall, and there is a variable amount of brosis. Characteristically, the elastin layer of the wall is decient or absent, and the muscle and cartilage layers show signs of destruction. These changes weaken the wall and lead to the distortion of the normal architecture. The process often involves bronchioles, and long-standing obstruction may result in complete brosis of small airways. The airway epithelium is damaged and ciliated cells are lost. There is goblet cell hyperplasia and mucus gland hypertrophy. There may be peribronchial pneumonic changes with evidence of parenchymal damage. The pulmonary arteries may thrombose and can recanalize. With long-standing disease there is hypertrophy of the bronchial arteries with anastamosis and sometimes considerable shunting of blood to the pulmonary arteries. There is usually chronic inammation, in which lymphocytes predominate in the bronchial wall and neutrophils in the lumen. The walls of bronchi and bronchioles contain lymphoid follicles and nodes containing B lymphocytes, CD4 and CD8 T lymphocytes, and mature macrophages.

Bacteriology
The bacterial species isolated in bronchiectasis are listed in Table 3. Mixed infections are common. Bronchiectatic airways are often chronically colonized, and for long periods a balance may be struck in which the bacteria colonize the mucosal surface without stimulating a systemic inammatory response. Patients carry the same strain for long periods, and acquisition of a new strain is not necessarily associated with an exacerbation. The species

of bacteria and their concentration in the lumen are both important determinants of the level of inammation. Host factors and the presence of additional viral infection will also inuence when an exacerbation occurs, and it may be a combination of events that upsets the balance and provokes an exacerbation. This may lead to invasion of the mucosa and peribronchial pneumonia. The etiology and severity of the bronchiectasis inuence the type of bacterial infection. For example, S. aureus is associated with CF and ABPA, and Pseudomonas aeruginosa is associated with extensive lung disease and severe airow obstruction. The initial infection with P. aeruginosa is usually a nonmucoid strain, which becomes a mucoid phenotype when chronic infection occurs. More than any other species, P. aeruginosa is likely to establish chronic infection, and once this has occurred it usually persists for the rest of the patients life. However, compared to CF, there is less evidence that this is associated with more rapid disease progression. Patients with chronic P. aeruginosa infection do have worse quality of life, and this may be due to several factors: their underlying disease is worse; treatment is more often required in hospital for intravenous antibiotics because ciprooxacin is the only active oral antibiotic; because of antibiotic resistance, treatment is less effective and therefore exacerbations occur more frequently; and long-term antibiotic prophylaxis is used more frequently. Tuberculosis is a rare complication of bronchiectasis, but when it does occur the classic clinical and radiological features may not be present. A high index of suspicion should be maintained in patients presenting with persistent low-grade fever, weight loss, or hemoptysis. Environmental nontuberculous mycobacterial species are sometimes isolated from bronchiectatic sputum. This may be a one-off isolate indicating environmental exposure just before the sputum sample was obtained. The mycobacterium may establish itself in the airway without apparently affecting the patients condition. However,

BRONCHIECTASIS 265

long-term follow-up is required to ensure this is true colonization and not low-grade infection. Some species are more pathogenic and therefore more likely to require treatment (e.g., M. avium complex and M. kansasii). These species can cause infection in patients without pre-existing lung disease or demonstrable immune deciency. The infection may cause bronchiectasis, and in such cases it can be difcult to determine if the infection is primary or secondary. When there is a combination in a CT scan of mild to moderate cylindrical bronchiectasis and peripheral nodules, some of which may be cavitating, sputum samples should be sent for mycobacterial microscopy and culture.

Investigations

Clinical Features
Symptoms

The most common symptoms are chronic cough and sputum production. Exacerbations are caused by bronchial infections, which may be viral or bacterial or both, or there may be chronic bacterial infection causing daily purulent sputum production. Usually, an exacerbation is associated with increased sputum volume and purulence, although the volume can decrease because the sputum becomes more viscous and therefore difcult to clear. Low-grade temperature is common, whereas a high temperature is suspicious of pneumonia. Chronic rhinosinusitis is very common, and most patients have expiratory airow obstruction, the severity of which correlates with the severity of bronchiectasis. This is usually largely irreversible, although some patients do have an asthmatic component. Chest pains are common and are usually described as an ache, but they may be pleuritic during exacerbations. Hemoptysis is usually minor and complicates an exacerbation; serious hemoptysis requiring selective embolization or surgery is rare, probably because of the ready availability of antibiotics and because cystic bronchiectasis is less common. Undue tiredness and difculty concentrating occur in poorly controlled disease.
Physical Signs

These are listed in Table 4. Suspicion of bronchiectasis should lead to a high-resolution thin-section (e.g., fast scan time of 1 s or less, 1 mm sections, and 10-mm spacing) CT scan. Investigation may discover a treatable cause of bronchiectasis. Younger patients, those with associated conditions, and those in whom respiratory function is deteriorating and/or infective exacerbations are becoming more frequent or prolonged should be seen by a respiratory physician with a special interest in bronchiectasis who has access to all the investigations listed in Table 4. Chest radiographs are an insensitive test for bronchiectasis; in one study, they detected less than half of patients who subsequently had positive bronchography. The CT ndings are related to the presence of dilated air-lled bronchi, dilated secretion-lled bronchi, and loss of volume resulting from parenchymal loss, which leads to crowding of the bronchi. The appearance depends on the orientation of the bronchi to the scanning plane. When they lie in the same plane, they appear as tram lines that do not decrease (taper) in diameter in the usual way as they progress to the outside of the lung (Figure 2(a)). The easiest way to determine whether a bronchus is dilated is to compare it to the adjacent pulmonary artery. Dilated bronchi that are perpendicular to the
Table 4 Investigation of bronchiectasis All patients Chest radiograph PA and lateral Sinus radiograph Lung function tests High-resolution thin-section CT scan Sputum microscopy to determine cell type (neutrophils or eosinophils) Sputum culture and sensitivities Sputum smear and culture for acid fast bacilli Skin tests (atopy, aspergillus) Sweat test, nasal potentials, CF genotyping Cilia studies (if nasal mucociliary clearance is prolonged or nasal nitric oxide is low, proceed to light microscopy of ciliary beat frequency and then electron microscopy) Blood investigationsa Selected patients Fiberoptic bronchoscopy Videouoroscopy of swallowing to detect aspiration Ph study for acid reux Semen analysis Tests for associated conditions Blood tests for rarer immune deciencies
a Full blood count with differential white cell count; C-reactive protein; erythrocyte sedimentation rate; total immunoglobulin (Ig) levels of IgG, IgM, IgA, and IgE and specic antibodies to pneumococcus and tetanus antigens; aspergillus radioallergosorbent test (IgE) and precipitins (IgG); rheumatoid factor; protein electrophoretic strip; and a1-antiproteinase.

There may be coarse inspiratory crackles heard over the site of bronchiectasis, but sometimes there are no signs to suggest the diagnosis. Airow obstruction can cause wheezes, and there may be late inspiratory squeaks indicating small airway disease. Clubbing is unusual in modern bronchiectasis because it is associated with more severe disease. A 24 h sputum collection can be very informative because patients tend to be inaccurate in their description of the color and volume of what they produce.

266 BRONCHIECTASIS

Figure 2 High-resolution thin-section computed tomography of bronchiectasis demonstrating (a) a nontapering bronchus, (b) the signet ring sign, (c) tree-in-bud pattern, and (d) mosaic perfusion.

scanning plane have a circular appearance, and then the smaller pulmonary artery gives a signet ring appearance (Figure 2(b)). Mucus-lled bronchi appear as branching tubes or nodules, and many lled small airways (exudative bronchiolitis) give a treein-bud appearance (Figure 2(c)). End-expiratory scans exaggerate increased transradiency in areas where gas trapping has occurred because of obstruction of small airways. Since gas trapping is usually patchy, this gives a pattern of mosaic perfusion (Figure 2(d)). Peripheral blood inammatory markers (neutrophil count and C-reactive protein) and sputum appearance can be used to monitor the response of an exacerbation to treatment. Most patients with bronchiectasis require long-term follow-up because disease progression can occur insidiously or in a stepwise rather than gradual manner. A history of more frequent and prolonged exacerbations, not responding well to treatment, is usually associated with an increase in sputum volume and purulence, increased breathlessness, as well as increased tiredness. CT scan features associated with subsequent disease progression are bronchial wall thickening, mucus plugging, and tree-in-bud pattern. Lung function may show a decrease in FEV1 and gas transfer. These changes are reversible, but mosaic perfusion is not. Health status questionnaires, such as the St Georges Respiratory Questionnaire, and measures of exercise capacity, such as the shuttle walking test, can also be used to monitor the patients progress. A

deterioration should lead to a review of sputum bacteriology (including mycobacteria) and aspergillus serology, as well as physiotherapy practice, before considering a change to the management regimen. P. aeruginosa is often isolated for the rst time in these circumstances, but it may reect the patients poor condition rather than be the cause of it.

Pathogenesis
Excess mucus is produced in the dilated, tortuous bronchiectatic airways. The mucus is poorly cleared partly because of the abnormal anatomy but also because cilia are lost when the epithelium is damaged, and the mucus is less elastic and more viscous largely due to its inammatory cell DNA content. Bacteria adhere avidly to the stationary mucus and multiply so that they are often present in very high concentrations. Large numbers of neutrophils are attracted into the bronchial lumen from the circulation by chemotactic products of the bacteria and also mediators released by host cells (e.g., interleukin (IL)-8, C5a, and leukotriene B4). Serum levels of adhesion molecules are elevated, suggesting that endothelial activation also occurs in the lung. Activated neutrophils spill proteolytic enzymes and reactive oxygen species while trafcking through the lung and during phagocytosis. These may overwhelm the bodys ability to neutralize them and cause tissue damage in the affected area. The exuberant inammation contains infection within the

BRONCHIECTASIS 267

Impaired host defences

Tissue damage

Microbial infection

agar beads to establish chronic infection after they are instilled into the airway, but a considerable amount of pneumonia also occurs. Another model achieved the same result by partially ligating a bronchus and instilling bacteria distally, but this was technically difcult. Future work may utilize candidate genes to generate inducible transgenic animal models that mimic the abnormal inammatory responses seen in the lung in bronchiectasis.

Management and Therapy

Surgical Treatment
Inflammation

Figure 3 A vicious cycle of bacterial infection-driven, host inammation-mediated lung tissue damage in bronchiectasis.

lung, so bacteremia and spread of infection outside the lung are very rare. However, the inammation may fail to eradicate the infection due to impaired local host defenses, so the infection becomes chronic, and it may slowly spread to involve adjacent normal bystander airways. Immune complexes form between bacterial antigens and antibodies that are produced locally and arrive by transudation. These stimulate other inammatory processes. Epithelial cells, lymphocytes, and macrophages release cytokines and other factors that orchestrate and perpetuate the inammation. Bronchiectatic secretions contain large amounts of IL-8, IL-1a, IL-1b, tumor necrosis factor-a, IL-6, and granulocyte colony-stimulating factor. The lung defenses are impaired by the damage caused directly by bacterial products and by inammation, which in turn promotes continued infection. This has been termed a vicious cycle of events (Figure 3). Bronchiectasis may occur because of an acute insult to the bronchial wall (e.g., whooping cough in a young child or inhalation of a toxic gas), and the damaged area may subsequently become infected. In other cases, there may be a recognized deciency of the host defenses that renders the patient prone to infection (e.g., primary ciliary dyskinesia or common variable immune deciency). However, in many cases the starting point of the vicious cycle may be obscure. It seems more likely that as yet unknown deciencies in the hosts immune defense system, or an abnormality in the bodys ability to control the inammatory response, will explain these cases, and cases in which bronchiectasis progresses rapidly, rather than the direct effect of a virulent infectious process. There are no satisfactory animal models of bronchiectasis. A rat model uses bacteria embedded in

The only curative treatment of bronchiectasis is surgical resection. Surgery is only appropriate when the bronchiectasis is localized and there is no underlying condition that predisposes to generalized bronchiectasis, such as primary ciliary dyskinesia. Good results are obtained from resection of severe localized bronchiectasis when the bronchiectatic area contributes little to lung function, which is otherwise reasonably preserved. Modern cylindrical bronchiectasis is usually bilateral, and surgery is rarely considered for this reason. Palliative surgical resection may be considered if a localized area of severe bronchiectasis dees medical management and acts as a sump for infection of other areas, even if less severe bronchiectasis is present elsewhere. Lung transplantation (usually two lungs or heartlung) is used successfully to treat respiratory failure due to bronchiectasis.
Medical Treatment

Patients with daily sputum production must perform postural drainage at least once daily and do this two or three times daily during exacerbations. They are taught to adopt the correct position to drain affected areas and to clear mucus by controlled breathing techniques sometimes aided by chest percussion. Patients need to be reminded about and encouraged to perform physiotherapy, and their technique should be regularly reviewed. Physical exercise should also be encouraged because it aids mucus clearance. A number of conditions that are recognized as causes or associations of bronchiectasis may require specic treatment (e.g., hypogammaglobulinemia and allergic bronchopulmonary aspergillosis). In patients with an asthmatic component, inhaled bronchodilators and corticosteroids are used in the usual way, and beta-agonists are often used to improve mucociliary clearance. However, there is little evidence to support the widespread use of inhaled corticosteroids in the absence of asthma. If this approach is used, then careful measure of objective benet should be sought (e.g., lung function). Short

268 BRONCHIOLITIS

courses of systemic corticosteroids may be used during severe exacerbations. Patients with chronic respiratory failure require long-term oxygen treatment. Carbon dioxide retention is sometimes a problem. Nasal intermittent positive-pressure ventilation is effective in these cases and surprisingly well tolerated despite a history of sinusitis. This approach is also effective in assisting mucus clearance. Antibiotics are given during infective exacerbations associated with purulent sputum, breathlessness, and malaise. The choice of antibiotic is inuenced by the likely bacterial species (Table 3), (previous) sputum culture results, and the presence or absence of P. aeruginosa. As a general rule, because of the damaged airways and the level of infection, the antibiotic dosage has to be higher and the course longer than usual. The amount of bacterial resistance is also high due to previous antibiotic prescription (e.g., b-lactamase production in Hemophilus inuenzae). Intravenous antibiotics are used for severe exacerbations, resistant species, and when oral antibiotics have failed. Continuous antibiotic treatment can be given as prophylaxis by the oral or nebulized route or as regular planned courses of intravenous treatment. This improves patient well-being, probably by reducing the frequency of exacerbations and the number of bacteria in the lung (and thus the level of inammation). However, there is no evidence that it alters disease progression, and there is a denite risk that it will drive the lung ora toward more resistant strains and species, such as P. aeruginosa and Stenotrophomonas maltophilia. This approach should be reserved for patients who, despite optimum medical management, continue to have very frequent (more than six per year) exacerbations, some of which involve admission to the hospital. There has been great interest in the past few years in macrolide antibiotic prophylaxis. The benet is thought not to be due to their action of killing bacteria but via effects on inammatory cells and possibly mucus production. Several studies have

provided encouraging results, and this is a promising area of future research.

See also: Bronchiolitis. Bronchomalacia and Tracheomalacia. Cilia and Mucociliary Clearance. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defense Systems. Immunoglobulins. Panbronchiolitis. Pneumonia: Mycobacterial. Primary Ciliary Dyskinesia.

Further Reading
Amsden GW (2005) Anti-inammatory effects of macrolides an underappreciated benet in the treatment of community-acquired respiratory tract infections and chronic inammatory pulmonary conditions? Journal of Antimicrobial Chemotherapy 55: 1021. Barker AF (2002) Bronchiectasis. New England Journal of Medicine 346: 13831393. Bush A, Cole PJ, Hariri M, et al. (1998) Primary ciliary dyskinesia diagnosis and standards of care. European Respiratory Journal 12: 982988. Evans D, Bara AI, and Greenstone M (2005) Prolonged Antibiotics for Purulent Bronchiectasis, the Cochrane Collaboration. New York: Wiley. Hansell DM (1998) Imaging of obstructive pulmonary disease. Bronchiectasis Radiology Clinics of North America 36: 107128. Hermaszewski RA and Webster ADB (1993) Primary hypogammaglobulinaemia: a survey of clinical manifestations and complications. Quarterly Journal of Medicine 86: 3142. Homma H, Yamanake A, Tanimoto S, et al. (1983) Diffuse panbronchiolitis. A disease of the transitional zone of the lung. Chest 83: 6369. Mukhopadhyay S, Singh M, Cater JI, et al. (1996) Nebulised antipseudomonal antibiotic therapy in cystic brosis: a metaanalysis of benets and risks. Thorax 51: 364368. Pasteur MC, Helliwell SM, Houghton SJ, et al. (2000) An investigation into causative factors in patients with bronchiectasis. American Journal of Respiratory and Critical Care Medicine 162: 12771284. Wilson R (2003) Bronchiectasis. In: Gibson GJ, Geddes DM, Costabel U, Sterk PJ, and Corrin B (eds.) Respiratory Medicine, pp. 14451464. London: Saunders/Elsevier. Wilson R and Abdallah S (1997) Pulmonary disease caused by non-tuberculous mycobacteria in immunocompetent patients. European Respiratory Monograph 12: 247272.

BRONCHIOLITIS
R L Smyth and S P Brearey, Alder Hey Childrens Hospital, Liverpool, UK
& 2006 Elsevier Ltd. All rights reserved. is characterized by wheeze, respiratory distress, and poor feeding. Respiratory syncytial virus (RSV) is the most common cause for bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. The healthcare burden of bronchiolitis is large, due to large numbers of hospitalized infants and the high risk of nosocomial spread during epidemics. Most children will suffer only mild, short-lived symptoms. A small proportion will need admission to hospital, where treatment is generally supportive until the

Abstract
Bronchiolitis is a common respiratory tract infection usually affecting infants and young children during annual epidemics. It

BRONCHIOLITIS 269
illness resolves. Some will require ventilatory support for which mortality can be up to 10%. Infants at high risk of severe disease include those born prematurely, those with chronic lung disease, and immunocompromised infants. RSV infects ciliated epithelial cells, causing sloughing of the epithelium, cytokine and inammatory mediator release, increases in mucus production, and interstitial edema. Clinical manifestations of bronchiolitis are a combined result of viral toxicity and the immune response to infection. Innate immune responses are important to the pathogenesis of bronchiolitis, as severe infection tends to occur after maternal antibody protection has waned and before the infants adaptive immune responses have matured. Immunoprophylaxis, in the form of intramuscular anti-RSV IgG1, is effective in reducing rates of hospitalization for high-risk infants.

Introduction
Bronchiolitis, meaning inammation of the bronchioles, is a clinical complex usually affecting children less than 2 years old. It is characterized by wheezing, dyspnea, tachypnea, and poor feeding. The clinical characteristics, originally termed congestive catarrhal fever, have been recognized for over 150 years. It was not until the late 1950s that the epidemiology and viral etiology of the illness were described. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. Epidemics occur during the winter months in temperate climates and during the rainy season in tropical climates (Figure 1). In the US, more than 120 000 infants are hospitalized annually with RSV infection, with more than 200 deaths
5000 4500 4000 3500 Number of reports 3000 2500 2000 1500 1000 500

attributed to RSV lower respiratory tract disease. Total hospital charges for RSV-coded primary diagnoses over the 4-year period from 1997 to 2000 have been estimated at US$2.6 billion. Within hospitals, there is a high risk of nosocomial spread of RSV during epidemics, which can put vulnerable infants at risk of severe disease. Infection in infancy also predisposes children to the development of recurrent wheeze, thus increasing the long-term healthcare burden of this disease. Prophylaxis is available as a recombinant monoclonal antibody, but is expensive and currently limited to infants at highest risk of severe disease. Development of an effective vaccine would have a dramatic effect on morbidity and healthcare costs, but this is unlikely to occur in the near future. Treatment is generally supportive until the infection runs its natural course.

Etiology
RSV is the primary cause of bronchiolitis. Seroepidemiological studies show that over 90% of children are infected with RSV by 2 years of age. Infection is spread by droplet exposure or direct contact with secretions. Fomites are infectious outside the body for up to 12 h. RSV was rst isolated from chimpanzees in 1955 and was originally called chimpanzee coryza agent (CCA). Subsequently, RSV has been shown to cause 7585% of cases of bronchiolitis. Other pathogens known to cause bronchiolitis are shown in Table 1. Historically, in over 10% of cases of bronchiolitis, no pathogen is detected. More

0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year
Figure 1 Laboratory reports to Communicable Disease Surveillance Centre, Health Protection Agency, UK of infections due to respiratory syncytial virus in England and Wales, 19902004 (4 weekly). Reproduced with permission from HPA.

270 BRONCHIOLITIS

recently, polymerase chain reaction (PCR) techniques have identied other pathogens, such as human metapneumovirus (hMPV) and coronavirus, which may have gone undetected previously. Co-infection with
Table 1 Bronchiolitis pathogens Respiratory syncytial virus Rhinovirus Parainuenza viruses (1,2,3) Inuenza viruses (A and B) Human metapneumovirus Coronavirus (NL-63) Adenovirus Bordatella pertussis Mycoplasma pneumoniae

hMPV and RSV may be associated with a higher risk of severe disease. RSV is a negative-sense single-stranded RNA pneumovirus from the Paramyxoviridae family (Figures 2 and 3). The viral genome contains only 10 genes that encode for 11 viral proteins. Nine of these are structural proteins and glycoproteins that form the viral coat and bring about attachment to host cells, whilst the other two direct viral replication within the host cell. Table 2 describes the functions of each protein. Based on immunological techniques, two different strains of the virus have been identied: A and B. Subsequently, it has been demonstrated that the two strains are also genetically distinct. Most variability between strains is due to differences in the amino acid sequence of the G protein on the viral coat. Strain A is seen more commonly in the UK and North America. There is some evidence that strain A results in more severe infections. The F protein, responsible for fusion of infected cells with adjacent uninfected cells, facilitates cell-to-cell transmission of the virus and results in epithelial cell syncytia (appearance of apparently large multinucleate cells), which give the virus its name.

Pathology
RSV initially causes inammation of the bronchioles and destruction of ciliated epithelial cells. The submucosa becomes edematous, whilst cellular debris and mucus form plugs within the bronchioles. Neutrophils and alveolar macrophages are the

Figure 2 Negative stain electron micrograph of RSV. Scale 100 nm. Reproduced with permission from Professor C A Hart.

Envelope proteins

G SH F

Nucleocapsid complex (N, P, and L proteins)

Matrix M1 proteins M2 Negative-sense RNA genome Lipid envelope

Figure 3 A schematic diagram of the RSV virion.

BRONCHIOLITIS 271
Table 2 RSV proteins and their functions Protein Surface glycoproteins F Function Virus penetration and fusion of infected cells with uninfected cells Virus attachment Unknown

G SH Nucleocapsid-associated proteins N P L M2-1 M2-2 Matrix protein M1

Encapsidates genome Phosphoprotein RNA polymerase Transcription elongation factor Regulation of transcription

May mediate association of nucleocapsid with envelope

Non structural proteins NS1 NS2

Antagonize interferon-induced antiviral response Unknown

predominant inammatory cells in the small airways. There is a peribronchiolar inltration with lymphocytes that is associated with edema uid accumulating within the alveoli. Severe disease involves destruction of the respiratory epithelium, parenchymal necrosis, and formation of hyaline membranes. The bronchiolar epithelium regenerates after 34 days, but cilia do not reappear until up to 15 days after the illness has resolved.

Clinical Features
The spectrum of severity for infected children is wide. After an incubation period of 12 days, most infants exhibit predominantly upper respiratory tract symptoms. These include rhinorrhea, cough, and low-grade fever that often persists for several weeks, before resolving, without any other symptoms. Thirty to fty percent of infants will progress to develop lower respiratory tract signs and symptoms within 23 days of infection. Infants may develop a rapid respiratory rate, wheeze, and other signs of respiratory distress (subcostal and intercostal recession, tracheal tug, and nasal aring). Apnea at presentation is common, especially in infants less than 6 weeks old, those with a history of apnea of prematurity, and those with congenital heart disease. Approximately 23% of infected children require admission to hospital. Reasons for admission include hypoxia, inadequate uid intake, apnea, and signs of imminent respiratory failure. A more severe cough,

pharyngitis, conjunctivitis, and otitis media may also be present. Irritability, signs of cardiovascular compromise, lethargy, and exhaustion are late signs and may be followed by respiratory arrest if no clinical interventions are made. One to two percent of infants hospitalized with bronchiolitis require ventilatory support and mortality in this group may be up to 10%. Premature birth, chronic lung disease of prematurity, congenital heart disease, cystic brosis, and immunodeciency all predispose infants to a high risk of severe disease. In addition to this, boys are more likely to be hospitalized than girls. Infants exposed to high levels of particulate air pollution or cigarette smoke are also more likely to suffer from severe bronchiolitis. A chest radiograph will typically show hyperination, a attened diaphragm, and patchy peribronchial inltration. The chest radiograph can be normal even in severe cases. A reduction in oxygen saturations is associated with increased respiratory rate and is an accurate indicator for reduced gas exchange that is seen in severe disease. Arterial or capillary blood gas analysis is helpful in assessing changes in the clinical status, particularly if ventilatory support is being considered. Most infants, given adequate supportive care, improve clinically in 34 days. Within 2 weeks of the height of their illness most infants will have a normal respiratory rate and any radiological abnormalities will have cleared. However, up to 20% of infants may suffer from persistent wheezing and airway obstruction for several months, especially those that required hospital admission. In the longer term, airway reactivity is increased in children after infection and persists for at least 58 years. It is still uncertain whether RSV causes asthma in later life. RSV infection does not confer lasting immunity to reinfection. However, subsequent infections are usually less severe and are more likely to be limited to the upper respiratory tract.

Pathogenesis
RSV rst infects the ciliated epithelial cells of the respiratory tract. Subsequent disease manifestations are caused by a combination of viral cytotoxicity and the immune response to infection. These effects are summarized in Figure 4. On contact with epithelial cells, the G protein attaches the virus to epithelial cells and allows viral RNA and enzymes to enter the cell and initiate production of new viral RNA and proteins. The F protein mediates the formation of syncytia that enable the virus to spread rapidly. Multiple new viruses are assembled within cells, which are ultimately

272 BRONCHIOLITIS
Soluble mediators: surfactant protein, immunoglobulin, sCD14 Opsonization Virus aggregation Increased phagocytosis

RSV

Epithelial cell sloughing Epithelial cell Macrophage

Airway inflammation Bronchoconstriction Mucus accumulation Surfactant inhibition

Inflammatory cytokines and chemokines

Dendritic cell

Neutrophil Eosinophil

Macrophage

NK cell

+ B lymphocyte CD4 T-helper cell

Th2 response Th1 response

CD8+ cytotoxic T cell

Figure 4 Pathogenesis of RSV bronchiolitis. RSV rst infects ciliated epithelial cells of the respiratory tract. Inammatory cytokines and chemokines, secreted by infected epithelial cells and macrophages, attract inammatory cells and cause inammation and bronchoconstriction in the airway. The predominant inammatory cells in the airway are neutrophils and alveolar macrophages. Dendritic cells are the main antigen-presenting cells that stimulate T-lymphocyte function. Neutrophil survival is prolonged and IL-9 secretion promotes mucus production. CD4 T-helper lymphocytes may be skewed to produce proinammatory (Th2) cytokines. The combination of epithelial cell sloughing, mucus plugging, and airway inammation leads to the clinical presentation of acute bronchiolitis.

destroyed, leading to sloughing of the epithelium. As the cells are destroyed they release proinammatory substances and cytokines that increase capillary permeability and attract inammatory cells such as neutrophils (interleukin-8 (IL-8) secretion), macrophages (IL-1b, MIP-1a), eosinophils (RANTES, MIP1a), and natural killer cells (interferon (IFN)-a/b). Some secreted mediators, such as IL-9 stimulate mucus production, whilst others, such as leukotrienes, cause bronchoconstriction. Pulmonary surfactant function is also inhibited. Increased production and impaired clearance of cellular debris and mucus results in plugging of the small airways, air trapping, and atelectasis. For infants with preexisting poor lung function, such as those with chronic lung disease, this airway obstruction is likely to have more clinical signicance and cause severe disease. The immune response to RSV includes innate and adaptive components. Innate immune responses are now recognized as more important than previously thought and are capable of inuencing the subsequent adaptive immune response.

Innate immunity is important in defense against RSV and other viruses that cause bronchiolitis as the adaptive immune responses of infants are relatively immature. The innate immune response is rapid and recruits effector molecules and phagocytic cells to the site of infection through the release of cytokines. Pulmonary surfactant provides early defense from viral infection. Surfactant proteins A and D are members of the collectin family that can bind to the surface of a number of pathogens including RSV. The virus is thus opsonized and capable of binding to complement and receptors on phagocytic cells (neutrophils and macrophages), eosinophils, and natural killer cells. Toll-like receptors (TLRs) are the most important of these receptors. They are present on epithelial cells and phagocytic cells and recognize distinctive nonhuman pathogen-associated molecular patterns (PAMPs). Although their precise role is still being elucidated, TLRs are important receptors to the innate and adaptive immune responses to RSV. TLR2 and TLR4 are receptors for surfactant proteins A and D, both of which bind to RSV. TLR4 and CD14 have been shown to be co-receptors for the

BRONCHIOLITIS 273

RSV F protein. Genetic polymorphisms in the TLR4 gene are associated with a risk of severe disease. TLR3 recognizes double-stranded RNA in endosomes of the cell and initiates an immune response to RSV. Pulmonary dendritic cells also express TLRs and are the predominant antigen-presenting cells in RSV infection. Circulating dendritic cells are recruited to sites of viral replication in the lungs. They produce large amounts of type I interferon (IFN-a and -b) and stimulate subsequent T-lymphocyte responses to RSV infection. Regulatory T lymphocytes (Treg) are known to dampen inammatory responses and could be stimulated by RSV either via TLRs expressed on their cell surface or via pulmonary dendritic cells. Therefore, they provide a way by which the T-lymphocyte response to RSV can be inuenced by both innate and adaptive immunity. Alveolar macrophages have phagocytic functions, but act mainly as antigen-presenting cells, interacting with helper and cytotoxic T lymphocytes. Along with respiratory epithelial cells they respond, via TLR and other signaling pathways, by secreting cytokines that increase tissue permeability as well as attracting and activating additional inammatory cells. Neutrophils are the predominant airway leukocytes in RSV bronchiolitis, representing about 90% of cells in the upper airway and 80% of cells in the lower airway. Neutrophil chemotaxis is dependent on the production of the potent chemotractant, IL-8, by airway epithelial cells and macrophages. IL-8 is secreted very soon after infection, leading to an almost immediate inammatory response before the infection is established. A later peak in IL-8 secretion is dependent on viral replication. Genetic polymorphisms in the IL-8 gene are associated with a risk of severe disease. Neutrophil recruitment and adherence to epithelial cells is increased by persistence of RSV infection. Neutrophils, whose survival is prolonged, secrete products such as myeloperoxidase and neutrophil elastase, which amplify viral cytotoxicity. Neutrophils have also been shown to secrete IL-9 in large quantities in the lungs of severely infected infants. IL-9 is a potent proinammatory cytokine that is known to cause eosinophilic inammation, bronchial hyperresponsiveness, and increased mucus production. Neutrophil function therefore plays an important role in the pathological changes that occur in bronchiolitis. Eosinophils might be expected to have an important role in the pathogenesis of bronchiolitis. They have antiviral activity and eosinophil chemoattractants are secreted by infected respiratory epithelial cells. Following the clinical trials of the formalininactivated vaccine, postmortem examinations revealed massive eosinophilic inltrates. However,

subsequent clinical studies have not reported signicant numbers of eosinophils in the airways of infants with RSV bronchiolitis. Adaptive immunity to RSV is generally composed of protective humoral immunity (B cells and antibodies) and viral clearance (T cells). The humoral response to RSV infection results in the production of IgG, IgM, and IgA antibodies in both blood and airway secretions. Protective benets of these antibodies are demonstrated by the reduced likelihood of infection in the rst month of life in term babies, due to the carriage of maternal IgG antibodies. Prophylaxis with monoclonal anti-RSV IgG (palivizumab), which has been shown to reduce the incidence and frequency of infections in high-risk infants, also demonstrates the role of the humoral response. Evidence from animal models conrms that antibody protects against RSV disease, but once infection is established, the T-cell response promotes viral clearance. The role of cell-mediated immunity is demonstrated in children with decient cellular immunity who shed the virus for many months after initial infection, compared to 2 weeks for immunocompetent infants. The cell-mediated response is composed of the CD8 cytotoxic T lymphocytes and CD4 T-helper lymphocytes. Cytotoxic lymphocytes are important in both recovery from and the pathogenesis of RSV bronchiolitis. They promote clearance of RSV from the lungs but also cause pulmonary injury. Functional studies in T-lymphocyte-depleted mice demonstrated prolonged RSV replication, yet no overt evidence of illness. CD4 T-helper cells have traditionally been subdivided further according to the cytokine proles they secrete: T-helper-1 (Th1) cells produce interferon gamma (IFN-g) and other antiviral cytokines. Th2 cells produce cytokines that induce eosinophil proliferation, and the release of leukotrienes and IgE antibodies leading to an enhanced inammatory response. The idea that RSV bronchiolitis might be a Th2-type disease, and that this may explain airway obstruction and postbronchiolitic wheeze, has somewhat limited evidence to support it. Multiple factors are more likely to inuence the T-lymphocyte response to RSV infection.

Animal Models
Several experimental animal models have been used to improve our understanding of the pathophysiology of RSV bronchiolitis. The variety of animal models used reects the diversity of clinical manifestations of RSV disease, dependent on an individuals age, genotype, phenotype, immune status, and concurrent disease. RSV has been shown to replicate in

274 BRONCHIOLITIS

animal models: primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters have been used. The need to investigate immunological responses to RSV, using good animal models, was highlighted after the unsuccessful development of the rst formalin-inactivated (FI) RSV vaccine in the 1960s. When natural RSV infection occurred in children given the vaccine, 80% of these children were hospitalized and two children died. Interpretation of the trial results was hampered by the lack of a small animal model, in which vaccine-enhanced disease could be studied. It is now apparent that other vaccine formulations are also capable of stimulating inappropriate immune responses to RSV. Therefore, comprehensive studies using animal models are essential before any further human vaccine trials are undertaken and are key to our understanding of the pathogenesis of bronchiolitis. Cotton rats have proved to be useful animal models, as they are uniformly susceptible to pulmonary infection through adulthood and are more immunologically responsive than mice to RSV. Studies using the cotton rat model were largely responsible for the development of RSV-neutralizing IgG in preventing pulmonary infection and treatment with ribavarin. Despite this, cotton rats have no congenic, transgenic, or knockout strains and there is only limited availability of reagents used to characterize immunological responses to RSV. Mice have been the most widely used model, due to a wide array of inbred, congenic, transgenic, and knockout strains. They also have the most reagents available, specic to RSV immunology, and have relatively low maintenance costs. Many insights have been gained into the immune response to RSV, which could not have been achieved using other models. The BALB/c mouse is particularly important, as it develops similar airway inammatory responses to humans after intranasal RSV infection. Mice studies have been useful in investigating the immunology of RSV infection, especially the cytokine responses to the virus and for experimental vaccines.

Despite considerable efforts to develop effective treatments for RSV bronchiolitis, no effective measures exist other than supportive care. The cornerstones of supportive care are supplemental oxygen to correct hypoxia and adequate uid administration. Infants with inadequate uid intake may need nasogastric feeding or, if this is unsuccessful, intravenous uids. Intravenous uids should be given with care due to the possibility of worsening lung uid accumulation and left ventricular overload. A large number of trials and meta-analyses have investigated the efcacy of b2 agonists and ipratropium for patients with bronchiolitis. There is no compelling evidence that bronchodilators are useful in the treatment of bronchiolitis. There may be a subgroup of patients for which bronchodilators are safe and efcacious. However, no criteria exist to identify this subgroup and a signicant number of studies found that patients deteriorated after receiving bronchodilators. The inconsistency of these results can be explained by our knowledge of the causes of wheeze in bronchiolitis. Bronchodilators will have no effect on increased mucus production, sloughed epithelial cells in the airway, or interstitial edema. Ribavarin is an antiviral preparation administered by aerosol and designed to inhibit the synthesis of viral structural proteins. Although early trials of ribavarin supported its use, later trials found no signicant positive effect. In addition, it is potentially teratogenic and labor intensive to administer. Other treatments that have failed to demonstrate consistent benets in the treatment of bronchiolitis include epinephrine, inhaled or systemic corticosteroids, recombinant human DNase, interferon-a, vitamin A, and antibiotics.

Prevention
Two immunoprophylactic agents are currently available that are recommended for infants at risk of severe infection. RSV immune globulin intravenous (RSV-IGIV) is a polyclonal hyperimmune globulin, prepared from donors selected for having high serum titers of RSV neutralizing antibody. Palivizumab is a humanized murine monoclonal IgG1 antibody against the RSV F-glycoprotein and is given as a monthly intramuscular injection during the RSV season. RSV-IGIV was the rst immunoprophylactic agent to be licensed for prevention of severe RSV bronchiolitis and clinical trials demonstrated a 4163% reduction in hospital admissions attributable to RSV lower respiratory tract infections. However, RSV-IGIV has a number of disadvantages that have resulted in a decline in its use in favor of palivizumab. Early trials reported an increase in postoperative mortality in patients with cyanotic

Management and Current Therapy

After admission to hospital, infants should be monitored and assessed regularly. Pulse oximetry, uid balance, and respiratory rate are useful in assessing changes in the infants condition. Barrier nursing and stringent hand-washing policies have been shown to reduce nosocomial spread of RSV. Rapid RSV antigen testing, by either immunouorescence or enzyme immunoassay, of nasopharyngeal secretions is useful in determining RSV status in hospital.

BRONCHOALVEOLAR LAVAGE 275

congenital heart disease who received RSV-IGIV. Administration required intravenous access and a 4-h infusion every month. There are concerns regarding volume overload, interference with live vaccines, and theoretical risks of infection from human donors. Trials of palivizumab have also demonstrated benets to high-risk infants, with early trials demonstrating a 4555% reduction in hospital admissions attributable to RSV lower respiratory tract infections. It is free from potential risk of infection from human donors, does not interfere with immune response to vaccines, and can be administered easily, without the need for hospital admission. Clinical trials also demonstrated its safety for infants with hemodynamically signicant congenital heart disease. However, no trial has demonstrated a signicant reduction in mortality due to RSV infection. Although palivizumab was shown to reduce rates of hospitalization and intensive care admissions in large double-blind randomized control trials, several studies have questioned its cost-effectiveness. The American Academy of Pediatrics recommends its use for certain high-risk groups: children under 2 years with chronic lung disease requiring medical therapy; infants born before 28 weeks gestation in the rst year of life; and infants born at 2932 weeks gestation up to 6 months of age. Infants born at 3235 weeks gestation have a lower risk of hospitalization and palivizumab is only recommended for these infants if they have two other risk factors such as child care attendance, school age siblings, parental smoking, or congenital abnormalities of the airways. Infants with hemodynamically signicant congenital heart disease are also likely to benet and palivizumab is recommended in this group, particularly those with cyanotic heart disease, congestive heart failure, or pulmonary hypertension. There is little knowledge regarding its benets for immunocompromised infants, children with cystic brosis, or prophylaxis in the second year of life.

See also: Antiviral Agents. Bronchodilators: Beta Agonists. Chemokines. Cilia and Mucociliary Clearance. Epithelial Cells: Type I Cells; Type II Cells. Genetics: Gene Association Studies. Immunoglobulins. Infant Respiratory Distress Syndrome. Leukocytes: Eosinophils; Pulmonary Macrophages. Mucus. Pediatric Pulmonary Diseases. Surfactant: Overview. Toll-Like Receptors. Vaccinations: Viral. Viruses of the Lung.

Further Reading
American Academy of Pediatrics Committee on Infectious Diseases and Committee of Fetus and Newborn (2003) Revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections. Pediatrics 112(6): 14421446. Arden KE, Nissen MD, Sloots TP, and Mackay IM (2005) New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia. Journal of Medical Virology 75: 455462. Black CP (2003) Systematic review of the biology and medical management of respiratory syncytial virus infection. Respiratory Care 48(3): 209233. Byrd LG and Prince GA (1997) Animal models of respiratory syncytial virus infection. Clinical Infectious Diseases 25: 13631368. McNamara PS and Smyth RL (2002) The pathogenesis of respiratory syncytial virus disease in childhood. British Medical Bulletin 61: 1328. Semple MG, Cowell A, Dove W, et al. (2005) Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. Journal of Infectious Diseases 191: 382386. Welliver RC (2003) The burden of respiratory syncytial virus (RSV) and the value of prevention. Journal of Pediatrics 143(5): S111S162. Welliver RC (2004) Bronchiolitis and infectious asthma. In: Feigin RD, Demmier GJ, Cherry JD, and Kaplan SL (eds.) Textbook of Pediatric Infectious Diseases, 5th edn., pp. 273285. Philadelphia: Elsevier. Wohl MB (1998) Bronchiolitis. In: Chernick V, Boat TF, and Kendig EL (eds.) Kendigs Disorders of the Respiratory Tract in Children, 6th edn, pp. 473485. Philadelphia: Harcourt. Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nature Reviews: Immunology 5: 5868.

Bronchiolitis Obliterans

see Interstitial Lung Disease: Cryptogenic Organizing Pneumonia.

BRONCHOALVEOLAR LAVAGE
M Drent and J A Jacobs, University Hospital Maastricht, Maastricht, The Netherlands
& 2006 Elsevier Ltd. All rights reserved. diseases. The analysis of cells obtained by BAL may be helpful in narrowing the differential diagnosis. The cellular analysis of the BAL uid (BALF) relies on an accurate identication and differential counting of the cells obtained. Universal standards are not available; consequently, each institution should ensure that the various readers of BALF samples have been properly trained in cellular analysis. Using a standard technique for performing the BAL and handling the sample will also reduce variability. To explore the possible role for BALF analysis in the

Abstract
Provided its limitations are kept in mind, there is a place for bronchoalveolar lavage (BAL) in the evaluation of diffuse lung

BRONCHODILATORS / Anticholinergic Agents 285

Bronchocentric Granulomatosis

see Systemic Disease: Sarcoidosis.

BRONCHODILATORS
Contents

Anticholinergic Agents Beta Agonists Theophylline

Anticholinergic Agents
N J Gross, Hines VA Hospital, Chicago, IL, USA
Published by Elsevier Inc.

the routine treatment of airways obstruction particularly for chronic obstructive pulmonary disease (COPD) but also for asthma in certain circumstances.

Chemistry Abstract
Anticholinergic agents have bronchodilator effects on the human airways and have a role in the treatment of obstructive airways diseases, particularly chronic obstructive pulmonary disease (COPD). Those in approved clinical use are synthetic quaternary ammonium congeners of atropine, and include ipratropium bromide and tiotropium bromide, each of which is very poorly absorbed when given by inhalation. Ipratropium has a relatively short duration of action and has been widely used for many years, either alone or in combination with the short-acting b-adrenergic agent albuterol for the maintenance treatment of stable COPD. Tiotropium, which was introduced in the early 2000s, has a duration of action of at least 12 days making it suitable for once-daily maintenance treatment of COPD. Both agents have a wide therapeutic margin and are very well tolerated by patients.

Introduction
Naturally occurring anticholinergic agents such as atropine and scopolamine which are present in many plants, for example, belladonna spp. have been used in traditional medical cultures to relieve breathlessness for millennia. These agents, which are all tertiary ammonium alkaloids, and which were smoked or chewed, are well absorbed into the systemic circulation and thus have multiple systemic side effects that limited their clinical usefulness as bronchodilators. In the last century it was discovered that synthetic quaternary ammonium analogs of atropine were very poorly absorbed but retained topical anticholinergic activity. This enabled the development of quaternary ammonium analogs of atropine such as ipratropium, oxytropium, and tiotropium bromides for clinical use as inhaled bronchodilators. In the last two decades, these agents have become important components in

The structure of some natural and synthetic anticholinergic agents is shown in Figure 1. The parent compound, atropine, is a tertiary ammonium alkaloid due to a 3-valent nitrogen atom on the tropine ring. Although widely used for respiratory purposes in previous eras, its rapid absorption and widespread systemic distribution resulted in unacceptable systemic effects. Atropine and other natural tertiary agents are no longer used for respiratory diseases and will not be further considered. Synthetic analogs such as ipratropium bromide and tiotropium bromide are quaternary ammonium compounds and carry a charge on the 5-valent nitrogen atom which renders them less able to cross tissue barriers. However, they are able to penetrate mucosal surfaces sufciently to exert pharmacologic actions on local structures such as smooth muscle.

Mode of Action
Anticholinergic agents compete with acetylcholine for muscarinic receptors, inhibiting the numerous
Br H2O + C H CH3 N 3 7 O CH2OH O C CH O CH2OH O C CH

CH3

Atropine

Ipratropium bromide

Figure 1 The molecular structure of tertiary and quaternary ammonium compounds.

286 BRONCHODILATORS / Anticholinergic Agents

housekeeping actions of the parasympathetic branch of the autonomic nervous system. When taken by the inhalational route, the poorly absorbed synthetic quaternary ammonium agents have actions that are limited to the sites of their deposition, namely the mouth where they inhibit salivary gland secretions, and the airways where they relax bronchial smooth muscle. For reasons that are not well understood, they tend not to inhibit respiratory gland secretions nor mucociliary clearance. Unlike other bronchodilators such as b-adrenergic agents or methyl xanthines, inhaled anticholinergic agents have no demonstrated effects other than as airway smooth muscle relaxants. They do not have clinically signicant anti-inammatory effects. They do not have signicant cardiac, or pulmonary vascular actions, nor systemic effects. They have not been shown to have long-term effects on disease progression (although trials with tiotropium that explore such an effect are underway at present).
Cholinergic Activity in the Lungs

receptors are present on bronchial smooth muscle cells. Both are believed to mediate bronchomotor tone and reex bronchoconstriction. M2 receptors, in contrast, are located on postganglionic autonomic bers and are believed to be autoreceptors whose stimulation provides feedback inhibition of further acetylcholine release from terminals of these nerves, tending to limit vagally mediated bronchoconstriction. An implication of this scheme is that M1 and M3 receptors would be appropriate targets for anticholinergic bronchodilators, while M2 receptors should be spared. Indeed, there is evidence that M2 receptors are selectively damaged by certain viruses as well as eosinophil products, perhaps accounting for the bronchospasm associated with viral infections and asthma.
Available Agents

In humans, airway caliber is largely controlled by the cholinergic parasympathetic system via branches of the vagus nerve, which stimulates smooth muscle contraction. At rest, parasympathetic activity provides bronchomotor tone, a low level of bronchial smooth muscle activity. There is evidence that resting bronchomotor tone is increased in both asthma and COPD. In addition to supplying bronchomotor tone, cholinergic activity can also be reexly increased by numerous stimuli such as inhalation of cold dry air, mechanical factors, irritant particles, aerosols and gases, and specic mediators such as histamine and bronchoconstricting eicosanoids. Clinically, the use of an anticholinergic agent aims both to abolish cholinergic tone of airway smooth muscle and to inhibit the phasic increases in reex bronchoconstriction.
Muscarinic Receptors in Lungs

In the US, ipratropium bromide is available as a metered-dose inhaler (MDI) and as a nubilizable solution. Combinations with albuterol are also available in both forms. Its duration of action, both as monotherapy and as a combination, is 46 h. Tiotropium bromide is available as a dry powder inhaler; its duration of action is greater than 24 h, probably at least 48 h. Oxitropium bromide is available in several countries outside the US, and has a duration of action of 68 h. All of these agents are functionally selective for M1 and M3 muscarinic receptor subtypes, and tend to spare M2 receptors by dissociating rapidly from the latter. The half-life of the tiotropium M3 receptor complex is 35 h compared with 0.3 h for ipratropium. Apart from tiotropiums somewhat slower onset of action, much longer duration of action, and possibly slightly greater bronchodilator action at peak effect, the actions and side effects of each of these agents are very similar.

Clinical Role and Uses

Stable COPD

Three muscarinic receptor subtypes, called Ml, M2, and M3, are expressed in human lungs (Figure 2). M1 receptors are found in peribronchial ganglia, and M3
M1 M2 M3

Preganglionic nerve

Ganglion

Postganglionic nerve

Smooth muscle

Figure 2 The presumed location of muscarinic receptors in the parasympathetic pathway of human lungs.

The major clinical use of inhaled anticholinergic agents is for the routine treatment of stable COPD. Both ipratropium, given three to four times daily, and tiotropium given once daily qam, are approved and recommended by current guidelines as options for rst-line therapy for stable COPD of all degrees of severity. In practice, tiotropium is generally preferred because its prolonged duration of action provides more consistent bronchodilation around the clock, and its once-daily use is more convenient for patients. The onset of bronchodilation is slower than with inhaled short-acting b-adrenergic agents, peak effects being reached at 3060 min with ipratropium

BRONCHODILATORS / Anticholinergic Agents 287

and at about 34 h with tiotropium. The magnitude of bronchodilation, measured as the peak of FEV1 improvement, is very approximately 0.150.25 l following a single dose of 40 mg ipratropium by MDI, 0.30 l following 400 mg of ipratropium by nebulization, and 0.30 l following 18 mg of tiotropium dry powder inhaler (DPI). Uniquely, regular use of tiotropium results in a rise in the prebronchodilator (trough) FEV1 of 0.10.15 l after about 1 week of once-daily use, and a peak FEV1 level (3 h after the morning dose) that is about 0.05 l higher than that after the rst day of use, indicating a further benet with regular use. These improvements with tiotropium compare favorably with those obtained with long-acting b-adrenergic bronchodilators. In parallel with improvements in FEV1, a decrease in functional residual capacity (a marker of hyperination) occurs by 4 weeks of regular treatment with tiotropium. No tachyphylaxis has been observed with long-term use of anticholinergic agents. In addition to these effects on lung function, measurable symptomatic improvements have been consistently reported with regular use of tiotropium such as clinically signicant improvements in dyspnea, exercise capacity, health status (quality of life), and most importantly, an approximately 2025% reduction in the frequency of acute exacerbations of COPD. The mechanism of the latter, which is shared by some other treatments for COPD, is not well understood.
Acute Exacerbations of COPD

agent. It has not been possible to predict reliably which asthmatics will benet from ipratropium other than by an individual trial. Ipratropium may also be a useful alternative bronchodilator for those rare asthmatic patients who cannot tolerate the palpitations or tachycardia that a b-adrenergic agent may produce. The role of tiotropium has not been studied in asthma.
Acute Severe Asthma

Although short-acting b-adrenergic agents are the bronchodilators of choice in the initial management of acute severe asthma, several studies and a metaanalysis suggest that the inclusion of ipratropium in the initial treatment of these serious events provides more rapid improvement in lung function and may avoid prolonged emergency room treatments and hospitalization. Current guidelines recommend that ipratropium (usually by nebulization) be added when the response to initial treatments with a short-acting b-agonist is less than complete or poor. In practice, it is very common for the combination of albuterol and ipratropium to be given routinely as initial treatment for episodes of acute severe asthma, at least for the rst few treatments.
Pediatric Airways Disease

Studies comparing bronchodilator treatments for acute exacerbations of COPD do not show consistent benet for treatments that include anticholinergic agents. Nevertheless, most current guidelines recommend the addition of ipratropium to a short-acting b-agonist in the initial treatment regimen for these serious events. Tiotropium is not appropriate as monotherapy for these events; however, it should be continued through the exacerbation when patients have been receiving it already.
Stable Asthma

For pediatric asthma, both stable and acute severe forms, the role of ipratropium is similar to that in adults. Although studies with adequate statistical power or long duration in children are lacking, adult doses have been used without adverse effects in children down to 4 years of age. As in adults, anticholinergic agents have a very limited role in asthma. The role of tiotropium has not been studied in pediatric asthma. There are occasional reports of the use of ipratropium in other pediatric diseases such as cystic brosis, viral bronchiolitis, exercise-induced asthma, and bronchopulmonary dysplasia. Although these sometimes suggest a benet from the use of ipratropium, the body of evidence is insufcient to make any recommendation in these conditions.

Numerous studies have compared ipratropium with a variety of short-acting b-adrenergic agents for the regular treatment of stable asthma. Almost uniformly, these reports show that ipratropium is a less potent bronchodilator than a short-acting b-adrenergic agent in asthmatic patients. No anticholinergic agent has been approved for use in asthma in the US. However, a few patients with otherwise typical asthma respond well to it, and it may occasionally add to the bronchodilation obtained with an adrenergic

Side Effects and Adverse Events

Being very poorly absorbed, inhaled anticholinergic agents have a very wide therapeutic margin and are very well tolerated, even in rare instances when massive doses have accidentally been given. In normal clinical use, dryness of the mouth is common to all agents in this class but is rarely sufciently severe for the patient to discontinue use. Bad taste is an occasional complaint, as is a brief coughing spell shortly

288 BRONCHODILATORS / Beta Agonists

after inhalation. One serious but rare idiosyncratic effect is paradoxical bronchospasm whose mechanism is unknown. It has been reported to occur in perhaps 0.3% of patients following use of any of the above anticholinergic bronchodilators. The drug should be discontinued in patients who experience wheezing, chest tightness, and dyspnea within an hour of the inhalation. Ipratropium was extensively studied for possible adverse effects on mucociliary clearance from the lungs, urinary outow, and increased intraocular pressure (well-known side effects of atropine). These were found not to be problems with inhaled ipratropium, nor have they been found with oxitropium or tiotropium. However, each of these agents can cause dilatation of the pupil and, possibly, precipitate acute glaucoma, if they are placed directly onto the eye. Care should be taken that neither the mist from a nebulized anticholinergic treatment nor even minute amounts of tiotropium dry powder accidentally nd their way into the eye.
See also: Acetylcholine. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview.

airway epithelial cells. Drugs targeted at b2-adrenoceptors fall into two main classes: short-acting b-agonists (SABAs) such as salbutamol and terbutaline and long-acting b2-agonists (LABAs) such as salmeterol and formoterol. These agents are in general composed of a benzene ring with a chain of two carbon atoms and either an amine head group or a substituted amine head group. Beta2-agonists interact selectively at the b2-adrenoceptor to stimulate elevation in cell cyclic AMP content which is responsible for the majority of downstream signaling events following from receptor stimulation. The major clinical effect of b2-agonists is airway smooth muscle relaxation which produces the acute bronchodilator effects of these drugs. In addition, b2adrenoceptor agonists are bronchoprotective agents, that is, they inhibit bronchoconstriction induced by irritant challenge, for example, with histamine or methacholine. The b2-adrenoceptor is highly polymorphic and potential functional effects which may be of clinical relevance have been described for some of these polymorphisms. SABAs are rst-line bronchodilators agents used in the management of asthma: LABAs should be used in the management of moderately severe asthma in combination with inhaled steroids. Side effects of these agents include tremor, tachycardia, and hypokalemia: there have also been concerns that regular use of some b2-adrenoceptor agonists (e.g., fenoterol) may be associated with asthma exacerbation and very rarely death.

Introduction
Beta-adrenoceptors were initially separated into b1- and b2-subtypes based upon differences in physiological response proles in the heart, blood vessels, and airways. Subsequently a b3-subtype was identied and shown to be present in a number of tissues, most notably adipocytes. The main consequences of activation of these three subtypes are shown in Table 1. Given the prole of receptor mediated effects shown in Table 1, the most effective b-agonists in the management of airway disease can be seen to be selective b2-adrenoceptor agonists. These fall into two main groups: the short-acting b2-agonists (SABAs) and long-acting b2-agonists (LABAs). Frequently used SABAs include salbutamol (albuterol) and terbutaline, whilst the most frequently used LABAs

Further Reading
Cattapan SE and Gross NJ (2002) Anticholinergic agents. In: Barnes PJ, Drazen JM, Rennard S, and Thomson NC (eds.) Asthma and COPD: Basic Mechanisms and Clinical Management, pp. 527534. New York: Academic Press. Celli BR and MacNee W (2004) Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. European Respiratory Journal 23: 932946. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology & Therapeutics 98: 5969. Global Initiative for Chronic Obstructive Lung Disease (2003) Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: Update. Bethesda: National Institutes of Health, National Heart, Lung, and Blood Institute. Gross NJ (2004) Tiotropium bromide. Chest 126: 19461953.

Table 1 Effects of the three subtypes of b-adrenoceptor

Beta Agonists
I P Hall, Nottingham University, Nottingham, UK
& 2006 Elsevier Ltd. All rights reserved.

b1-Adrenoceptor Tachycardia Positive inotropy

b2-Adrenoceptor Bronchodilatation Inhibition of mediator release from mast cells Mucus secretion Increased ciliary beat frequency Relaxation of uterine smooth muscle Venous and arterial dilatation

b3-Adrenoceptor Lipolysis

Abstract
Beta2-adrenoceptor agonists are the mainstay bronchodilator agents used in the treatment of asthma and chronic obstructive pulmonary disease. Three b-adrenoceptors have been characterized using molecular pharmacology and molecular physiological approaches: b2-adrenoceptors are the major receptor subclass mediating both bronchodilatation and effects on

BRONCHODILATORS / Beta Agonists 289

are formoterol and salmeterol. In the 1960s70s other drugs were used, including isoprenaline and fenoterol. However, because of concerns over an increase in asthma deaths associated with use of the nonselective b-agonist isoprenaline, this is no longer in general use in clinical practice: similar concerns regarding asthma deaths associated with fenoterol led to its withdrawal from the market in New Zealand and some other countries in the 1990s. Beta2-adrenoceptor agonists can also be classied according to whether they are partial agonists or full agonists. In most assay systems salbutamol and salmeterol act as partial agonists whereas terbutaline and formoterol are generally full or near full agonists. A number of novel b-adrenoceptor agonists are currently in development. In general these are full agonists with prolonged duration of action.

kinase A activation. In airway smooth muscle, these effects include phosphorylation of key targets, including myosin light chain kinase and calcium activated potassium channels, increased calcium sequestration and/or removal and inhibition of contractile signaling pathways (Figure 2). However, the correlation between whole cell cyclic AMP content and physiological effects (e.g., smooth muscle relaxation) is not precise which has led to the suggestion that some effects of receptor stimulation are cyclic AMP independent. These effects could potentially be mediated directly by the a-subunit of Gs or by non-Gs coupled pathways. This may be relevant to some of the longer-term effects of b2-adrenoceptor stimulation on gene transcription.

Role of b2-Adrenoceptor Agonists in Respiratory Medicine

Beta2-adrenoceptor agonists have two main clinically relevant effects. First, they provide the mainstay bronchodilator therapy used in the management of asthma and other airway diseases where reversible airow obstruction may be present, for example, chronic obstructive pulmonary disease (COPD). As required SABAs are rst-line agents for the management of mild asthma: most guidelines (e.g., those produced by the British Thoracic Society) suggest as required usage is appropriate as monotherapy as long as patients are not requiring more than an average of one dose per day. For those patients requiring frequent doses (i.e., more than one daily of SABAs), an inhaled steroid should be added to the treatment. In general, the LABAs should be used for those patients who are inadequately controlled on moderate doses of inhaled steroid (typically 400 800 mg of inhaled beclomethasone or equivalent) where control is inadequate. These drugs should not be used as monotherapy. Salbutamol, terbutaline, salmeterol, and formoterol are all available in both aerosol form administered via metered dose inhalers (MDIs) and also as dry powder formulations in most countries. Beta2-agonists are also used in the management of acute asthma where they are generally given by nebulizer (salbutamol or terbutaline) or in severe acute asthma by intravenous infusion. In the past, oral preparations of both salbutamol and terbutaline were used in the management of asthma but these have been superseded by long-acting inhaled formulations. A very small number of patients have been treated with subcutaneous b2-agonists (usually terbutaline): whilst there are anecdotal reports of benet there are no high-quality randomized control trials utilizing this

Chemical Structure
The chemical structure of the four most frequently used b-agonists is shown in Figure 1. Most are based on the naturally occurring catecholamine epinephrine. The structure activity relationship of these drugs depends on the presence or substitution of hydroxyl (OH) groups on the benzene ring and on the substituted head group. Substitution of the hydroxyl groups at position 3 and 4 on the benzene ring generally results in production of a less potent catecholamine: however, these drugs are more resistant to breakdown by catechol-O-methyl transferase (COMT). Substitutions on the a carbon atom prevent oxidation by monoamine oxidase (MAO). The action of b-agonists within tissue is terminated as a consequence of uptake into sympathetic nerve endings (uptake 1) or uptake into other innervated tissues such as smooth muscle (uptake 2) and subsequent degradation by COMT and MAO. Beta-adrenoceptor agonists can also be conjugated to sulfates or glucuronides in the liver, gut wall, and lung.

Mode of Action
Beta2-adrenoceptor agonists mediate clinical effects by stimulation of the b2-adrenoceptor. This receptor is one of the superfamilies of G-protein-coupled receptors (GPCRs). The b2-adrenoceptor is coupled via the stimulatory G protein Gs to adenylate cyclase. Increases in adenylate cyclase activity result in elevation of intracellular cyclic AMP content and subsequently activation of protein kinase A. The majority of the downstream events stimulated by b2-agonists are therefore a consequence of protein

290 BRONCHODILATORS / Beta Agonists

HO OH HO CHCH2NHCH3

Epinephrine H H HO CHOHCH2NHCCH2 CH3 OHCHN Formoterol HOH2C OH HO CH3 OCH3

CHCH2NHCCH3 CH3

Salbutamol CH(OH)CH2NH(CH2)6O(CH2)4Ph

CH2OH OH Salmeterol HO OH CH3

CHCH2NHCCH3 CH3 HO Terbutaline

Figure 1 Structure of a range of b-agonists.

approach which should only be initiated by specialist asthma clinics dealing with severe patients. The use of SABAs and LABAs is also recommended in most COPD guidelines: again SABAs should be used for rapid relief of symptoms and

LABAs reserved for patients with more severe disease. In general, the therapeutic effect in terms of both bronchodilatation and symptom relief is much smaller than in true asthma. Some patients with severe COPD have been treated with regular (four

BRONCHODILATORS / Beta Agonists 291

2AR

Gs ATP

AC

cAMP

PKA activation

Downstream effects (e.g., relaxation of airway smooth muscle)

5AMP Phosphodiesterases
Figure 2 Activity of b2-agonists. AC, adenylate cyclase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; b2AR, b2-adrenoceptor; Gs, stimulatory G protein; PKA, protein kinase A.

times daily) nebulized b2-agonists (sometimes in combination with an anticholinergic); this approach should be reserved for patients with severe disease and where there is objective evidence of benet. In addition to the bronchodilator effects of b2-agonists, this class of drugs also protects against the actions of bronchoconstrictor stimuli; this may be part of the basis for the benecial effects of LABAs seen when used regularly. These bronchoprotective effects can be readily demonstrated in the lung function laboratory by the administration of b2-agonists before bronchial provocation tests. The degree of protection seen is maximal within the rst 1224 h when regular dosing is instituted with both SABAs and LABAs; subsequently, the degree of bronchoprotection afforded falls somewhat although still remains above placebo levels.
Contraindications

In general, b2-agonists are safe and effective drugs in the management of reversible airow obstruction. However, there have been a number of concerns over the last 50 years regarding their usage. Arrhythmias have been reported following both intravenous and inhaled administration of b2-agonists. These are probably due to a direct effect of b2-adrenoceptor agonist stimulation in the heart and in addition hypokalemia which occurs as a consequence of stimulation of sodium potassium ATPase. These effects are likely to be related to relative selectivity for b2- over b1-receptors and dose; it is possible that arrhythmias were related to the excess of asthma deaths seen following the introduction of isoprenaline and fenoterol in the 1960s70s. Troublesome side effects with b2-agonists are relatively rare. The main limiting factor is tremor which again is dose related. There have been persistent concerns regarding regular usage of SABAs and LABAs in terms of over all

asthma control. These concerns arose from a number of studies where regular (generally four times a day) SABAs were used: in these studies overall asthma control and/or lung function deteriorated in groups taking regular as opposed to as required treatment. These studies were in part responsible for the advice to use SABAs on an as-required rather than regular basis in the management of asthma. Very recently these concerns have extended to LABAs resulting in a black box warning from the FDA. One possible explanation underlying this effect is the presence of polymorphic variation within the b2-adrenoceptor. The receptor is highly polymorphic, with nine known single nucleotide polymorphisms (SNPs) in its coding region. Four of these code for amino acid substitutions of which three may have functional relevance (Arg/Gly16, Gln/ Glu27, Thr/Ile164). The Thr164 polymorphism alters agonist-binding properties of the receptor but is rare (allelic frequency 3% in Caucasian populations). The Arg16 polymorphism has been associated in several studies with worse overall asthma control and in a recent prospective trial individuals given regular salbutamol who were Arg16 homozygous showed markedly reduced responses compared with individuals homozygous for the Gly16 form of the receptor. Arg16 homozygous individuals account for roughly 15% of the Caucasian population.
See also: Adrenergic Receptors. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. G-Protein-Coupled Receptors. Leukocytes: Mast Cells and Basophils.

Further Reading
British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guideline on the management of asthma. Thorax 58(supplement I): 194. Drazen JM, Israel E, Boushey HA, et al. (1996) Comparison of regularly scheduled with as-needed use of albuterol in mild asthma. New England Journal of Medicine 335: 841847.

292 BRONCHODILATORS / Theophylline

Fenech A and Hall IP (2002) Pharmacogenetics of Asthma. British Journal of Clinical Pharmacology 53(1): 315. Hall IP (2004) The beta-agonist controversy revisited. Lancet 363(9404): 183184. Hall IP and Tatterseld AE (1992) b-agonists. In: Clark TJ, Godfrey S, and Lee T (eds.) Asthma, 3rd edn., pp. 341366. London: Chapman and Hall. Israel E, Chinchilli VM, Ford JG, et al. (2004) Use of regularly scheduled albuterol treatment in asthma: genotype-stratied, randomised, placebo-controlled cross-over trial. Lancet 364: 15051512. Kobilka BK, Dixon RAF, Frielle T, et al. (1987) cDNA for the human b2 adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proceedings of the National Academy of Sciences of the United States of America 84: 4650. National Collaborating Centre for Chronic Conditions (2004) Chronic obstructive pulmonary disease: national clinical guideline on management of chronic obstructive pulmonary disease in adults in primary and secondary care. Thorax 59(supplement I): 1232. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334339. Sears MR, Taylor DR, Print CG, et al. (1990) Regular inhaled bagonist treatment in bronchial asthma. Lancet 336: 13911396. Tatterseld AE, Knox AJ, Britton JR, and Hall IP (2002) Asthma. Lancet 360: 13131322. van Schayck CP, Dompeling E, van Herwaarden CLA, et al. (1991) Bronchodilator treatment in moderate asthma or chronic bronchitis: continuous or on demand? A randomized controlled study. British Medical Journal 303: 14261431. Vathenen AS, Higgins BG, Knox AJ, et al. (1988) Rebound increase in bronchial responsiveness after treatment with inhaled terbutaline. Lancet 1: 554558. not controlled by bronchodilator therapy. Side effects are related to plasma concentrations and include nausea, vomiting, and headaches due to phosphodiesterase inhibition and, at higher concentrations, to cardiac arrhythmias and seizures due to adenosine A1 receptor antagonism.

Theophylline has been used in the treatment of airway disease since 1930. Indeed, theophylline is still the most widely used antiasthma therapy worldwide because it is inexpensive. Theophylline became more useful with the availability of rapid plasma assays and the introduction of reliable slow-release preparations. However, the frequency of side effects and the relatively low efcacy of theophylline have recently led to reduced usage since inhaled b2 agonists are far more effective as bronchodilators and inhaled steroids have a greater anti-inammatory effect. However, in patients with severe asthma and chronic obstructive pulmonary disease (COPD), it remains a very useful drug. There is increasing evidence that theophylline has an anti-inammatory or immunomodulatory effect.

Chemical Structure
Theophylline is a methylxanthine similar in structure to the common dietary xanthines, caffeine and theobromine (Figure 1). Several substituted derivatives have been synthesized but none has any advantage over theophylline, apart from the 3-propyl derivative enprofylline, which is more potent as a bronchodilator and may have fewer toxic effects. Many salts of theophylline have also been marketed, with the most common being aminophylline, which is the ethylenediamine salt used to increase solubility at neutral pH. Salts such as choline theophyllinate do not have any advantage and others, such as acepifylline, are virtually inactive so that theophylline remains the only methylxanthine in clinical use.

Theophylline
P J Barnes, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Mode of Action Abstract

Theophylline is a methylxanthine that has been used to treat airway diseases for more than 70 years. It was originally used as a bronchodilator, but the relatively high doses required were associated with frequent side effects, so its use declined as inhaled b2 agonists became more widely used. Recently, it has been shown to have anti-inammatory effects in asthma and chronic obstructive pulmonary disease (COPD) at lower concentrations. The molecular mechanism of bronchodilatation is non-specic phosphodiesterase inhibition, but the anti-inammatory effect may be due to histone deacetylase activation, resulting in switching off of activated inammatory genes. Theophylline is given systemically (orally as slow-release preparations for chronic treatment and intravenously for acute exacerbations of asthma), and blood concentrations are related to hepatic metabolism. This may be increased or decreased in several diseases and by concomitant drug therapy. Theophylline is usually used as an add-on therapy in asthma patients not well controlled on inhaled corticosteroids and in COPD patients with severe disease

Although theophylline has been in clinical use for more than 70 years, its mechanism of action is still uncertain. In addition to its bronchodilator action, theophylline has many other actions that may be relevant to its antiasthma effect (Figure 2). Many of these effects are seen only at high concentrations that far exceed the therapeutic range. There is increasing evidence that theophylline has anti-inammatory effects, with a reduction in eosinophils and T lymphocytes in asthma and of neutrophils in COPD. Conversely, withdrawal of theophylline results in clinical deterioration and increased inammation in patients with severe asthma and COPD. Several molecular mechanisms of action have been proposed for theophylline (Table 1). Phosphodiesterases, which break down cyclic nucleotides (cyclic

BRONCHODILATORS / Theophylline 293

O H3C O N N CH3 Theophylline
Figure 1 Structures of naturally occurring methylxanthines.

H N N H3C N

CH3 N H N

CH3 N

N CH3 Caffeine

N CH3

Theobromine

Agonist

Agonist

R G AC Theophylline

GC

PDE3,4,7 ATP 3,5cAMP + AMP GMP

PDE5 +

3,5cGMP

GTP

Bronchodilatation

Inflammatory cells

Figure 2 The inhibitory effect of theophylline on phosphodiesterases (PDE) may result in bronchodilatation and inhibition of inammatory cells. ATP, adenosine triphosphate; AMP, adenosine monophosphate; PKA, protein kinase A; GTP, guanosine triphosphate; GMP, guanosine monophosphate; PKG, protein kinase G.

Table 1 Mechanisms of action of theophylline Phosphodiesterase inhibition (nonselective) Adenosine receptor antagonism (A1, A2A, and A2B receptors) Increased interleukin-10 release Stimulation of catecholamine (epinephrine) release Mediator inhibition (prostaglandins, tumor necrosis factor-a) Inhibition of intracellular calcium release Inhibition of NF-kB (decreased nuclear translocation) Increased apoptosis Increased histone deacetylase activity (increased efcacy of corticosteroids)

AMP and cyclic GMP) in the cell, are weakly inhibited by theophylline, and this accounts for the bronchodilator effect of theophylline (predominantly phosphodiesterase-3 (PDE3) and PDE5) (Figure 2). However, it is unlikely to account for the nonbronchodilator effects of theophylline that are seen at subbronchodilator doses. Theophylline inhibits adenosine receptors at therapeutic concentrations and an inhibitory action on A2B receptors may account for its inhibitory effect on mast cells. Adenosine antagonism is unlikely to account for the anti-inammatory effects

of theophylline but may be responsible for serious side effects, including cardiac arrhythmias and seizures. Theophylline enhances the release of interleukin-10, which has anti-inammatory effects from inammatory cells, but this is secondary to PDE inhibition and requires high concentrations. It inhibits the proinammatory transcription factor nuclear factor kappa B (NF-kB) but this also requires concentrations above the therapeutic range. Theophylline promotes apoptosis in eosinophils, neutrophils, and T lymphocytes associated with a reduction in the antiapoptotic protein Bcl-2. Recruitment of histone deacetylase-2 (HDAC2) by glucocorticoid receptors switches off inammatory genes. Theophylline is an activator of HDAC at therapeutic concentrations, thus enhancing the anti-inammatory effects of corticosteroids (Figure 3). This mechanism is independent of PDE inhibition or adenosine antagonism, and the antiinammatory effects of theophylline are inhibited by an HDAC inhibitor trichostatin A. Low doses of theophylline increase HDAC activity in bronchial biopsies of asthmatic patients and correlate with the reduction in eosinophil numbers in the biopsy.

294 BRONCHODILATORS / Theophylline

Inflammatory stimuli (e.g., IL-1 , TNF- )

Corticosteroids

Theophylline

NF- B

p65 p50 Co-activators p65 p50 CBP

GR

Inflammatory protein (e.g., GM-CSF, IL-8)

GR

HDAC Deacetylation

HAT Acetylation

Inflammatory gene transcription

Gene activation

Gene repression

Inflammatory gene transcription

Figure 3 Theophylline directly activates histone deacetylases (HDACs), which deacetylate core histones that have been acetylated by the histone acetyltransferase (HAT) activity of coactivators such as CREB-binding protein (CBP). This results in suppression of inammatory genes and proteins, such as granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8), that have been switched on by proinammatory transcription factors such as nuclear factor kappa B (NF-kB). Corticosteroids also activate HDACs, but they do so through a different mechanism resulting in the recruitment of HDACs to the activated transcriptional complex via activation of the glucocorticoid receptors (GR), which function as a molecular bridge. This predicts that theophylline and corticosteroids may have a synergistic effect in repressing inammatory gene expression.

Pharmacokinetics
There is a close relationship between improvement in airway function and serum theophylline concentration. Below 10 mg l 1 bronchodilatation is minimal and above 25 mg l 1 additional benets are outweighed by side effects, so the therapeutic range for bronchodilatation was usually taken as 10 20 mg l 1. It is now clear that theophylline has antiasthma effects other than bronchodilatation and that these may be seen below 10 mg l 1. A more useful therapeutic range is 515 mg l 1. The dose of theophylline required to give these therapeutic concentrations varies between subjects, largely because of differences in clearance. In addition, there may be differences in bronchodilator response to theophylline and, with acute bronchoconstriction, higher concentrations may be required to produce bronchodilatation. Theophylline is rapidly and completely absorbed, but there are large interindividual variations in clearance due to differences in hepatic metabolism (Table 2). Theophylline is metabolized in the liver by the cytochrome P450 microsomal enzyme system (mainly CYP1A2), and a large number of factors may inuence hepatic metabolism. Increased clearance is seen in children (116 years old) and in cigarette and marijuana smokers. Concurrent administration of phenytoin and phenobarbitone increases activity of P450, resulting in increased metabolic breakdown so that higher doses may be required.

Table 2 Factors affecting clearance of theophylline Increased clearance Enzyme induction (rifampicin, phenobarbitone, and ethanol) Smoking (tobacco and marijuana) High-protein, low-carbohydrate diet Barbecued meat Childhood Decreased clearance Enzyme inhibition (cimetidine, erythromycin, ciprooxacin, allopurinol, zileuton, and zarlukast) Congestive heart failure Liver disease Pneumonia Viral infection and vaccination High-carbohydrate diet Old age

Reduced clearance is found in liver disease, pneumonia, and heart failure, and doses need to be reduced to half and plasma levels monitored carefully. Increased clearance is also seen with certain drugs, including erythromycin, certain quinolone antibiotics (ciprooxacin but not ooxacin), allopurinol, cimetidine (but not ranitidine), uoxamine, and zarlukast, which interfere with cytochrome P450 function. Thus, if a patient on maintenance theophylline requires a course of erythromycin, the dose of theophylline should be halved. Viral infections and vaccination may also reduce clearance, and this may be particularly important in children. Because of these variations in clearance, individualization of

BRONCHODILATORS / Theophylline 295

theophylline dosage is required and plasma concentrations should be measured 4 h after the last dose with slow-release preparations when steady state has usually been achieved. There is no signicant circadian variation in theophylline metabolism, although there may be delayed absorption at night, which may relate to the supine posture.

Theophylline is a less preferred option than inhaled corticosteroids and is recommended as a second-line choice of controller in the management of asthmatic patients. Although LABAs are more effective as an add-on therapy, theophylline is considerably less expensive and may be the only affordable add-on treatment when the costs of medication are limiting.
Chronic Obstructive Pulmonary Disease

Role of Theophylline in Respiratory Medicine

Acute Severe Asthma

In patients with acute severe asthma, intravenous aminophylline is less effective than nebulized b2 agonists and should therefore be reserved for those patients who fail to respond to b agonists. Theophylline should not be added routinely to nebulized b2 agonists since it does not increase the bronchodilator response and may only increase their side effects. It is of little proven benet in exacerbations of COPD.
Asthma

Theophylline is still used as a bronchodilator in COPD, but inhaled anticholinergics and b2 agonists are preferred. Theophylline tends to be added to these inhaled bronchodilators in more severe patients and has been shown to give additional clinical improvement when added to a LABA. A theoretical advantage of theophylline is that its systemic administration may have effects on small airways, resulting in a reduction of hyperination and thus a reduction in dyspnea.
Sleep Apnea

Theophylline has little or no effect on bronchomotor tone in normal airways but reverses bronchoconstriction in asthmatic patients, although it is less effective than inhaled b2 agonists and is more likely to have unwanted effects. Theophylline and b2 agonists have additive effects, even if true synergy is not seen, and there is evidence that theophylline may provide an additional bronchodilator effect even when maximally effective doses of b2 agonist have been given. This means that if adequate bronchodilatation is not achieved by a b agonist alone, theophylline may be added to the maintenance therapy with benet. Addition of low-dose theophylline to either high- or low-dose inhaled corticosteroids in patients who are not adequately controlled provides better symptom control and lung function than doubling the dose of inhaled steroid, although it is less effective as an add-on therapy than a long-acting b2 agonist (LABA). Theophylline may be useful in patients with nocturnal asthma since slow-release preparations are able to provide therapeutic concentrations overnight, although it is less effective than a LABA. Studies have also documented steroid-sparing effects of theophylline. Although theophylline is less effective than a b2 agonist and corticosteroids, a minority of asthmatic patients appear to derive unexpected benet, and even patients on oral steroids may show a deterioration in lung function when theophylline is withdrawn. Theophylline has been used as a controller in the management of mild persistent asthma, although it is usually found to be less effective than low doses of inhaled corticosteroids.

Theophylline has been used to treat sleep apnea in adults and apnea in neonates because it increases ventilation rate. However, there is no convincing evidence that it is effective for these indications, and it cannot be routinely recommended.

Adverse Effects and Contraindications

Unwanted effects of theophylline are usually related to plasma concentration and tend to occur when plasma levels exceed 20 mg l 1. However, some patients develop side effects even at low plasma concentrations. To some extent, side effects may be reduced by gradually increasing the dose until therapeutic concentrations are achieved. The most common side effects are headache, nausea, and vomiting (due to inhibition of PDE4), abdominal discomfort, and restlessness (Table 3). There may also be increased acid secretion and diuresis (due to inhibition of adenosine A1 receptors). There was concern that theophylline, even at therapeutic concentrations, may lead to behavioral disturbance and learning difculties in schoolchildren, although

Table 3 Side effects of theophylline Nausea and vomiting Headaches Gastric discomfort Diuresis Behavioral disturbance (?) Cardiac arrhythmias Epileptic seizures

296 BRONCHOMALACIA AND TRACHEOMALACIA

it is difcult to design adequate controls for such studies. At high concentrations, cardiac arrhythmias may occur as a consequence of PDE3 inhibition and adenosine A1 receptor antagonism, and at very high concentrations seizures may occur (due to central A1 receptor antagonism). Use of low doses of theophylline that give plasma concentrations of 510 mg l 1 largely avoids side effects and drug interactions and makes it unnecessary to monitor plasma concentrations (unless checking for compliance). Care must be taken when using theophylline in children, elderly patients, those taking drugs that inhibit cytochrome P450, and those with heart and liver disease because of the pharmacokinetic considerations discussed previously.

account for the add-on benets of theophylline in asthma, whereas in COPD theophylline appears to overcome corticosteroid resistance in vitro and therefore may be a useful anti-inammatory therapy in the future. New drugs based on this molecular action of theophylline may also be developed in the future.
See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cyclic Nucleotide Phosphodiesterases. Gene Regulation.

Further Reading
Barnes PJ (1996) The role of theophylline in severe asthma. European Respiratory Review 6: 154S159S. Barnes PJ (2003) Theophylline: new perspectives on an old drug. American Journal of Respiratory and Critical Care Medicine 167: 813818. Barr RG, Rowe BH, and Camargo CA Jr (2003) Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials. British Medical Journal 327: 643. Cosio BG, Tsaprouni L, Ito K, et al. (2004) Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. Journal of Experimental Medicine 200: 689695. Hansel TT, Tennant RC, Tan AJ, et al. (2004) Theophylline: mechanism of action and use in asthma and chronic obstructive pulmonary disease. Drugs of Today (Barcelona) 40: 5569. Kankaanranta H, Lahdensuo A, Moilanen E, and Barnes PJ (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respiratory Research 5: 17. Rabe K, Magnussen H, and Dent G (1996) Theophylline and selective PDE inhibitors as bronchodilators and smooth muscle relaxants. European Respiratory Journal 8: 637642. Ram FS, Jones PW, Castro AA, et al. (2002) Oral theophylline for chronic obstructive pulmonary disease. Cochrane Database of Systematic Reviews CD003902. Shah L, Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews CD001281. Weinberger M and Hendeles L (1996) Theophylline in asthma. New England Journal of Medicine 334: 13801388.

Future Directions
The use of theophylline has been declining, partly because of the problems with side effects but mainly because more effective therapy with inhaled corticosteroids has been introduced. Oral theophylline is still a useful treatment in some patients with difcult asthma and appears to have effects beyond those provided by steroids. Rapid-release theophylline preparations are cheap and are the only affordable antiasthma medication in some developing countries. Theophylline has anti-inammatory effects at doses that are lower than those needed for bronchodilatation, and plasma levels of 510 mg l 1 are now recommended instead of the previously recommended 1020 mg l 1. Because the molecular mechanisms for the anti-inammatory effects of theophylline are better understood (HDAC activation), there is a strong scientic rationale for combining low-dose theophylline with inhaled corticosteroids, particularly in patients with more severe asthma. The synergistic effect of low-dose theophylline and corticosteroids on inammatory gene expression may

BRONCHOMALACIA AND TRACHEOMALACIA

P N Mathur and J R Ladwig, Indiana University Hospital, Indianapolis, IN, USA
& 2006 Elsevier Ltd. All rights reserved. tracheobronchomalacia. Diagnosis is best performed by bronchoscopy but improvements in computed tomography has made radiographic diagnosis easier. Treatment varies from various means of stenting the airway to surgical correction.

Abstract
Tracheomalacia and bronchomalacia are a loss of cartilaginous support of the trachea and large airways. This can occur as a congenital defect or as a result of acquired disease processes. The loss of cartilaginous support leads to airway obstruction upon expiration. Symptoms of dyspnea and cough result from

Introduction
Tracheomalacia is a condition of weakened cartilaginous support of the trachea. The term bronchomalacia refers to loss of supportive structure for

BRONCHOMALACIA AND TRACHEOMALACIA 297

the large airways. With the loss of structural support, the trachea does not maintain its diameter and the membranous portion of the trachea collapses anteriorly, causing obstruction of airow during expiration.

Clinical Features
Diagnosis of tracheobronchomalacia includes a thorough history and physical examination to exclude other conditions or elicit clinical features typical of tracheobronchomalacia. Clinical ndings are nonspecic and often manifest in dyspnea on exertion and cough. The cough of tracheobronchomalacia has been characterized as seal-like. Diagnosis of tracheobronchomalacia is aided by pulmonary function testing, imaging, and direct visualization via bronchoscopy.
Pulmonary Function Testing

Etiology
Tracheomalacia and bronchomalacia can be the result of multiple etiologies. In infants, the cause is often a congenital defect in cartilaginous development which usually resolves spontaneously as the child reaches 6 months of age and older. It may also be the result of congenital vascular rings causing compression of the airways resulting in abnormal development or erosion of cartilaginous rings. In adults, tracheobronchomalacia may also be the result of previously unrecognized congenital abnormalities, or acquired anatomic or pathologic processes. Substernal goiter may present with cough in tracheomalacia. The enlargement of thyroid tissue can lead to compressive erosion of tracheal rings. Neoplasm may also exert pressure on the tracheal rings or cause destruction by local invasion resulting in tracheobronchomalacia. Inammatory processes may also be a cause of tracheomalacia. Relapsing polychondritis, an autoimmune disease directed against cartilage, may affect the larynx and cartilaginous rings of the large airways. It is most often seen in the fourth decade; airway symptoms are more common amongst women with the disease. Patients with severe chronic obstructive pulmonary disease (COPD) may develop atrophy of cartilage in the airway, leading to easy collapsibility of airways. The diagnosis of tracheomalacia in a patient with COPD may go unrecognized until progression of disease prompts bronchoscopy or other imaging. Patients who have had prolonged intubation or require tracheostomy may also develop tracheomalacia. The local pressure effect of the inated balloon at the distal end of the tube can cause local inammation, overdistention, or local tissue ischemia resulting in erosion of the cartilaginous rings.

Patients with tracheomalacia show reduced expiratory ow measurements. Flowvolume curves often demonstrate expiratory ow limitation (Figure 1). Inspiratory ow is preserved while the expiratory loop is similar to that of COPD.
Diagnostic Imaging of the Trachea

As a general rule, plain chest radiographs are not useful for diagnosis of tracheomalacia. In extreme cases of airway narrowing, a large air column above the obstruction may be seen. A simple computed tomography (CT) scan of the chest may not be useful for tracheobronchomalacia. However, it is very useful for identication of
Flow (l s1)

16.00

8.00

0.00 8.00

8.00

Pathology
Congenital tracheobronchomalacia represents a failure or incomplete development of the cartilaginous rings of the airway. In adults, tracheobronchomalacia is acquired more commonly as described above. The acquired insult often leads to compression and local destruction of the tracheal rings.
16.00 Pre

Volume (l) Post

Figure 1 Expiratory ow measurements. Courtesy of C Mayer, University of Cincinnati.

298 BRONCHOMALACIA AND TRACHEOMALACIA

extrinsic causes of tracheal compression which may result in tracheobronchomalacia such as substernal goiter, vascular abnormalities, and neoplasm. One method of making CT more benecial in diagnosing tracheobronchomalacia is to compare inspiratory and expiratory images. This aids in demonstrating the dynamic narrowing of the trachea and it may then be more obvious on the CT scan (Figures 2(a) and 2(b)). Newer advances in CT imaging show signicant sensitivity and specicity for identifying airway stenosis. A virtual bronchoscopy can be performed with the images acquired from a routine CT scan that is then reconstructed through computer software (Figures 2(c) and 2(d)). The imaging is enhanced when a multidetector CT image is used instead of a single detector one. There are different methods to render the data received by the CT scan. Data may be surface or volume rendered. Surface rendering is faster and requires less computer power. However, volume rendering provides more

detail of the mucosal-airway interface allowing more detail of the mucosa. Virtual bronchoscopy has been found to have high sensitivity (90%) and specicity (96.6%) for central airway stenosis. The virtual bronchoscopy provides the benet of detailed imaging in patients that might not be candidates for routine bronchoscopy. It also provides the bronchoscopist a noninvasive method to acquire information allowing for planning prior to an interventional procedure or foregoing a procedure for lesions not amenable to repair. The gold standard diagnostic tool for tracheobronchomalacia is bronchoscopy. If a patient is able to tolerate bronchoscopy with minimal sedation, various respiratory maneuvers can be performed (forced expiration, cough) to exaggerated or elicit airway obstruction. Once the diagnosis of tracheobronchomalacia is made, consideration of therapeutic options is undertaken. Correction of underlying extrinsic processes (substernal goiter, vascular rings, and neoplasm),

Figure 2 (a) Inspiration, (b) expiration, (c) virtual bronchoscopy inspiration, (d) virtual bronchoscopy expiration. Courtesy of C Mayer, University of Cincinnati.

BRONCHOMALACIA AND TRACHEOMALACIA 299

systemic illness (relapsing polychondritis), and airway disease (COPD) should be treated appropriately. If the causative process cannot be eliminated or when the tracheomalacia already results in airway compromise, several options exist to palliate the obstruction.

airway pressure (CPAP) to maintain a patent airway. In one case of relapsing polychondritis, it provided a temporary measure, delaying the time to invasive procedures. If further investigation identies the type of patient most likely to benet from CPAP, it may prove a useful bridge prior to a more invasive therapy.
Surgery

Management and Current Therapy

Balloon Bronchoplasty

Airways may be dilated by rigid or inatable balloon dilators. The modality is best used for extrinsic or intrinsic compression of the airways. Balloon bronchoplasty is easily performed through a exible bronchoscope. Alone, the modality does not provide sustained benet. However, it has been described as a useful tool in optimizing the airway caliber prior to stent placement.
Airway Stents

Tracheobronchomalacia is one of the indications for airway stenting, as described by the ERS/ATS statement on interventional pulmonology. There are a number of different types of airway stents, each with pros and cons. Generally stents can be categorized as silicone, metal, or a hybrid of the two materials. Silicone stents have the benet of being cheaper and more easily removed. However, they require knowledge of rigid bronchoscopy for placement and have higher rates of migration. Currently, the most widely used stent is the Dummon stent, which is a silicone stent that has studs on the outer surface to retard migration. Metal stents have the advantage of being placed via exible bronchoscopy and are less prone to migration. Metal stents may be uncovered or covered by a silastic or polyurethane coating. However, uncovered stents should not be used in benign disease such as tracheobronchomalacia, as they allow ingrowth of malignant or granulation tissue. Metal stents are very difcult to remove, may fracture, and pose a risk if future interventional bronchoscopy is needed (laser, APC) because of the potential for conduction of current/heat or airway re. In choosing a stent, the airway should be evaluated by bronchoscopy and/or CT scan to determine the extent of the obstruction. The optimal stent is one that exceeds the margins of the stenosis and whose diameter is larger than that of the airway. Placement of a stent is expected to bridge the extent of the abnormal airway. Inadequate stenting may only result in moving the choke point, the area of airow obstruction, distal to the stent. One method of noninterventional stenting still under investigation is the use of continuous positive

Multiple surgical approaches to tracheomalacia have been described. One surgical procedure used for treatment of tracheobroncomalacia is a membranous-wall tracheoplasty. This is done through a right thoracotomy. The procedure involves sewing a polypropylene mesh to the membranous wall of the trachea which then provides external support of the tracheal wall. Another external wall stent procedure has been investigated in an animal model using reabsorbable stents. Although still experimental, this may provide another alternative in the right patient population.
See also: Bronchiectasis. Bronchoscopy, General and Interventional. Relapsing Polychondritis. Trauma, Chest: Postpneumonectomy Syndrome. Upper Airway Obstruction.

Further Reading
Adliff M, et al. (1997) Treatment of diffuse tracheomalacia secondary to relapsing polychondritis with continuous positive airway pressure. Chest 112: 17011703. Aquino SL, Shepard J-AO, Ginns LC, et al. (2001) Acquired tracheomalacia: detection by expiratory ct scan. Journal of Computer Assisted Tomography 25(3): 394399. Bolliger CT and Mathur PN (2002) ERS/ATS statement on interventional pulmonology. European Respiratory Journal 19: 356373. Davis S, et al. (1998) Effect of continuous positive airway pressure on forced expiratory ows in infants with tracheomalacia. American Journal of Respiratory and Critical Care Medicine 158: 148152. Ernst A, et al. (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169: 12781297. Hoppe H, et al. (2004) Grading airway stenosis down to the segmental level using virtual bronchoscopy. Chest 125: 704711. Hopper KD, et al. (2000) Mucosal detail at CT virtual reality: surface versus volume rendering. Radiology 214: 517522. Miyazawa T, et al. (2004) Stenting at the ow-limiting segment in tracheobronchial stenosis due to lung cancer. American Journal of Respiratory and Critical Care Medicine 169: 10961102. Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 13681373. Sewall A, Gregory K, et al. (2003) Comparison of resorbable poly-L-lactic acid polyglycolic acid and internal palmaz stents for the surgical correction of severe tracheomalacia. The Annals of Otology, Rhinology, and Laryngology 112: 515521. Wright CD (2003) Tracheomalacia. Chest Surgery Clinics of North America 13: 349357.

300 BRONCHOPULMONARY DYSPLASIA

BRONCHOPULMONARY DYSPLASIA
E Bancalari, University of Miami School of Medicine, Miami, FL, USA
& 2006 Elsevier Ltd. All rights reserved.

90 80 70 BPD (%) 60 50 40 30 20 10 0
501750 7511000 10011250 12511500 5011500

Oxygen at 28 d Oxygen at 36 wks PMA

Abstract
Bronchopulmonary dysplasia (BPD) is one of the most common sequelae in premature infants who survive after prolonged mechanical ventilation. Its pathogenesis is multifactorial and includes prematurity, perinatal infections, pulmonary volutrauma, oxygen toxicity, and increased pulmonary blood ow due to patent ductus arteriosus. It is characterized by chronic respiratory failure with abnormalities in lung function that can persist into adulthood. This is due to severe alterations in lung development characterized by decreased alveolar and capillary formation plus emphysema, brosis, and airway obstruction in more severe cases. The prevention of BPD is based on the avoidance of all the factors that are implicated in its pathogenesis.

Birth weight (g)

Figure 1 Incidence of BPD (% of total births) in the NICHD Neonatal Research Network, Jan. 1995 to Dec. 1996. PMA, post menstrual age. Reproduced from Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 18.

Bronchopulmonary dysplasia (BPD) was rst described by Northway and co-workers in 1967 and has become a major complication in premature infants who require prolonged mechanical ventilation. The natural course of severe respiratory distress syndrome (RDS) in the premature infant was altered in the 1960s by the introduction of mechanical ventilation. As a result, smaller and sicker infants survive, but many of these survivors are left with chronic lung damage. These infants frequently remain oxygen- and ventilator-dependent for prolonged periods of time, and the mortality rate can be as high as 3040%. In surviving infants, pulmonary function may remain abnormal for years, and they have increased risk of neurodevelopmental sequelae and impaired growth curves. The incidence of BPD is closely and inversely related to both, birth weight and gestational age (Figure 1). For this reason, with increasing survival of extremely premature infants, the number of patients at risk for developing BPD has increased.

Figure 2 Low-magnication view from a lung with BPD showing areas of emphysema alternating with areas of partial collapse. Reproduced from Kluwer Academic Publishers Advances in Perinatal Medicine, 1982, p. 181, Barotrauma to the lung, Bancalari E and Goldman SL, gure 29, with kind permission of Springer Science and Business Media.

Pathology
Most of the pathologic descriptions of BPD represent the most severe form of chronic lung damage since those infants with milder forms of BPD rarely die. Macroscopically, the lungs are rm, heavy, and dark in color, with a grossly abnormal appearance showing emphysematous areas alternating with areas of collapse. Histologically, the lungs show areas of emphysema, with abnormal alveoli that have coalesced into larger cystic areas, surrounded by areas of atelectasis (Figure 2). Widespread bronchial and

bronchiolar mucosal hyperplasia and metaplasia reduce the lumen in many small airways. In addition, there is interstitial edema and an increase in brous tissue with focal thickening of the interstitial spaces. Lymphatics may be dilated and torturous. There may be vascular evidence of pulmonary hypertension, such as medial muscle hypertrophy, elastic degeneration, and reduction in the branching of the pulmonary vascular bed. The heart frequently shows evidence of biventricular hypertrophy. Infants dying with severe BPD show a marked reduction in the number of alveoli and capillaries, with a reduction in the gas exchange surface area. Infants who have mild

BRONCHOPULMONARY DYSPLASIA 301

BPD at the time of death have only mild or moderate alveolar septal brosis, lack of severe airway epithelial lesions, and normal-appearing pulmonary vessels. Nonetheless, they show evidence of inhibition of acinar and capillary development.

Clinical Features
The diagnosis of BPD is based on clinical and radiographic manifestations, which are not specic. With rare exceptions, BPD occurs in preterm infants and is preceded by the use of prolonged mechanical ventilation. Radiographic ndings include hyperination and nonhomogeneity of pulmonary tissues, with ne or coarser densities extending to the periphery in the more severe forms (Figure 3). In milder forms, the radiographic changes are also milder, revealing mainly diffuse haziness. Once lung damage has occurred, these infants continue to require mechanical ventilation and increased oxygen concentration for months or sometimes years. Because of the increased use of antenatal steroids and exogenous surfactant, many small infants have mild initial respiratory disease and require ventilation with low pressures and oxygen concentration. After a few days or weeks of mechanical ventilation, these infants frequently show progressive deterioration in lung function, an increase in their ventilatory and oxygen requirements accompanied by signs of respiratory failure, and ultimately develop BPD.

This deterioration is frequently triggered by bacterial or viral infections or respiratory failure secondary to a patent ductus arteriosus (PDA). Infants with BPD who survive show a slow but steady improvement in lung function and roentgenographic changes, with gradual weaning from ventilator and oxygen therapy. Signs of respiratory distress, such as chest retractions and tachypnea, frequently persist long after extubation. More severe BPD may evolve into progressive respiratory failure, and even death, as a result of severe lung damage and pulmonary hypertension or intercurrent infections. Some of these infants may also develop anastomoses between systemic and pulmonary circulations, which may aggravate their pulmonary hypertension. Acute pulmonary infection, either bacterial or viral, frequently complicates the course of BPD and, in many cases, is the precipitating cause of death. Because of chronic hypoxia and higher energy expenditure required by their increased work of breathing, weight gain in infants with BPD is usually below normal, even when receiving appropriate caloric intake for their age. In addition, poor feeding tolerance and a tendency toward uid retention and pulmonary edema often requires uid restriction as well as diuretic therapy, further limiting the calories that can be provided.

Pathogenesis
Because BPD occurs almost exclusively in premature infants who have received mechanical ventilation and increased oxygen concentrations, prematurity, baro/volutrauma, and oxygen toxicity have been considered crucial factors in BPD pathogenesis. Other factors that may play an important role in BPD pathogenesis include perinatal infections, pulmonary edema resulting from a PDA or excessive uid administration, and nutritional deciencies.
Prematurity

BPD occurs almost exclusively in the extremely premature infant. This suggests that the vulnerability of the very preterm infant may be related to the immature state of lung development. It is likely that premature birth followed by therapeutic interventions disrupts lung development during this crucial period, producing an interruption and/or disruption in the development of alveoli, capillaries, and lung matrix.
Barotrauma/Volutrauma
Figure 3 Chest radiograph from an infant with bronchopulmonary dysplasia, showing areas of mild hyperination alternating with coarse densities.

Almost all infants who develop BPD have received prolonged mechanical ventilation; therefore, it is

302 BRONCHOPULMONARY DYSPLASIA

likely that mechanical trauma also plays an important role in the pathogenesis of BPD. The presence of an endotracheal tube may also contribute to BPD pathogenesis by increasing the risk of pulmonary infections. Studies on preterm experimental animals have demonstrated that only a few breaths with excessive tidal volumes given prior to surfactant replacement produce lung damage with a decrease in lung compliance.
Oxygen Toxicity

BPD is supported by the documented benecial effects of steroids in these infants.

Patent Ductus Arteriosus and Pulmonary Edema

Pulmonary oxygen toxicity is also considered an important factor in the pathogenesis of BPD. High inspired oxygen concentrations can increase the production of cytotoxic oxygen free radicals, which overwhelm the antioxidant defenses in the capillary endothelial cells and the alveolar epithelial cells of the premature lung because of incomplete development of the pulmonary antioxidant enzyme system. The precise concentration of oxygen that is toxic to the premature lung is unknown, but it is possible that any concentration in excess of room air may increase the risk of lung damage when administered over prolonged periods of time.
Infection and Inammation

Evidence has been mounting to support a role for infection and inammation in the pathogenesis of BPD, particularly in very small infants who develop BPD after receiving prolonged mechanical ventilation for poor respiratory effort rather than because of severe underlying lung disease. The evidence of an association between ureaplasma urealyticum tracheal colonization and the development of BPD has been inconsistent. However, maternal infections, specically chorioamnionitis, are associated with an increased risk of BPD. Several inammatory cytokines are present in higher concentrations in fetal cord blood and in the amniotic uid of mothers who deliver infants who subsequently develop BPD. An inammatory response in the lung can also be triggered by other factors, including oxygen free radicals, ventilation with excessive tidal volumes, and increased pulmonary blood ow due to a PDA. Among the markers of inammation found in high concentrations in tracheobronchial secretions in infants who develop BPD are neutrophils, macrophages, leukotrienes, platelet-activating factor (PAF), interleukin (IL)-6, IL-8, and tumor necrosis factor. The increased concentration of inammatory mediators may be responsible for the bronchoconstriction, vasoconstriction, and increased vascular permeability characteristic of these infants. The potential role of inammation in the pathogenesis of

Infants with RDS who receive greater uid intake or do not have a diuretic phase in early life have a higher incidence of BPD. High uid intake increases the incidence of PDA, which then produces increased pulmonary blood ow, an increase in interstitial lung uid, and causes a decrease in pulmonary compliance and increased resistance. This deterioration in lung mechanics may prolong the requirement for mechanical ventilation and high inspired oxygen concentrations, thereby increasing the risk of BPD. In addition, the increased pulmonary blood ow can induce endothelial damage with neutrophil margination and activation in the lung and contribute to the progression of the inammatory cascade. These interrelated factors may explain the strong association between the duration of the PDA and the increased risk of BPD. Infants with established BPD have a predisposition for uid accumulation in their lungs. Capillary permeability may be increased due to the oxygen toxicity, volutrauma, or infection. Infants with BPD can also have increased plasma levels of vasopressin with a reduced urine output and decrease in free water clearance. With excessive accumulation of lung uid, lung function is further compromised, perpetuating a cycle in which more aggressive respiratory support is required, thereby resulting in additional lung injury. Figure 4 shows the relative inuence of different factors in the pathogenesis of BPD.
Increased Airway Resistance

Infants with severe BPD frequently have a marked increase in airway resistance. This can impair distribution of the inspired gas and thereby favor uneven lung expansion. The airway obstruction may be secondary to bronchiolar epithelial hyperplasia, metaplasia, and mucosal edema, and it may also relate to pulmonary edema secondary to PDA or uid overload. These infants may also have bronchoconstriction resulting from smooth muscle hypertrophy. Other possible factors producing increased airway resistance in infants with BPD include inammatory mediators such as leukotrienes and PAF, which have been found in high concentrations in the airways of infants with BPD. Finally, tracheobronchomalacia, which may be present in some infants with severe BPD, can produce marked airway obstruction, particularly during periods of agitation and increased intrathoracic pressure.

BRONCHOPULMONARY DYSPLASIA 303

100 OR for BPD 10 1 0.1
ps is PD A ps is s rw ee Fe m al r1 Se Se am ni on pe pe ve r + e R D S g e k 00 iti

Other Factors

Figure 4 Perinatal and postnatal risk factors for the development of BPD dened as X28 days duration of oxygen-dependency during hospitalization. Obtained by logistic regression analysis from all extremely premature infants born at UM/JMH during the period 19952000 (N 505 alive at 28 days). Birth weight (Bw), 5001000 g; gestational age (GA), 2332 weeks; PDA, patent ductus arteriousus; RDS, respiratory distress syndrome; OR, odds ratio. Reproduced from Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and denition. Seminars in Neonatology 8: 63, with permission from Elsevier.

Other factors have been proposed as having a pathogenetic role in BPD. One of these factors is a genetic predisposition to abnormal airway reactivity since there have been reports of a stronger family history of asthma in infants with BPD. Vitamin A deciency may also play a role because infants who develop BPD have lower vitamin A levels than those who recover without BPD. This possible association is supported by the similarities between some of the airway epithelial changes observed both in BPD and in vitamin A deciency and by clinical evidence that vitamin A administration in the rst weeks of life reduces the incidence of BPD. Another factor suggested to play a role in the development of BPD is early adrenal insufciency because infants with BPD have lower cortisol levels in the rst week of life. Figure 5 summarizes the most important factors that contribute to the pathogenesis of BPD.

PD A

Bw

C ho

rio

Prematurity-respiratory failure mechanical ventilation

Excessive tidal volume Decreased lung compliance Volutrauma

Increased inspired oxygen Deficient antioxidant systems Nutritional deficiencies Oxygen toxicity

Se

Pre/postnatal infections PMNs activation Inflammatory mediators elastase/proteinase inhibitors imbalance

Patent ductus arteriosus Excessive fluid intake

Increased pulmonary blood flow-lung edema

Acute lung injury inflammatory response

Airway damage Metaplasia Smooth muscle hypertrophy Mucus secretion

Vascular injury Increased permeability Smooth muscle hypertrophy

Interstitial damage
Fibronectin Elastase Alveolar septation Vascular development

Airway obstruction Emphysema-atelectasis

Pulmonary edema Pulmonary hypertension

Matrix damage Fibrosis Decreased number of alveoli and capillaries

Bronchopulmonary dysplasia
Figure 5 Factors contributing to the pathogenesis of bronchopulmonary dysplasia.

304 BRONCHOPULMONARY DYSPLASIA

Animal Models
Because BPD is a chronic process with multiple factors involved in its pathogenesis, it has been extremely difcult to develop suitable animal models. However, there are two models that closely resemble human BPD. The rst model was developed by Drs Robert de Lemos and Jacqueline Coalson in preterm baboons. When these animals are delivered at approximately 75% gestation and are subsequently ventilated over several weeks with positive pressure and high oxygen concentrations, they develop physiologic and morphologic changes that closely resemble those of human BPD. This is a very labor-intensive and expensive model that requires a full intensive care setting for these animals to survive. A similar model has been developed in preterm lambs that, after several weeks of mechanical ventilation, also develop histopathological changes very similar to human BPD. These models are used extensively by investigators who are exploring various aspects of lung development, injury, and repair as well as possible strategies to prevent BPD.

Prevention and Management

Strategies to prevent BPD focus on eliminating or reducing the multiplicity of factors known to contribute to lung injury (Figure 5). Since lung immaturity is the main predisposing factor in the pathogenesis of BPD, prevention of BPD should start prenatally by attempting to avoid premature birth. When preterm birth is imminent, administration of antenatal steroids reduces the incidence and severity of RDS and the subsequent development of severe BPD. Minimizing volutrauma and exposure to high inspired oxygen concentrations is also critical to reduce BPD. Surfactant replacement therapy in infants with RDS improves their respiratory course, enabling mechanical ventilation with lower inspiratory pressures and oxygen concentrations. The use of positive end expiratory pressure (PEEP) is critical because insufcient PEEP is associated with increased ventilatorassociated lung damage. Ventilation with high frequency has not been conclusively shown to reduce BPD, although data from experimental animals suggest that this ventilation modality may reduce lung injury. Data demonstrating an increased risk of BPD in infants exposed to prenatal and postnatal infections suggest that prevention and aggressive management of these infections should also play a preventive role in BPD. Avoidance of excessive uid intake is also important in the prevention of BPD. Diuretics, specically loop diuretics, may be indicated to improve pulmonary uid balance and reduce interstitial lung

water. Prompt closure of a PDA using prostaglandin inhibitors or by surgical ligation is important in reducing the risk and/or severity of BPD. Bronchodilators, most of them b-agonists, administered by inhalation have been shown to reduce airway resistance in infants with BPD. Because their effect is short-lived and often associated with cardiovascular side effects (e.g., tachycardia, hypertension, and possible arrhythmias), their use tends to be limited to acute exacerbations of airway obstruction. Adequate nutrition is important in BPD prevention. General undernutrition, particularly an insufciency in dietary protein, may increase the vulnerability of the preterm infant to oxidant lung injury and the development of BPD. Additional nutrients, including those that may increase intracellular glutathione (e.g., sulfur-containing amino acids), inositol to serve as substrate for surfactant, and selenium and other trace minerals to function as essential cofactors for the pulmonary antioxidant enzymes, may provide added protection to the premature infant against the development of BPD. Clinical trials in preterm infants with severe RDS have shown that supplemental vitamin A administration reduces the incidence and severity of BPD. Although the use of corticosteroids clearly has short-term benets in lung function in infants with BPD, the optimal age of treatment, dose schedule, and duration of therapy have not been established. Most alarming are recent follow-up data demonstrating that infants who received short or prolonged steroid therapy had worse neurological outcome compared to controls. Table 1 summarizes some of the strategies to prevent BPD. Future directions in BPD-prevention may include exogenous administration of specic antioxidants, genetic manipulation, and strategies for maturing the preterm lung that have greater selectivity and fewer adverse effects than the use of glucocorticoids.
Table 1 Potential strategies to prevent BPD Avoid premature birth Antenatal steroids Surfactant replacement Minimize volutrauma and duration of mechanical ventilation Minimize oxygen exposure Prevention/aggressive management of pre- and postnatal infections Avoid excessive uid administration Prompt patent ductus asteriosus closure Maintain normal serum vitamin A levels Investigational Corticosteroids and other anti-inammatory drugs Exogenous administration or induction of antioxidant enzymes Gene therapy/genetic manipulation

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 305 See also: Epithelial Cells: Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Development: Overview. Pediatric Pulmonary Diseases. Peripheral Gas Exchange.
Husain AN, Siddiqui NH, and Stocker JT (1998) Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Human Pathology 29: 710717. Jobe AH and Bancalari E (2001) Bronchopulmonary dysplasia. American Journal of Respiratory and Critical Care Medicine 163: 17231729. Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 18. Northway WH, Moss RB, Carlisle KB, et al. (1990) Late pulmonary sequelae of bronchopulmonary dysplasia. New England Journal of Medicine 323: 17931799. Northway WH Jr, Rosen RC, and Porter DY (1967) Pulmonary disease following respirator therapy of hyaline membrane disease: bronchopulmonary dysplasia. New England Journal of Medicine 276: 357368. Pierce MR and Bancalari E (1995) The role of inammation in the pathogenesis of bronchopulmonary dysplasia. Pediatric Pulmonology 19: 371378. Rojas MA, Gonzalez A, Bancalari E, et al. (1995) Changing trends in the epidemiology and pathogenesis of neonatal chronic lung disease. Journal of Pediatrics 126: 605610. Sosenko I and Bancalari E (2003) Bronchopulmonary dysplasia (BPD). In: Greenough A and Milner A (eds.) Neonatal Respiratory Disorders, 2nd edn., pp. 399422. London: Arnold. Watterberg KL, Demers LM, Scott SM, et al. (1996) Chorioamniotis and early lung inammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 97: 210215. Watterberg KL, Scott SM, Backstrom C, et al. (2000) Links between early adrenal function and respiratory outcome in preterm infants: airway inammation and patent ductus arteriosus. Pediatrics 150: 320324. Yoon BH, Romero R, Jun JK, et al. (1997) Amniotic uid cytokines (interleukin-6, tumor necrosis factor-a, interleukin-1b, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. American Journal of Obstetrics and Gynecology 177: 825830.

Further Reading
Bancalari E (2004) Pathophysiology of chronic lung disease. In: Polin R, Fox W, and Abman S (eds.) Fetal and Neonatal Physiology, 3rd edn., vol. 1, pp. 954961. Philadelphia: Saunders. Bancalari E (2005) Neonatal chronic lung disease. In: Fanaroff AA and Martin RJ (eds.) NeonatalPerinatal Medicine Diseases of the Fetus and Infant, 8th edn. St. Louis: Mosby. Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and denition. Seminars in Neonatology 8: 6371. Bancalari E and Goldman SL (1982) Barotrauma to the lung. Advances in Perinatal Medicine 181. Bancalari E, Wilson-Costello D, and Iben SC (2005) Management of bronchopulmonary dysplasia. In: Maalouf E (ed.) Best Practice Guidelines, Early Human Development. Oxford: Elsevier. Bland RD and Coalson JJ (2000) Chronic Lung Disease in Early Infancy. New York: Dekker. Coalson JJ, Winter VT, Gerstmann DR, et al. (1992) Pathophysiologic, morphologic, and biochemical studies of the premature baboon with bronchopulmonary dysplasia. American Review of Respiratory Disease 145: 872881. DAngio CT and Maniscalco WM (2004) Bronchopulmonary dysplasia in preterm infants: pathophysiology and management strategies. Paediatric Drugs 6: 303330. Gonzalez A, Sosenko IRS, Chandar J, et al. (1996) Inuence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighting 1000 grams or less. Journal of Pediatrics 128: 470478. Grier DG and Halliday HL (2003) Corticosteroids in the prevention and management of BPD. Seminars in Neonatology 8: 8391.

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

D J Feller-Kopman and R Bechara, Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Bronchoscopy refers to examination of the tracheobronchial tree via a rigid or exible bronchoscope. This chapter will review both diagnostic and therapeutic bronchoscopy, with a focus on the available technology and procedural techniques used to diagnose and treat a variety of lung diseases. As Bronchoscopy: Regular and Interventional is a topic for which textbooks are available, the reader is encouraged to explore the relevant literature, with some pertinent references listed in the further reading section.

Introduction
Gustav Killian has been described as the Father of Bronchoscopy. With the vision of using a metallic

tube, electric light, and topical cocaine to prevent glottic closure, Killian removed a pork bone from a farmers airway in 1897. Over the subsequent years, Killian went on to develop bronchoscopes, laryngoscopes, and endoscopes, as well as describe techniques such as using uoroscopy and X-ray to dene endobronchial anatomy. Over the next 150 years, bronchoscopic techniques and instruments continued to be rened. In 1966, Shigeto Ikeda, presented the rst prototype exible beroptic bronchoscope at the 9th International Congress on Diseases of the Chest in Copenhagen. In 1968 Machita and Olympus introduced the rst commercially available beroptic bronchoscopes. In 1980, Dumon presented his use of the neodymium:yttrium aluminum garnet (Nd:YAG) laser via the beroptic bronchoscope, and since that time the exible bronchoscope has been widely utilized as both a diagnostic and therapeutic tool for both diseases of the parenchyma and central airways.

306 BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

With the miniaturization of electronic devices, Ikeda was able to incorporate the video chip into the bronchoscope, and Pentax introduced the rst video bronchoscope in 1987. Suddenly, endoscopic pictures could be printed out and shared, and the physicians no longer needed to look through an eyepiece, but instead the endoscopic image could be projected onto monitors, allowing everyone in the room to visualize what was happening in the airway.

Modern Video Bronchoscopes

Little has changed in the appearance of bronchoscopes since 1968. The external diameter of the exible bronchoscope varies from 2.7 to 6.3 mm diameter. The diameter of the working channel ranges from 1.2 to 3.2 mm. A working channel X2:8 mm is recommended for more therapeutic exible bronchoscopy, as well as endobronchial ultrasound (EBUS), as it allows for better suction and the passage of larger instruments. Most exible bronchoscopes can ex 1801 up and 1301 down. It is important to note the relative anatomy at the tip of the bronchoscope. By convention, as viewed from the operators perspective, the camera is at 9:00, suction at 6:00 and the working channel at 3:00. These landmarks play a role when navigating the airways as the bronchoscope may need to be rotated in order to visualize the intended target or guide a tool to its intended target.

physical examination, and imaging studies. A distinct plan should be in place regarding the sequence of sampling techniques, and everyone participating in the procedure should be familiar with both this plan and also their particular roles during the procedure. Topical anesthesia is crucial, and we typically use 1% lidocaine, keeping the total dosage o400 mg. Stronger concentrations do not provide additional sensory anesthesia and will only limit the volume of lidocaine one can apply to the airways. Most procedures are performed under conscious sedation with a narcotic and a benzodiazepine. All practitioners involved in the administration of conscious sedation are required to undergo formal training by their institution and should have a thorough understanding of the effects, side effects, and typical dosages, as well as antagonist drugs. The physician should also be comfortable in airway management including the use of bag-valve mask, oral airways, and endotracheal intubation. If one plans to perform bronchoscopy through an endotracheal tube (or tracheostomy), the internal diameter should ideally be 47.5 mm in order to accommodate the bronchoscope and maintain patient ventilation. Depending on the length of the procedure, it may be necessary to remove the bronchoscope intermittently to ensure adequate ventilation.

Diagnostic Bronchoscopy
There are many indications for diagnostic bronchoscopy. The most common indication is for the diagnosis and staging of suspected lung cancer. Other indications include evaluation of diffuse lung disease, inltrates in the immunocompromised host, hemoptysis, and cough. Additionally, bronchoscopy with bronchoalveolar lavage (BAL) and/or brushing is useful for the diagnosis of community and healthcare associated pneumonia.
Cancer Diagnosis and Staging

Airway Anatomy
It is crucial that the bronchoscopist become an expert in airway anatomy. Anatomical knowledge should include the naso and oropharynx, as well as the larynx as these structures are visualized, yet often overlooked, by the pulmonologist, who is typically more concerned about lower airway/parenchymal disease. Additionally, the segmental anatomy of the lungs, from both an external and internal perspective, is required, and we recommend knowledge of both the name and number system. One must also be familiar with the anatomy external to the airway, primarily the intrathoracic vessels and lymph nodes, as these will serve as reference points for transbronchial needle aspiration and EBUS, and will help avoid injury should the bronchoscopist use therapy such as the Nd:YAG laser or brachytherapy. The use of endoscopic simulators has been associated with a more rapid acquisition of bronchoscopic expertise and we recommend the novice bronchoscopist use a simulator as much as possible.

Preparation
Prior to the procedure, it is crucial that the bronchoscopist review the patients relevant history,

Lung cancer is the leading cause of cancer deaths in the US, and the incidence and number of lung cancer deaths continues to increase amongst women in the US. Bronchoscopy provides a minimally invasive approach for the diagnosis of tumors in the central airways. Though the yield is somewhat lower for solitary parenchymal lesions, advances in navigation with techniques such as computed tomography (CT) uoroscopy and electromagnetic guidance (to be discussed later) have signicantly improved the yield for peripheral tumors. The bronchoscopic evaluation of patients with suspected malignancy is guided by clinical symptoms as well as radiographic ndings. Depending upon the

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 307

history and chest CT ndings, bronchoscopy may be the diagnostic modality of choice as it can both make the diagnosis and stage the patient at the same time. If appropriate staging is to be performed, biopsy of the lesion that will place the patient in the highest clinical stage should occur prior to other biopsies. For example, if a patient presents with a left lower lobe mass and a right paratracheal lymph node (4R), the appropriate procedure would be a bronchoscopy with transbronchial needle aspiration (TBNA) of the 4R node, with attention then turned to the left lower lobe lesion, as a positive TBNA would stage the patient as IIIb and therefore change the treatment plan. Initial biopsy of the mass could contaminate the working channel and make the TBNA a false positive, precluding the patient from curative surgery. There are several available tools by which to obtain specimens during bronchoscopy including forceps biopsy, brushing, bronchial wash/lavage, and TBNA. Electrocautery snare forceps removal of a pedunculated airway lesion can also provide excellent tissue for the pathologist. The choice of the above modalities is primarily determined by the location of the pathology; however, data support the use of the combination of techniques to improve diagnostic yield, as opposed to using them in isolation. Endobronchial needle aspiration should be used with all visible lesions, as its use in combination with conventional techniques has been associated with an improvement in the diagnostic yield. If the lesion is not visible endoscopically, BAL can be performed. Briey, the bronchoscope is wedged in the target segmental or subsegmental bronchus leading to the lesion. Two or three aliquots of 4060 ml of normal saline solution are instilled, and then aspirated. Ideally, the return should be between 40% and 60% of the instilled volume, however this is dependent upon the segment lavaged, with better return coming from less dependant locations. BAL is thought to sample approximately 1 million alveoli and the cellular and noncellular contents of the lavage uid have been shown to closely correlate with the inammatory nature of the entire lower respiratory tract. Transbronchial biopsy, brushing, and TBNA can also be performed for peripheral lesions. The diagnostic yield of bronchoalveolar lavage for peripheral cancer ranges from 4% to 68%. Without advanced guidance (discussed below), the yield for transbronchial biopsy ranges from 49% to 77%. The yield from brushing alone is 2657%. The combination of all the three techniques results in a combined yield of upto 68%. TBNA of peripheral nodules has been shown to have a higher diagnostic yield than other sampling techniques, and should be used to biopsy

peripheral lesions, as well as mediastinal and hilar lymph nodes. Despite TBNA being introduced more than 25 years ago, it remains an underutilized technique, with only 12% of pulmonologists reporting its routine use for the diagnosis and staging of lung cancer. The technique, however, is incredibly useful, and may preclude further invasive surgery in 29% of patients. The yield with TBNA has been associated with tumor cell type (small cell 4 non-small cell 4 lymphoma), lymph node size, and lymph node location. The use of CT uoroscopy to guide TBNA and transbronchial biopsy has several advantages over standard uoroscopy. As opposed to standard uoroscopy, which is typically used only in two dimensions, CT provides the ability to visualize the target in three dimensions. With standard uoro, either the patient or the C-arm needs to be rotated to conrm the biopsy tool is not anterior or posterior to the target. CT uoro provides real-time three dimensional conrmation of the appropriate (Figure 1) and inappropriate (Figure 2) biopsy sites. The use of CT uoro to guide TBNA has been associated with an accuracy of 88%, including patients who have previously undergone a nondiagnostic bronchoscopy with standard TBNA. Endobronchial ultrasound is another important modality used to help improve accuracy of TBNA. With the development of a miniaturized 20 MHz transducer that can be inserted via a 2.8 mm working channel, lymph nodes and masses adjacent to the airway can now be visualized (Figure 3). Like CT uoro, EBUS has also been shown to improve the yield of TBNA. Recently, a bronchoscope with a dedicated ultrasound probe and distinct working TM channel has been developed (Puncturescope , Olympus Corporation, Tokyo, Japan), and has the benet

Figure 1 CT uoroscopy showing the biopsy forceps in the target.

308 BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

Several studies have shown that the use of autouorescence increases the detection of early stage lung cancer by up to sixfold when compared to white light bronchoscopy. The denitive role of AF bronchoscopy in the early detection of lung cancer remains to be dened.
Diffuse Parenchymal Lung Disease

Figure 2 CT uoroscopy showing the biopsy forceps anterior to the target, near the pleura. Note that without rotation of the patient/C-arm, the biopsy forceps may appear to be within the target on standard uoroscopy.

Figure 3 Endobronchial ultrasound image showing tumor invading airway wall from the 12:002:00 position.

of providing real-time guidance for TBNA of mediastinal and hilar lymph nodes, with excellent results. EBUS has been shown to better differentiate airway invasion versus compression by adjacent tumor when compared to CT, and can suggest the histology of a solitary pulmonary nodule based on the ultrasound morphology. Autouorescence (AF) bronchoscopy is an increasingly popular tool used for the early detection of cancer in the central airways, primarily carcinoma in situ (CIS), and squamous cell carcinoma. When exposed to light in the violetblue spectrum (400450 nm), the normal airway uoresces green. As submucosal disease progresses from normal, to metaplasia, to dysplasia, to CIS, there is a progressive loss of the green AF, causing a redbrown appearance of the airway wall.

Diffuse parenchymal lung disease describes a group of infectious, inammatory, and brotic disorders which may involve the interstitial, alveolar, bronchial, and vascular structures of the tracheobronchial tree. The most commonly used sampling techniques for patients with diffuse disease include BAL, bronchial brushing, transbronchial biopsy (TBBx), and occasionally endobronchial biopsy (EBBx) and TBNA. Though associated with a low morbidity, transbronchial biopsy should be used only when the potential results will impact on treatment decisions. Diseases in which transbronchial biopsy can prove diagnostic or has been shown to signicantly increase the diagnostic yield as compared to less invasive means include lymphangitic carcinomatosus, sarcoidosis, rejection after lung transplantation, hypersensitivity pneumonitis, and sometimes, invasive fungal infection. The overall diagnostic yield for transbronchial biopsy in this category depends on the disease entity. For example, the yield for sarcoidosis can approach 90%, but is much lower for patients with vasculitis or cryptogenic organizing pneumonia. Aside from providing a specic diagnosis in cases of cancer or infection, the results of BAL can serve to limit the differential diagnosis considerably. For example, a BAL with lymphocyte predominance suggests granulomatous disease such as sarcoidosis, berylliosis, or a lymphoproliferative disorder. Neutrophil predominance suggests bacterial infection, acute interstitial pneumonia, and can be seen in patients with asbestosis or usual interstitial pneumonitis. Eosinophils are seen in patients with eosinophilic pneumonias, hypereosinophilic syndromes, or ChurgStrauss syndrome. Patients with pulmonary alveolar proteinosis (PAP), have a unique appearance to the BAL uid that is described as milky or opaque. The alveolar macrophages are lled with PAS-positive material, and lamellar bodies can be seen under electron microscopy.
Infectious Diseases

Community acquired pneumonia The role of bronchoscopy in community acquired pneumonia (CAP) remains controversial. When used, BAL and protected brush are the main diagnostic procedures, and the specimens should ideally be sent for

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 309

respiratory tract culture prior to the initiation of antibiotics, the use of semiquantitative or quantitative culture data, and the use of negative culture data to discontinue antibiotics in patients who have not had changes in their antibiotic regimen within the last 72 h. Additionally, the use of a bronchoscopic strategy was supported as a way to reduce 14-day mortality. Immunocompromised host The early diagnosis and initiation of the appropriate antibiotic is the cornerstone of successful treatment of the immunocompromised patient with pneumonia. Additionally, it is important to note that multiple diagnoses can often be present simultaneously in these patients, and noninfectious conditions may have a similar presentation of cough, dyspnea, fever, and an inltrate on imaging. Bronchoscopy is an excellent method of evaluating these patients as less invasive techniques can miss the diagnosis in approximately 30% of patients, and earlier diagnosis may improve mortality. BAL is the most commonly used bronchoscopic technique used to obtain a diagnosis in immunocompromised patients, with an overall diagnostic yield of approximately 6070%. In comparative studies, the sensitivity of TBBx has been shown to be roughly the same, though the combined use of both techniques may increase the yield. The results of BAL have been shown to change management in up to 84% of cases. Even in the immunocompromised host, bronchoscopy with BAL remains a safe procedure. Brushing and TBBx, however, have been associated with a higher incidence of bleeding complications in patients who are thrombocytopenic. The gold standard for tissue diagnosis remains open lung biopsy, which can yield a specic diagnosis in 62% of patients, and result in a signicant increase in survival. There are no data suggesting that bronchoscopy reduces mortality in this patient population. Hemoptysis There are many causes of hemoptysis including infectious, inammatory, vascular, and neoplastic processes. Though one would think that bronchoscopy can often make the diagnosis of a radiographic occult neoplasm in a patient with hemoptysis, a bronchoscopic diagnosis of malignancy is made in o5% of patients. Indications for bronchoscopy in patients with normal chest imaging include age 440 years, male gender, and a 440 pack-year smoking history. Though the appropriate timing for bronchoscopy is controversial, there is a greater likelihood of identifying the bleeding source when performed within the rst 48 h of symptoms. The combined use of bronchoscopy and CT is also recommended. If patients are clinically stable, we prefer

Figure 4 (a) Protected specimen brush; (b) protected specimen brush with the protective wax cap expelled.

quantitative culture, with a threshold of 104 colonyforming units (CFU) for the BAL and 103 CFU for the protected brush (Figure 4). In addition to providing a microbiologic diagnosis, another important indication for bronchoscopy in patients with CAP is to rule out an obstructing endobronchial lesion in the right clinical setting. Obviously, it is crucial to avoid contamination with upper airway secretions when performing bronchoscopy in patients with pneumonia. Key procedural aspects include minimizing suctioning, as well as minimizing the instillation of lidocaine, as secretions in the working channel will be ushed back into the airways, and high concentrations can be bacteriostatic. Healthcare and ventilator associated pneumonia The American Thoracic Society and Infectious Disease Society have recently reviewed these topics in great detail, and used the techniques of evidence based medicine to guide their recommendations. Some major points included the collection of a lower

310 BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

to obtain the CT rst, as it can serve as a road map to guide bronchoscopy.

Therapeutic Bronchoscopy
Bronchoscopy in Hemoptysis

The denition of massive hemoptysis has ranged from 1001000 cm3 expectorated in a 24 h period. As the majority of patients with massive hemoptysis die from asphyxia, and not exanguination, and the anatomic dead-space is approximately 150 cm3, we consider any amount over 100 cm3 in 24 h as massive. In addition to identifying the source and cause of bleeding, bronchoscopy clearly plays an important therapeutic role in patients with massive hemoptysis. If available, we strongly recommend rigid bronchoscopy as the procedure of choice in these patients. In addition to securing an airway, and providing oxygenation and ventilation, the rigid bronchoscope allows the passage of large-bore suction catheters as well as a variety of other tools that can help stop the bleeding including cryotherapy, electrocautery, argon plasma coagulation (APC), and Nd:YAG laser, as well as bronchial blockers. If rigid bronchoscopy is not available, we recommend tracheal intubation with the largest endotrachcal tube available, with selective right or left-mainstem intubation to protect the non-bleeding lung as needed. Double-lumen endotracheal tubes, or specialized tubes that come with an endobronchial blocker (e.g., Univent, Vitaid, Williamsville, NY), can be more difcult to place, especially in the setting of massive hemoptysis. The main role of exible bronchoscopy in the patient with massive hemoptysis lies in obtaining lung isolation, and protecting the good lung, as the suction channel of a exible scope is relatively small. All bronchoscopists should become familiar with bronchial blockers (e.g., Arndt Bronchial Blocker, Cook Critical Care, Bloomington, IN), which can be passed in parallel to the exible scope, and some, even inserted through the working channel. These catheters are guided to the culprit segmental, lobar, or mainstem bronchus and the balloon is inated to the recommended volume/pressure. After a maximum of 24 h, the balloon should be deated under bronchoscopic visualization. Other bronchoscopic techniques used to control hemoptysis include the topical application of iced saline, epinephrine (1 : 20 000), thrombin/thrombinbrinogen, or cyanoacrylate solutions.
Other Therapeutic Bronchoscopic Techniques

argon gas (plasma) to achieve tissue coagulation and hemostasis. As the plasma is directed to the closest grounded source, APC has the ability to treat lesions lateral to the probe, or around a bend, that would not be suitable for laser therapy. The depth of penetration for APC is approximately 23 mm, and hence the risk of airway perforation is also less when compared to lasers. Laser therapy The Nd:YAG laser is the most widely used laser in the lower respiratory system, and has been used for both benign and malignant disease. The primary advantage of laser photoresection includes rapid destruction/vaporization of tissue. Lesions most amenable to laser therapy are central, intrinsic, and short (o4 cm), with a visible distal endobronchial lumen. When lesions meet these criteria, patency can be re-established in more than 90% of cases. Care must be taken however, as the depth of penetration can approach 10 mm and airway perforation with resultant pneumothorax, pneumomediastinum, and vascular injury have been reported. In view of this, we encourage its use only by experienced interventional bronchoscopists. Nevertheless, the safety record of laser bronchoscopy is excellent, with an overall complication rate of o1%. Cryotherapy Cryotherapy is a safe and effective tool for a variety of airway problems. By releasing nitrous oxide stored under pressure, the tip of the cryoprobe rapidly cools to 891C. We primarily use cryotherapy for the removal of organic foreign bodies with high water content such as grapes and vegetable matter, in addition to facilitating the removal of tenacious mucus or blood clots when performing exible bronchoscopy. Compared to the other techniques described in this chapter, cryotherapy results in delayed tumor destruction, requiring a repeat bronchoscopy to remove the necrotic tumor. The distinct advantage of cryotherapy lies in the fact that the normal cartilage and brous tissue of the airway are relatively cryoresistant, in addition to the lack of risk of airway res. Electrocautery Electrocautery uses alternating current at high frequency to generate heat, which cuts, vaporizes, or coagulates tissue depending on the power. Electrocautery is a contact mode of tissue destruction and a variety of cautery probes are available including blunt tip probes, knifes, and snares. We favor the use of the cautery snare for pedunculated lesions of the airway as the stalk can be cut and coagulated while preserving the majority of the tissue for pathologic interpretation. As with laser therapy,

Argon plasma coagulation Argon plasma coagulation (APC) is a noncontact method using ionized

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the risks of electrocautery include airway perforation, airway res, and damage to the bronchoscope. Photodynamic therapy Photodynamic therapy (PDT) involves the intravenous injection of a photosensitizing agent, then activating the drug with a nonthermal laser to produce a phototoxic reaction and cell death. As tumor cells retain the drug longer than other tissues, waiting approximately 48 h after drug injection will lead to preferential tumor cell death as compared to normal tissue injury. As with cryotherapy, maximal effects are delayed, and a repeat, clean-out bronchoscopy should be performed 2448 h after drug activation. The primary side effect from PDT is systemic phototoxicity, which can last up to 6 weeks after injection. Newer drugs are being developed with the hopes of increasing tumor selectivity and reducing the duration of skin phototoxicity. As laser activation uses a nonthermal laser source, airway res are not an issue. PDT has been shown to be curative for early stage lung cancer of the airways and is an especially attractive option for patients with endobronchial CIS who are not surgical candidates due to other comorbidities. Brachytherapy Brachytherapy refers to endobronchial radiation, primarily used for the treatment of malignant airway obstruction. The most commonly used source of radiation is iridium-192 (192Ir), which is inserted bronchoscopically via a catheter. Brachytherapy may be delivered by either low-dose rate (LDR), intermediate-dose rate (IDR), or high-dose rate (HDR) methods, with most authors currently recommending the afterloading HDR technique. This allows the bronchoscopist to place the catheter in the desired location and the radiation oncologist to deliver the radiation in a protected environment. The main advantage of HDR is patient convenience, as each session lasts o30 min; however, multiple bronchoscopies are required to achieve the total 1500 cGy dose that is currently recommended. The LDR technique may be appropriate for patients who live far from the hospital or who are otherwise hospitalized as it only requires one bronchoscopy, but the catheter has to stay in place for 2060 h. The main advantage of brachytherapy as compared with external-beam radiation is the fact that less normal tissue is exposed to the toxic effects of radiation. The most common side effects include intolerance of the catheter, radiation bronchitis, airway perforation, and, occasionally, massive hemorrhage. Treatment of tumors in the right and left upper lobes has the highest incidence of hemorrhage, as these are located near the great vessels.

Airway stents Montgomery is credited as initiating the widespread use of airway stents after his development of a silicone T-tube in 1965 for use in patients with tracheal stenosis. Dumon, however, introduced the rst completely endoluminal airway stent in 1990. Airway stents are the only technology that can alleviate extrinsic airway compression. They are commonly used in conjunction with the other modalities for patients with intrinsic or mixed disease. Over the last 15 years, there has been an explosion in both stent design and the number of endoscopists who place airway stents. As with any procedure, it is crucial to understand the indications and contraindications of the procedure as well as be able to anticipate, prevent and manage the associated complications. Unfortunately, the ideal stent has

Figure 5 Metal stent in the trachea.

Figure 6 Some available silicone stents ex vivo.

312 BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

not yet been developed. This stent would be easy to insert and remove, yet would not migrate; would be of sufcient strength to support the airway, yet be exible enough to mimic normal airway physiology and promote secretion clearance; biologically inert to minimize the formation of granulation tissue; and available in a variety of sizes. There are currently two main types of stents: metal, generally Nitinol (Figure 5), and silicone (Figure 6). Though metal stents are easily placed, they can be extremely difcult to extract, and may cause excessive granulation tissue formation (Figure 7). They are

available in covered and uncovered varieties. For malignant airway obstruction, the only appropriate metal stents are covered models, which minimize tumor ingrowth. Some authors feel that there is no indication for an uncovered metal stent. The main advantage of metal stents is their larger internal : external diameter ratio as compared to that of silicone stents. Though silicone stents require rigid bronchoscopy for placement, they are more easily removed and are signicantly less expensive. The future of airway stenting likely lies in the creation of bioabsorbable stents, made out of materials such as vicryl laments or poly-L-lactic acid (SR-PLLA). In addition to malignant airway obstruction, airway stents can be helpful in patients with tracheobronchomalacia and tracheoesophageal stula. In patients with tracheoesophageal stula, doublestenting of the esophagus and airway is recommended to maximally prevent aspiration.

Figure 7 Metal stent removal.

Powered instrumentation The microdebrider is a tool consisting of a hollow metal tube with a rotating blade coupled with suction. We have had excellent results with this technology in the treatment of both benign and malignant central airway obstruction. A primary advantage of the microdebrider is the rapidity of obtaining a patent airway and the lack of risk of airway res as compared to modalities using heat.

Figure 8 Super dimension bronchoscopy showing the locatable guide in the target in the axial, coronal, and sagittal planes on CT, as well as the ghter-pilot view in the lower right, with the target 0.2 cm directly ahead.

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Future Directions in Bronchoscopy

In the appropriate patient, lung volume reduction surgery has been shown to improve both quality and quantity of life. The major drawback to this surgery is the associated morbidity and cost of the procedure. Recent studies have suggested that lung volume reduction can be obtained bronchoscopically, either by creating channels in the distal airways/parenchyma, or by the placement of one-way endobronchial valves to promote deation of hyperinated lung that does not signicantly contribute to gas exchange. The results of large-scale, multicenter trials will become available within the next several years, but the preliminary data are promising. The use of electromagnetic navigation is a novel technology that has been shown to allow accurate sampling of peripheral solitary pulmonary nodules o1 cm in diameter. Briey, a virtual bronchoscopy is generated from the patients CT scan. Anatomic landmarks that are easily identied, such as the carinae, are marked, as is the target. At the time of bronchoscopy, the patient lies in an electromagnetic eld and a steerable, locatable guide is placed through an extended working channel of the bronchoscope. The location of the guide in the electromagnetic eld is accurate to o5 mm in the x, y, and z axes, as well as yaw, pitch, and roll. The points previously identied in the virtual bronchoscopy are then marked with the locatable guide, which in essence, marries the CT scan with the bronchoscopic image. Navigation is then performed by looking at the CT in the axial, sagittal, and coronal planes, and the guide is steered toward the target (Figure 8). Once found, the guide is removed, and standard bronchoscopic tools such as TBNA needles and forceps are placed through the extended working channel. This technology not only has the potential to revolutionize diagnostic bronchoscopy,

but therapeutic bronchoscopy as well. For example, if a patient with a 2.5 cm, stage Ia, nonsmall cell cancer is not an operative candidate, this technology may allow for bronchoscopic treatment by either radiofrequency ablation or the implantation of ducials to allow stereotactic radiosurgery.
See also: Alveolar Hemorrhage. Bronchiectasis. Bronchiolitis. Bronchomalacia and Tracheomalacia. Drug-Induced Pulmonary Disease. Interstitial Lung Disease: Overview. Lung Anatomy (Including the Aging Lung). Mediastinal Masses. Panbronchiolitis. Pneumonia: Overview and Epidemiology; The Immunocompromised Host. Tumors, Malignant: Overview. Upper Airway Obstruction. Upper Respiratory Tract Infection.

Further Reading
Becker HD (1991) Atlas of Bronchoscopy: Technique, Diagnosis, Differential Diagnosis, Therapy. Philadelphia: BC Decker. Bolliger CT and Mathur PN (2000) Interventional Bronchoscopy. Basel: Karger. Detterbeck FC, DeCamp MM Jr, Kohman LJ, and Silvestri GA (2003) Invasive staging: the guidelines. Chest 123(90010): 167S175S. Ernst A, Feller-Kopman D, Becker HD, and Mehta AC (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169(12): 12781297. Ernst A, Silvestri GA, and Johnstone D (2003) Interventional pulmonary procedures: guidelines from the American College of Chest Physicians. Chest 123(5): 1693. Fagon JY, Chastre J, Wolff M, et al. (2000) Invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia: a randomized trial. Annals of Internal Medicine 132(8): 621630. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. (2005) American Journal of Respiratory and Critical Care Medicine 171(4): 388416. Prakash USB (1994) Bronchoscopy. New York: Raven Press.

C
Calcium Channels
see Ion Transport: Calcium Channels.

CAPSAICIN
M G Belvisi and D J Hele, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The pungent ingredient in red pepper fruits of the genus Capsicum, which includes paprika, jalapen o, and cayenne, is called capsaicin. Capsaicin is known to stimulate sensory nerves leading to the activation of nociceptive and protective reex responses (e.g., cough, bronchospasm) and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inammatory responses often referred to as neurogenic inammation, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion. The capsaicin receptor has recently been identied and has been named the type 1 vanilloid receptor (VR1; TRPV1). It has previously been suggested that there is an upregulation of TRPV1 expression in inammatory diseases and that inappropriate activation of this receptor may lead to sensory nerve hyperresponsiveness. Thus, it would appear that airway inammatory diseases (e.g., asthma and chronic obstructive pulmonary disease) may respond to treatment with an effective and selective inhibitor of TRPV1 and to this end much work is being carried out to develop novel inhibitors.

via the peripheral release of neuropeptides, a phenomenon known as neurogenic inammation. A characteristic feature of many nociceptive sensory bers is their sensitivity to capsaicin. However, until recently the molecular mechanisms involved in activation of sensory nociceptive bers were unknown. Pharmacological evidence for the presence of a capsaicin receptor in sensory nerves was provided by the discovery of two capsaicin analogs, resiniferatoxin (potent agonist) and capsazepine (an antagonist). First, specific binding sites for resiniferatoxin were demonstrated on dorsal root ganglion membranes and second, capsazepine has been found to inhibit numerous capsaicin-evoked neuronal responses including those in the airways. The capsaicin receptor has recently been identied and has been named the type 1 vanilloid receptor (VR1; transient receptor potential vanilloid 1 (TRPV1)).

Capsaicin and Functional Responses in the Airways

Cough

Introduction
Sensory nerves in the airways regulate central and local reex events such as bronchoconstriction, airway plasma leakage, and cough. Sensory nerve activity may be enhanced during inammation such that these protective reexes become exacerbated and deleterious. Sensory nerve reexes are under the control of at least two different classes of sensory ber, the myelinated rapidly adapting stretch receptors (RARs) and nonmyelinated capsaicin-sensitive C bers. In the airways, activation of RARs and C bers elicits cough, bronchoconstriction, and mucus secretion via an afferent central reex pathway. Activation of C bers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as bronchoconstriction, mucus secretion, plasma exudation, and vasodilatation

Inhalation challenge with capsaicin and low pH (e.g., citric acid) has been shown to evoke cough and has been used as a model for the last 50 years to investigate the action of potential antitussive therapies in clinical trials. In fact, agonists at TRPV1 such as capsaicin and resiniferatoxin are the most potent stimulants of the cough reex so far described in man. Therefore, it has been suggested that TRPV1 activation may be one of the primary sensory mechanisms in cough. However, if activation of TRPV1 is an important initiating factor for the cough reex then an endogenous capsaicin-like ligand must be present. A number of putative endogenous ligands that are known to activate TRPV1 have been demonstrated to cause cough. TRPV1 has been shown to be sensitive to a fall in the extracellular pH, and H ions not only stimulate the receptor directly but also

316 CAPSAICIN

increase the sensitivity of the receptor to capsaicin and low pH solutions, which are known to elicit cough in animals and humans. Interestingly, the TRPV1 antagonists capsazepine and iodo-resiniferatoxin have been shown to inhibit capsaicin, citric acid, and anandamide-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, that is, activation of TRPV1 (Figure 1). However, it is unlikely that TRPV1 activation is the only stimulus for cough since some tussigenic agents such as hypertonic saline are not inhibited by TRPV1 antagonism.

often referred to as neurogenic inammation, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion (Figure 2).
Bronchoconstriction

Neurogenic Inammation
Capsaicin activates sensory nerves leading to the activation of nociceptive and protective reex responses and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inammatory responses
2 Capsazepine (10 M) Control

Coughs min1

1.5

0.5

30 M Capsaicin

80 M

Citric acid (0.25 M)

Figure 1 Representation of data demonstrating that the TRPV1 antagonist capsazepine inhibits capsaicin and citric acid-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, i.e., activation of TRPV1.

Early experiments using the relatively weak TRPV1 antagonist capsazepine provided pharmacological validation that capsaicin-induced bronchospasm in guinea pig bronchi did indeed involve the activation of TRPV1. Contractile responses to resiniferatoxin and capsaicin were unaffected by the neurokinin (NK)-1 antagonist CP 96345, partially inhibited by the NK-2 antagonist SR 48968, but nearly abolished by a combination of the antagonists. These data suggest that resiniferatoxin and capsaicin both release tachykinins that act on both NK-1 and NK-2 receptor subtypes in a TRPV1-dependent manner. More recently, a more potent and selective agent has been used to conrm these observations. Interestingly, contractile and relaxant responses to capsaicin and resiniferatoxin have also been examined in human isolated bronchus (512 mm outside diameter). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The capsaicin-induced contractions were mimicked by resiniferatoxin and inhibited by capsazepine. The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968, whereas responses to concentrations of NKA, NKB, substance P, neuropeptide g, and neuropeptide K, which produced contractions of a similar size, were almost abolished by SR 48968. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone in a TRPV1-dependent manner, but this response does not appear to involve substance P or related neurokinins. Airway hyperresponsiveness (AHR) to bronchoconstrictor agents is recognized as a critical feature of

Capsaicin

Vasodilatation

Plasma exudation

C fibers

Central reflexes, e.g., cough

Mucus secretion

Neuropeptide release SP, NKA, CGRP

Bronchoconstriction
Figure 2 Activation of sensory nerves by capsaicin. SP, substance P; CGRP, calcitonin gene-related peptide; NKA, neurokinin A. Adapted from Barnes PJ (2001) Neurogenic inammation in the airways. Respiration Physiology 125: 145154.

CAPSAICIN 317

bronchial asthma and sensory nerves in the airway are strongly implicated in the hyperresponsiveness. Several studies have demonstrated that pretreatment with capsaicin (which depletes sensory neuropeptides) significantly inhibited the late bronchial response that was observed after ovalbumin inhalation, AHR, and eosinophil accumulation in an allergic guinea pig model, and AHR to histamine in a rabbit model. It remains to be seen whether TRPV1 antagonists are effective in this regard.
Plasma Extravasation

Capsazepine Airway C fiber

Capsaicin, RTx H+ (citric acid, low pH), heat +

TRPV1

Cholinergic reflex activation

Cough Chest tightness

AHR

Mucus secretion

Figure 3 Events mediated via TRPV1 following the activation of nociceptive airway C bers by noxious stimuli.

Activation of C bers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as plasma exudation and vasodilatation via the peripheral release of neuropeptides. Plasma extravasation has been shown to be induced in rats or guinea pigs by intravenous injections of substance P (SP) and capsaicin. The effect of intravenous capsaicin was absent in capsaicin-desensitized animals and in those pretreated with capsazepine. Capsaicin-induced plasma extravasation was also markedly inhibited by CP 96345, a nonpeptide antagonist of tachykinin NK-1 receptors. These data suggest that capsaicin-induced plasma leakage is mediated by the release of neuropeptides and the activation of NK-1 receptors via a TRPV1dependent mechanism.
Mucus Secretion

When stimulated capsaicin-sensitive C bers (afferents) containing the neuropeptides SP, NKA, and calcitonin gene-related peptide have also been shown to evoke neurogenic secretion from airway mucussecreting cells. However, although capsaicin has been shown to elicit mucus secretion, there is no data available describing the effect of TRPV1 antagonists on this response.

Molecular Mechanism of Action of Capsaicin

The Type 1 Vanilloid Receptor

(PMg/PNaB5). TRPV1 is activated by heat (4431C), but is effectively dormant at normal body temperature and low pH (o5.9) and may act as an integrator of chemical and physical pain-eliciting stimuli. When activated, TRPV1 produces depolarization through the inux of Na , but the high Ca2 permeability of the channel is also important for mediating the response to pain. Gating by heat is direct but the receptor can be opened by ligands or stimuli such as mild acidosis, which also reduces the threshold for temperature activation and potentiates the response to capsaicin. To summarize, TRPV1 mediates nociception and contributes to the detection and integration of diverse chemical and thermal stimuli. The expected role for TRPV1 is in pain pathways and recent data from a study with TRPV1 knockouts showed impaired inammatory thermal hyperalgesia. This has led to a growing interest in developing small molecule antagonists for this target. Recent preclinical data demonstrating efcacy in rodent models of both thermal and mechanical neuropathic or inammatory pain has fuelled this enthusiasm. The established role of sensory nerve activation in the cough reex and the role of TRPV1 in inammatory pain has also alerted the respiratory community to the therapeutic potential of TRPV1 antagonists as antitussives and as therapy for airway inammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD).
Ligands for TRPV1

TRPV1 is a membrane-associated vanilloid receptor. It is a nociceptor-specific ligand-gated ion channel expressed on the neuronal plasma membrane of nociceptive C bers and is required for the activation of sensory nerves by vanilloids such as capsaicin, the pungent extract from plants in the genus Capsicum (e.g., hot chilli peppers) (Figure 3). TRPV1 also mediates the response to painful heat, extracellular acidosis, protons, and tissue injury. TRPV1 is an outwardly rectifying cation-selective ion channel with a preference for calcium (PCa/PNaB10) and magnesium

Exogenous agonists of TRPV1 include capsaicin and resiniferatoxin. Endogenous agonists include the cannabinoid receptor agonist anandamide, N-arachidonoyl-dopamine, inammatory mediators (bradykinin, 5-HT, and prostaglandin E2 (PGE2)), hydrogen ions, heat, arachidonic acid, lipoxin A4, prostacyclin, ethanol, and several eicosanoid products of lipoxygenases including 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-(S)-hydroxyeicosatetraenoic acid and leukotriene B4. Interestingly, the effect of bradykinin

318 CAPSAICIN

may be indirect given that data exists suggesting that it appears to activate bradykinin B2 receptors on afferent neurons leading to the generation of lipoxygenase metabolites that have agonist activity at TRPV1. The evidence to support the suggestion that TRPV1 can be gated not only by vanilloids such as capsaicin, but also by protons, heat, agonists at certain G-protein-coupled receptors, ethanol, cannabinoids, and lipoxygenase products of arachidonic acid has come from electrophysiological patch-clamp recording studies on the cell bodies of TRPV1-expressing cells. Furthermore, pharmacological antagonism of TRPV1 has been shown to inhibit action potential discharge evoked by each of these stimuli. However, due to the nonselective effects of several of these tool compounds, this pharmacological approach has its limitations. Furthermore, if the antagonist inhibits but does not completely block a given response, it is not clear whether activation of the TRPV1 is obligatory for the response or merely contributes to the end response. In studies using TRPV1/ mice it was concluded that whereas TRPV1 is required for action potential discharge of C ber terminals evoked by capsaicin and anandamide, it plays a contributory role in responses evoked by low pH of bradykinin. These data suggest that TRPV1 is one of multiple ion channels responsible for the bradykinin-evoked generator potentials (i.e., membrane depolarization) in C ber terminals.
TRPV1 Receptor Antagonists

antagonist as a pharmacological tool in order to explore the role for TRPV1 in various physiological settings. Recently, however, although the in vitro antagonistic activity of iodo-resiniferatoxin has been conrmed, in vivo studies have demonstrated some agonist activity of this agent at high doses. Therefore, there is a possibility that this agent retains some agonistic activity and that an agonist-dependent desensitization effect on sensory nerves may well contribute to some of the antagonistic activity observed. The GlaxoSmithKline lead antagonist SB-366791, which has a good selectivity prole in a series of assays, and potent vanilloid agonist AM-404 should provide useful tools for probing the physiology and pharmacology of TRPV1. Several TRPV1 receptor antagonists that share no obvious structural resemblance to any class of vanilloid agonist have also been developed. These compounds have been discovered by high-throughput random screening projects. At present this group of antagonists includes: (1) N-(haloanilino)carbonyl-N-alkyl-N-arylethylendiamines discovered at SKB; (2) N-diphenyl-Nnapthylureas discovered at Bayer; and (3) pyridol [2,3-d]-pyrimidin-4-ones discovered at Novartis.

Localization of TRPV1
The capsaicin-sensitive vanilloid receptor is expressed mainly in sensory nerves including those emanating from the dorsal root ganglia and afferent bers that innervate the airway, which originate from the vagal ganglia. In the dorsal root and trigeminal ganglion, TRPV1 is localized to small and mediumsized neurons. Somatic sensory neurons of the vagus nerve are located in the jugular ganglion, whereas visceral sensory neurons of the nerve are located in the nodose ganglion. Previous studies have demonstrated that TRPV1-containing neurons are abundant in these ganglia. The traditional view that TRPV1 is simply a marker of primary sensory nerves is now being challenged. The TRPV1 receptor-has been detected in guinea pig and human airways by receptor-binding assays and has been identied in nonneuronal cell types such as mast cells, broblasts, and smooth muscle.

Antagonists of TRPV1 include ruthenium red, a dye that exhibits properties of noncompetitive antagonism for the TRPV1, and has a poorly dened mechanism of action and limited selectivity. Capsazepine is another agent characterized as a weak but relatively selective, competitive TRPV1 receptor antagonist (over other TRPVs). However, at the concentrations required to antagonize the TRPV1 (approximately 10 mM), capsazepine has demonstrated non-specific effects such as inhibition of voltage-gated calcium channels and nicotinic receptors. Furthermore, this ligand has poor metabolic and pharmacokinetic properties in rodents, where it undergoes extensive rstpass metabolism when given orally. Recently, a number of antagonists with improved potency and/or selectivity have been described and these include antagonists that are structurally related to agonists such as iodo-resiniferatoxin, which is 100-fold more potent than capsazepine. Previous evidence has indicated that iodo-resiniferatoxin is a potent antagonist at the TRPV1 both in vitro and in vivo. Since then several studies have used this

Role of TRPV1 in Airway Inammatory Disease

A role for TRPV1 in airway inammatory disease would depend on the presence of endogenous activators of this channel under physiological and pathophysiological conditions. In fact, it is quite possible that this situation does, in fact, exist in the disease scenario given that TRPV1 activation can be initiated

CAPSAICIN 319

and responses to other activators potentiated in an acidic environment, which has been shown to be present in diseases such as asthma and COPD. Interestingly, it has previously been established that the airway response to TRPV1 activation is enhanced in certain disease conditions. In particular, it has been reported that the cough response elicited in response to capsaicin is exaggerated in diseases such as asthma and COPD. Anandamide is also an endogenous ligand known to be produced in central neurons. However, more recent studies have suggested that anandamide is synthesized in lung tissue as well as a result of calcium stimulation suggesting that this mediator could also be involved in the activation of TRPV1 under normal and disease conditions. Inammatory agents (e.g., bradykinin, ATP, PGE2, and nerve growth factor (NGF)) can indirectly sensitize TRPV1 to cause hyperalgesia. Thus, bradykinin and NGF, which activate phospholipase C (PLC), release TRPV1 from phosphatidylinositol 4,5-biphosphate (PIP2)-mediated inhibition. However, PLC also regulates TRPV1 by diacylglycerol (DAG) formation and subsequent activation of protein kinase C (PKC), which in turn phosphorylates and sensitizes TRPV1. Inammatory stimuli, including prostaglandins and NGF among others, have also been shown to upregulate the expression and function of TRPV1 via the activation of p38 mitogen-activated protein-kinase (MAPK)- and protein kinase A (PKA)-dependent pathways. Previous data has suggested that there is an upregulation of TRPV1 in inammatory diseases. For example, TRPV1 immunoreactivity is greatly increased in colonic nerve bers of patients with active inammatory bowel disease. Furthermore, a recent study has found an increase in TRPV1 expression in sensory nerves found in the airway epithelial layer in biopsy specimens from patients with chronic cough compared to noncoughing, healthy volunteers. There was a significant correlation between the tussive response to capsaicin and the number of TRPV1positive nerves in the patients with cough. Therefore, it has been postulated that TRPV1 expression may be one of the determinants of the enhanced cough reex found in patients with chronic cough and that the recently described TRPV1 antagonists could be effective in the treatment of chronic persistent cough due to diverse causes.

work is being carried out to develop novel inhibitors. Interestingly, capsaicin-sensitive nerve stimulation in subjects with active allergic rhinitis produces reproducible and dose-dependent leukocyte inux, albumin leakage, and glandular secretion. These results provide in vivo evidence for the occurrence of neurogenic inammation in the human upper airway with active allergic disease and may therefore suggest the therapeutic utility of TRPV1 antagonists in the management of this disease. In addition, the treatment of persistent cough is a facet of airway diseases that is sorely in need of effective treatment and a TRPV1 inhibitor may prove extremely effective against cough induced by gastroesophageal acid reux, for example, as well as that associated with asthma and other diseases of the airways described above.
See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy.

Further Reading
Barnes PJ (2001) Neurogenic inammation in the airways. Respiratory Physiology 125: 145154. Belvisi MG (2003) Airway sensory innervation as a target for novel therapies: an outdated concept? Current Opinion in Pharmacology 3: 239243. Belvisi MG (2003) Sensory nerves and airway inammation: role of A delta and C-bres. Pulmonary Pharmacology and Therapeutics 16: 17. Belvisi MG and Geppetti P (2004) Cough * 7: current and future drugs for the treatment of chronic cough. Thorax 59: 438440. Caterina MJ and Julius D (2001) The vanilloid receptor: a molecular gateway to the pain pathway. Annual Review of Neuroscience 24: 487517. Coleridge HM and Coleridge JC (1994) Pulmonary reexes: neural mechanisms of pulmonary defence. Annual Review of Physiology 56: 6991. Holzer P (1991) Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacological Reviews 43: 143201. Hwang SW and Oh U (2002) Hot channels in airways: pharmacology of the vanilloid receptor. Current Opinion in Pharmacology 2: 235242. Karlsson JA (1993) A role for capsaicin sensitive, tachykinin containing nerves in chronic coughing and sneezing but not in asthma: a hypothesis. Thorax 48: 396400. Maggi CA and Meli A (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. General Pharmacology 19: 143. Morice AH and Geppetti P (2004) Cough 5: the type 1 vanilloid receptor: a sensory receptor for cough. Thorax 59: 257258. Rogers DF (2002) Pharmacological regulation of the neuronal control of airway mucus secretion. Current Opinion in Pharmacology 2: 249255. Szallasi A and Blumberg PM (1999) Vanilloid (Capsaicin) receptors and mechanisms. Pharmacological Reviews 51: 159212. Szolcsanyi J (2004) Forty years in capsaicin research for sensory pharmacology and physiology. Neuropeptides 38(6): 377384.

Conclusions
Thus, it would appear that airway inammatory diseases (e.g., asthma and COPD) may respond to treatment with an effective and selective inhibitor of the capsaicin receptor TRPV1 and to this end much

320 CARBON DIOXIDE

CARBON DIOXIDE
R A Klocke, University at Buffalo, Buffalo, NY, USA
& 2006 Elsevier Ltd. All rights reserved. Table 1 Relative contributions of dissolved CO2, bicarbonate ion, and carbamate compounds to carbon dioxide transport in arterial blood and excretion in the lungs Transport (%) Dissolved CO2 Bicarbonate ion Carbamate compounds 5 88 7 Excretion (%) 8 79 13

Abstract
Large quantities of CO2 produced in tissues must be efciently transferred into blood and transported to the lungs where CO2 is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements. Carbon dioxide is transported as three different, but interrelated, entities in blood. A small portion of total CO2 content in blood exists as dissolved carbon dioxide. Although small in absolute quantity, dissolved CO2 has a key role in exchange because it is the only form that rapidly crosses membranes separating blood from tissues and alveolar gas. The largest quantity of CO2 exists in blood as bicarbonate ion. This ion is a product of the dissociation of carbonic acid, a compound formed when CO2 combines with water. A modest amount of carbon dioxide is transported as carbamate, a compound formed by binding of CO2 to amino groups of the hemoglobin molecule. Exchange of oxygen augments simultaneous CO2 exchange through changes in the molecular conguration of hemoglobin. This structural change increases buffering of hydrogen ions and binding of CO2 as carbamate. The time required for completion of these interrelated processes is critical because blood remains in the pulmonary capillaries for less than 1 s. Two processes overcome this limitation. The natural rate of conversion between CO2 and carbonic acid is quite slow, but is catalyzed in vivo by carbonic anhydrase. In addition, a specialized transporter protein facilitates bicarbonate transfer across the erythrocyte membrane. Reactions of CO2 play a major compensatory role in diseases that cause metabolic acidosis. Hydrogen ions combine with bicarbonate ions and are converted into CO2, which is subsequently excreted in the lungs. This process reduces circulating hydrogen ions and is an important factor in defense of acidbase homeostasis. Although the rare congenital absence of isomers of carbonic anhydrase have been previously associated with abnormalities in the brain, bones, and kidneys, recent evidence raises the possibility that CO2 excretion may also be impaired in these patients.

Dissolved CO2

At normal CO2 tensions present in blood, dissolved CO2 has an approximate concentration of 1.2 mM. The difference between arterial and venous blood is small and accounts for only 8% of the total CO2 excreted in the lungs (Table 1). However, dissolved CO2 is crucial to gas exchange since it is the only form that can rapidly traverse the capillary membranes separating circulating blood from tissues and alveolar gas.
Bicarbonate Ion

Carbon dioxide combines with water to form carbonic acid, a weak acid with a pH of 3.5. At the pH range present in the body, this acid almost completely dissociates into hydrogen ions and bicarbonate ions: H2 O CO2 2 H2 CO3 2H HCO 3
CA

Introduction
Carbon dioxide (CO2) and water are the end products of oxidative metabolism that generates the energy required for maintenance of body homeostasis. Large quantities of CO2 produced in tissues must be efciently transferred into blood and transported to the lungs where it is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements.

CO2 Transport in Blood

Carbon dioxide is transported in three forms: as dissolved CO2, as bicarbonate ion, and bound to hemoglobin as a carbamate compound.

The natural rate of formation of carbonic acid from CO2 is very slow and requires 6090 s to reach completion. However, erythrocytes contain large quantities of carbonic anhydrase (CA), an enzyme that catalyzes this reaction. The hydrogen ions formed in this reaction are buffered effectively by hemoglobin, which is the dominant nonbicarbonate buffer in blood. Release of oxygen from the hemoglobin molecule alters the quaternary structure of hemoglobin, thereby increasing the afnity of binding sites for hydrogen ions (the Bohr effect). This increases the buffering capacity of deoxygenated hemoglobin. Figure 1 illustrates the buffering curves of oxygenated and reduced hemoglobin. As hemoglobin is reduced in the tissues, its buffering curve shifts upward and significant amounts of hydrogen ion can be buffered without any change in pH. It is estimated that approximately one half of the hydrogen ions released in aerobic metabolism are buffered in this manner. The majority of CO2 in blood is carried as bicarbonate (Table 1).

CARBON DIOXIDE 321

3.0 H+ added (mEq) 2.0 1.0 0.0 pH without H+ binding 7.30 7.35 pH 7.40 H+ from CO2
Ar Ve

ter

ial

no

us

Actual pH

Figure 1 Buffering curves of hemoglobin in arterial and venous blood. With release of oxygen bound to hemoglobin, buffering sites on hemoglobin are able to bind more hydrogen ions (the Bohr effect). The increased binding of hydrogen ions occurs as bloodacid base status shifts from the arterial to venous buffering curve. Hydrogen ions generated from CO2 exchange in the tissues are buffered with less change in pH than would occur if buffering took place along the arterial curve.

two N-terminal portions of the b-chains on the surface of hemoglobin are more widely separated in the reduced state as a result of the accompanying change in the quaternary structure. Positive charges on the b-chains induce the negatively charged 2,3DPG molecule to enter the enlarged cavity between the two separated b-chains of reduced hemoglobin. The positioning of 2,3-DPG near the N-terminal a-amino groups alters the equilibrium constants of reactions [2] and [3], and reduces the quantity of the uncharged form of the a-amino groups that binds CO2.
Haldane Effect

Carbamate Compounds

Amino groups of proteins can reversibly bind hydrogen ions:

R-NH 3 2H R-NH2

where R represents the remainder of the protein moiety. Carbon dioxide can bind to the uncharged amino groups: R-NH2 CO2 2R-NHCOOH 2R-NHCOO H 3

to form carbamic acids that dissociate into carbamate and hydrogen ions at normal blood pH. The quantity of CO2 bound to serum proteins and the e-amino groups of hemoglobin is minimal and does not change significantly between arterial and venous CO2 tensions. However, the a-amino groups of the N-termini of the hemoglobin molecule contribute substantially to CO2 transport. The changes in molecular conguration accompanying reduction of hemoglobin alter the equilibrium constants of reactions [2] and [3], favoring binding of CO2 as carbamate. As a result, the relative contribution of carbamate to the exchange of CO2 is approximately twice as great as its relative concentration in blood (Table 1). The a-amino groups of the b-chains of hemoglobin are affected to a greater extent by conformational changes of the molecule and are responsible for three-quarters of the carbamate contribution to exchange. The concentration of 2,3-diphosphoglycerate (2,3DPG) within the erythrocyte inuences the contribution of carbamate compounds to CO2 exchange. The

The carbon dioxide dissociation curve of normal human blood is illustrated in Figure 2. The total concentration of CO2 in all forms is plotted as a function of the carbon dioxide partial pressure in blood. As seen in the gure, at any given PCO2 the total CO2 content is greater in blood with a reduced oxygen content compared to fully oxygenated blood. This is known as the Haldane effect, named after one of the investigators who rst described this phenomenon. The difference in CO2 content between the two curves at the same PCO2 is termed oxylabile CO2. It is produced by the changes in the quaternary structure of hemoglobin that accompany oxygenation and reduction. Oxylabile CO2 has two components. The rst is an increase in bicarbonate concentration resulting from greater buffering of hydrogen ions by reduced hemoglobin. The second component is the result of increased carbamate formation associated with reduced hemoglobin. The Haldane effect enhances the transport of carbon dioxide. The shift of the CO2 dissociation curve caused by release of oxygen allows for transport of CO2 with a lower CO2 tension in venous blood than would occur if there were no shift in the position of the dissociation curve (Figure 2). The synergism between oxygen and carbon dioxide exchange facilitates transport of CO2 to a much greater extent than the transport of O2.

Time-Dependent Processes in CO2 Exchange

The actual exchange of CO2 in the pulmonary capillaries involves a series of complicated processes. Exchange of each form in which CO2 is transported is discussed separately, but the processes occur simultaneously (Figure 3). The reactions involved in conversion of bound CO2 to dissolved CO2 are complex and require nite time. These reactions probably reach completion in 0.30.4 s, which is

322 CARBON DIOXIDE

60

40 CO2 content (ml dl1)

70% HbO2

54

PCO2

52 20 97.5% HbO2 50 a 48 40 45 50 PCO2 without Haldane effect

0 0 20 PCO (mmHg) 2
Figure 2 Carbon dioxide dissociation curves of blood. The two curves represent the relationship between CO2 content and PCO2 of arterial (oxygen saturation of 97.5%) and venous (oxygen saturation of 70%) blood. Binding of oxygen to hemoglobin causes a shift from the venous to arterial CO2 dissociation curve. The inset in the gure illustrates the effect of this shift on blood PCO2 . The difference in PCO2 between arterial (a) and venous blood (v ) is substantially less than would occur if the Haldane effect were not operative and CO2 exchange took place along a single dissociation curve (a and v 0 ). Data used to construct the dissociation curves taken from Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, p. 238. Chicago: Yearbook Medical Publishers.

40

60

sufcient for excretion of CO2 to occur during the time the blood remains in the pulmonary capillary (0.7 s at rest, 0.5 s during exercise). Although the process of diffusion of CO2 across the alveolarcapillary membrane is quite rapid (B0.01 s), the chemical reactions and other transport processes necessary for CO2 exchange appreciably slow the actual rate of exchange. In peripheral tissues, these same processes occur in an opposite manner.

Bicarbonate Ion

Dissolved CO2

When blood reaches the pulmonary capillaries, dissolved CO2 diffuses from the plasma and the interior of the erythrocyte across the alveolarcapillary membrane into the alveoli. The reduction in dissolved CO2 rapidly lowers capillary PCO2, and disturbs the equilibrium of carbamate and bicarbonate reactions. This promotes conversion of the latter two compounds into CO2 which, in turn, diffuses out of the capillary into the alveoli.

As blood PCO2 falls, equation [1] is reversed and bicarbonate is converted rst into carbonic acid and then into free CO2. Carbonic anhydrase inside the erythrocyte catalyzes this reaction by a factor of 13 00015 000. Hydrogen ions required for conversion of bicarbonate to CO2 are largely supplied by hemoglobin. The simultaneous binding of oxygen to hemoglobin causes the release of hydrogen ions. In the plasma, the conversion of bicarbonate ion to CO2 occurs at a much slower rate. Plasma proteins are much less effective buffers than hemoglobin and are not affected by simultaneous oxygen transfer. Therefore, fewer hydrogen ions are available for bicarbonate conversion. There is a modest amount of carbonic anhydrase attached to the capillary endothelium, but this is sufcient to provide catalysis that is only 1/100 of that inside the red cell. As a result of the differences in buffering power and catalysis, the concentration of bicarbonate inside the erythrocyte decreases much more rapidly than that in plasma.

CARBON DIOXIDE 323

CO2 Alveolus
CA CA CA CA CA CA CA

Carbonic Anhydrase

Plasma

Cl HCO + H+ 3 3 Cl
+ H+ HCO3

H2CO3

CO2 + H2O

H2CO3

HHb

Hb + H+ RNHCOO + H+

CO2 + H2O + H+ + RNH2 RNH+ 3 RNHCOOH

CA

Figure 3 Carbon dioxide exchange in the lung. Solid lines indicate reactions that take place rapidly. The dashed line represents the slower reaction of formation of CO2 from H2CO3 as catalyzed by carbonic anhydrase (CA) localized to the capillary endothelium. The dotted lines indicate buffering reactions of hemoglobin. The number 3 indicates the band 3 anion transporter protein that exchanges bicarbonate and chloride ions between the erythrocyte and plasma. Reproduced from Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 16331642. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

This difference in bicarbonate concentration leads to transfer of bicarbonate from the plasma into the erythrocyte. Bicarbonate ions cannot enter the red cell independently because they are negatively charged. They exchange simultaneously in the opposite direction with intracellular chloride ions. This paired bicarbonatechloride exchange is accomplished in an electrically neutral fashion by a transmembrane protein known as the band 3 anion transporter protein. Through this process of anionic exchange, more than one-half of the total bicarbonate converted to CO2 in the lung originates in plasma, but enters the erythrocyte to be converted into CO2 before being excreted.
Carbamate Compounds

Five of the more than one dozen identied isomers of the enzyme carbonic anhydrase (CA) are potentially involved in CO2 exchange in the lungs and tissues. Both CA I and II are present in large quantities within the erythrocytes of humans and many mammals. This enzymatic activity is much greater than that thought necessary for efcient CO2 exchange, but the reason for the excess enzyme is not apparent. CA I is a less potent enzyme and its activity is partially inhibited by chloride ion. Although CA I comprises 8590% of the total enzyme inside human erythrocytes, CA II is responsible for the majority of total enzymatic activity. Some erythrocytic CA II is bound to intracellular portions of the band 3 anion transporter protein, providing a highly efcient com plex for interconversion of CO2 and HCO3 and transmembrane exchange of the bicarbonate ion. CA IV is a high-potency isomer that is localized to membranes, especially the sarcolemma of muscle tissue and the capillary endothelium of most tissues. Its impact on gas exchange in the lung is probably limited by its low concentration and the lack of substantial plasma buffering compared to that inside the red cell. Mathematical models of CO2 exchange in muscle suggest sarcolemmal CA IV may play a role in maintaining acid-base balance when large quantities of lactic acid are produced during exercise. CA III and V isomers also are present in some striated muscle, but their role, if any, in CO2 exchange is unclear.

Carbon Dioxide in Disease

Carbon dioxide elimination can be impaired in multiple disease states. However, this derangement is rarely caused by abnormal transport, but is usually the result of reduced ventilation or mismatching of ventilation and blood ow within the lung. Genetic deciencies of CA II are accompanied by renal tubular acidosis, osteopetrosis, and cerebral calcications. Most studies have failed to detect abnormalities in CO2 exchange in patients at rest, presumably because CA I present in erythrocytes is sufcient to support CO2 exchange. However, studies have not been conducted during exercise when abnormalities would be more likely to occur. A recent description of patients with carbonic anhydrase deciencies suggests that even CO2 exchange at rest may not be completely normal. The difference between end-tidal gas (a surrogate for alveolar gas) and arterial blood CO2 tensions was increased, implying that CO2 exchange did not reach equilibrium during transit through the pulmonary capillary bed. Further studies are required to validate this possible impairment.

As PCO2 decreases within the erythrocyte, through reactions [2] and [3] carbon dioxide bound to the a-amino groups is released as free CO2 and diffuses into the alveoli. The hydrogen ions required for these reactions are supplied through the buffering reactions of hemoglobin. Simultaneous binding of oxygen facilitates the release of CO2 bound to hemoglobin. Oxygenation reduces the afnity of the amino groups for CO2 and promotes CO2 release. Because this portion of CO2 exchange is dependent on oxygenation of hemoglobin, this reaction is delayed until oxygen is rst bound to hemoglobin.

324 CARBON MONOXIDE

The carbon dioxide dissociation curve of blood is markedly affected by acidbase alterations. Abnormalities that increase blood bicarbonate concentration, i.e., metabolic alkalosis and compensated respiratory acidosis, increase the total quantity of carbon dioxide in all forms at any given CO2 tension. The opposite is true in metabolic acidosis, which is characterized by a marked reduction in bicarbonate, and therefore total CO2 content, at any give PCO2 . The ratio of the arterialvenous difference in CO2 content divided by the arterialvenous difference in PCO2 provides an index of the efciency of carbon dioxide transport. Although this ratio has varied in studies of patients with acidbase abnormalities, it is signicantly reduced only in metabolic acidosis. This decreased efciency of CO2 transport is caused by a reduction in the Haldane effect in metabolic acidosis. This results in elevation of venous, and therefore tissue, PCO2 . However, it is important to note that there are few studies of CO2 transport in clinical circumstances and much more data is needed to characterize fully the effects of acidbase disturbances on CO2 exchange. Reactions of carbon dioxide have a prominent role in acidbase balance that is particularly emphasized in metabolic acidosis. This common condition occurs most frequently when oxidation of glucose is impaired and energy is generated via biochemical pathways that produce strong acids. Two common conditions are impairment of oxygen delivery to tissues leading to production of lactic acid and diabetic ketoacidosis that results in generation of acetoacetic and b-hydroxybutyric acids. In both situations, these strong acids ionize completely and release large quantities of hydrogen ions. With the resultant increase in hydrogen ion concentration, bicarbonate ion binds to hydrogen ion to form carbonic acid, which is further converted into CO2 and water. The CO2 formed in this buffering reaction is excreted in

the lungs, completing the removal of free hydrogen ions from the circulating blood. Thus, the reactions of carbon dioxide play a major role in defending acidbase balance in these circumstances.
See also: AcidBase Balance. Hemoglobin. Ventilation, Perfusion Matching. Ventilation: Overview.

Further Reading
Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, pp. 235242. Chicago: Yearbook Medical Publishers. Geers C and Gros G (2000) Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiological Reviews 80(2): 681715. Henry RP and Swenson ER (2000) The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Respiratory Physiology 121(1): 112. Klocke RA (1987) Carbon dioxide transport. In: Farhi LE and Tenney SM (eds.) Handbook of Physiology: The Respiratory System, pp. 173197. Bethesda: American Physiological Society. Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 16331642. New York: Raven Press. Lindskog S and Silberman DN (2000) The catalytic mechanism of mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 175195. Basel: Birkhauser. Swenson ER (2000) Respiratory and renal roles of carbonic anhydrase in gas exchange and acidbase regulation. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 281341. Basel: Birkhauser. Tanner MJA (2002) Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders. Current Opinion in Hematology 9(2): 133139. Venta PJ (2000) Inherited deciencies and activity variants of the mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 403412. Basel: Birkhauser. Wetzel P and Gros G (2000) Carbonic anhydrases in striated muscle. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 375399. Basel: Birkhauser.

CARBON MONOXIDE
D Morse, University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved. including inammation, cellular proliferation, and apoptotic cell death. This article offers a broad overview of our current understanding of the role of CO in respiratory disease.

Abstract
Carbon monoxide (CO) is generated in the human body by the catabolism of heme. This reaction is catalyzed by an enzyme known as heme oxygenase, which has both an inducible (heme oxygenase-1) and constitutive (heme oxygenase-2) form. CO was once believed to be a mere byproduct of heme breakdown, but is now known to modulate a number of cellular functions

Introduction
Carbon monoxide (CO) is known to most pulmonologists as an air pollutant arising from the partial combustion of organic molecules and as a potentially lethal gas when inhaled in high concentrations. The avid binding of CO to heme iron results

CARBON MONOXIDE 325

CO Heme oxygenase Biliverdin reductase

Heme

Biliverdin

Bilirubin

Fe2+ Ferritin
Figure 1 The enzymatic reaction catalyzed by heme oxygenase results in the release of CO.

in displacement of oxygen from hemoglobin, thus reducing the oxygen carrying capacity of blood. This phenomenon in turn leads to tissue ischemia and the familiar symptoms of CO poisoning. It is less widely appreciated that CO, in addition to being an environmental pollutant, is a biological product of ordinary metabolism. CO is generated in the human body by the catabolism of heme. This endogenously produced CO results in the normal baseline human carboxyhemoglobin level of 0.41%, and CO can be measured in the breath as it is excreted. The enzyme that releases CO from the breakdown of heme is known as heme oxygenase. There is a constitutively expressed form of this enzyme (heme oxygenase-2 (HO-2)) and an inducible form (heme oxygenase-1 (HO-1)). As one of the three byproducts of heme degradation (Figure 1), CO was initially considered a catabolic waste product. In fact, it was suggested in 1969 that CO production from humankind could contribute significantly to air pollution. We now know that this concern was misplaced, and it has more recently become evident that CO production serves a number of biological purposes. This article focuses on the functions of CO that relate most closely to pulmonary medicine, but it should be noted that a large body of literature exists describing functions for CO such as neurotransmission and vasodilation that cannot be discussed in detail here.

form of heme oxygenase, HO-1, is widely distributed throughout the body and is highly evolutionarily conserved. Its activity is transcriptionally regulated, and it is induced by more stimuli than any other known gene. The inducers of HO-1 include mainly conditions that would cause stress to cells, such as increased oxidant burden, radiation, heavy metals, and hypoxia. Not surprisingly, there are a number of cis-acting DNA sequence elements that serve as potential binding sites for transcription factors in the proximal promoter region and at distal enhancer sites. The dominant sequence element in the distal enhancer regions of mouse and human ho-1 is the stress-responsive element (StRE). The StREs represent targets of multiple dimeric proteins generated by intrafamily homodimerization or intra- and interfamily heterodimerization of individual members of the Jun, Fos, CREB, ATF, Maf, and the Capncollar/ basic-leucine zipper subclasses of the basic-leucine zipper superfamily of transcription factors.

Biological Functions
Anti-Inammatory Effects

Structure
CO is a colorless, odorless diatomic gas composed of a single carbon atom that is triply bonded to a single oxygen atom. It is an inert gas that does not readily engage in chemical reactions, but its afnity for heme is approximately 200 times that of oxygen. CO is slightly soluble in water (and therefore plasma).

Regulation of Activity
The rate-limiting step in the generation of CO is heme catalysis by heme oxygenase. The inducible

Several lines of evidence suggest that one of the reasons CO may be generated under conditions of stress is for its ability to dampen inammation. There has been one reported case of human HO-1 deciency, and the affected child exhibited signs of chronic inammation and was highly vulnerable to oxidative stress. Experimental studies have shown that when mice or macrophages are stimulated with lipopolysaccharide, a component of bacterial cell walls, the production of proinammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and interleukin-1b) is greatly increased. This model is frequently used to simulate human sepsis, a condition that is associated with variable elevation of these same cytokines. When these same mice or cells are administered low concentrations of CO (250 ppm) along with lipopolysaccharide, the production of the proinammatory cytokines is inhibited. Furthermore, the production of the anti-inammatory cytokine interleukin-10 is augmented by CO treatment. CO has also been shown to affect the expression of granulocytemacrophage colony-stimulating factor (GM-CSF), a glycoprotein that promotes the proliferation and the differentiation of hematopoietic progenitor cells into neutrophils and macrophages. A number of chronic inammatory pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, and sarcoidosis are associated with elevated levels of GM-CSF. The anti-inammatory effects of CO are mediated primarily via the mitogen-activated proteinkinase (MAPK) signaling pathways.

326 CARBON MONOXIDE Antiapoptotic Effects

Programmed cell death, or apoptosis, must be nely balanced for the maintenance of health and resolution of disease. Under conditions of stress, excessive apoptosis may lead to worsening organ dysfunction, and one could postulate that a stress-induced antiapoptotic molecule could provide protection. This thinking led to the examination of an antiapoptotic role for CO. The rst experiments performed in vitro demonstrated that applying CO to broblasts or endothelial cells could prevent cell death in response to tumor necrosis factor alpha. This effect was conrmed in animal models of disease, including ischemia/reperfusion injury and transplantation. The antiapoptotic effect of CO is not universal, however. In Jurkat T cells, CO has been shown to increase Fas/ CD95-induced apoptosis. Additionally, high concentrations of CO lead to apoptosis of tissue in rodents, associated with CO poisoning and tissue injury. As with the anti-inammatory effects, the antiapoptotic effects of CO are primarily mediated by the MAPK signaling pathways.
Antiproliferative Effects

antiproliferative effects of CO are generally mediated by increased intracellular cyclic GMP levels, although in some cell types the MAPK pathways may participate as well.

Carbon Monoxide in Respiratory Diseases

The lung is an organ with a particular vulnerability to oxidative injury as the interface between the atmosphere and the rest of the body. Oxidative stress may also be caused by pathologic conditions such as infection, inammation, and ischemia reperfusion. Lungs therefore require potent defense mechanisms and possess high levels of antioxidant enzymes. HO-1 is one of the few inducible molecules that can protect the lungs from an increased oxidant burden under stressful circumstances. In the lungs, HO-1 is highly expressed in alveolar macrophages, but is also found in epithelial cells, broblasts, and endothelial cells. The CO produced by HO-1 activity is primarily excreted in the breath, and the level of exhaled CO has been shown to increase in disease states and varies with therapy or exacerbations. Our current knowledge about the role of CO in lung disease is derived mainly from preclinical animal and cell culture studies; highlights of this work are summarized below and in Figure 2. Increasingly, the focus of CO research has been directed at potential therapeutic applications, such as using inhaled CO to limit lung injury due to a variety of insults. The CO animal and cell studies alluded to in the sections that follow generally use a concentration range of 50250 ppm. For reference, this inhaled concentration would result in carboxyhemoglobin levels ranging from 8% to 25% at equilibrium (after 8 h of exposure) in humans.
Acute Lung Injury

Regulation of cell proliferation is important not only for the maintenance of homeostasis, but in the bodys response to disease. It appears likely that stress-induced production of CO modulates cell growth in disease states, thereby potentially altering the progression of a variety of illnesses. The antiproliferative function of CO has been established in vitro in a number of different cell types. CO applied directly to smooth muscle cells causes growth arrest, and endogenous CO released from vascular smooth muscle cells as a consequence of hypoxiainduced HO-1 expression has the same effect. This endogenously produced CO also results in increased production of guanosine 30 ,50 -(cyclic)phosphate (cyclic GMP) in co-cultured endothelial cells, which in turn leads to downregulation of the expression of endothelial-derived mitogens such as platelet-derived growth factor and endothelin-1. The antiproliferative effect of CO has been implicated in the protection conferred by CO against vascular stenosis associated with balloon catheter injury. CO has also been shown to suppress the proliferation of human airway smooth muscle cells, which may be relevant to the pathogenesis of asthma, where inammatory cell-derived mediators stimulate smooth muscle proliferation. CO has been further shown to arrest the growth of cultured T lymphocytes, macrophages, and broblasts, which may account in part for the inuence of CO on the progression of immune-mediated and brotic lung diseases. The

Many of the initial studies done to characterize the location and function of HO-1 in the lung used rodent models of acute lung injury such as hyperoxia. In rodents, high concentrations of inhaled oxygen result in lung edema and death within several days. This oxidant injury results in increased expression of HO-1 in the lung, and overexpression of HO-1 in the bronchiolar epithelium by adenoviral gene transfer results in enhanced protection from the lethal effects of hyperoxia. Inhaled CO confers similar protection against hyperoxic lung injury. In vitro studies suggest that a part of the mechanism of protection by CO is due to prevention of cell death in response to increased oxygen tension. Ventilator-induced lung injury may complicate acute lung injury in humans, and recent rodent studies suggest that inhaled CO also exerts anti-inammatory effects in the setting of high tidal volume ventilation.

CARBON MONOXIDE 327

le c

CO
oot hm

us c

Bron

choc

onst

rictio

thought to be major contributors to the pathogenesis of COPD. Patients with COPD have lower HO-1 expression in alveolar macrophages than controls, and a microsatellite polymorphism in the promoter for HO-1 has been associated with the development of emphysema. This suggests that an abnormal response of HO-1 to oxidative stress may be linked to the development of COPD.
Pulmonary Fibrosis

Sm

ell p

roli fe

rati o

CO

Apoptosis

CO

Inflammation

CO

Immunohistochemical analysis of lung tissue from patients with various forms of interstitial lung disease reveals increased expression of HO-1, primarily in alveolar macrophages. Increased expression of HO-1 by adenoviral transfer has been shown to suppress lung brosis in a murine bleomycin model, and inhaled CO has similar effects. The mechanism by which HO-1 and CO confer protection against brosis has not been fully elucidated, but CO has been shown to alter the cytokine milieu, decrease matrix production by broblasts, slow broblast proliferation, and inhibit epithelial apoptosis, any of which could contribute to a modulatory effect on brosis.
Pulmonary Vascular Disease

CO

Fibroproliferation

Figure 2 Schematic summary of CO effects in lung disease. CO has been shown to suppress cell proliferation, bronchoconstriction, apoptosis, inammation, and broproliferation.

Obstructive Lung Disease

Asthma is a disease characterized by inammation, and so it is not surprising that exhaled CO levels have been shown to increase during asthma exacerbations. Studies in mice and cell culture suggest that the CO generated in the lung of asthmatics may affect the course of disease. Low-concentration inhaled CO has been shown to attenuate aeroallergeninduced inammation in mice, resulting in decreased interleukin-5 and eicosanoid mediator levels, as well as decreased bronchoalveolar lavage inammatory cell counts. Exogenously administered CO has also been shown to decrease airway hyperresponsiveness in mice, and as previously noted, CO may modulate the lung remodeling in asthma due to its inhibitory effect on airway smooth muscle cell proliferation. Exposure to reactive oxygen species and an imbalance in oxidant/antioxidant status are also

Following the discovery that nitric oxide could mediate vascular relaxation by increasing intracellular levels of cyclic GMP, it was natural to postulate that CO would have a similar effect, as CO was also known to induce cyclic GMP. We now know that CO causes vasodilation directly via activation of soluble guanylate cyclase in smooth muscle cells and indirectly through inhibition of the vasoconstrictors endothelin-1 and platelet-derived growth factor-B. In addition to these vasodilatory effects, CO may also prevent vascular remodeling by inhibiting smooth muscle cell proliferation. Enhanced activity of HO-1 in the lung has been shown to prevent the development of hypoxic pulmonary hypertension and inhibit structural remodeling of the pulmonary vessels in rats. Inhaled CO has also been shown to attenuate experimental pulmonary hypertension in rats. CO has also been strongly implicated in the development of hepatopulmonary syndrome in experimental models, and patients with hepatopulmonary syndrome have been shown to have higher carboxyhemoglobin levels than control subjects. In summary, CO production under conditions of physiological stress results in a wide range of adaptive responses that are relevant to pulmonary disease. Improving our understanding of the biological activities of CO will continue to enhance our knowledge of pulmonary pathophysiology. The goals of current research include such things as monitoring disease

328 CAVEOLINS

activity via measurement of exhaled CO, or even manipulating levels of CO in the body therapeutically. If successful, such research could bring CO fully into the day-to-day practice of pulmonary medicine.
See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Pulmonary Fibrosis.

Further Reading
Ameredes BT, Otterbein LE, Kohut LK, et al. (2003) Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 285(6): L1270L1276. Brouard S, Otterbein LE, Anrather J, et al. (2000) Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. The Journal of Experimental Medicine 192(7): 1015 1026. Dolinay T, Szilasi M, Liu M, and Choi AM (2004) Inhaled carbon monoxide confers antiinammatory effects against ventilatorinduced lung injury. American Journal of Respiratory and Critical Care Medicine 170(6): 613620. Fujita T, Toda K, Karimova A, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of brinolysis. Nature Medicine 7(5): 598604. Horvath I, Donnelly LE, Kiss A, et al. (1998) Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668672.

Maines MD (1993) Carbon monoxide: an emerging regulator of cGMP in the brain. Molecular and Cellular Neurosciences 4: 389397. Morita T and Kourembanas S (1995) Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. The Journal of Clinical Investigation 96: 26762682. Morita T, Mitsialis SA, Hoike H, Liu Y, and Kourembanas S (1997) Carbon monoxide controls the proliferation of hypoxic smooth muscle cells. Journal of Biological Chemistry 272: 3280432809. Otterbein LE, Bach FH, Alam J, et al. (2000) Carbon monoxide has anti-inammatory effects involving the mitogen-activated protein kinase pathway. Nature Medicine 6: 422428. Otterbein LE, Haga M, Zuckerbaun BS, et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nature Medicine 9: 183190. Otterbein LE, Mantell LL, and Choi AM (1999) Carbon monoxide provides protection against hyperoxic lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 276: L688L694. Yachie A, Niida Y, Wada T, et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deciency. The Journal of Clinical Investigation 103: 129135. Yet SF, Perrella MA, Layne MD, et al. (1999) Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. The Journal of Clinical Investigation 103: R23R29. Zhou Z, Song R, Fattman CL, et al. (2005) Carbon monoxide suppresses bleomycin-induced lung brosis. American Journal of Pathology 166(1): 2737.

Carcinoma

see Tumors, Malignant: Bronchogenic Carcinoma; Bronchial Gland and Carcinoid Tumor;

Carcinoma, Lymph Node Involvement.

CAVEOLINS
M P Lisanti and J-F Jasmin, Albert Einstein College of Medicine, New York, NY, USA
& 2006 Elsevier Ltd. All rights reserved. of Cav-1 and Cav-2 protein expression, a loss of caveolae formation, thickened alveolar septa, lung hypercellularity, pulmonary endothelial cell proliferation, brosis, and exercise intolerance. Cav-2 ( / ) mice show similar pulmonary abnormalities to the Cav-1 ( / ) mice, but without a loss of Cav-1 expression and normal caveolae formation. Furthermore, Cav-1 ( / ) mice develop pulmonary hypertension and right ventricular hypertrophy. Interestingly, the endogenous levels of caveolin proteins appear to be altered in numerous pulmonary disorders, such as pulmonary hypertension, lung cancers, pulmonary brosis, and pulmonary interstitial edema.

Abstract
Caveolae are small invaginations of the plasma membrane that are implicated in endocytosis, vesicular trafcking, and signal transduction. Caveolin proteins (Cav), the principal structural proteins of caveolae, consist of three distinct genes, namely Cav1, Cav-2, and Cav-3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, broblasts, smooth muscle cells, and epithelial cells. In contrast, Cav-3 is muscle-specic and is solely expressed in smooth, cardiac, and skeletal muscle cells. Caveolin proteins appear to play a role as key regulators of pulmonary structure and function. Homozygous deletion of the Cav-1 gene (Cav-1 ( / )) results in numerous pulmonary abnormalities, characterized by a loss

Introduction
Caveolae, which were rst identied in the 1950s as smooth uncoated pits, are now dened as 50 100 mm ask-shaped invaginations of the plasma

CAVEOLINS 329

membrane; they are implicated in endocytosis, vesicular trafcking, and signal transduction (Figure 1). Caveolae can either be observed as curved U-shaped invaginations of the plasma membrane, fully invaginated caveolae, grape-like clusters of interconnected caveolae (caveosome), or as a transcellular channel (as a consequence of the fusion of individual caveolae) (Figure 2). These caveolar domains are a subset of lipid rafts that contain the essential coat protein, named caveolin (Cav). As shown in Figure 3, caveolins appear as membrane-bound proteins that form homo-oligomers and show an unusual topology, with both their NH2 and COOH termini facing the cytoplasm. The caveolin gene family consists of

three distinct genes, namely Cav-1, Cav-2, and Cav3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, adipocytes, broblasts, and smooth muscle cells. On the other hand, Cav-3 appears to be muscle-specic and is thus solely expressed in smooth, cardiac, and skeletal muscle cells. Cav-1 further encodes two isoforms, namely Cav-1a and Cav-1b. These two Cav-1 isoforms derive from two distinct Cav-1 mRNAs generated by alternative transcription initiation sites.

Expression of Caveolin Proteins in the Lungs

Although all three caveolin isoforms are expressed in the lungs, Cav-1 still remains the most extensively studied isoform, with wide expression in endothelial cells, broblasts, smooth muscle cells, bronchial epithelial cells, and alveolar type I cells. However, Cav-1 is poorly expressed in alveolar type II cells. Cav-1 is also present in the developing lung, suggesting important roles for caveolin proteins in lung development. Interestingly, in the developing lung, the expression of the two Cav-1 isoforms appears to be cell-type specic, with Cav-1a being expressed in endothelial cells and Cav-1b being expressed in alveolar type I cells. This cell-type specic expression could be ascribed to differential transcriptional regulation of Cav-1 in lung epithelial and endothelial cells. Whether this cell-type specic expression of Cav-1 isoforms also occurs in adult mouse lung remains to be claried.

RBC

Figure 1 Electron micrograph (EM) depicting endothelial cell caveolae (arrows) from a mouse lung endothelial cell. RBC, red blood cell. Scale 200 nm. Adapted from Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2-decient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22(7): 23292344, with permission of the American Society for Microbiology.

Caveolin Proteins and Pulmonary Endocytosis and Transcytosis

Based on their appearance and membrane localization, caveolae were initially proposed as transport vesicles. These vesicles were then thought to either

6 2

1 5

3 4

Nucleus

Figure 2 Schematic representation of a cell illustrating morphological variants of caveolae. These include: 1, traditional caveolae; 2, fully invaginated caveolae; 3, cavicles (internalized caveolae not associated with the plasma membrane); 4, caveosome or rosette-like structures; 5, grape-like clusters of interconnected caveolae; 6, transcellular channels.

330 CAVEOLINS

internalize macromolecules (endocytosis) or transport them across the cell (transcytosis). The presence of several proteins implicated in vesicle formation, docking, and fusion in lung caveolae strongly supports a role for caveolae in endocytosis and transcytosis. In accordance with this idea, several viruses, bacteria, and toxins, such as simian virus 40 and cholera toxin, are thought to be preferentially internalized by caveolae. Furthermore, transcytosis of gold-conjugated albumin appears to be restricted to caveolae in the lungs. The caveolar localization of gp60, a 60 kDa albumin-binding protein, supports the role of caveolar domains in the transcytosis of albumin. The recent generation of caveolin-decient mice elegantly conrmed the implications of lung caveolae in albumin endocytosis. Indeed, lung endothelial cells of Cav-1 ( / )-decient mice show defects in the uptake and transport of gold-conjugated albumin.

Caveolin Proteins and Pulmonary Signal Transduction

Besides their roles as transport vesicles, caveolae microdomains also function in signal transduction via their compartmentalization of signal transduction

cascades. Indeed, lung caveolae are known to compartmentalize several signaling molecules, such as protein kinase C (PKC), phosphatidylinositol-3 (PI3) kinase, heterotrimeric G-protein subunits, extracellular signal-related kinase (ERK), endothelial nitric oxide synthase (eNOS), signal transducer and activator of transcription-3 (STAT3), and Ras-related GTPases. In accordance with a role in signal transduction, lung caveolae are also enriched in the calcium ATPase and inositol triphosphate receptors. Most of the proteins sequestered within caveolar domains possess caveolin-binding motifs and consequently interact with Cav-1. These proteinprotein interactions are mediated by the interaction of a caveolin-binding motif (within specic target proteins) with a specic region of Cav-1 (residues 82101), named the scaffolding domain (Figure 3). Interestingly, Cav-1 also functions as a negative regulator of many of these signaling molecules (Figure 4). The generation of Cav-1 ( / )-decient mice supports the Cav-1mediated negative regulation of several proteins, such as eNOS, ERK1/2, and STAT3. For example, lungs of Cav-1 ( / )-decient mice show increased NO production, as well as microvascular hyperpermeability. Interestingly, administration of an NOS inhibitor

C-terminal

Transmembrane domain Scaffolding domain Oligomerization domain

N-terminal
Figure 3 Schematic representation of the topology of caveolins as the principal structural proteins of caveolae. Some important domains of Cav-1, such as the oligomerization, scaffolding, and transmembrane domains, are illustrated.

CAVEOLINS 331
Caveolin-1

82

101

178

DGIWKASFTTFTVTKYWFYR

(Scaffolding domain of Cav-1)

XXXXXXX

(Caveolin-binding sequence motif)

Inactivation

PI3 Kinase

eNOS

STAT3

ERK1/2

G-protein subunits

PKC

Modulation of pulmonary signal transduction

Figure 4 Schematic representation of the interaction of the Cav-1 scaffolding domain with the caveolin-binding sequence motifs of PI3 kinase, eNOS, STAT3, ERK1/2, G-protein subunits, and PKC. Reproduced from Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing preassembled signaling complexes at the plasma membrane. Journal of Biological Chemistry 273: 54195422, copyright & 1998 by the American Society for Biochemistry and Molecular Biology.

(L-NAME) to Cav-1 ( / )-decient mice reverses their microvascular hyperpermeability, thus supporting Cav-1-mediated inhibition of eNOS activity. Similarly, Cav-1 ( / )-decient mice show hyperactivation of cardiac ERK1/2 and pulmonary STAT3 signaling.

Pulmonary Phenotypes of Caveolin-Decient Mice

Surprisingly, mice with homozygous deletion of the different caveolin genes are viable and fertile. Yet, Cav-1 ( / )-decient mice show numerous abnormal phenotypes, such as a reduction in life span, loss of caveolae formation in tissues that usually express Cav-1, loss of Cav-2 expression, resistance to dietinduced obesity, decreased vascular tone secondary to eNOS hyperactivation, and the development of cardiomyopathy. On the other hand, Cav-3 ( / )decient mice show a loss of caveolae formation in their cardiac and skeletal muscles and develop progressive cardiomyopathy. Importantly, the generation of caveolin-decient mice identied caveolin proteins as key regulators of pulmonary structure and function. Indeed, Cav-1 ( / )-decient mice demonstrate pulmonary abnormalities, characterized by thickened alveolar septa, hypercellularity, brosis, endothelial cell

proliferation, and exercise intolerance. Importantly, selective homozygous deletion of the Cav-2 gene results in similar thickening of the alveolar septa, hypercellularity, and endothelial cell proliferation as in Cav-1 ( / )-decient mice, but without any defects in caveolae formation or Cav-1 expression. These ndings suggest a selective role for Cav-2 in lung structure and function. Interestingly, Cav-1 ( / )decient mice also develop pulmonary hypertension and right ventricular hypertrophy, as shown by increases in the right ventricular systolic pressures and the ratio of the right ventricular weight over the left ventricular weight, respectively. Whether Cav-2 ( / )-decient mice also develop pulmonary hypertension and right ventricular hypertrophy still remains to be determined. Similarly, although Cav-3 is highly expressed in the smooth muscle cells of the lungs, the potential pulmonary phenotypes of Cav-3 ( / )-decient mice still remain to be assessed.

Pathological Implications of Caveolin Proteins

The endogenous expression of caveolin proteins appears to be altered in numerous lung diseases, such as pulmonary hypertension, lung cancers, pulmonary brosis, and pulmonary interstitial edema.

332 CAVEOLINS Pulmonary Hypertension

Given the pulmonary phenotype of caveolin-decient mice and their roles as negative regulators of several pro-proliferative signaling pathways, caveolin proteins could be important regulators of pulmonary hypertension development. Accordingly, both Cav-1 and Cav-2 protein levels appear to be decreased in the lungs of rats subjected to myocardial infarction (MI)-induced pulmonary hypertension. The downmodulation of Cav-1 and Cav-2 expression is associated with the increased tyrosine-phosphorylation of STAT3, as well as the upregulation of Cyclin D1 and D3 in the lungs of MI rats. The decreased expression of caveolin proteins could thus represent an initiating mechanism leading to the activation of several proproliferative pathways, such as the STAT3/cyclinsignaling cascades and consequently leading to the development of pulmonary hypertension. This hypothesis is supported by the ndings of increased STAT3 phosphorylation and increased cyclin D1/D3 expression in the lungs of Cav-1 ( / )- and Cav-2 ( / )-decient mice. Furthermore, pulmonary Cav-1a expression was shown to be decreased in rats with monocrotaline-induced pulmonary hypertension as early as 48 h after monocrotaline treatment. Accordingly, monocrotaline treatment of cultured pulmonary arterial endothelial cells also results in decreased Cav-1a expression, increased STAT3, and ERK1/2 phosphorylation, and the stimulation of DNA synthesis within 48 h.
Lung Cancers

metastases isolated from patients with lung carcinoma. The upregulation of Cav-1 even correlates with the cancer stage and mortality rate in patients with lung adenocarcinoma and pulmonary squamous cell carcinoma. Although the increased expression of Cav-1 might promote the inhibition of apoptosis, the induction of lopodia formation, or even the stimulation of angiogenic processes, the exact mechanisms underlying the prometastatic effects of Cav-1 remain puzzling and may well be cell-type specic.
Pulmonary Fibrosis

Caveolin proteins are often perceived as tumor-suppressor genes. Accordingly, the Cav-1 gene is localized to a suspected tumor-suppressor locus on human-chromosome 7 (7q31.1/D7S522). Caveolin1s well-known negative regulation of several proproliferative proteins, such as ERK1/2, H-Ras, c-Src, and cyclin D1, strongly supports the tumor-suppressor role of Cav-1. Accordingly, a reduction of Cav-1 expression was reported in numerous lung carcinoma cell lines. Caveolin-1 gene transcriptional activity also appears to be downregulated in human lung adenocarcinomas. The generation of caveolin-decient mice strongly supports the tumor-suppressor roles of Cav-1. Indeed, Cav-1 ( / )-decient mice interbred with tumor-prone transgenic mice show accelerated development of multifocal dysplastic mammary lesions, as well as enhanced lung metastasis. Although Cav-1 definitely appears to act as a tumor-suppressor gene in numerous cancers, its precise role in the development of metastasis remains obscure. Indeed, the expression of Cav-1 conversely increases in ipsilateral hilar/peribronchial lymph node

Given the pulmonary phenotype of caveolin-decient mice, caveolin proteins could also be implicated in the development of lung brosis. Accordingly, the expression of Cav-1 is altered in several models of lung brosis, such as irradiation, CdCl2/TGF-b1, and bleomycin. Western blot analysis of total lung homogenates indicates an overall decrease in Cav-1 expression in mice with irradiation- and bleomycininduced brosis. This decrease in pulmonary Cav-1 expression is associated with hyperactivation of the Ras-p42/44 MAP kinase pathway (ERK signaling), which is thought to favor collagen deposition. Importantly, lung broblasts isolated from scleroderma patients with pulmonary brosis also show a marked decrease in Cav-1 expression and hyperactivation of ERK signaling. Interestingly, the expression of Cav-1 appears to be differentially regulated in alveolar type I cells and endothelial cells. Indeed, although the expression of Cav-1 appears to decrease in lung epithelial cells, it conversely might increase in lung endothelial cells. Whereas an upregulation of endothelial Cav-1 expression could indicate an increase in endothelial caveolar transcytosis, the downregulation of epithelial Cav-1 expression might impair the metabolic function and reduce the signal transduction capacity of alveolar type I cells. Thus, the loss of lung epithelial Cav-1 expression appears as an indicator of subcellular alterations in lung brogenesis.
Pulmonary Interstitial Edema

Caveolin proteins might also be implicated in the development of pulmonary interstitial edema. Indeed, slight increases (B5%) in extravascular water are sufcient to drive the recruitment of endothelial caveolae and the upregulation of Cav-1 protein levels at the airblood barrier plasma membrane. These effects could be subsequent to increased hydraulic interstitial pressures and shear stress. The recruitment of endothelial caveolae might thus represent an important mechanotransduction response to ow increases.

CD1 333

Conclusions
Caveolin proteins represent novel regulators of pulmonary structure and function, which may play essential roles in the development of a number of pulmonary diseases. Indeed, caveolin-decient mice show numerous abnormalities in pulmonary structure and function. Furthermore, caveolin protein expression appears to be altered in several pulmonary disorders such as pulmonary hypertension, lung cancers, pulmonary brosis, and pulmonary interstitial edema. Thus, tight regulation of caveolin protein expression appears fundamental for normal pulmonary functioning. Whether in vivo modulation of the caveolin protein levels could have therapeutic effects on pulmonary diseases remains to be determined. Interestingly, in vivo administration of a cell-permeable Cav-1 mimetic peptide was previously shown to reduce microvascular hyperpermeability, inammation, and tumor progression in mice. Whether the in vivo administration of such a cell-permeable Cav-1 peptide could complement the decreased expression of endogenous Cav-1 and prevent the development of pulmonary hypertension, pulmonary brosis, and some lung cancers remains to be investigated.
See also: Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Pulmonary Edema. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Vascular Disease.

Further Reading
Cohen AW, Hnasko R, Schubert W, and Lisanti MP (2004) Role of caveolae and caveolins in health and disease. Physiological Reviews 84: 13411379. Gratton JP, Lin MI, Yu J, et al. (2003) Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice. Cancer Cell 4: 3139. Ho CC, Huang PH, Huang HY, et al. (2002) Up-regulated caveolin-1 accentuates the metastasis capability of lung

adenocarcinoma by inducing lopodia formation. American Journal of Pathology 161: 16471656. Jasmin JF, Frank PG, and Lisanti MP (2005) Caveolin proteins in cardiopulmonary disease and lung cancers. Advances in Molecular and Cell Biology 36: 211233. Jasmin JF, Mercier I, Hnasko R, et al. (2004) Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression. Cardiovascular Research 63: 747755. Mathew R, Huang J, Shah M, Patel K, Gewitz M, and Sehgal PB (2004) Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 110: 14991506. Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing preassembled signaling complexes at the plasma membrane. Journal of Biological Chemistry 273: 54195422. Razani B, Engelman JA, Wang XB, et al. (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. Journal of Biological Chemistry 276: 3812138138. Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2decient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22: 23292344. Razani B, Woodman SE, and Lisanti MP (2002) Caveolae: from cell biology to animal physiology. Pharmacological Reviews 54: 431467. Schubert W, Frank PG, Razani B, et al. (2001) Caveolae-decient endothelial cells show defects in the uptake and transport of albumin in vivo. Journal of Biological Chemistry 276: 48619 48622. Schubert W, Frank PG, Woodman SE, et al. (2002) Microvascular hyperpermeability in caveolin 1 ( / ) knock-out mice. Journal of Biological Chemistry 277: 4009140098. Tourkina E, Gooz P, Pannu J, et al. (2005) Opposing effects of protein kinase C alpha and protein kinase C epsilon on collagen expression by human lung broblasts are mediated via MEK/ ERK and caveolin-1 signaling. Journal of Biological Chemistry 280: 1387913887. Williams TM, Medina F, Badano I, et al. (2004) Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo. Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion. Journal of Biological Chemistry 279: 5163051646. Zhao YY, Liu Y, Stan RV, et al. (2002) Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proceedings of the National Academy of Sciences, USA 99: 1137511380.

CD1
S M Behar, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved. cells. The human CD1 locus contains ve distinct genes, CD1A E, which have an overall structure similar to class I major histocompatibility complex (MHC) including their association with b2 microglobulin. However, unlike class I MHC, the CD1 antigen-binding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. The ability of CD1 to present lipid antigens to T cells significantly expands the universe of antigens that can be recognized by T cells. CD1 proteins are divided into two groups: group 1 CD1 (CD1a, -b, and -c) and

Abstract
CD1 proteins are a distinct lineage of antigen-presenting molecules that evolved to present lipid and glycolipid antigens to T

334 CD1
group 2 CD1 (CD1d). Group 1 CD1-restricted human T cells can recognize mycobacterial cell wall lipids and are elicited as part of the immune response to Mycobacterium tuberculosis. Some CD1d-restricted T cells, also known as natural killer T (NKT) cells, recognize endogenous self-antigens while others recognize foreign microbial antigens. CD1d-restricted NKT cells rapidly secrete cytokines following activation and are thought to be an important link between innate and adaptive immunity. NKT cells appear to enhance pulmonary immunity against several pulmonary pathogens. Conversely, NKT cells are required for the development of airway hyperreactivity and inammation in rodent models of allergic pulmonary hypersensitivity. Thus, CD1-restricted T cells are an important component of the T cell response to infection and tumors, and have an important role in the regulation of many diverse immune responses.

CD1 locus makes it more likely that such antigens would elicit a consistent immune response, even in genetically diverse human populations.

Structure
The human CD1 genetic locus is located on chromosome 1 and is unlinked to the major histocompatibility complex (MHC) locus, which is found on chromosome 6. Five distinct genes are encoded within the human CD1 locus, CD1A, -B, -C, -D, and -E, and in contrast to MHC, there is only limited polymorphism between unrelated individuals. CD1A, -B, and -C have been designated group I CD1 based on their primary sequence homology. Genes encoding CD1 proteins have been detected in all mammals studied to date, and these studies have observed tremendous variation in the number and type of CD1 genes found in different species. Importantly, mice and rats only have orthologs of CD1D (group II CD1). The murine CD1 genes, known as CD1d1 and CD1d2, are more than 95% homologous to one another and may have arisen by gene duplication. The homology between human and mouse CD1d is striking as human CD1d-restricted T cells recognize murine CD1d, and vice versa. Thus, rodents are a useful experimental model to ascertain the function of CD1d and CD1d-restricted T cells. Small animal models that are suitable for the study of group I CD1 also exist and include the rabbit and the guinea pig. Thus, the CD1 genetic locus is evolutionarily conserved in mammals and is distinct from the MHC locus. While CD1 and MHC may share a common ancestor, CD1 has evolved into a distinct lineage of antigen-presenting molecules specialized for the presentation of lipid antigens to T cells. Despite only limited amino acid sequence homology to MHC, the CD1a, -b, -c, -d, and -e polypeptides all associate with b2 microglobulin and form a tertiary structure similar to class I MHC (Figure 1). However, unlike class I MHC, the CD1 antigenbinding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. These properties make CD1 particularly well suited to bind lipid and glycolipid molecules. Nearly all of the lipid antigens presented by CD1 have a polar head group and one or two lipid tails. Combining crystallographic and structure-function data, a model emerges in which the hydrophobic acyl chains of the lipid antigen are buried deep within the antigenbinding groove, while the polar head group is closer to the molecular surface and just emerges from the antigen-binding groove. The T cell receptor (TCR) recognizes the combination of the polar head group and the surface of the CD1 protein. The crystal structures of human CD1a and CD1b, and murine

Introduction
CD1 was rst recognized as a human cell surface antigen frequently expressed by thymocytes and certain neoplasms of T cell origin. Later, it was found to be highly expressed by Langerhans cells and dendritic cells (DCs). Even today, CD1 remains a useful phenotypic marker of these cells. However, the nding that CD1 was a target of certain autoreactive human T cells led to the insight that CD1 may be an antigen-presenting molecule. We now understand that while certain CD1-restricted T cell clones are autoreactive, presumably because they recognize endogenous self-lipid antigens, other CD1-restricted T cells recognize foreign antigens. In a series of landmark studies, Porcelli, Beckman, and Brenner demonstrated that CD1b and CD1c present microbial antigens to human T cells. Eventually, the antigens presented by CD1 began to be identied. The rst antigen to be puried was mycolic acid, a unique lipid molecule found in the cell wall of Mycobacterium tuberculosis, which was recognized by a CD1brestricted T cell clone. Mycolic acid, which is responsible for the acid-fastness of M. tuberculosis and is thought to be a bacterial virulence factor, belongs to a family of a-branched b-hydroxy fatty acids and its two acyl chains are approximately 90 carbons long. This discovery shattered the paradigm that T cells could only recognize short peptide sequences and was the rst evidence that CD1 presented microbial lipid antigens to T cells. These ndings have generated considerable interest in determining whether CD1-restricted T cells contribute to host defense against infection, as the lipid antigens recognized by them may be potential vaccine candidates. Microbial pathogens are unable to rapidly vary lipid antigen structure when subjected to immune selective pressure because lipids are products of multistep biosynthetic pathways. CD1-presented antigens are also attractive vaccine candidates because the limited degree of polymorphisms in the

CD1 335

(a)

(b)

(c)

Figure 1 Crystal structure of CD1b. In 2002, Gadola et al. solved the three-dimensional structure of CD1b with phosphatidylinositol in the antigen-binding groove. The CD1b heavy chain is displayed in blue and b2-microglobulin is displayed in yellow. The phosphatidylinositol phospholipid ligand is colored according to the CPK scheme. In (a) and (b), CD1b is observed as it might appear on the cell surface, looking either parallel (a) or perpendicular (b) to the two a-helices formed by the a1 and a2 domains that create the antigen-binding pocket. In (c), the perspective is looking down at the top of CD1b, which is the portion of the molecule that interacts with the T cell receptor. The view in (c) is enlarged compared to (a) and (b), and the a3 domain and b2 microglobulin have been removed for clarity.

CD1d, have all been solved and reveal some interesting differences. The antigen-binding groove of CD1a is shallower and less complex than that observed for CD1b or CD1d. CD1b has the most complicated antigen-binding pocket consisting of a maze of channels, giving it the capacity to bind lipid antigens with very long acyl chains. Thus, the antigenbinding groove of the different CD1 molecules may affect the binding and selection of the antigens that are presented to CD1-restricted T cells.

Regulation of Production and Activity

Group I CD1 is strongly expressed on cortical thymocytes and on a variety of APC subtypes in several different tissues. For example, Langerhans cells in the skin express very high levels of CD1a, and other DCs, particularly in inamed tissue, express CD1a, -b, and -c. In addition, CD1c and CD1d are found on certain B cell subsets. Murine CD1d is more widely expressed, and is detected on nearly all lymphocytes and myeloid-lineage cells. Although little is known about CD1 regulation, cell surface expression of group I CD1 increases during cytokine-induced differentiation and maturation of monocyte-derived dendritic cells. Similarly, CD1d is upregulated during macrophage activation by cytokines and microbial products. Modulation of CD1 surface expression may be one way that the immune system regulates activation of CD1-restricted T cells. This could be particularly important for the regulation of immune responses by CD1d-restricted natural killer (NK) cells. Following protein translation, CD1 associates with b2m in the endoplasmic reticulum. Processing and loading of lipid antigens requires a unique set of accessory molecules, which are beginning to be

delineated. These include glycosidases to trim sugars from glycolipid antigens and microsomal triglyceride transfer protein and saponins that facilitate lipid transfer between membranes and CD1. Following transportation to the cell surface, CD1 proteins undergo extensive recycling through the endocytic pathway, which is dependent on a tyrosine-containing motif found in the cytoplasmic tail of CD1. For example, CD1a, which has a truncated cytoplasmic tail lacking the tyrosine-based motif, is primarily expressed on the cell surface although it can also be detected in the recycling endosome. In contrast, CD1b is expressed on the cell surface but also has an extensive distribution in the endocytic compartment and colocalizes with class II MHC. Such differences are likely to affect the source and type of lipid each CD1 molecule encounters. For example, CD1b may have evolved to sample lipids from the late endocytic compartment while CD1a may have evolved to sample lipids from the extracellular milieu. Thus, each CD1 protein follows a unique recycling pathway and has a different steady state distribution.

Biological Function
The main function of both group I and group II CD1 is antigen presentation of lipids and glycolipids to T cells. Although there are likely to be certain constraints on the type of lipids presented by CD1, a surprisingly diverse number of lipids have been shown to bind or be presented by CD1 to T cells. Group I CD1 presents a number of lipids derived from the cell wall of M. tuberculosis to human T cells including the lipopeptide didehydroxymycobactin (CD1a), mycolic acid and glucose monomycolate (both CD1b), and isoprenoid glycolipids (CD1c).

336 CD1

Both self and foreign antigens have been identied that are presented by CD1d. CD1d-restricted T cells can recognize phospholipids (including phosphatidylinositol and phosphatidylethanolamine) and certain ceramides, although whether these antigens are important in immunoregulation or autoimmune disease is not yet certain. In addition, certain lipids from Leishmania donovani and M. tuberculosis bind to CD1d and activate NKT cells. Finally, synthetic lipid antigens are presented by CD1d and activate CD1drestricted T cells. The prototype of these antigens is a-galactosylceramide. Although their physiological relevance is unknown, these synthetic antigens have been extremely useful tools. Because these antigens are potent and specic activators of CD1d-restricted T cells, they have been used in vitro and in vivo to study the function of CD1d-restricted T cells, particularly in whole animal models, and may ultimately be useful pharmacologically as a way to exploit the therapeutic potential of these T cells. As more CD1presented antigens are discovered, their apparent diversity substantiates the hypothesis that antigen presentation by CD1 to T cells significantly expands the number of microbial antigens that can be recognized by T cells and enhances their capacity to mediate antimicrobial immunity.

CD1-Restricted T Cells
Similar to the MHC/peptide complex, recognition of the CD1/lipid antigen complex is mediated by the T cell receptors (TCRs) (Figure 2). Both the ab and the gd TCRs can recognize the CD1/antigen complex, and although the initial descriptions of CD1-restricted T cells were distinguished by their lack of CD4 or CD8 accessory molecules, examples of CD4 and CD8 CD1-restricted T cells have been reported. Functional differences among these various CD1-restricted T cell subsets exist. For example, human CD4 and CD4 8 CD1d-restricted T cells differ in the spectrum of cytokines produced following activation. Another difference is that while CD1restricted M. tuberculosis antigen-specic T cells can kill infected cells, CD8 T cells accomplish this using cytotoxic granules, whereas killing by CD4 8 T cell-mediated killing is CD95/CD95L dependent (Figure 2). Like other T cells, CD1-restricted T cells require costimulation for their proliferation. However, they are typically CD28 negative and their costimulatory requirement appears to be independent of the CD28-CD80/CD86 axis. The V, D, and J gene segments encoding group 1 CD1-restricted TCRs are similar to those used by MHC-restricted T cells. Interestingly, many CD1-restricted TCRs have an increased frequency of basic residues in the

CDR3 region. These positively charged residues are hypothesized to interact with the polar head group of lipid antigens, which usually carry a negative charge. An important advance in the biology of CD1d-restricted T cells was the nding that many NKT cells are CD1d-restricted. NKT cells are a unique subset of lymphocytes found in humans and other species that not only express the T cell antigen receptor (i.e., TCR), but also cell surface antigens that are characteristic of NK cells. NKT cells are not abundant in lymphoid organs but are increased in frequency in the bone marrow and certain peripheral organs such as the liver. Two distinct subsets of CD1d-restricted NKT cells have been described. The best-described subset is referred to as invariant NKT cells (iNKT cells), because they express an invariant TCR alpha (a) chain. Murine iNKT cells use a canonical Va14Ja281 TCRa chain, which preferentially pairs with TCR beta (b) chains encoded by Vb2, -7, or -9. Human iNKT cells are similar and are encoded by an invariant Va24-JaQ TCRa chain. Thus, the TCR repertoire of iNKT cells has only limited diversity. A second subset of CD1d-restricted T cells uses a more diverse repertoire, although it may be less varied than MHC-restricted T cells. Although NKT cells have become synonymous with CD1d-restricted T cells, caution is required as conventional T cells can also express NK cell markers, particularly when activated. Thus, more precise methods are required to identify CD1-restricted T cells. The specicity of most iNKT cells for the synthetic antigen a-galactosylceramide provides one way to identify these T cells. Thus, ow cytometry has been used to detect binding of CD1d multimers loaded with a-galactosylceramide to iNKT cells. When identied this way, the phenotype of iNKT cells resembles memory T cells. Even their pattern of chemokine receptor expression is typical of T cells that home to peripheral tissues rather than lymphoid organs. Consistent with their activated phenotype, CD1d-restricted iNKT cells rapidly produce a burst of IL-4 within hours of stimulation, followed secretion of large amounts of interferon gamma (IFN-g). Not surprisingly, iNKT cells can inuence whether CD4 T cell responses acquire a Th1- or Th2-like phenotype. Thus, iNKT cells are increasingly viewed as a link between innate and adaptive immunity, because of the kinetics of their response, as well as their ability to inuence conventional T cell immunity (Figure 2).

CD1 in Respiratory Diseases

How CD1 and CD1-restricted T cells affect the expression of respiratory diseases is still being dened. As antigen-presenting molecules, CD1 and

CD1 337
Perforin Granzyme Granulysin

IL - 12 T cell Activation of adaptive immunity APC T cell CD1b TCR IFN TNF NK cell M

Fas - FasL T cell TCR CD1b

Activation of innate immunity IFN TNF IFN TNF -

(a)

Effector functions Perforin Granzyme Granulysin

Immunoregulation

IL - 12 T cell Activation of adaptive immunity

Fas - FasL NKT cell TCR CD1d APC

CD40CD40L NKT cell CD1d TCR IFN TNF-

NK cell

IFN TNF -

IFN -

Activation of innate immunity

(b)

Effector functions

Immunoregulation

Figure 2 The effector and immunoregulatory functions of CD1-restricted T cells. (a) Presentation of microbial or self-lipid antigens by CD1a, -b, or -c expressed on DCs leads to activation of group I CD1-restricted T cells. When stimulated, these T cells secrete IFN-g and TNF-a, which can lead to activation of macrophages (Mf). CD1-restricted T cells are also potent cytolytic T cells and kill target cells either by granule-dependent mechanisms or by Fas-mediated killing. CD1-restricted T cells also stimulate DC maturation and IL-12 production. Thus, by inducing DC maturation and secreting cytokines, group 1 CD1-restricted T cells can modulate other innate and adaptive immune responses. (b) CD1d presentation of exogenous antigens such as aGalCer or endogenous ligands leads to the activation of CD1d-restricted NKT cells. IL-12 is an important costimulatory signal for NKT cell activation, which is produced by DCs after CD40CD40L interaction or after TLR activation by microbial ligands. Activated NKT cells secrete IFN-g, TNF-a, and IL-4, which can stimulate other cell types and inuence the polarization of CD4 T cells. In addition, NKT cells are potent cytolytic cells, and when ld M and Behar SM (2005) The role of group 1 and group 2 activated can kill target cells by the mechanisms shown. Adapted from Sko CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544551, with permission from Elsevier.

CD1-restricted T cells potentially play a role in immunity to pathogens, tumors, and a variety of immunologically mediated diseases. Lower numbers of circulating CD1d-restricted iNKT cells are found in patients with several types of cancers, including multiple melanomas and prostate cancer, and in patients with autoimmune conditions such as systemic sclerosis and rheumatoid arthritis. Patients with sarcoidosis and autoimmune diabetes not only have fewer iNKT cells, but those that are present are dysfunctional. These ndings support a role for NKT cells in natural tumor immunosurveillance and in

preventing the emergence of autoimmune disease, but do not delineate how NKT cells exert their protective role. The ability of CD1 to present microbial antigens to T cells has strongly implicated CD1restricted T cells in microbial immunity. Not only can CD1-restricted T cells recognize mycobacterial lipids, but these lipid antigens are processed and presented by M. tuberculosis infected cells, demonstrating that the CD1 antigen-presentation pathway is physiologically relevant during infection. Furthermore, recognition of infected cells leads to activation of CD1-restricted T cells, which induces IFN-g

338 CD11/18

and tumor necrosis factor alpha secretion and target cell lysis. Thus, CD1-restricted T cells express effector functions that are benecial during infection. Finally, to address the relevance of these observations during human tuberculosis, it should be noted that CD1-restricted responses to mycobacterial lipid antigens are detected in the peripheral blood of people infected with M. tuberculosis, indicating that CD1restricted T cell responses are physiologically primed following infection. Mouse models for human diseases are powerful tools to identify the mechanisms by which the immune system combats disease. As CD1d orthologs exist in mice, more information has been obtained on the role of CD1d and CD1d-restricted NKT cells in murine models of human disease. For example, it has been shown that iNKT cells contribute to natural tumor immunosurveillance. Although NKT cells prevent the induction of certain autoimmune diseases, they are an important source of Th2 cytokines during the induction of allergen-induced airway hyperreactivity. Lastly, CD1d-restricted NKT cells are important for host protection against pathogenic viruses, bacteria, fungi, and parasites. Mice lacking CD1d are more susceptible to certain pulmonary infections including Pseudomonas aeruginosa, Pneumococcus pneumoniae, and Cryptococcus neoformans. In other models, specic activation of CD1d-restricted NKT cells can enhance immunity and prolong survival of mice infected with virulent M. tuberculosis. NKT cells are thought to make an early contribution to host immunity, which is not surprising given their rapid activation and production of various cytokines. Less is known about the in vivo role of group 1 CD1-restricted T cells but more information is becoming available with the development of alternative small animal models such as the guinea pig and the

potential to make transgenic mice expressing human group I CD1 genes.

See also: Interferons. Leukocytes: T cells; Pulmonary Macrophages. Systemic Disease: Sarcoidosis.

Further Reading
Bollyky PL and Wilson SB (2004) CD1d-restricted T-cell subsets and dendritic cell function in autoimmunity. Immunology and Cell Biology 82: 307314. Brigl M and Brenner MB (2004) CD1: antigen presentation and T cell function. Annual Review of Immunology 22: 817890. Dascher CC and Brenner MB (2003) Evolutionary constraints on CD1 structure: insights from comparative genomic analysis. Trends in Immunology 24: 412418. Gadola SD, Zaccai NR, Harlos K, et al. (2002) Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains. Nature Immunology 3: 721726. Godfrey DI and Kronenberg M (2004) Going both ways: immune regulation via CD1d-dependent NKT cells. Journal of Clinical Investigation 114: 13791388. Kronenberg M and Gapin L (2002) The unconventional lifestyle of NKT cells. Nature Reviews: Immunology 2: 557568. Moody DB and Porcelli SA (2003) Intracellular pathways of CD1 antigen presentation. Nature Reviews Immunology 3: 1122. Porcelli SA (1995) The CD1 family: a third lineage of antigen presenting molecules. Advances in Immunology 59: 198. Shinkai K and Locksley RM (2000) CD1, tuberculosis, and the evolution of major histocompatibility complex molecules. Journal of Experimental Medicine 191: 907914. Sko ld M and Behar SM (2003) Role of CD1d-restricted NKT cells in microbial immunity. Infection and Immunity 71: 54475455. Sko ld M and Behar SM (2005) The role of group 1 and group 2 CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544551. van der Vliet HJ, Molling JW, von Blomberg BM, et al. (2004) The immunoregulatory role of CD1d-restricted natural killer T cells in disease. Clinical Immunology 112: 823. Watts C (2004) The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nature Immunology 5: 685692.

CD11/18
D S Wilkes and T J Webb, Indiana University School of Medicine, Indianapolis, IN, USA
& 2006 Elsevier Ltd. All rights reserved. leukocyte adherence has been shown using mAb blocking experiments, knockout animal models, and studies of individuals with a deciency in these glycoproteins. The use of mAbs against these adhesion receptors results in defective adherence, which suggests that this approach may have therapeutic potential in treating certain pulmonary diseases.

Abstract
Integrins are cell membrane receptors that assemble as heterodimers by the noncovalent association of the a and b subunits. The b2 family of leukocyte integrins comprises four a subunits (CD11ad) with a common b2 subunit (CD18). These adhesion molecules have been shown to play an important role in host defense. The critical role of the CD11/CD18 receptors in

Introduction
Lymphocyte migration from the bloodstream into secondary lymphoid organs is essential for maintaining homeostasis and for providing efcient defense

CD11/18 339

against pathogens. This migration occurs mostly through high endothelial venules and is partially regulated by signaling cascades involving adhesion and activation. Integrins mediate the rm adhesion of leukocytes to the endothelium that allows for the transmigration of cells into tissue. The CD11/CD18 leukocyte adhesion molecules consist of four cell surface membrane glycoproteins, namely LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150, 95 (CD11c/CD18), and adb2 (CD11d/CD18). The a subunits form a noncovalent association with a common b subunit (CD18) of 94 kDa to form a1b2 heterodimers. The divalent cations Ca2 and Mg2 are essential for the stabilization and function of the a1b2 complex. Signaling events through the b2 integrins lead to full activation status of the molecules. The active form of integrin is usually characterized by lateral clustering of the proteins on the cell membrane and by a change in structural conformation.

Cellular Location and Structure

The four distinct genes encoding for the a subunits occur in a cluster on chromosome 16, band p11 p13.1. This region also contains the gene encoding sialophorin (CD43), a leukocyte adhesion receptor, and the b isoform protein kinase C (PKC), suggesting the presence of a gene cluster involved in leukocyte adhesion and activation. The gene encoding CD18 is on chromosome 21, band q22. The major structural features of CD11/CD18 are illustrated in Figure 1. CD11ad and CD18 are transmembrane proteins spanning the plasma membrane once, with a short C-terminal cytoplasmic region and a large N-terminal extracellular domain. In the a subunit there are seven repeating homologous sequences at the N terminus. This part of the a subunit has been modeled as a b propeller fold and is a major contributor to the
Signal sequence

globular head of an integrin. Inserted into the b propeller fold is the inserted or I domain. The I domain is present in 9 out of 18 a subunits and is homologous to the A domain of the von Willebrand factor protein. Consequently, this region is frequently referred to as the I/A domain. A key feature of the I/A domain is the metal ion-dependent adhesion site or the MIDAS motif, which is composed of D-X-S-X-S. It is the MIDAS motif plus surrounding residues that form the actual ligand binding site. The b2 subunit (CD18) also contains a MIDAS motif, but it has not been directly shown to bind metal ions. The short and highly conserved cytoplasmic region of CD18 contains tyrosine and several serine and threonine residues, which account for stimulus-induced phosphorylation (Figure 2). An interesting feature of the extracellular region of CD18 is a highly conserved cysteine-rich region (CRR) consisting of four tandem repeats of an eight-cysteine motif. Notably, a point mutation in this region prevents cell surface expression of CD18. The a subunits of CD11/CD18 are highly homologous but distinct from the common b subunit. Mac-1 and p150, 95 are more related to each other (63% identity at the amino acid level) than to LFA-1 (36% homology), and this is reected by their close functional similarity. Polymorphisms in LFA-1 and Mac-1 have been reported. Accordingly, monoclonal antibodies (mAbs) have been shown to recognize specic activation or conformation states of CD11/CD18 receptors.

Biosynthesis
The a and b subunits of CD11/CD18 are synthesized in leukocytes from independent precursors. The two subunits associate as precursors prior to further carbohydrate processing in the Golgi. Association of ab

I/A domain MIDAS motif D-X-S-X-S 1 22 110 M 1 2 MIDAS motif D-X-S-X-S 3 4 5 M 6 M 7 361

Cysteine rich repeats

1 449

4 628 700 723 769 TM C

Figure 1 Schematic diagram of the primary structure of CD11/CD18. The main structural features of the a (CD11) and b (CD18) subunits are illustrated. In the b subunit, the red regions 14 outline the four cysteine-rich repeats. The yellow region represents the transmembrane (TM) site, and C represents the cytoplasmic end. In the a subunit, the seven tandem repeats in the extracellular portion are shown in teal, and repeats 57 contain a metal binding site (M).

340 CD11/18

CD18
2-integrins

LPS

Mac-1

CD11b

Tyrosine kinases Cytoskeleton rearrangement & cell motility Ca2+ influx

Monocyte Lymphocyte CD11a LFA-1 ICAM-1 CD18

Endothelial cells
Figure 2 Overview of b2 integrin signaling. b2 integrin engagement induces phosphorylation of tyrosine kinases. This can result in cytoskeletal rearrangement, an event that controls cell motility. Also, Ca2 signals are generated as described in detail in the legend to Figure 3.

Table 1 b2 adhesion molecules and their ligands Name CD11a/CD18 (LFA-1) CD11b/CD18 (Mac-1) Structure aLb2 aMb2 Ligand ICAM-1 (CD54), ICAM-2 (CD106) iC3b, factor w, brinogen, ICAM-1?, LPS, Leishmania gp63 iC3b Cell distribution All leukocytes Monocytes, macrophages, some lymphocytes, natural killer cells, neutrophils, granulocytes Monocytes, granulocytes, macrophages, natural killer cells, certain lymphocytic tumor cell lines Monocytes, macrophage foam cells, splenic red pulp macrophages, lymphocytes

CD11c/CD18 (p150,95)

axb2

CD11d/CD18

adb2

LFA-1, lymphocyte function-associated antigen; Mac-1, macrophage-1 antigen; ICAM, intercellular adhesion molecules; iC3b, proteolytic fragment of complement protein C3; factor w, coagulation factor w.

precursors appears to be important for further carbohydrate processing and cell surface expression of the heterodimers. Following association, the CD11 and CD18 subunit precursors undergo an increase in their molecular mass that is accompanied by a conversion on N-linked carbohydrates to their complex form. As expected, more CD18 precursors are synthesized in lymphocytes and monocytes than each of the three a subunits. Interestingly, the type of a subunit affects the glycosylation pattern of CD18.

Expression and Biological Function

The CD11/CD18 glycoproteins are differentially expressed (Table 1). The level of CD11/CD18

expression is dependent on the cell type and the state of cell activation and differentiation. The CD11/ CD18 glycoproteins are only expressed in leukocytes. Whereas CD11a/CD18 is present on all leukocytes, CD11b, c/CD18 is normally restricted to expression on monocytes, macrophages, polymorphonuclear lymphocytes (PMNs), and natural killer (NK) cells. However, p150, 95 and the recently described adb2 are mainly expressed by tissue macrophages. In B and T cells, all CD11/CD18-dependent functions such as mitogen, antigen, and alloantigeninduced proliferation, T cell-mediated cytotoxicity, B cell aggregation, and Ig production are inhibited by anti-LFA-1 mAbs. This is consistent with LFA-1 being the only heterodimer normally expressed on these

CD11/18 341

cells. Granulocytes, monocytes, and NK cells express all three CD11/CD18 heterodimers, which accounts for the wider range of CD11/CD18-dependent functions mediated by these cells. LFA-1 seems to mediate an early Mg2 -dependent adhesion step in cellcell interaction. Studies have shown that LFA-1 is primarily responsible for adhesion and signaling at the immunological synapse, involved in the clustering of proliferating lymphocytes, and found between CTLs and their targets. Anti-LFA-1 mAbs inhibit T-cell proliferation in response to stimuli that require cellcell contact. Notably, APC-independent T-cell responses do not require LFA-1. Also, at high effector:target ratios or during secondary stimulation, the role of LFA-1 is significantly diminished, perhaps as a result of increased expression of several other adhesion molecules. Mac-1 is essential for binding iC3b and other ligands, spreading, homotypic adhesion, and phagocytosis of opsonized particles in neutrophils, a role that may also be fullled by p150, 95 in resident macrophages. Following activation, Mac-1 can initiate signaling via its linkage to the actin cytoskeleton and associated signaling proteins. Also, Mac-1 is thought to act as a signaling partner for other leukocyte receptors, including lipopolysaccharide (LPS)/LPS binding protein receptors (CD14), formyl-methionylleucyl-phenylalanine (FMLP) receptors, urokinase plasminogen activator receptors (CD87), and FcRs.
Endothelial cells

Whereas MAC-1 is a phagocytic receptor, used by neutrophils to engulf bacteria, yeast, and other microorganisms, the roles of p150, 95 and adb2 have not been well documented. All CD11/CD18 receptors contribute to chemotaxis and adhesion to cytokine-activated endothelium by neutrophils and monocytes and to antibody-dependent cellular cytotoxicity (ADCC).

Ligands
Several ligands have been shown to interact with CD11/CD18 molecules in a divalent cation-dependent manner, as shown in Table 1. The rst wellcharacterized ligand was iC3b, a component of the complement system. iC3b binds to Mac-1 and p150, 95 at 37oC in resting cells, but it is greatly enhanced in cells activated by phorbol esters. LFA-1 binds to the two N-terminal domains of intracellular adhesion molecules (ICAM)-1 and -2. ICAM-1 is inducible and expressed on leukocytes and endothelial and epithelial cells in response to inammatory cytokines (Figure 3). In contrast to ICAM-1, ICAM-2 is constitutively expressed on endothelial cells and a variety of B and T lymphoblastoid cell lines, and its expression is not enhanced by inammatory stimuli. However, unlike ICAM-2, ICAM-1 was uniquely found to be involved in migration of lymphocytes into inamed tissue and in trapping lymphocytes in the lung. Concurring with

ICAM-1 CD18 CD11a

Lymphocyte

Tyrosine kinases

PLC- 1/2 PIP2 DAG PKC

PLC- 1/2

Ins(1,4,5)P3 Ca2+ release

Figure 3 b2 integrins can induce calcium signaling in leukocytes. Ligand binding of b2 integrins causes activation of tyrosine kinases. The tyrosine kinases are phosphorylated, which increases the catalytic activity of PLC-g. Then, PLC-g catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP2) into both diacylglycerol (DAG) and inositol triphosphate (Ins(1,4,5)P3). Next, Ins(1,4,5)P3 stimulates the release of Ca2 from internal stores, which is followed by an inux of Ca2 .

342 CD11/18

those data, studies using a murine model of asthma found that ICAM-1 is important in the migration of lymphocytes to the lungs during an allergic inammatory response. Also, in another study examining the homing mechanisms of cytotoxic T cells, LFA-1 was shown to be critical for the retention of effector CD8 T cells in the lungs. Moreover, in an adoptive transfer model involving alloreactive CD4 T cells, adherence of T cells in the lungs was shown to be partially dependent on LFA-1 and its receptor ICAM-1. Conicting data exist regarding whether Mac-1 also binds to ICAM-1; however, binding of two other ligands, coagulation factor w and brinogen, to an activated form of Mac-1 has been shown. All three heterodimers (CD11ac/CD18) bind to LPS, as determined by blocking studies using mAbs, suggesting that binding is mediated by homologous regions in the CD11 subunits or by the common CD18 subunit.

CD11/CD18 in Respiratory Disease

Leukocyte adhesion deciency (LAD-1) is a disorder caused by the lack of surface expression of CD11/ CD18, which is due to heterogeneous defects in the CD18 subunit. The classical disease is characterized by recurring necrotic soft tissue infections, impaired wound healing with a lack of pus, and severe gingivitis. Also, phagocytes from patients with this disorder have functional defects in aggregation, chemotaxis, phagocytosis, endothelial cells, and complement iC3b binding and ADCC. Two phenotypes of LAD-1 have been reported based on the level of CD18 expressed. In the severe form, CD11/CD18 is not detectable, and death from bacterial infections usually occurs during the rst few years of life. However, in the moderate phenotype cells express 25% of the normal level of CD18 and the clinical course is much milder. In CD18decient mice, as with LAD-1 patients, marked neutrophilia was found. However, in contrast to LAD-1 or CD18-decient mice, CD11b-decient mice do not exhibit any leukocytosis or display a significant increase in bacterial infections. Nevertheless, in vivo studies have shown that these mice have defective rm adhesion, and in vitro studies have shown that neutrophils from these mice have defects in adhesion, iC3b-mediated phagocytosis, phagocytosis-induced respiratory burst, and homotypic aggregation. Remarkably, although CD11a-decient mice displayed normal CTL responses to a systemic virus infection, they failed to develop a DTH response to 2,4-dinitrouorobenzene sensitization and challenge. Because ICAM-1 is a major ligand for LFA-1 and perhaps Mac-1, ICAM-1-decient mice are

particularly interesting. Two groups have reported ICAM-1 knockout mice. Although both ICAM-1decient murine lines develop normally and have moderate leukocytosis, the mice displayed multiple abnormalities in their inammatory responses, specifically impaired neutrophil emigration in response to chemical peritonitis, resistance to septic shock, and decreased contact hypersensitivity reaction. Intriguingly, it has been shown that during allergic lung inammation, there is a delayed increase in eosinophils in the airway lumen and a prolonged presence of eosinophil inltrates in lung tissue in ICAM-2decient mice. In contrast, the importance of ICAM2 in binding T cells has been demonstrated in frozen section assays of chronically inamed human airway epithelium; however, inammation of the lungs in ICAM-2-decient mice was due to an accumulation of eosinophils, not lymphocytes. Overall, it has been suggested that ICAM-2 expressed in vascular endothelium, alveolar walls, or large airway epithelium participates in the trafc of eosinophils from the bloodstream to the airway lumen.

Conclusion
Lymphocyte homing is tightly regulated; however, the molecular basis of lymphocyte migration to the lungs is not well understood. Because the lung is the portal of entry for airborne pathogens, lymphocytes must constantly recirculate. Circulating lymphocytes become transiently trapped within the lung. This has been shown for normal lymphocytes and even more so for activated/memory lymphocytes. Notably, retention is mediated, at least in part, by LFA-1. Interestingly, studies have shown that effector and memory cytotoxic T lymphocytes traverse to the lungs and reside in the lung parenchyma for extended periods. In addition, it has been suggested that normal lung microvessels can constitutively support the retention of activated CD8 T cells. Moreover, murine studies investigating the role of LFA-1 in an acute RSV infection showed that treatment of mice with neutralizing Abs to LFA-1 resulted in diminished illness and delayed viral clearance. These data further support the hypothesis that using blocking mAbs against b2 integrins could be an immunotherapeutic strategy. Many diseases that affect the lungs involve inammation. Following sequestration and initial adhesion, neutrophils migrate along the capillary endothelium and into the interstitium and alveoli. This emigration can occur through either a CD11/CD18-dependent or -independent pathway. The adhesion pathway used depends on the stimulus. Stimuli that have been shown to induce CD18-dependent emigration include Escherichia coli, E. coli lipopolysaccharide,

CD14 343

Pseudomonas aeruginosa, IgG immune complexes, interleukin-1, and phorbol myristate acetate. These stimuli appear to act by inducing the translocation of NF-kB, resulting in the production of inammatory cytokines and ICAM-1 on the pulmonary capillary endothelial cells. Stimuli that have been shown to induce the CD11/CD18-independent neutrophil emigration include Streptococcus pneumoniae, group B Streptococcus, Staphylococcus aureus, hydrochloric acid, hyperoxia, and C5a. Previous studies have shown that the absence of CD11/CD18 in granulocytes and monocytes attenuates the acute cellular inammatory responses in vivo; therefore, these data suggest that blocking CD11/CD18 may ablate the tissue damage induced by these cells. Consequently, this was shown to be the case in several ischemiareperfusion injury animal models. In fact, giving anti-Mac-1 mAbs before or soon after the induction of ischemiareperfusion injury significantly reduced tissue injury, microvascular permeability, and acidosis. However, the lack of protection of the lung tissue in some of these models may be an indication of the importance of other adhesion pathways mediating PMN migration. Lastly, detailed investigations of the LAD-1 syndrome in humans and studies involving adhesion molecule-decient mice have provided novel insights into the cell and molecular biology of leukocyte emigration. However, further studies must be done to address the discrepancies observed between decient animals and wild-type animals treated with blocking mAbs.

Further Reading
Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75: 10371050. Buyon JP, Slade SG, Reibman J, et al. (1990) Constitutive and induced phosphorylation of the a- and b-chains of the CD11/CD18 leukocyte integrin family: relationship to adhesion-dependent functions. Journal of Immunology 144: 191197. Dayer JM, Isler P, and Nicod LP (1993) Adhesion molecules and cytokine production. American Review of Respiratory Disease 148: S70S74. Doerschuk CM (2000) leukocyte trafcking in alveoli and airway passages. Respiratory Research 1: 136140. Doerschuk CM, Mizgerd JP, Kubo K, et al. (1999) Adhesion molecules and cellular biomechanical changes in acute lung injury. Chest 116: 37S43S. Etzioni A, Doerschuk CM, and Harlan JM (1999) Of man and mouse: leukocyte and endothelial adhesion molecule deciencies. Blood 94: 32813288. Graham IL, Gresham HD, and Brown EJ (1989) An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors. Journal of Immunology 142: 23522358. Hogg N and Bates PA (2000) Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biology 19: 211 222. Lehmann JCU, Jablonski-Westrich D, Haubold U, et al. (2003) Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafcking. Journal of Immunology 171: 25882593. Pilewski JM and Albelda SM (1993) Adhesion molecules in the lung: an overview. American Review of Respiratory Disease 148: 531537. Thatte J, Dabak V, Williams MD, et al. (2003) LFA-1 is required for retention of effector CD8 T cells in mouse lungs. Blood 101: 49164922. van Spriel AB, Keusen JHW, van Egmond M, et al. (2001) Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation. Blood 97: 24782486. van Spriel AB, van Ojik HH, Bakker A, et al. (2003) Mac-1 (CD11b/CD18) is crucial for effective Fc-receptor mediated immunity to melanoma. Blood 101: 253258. Xu B, Wagner N, Pham LN, et al. (2003) Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, a4b1 integrin/VCAM, and LFA-1 adhesion pathways. Journal of Experimental Medicine 197: 1255 1267.

Acknowledgments
This work was supported by National Institutes of Health grants HL60797 and HL/AI67177 to D S Wilkes.
See also: Adhesion, CellMatrix: Integrins. Coagulation Cascade: Fibrinogen and Fibrin. Complement. Dendritic Cells. Leukocytes: Eosinophils; Neutrophils; Monocytes; Pulmonary Macrophages.

CD14
Y Tesfaigzi and M Daheshia, Lovelace Respiratory Research Institute, Albuquerque, NM, USA
& 2006 Elsevier Ltd. All rights reserved. monocytes, macrophages, and some granulocytes. CD14 is a key molecule in the activation of innate immune cells and exists as a membrane-anchored or soluble form. It is encoded by a gene on human chromosome 5q31.1, a region where several genes implicated in asthma pathogenesis are localized. CD14 is expressed in a variety of hematopoietic and parenchymal cells and it has a range of biological activity including cell differentiation, immune response, and host-pathogen interactions. While CD14 is an essential part of the lipopolysaccharide

Abstract
CD14 is a glycolipid-anchored membrane glycoprotein expressed on cells of the myelomonocyte lineage including

344 CD14
(LPS) receptor complex, it requires interaction with long-terminal repeat 4 to successfully transmit LPS-induced signals to the cell. In addition, CD14 is an integral part of the mechanism by which macrophages interact and engulf apoptotic cells. A functional single nucleotide polymorphism within the promoter region of CD14 that affects expression levels is associated with a higher risk of developing atopy. The interactions of CD14 with other receptors are important for the normal signaling of LPS and host-pathogen interactions and affect the susceptibility or resistance to a variety of diseases including atopy and septic shock.

levels of sCD14 have been associated with inammatory/infectious diseases such as dermatitis, septic shock, malaria infection, human immunodeciency virus (HIV), and severe burns.

Biological Functions
CD14 and LPS Binding

Introduction
CD14 was originally discovered through reactivity of monoclonal antibodies on human peripheral blood monocytes and was cloned in 1988. Subsequently, the protein was puried, and our understanding of its role in inammatory responses has been increasing steadily. While this protein is best known for its ability to act as a receptor for bacterial endotoxin, lipopolysaccharides (LPS), and to elicit proinammatory host responses, other functions for CD14 such as those related to the recognition of apoptotic cells and clearance by phagocytosis have also gained importance. This latter role involves a process that suppresses inammation and appears to contradict its proinammatory function, making this protein highly interesting to study.

Structure
The CD14 protein is encoded by a gene localized on the human chromosome 5q31.1 or on the syntenic mouse chromosome 18. In humans and mice, CD14 has an amino acid length of 375 and 366, respectively, that is transcribed by a 1367-nucleotide mRNA. CD14 exists in two different forms: a membrane-anchored and a soluble (sCD14) form. The membranebound CD14 is a glycoprotein with a molecular weight of 5355 kDa, attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The sCD14 is generated when the membrane-bound CD14 is shed and released into the bloodstream.

While LPS is the main ligand for CD14, several other molecules such as peptidoglycans, phospholipids, and dextrans, can interact with CD14. Several lines of evidence suggest a crucial role for CD14 in the biological effects of LPS: (1) The neutralizing antibody to CD14 inhibits the interaction of LPS with the receptor and protects primates from endotoxininduced shock; (2) CD14-decient mice are 10100 times less sensitive to LPS than their wild-type counterparts because they produce very low amounts of proinammatory cytokines and are resistant to a lethal dose of Gram-negative bacteria; (3) overexpression of CD14 renders mice more susceptible to LPS-induced septic shock or to Gram-negative bacteria-induced death. The susceptibility of CD14-decient mice is specic to Gram-negative bacteria because responses to infection by Gram-positive bacteria such as Staphylococcus aureus are not affected by the absence of CD14. The interaction between LPS and CD14 is facilitated by the soluble LPS-binding protein (LBP), which increases the afnity of LPS to CD14 by more than 100- to 1000-fold. Therefore, LBP-decient mice are more resistant to LPS than wild-type mice and are defective in their ability to mount appropriate inammatory responses to bacterial infection. For example, LBP-decient mice are incapable of controlling infection by Gram-negative bacteria such as Salmonella and succumb to a dose of pathogen, which is well tolerated by their heterozygous or wild-type littermates. It has also been reported that LBP may be incorporated as a transmembrane protein in the cytoplasmic membrane of mononuclear cells to mediate activation of these cells by LPS, revealing that LBP has a different mode of action in LPS signaling.
CD14 as a Component of LPS Receptor Complex

Regulation of Expression
The regulatory sequence of CD14 contains multiple consensus-binding sites for CAAT/enhancing and binding protein (C/EBP) and Sp transcription factors. The gene expression is induced by transforming growth factor beta (TGF-b) and vitamin D and is downregulated by interleukin (IL)-4. Membranebound CD14 is expressed by a variety of cells, including monocytes/macrophages, neutrophils, granulocytes, and hepatocytes. Increased serum

Although CD14 is the major membrane receptor for binding LPS, it is incapable of inducing intracellular signaling because it lacks an intracellular domain. CD14 must form a complex with Toll-like receptor 4 (TLR4), which is bound to myeloid differentiation protein-2 (MD-2) to transmit intracellular signaling in response to LPS binding (Figure 1). The mature human MD-2 and TLR4 proteins consist of 160 and 839 amino acid residues, respectively. After LPS

CD14 345

LPS LPS LBP LBP

LPS

TLR4 TIR

CD14

of NF-kB, but it does so via an alternative pathway that does not require MyD88 and TRAF6. Additionally, TRIF/TICAM can activate the interferon related factor 3 (IRF3) pathway and increase interferon beta (IFN-b) to turn on the JAK/STAT pathway. LPS signaling is decreased in the absence of CD14 or TLR4, revealing the primordial importance of these molecules for LPS effects. Although MyD88 ( / ) mice showed a significant reduction in tumor necrosis factor alpha (TNF-a) production, these mice still exhibit NF-kB and MAPK activation and IFN-b release in response to LPS. Additionally, TRIF/TICAM-1 ( / ) mice exhibit suppressed expression of IFN-b as well as lower IFN-g production; however, the MyD88-dependent activation of IRAK-1, NF-kB, and MAPK are not affected. Complete loss of LPS-induced responses are only observed in mice decient for both MyD88 and IRIF/TICAM-1 genes, suggesting that LPS acts entirely through these two signaling pathways.
CD14 and Phagocytosis

MD-2

Figure 1 Interaction of CD14 with TLR-4 for signaling through the intracellular TIR domain. LPS, lipopolysaccharide; LBP, LPSbinding protein; TLR4, Toll-like receptor 4; MD-2, myeloid differentiation protein-2.

interacts with the LBP, the heterodimer binds to CD14, which subsequently activates TLR4/MD-2 and initiates signaling through the Toll/IL-1R (TIR) intracellular domain. However, airway epithelial, endothelial, and dendritic cells respond to LPS even though they lack the membrane-bound CD14 because the soluble CD14 is believed to form the LPS complex and activate TLR4. Activation of LPS receptor complex (LPSRC) causes many cellular changes, including increased transcription of multiple genes, release of a variety of cytokines, cell proliferation, and cell death. LPSRC activation results in the recruitment of at least two major cytoplasmic proteins to TLR4: the myeloid differentiation factor 88 (MyD88) and the TIR-containing adapter molecule (TRIF, also known as TICAM). The MyD88-dependent pathway leads to the recruitment of the MyD88 adaptor-like protein (Mal, also known as TIRAP), then the IL-1R-associated kinase (IRAK) and TRAF6, which can either activate mitogen-activated protein kinase kinase (MAPKK) with the ultimate activation of activation protein 1 (AP-1), or can activate IKK and phosphorylate IKba, causing the rapid ubiquitin-dependent proteolysis of the IKK and translocation of free nuclear factor kappa B (NF-kB) to the nucleus. The TRIF/ TICAM pathway can also activate IKK and induction

Cells undergoing apoptosis are cleared rapidly by phagocytosing macrophages, thus preventing the release of intracellular material from dying cells, which would cause inammation and tissue damage. The removal of apoptotic cells in the body involves a complex array of macrophage receptors, including CD14. CD14 is upregulated in migratory macrophages inltrating Burkitts lymphoma tumors to engage in clearance of apoptotic cells. The monoclonal antibody 61D3 that binds to CD14 on the surface of human macrophages markedly inhibits the capacity of macrophages to interact with and engulf cells undergoing apoptosis. The CD14-mediated interaction of cells with macrophages is specic for apoptotic cells because interaction of viable cells with macrophages is not affected by the monoclonal antibody 61D3. When transiently expressed on the surface of COS cells (African green monkey kidney broblast-like cell line), CD14 was found to impart an increase in both of apoptotic lymphocytes by these cells, and both were inhibited by the monoclonal antibody against CD14. Quantitative histological studies indicated that CD14-decient mice have defective clearance capacity leading to persistence of apoptotic cells in all tissues studied. Additionally, TL-9 mouse macrophages decient in CD14 expression were completely unable to phagocytose apoptotic thymocytes, and removal of CD14 from J774 macrophages significantly reduced the phagocytic ability of these cells. These studies show that CD14 is an integral part of the phagocytosis process. There is growing evidence that CD14 is a crucial component of the mechanism by which apoptotic

346 CD14

cells are recognized. CD14 interacts with phosphatidylserine, whose exposure to the outer leaflet of the cell membrane is a universal feature of apoptotic cells. CD14 acts as a tethering receptor for apoptotic cells, and when CD14 was removed from macrophages by cleaving its glycosylphosphatidylinositol tether, the phagocytosis of apoptotic lymphocytes by macrophages was substantially reduced. The intracellular adhesion molecule-3 (ICAM-3) is one of many molecules on the surface of apoptotic cells that supports the essential recognition by macrophages. Interestingly, the ICAM-3-mediated interaction of apoptotic leukocytes involves CD14 but is independent of macrophage lymphocyte function-associated antigen-1 (LFA-1), a2b2, and avb3. Several models have suggested that the engagement of CD14 and apoptotic cells would not induce generation of inammatory responses, as is the case following CD14 and LPS interaction. The size and structural differences between the two ligands could explain why the distinct biological outcome of CD14 activation and the interaction of CD14 and the apoptotic cells do not need the presence of LBP. It is possible that when CD14 binds to apoptotic cells, it does not engage TLR4 or if TLR4 is activated, CD14 interaction with the dying cells, may interact with yet unknown receptor(s) to block the TLR4 responses.
CD14 and Respiratory Disease

T Regulatory sequence Coding sequence DC +

sCD14 + Macrophage

CD14 locus

IL-12 Th0 + Th1 B cells

IFN IFNIgE

Figure 2 A T allele within the regulator promoter region of CD14 is associated with increased sCD14 levels, which is known to mediate interleukin-12 (IL-12) and interferon gamma (IFN-g) production by dendritic cells (DC) and macrophages, respectively. These cytokines cause a Th1 differentiation of T lymphocytes and suppress the production of IgE by B cells.

Several lines of evidence have led to the hypothesis that the CD14 gene may be associated with asthma. Engagement of CD14 by LPS and other bacterial cell wall components enhances IL-12 secretion from dendritic cells, which is believed to be an obligatory signal for the differentiation of naive T cells into Th1-like cells (Figure 2). Activation of CD14 by LPS leads to the release of IL-12, IL-18, and IFN-g, which can deviate the Th2 immune response and decrease the production of immunoglobulin E (IgE) by the B cells. It is also conceivable that upregulation of sCD14 could induce apoptosis in the pathogenic cells involved in asthma and/or induce the induction of suppressive factors such as IL-10 or TGF-b, leading to a decreased severity of the disease. These hypotheses are supported by reports in several British cohorts that reduced sCD14 levels in amniotic uid and breast milk are associated with the subsequent development of atopy and that high levels of sCD14 are protective against development of recurrent wheezing in hospitalized children with respiratory syncytial virus infection. Furthermore, rhinovirus infection, the most common trigger of

acute asthma exacerbations, significantly reduces CD14 levels. The CD14 gene is located in a chromosomal locus proximal to several genes implicated in the pathogenesis of asthma, including IL-3, IL-4, IL-5, IL-9, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF). C-159 T, a common single nucleotide polymorphism (SNP) in the proximal promoter region of CD14, has been associated with different levels of sCD14 expression, and homozygous carriers of the T allele have significantly increased sCD14 and decreased total serum IgE. Increased sCD14 levels are associated with low IL-4 and high IFN-g production. The underlying mechanism is believed to be the decreased binding of transcription factor Sp3 to the GC box that includes the polymorphic nucleotide, resulting in enhanced transcriptional activity. Many population-based studies have found associations of the functional promoter polymorphism C159 T with a severity or susceptibility to atopy and serum IgE levels. However, different studies have reported conicting results as to whether the C or the T allele is associated with atopy, and two studies in Germany involving a large cohort of children found no association with asthma. It is still unclear whether these differences are caused by other SNPs within the same gene or differences in LPS concentrations within various studies. A role for conferring increased sensitivity of this SNP with LPS exposure was supported by the nding in a study of farmers that those homozygous for the -159 T allele have significantly lower lung function and an increased prevalence of wheezing compared to those with the C allele.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 347 See also: Apoptosis. Asthma: Overview. Dendritic Cells. Endotoxins. Interferons. Leukocytes: Monocytes; Pulmonary Macrophages. Toll-Like Receptors.
immune responses. National Review of Immunology 2(12): 965975. Schmitz G and Orso E (2002) CD14 signalling in lipid rafts: new ligands and co-receptors. Current Opinion in Lipidology 13(5): 513521. Takeda K and Akira S (2004) TLR signaling pathways. Seminars in Immunology 16(1): 39. Triantalou M and Triantalou K (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends in Immunology 23(6): 301304. Ulevitch RJ, Mathison JC, and da Silva Correia J (2004) Innate immune responses during infection. Nature Immunology 5(10): 987995. Ulevitch RJ and Tobias PS (1999) Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology 11(1): 1922. Wright SD (1995) CD14 and innate recognition of bacteria. Journal of Immunology 155(1): 68. Zhang G and Ghosh S (2001) Toll-like receptor-mediated NFkappaB activation: a phylogenetically conserved paradigm in innate immunity. Journal of Clinical Investigation 107(1): 1319.

Further Reading
Baldini M, Vercelli D, and Martinez FD (2002) CD14: an example of gene by environment interaction in allergic disease. Allergy 57(3): 188192. Cook DN, Pisetsky DS, and Schwartz DA (2004) Toll-like receptors in the pathogenesis of human disease. Nature Immunology 5(10): 975979. Gregory CD (2000) CD14-dependent clearance of apoptotic cells: relevance to the immune system. Current Opinion in Immunology 12(1): 2734. Koppelman GH and Postma DS (2003) The genetics of CD14 in allergic disease. Current Opinion in Allergy and Clinical Immunology 3(5): 347352. Savill J, Dranseld I, Gregory C, and Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates

CELL CYCLE AND CELL-CYCLE CHECKPOINTS

A Carnero, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in the cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specic events and coupling them to further progression. Cell-cycle checkpoints ensure that the system only proceeds to the next stage when the previous stage has been completed. Correct control of the cell cycle results from the coordinated and sequential activationinactivation of a family of key regulators known as cyclin-dependent kinases. In this article, the mechanisms that ultimately regulate the checkpoints and their contribution to the cell cycle are discussed.

phenotypes of the cdc mutants revealed that the execution of late events in the cell cycle depended on the prior completion of early events. Cell division occurs by an ordered series of metabolic and morphogenic changes that are collectively called the cell cycle. The cell cycle is divided into four distinct phases (Figure 1):
*

Synthetic (S) phase, in which the replication of DNA takes place Mitotic (M) phase, in which equal distribution of duplicated DNA and cellular division take place Gap 1 (G1) phase: the gap between completion of the M phase and the beginning of the S phase Gap 2 (G2) phase: the gap between completion of the S phase and the beginning of the M phase

Checkpoints and the Cell Cycle

In 1970, Lee Hartwell and colleagues recognized the rst cell division cycle (cdc) mutants among a large collection of temperature-sensitive lethal mutants of Saccharomyces cerevisiae. These clones were further identied as mutants that arrested division at a unique stage of the cell cycle regardless of their stage at the time they were shifted to restrictive temperature. The

Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specic events and coupling them to further progression. Cell cycle checkpoints ensure that the system only proceeds to the next stage when the

348 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

Spindle checkpoint

G2/M checkpoint

lin B

Cyc

CDK1 G2 CDK4

Cyclin D

G1

CDK2 CDK2 S Cyclin A

DNA damage checkpoint

Figure 1 Cell cycle and cell-cycle checkpoints.

previous stage has been completed. The key checkpoints appear to be as follows:
* *

G2/M: No entry to M without completion of S. Spindle checkpoint: Prevents anaphase onset until completion of mitotic spindle assembly. Start or restriction (R) point: Blocks progress for nonproliferating or differentiated cells; integrates external signals to proceed with the cell cycle. DNA damage: monitoring of DNA damage and block of cell-cycle progress; allows repair mechanisms to proceed; if DNA repair fails, the cell may be directed into apoptosis.

The coordination of these complex processes is achieved by a series of coordinated and sequential phase transitions of key regulators known as cyclin-dependent kinases (CDKs), which are one of the major targets detected in the cdc mutants (Figure 1).

Mammalian Cell Cycle Regulators

The CDKs are a family of closely related Ser/Thr kinases that are activated by association with a regulatory subunit called cyclin (Table 1). The typical catalytic subunit contains a 300-amino acid catalytic core that is inactive when monomeric and unphosphorylated at certain positions. Cyclins are members of structurally related proteins whose levels usually oscillate during the cell cycle. The homology among cyclins is mostly limited to the cyclin box, the domain responsible for

CDK binding and activation. Mutations in this region inhibit both binding and activation. Each CDK interacts with a specic subset of cyclins, although the size of the subset varies (Table 1). The cyclin function is primarily controlled by changes in cyclin levels. In mammalian cells, sequential oscillation in the levels of major cyclins (E, A, and D) reects levels of messenger RNA and specic proteolysis. Whereas cyclins are essential to activate CDKs, the specic destruction of cyclins is central to the proper regulation of the cell cycle. In terms of proteolysis, the cyclins can be roughly divided into two classes those that are constitutively unstable through the cell cycle and whose level is therefore determined by the ratio of transcription (cyclins D and E) and those that are unstable in only one phase of the cell cycle. The G2 cyclins (A and B type) are stable throughout interphase and specifically destroyed at mitosis. Cyclin degradation involves the ubiquitin-dependent degradation machinery. However, the details of the conjugation cyclinproteasome differ for the different cyclins. E and D cyclins are ubiquitinated by the SCF complex, whereas G2 cyclins are ubiquitinated by the anaphase-promoting complex (APC). The SCF complex is active throughout the cell cycle, and the destruction of its substrates depends on their phosphorylation, with different phosphate-binding proteins (F box proteins) guiding different sets of substrates to destruction. The APC is activated at the onset of anaphase and degrades its substrates as the cell exits mitosis.

Cyc lin E

G1 checkpoint

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 349

Table 1 Complexes of cyclin-dependent kinases (CDKs) Kinase CDK1 CDK2 CDK3 CDK4 CDK5 CDK6 CDK7 CDK8 CDK9 CDK10 CDK11 Regulatory subunit Cyclin A and B Cyclin A, E, and D Cyclin E Cyclin D p35, cyclin D Cyclin D Cyclin H Cyclin C Cyclin T Cyclin L Substrate pRb, NF, Histona H1 pRb, p27 E2F1/DP1 pRb, Smad3 NF, Tau pRb CDK1/2/4/6 RNA pol II pRb, MBP Ets2 RNA pol II Function G2/M G1/S, S G1/S G1/S Neuronal differentiation G1/S CAK Transcriptional regulation G1/S Transcription Transcription

Degradation Cyclin synthesis

CKIs Cyclin Inactive Cyclin CDK CDK thr161 thr14 tyr15 thr161 thr14 tyr15 thr161 Active Cyclin CDK P P P pRb E2F

P P P pRb

E2F

Cyclin H CDK7 thr161

CDC25 WEE 1 DPI E2F Transcription

Figure 2 CDK regulation at the G1 checkpoint.

In addition to cyclin-binding, complete CDK activation requires phosphorylation at a conserved 160/ 161 threonine residue. This phosphorylation may affect the cyclin-binding site because it enhances the binding of some CDK/cyclin complexes. This activating phosphorylation is performed by another CDK/ cyclin complex termed CAK, which consists of CDK7 bound to cyclin H. Mammalian CDK7/cyclin H complexes can phosphorylate CDK1 and CDK2 complexed with various cyclins as well as CDK4 (Figure 2). CDK/cyclin complexes can also be inhibited by phosphorylation of the CDK subunit at the amino terminus (Thr14 and Tyr15 residues in human CDK1 and CDK2). Inactivation via tyrosine 15 phosphorylation is important in the control of the initiation of mitosis (Figure 2). The phosphate in tyrosine 15 is removed by a specic phosphatase, CDC25. There are at least three CDC25 family members in

mammalian cells. CDC25C is more likely to activate cyclin B/CDC2 at mitosis, whereas CDC25A is phosphorylated and activated by cyclin E/CDK2 at the initiation of DNA synthesis. Finally, numerous studies have identied additional regulatory subunits for the CDKs, the cyclindependent kinase inhibitory proteins (CKIs). These proteins bind and inhibit CDK activity. There are two families of CKIs: the CIP/KIP family and the INK4 family (Table 2). The CIP/KIP family includes p21waf1/cip1/sdi1(p21waf1), p27kip1, and p57kip2 and seems to be central in differentiation processes. The members of this family are general inhibitors of all the cyclin/CDK complexes and accumulate in response to a broad range of antiproliferative stimuli. These proteins preferentially associate with the cyclin/CDK complex rather than with the individual CDK subunits. The overexpression of these proteins

350 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

Table 2 Involvement of cell-cycle regulatory elements in human cancer Protein CDK4 Cyclin Cyclin Cyclin Cyclin D1 D2 E A Alteration Mutation Overexpression Overexpression Overexpression Overexpression Activation Activation Inactivation/degradation Deletion/inactivation Deletion/inactivation, mutation Deletion/inactivation Overexpression Overexpression Overexpression Deletion/inactivation Mutation, deletion Tumor Melanoma Breast, prostate, gastric, parathyroid carcinoma, lung cancer Colorectal carcinoma Breast, ovarian, and gastric carcinoma Hepatocellular carcinoma, lung cancer Head and neck cancer, lung cancer Breast cancer, lymphoma, head and neck cancer, lung cancer Breast, prostate, and colorectal carcinoma, lung cancer BeekwithWiedemann syndrome Melanoma, lymphoma, lung cancer, pancreatic carcinoma Leukemia, lymphoma Lung cancer, head and neck squamous carcinoma Several tumors, lung cancer Several tumors, lung cancer Retinoblastoma, lung cancer Many tumors, lung cancer

CDC25A CDC25B p27KIP1 p57KIP2 p16INK4a p15INK4b Plk1 Aurora A Aurora B pRb p53

arrests cells in different states of the cell cycle according to their biochemical function. The INK4 family of CKIs consists of four related proteins: p16ink4a, p15ink4b, p18ink4c, and p19ink4d. These proteins specifically bind and inactivate CDK4 and CDK6 kinases, inducing G1 arrest. These proteins appear to bind the CDK subunit alone, competing with their activating partner, cyclin D. It is possible that these proteins inhibit CDK activity by reducing the afnity for cyclin D or by competitively preventing the binding of cyclin D to the CDK subunit.

The Cell Cycle

Progression through the cell cycle results from the coordinated and sequential activationinactivation of the previously described elements.
Restriction Point

After the proliferative stimuli (growth factors and oncogenes) induce cell proliferation, a rst checkpoint (the R point) at late G1 integrates both positive and negative external and internal signals before the cell commits to another round of DNA replication (Figure 1). In mammalian cells the R point is regulated mainly by the CDKs bound to the D type of cyclins, and its activation is mainly driven by transcriptional activation of cyclin D synthesis. Most growth factors and oncogenes trigger cyclin D transcription. There are three types of D cyclins, and they are in part cell-type specic, with most cells expressing D3 and either D1 or D2. The D type of cyclins

associate and activate CDK4 and, in some cells, also CDK6. These cyclins have a very short half-life and their expression is highly growth factor inducible. Dtype cyclins are fundamental in cell-cycle regulation of the retinoblastoma protein (pRb; the major regulator of the R checkpoint). The phosphorylation of pRb is controlled by two opposing reactions: phosphorylation by CDKs in G1, which converts pRb into its inactive form in terms of ligand-binding activity, and dephosphorylation in late M by phosphatases, which reconstitute active pRb. As a result of this interplay of enzymatic reactions, the pRb phosphorylation state uctuates as the cell passes through the cell cycle. In cycling cells, pRb is found in its active, underphosphorylated form only during the early period of the G1 phase. Although changes in the array of phosphorylation events may occur at other phases and inactivating phosphorylation of newly synthesized pRb may occur throughout the cycle, pRb phosphorylation in late G1 phase and its dephosphorylation in late M phase are the two critical events regulating pRb growth-restraining activities. In vitro, cyclin E- and cyclin D-activated kinases phosphorylate pRb differentially producing an overlapping subset of sites. This has led to the development of three different models of how different phosphorylation events may contribute to pRb control in cells. The rst possibility is that both CDK complexes have identical effects, inactivating pRb by phosphorylating in different positions. Alternatively, phosphorylation by different complexes could have different effects by regulating the binding to a different subset of effectors. Finally, combined phosphorylation of both kinases may be necessary to

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 351

completely inactivate pRb. It is difcult to determine which model is correct. Cyclin D1 and pRb functions seem to be interdependent. When cyclin D1 is ectopically expressed in G1 phase, pRb is phosphorylated earlier than in the normal cycle and G1 progression is accelerated. Antibodies to cyclin D1 arrest most cell types before S phase; however, they do not arrest if they lack functional pRb. The effect of the INK4 family of CKIs also seems to be dependent on the presence of functional pRb since the absence of pRb makes the cells insensitive to INK4induced G1 arrest. Unlike cyclin D, cyclin E is both rate-limiting and required for the G1/S transition in both pRb-positive and -negative cells. It has been shown that combined expression of c-myc and oncogenic ras allows entry of growth factor-deprived cells into S phase without pRb phosphorylation. Activation of cyclin E, but not cyclin D, was seen in these cells. A possible target of this pRb-independent pathway is the other members of the pRb family of pocket proteins, p107 and p130, that form stable complexes with cyclin E/CDK2 and cyclin A/CDK2 and whose expression is dramatically regulated at the end of G1 or S phase, respectively. Sequential phosphorylation of particular sites may result in the orchestrated disruption of pocket protein complexes containing different transcription factors, leading to the sequential succession of events required to promote progression through the cell cycle. However, CDK4 is dispensable in most cell types, probably due to a compensatory role by CDK6. Both CDK4 and CDK6 null mice develop normally with minor problems, such as diabetes or some impaired hematopoiesis, respectively. Double CDK4/CDK6 null mice die in early stages of embryogenesis due to severe anemia, but they show normal organogenesis. Double null MEFS proliferate normally and become immortal upon serial passage. These results indicate that in certain circumstances, D-type CDKs are not essential for cell-cycle entry. However, it still has to be conrmed whether a true redundancy for the CDK4/6 activity exists or whether it is an effect of molecular plasticity in early embryogenesis that will never take place in normal developing embryos. One of the consequences (in addition to chromatin remodeling) of pRb phosphorylation is the release of sequestered E2F proteins. The E2F proteins heterodimerize with DP1, forming the active transcription factor. The activity of E2F/DP1 induces the transcription of a number of genes involved in DNA synthesis and cell proliferation. High levels of INK4 proteins lead to inhibition of CDK4/6 kinases and keep pRb unphosphorylated and bound to the E2F transcription factor. The sequestered factor is thus inactivated, inducing a block in G1. This scheme is

much more complex in its details. First, there are six members of the E2F transcription factor family (ve activators and a negative regulator, E2F6), and only three of them (E2F1, -2, and -3) are under direct control of pRb. Hypophosphorylated pRb binds the other two members only weakly. E2F4 and E2F5 appear to bind preferentially to p107 and p130, which have a weak afnity for the other three. This suggests the possible existence of two parallel pathways: pRb/E2F1, -2, or -3 and p107/p130/E2F4 or -5. Also, pRb may regulate a number of downstream effectors besides E2Fs, including Elf-1, Myo-D, PU.1, ATF-2, and c-Abl proteins; however, the effector functions of most of these are not well-known.
S Phase

Once external signals have been integrated at the R checkpoint and the cells are committed to duplication, the cells initiate the S phase, during which other CDKs take control of DNA replication. CDK2 is considered to be most directly involved in DNA replication. Cyclins E and A sequentially activate CDK2, and both are thought to be essential for initiating DNA replication. Ectopic overexpression of cyclin E in mammalian broblasts causes premature entry into S phase, and microinjection of antibodies against cyclin E prevents S phase initiation. Cyclin A activates CDK2 soon after cyclin E does, concomitant with DNA synthesis. Ablation of cyclin A synthesis blocks entry into S phase. Cyclin A co-localizes with sites of DNA replication in S phase nuclei, suggesting that it may be directly involved in the assembly, activation, or regulation of replication structures. In vitro studies using SV40 as a model system have demonstrated that CDKs are necessary for the assembly of replication origin initiation complexes containing unwound DNA. Multiple experiments demonstrate the essential role of CDK2 in the cell cycle. Overexpression of p27 or dominant-negative CDK2 mutants inhibit proliferation. Furthermore, injection of antibodies against CDK2 subunit or treatment of the cells with CDK2 inhibitors also block proliferation. Moreover, a central role for CDK2/cyclin E has been indicated from observations of mice engineered to express cyclin E in place of cyclin D1. Animals lacking cyclin D1 have proliferative defects and fail to activate CDK2 in a subset of tissues. These phenotypes were suppressed in a knock-in animal that expresses cyclin E from the cyclin D1 promoter, suggesting that loss of CDK/cyclin D phosphorylation of pRb has no effect if cyclin E is no longer dependent on pRb inactivation.

352 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

However, recent ndings challenge this view. The inhibition of CDK2 by several ways has no effect on proliferation in some cell lines. High levels of CDK4 may compensate for the absence of CDK2. Moreover, both CDK4 and CDK2 activities may be unnecessary for proliferation in pRb-negative cells. Similar conclusions were derived using CDK2 null mice, which develop normally except for some defects in meiosis.
DNA Damage Checkpoint

The cells will arrest at the G1/S checkpoint in response to DNA damage to prevent the replication of mutated DNA. p53 tumor suppressor plays a critical role during this damage-induced checkpoint because p53-decient cells fail to undergo G1/S arrest after genotoxic stress. After exposure to genotoxic insults, p53 is activated and transcriptionally regulates many targets (e.g., p21 waf1, GADD45, reprimo, and 143-3 d) that directly provoke cell-cycle arrest.
G2/M Checkpoint and Early Mitosis

The orchestration of mitotic events requires exquisite regulation, and the penalty for errors in chromosome segregation is severe. Chromosomal imbalances are responsible for many cases of abortions, birth defects, and cancer. Once S phase is complete, the cell duplicates cell structures and separates the chromosomes. For this process, the cell must establish another checkpoint at the onset of G2, before the initiation of mitosis. The key component is a mitotic regulator composed of CDK1 and cyclin B. Together, these proteins form the mitosis-promoting factor (MPF), the activity of which initiates mitosis. MPF is regulated by periodic accumulation and destruction of cyclins, although additional controls regulate the complex. The mitotic phase can be dened by three transitions that involve cyclin B. First, cyclin B activates MPF and initiates prophase. CDK1 bound to cyclin B must also be phosphorylated by CAK in threonine 161 and dephosphorylated in tyrosine 15 and threonine 14. This activation will induce the specic phosphorylation of mitotic targets triggering downstream events. Nuclear lamins, kinesin-related motors and other microtubule-binding proteins, condensins, and Golgi matrix components are substrates of CDK1/cyclin B important for nuclear envelope breakdown (NEBD), chromosome separation, spindle assembly, chromosome condensation, and Golgi fragmentation. Second, MPF activates a ubiquitinproteolytic system that degrades cyclin B and initiates anaphase. Finally, the cyclin B destruction machinery is turned off and the cycle is reset.

Centrosomes are the major microtubule-organizing center of animal cells. In most cell types, duplicated centrosomes remain closely paired and continue functioning as single microtubule organizing centers during G2. After G2, however, they separate and migrate apart. The separation of centrosomes seems to be regulated by several kinases. Nek2, a member of the NIMA family, is thought to phosphorylate the centrosomal protein c-Nap1 causing the dissolution of a dynamic structure that tethers duplicate centrosomes to each other. An abrupt increase in c-Nap1 phosphorylation at the G2/M transition is caused by the cell-cycle-regulated inhibition of a type 1 phosphatase. At a later step, several kinesinrelated motor proteins and cytoplasmic dynein are required for centrosome separation. In organisms undergoing open mitosis, NEBD occurs soon after centrosome separation. During interphase, the nuclear envelope is stabilized by the nuclear lamina, but at the onset of mitosis, this structure disassembles as a consequence of lamin hyperphosphorylation. The predominant kinase triggering mitotic lamina depolymerization in vivo is CDK1/cyclin B. PLKs are key regulators of mitosis and cytokinesis because they are subject to complex temporal and spatial control. PLK1 seems to be involved in mitotic entry, centrosome maturation, spindle-assembly, and APC regulation. PLK1 is involved in the recruitment of a-tubulin during centrosome maturation, a process by which centrosomes increase their microtubule nucleation activity required for spindle formation. The association of PLKs with the centrosome also has implications for the regulation of mitotic entry. The initial activation of CDK1/cyclin B occurs at the centrosome, probably through phosphorylation of both CDC25 and Myt1. In mitotic cells, PLK1 also associates with the kinetochores. These structures play an important role in the spindle checkpoint. This checkpoint operates to ensure that all chromosomes have undergone bipolar attachment to the spindle before sister chromatids are separated. Another family of kinases that plays a critical role in chromosomal segregation and cell division is the Aurora family. Aurora kinases are implicated in the centrosome cycle, spindle assembly, chromosome condensation, chromosomemicrotubule attachment, the spindle checkpoint, and cytokinesis. Aurora kinases are regulated through phosphorylation, the binding to specic partners, and proteolysis. In mammals, there are three Aurora members that differ in expression patterns, localization, and timing of activity. Aurora A is upregulated at the onset of mitosis. It localizes at centrosomes during interphase and at both spindle poles and microtubules during early mitosis. Aurora B

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 353

reaches maximal levels later in mitosis, rst associated with the centromeres/kinetochores, then relocalizes to the midzone of the central spindle and nally concentrates at the midbody between dividing cells. Aurora A is implicated primarily in centrosome maturation and spindle assembly, whereas Aurora B is thought to regulate chromosome condensation and cohesion, kinetochore assembly, the spindle checkpoint, and the coordination between chromosome segregation and cytokinesis. Aurora B constitutes a key link between structural aspects of the microtubulekinetochore interactions and checkpoint signaling. The third member of the family, Aurora C, has a more restricted expression pattern and its functions are mostly unknown.
Spindle Checkpoint

Proper distribution of chromosomes between daughter cells is controlled by the spindle-assembly checkpoint that separates metaphase from anaphase, two clearly dened stages within mitosis. At this stage (metaphase), sister chromatids (pairs of identical chromosomes to be segregated between the daughter cells) are aligned at the midzone of the mitotic spindle. The spindle checkpoint, a safeguard mechanism that prevents defects in chromosome segregation, inhibits anaphase onset until the mitotic spindle is correctly assembled.
Metaphaseanaphase transition

At the start of anaphase, cohesins, the molecules responsible for sister chromatid cohesion, are degraded by a protease known as separase. In turn, separase is activated by degradation of a specic inhibitor, securin, by APC, a ubiquitinligase multiprotein complex (Figure 3). APC is the ultimate gatekeeper of the spindle-assembly checkpoint, ensuring that sister chromatids do not segregate unless an identical set of chromosomes are properly aligned at the spindle midzone. APC activity is regulated primarily by Mad2, a sensor of microtubule attachment to the kinetochores that sequesters Cdc20, a molecule required for APC activation. Mad2 binds to Bub3 and BubR1. The Mad/Bub complex is recruited to unattached kinetochores and inhibits the activation of the APC/Cdc20 complex, which is necessary for sister chromatid separation. This results in metaphase arrest until all kinetochores are correctly aligned. Completion of kinetochore attachment leads to the dissociation of Mad2 from Cdc20 (Figure 3). The spindle checkpoint requires mitotic CDK activity. Inhibiting CDK activity overrides checkpointdependent arrest. Following inhibition, the interaction between APC and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. CDK inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro

Improper spindle Aur Plk

Normal spindle

Cdc20 APC Separase

Bub

Cdc20 Mad2

APC

Cohesin cleaved Sister chromatid separation

Securin

UbUbUbUb

Cohesin intact

Metaphase arrest

Figure 3 Spindle checkpoint.

354 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

CDK1-phosphorylated Cdc20 interacts with Mad2 rather than APC. Thus, CDK1 activity seems to be required to restrain APC/Cdc20 activation until completion of spindle assembly. In addition, evidence indicates that CDK1 activity is required for the efcient function of the spindle checkpoint protein Bub1. Phosphorylation of key elements may play a role in the timing of the events that lead to mitosis exit and in regulating the spindle checkpoint function. Phosphorylation of APC subunits by PLKs appears to help APC activity. PLK-1 recruits spindle checkpoint proteins to centromeres and creates the tension-sensing phosphoepitope on mitotic kinetochores and APC. Aurora B, Survivin, and INCENP physically interact and form a complex that localizes to the kinetochores in prometaphase, to the cell equator during metaphase, and to the midbody during cytokinesis. The absence of function of these proteins has revealed their involvement in the spindle checkpoint. These proteins are also required to establish bipolar attachment of chromosomes, probably by destabilizing kinetochoremicrotubule attachments that lack tension. Aurora communicates with the other downstream components probably through direct phosphorylation of BubR1 and Mps1.

Cell Cycle and Cell-Cycle Checkpoints in Respiratory Diseases

Hanahan and Weinberg suggested that all cancer genotypes are the manifestation of six essential physiological alterations shown by most, and perhaps all, types of human tumors. Although breaching of these six physiological barriers seems to be necessary for most tumors to achieve full malignancy, cancer is increasingly viewed as a cell-cycle disease. This view reects evidence that the vast majority of tumors, and likely all tumors, have suffered one or more defects that derail the cell-cycle machinery (Table 2). Such defects can target either components of the cell cycle, including checkpoint mechanisms, or elements of the upstream signaling cascades, whose events eventually converge to trigger cell-cycle events. Lung cancer remains a worldwide major health challenge. Despite improvements in treatment, the 5year survival rate for individuals with lung cancer is only approximately 15%. Histologically, 80% of lung cancers are non-small cell lung cancer (NSCLC), whereas the remaining 20% of cases are small cell lung cancer (SCLC). On the basis of cell morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC.

The pRb protein is inactivated in more than 90% of SCLCs as a result of different mechanisms, including point mutations and abnormal mRNA expression. Changes in the other Rb family members (p107 and p130) have been detected in only a few cases. In contrast to SCLC, the majority of NSCLC cases exhibit abnormalities in the upstream regulators of the pRb pathway, including inactivation of p16ink4a, reduced levels of p27Kip, and enhanced expression of cyclin D1. p16ink4a abnormalities are frequently found in NSCLCs but are rare in SCLCs. Perhaps 3050% of early stage primary NSCLCs do not express p16ink4a (Table 2). The overexpression of cyclin A has been consistently shown to be a negative prognostic indicator of lung cancer. Also, cdc25A, one of the rate-limiting mechanisms for G1 progression into S phase, is frequently overexpressed in NSCLC. The gene cdk10 is essential for cellular proliferation, and its effect is also exerted in the G2/M transition. Only recently was this gene found to be overexpressed in lung adenocarcinomas. Also, cyclin B1 and CDK1, regulators of the G2/M transition, are overexpressed in lung cancer. Following DNA damage, a series of events is initiated that ends with p53 activation and the transcriptional regulation of many cell-cycle regulators. The fundamental importance of p53 in lung cancer is highlighted by the frequency of its mutations80% in SCLC and 50% in NSCLC. Once the p53 gene is deleted or mutated, cells become susceptible to DNA damage and deregulated cell growth.

Ceramide and the Cell Cycle

The sphingolipid ceramide is generated from the membrane-associated sphingomyelin by the activation of neutral sphingomyelinase in response to a variety of extracellular inducers. Ceramide acts as a second messenger to mediate many of the effects of these inducers. Ceramide is an important molecule that regulates diverse signaling pathways involving apoptosis, cell senescence, cell cycle, and differentiation. For the most part, ceramides effects are antagonistic to growth and survival. Interestingly, ceramide and the progrowth agonist, diacylglycerol (DAG), appear to be regulated simultaneously but in opposite directions in the sphingomyelin cycle. Whereas ceramide stimulates signal transduction pathways that are associated with cell death or at least are inhibitory to cell growth (e.g., stress-activated protein kinase or mitogen-activated protein kinase pathways), DAG activates the classical and novel isoforms of the protein kinase C (PKC) family. These PKC isoforms are associated with cell growth

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 355

and cell survival. Thus, ceramide and DAG generation may serve to monitor cellular homeostasis by inducing prodeath or progrowth pathways, respectively. Ceramide levels are elevated in response to diverse stress challenges, including chemotherapeutic drug treatment, irradiation, or treatment with prodeath ligands such as tumor necrosis factor-a. Consistent with this notion, ceramide is a potent apoptogenic agent. Ceramide activates stress-activated protein kinases such as c-jun N-terminal kinase and thus affects transcription pathways involving c-jun. Ceramide activates protein phosphatases such as protein phosphatase-1 and protein phosphatase-2. Ceramide activation of protein phosphatases has been shown to promote inactivation of a number of progrowth cellular regulators, including the kinases PKC-a and Akt, Bcl-2, and the retinoblastoma protein. Ceramide levels remain relatively constant throughout the life span of broblasts but increase with the onset of cellular senescence (a terminally arrested state that somatic cells acquire after a limited round of replication), increasing severalfold compared to the levels of young cells. Whereas in differentiation and apoptosis ceramide levels increase transiently, in senescence the increase in ceramide levels is permanent. The addition of a cell-permeable ceramide to young broblasts to concentrations usually seen in senescent cells mimics the senescent phenotype, with many of its markers. Higher concentrations of ceramide induce apoptosis. Cells treated with ceramide are unable to produce the AP-1 transcription factor and progressively inhibit pRb phosphorylation. It has been found that ceramide levels increase as cells exit mitosis to G1 phase. This increase precedes the dephosphorylation of pRb that occurs as the cells exit G2/M. In contrast, no changes in ceramide have been observed during other cell-cycle phase transitions, suggesting a role for the endogenous ceramide in the transition from G2/M to G1. In agreement with this, microinjection of ceramide or sphingosine was sufcient to reinitiate the cell cycle by reactivating G2/M transition in Xenopus oocytes. Ceramide also inhibits phospholipase D and PKC. Phospholipase D and PKC transduce mitogenic signals, and their progressive inactivation has been observed during cellular senescence, probably due to the increased in ceramide.

Acknowledgments
This work was supported by grant BIO-01-0069 from the Spanish Ministry of Science and Technology and grant PI020126 from the Spanish Ministry of Health.
See also: DNA: Repair; Structure and Function. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Further Reading
Adams PD (2001) Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochimia et Biophysica Acta 1471(3): M123M133. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333374. Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100: 5770. Harris SL and Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24: 28992908. Hartwell LH, Culotty J, Pringle JR, and Reid BJ (1974) Genetic control of the cell division cycle in yeast. V. Genetic analysis of mutants. Genetics 74: 267286. Hartwell LH, Culotty J, and Reid BJ (1970) Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proceedings of the National Academy of Sciences USA 66: 352359. Hartwell LH and Kastan MB (1994) Cell cycle control and cancer. Science 266: 18211828. Jeffrey PD, Russo AA, Polyak K, et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclin ACDK2 complex. Nature 376: 313320. Kelly TJ and Brown GW (2000) Regulation of chromosome replication. Annual Review of Biochemistry 69: 829880. Malumbres M and Carnero A (2003) Cell cycle deregulation: a common motif in cancer. Progress in Cell Cycle Research 5: 518. Meijer L, Jezequel A, and Ducommun B (2000) Progress in Cell Cycle Research, vol. 4. Norwell, MA: Kluwer. Murray AW (2004) Recycling the cell cycle: cyclins revisited. Cell 116: 221234. Murray AW and Hunt T (1993) The Cell Cycle: An Introduction. Oxford: Oxford University Press. Osada H and Takahasi T (2002) Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 21: 74217434. Peters JM (2002) The anaphase-promoting complex: proteolysis in mitosis and beyond. Molecular Cell 9: 931943. Sherr CJ (2004) Principles of tumor suppression. Cell 116(2): 235246. Sherr CJ and Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development 13: 15011512. Stein GS, Baserga R, Giordano A, and Denhardt DT (eds.) (1999) The Molecular Basis of Cell Cycle and Growth Control. New York: WileyLiss.

356 CHEMOKINES

CHEMOKINES
O Morteau, Childrens Hospital, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Chemokines are an extended family of small size chemoattractant proteins that mediate leukocyte migration and positioning. They have been classied into the C, CC, CXC, and CX3C subfamilies based on the position of a cysteine residue in their primary structure. Chemokines interact with structurally related G-protein-coupled receptors located on the surface of leukocytes, and trigger a cascade of intracellular events involving several distinct signaling pathways. The biological functions of chemokines include chemoattraction, integrin activation, leukocyte degranulation and mediator release, and angiogenesis, or angiostasis. They are also involved in various ways in the establishment and maintenance of the innate and adaptive immune systems. The importance of chemokines and their receptors in the pathogenesis of respiratory diseases is exemplied in conditions as diverse as airway allergy (asthma), bacterial and viral infections (tuberculosis, respiratory syncytial virus infection), allograft complications (acute lung allograft rejection, graft-versus-host disease), lung cancer, and other pathologies including chronic obstructive pulmonary disease, idiopathic pulmonary brosis, and acute respiratory distress syndrome.

1996, some chemokine receptors were found to act as co-receptors for human immunodeciency virus (HIV)-1, and in 1998, viral chemokines were identied and characterized. The role of chemokines and their receptors (including CCR4, CCR9, and CCR10) in tissue-specic homing was conrmed in 1999. The fast-expanding chemokine family was designated by various successive terms, including the platelet factor-4 family, the small inducible cytokine family, and the intercrines. The standard term chemokines (a neologism for chemotactic cytokines) was eventually coined in 1992 at the Third International Symposium on Chemotactic Cytokines in Baden. In 1995, the International Union of Pharmacology Committee on Receptor Nomenclature and Classication (NC-IUPHAR) created a subcommittee on chemokine receptor nomenclature. In 1999, a chemokine nomenclature system that parallels the chemokine receptor nomenclature was proposed at the Keystone Symposium on Chemokines and Chemokine Receptors.

Introduction
Chemokines are an extended family of small size chemoattractant cytokines that facilitate leukocyte migration and positioning, and exert their effects via binding to receptors located at the target cell surface. To date, 43 chemokines have been identied, binding to 19 different receptors. The rst chemokine was discovered by Walz and co-workers in 1977. It was a procoagulant and angiostatic factor called platelet factor 4, now renamed CXCL4. From 1984 to 1989, several investigators cloned the cDNAs for structurally related proteins, including IP-10 (IP10 10 kDa IFN (interferon)-ginducible protein), JE, Mig, RANTES (RANTES regulated on activation normal T cell expressed and secreted), I-309, KC, and MIP-1 a (MIP macrophage inammatory protein). At the time a function had yet to be attributed to this growing gene family, and it is only with the characterization of the neutrophil chemoattractant interleukin 8 (IL-8), also called CXCL8, that chemokines were recognized as chemotactic molecules. Between 1989 and 1994, interest in the chemokine eld grew dramatically as MCP-1 (MCP monocyte chemoattractant protein), RANTES, and eotaxin were reported to target monocytes, T cells, and eosinophils, respectively. In

Chemokine Structure and Nomenclature

Most chemokines are secreted proteins of 67 to 127 amino acids (only CXCL16 and CX3CL1 are membrane-bound), and contain a conserved tetra-cysteine motif located at the NH2 terminus. Chemokine nomenclature is based on the relative position of the rst two of these four consensus cysteines (Table 1). The separation of the cysteines by a nonconserved amino acid denes the CXC chemokine subclass, while the presence of two adjacent cysteines denes the CC chemokine subclass. Two minor chemokine subclasses (C and CX3C) either lack two out of four canonical cysteines (XCL1 and XCL2 chemokines), or contain three intercalated nonconsensus amino acids (CX3CL1 chemokine). CXC chemokines can be further discriminated into two structural subgroups (ELR and ELR), based on the presence or absence of the tripeptide motif glutamic acidleucinearginine (ELR) N-terminal to the rst cysteine. One structure/function correlate is the specicity of ELR CXC chemokines for neutrophils. Another one is the ability of ELR CXC chemokines to promote the neoformation of blood vessels (angiogenesis), while ELR CXC chemokines exert the opposite effect (angiostasis), as we will see later. The CXC, CC, CX3C, and C chemokine subclasses are

CHEMOKINES 357
Table 1 Chemokines and their receptors Class CXC (a) Systemic name CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15 CXCL16 CCL1 CCL2 CCL3 CCL4 CCL5 CCL6 CCL7 CCL8 CCL9 CCL10 CCL11 CCL12 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28 XCL1 XCL2 CX3CL1 Common synonyms GROa, MGSA, KC (mouse), MIP-2 (mouse) GROb, MIP-2a GROg, MIP-2b Platelet factor 4 ENA-78 GCP-2 PBP, CTAP-III, b-TG, NAP-2 IL-8, MDNCF, GCP, LIF, NAP-1, LYNAP Mig, CGR-10 (mouse) IP-10, CGR2 (mouse) I-TAC, b-R1, IP9, H174 SDF-1a, SDF-1b, PBSF, TLSF, TPAR1 BCA-1, BLC BRAK, Bolekine Receptor CXCR2 CXCR2 CXCR2 Unknown CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5 Unknown Unknown CXCR6 CCR8 CCR2 CCR1, CCR5 CCR5 CCR1, CCR3, CCR5 Unknown CCR1, CCR2, CCR3 CCR3 Unknown Unknown CCR3 CCR2 CCR2, CCR3 CCR1 CCR1, CCR3 CCR1 CCR4 Unknown CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR10 XCR1 XCR2 CX3CR1

CC(b)

1-309, TCA-3 (mouse), SIS-f (mouse) MCP-1, MCAF, HC11, JE (mouse) MIP-1a, LD-78a, PAT464, GOS19, hSISa MIP-1b, ACT-2, HC21, MAD-5, LAG-1 RANTES, SIS-d C10 (mouse), MRP-1 (mouse) MCP-3, NC28, FIC, MARC (mouse) MCP-2, HC14 MRP-2 (mouse), MIP-1g (mouse) Eotaxin MCP-5 (mouse) MCP-4, ckb10, NCC-1 CC-1, HCC-1, NCC-2, CCCK-1, Ckb1 HCC-2, Lkn-1, MIP-5, CC-2, NCC-3 HCC-4, LEC, NCC-4, LMC, Mtn-1, LCC-1 TARC DC-CK-1, PARC, MIP-4, AMAC-1, ckb7 MIP-3b, ELC, exodus-3, ckb11 MIP-3a, LARC, exodus-1, ST38 (mouse) 6Ckine, SLC, exodus-2, TCA4, ckb9 MDC, STCP-1, dc/bck (mouse) MPIF-1, MIP-3, ckb8-1 Eotaxin-2, MPIF-2, ckb6 TECK, ckb15 Eotaxin-3, MIP-4a ESkine, CTACK, skinkine, ILC (mouse) MEC Lymphotactin a, SCM-1a Lymphotactin b, SCM-1b Fractalkine, neurotactin (mouse)

C (g)

CX3C (d)

sometimes referred to as a, b, g, and d chemokines, respectively. Aside from the ofcial nomenclature, chemokines can be divided on the basis of their biological functions into inammatory chemokines, homeostatic chemokines, and dual chemokines (Table 2). Inammatory/inducible chemokines are expressed in inamed tissues in response to proinammatory

cytokines or pathogens. They orchestrate innate and adaptive immune responses via recruitment of effector leukocytes in infection, inammation, tissue injury, and tumors. By contrast, homeostatic/ constitutive chemokines are involved in immune surveillance through regulation of lymphocyte and dendritic cell trafcking. They guide leukocytes during hematopoiesis in the bone marrow and thymus,

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Table 2 Inammatory, homeostatic, and dual chemokines Chemokines Inammatory CXCL6 CXCL8 CXCL1 CXCL2 CXCL3 CXCL5 CX3CL1 CCL2 CCL3 CCL5 CCL7 CCL8 CCL11 CCL13 CCL14 CCL15 CCL24 CCL26 CCL27 CCL28 XCL1 XCL2 Homeostatic CXCL12 CXCL13 CXCL14 CCL18 CCL19 CCL21 Dual function CXCL9 CXCL10 CXCL11 CXCL16 CCL1 CCL17 CCL20 CCL22 CCL25 Receptors CXCR1, CXCR2 CXCR1, CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CX3CR1 CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR3 CCR3 CCR2, CCR3 CCR1 CCR1, CCR3 CCR3 CCR3 CCR10 CCR10 XCR1 XCR1 Functions Innate immunity

Extravasation Innate and adaptive immunity

CXCR4 CXCR5 Unknown Unknown CCR7 CCR7

Hematopoiesis Homeostasis of leukocytes Development of antigen-presenting cells T celldendritic cell interaction (spleen, lymph node) T lymphopoiesis Spleen and lymph node T cell homing

CXCR3 CXCR3 CXCR3 CXCR6 CCR8 CCR4 CCR6 CCR4 CCR9

T lymphopoiesis Adaptive immunity (Th1 responses) T lymphopoiesis, extravasation T lymphopoiesis, adaptive immunity T lymphopoiesis Development of dendritic cells, adaptive immunity Adaptive immunity (cutaneous T cells) T lymphopoiesis, adaptive immunity, T cell and B cell trafcking in small intestine

Adapted from Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 7584.

during initiation of adaptative immune responses in the spleen, lymph nodes, and Peyers patches, and in immune surveillance of healthy peripheral tissues. Sharing both functions are the dual chemokines, which participate in immune defense functions and also target noneffector leukocytes, such as precursor and resting mature leukocytes, at sites of leukocyte development and immune surveillance. Genes encoding inammatory chemokines are found in two major clusters on human chromosomes 4 (CXC chemokines) and 17 (CC chemokines), while genes for homeostatic chemokines are located alone

or in small clusters on chromosomes 1, 2, 5, 7, 9, 10, and 16.

Chemokine Receptors
Human formyl-peptide receptor was the rst chemokine receptor cloned in 1990. Two human receptors for CXCL8 (IL-8) were subsequently cloned in 1991: CXC chemokine receptor 1 (CXCR1) and CXCR2. As of today, 19 receptors have been identied (Table 1). The protein sequences of chemokine receptors share 25% to 80% amino acid identity,

CHEMOKINES 359

which suggests a common ancestor. All chemokine receptors are G-protein-coupled receptors (GPCRs) with seven transmembrane domains. However, chemokine receptors share a few features that are less commonly found in other GPCRs. They include a length of 340 to 370 amino acids; an acidic N-terminal segment; the sequence DRYLAIVHA (or a variation of it) in the second intracellular loop; a short basic third intracellular loop; and a cysteine in each of the four extracellular domains. An additional characteristic is the presence of a tyrosine sulfation in the N-terminus, which is critical for CCR5 activity as an HIV co-receptor. Even though the three-dimensional structure of chemokine receptors is unknown, the presence of seven transmembrane domains was inferred from the structure of rhodopsine. However, the presence of extracellular and intracellular loops is more speculative. It is commonly accepted that chemokine receptors contain seven transmembrane domains separated by amino acid loops directed toward the outside (extracellular loops) and inside (intracellular loops) of the cell (Figure 1). While extracellular domains serve as binding sites for the chemokine ligands, intracellular loops interact with G proteins and scaffolding proteins to trigger a cascade of intracellular changes (see chemokinechemokine receptor interactions). The ligand-binding site of chemokine receptors is highly complex. It is composed of multiple noncontiguous domains and at least two distinct subtypes: one for docking and the other for triggering.

The classication systems of chemokines and chemokine receptors parallel each other. As illustrated in Table 1, human CC and CXC chemokine receptor names consist of the CCR and CXCR roots (where R stands for receptor) followed by numbers. Similarly, CC and CXC chemokine names consist of the CCL and CXCL roots (where L stands for ligand) followed by numbers. The lymphotactin and fractalkine receptors are named XCR1 and CX3CR1, respectively. A chemokine receptor has a distinct chemokine and leukocyte specicity, but may bind multiple chemokines. Reciprocally, a chemokine may bind multiple receptor subtypes. However, a number of chemokinechemokine receptor relationships are monogamous (SDF-1/CXCR4, TECK/CCR9, BCA1/CXCR5, MIP-3a/CCR6, lymphotactin/XCR1, fractalkine/CX3CR1) (where SDF represents stromal cell-derived factor, TECK represents thymus-expressed chemokine, BCA represents B-cell attracting chemokine). Importantly, distinct receptor subtypes specic for the same chemokine and same function can be coexpressed on the same cell. In addition, some nonchemokine ligands (including several HIV proteins) can bind chemokine receptors as well. For example, CCR3 and mainly CCR5 and CXCR4, serve as co-receptors for HIV infection of CD4 cells.

ChemokineChemokine Receptor Interaction

Chemokine Binding

Extracellular space NH2

Cell membrane

III II

IV

V VII

VI

Chemokine receptors are allosteric molecules, which means that their conformation can be modied by the interaction with another molecule (ligand), allowing further binding to a third molecule (G-protein). More specifically, chemokine binding to the extracellular domains of the chemokine receptor triggers a change in tertiary structure of the receptor. This modication allows the intracellular domains to bind and activate heterotrimeric G-proteins. In response, the activated G-proteins exchange guanosine diphosphate for guanosine triphosphate (GTP), and dissociate into a- and bg-subunits. Chemokine receptors can couple to several Ga isotypes. For example, chemotaxis, the main function of most chemokines, requires coupling of the receptor to Gai, a Ga isotype sensitive to pertussis toxin. This was demonstrated by the complete inhibition of cell migration following cell treatment with pertussis toxin.
Intracellular Signaling

Cytoplasm

COOH

Figure 1 Schematic structure of a chemokine receptor. The seven transmembrane domains and the extracellular and intracellular loops are represented.

The bg-subunits that dissociate from Gai mediate chemokine-induced signals by activating the

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phosphoinositide-3 kinases (PI3K), which produce phosphoinositol-3, 4, 5-triphosphate (PIP3). Chemokine stimulation triggers a change in leukocyte shape associated with the formation of an elongated edge, or pseudopod. PIP3K and its product PIP3 translocate to the pseudopod, where they colocalize with GTPase Rac. PIP3 not only activates Rac via specic guanine nucleotide exchange factors (GEFs), but also serves as a docking site for protein kinase B, which translocates to the pseudopod and can induce actin polymerization. Rac, a protein required for leukocyte migration, activates downstream effectors (p21-activated kinase, and WAVE), which stimulate actin-related protein (Arp) 2/3. The actin polymerization induced by Arp allows further pseudopod extension. In moving leukocytes, formyl peptide activates a different set of mediators. Parallel to the sequence of events triggering Rac activation, another pathway leads from the pertussis toxin-sensitive Ga12/13 to the activation of Rho by Rho GEFs at the uropod (extended pole of the leukocyte opposite to the pseudopod). Activated Rho induces focal activation of myosin II and contraction of actinmyosin complexes via its effector p160ROCK, a serine/threonine kinase. This process triggers the retraction of the pseudopod. The Rac and Rho pathways, which occur at opposite edges of the leukocyte, inhibit each other. This mutual inhibition is critical to induce and maintain functional and morphological cell polarity, and drive locomotion. At least one additional signaling pathway remains to be characterized. It is the one leading to the rapid integrin activation involved in leukocyteendothelial cell adhesion.
Chemokine Receptor Silencing

the binding of sulfated sugars of the glycosamine family to a few chemokines, such as CCL5 and CXCL4.

Biological Functions of Chemokines

At least four major biological functions can be attributed to chemokines: leukocyte chemoattraction, integrin activation, leukocyte degranulation, and angiogenesis or angiostasis.
Chemoattraction

Chemokines attract leukocytes that bear the appropriate chemokine receptors via formation of a chemokine gradient. This gradient acts as a road map for the migrating cells, guiding them toward their nal destination. As described earlier (see chemokine chemokine receptor interactions), the signaling of cell movement by chemokine receptors is a complex phenomenon and is not yet fully elucidated.
Integrin Activation

Leukocyte migration involves passage from the tissues to the blood and lymphatic vessels and from the vessels to the tissues (extravasation). Cells undergo a multistep process to bind the vessel endothelium. They tether to vessel walls via activation of selectins, then bind the endothelium via activation of integrins, which are able to regulate their afnity through rapid conformational change. This conformational change in the integrin structure leads to cell arrest, a mechanism that allows cells to subsequently cross the endothelium. Chemokines can activate integrins, and are involved in the multistep process for neutrophils, T cells, eosinophils, and monocytes.
Leukocyte Degranulation and Mediator Release

Chemokine receptor signaling is a transient process, due to the presence of GTPase activity in the Ga subunits. GTP hydrolyzation catalyzed by GTPase induces the reunion of Ga with Gbg, allowing the return to the initial conformation of inactive heterotrimers. Molecules called regulators of G-protein signaling can, depending on the Ga isotype, either stimulate or inhibit the GTPase activity of the Ga subunits. Chemokine receptor silencing can also be mediated by desensitization triggered by receptor phosphorylation by G-protein-coupled receptor kinases, and by downregulation induced by receptor sequestration and internalization.
Chemokine Receptor-Independent Pathways

Chemokines can stimulate leukocyte degranulation or release of inammatory mediators, which can be features of allergic reactions. For example, CCL2 (MCP-1) stimulates histamine release by basophils and mast cells, and CXCL8 (IL-8) stimulates neutrophil granule exocytosis. In addition, chemokines stimulate the macrophage respiratory burst leading to the release of reactive oxygen intermediates.
Angiogenesis or Angiostasis

Alternative GPCR-independent (and chemokine receptor-independent) signaling pathways result from

The formation of new blood vessels (angiogenesis) is a physiological feature associated with embryonic development, wound healing, chronic inammation, and the growth of malignant tumors. Some chemokines (mostly CXC chemokines) promote angiogenesis, while others mediate its inhibition (angiostasis).

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As stated earlier (see section Chemokine structure and nomenclature), the angiogenic property of the CXC chemokines correlates with the presence of the tripeptide motif glutamic acidleucinearginine (ELR). Proangiogenic chemokines include the ELR CXC chemokines CXCL1, 2, 3, 5, 6, 7, 8, and 12, and CCL2 (MCP-1). Antiangiogenic chemokines include the ELR CXC chemokines CXCL 4, 9, 10, 11, and 13, and CCL21 (SLC)(SLC secondary lymphoid-tissue chemokine). As we will see later, the angiogenic and angiostatic properties of chemokines are of major relevance in lung cancer and inammation. The four biological functions reviewed above are used in combination for various biological responses. For example, tumor rejection involves leukocyte chemotaxis to the tumor and angiostasis; allergic reactions involve leukocyte migration and degranulation.

Chemokines in Respiratory Diseases

Asthma and Airway Allergy

Asthma is characterized by airway hyperresponsiveness (AHR) and T-helper-2 (Th2) cell, eosinophil, and mast cell inltration (see Asthma: Overview; Extrinsic/Intrinsic). A number of chemokines and receptors are associated with the development of asthma (Table 3). It is important to bear in mind that most studies on chemokines in asthma are based on the use of mouse models. Although these murine models are useful tools, their relevance to the human disease is not absolute, due partly to species differences. Moreover, mouse models are characterized by acute allergic reactions, whereas human asthma is a chronic disease. Finally, differences may arise between the various murine models, in terms of the cell populations and chemokines involved in the allergic response, and the relevance of a clearance mechanism. Eotaxins are a family of chemokines that attract eosinophils. Three forms (CCL11 or eotaxin, CCL24 or eotaxin-2, and CCL26 or eotaxin-3) were identied in humans, whereas two forms only were identied in mice. CCL11 (eotaxin) is a potent chemoattractant of eosinophils. It binds exclusively to the CCR3 receptor, which is expressed on the surface of eosinophils, mast cells, and basophils. Neutralization of CCL11 reduces both airways inammation and AHR, via reduction of eosinophil and Th2 trafcking. However, mice genetically decient in CCL11 were only partially protected in models of allergic airway inammation, which supports the idea of functional redundancy between chemokines. Although the CCL11 receptor CCR3 has been regarded as a potential therapeutic target, a model of

CCR3-decient (knockout) mouse gave new insights on the complexity of the mechanisms involved. CCR3 knockouts exhibited a dramatic reduction of eosinophil recruitment to the lung following allergen challenge. Unexpectedly though, AHR was increased in these mice, which was later attributed to an increased accumulation of mast cells in the trachea. An additional study showed a protection against allergen-induced AHR in CCR3-decient mice in a model that did not involve mast cell recruitment. CCL17 (TARC) (TARC thymus- and activationregulated chemokine) (see Chemokines, CC: TARC (CCL17)) CCL22 are chemokines that induce the selective migration of Th2 cells via binding to CCR4. Neutralization of either CCL17 or CCL22 leads to abrogation of lung eosinophilia and AHR in mice. Additional studies have conrmed that CCL22 and CCR4 contribute to the recruitment of Th2 cells to the lung during allergen-driven inammation in mice and humans. CCL1 is the ligand to the CCR8 chemokine receptor expressed on Th2 cells, and the CCL1/CCR8 tandem has been suspected to mediate the asthmatic reaction. However, studies using CCL1 and CCR8 knockout mice gave controversial results. It is suggested that CCL1/CCR8 might be involved in eosinophil recruitment in some models, but might not be critical to Th2 recruitment to the lung in vivo. CXCR4 expression on leukocytes is widespread, and this receptor is involved in B and T lymphopoiesis. In addition, CXCR4 mediates Th2-cell trafcking during allergic reactions in mice, as shown in studies where CXCR4 blockade with either an antibody or a receptor antagonist resulted in abrogation of both airway inammation and AHR. Increased levels of CCL2 (MCP-1), CCL7 (MCP3), and CCL13 (MCP-4) were observed in bronchoalveolar lavage (BAL) cells, and bronchial biopsies of asthmatic patients, but the role of these chemokines in asthma is still controversial (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)). Allergic diseases caused by the fungus Aspergillus fumigatus are collectively referred to as allergic bronchopulmonary aspergillosis (ABPA) in humans, and allergic aspergillosis or fungal asthma in rodents. ABPA is characterized by severe lung eosinophilia, increased mucous secretion, enhanced serum IgE production, and chronic lung brosis. Specic chemokines are associated with ABPA. CCR2 and its major ligand CCL2 (MCP-1) (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)) are the key factors in the clearance of A. fumigatus spores in mice. Genetic deciency in either CCR2 or CCL12 leads to exacerbated allergic disease in mice. By contrast, the absence of the CXCL8 (IL-8) receptor

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Table 3 Chemokines and chemokine receptors in respiratory diseases Clinical condition Asthma Chemokines potentially involved CCL11 (eotaxin) CCL17 (TARC) CCL22 CCL1 CCL2 (MCP-1) CCL7 (MCP-3) CCL13 (MCP-4) CCL5 (RANTES) CXCL8 (IL-8) Chronic obstructive pulmonary disease CXCL8 (IL-8) CCL2 (MCP-1) CXCL10 (IP-10) CCL2 (MCP-1) CXCL8 (IL-8) CXCL1 (GROa) CXCL2 (GROb) CXCL5 (ENA-78) KC (mouse CXCL1) CCL5 (RANTES) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL2 (GROb) CCL5 (RANTES) CCL2 (MCP-1) CXCL9 (Mig) CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CXCL9 (Mig) CXCL10 (IP-10) CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CCL7 (MCP-3) CCL12 (MCP-5, mouse) CXCL5 (ENA-78) CXCL8 (IL-8) CXCL10 (IP-10) CCL5 (RANTES) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL8 (IL-8) CX3CL1 (fractalkine) CXCL5 (ENA-78) CXCL9 (Mig) CXCL10 (IP-10) CXCL11 (I-TAC) CXCL12 (SDF-1) Chemokine receptors potentially involved CCR3 CCR4 CCR4 CCR8 CXCR4 CCR2 CCR2 CCR2 CCR5 CXCR2 CXCR2 CCR2 CXCR3 CCR2 CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CCR1, CCR3, CCR5 CCR2 CCR5 CCR5 CXCR2 CCR1, CCR3, CCR5 CCR2 CXCR3 CCR2 CCR5 CCR5 CXCR3 CXCR3 CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR2 CXCR2 CXCR1, CXCR2 CXCR3 CCR1, CCR3, CCR5 CCR2 CCR1, CCR5 CCR5 CXCR1, CXCR2 CXCR1 CXCR2 CXCR3 CXCR3 CXCR3 CXCR4

Acute respiratory distress syndrome

Idiopathic pulmonary brosis

Acute lung allograft rejection

Graft-versus-host disease

Tuberculosis

Respiratory syncytial virus infection

Lung cancer

CHEMOKINES 363

CXCR2 or of CCR4 in mice enhances fungal clearance dramatically. CCL5 (RANTES) and its receptor CCR5 inhibit the innate response of alveolar macrophages against A. fumigatus.
Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) (see Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General; and Smoking Cessation) is characterized by neutrophil, macrophage, and CD8 T-cell inltration. CXCL8 is found in very high concentrations in the sputum of COPD patients, and these levels correlate with disease severity. In addition, CXCL8 neutralization by blocking antibodies reduces the chemotactic response of neutrophils to sputum from COPD patients. CXCL8 activates neutrophils via the CXCR1 and CXCR2 receptors. CXCR2 is also expressed on monocytes, which makes it a more attractive therapeutic target than CXCR1. Smallmolecule inhibitors of CXCR2 are currently being tested in clinical trials. The expressions of CCL2 (MCP-1) and its receptor CCR2 are increased in macrophages and epithelial cells of COPD patients. The CCL2/CCR2 couple might play a role in the recruitment of blood monocytes to the lung. Similarly, the expressions of some CXCR3 ligands, such as CXCL10 (IP-10), are elevated in CD8 T cells in peripheral airways of COPD patients.
Acute Respiratory Distress Syndrome

levels correlate with neutrophil concentration in BAL, but not with the severity of lung injury or subsequent clinical course. CCL2 is detectable in BAL of ARDS patients at the onset of ARDS, and persists in the lungs of patients with sustained ARDS. CCL2 concentrations are increased in ARDS patients with sepsis and shock, and this augmentation correlates with increased survival. No correlation was found between CCL2 levels and leukocyte numbers, which suggests that CCL2 may play another function than promoting monocyte recruitment, in ARDS. A number of animal models have been used to mimic ARDS, resulting in a short list of chemokines potentially involved in the disease. In a model of ischemiareperfusion injury, CXCL5 production correlated with the occurrence of neutrophil-dependent lung injury, and treatment with anti-CXCL5 antibodies were able to attenuate lung damage. In a model of bacterial pneumonia, depletion of CXCL2 was associated with a higher mortality, suggesting a protective role for that chemokine in ARDS. Similarly, lung-specic transgenic expression of KC, the rodent homolog of human CXCL1, was shown to enhance resistance to Klebsiella-induced pneumonia in mice. CCL5 (RANTES) mediates T cell and monocyte chemotaxis via the CCR1, CCR3, and CCR5 receptors (see Chemokines, CC: RANTES (CCL5) for review). In a murine model of pancreatitis-associated lung injury, treatment with the CCL5 (RANTES) receptor antagonist Met-RANTES protected against lung damage but provided little or no protection against pancreatitis.
Idiopathic Pulmonary Fibrosis

Acute (or adult) respiratory distress syndrome (ARDS) is characterized by the rapid onset of severe respiratory failure due to injury of the lung alveoli and the surrounding capillaries. ARDS can be triggered by clinical events as diverse as major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Several chemokines have been found in increased concentrations in the BAL uid from patients with established, or at risk for, ARDS. They include CCL2 (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)), and the CXCR2 ligands CXCL8 (which also bind CXCR1), CXCL1 (GROa) (GRO growth-related oncogene), CXCL2 (GROb) (see Chemometrics, CXC: CXCL1 (GRO1)CXCL3 (GRO3)) and CXCL5 (ENA-78) (ENA epithelial neutrophil-activating protein). Levels in CXCL8 were reportedly higher in patients with ARDS plus pneumonia, than in those with ARDS or pneumonia alone. In patients with established ARDS, CXCL8

The BAL levels of CCL2 (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)), CCL3, and CCL4 were increased in patients with idiopathic pulmonary brosis (IPF), although the expression of CCR5 (which binds CCL3 and CCL4) was markedly reduced in lymphocytes from IPF patients. This is in agreement with the hypothesis of a downregulation of the Th1 response in IPF. CXCL2 concentration was also elevated in the BAL of IPF patients.
Acute Lung Allograft Rejection

Acute lung allograft rejection is a major complication of lung transplantation and can lead to bronchiolitis obliterans syndrome (BOS), which is characterized by the progressive obliteration of the small airways. Several chemokines were detected in elevated concentrations in the BAL of patients with acute lung

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allograft rejection or BOS. They include CCL5 (RANTES), CCL2, CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), and CXCL8. In a rat model, in vivo neutralization of either CCL5 or CXCR9 attenuated acute lung allograft rejection via a decrease in mononuclear cell recruitment, and (in the case of CXCL9) a reduction in the expression of the CXCL9 receptor CXCR3. In a murine model, loss of CCL2/CCR2 signaling reduced the recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)).
Graft-versus-Host Disease

The production of the CCR2 ligand CCL2 (MCP1) in the lung correlated with host macrophage recruitment, and elimination of CCR2 but not of CCL2 expression decreased IPS severity.
Tuberculosis

Graft-versus-host disease (GVHD) is a complication of allogeneic bone marrow transplantation (allo-BMT), in which the immune cells of the donors bone marrow attack the recipients organs and tissues, particularly the skin, eyes, stomach, intestines, liver, and lung. In the lung, GVHD can be associated with idiopathic pneumonia syndrome (IPS), an inammatory disease characterized by diffuse interstitial pneumonitis and alveolitis leading to interstitial brosis. Host macrophages are recruited to the lung during the peritransplantation period, and thought to contribute to alloantigen presentation to donor T cells inltrating the lung, and to lung damage via cytokine production. Several studies using mouse models have focused on the role of chemokines in the recruitment of effector cells to the lung during GVHD and IPS. The chemokines expressed in the lung after allo-BMT include CCL2, CCL3, CCL4, CCL5, CCL11, and the CXCR3 ligands CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), CXCL10, and CXCL11. The roles of these chemokines differ, depending on when they are evaluated after allo-BMT. For example, CCL2, CXCL10, and CXCL11 are expressed prior to CCL3 and CCL4. Neutralization of CXCL9 and CXCL10 with antibodies reduced IPS severity and CD8 T-cell inltrates. Further inhibition occurred with the elimination of CXCR3 expression from the donor T cells, showing that CXCR3 is a key component of T-cell recruitment to the lung after allo-BMT. The CCL3/CCR5 couple also inuences the recruitment of donor T cells to the lung, and a differential role was established for CCR5, depending on the conditioning (lethal irradiation versus no irradiation) of the recipient. The elimination of CCL5 expression from donor T cells inhibited their recruitment to the lung at late time points after transplantation, probably via a decrease in CCR5 expression.

Mycobacterium tuberculosis is a strong inducer of chemokine expression. Human macrophages produce CCL2, CCL3, CCL4, and CCL5 in response to virulent strains of M. tuberculosis. Additional in vitro studies suggest that the chemokines interacting with CCR1, CCR2, and CCR5 may mediate leukocyte recruitment to the site of M. tuberculosis infection. However, direct evidence for a role of these chemokines in tuberculosis is still missing. Expression of CXCL8 and CCL2, but not CCL3, CCL4, and CCL5 was detected in a human alveolar epithelial cell line in response to virulent strains of M. tuberculosis. Granulocytes from the blood of tuberculosis patients produced CXCL8 and CXCL1 in response to a M. tuberculosis antigen. CXCL10 concentration was elevated in the bronchial epithelium of tuberculosis patients, and BAL levels of CCL2, CCL5, CCL7, CCL12, CXCL8, and CXCL10 were increased in tuberculosis patients (see Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12)). It was hypothesized that the increase in expression of the HIV co-receptors CCR5 and CXCL4 on M. tuberculosis-infected monocytes may result in increased productivity of HIV infection. Expression of CCL2, CCL3, CCL7, CCL12, CXCL5, and CXCL10 was observed in response to M. tuberculosis infection in mice. However, CCL2decient mice were able to clear M. tuberculosis infection, despite an initial increase in bacterial burdens in the lung and spleen. Yet, mice decient in the CCL2 receptor CCR2 were unable to control infection induced by high M. tuberculosis doses and succumbed. CCR5-decient mice controlled M. tuberculosis aerosol and intravenous infection and formed granulomas. It is possible that CCR1 compensates for the absence of CCR5 in those mice. Finally, expression of chemokines by macrophages in response to M. tuberculosis appears to be inuenced by the cytokine tumor necrosis factor (TNF)-a, which is a major product of M. tuberculosis infection in macrophages.
Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Some RSV-infected lung epithelial cells were shown to express increased amounts of CC and

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CXC chemokines, including CCL1 (I-309), CCL20 (Exodus-1), CCL17 (TARC), CCL5 (RANTES), CCL2 (MCP-1), CCL22 (MDC) (MDC macrophage-derived chemokine), CCL3 (MIP-1a), CCL4 (MIP-1b), CXCL1 (GROa), CXCL2 (GROb), CXCL3 (GROg), CXCL5 (ENA-78), CXCL8, CXCL11 (I-TAC) (I-TAC IFN-g-inducible T-cell a-chemoattractant), and CX3CL1 (fractalkine). The expression of CCL5, which mediates eosinophil and monocyte recruitment to the lung, is frequently enhanced during RSV infection in patients and murine models. Productions of CCL2, CCL3, and CCL4 are elevated in monocytes and eosinophils exposed to RSV. Infection of eosinophils with RSV leads to the release of CCL5, and CCL3, which is a chemoattractant for human blood eosinophils. CCL3 levels were found highly elevated in the respiratory tract of children with RSV-associated severe bronchiolitis. CXCL8 is a powerful chemoattractant for neutrophils, the main inammatory cell type to migrate to the lung during RSV infection. CXCL8 expression is markedly increased during RSV infection. A surface protein of RSV exhibits mimicry with the chemokine CX3CL1, and binds the CX3CR1 receptor, which induces leukocyte migration and facilitates RSV infection of cells.
Lung Cancer

lung metastases to other organs (see Chemokines, CXC: CXCL12 (SDF-1)).

See also: Asthma: Overview; Extrinsic/Intrinsic. Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1); CXCL9 (MIG); CXCL1 (GRO1)CXCL3 (GRO3). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General; Smoking Cessation.

Further Reading
Algood HMS, Chan J, and Flynn JAL (2003) Chemokines and tuberculosis. Cytokine & Growth Factor Reviews 14: 467477. Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108115. Hoffman SJ, Laham FR, and Polack FP (2004) Mechanisms of illness during respiratory syncytial virus infection: the lungs, the virus and the immune response. Microbes and Infection 6: 767 772. Humbles AA, Lu B, Friend DS, et al. (2002) The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inammation and hyperresponsiveness. Proceedings of the National Academy of Science, USA 99: 14791484. Lloyd CM and Rankin SM (2003) Chemokines in allergic airway disease. Current Opinion in Pharmacology 3: 443448. Mackay CR (2001) Chemokines: immunologys high impact factor. Nature Immunology 2: 95101. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 7584. Murphy PM (2002) International union of pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227229. Puneet P, Moochhala S, and Bathia M (2005) Chemokines in acute respiratory distress syndrome. American Journal of Physiology Lung Cellular and Molecular Physiology 288: L3L15. Rosenkilde ME and Schwartz TW (2004) The chemokine system a major regulator of angiogenesis in health and disease. APMIS 112: 481495. Rot A and von Andrian UH (2004) Chemokines in innate and adaptive host defense: basic chemokine grammar for immune cells. Annual Reviews in Immunology 22: 891928. Schuh JM, Blease K, Kunkel SL, and Hogaboam CM (2003) Chemokines and cytokines: axis and allies in asthma and allergy 14: 503510. Strieter RM, Belperio JA, Burdick MD, et al. (2004) CXC chemokines: angiogenesis, immunoangiostasis and metastases in lung cancer. Annals of the New York Academy of Science 1028: 351360. Thelen M (2001) Dancing to the tune of chemokines. Nature Immunology 2: 129134. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, and Serody JS (2005) Leukocyte migration and graft-versus-host disease. Blood 105: 41914199.

Cell transformation from preneoplastic to neoplastic, tumor growth, invasion, and metastases are events that depend on the formation of new blood vessels (angiogenesis). Angiogenesis is the product of an imbalance in the expression of angiogenic versus angiostatic factors. As noted earlier, ELR CXC chemokines promote angiogenesis, while ELR CXC chemokines mediate angiostasis. ELR CXC chemokines are important mediators of angiogenesis during tumorigenesis of non-small cell lung cancer (NSCLC), and their presence correlates with patient prognosis. In a murine model, CXCL5 showed a high degree of correlation with NSCLC-derived angiogenesis. By contrast, the ELR CXC chemokines CXCL4, CXCL9, CXCL10, and CXCL11 are angiostatic factors. The angiostatic action of CXCL9, CXCL10, and CXCL11 in NSCLC is mediated via CXCR3. In addition, CXCL12 plays important roles in lung cancer invasion and metastasis, and in the spread of

366 CHEMOKINES, CC / C10 (CCL6)

CHEMOKINES, CC
Contents

C10 (CCL6) MCP-1 (CCL2)MCP-5 (CCL12) RANTES (CCL5) TARC (CCL17) TECK (CCL25)

C10 (CCL6)
R M Strieter and M P Keane, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

IL-3, IL-4, IL-13, GM-CSF, SCF Pulmonary fibrosis +

Abstract
CCL6 is a CC chemokine that was cloned in the mouse in 1991. Although a human homolog has not been described to date, this novel chemokine shares signicant homology with the human CC chemokine, CCL15. In contrast to the other members of the CC chemokine family, CCL6 has four instead of three exons. CCL6 is produced by macrophages, broblasts, keratinocytes, vascular smooth muscle cells, and skeletal muscle. CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intracerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. CCL6 has an important role in the development of pulmonary brosis and it mediates some of the pro-brotic effects of interleukin-13 (IL-13). CCL6 also has an important role in the airway remodeling seen in animal models of asthma. Interestingly, CCL6 has a protective effect in animal models of peritoneal sepsis and it improves macrophage phagocytic functions. Furthermore, CCL6 has been shown to have a role in tumor growth and invasion.

CCL6

M
, T cells

IFNAirway remodeling Bronchial hyperreactivity

Figure 1 Regulation and function of CCL6 in respiratory disease.

Introduction
CCL6 is a CC chemokine that was cloned in the mouse in 1991. It was identied along with a series of other hematopoietic specic mRNAs in mouse bone marrow cultures stimulated with granulocytemacrophase colon-stimulating factor (GM-CSF). Although a human homolog has not been described to date, this novel chemokine shares signicant homology with the human CC chemokine, CCL15. Both of these chemokines are chemotactic for monocytes and T cells.

heparin-binding proteins. The chemokines display four highly conserved cysteine amino acid residues. The CC chemokine family has the rst two NH2-terminal cysteines in juxtaposition, and designated the CC cysteine motif. In contrast to the other members of the CC chemokine family, CCL6 has a novel second exon. With the other members of the CC family, the second exon contains the rst three of four highly conserved cysteine residues and the third exon contains the fourth. In contrast, exons 3 and 4 of CCL6 contain the four conserved cysteines distributed in a similar manner to exons 2 and 3 of the other CC chemokines. The novel second exon of CCL6 codes for a large number of charged amino acids. The CCL6 gene (Scya6) is closely lined to the CCL2 gene (Scya2) locus on mouse chromosome 11. The genes for all of the other murine CC chemokines are also located on chromosome 11, suggesting that they all evolved from the same ancestral gene. The primary translation product of CCL6 contains 116 amino acids including an amino terminal signal peptide. CCL6 has an amino terminal region of 28 amino acids, which is in contrast to the other CC chemokines that have an amino terminal region length of 910 amino acids.

Structure
Chemokines in their monomeric form range from 7 to 10 kDa and are characteristically basic

Regulation of Production and Activity

CCL6 is expressed in hematopoietic cells, broblasts, keratinocytes, vascular smooth muscle cells, and

CHEMOKINES, CC / C10 (CCL6) 367

skeletal muscle and is stimulated by exposure to interleukin-3 (IL-3), IL-4, IL-13, and GM-CSF (see Figure 1). Furthermore, C10 is differentially regulated by T-helper-1 (Th1) and T-helper-2 (Th2) cytokines. Bone-marrow-derived macrophages produce CCL6 in response to IL-4, IL-10, and IL-13 in a dose-dependent manner. In contrast, interferon gamma (IFN-g) inhibits IL-3- and GM-CSF-induced expression of CCL6. Stem cell factor has been shown to induce CCL6 from eosinophils.

in skeletal muscle and may contribute to the mononuclear cell inux in muscle tissue that is associated with muscular dystrophy. In addition to its chemotactic properties, CCL6 also promotes local tissue apoptosis, thereby facilitating tumor invasion in a murine cancer model. CCL6 also directly stimulates tumor growth and the leukemogenic phenotype in 32D cells.

Receptors
The receptor for CCL6 is not known. Interestingly, deletion of CCR1 leads to a similar effect on IL-13induced inammation as neutralization of CCL6, suggesting that CCL6 may signal through CCR1.

Biological Function
CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intra-cerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. In a model of chronic peritoneal inammation, there is delayed (24 h) but sustained elevation in CCL6 levels in peritoneal uid. These ndings demonstrate an important role for CCL6 in chronic inammation and specically macrophage recruitment. Interestingly, despite its proinammatory actions, CCL6 has been shown to improve survival in a murine model of septic peritonitis. Following cecal ligation and puncture, there is a signicant increase in levels of CCL6 over baseline, and neutralization of CCL6 leads to increased mortality. In contrast, administration of systemic CCL6 immediately after surgery leads to a signicant improvement in survival. This is associated with an increase in TNF-a and CCL2. CCL6 and IL-1 can enhance TNF-a secretion from peritoneal macrophages and IL-13 induces CCL6 from these peritoneal macrophages. CCL6 also enhances the bacterial phagocytic capability of peritoneal macrophages and leads to a signicant decrease in bacteremia. In similar murine models of septic peritonitis Stat6 / mice are protected from lethality, and this is associated with increased peritoneal lavage levels of CCL6, CCL22, IL-12, and TNF-a. This in turn leads to enhanced bacterial clearance. Further support for the important role in mononuclear cell recruitment is seen in a model of skin healing. CCL6 has been shown to be strongly expressed in full-thickness excisional skin wounds as early as day 1 and to be localized to macrophages of the granulation tissue and the keratinocytes at the wound edge, suggesting an important role in the strong macrophage inux that is seen in healing skin. Furthermore, CCL6 has been shown to be expressed

CCL6 in Respiratory Disease

CCL6 is elevated in the pathogenesis of bleomycininduced pulmonary brosis. Similarly both IL-4 and IL-13, which are potent inducers of CCL6, are elevated during the pathogenesis of bleomycin-induced pulmonary brosis. Neutralization of IL-13, but not IL-4, attenuates the development of brosis and levels of CCL6. This is consistent with the known important role of IL-13 in the development of pulmonary brosis. Furthermore, neutralization of CCL6 also attenuates bleomycin-induced pulmonary brosis and intrapulmonary macrophage numbers. This suggests that in addition to its direct effect on broblasts, IL-13 has a role in the development of pulmonary brosis via the induction of CCL6 and the subsequent recruitment of macrophages. Further support for the role of CCL6 in lung remodeling and repair is seen in studies using IL-13 transgenic mice. The treatment of IL-13 / mice with anti-CCL6 leads to decreased levels of mRNA for matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4). Furthermore, neutralization of CCL6 leads to a decrease in the ability of IL-13 to stimulate production of CCL2, CCL3, MMP-2, MMP-9, and cathepsins K, L, and S. Interestingly, deletion of CCR1 leads to a similar effect on IL-13-induced inammation as neutralization of CCL6 suggesting that CCL6 may signal through CCR1. In a murine model of fungal-induced asthma, there is a signicant increase in CCL6 in bronchoalveolar lavage (BAL), and this is signicantly higher than levels of CCL2, CCL3, or CCL11. In this model the major source of CCL6 is alveolar macrophages, broblasts, and vascular smooth muscle cells. Neutralization of CCL6 prior to intratracheal challenge with Aspergillus fumigates leads to decreased airway inammation and bronchial hyperresponsiveness although it had no effect on IL-10 or

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IgE levels. In a chronic model of fungal allergic airway disease, CCR1 / mice have signicantly lower levels of CCL6, CCL11, CCL22, and IL-4 and IL-13 at 30 days as compared to CCR1 / mice. This is associated with a decrease in subepithelial brosis and fewer numbers of goblets cells. Interestingly, there is no difference in airway hyperresponsiveness at 30 days, suggesting that the Th2-inducible chemokines are most important in the airway remodeling of asthma rather than the bronchial hyperreactivity.
See also: Asthma: Overview; Allergic Bronchopulmonary Aspergillosis. Chemokines. Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12). Extracellular Matrix: Basement Membranes; Degradation by Proteases. Interleukins: IL-6. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells. Matrix Metalloproteinases. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

Orlofsky A, Wu Y, and Prystowsky MB (2000) Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines. Cytokine 12: 220. Steinhauser ML, Hogaboam CM, Matsukawa A, et al. (2000) Chemokine C10 promotes disease resolution and survival in an experimental model of bacterial sepsis. Infection and Immunity 68: 6108. Wang W, Bacon KB, Oldham ER, and Schall TJ (1998) Molecular cloning and functional characterization of human MIP-1 delta, a new CC chemokine related to mouse CCF-18 and C10. Journal of Clinical Immunology 18: 214. Yi F, Jaffe R, and Prochownik EV (2003) The CCL6 chemokine is differentially regulated by c-Myc and L-Myc, and promotes tumorigenesis and metastasis. Cancer Research 63: 2923. Zhu Z, Ma B, Zheng T, et al. (2002) IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13induced inammation and remodeling. Journal of Immunology 168: 2953.

MCP-1 (CCL2)MCP-5 (CCL12)

A D Luster, W K Hart, and A M Tager, Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Further Reading
Asensio VC, Lassmann S, Pagenstecher A, et al. (1999) C10 is a novel chemokine expressed in experimental inammatory demyelinating disorders that promotes recruitment of macrophages to the central nervous system. American Journal of Pathology 154: 1181. Berger MS, Kozak CA, Gabriel A, and Prystowsky MB (1993) The gene for C10, a member of the beta-chemokine family, is located on mouse chromosome 11 and contains a novel second exon not found in other chemokines. DNA and Cell Biology 12: 839. Berger MS, Taub DD, Orlofsky A, et al. (1996) The chemokine C10: immunological and functional analysis of the sequence encoded by the novel second exon. Cytokine 8: 439. Hogaboam CM, Gallinat CS, Taub DD, et al. (1999) Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis. Journal of Immunology 162: 6071. Keane MP, Belperio JA, Henson PM, and Strieter RM (2005) Inammation, injury and repair. In: Nadel M, et al. (eds.) Textbook of Respiratory Medicine, 4th edn. Philadelphia: Elsevier/ Saunders. Keane MP, Belperio JA, and Strieter RM (2003) Chemokines in pulmonary brosis. In: Strieter RM, Kunkel SL, Standiford TJ, and Lenfant C (eds.) Chemokines in the Lung, Lung Biology in Health and Disease, vol. 172. New York: Decker. Keane MP, Belperio JA, and Strieter RM (2003) Cytokine biology and the pathogenesis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, 4th edn. Hamilton, ON: BC Decker. Ma B, Zhu Z, Homer RJ, et al. (2004) The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inammation and remodeling. Journal of Immunology 172: 1872. Orlofsky A, Berger MS, and Prystowsky MB (1991) Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes. Cell Regulation 2: 403. Orlofsky A, Lin EY, and Prystowsky MB (1994) Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. Journal of Immunology 152: 5084.

Abstract
The attraction of leukocytes into tissues is essential for inammation and host response to infection. This process is controlled by chemotactic cytokines known as chemokines. The monocyte chemoattractant proteins (MCP) are an important subfamily of chemokines that share structural as well as functional features. By activating various G-protein-coupled chemokine receptors, the MCPs induce the recruitment and activation of multiple subsets of leukocytes, including monocytes and macrophages, effector CD4 and CD8 T cells, B cells, dendritic cells, basophils, mast cells, natural killer cells, and eosinophils. Increased expression of MCP chemokines has been detected in a variety of respiratory diseases involving the recruitment of these leukocytes into the lung, including both inammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in several rodent models of respiratory diseases, in which disease severity can be modulated by inhibition of MCP signaling. The MCPs and their receptors therefore are attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases.

Introduction
The monocyte chemoattractant proteins (MCP) constitute an important subfamily of chemotactic cytokine (CC) chemokines that share structural features and have overlapping functions. By activating G-protein-coupled CC chemokine receptors present on the surface of different classes of leukocytes, the MCPs participate in inammatory responses by

CHEMOKINES, CC / MCP-1 (CCL2)MCP-5 (CCL12) 369

directing the recruitment and activation of these cells. Four human MCP (hMCP) proteins, hMCP-1 (CCL2), hMCP-2 (CCL8), hMCP-3 (CCL7), and hMCP-4 (CCL13), and four mouse MCP (mMCP) proteins, mMCP-1, mMCP-2, mMCP-3, and mMCP5 (CCL12), have been identied that share substantial amino acid identity. The cross-species assignments of orthologs among these genes is not entirely clear, but it is generally accepted that mMCP-1, mMCP-2, and mMCP-3 are orthologs of hMCP-1, hMCP-2, and hMCP-3, respectively. However, no mouse ortholog has been described for human MCP-4 to date, and mMCP-5 has no ortholog in the human genome, likely reecting divergence of the human and mouse MCP gene clusters after speciation.

The secondary and tertiary structure of MCP-1 has been solved by both nuclear magnetic resonance (NMR) and crystal analyses. As is characteristic of the chemokine family, the N-terminal cysteines are followed by a long loop that leads into three

hMCP-2

hMCP-1

hMCP-3

Structure
As is characteristic of chemokines, the MCPs are small basic proteins. All the MCPs are members of the CC, or b-chemokine family, whose primary structures contain four conserved cysteine residues, of which the rst two are adjacent to each other. Substantial homology exists between all members of the MCP family, with amino acid identity ranging from 74% between hMCP-1 and hMCP-3, to 38% between mMCP-1 (excluding a unique C-terminal extension) and mMCP-2 (Figure 1). Phylogenetic analysis of the protein sequences of the MCP family (Figure 2) suggests that the human and mouse MCP gene clusters diverge by gene duplication after speciation.

hMCP-4

mMCP-1

mMCP-3

mMCP-2

mMCP-5
Figure 2 Phylogenetic analysis of protein sequences of human and mouse MCPs. Analysis was performed using the neighborjoining method of ClustalW.

-23

+1

+25

Hu Hu Hu Hu Mu Mu Mu Mu Hu Hu Hu Hu Mu Mu Mu Mu

MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5 MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5

MK VSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLAS MK VSAALLCLLLMAATFSPQGLAQPDSVSIPITCCFNVINRKIPIQRLES MK ASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLES MK VSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKS MQ VPVMLLGLLFTVAGWSIHVLAQPDAVNAPLTCCYSFTSKMIPMSRLES MK IYAVLLCLLLIAVPVSPEKLTGPD--KAPVTCCFHVLKLKIPLRVLKS MR ISATLLCLLLIAAAFSIQVWAQPDGPNA-STCCY-VKKQKIPKRNLKS MK IS-TLLCLLLIATTISPQVLAGPDAVSTPVTCCYNVVKQKIHVRKLKS

+26 +75

YRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT YT RITNIQCPKEAVIFKTQRGKEVCADPKERWVRDSMKHLDQIFQNLKP YRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL YV-ITTSRCPQKAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTLKT Y K R I T S S R C P K E A V V F V T K L K R E V C A D P K K E W V Q T Y I K N L D R N Q M R S E P>> YE R I N N I Q C P M E A V V F Q T K Q G M S L C V D P T Q K W V S E Y M E I L D Q K S Q I L Q P YR R I T S S R C P W E A V I F K T K K G M E V C A E A H Q K W V E E A I A Y L D M K T P T P K P YR R I T S S Q C P R E A V I F R T I L D K E I C A D P K E K W V K N S I N H L D K T S Q T F I L>>

Figure 1 Alignment of protein sequences of human and mouse MCPs. For comparison to the other MCPs, the unique C-terminal extension of mMCP-1 is not included, and the 98 amino acid N-terminal sequence is presented. Amino acids conserved between different MCPs are boxed.

370 CHEMOKINES, CC / MCP-1 (CCL2)MCP-5 (CCL12)

antiparallel b-pleated sheets in what is referred to as a Greek key motif. The protein then terminates in an a-helix that overlies the b-pleated sheets. With regard to quaternary structure, all MCPs exist as dimers under conditions required for NMR and Xray crystallographic structural analyses. However, the physiologic quaternary structures of the MCPs, for example, whether they function physiologically as monomers, dimers, or higher-order multimers, remains to be determined.

members of the CC chemokine family, therefore only bind CC chemokine receptors (CCRs). All of the MCP proteins described to date, with the exception of mMCP-2, activate CCR2 to varying degrees. In addition, hMCP-2, hMCP-3, hMCP-4, and mMCP-3 activate CCR3, hMCP-2 and hMCP-3 activate CCR1, and hMCP-2 activates CCR5 (Figure 3). The CC chemokine receptor(s) activated by mMCP-2 have not yet been determined.

Biological Function Regulation of Production and Activity

MCPs are generally considered to be inammatory chemokines, whose inducible expression directs the migration of leukocytes participating in inammatory responses, as opposed to homeostatic chemokines, whose constitutive expression directs the positioning of leukocytes under basal conditions. During inammatory responses, the MCPs are highly induced in multiple cell types and tissues by a variety of stimuli, such as lipopolysaccharide, tumor necrosis factor alpha, interleukin (IL)-1b, interferon gamma (IFN-g), and platelet-derived growth factor (Table 1). Conversely, expression of MCP family members can be suppressed by glucocorticoids or immunusuppressive cytokines such as transforming growth factor beta and IL-10. In addition to transcriptional regulation of MCP expression, MCP activity can also be regulated by posttranslational modication. For example, glycosylation of hMCP-1 reduces its ability to direct the migration of monocytes in vitro. Posttranslational truncation of the rst four or ve N-terminal amino acids of hMCP-1, or the rst ve amino acids of hMCP-2, abrogates their chemoattractant activity. Additionally, the truncated form of MCP-2 acts as an inhibitor of other chemokines, including MCP-1, MCP-2, MCP-3, and RANTES (regulated upon activation normally T cell expressed and secreted). MCPs stimulate the directed migration, or chemotaxis, and activation of leukocytes that express receptors specic for these chemokines. The induction of MCP expression in tissue sites of inammation consequently directs the recruitment of activated leukocytes into these sites as a critical feature of host inammatory responses to infection, injury, or allergy. The classes of leukocytes recruited by the MCPs can be predicted by the chemokine receptors expressed by these cells (Figure 3). As noted, all of the MCP proteins except mMCP-2 are active on CCR2, which is expressed on monocytes and macrophages, antigen-experienced (CD45RO ) T cells, B cells, immature dendritic cells, basophils, mast cells, and natural killer (NK) cells. hMCP-2, hMCP-3, hMCP-4, and mMCP-3 are active on CCR3, which is expressed by eosinophils, basophils, and TH2polarized CD4 T cells. hMCP-2 and hMCP-3 are active on CCR1, which is expressed by monocytes, T cells, dendritic cells, and eosinophils. hMCP-2 is also active on CCR5, which is expressed by TH1-polarized CD4 T cells and antigen-experienced (CD45RO ) CD8 T cells, monocytes and macrophages, and dendritic cells. Mice transgenically overexpressing MCP-1 driven by various different tissue-specic promoters have been generated. Consistent with MCP-1 directing the migration of monocytes and macrophages, these transgenic mice generally have increased accumulation of these cells specically in those tissues in which there is increased MCP-1 expression. For example, mice overexpressing MCP-1 driven by the surfactant protein C promoter, in which MCP-1 is constitutively secreted into the airspaces, have increased numbers of monocytes, as well as lymphocytes, recovered in bronchoalveolar lavage (BAL) uid. In contrast, normal numbers of monocytes and lymphocytes are present in the lung parenchyma of these mice. Mice genetically decient for MCP-1 have also been generated. These mice are viable, develop normally, and have normal numbers of monocytes in their circulation and macrophages in their tissues. These mice have reduced monocyte recruitment in models of

Receptors
The MCPs stimulate cells by binding to specic surface chemokine receptors, which belong to the G-protein-coupled seven transmembrane domain receptor superfamily. Binding of MCPs to these receptors activates multiple signaling pathways that regulate the cellular machinery that propels cells during MCP-directed migration. Each of the major chemokine families has its own specic set of chemokine receptors. Although many chemokines bind to more than one chemokine receptor, the members of each chemokine family bind only to the chemokine receptors specic for that family. The MCPs, as

CHEMOKINES, CC / MCP-1 (CCL2)MCP-5 (CCL12) 371

Table 1 MCP production MCP MCP-1 Cell type/organ Fibroblasts Endothelial cells Vascular smooth muscle cells Monocytes/macrophages (including cell lines HL60, U937, THP1) Neutrophils Keratinocytes Synovial cells Type II pneumocyte cell line Mesangial cells Retinal pigmented epithelial cells Malignant cell lines (glioma, sarcoma, melanoma, hepatoma) Luteal cells Secondary lymphoid organs Lung (epithelium, alveolar macrophages) Brain (astrocytes) Spinal cord Seminal vesicles Kidney Inducer PDGF / IL-1 / TNF-a / viruses / dsRNA / LPS / cholera toxin IL-1 / TNF / IFN-g / IL-4 / MM-LDL / stretch PDGF / MM-LDL / stretch LPS / IFN-g / PMA TNF IFN-g IL-1 IL-1 / TNF IL-1 / TNF / IFN-g / basic FGF / LIF / IL-6 IL-1 / TNF / LPS

Asthma and granuolma models EAE models of multiple sclerosis / seizure Contusion injury Inammation (e.g., glomerulonephritis)/hypoxia/transplant rejection IL-1 / IFN-g / dsRNA / measles virus IL-1 / IFN-g EAE / multiple sclerosis

MCP-2

Fibroblasts Neutrophils Osteosarcoma cell line Astrocytes Organs: small intestine, peripheral blood, heart, placenta, lung, skeletal muscle, ovary, colon, spinal cord, pancreas, thymus Porcine luteal cells Fibroblasts Monocytes Platelets Bronchial epithelium Kidney Astrocytes Skin Endothelial cells Dermal broblasts Bronchoalveolar lavage cells Bronchial epithelial cell lines (A549, BEAS-2B) PBMC Nasal epithelium Arterial plaques (endothelial cells/macrophages) Organs: small intestine, thymus, colon, heart, placenta Macrophage cell line (RAW 264.7) Lung (alveolar macrophages/smooth muscle cells) Spinal cord Lymph node stromal cells Thymic stromal cells

MCP-3

PDGF TNF / IL-1 / IFN-g / LPS / lipoarabinomannan Asthma models glomerulonephritis EAE / multiple sclerosis Atopy IL-1 / TNF Asthma IL-1 / TNF / IFN-g PHA / IL-2 Sinusitis Atherosclerosis

MCP-4

MCP-5

IFN-g / LPS Asthma models Spinal cord contusion injury

dsRNA, double stranded RNA; EAE, experimental allergic encephalomyelitis; IFN-g, interferon gamma; IL, interleukin; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; MM-LDL, minimally modied-low density lipoprotein, PDGF, platelet-derived growth factor; PHA, phytohemagglutanin; PMA, phorbol myristate acetate; PBMC, peripheral blood mononuclear cells. Reproduced from Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 11451160. San Diego: Academic Press, with permission from Elsevier.

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MCPs activating CCR2: hMCP-1 mMCP-1 hMCP-2 mMCP-3 hMCP-3 mMCP-5 hMCP-4

Cells expressing CCR2: Monocytes/ TH1 CD4+ macrophages T cells TH2 CD4+ T cells CD8+ T cells B cells Dendritic cells Basophils/ mast cells NK cells

Biologic activity:

Cell recruitment and activation

(a)
MCPs activating CCR3: hMCP-2 hMCP-3 hMCP-4 mMCP-3 MCPs activating CCR1: hMCP-2 hMCP-3

Cells expressing CCR3: TH2 CD4+ T cells Basophils/ Eosinophils mast cells

Cells expressing CCR1: Monocytes/ macrophages Dendritic cells Eosinophils

Biologic activity:

Cell recruitment and activation

Biologic activity:

Cell recruitment and activation

(b)

(c)

MCPs activating CCR5:

hMCP-2

Cells expressing CCR5: Monocytes/ macrophages TH1 CD4+ T cells CD8+ T cells Dendritic cells

Biologic activity:

Cell recruitment and activation

(d)
Figure 3 Key biological functions of MCPs. By signaling through the chemokine receptors (a) CCR2, (b) CCR3, (c) CCR1, and (d) CCR5, the MCPs induce the recruitment and activation of multiple classes of leukocytes, including monocytes and macrophages, effector CD4 and CD8 T cells, B cells, dendritic cells, basophils and mast cells, NK cells, and eosinophils.

tissue inammation, however, including models of peritonitis and delayed hypersensitivity reactions, indicating that MCP-1 has a nonredundant role in directing monocytes into inamed tissues.

Role of MCPs in Respiratory Diseases

As would be expected from their biologic activity, increased expression of MCP chemokines has been

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detected in a variety of respiratory diseases involving the recruitment of leukocytes into the lung, including inammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in a variety of respiratory disease models in rodents, in which disease severity has been modulated by targeting MCP/CCR2 signaling.
Asthma

Other Pulmonary Infections

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the airways of asthmatic subjects, and gene polymorphisms of MCP-1 have been associated both with asthma susceptibility and severity. Further, treatment resulting in improvements in forced expiratory volume has been associated with reductions in airway expression of MCP-3 and MCP4. Increased airway expression of the MCPs, including MCP-1, MCP-3, and MCP-5, has also been noted in mouse models of allergic pulmonary inammation. Inhibition or deletion of the MCPs, or one of their receptors, CCR2, has produced conicting results in these models, however, and consequently the role of the MCPs in asthma remains to be denitively established.
Tuberculosis

Elevated expression of MCP-1 in the lung has been demonstrated in mouse models of infection with Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, inuenza A, Aspergillus fumigatus, Cryptococcus neoformans, and Pneumocystis carinii. MCP-1 has been demonstrated to participate in the recruitment of both macrophages and NK cells in a mouse model of pneumococcal pneumonia, and to direct the recruitment of macrophages responsible for the phagocytosis and removal of dying neutrophils in a mouse model of pseudomonas pneumonia. In so doing, MCP-1 expression is thought to attenuate the development of lung injury in the pseudomonas model. In a model of inuenza pneumonia, CCR2-decient mice are protected from the early pathological manifestations of infection due to reduced macrophage recruitment. In this model, however, the delay in macrophage accumulation in the CCR2-decient mice caused a subsequent delay in T cell recruitment, and an increase in pulmonary viral titers at early time points following infection. Finally, in a model of invasive aspergillus pneumonia performed in neutropenic mice, treatment of infected mice with neutralizing antiMCP-1 antibody reduced NK cell recruitment at early time points, and led to a greater than threefold increase in pathogen burden in lungs and twofold greater mortality.
Lung Allograft Rejection

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the lungs of patients with tuberculosis. A requirement for CCR2, and presumably its MCP ligands, for control of Mycobacterium tuberculosis infection has been dramatically demonstrated in mouse models. Mice genetically decient for CCR2 have a rapidly progressive and fatal course following high- or moderate-dose M. tuberculosis infection, developing lung mycobacterial burdens 100-fold greater than wild-type mice controls, which control infection and survive. CCR2-decient mice demonstrated multiple defects in leukocyte trafcking following infection, including defects in early macrophage recruitment to the lung and subsequent defects in macrophage and dendritic cell recruitment to the mediastinal lymph nodes. T cell activation in the mediastinal lymph nodes was consequently delayed, resulting in reduced accumulation of T cells primed to produce IFN-g in the lungs of infected CCR2-decient mice. Though this remains to be demonstrated in humans, the cellular responses mediated by the activation of CCR2 by its MCP ligands appear essential for immune control of M. tuberculosis infection.

MCP-1 levels are elevated in the lungs of transplant recipients experiencing acute rejection, as well as those experiencing chronic rejection, that is, bronchiolitis obliterans syndrome (BOS), compared with healthy transplant recipients. Elevated BAL levels of MCP-1 further have been shown to predict the development of BOS. Additionally, in allograft recipients shifted from cyclosporine- to tracrolimus-based immunosuppression due to refractory acute rejection, clinical and functional stabilization was accompanied by signicant and sustained reductions in lavage MCP-1 levels. Induction of MCP-1 expression has also been noted in mouse and rat models of allograft rejection and BOS. In a mouse model of tracheal transplantation, both the recruitment of mononuclear phagocytes and the severity of BOS were signicantly reduced by the interruption of MCP-1/CCR2 signaling.
Idiopathic Pulmonary Fibrosis

Elevated levels of MCP-1 have been noted in the lungs of idiopathic pulmonary brosis patients. Similarly, the expression of MCP-1 is increased in the

374 CHEMOKINES, CC / MCP-1 (CCL2)MCP-5 (CCL12)

lungs in rodent models of pulmonary brosis. In the mouse model of pulmonary brosis induced by bleomycin, inhibition of endogenous MCP-1 function by the transgenic expression of a dominant negative N-terminal deletion MCP-1 mutant reduced pulmonary brosis, although interestingly, without affecting macrophage or lymphocyte recruitment into the lung. A similar result has been reported in experiments using CCR2-decient mice, which were protected from brosis despite having leukocyte recruitment in the lung equivalent to that of wild-type controls. However, another group of investigators has recently reported that in their experiments performing the bleomycin model of pulmonary brosis in CCR2-decient mice, lung macrophage recruitment was reduced. MCP-1/CCR2 signaling thus may contribute to the development of pulmonary brosis through both leukocyte-dependent and nonleukocyte-dependent effects.
Acute Lung Injury/Acute Respiratory Distress Syndrome

inltration into the lung, as well as right ventricular systolic pressure, right ventricular hypertrophy, and medial hypertrophy of the pulmonary arterioles.
Other Respiratory Diseases

Elevated expression of the MCPs has also been noted in several other respiratory diseases, including chronic obstructive pulmonary disease, pulmonary alveolar proteinosis, sarcoidosis, hypersensitivity pneumonitis, eosinophilic pneumonia, and coalworkers pneumoconiosis.

Concluding Remarks
By virtue of their ability to direct the migration of multiple classes of leukocytes into the lungs, the MCPs are importantly involved in multiple respiratory diseases. In inammatory pathologies in which leukocyte recruitment participates in disease pathogenesis, such as asthma, allograft rejection, or ARDS, inhibition of MCP signaling is an attractive strategy for therapeutic intervention. In infectious pathologies in which leukocyte recruitment participates in host immune responses, such as tuberculosis or other pneumonias, augmentation of MCP signaling may be benecial. The MCPs and their receptors therefore represent attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Dendritic Cells. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells; Pulmonary Macrophages. Vascular Disease.

Elevated MCP-1 levels are present in BAL uid of acute respiratory distress syndrome patients, and have been correlated both with alveolar monocyte recruitment and lung injury score. Elevated MCP-1 expression has also been noted in several rodent models of acute lung injury, including a model of gastric aspiration-induced pneumonitis. In this model, MCP-1-decient mice had decreased survival compared with wild-type controls. Wild-type mice in this model demonstrated areas of compartmentalized inammation in the lung with prominent granuloma formation following acid aspiration, whereas MCP1-decient mice demonstrated severe diffuse pneumonitis without granulomas, suggesting that MCP-1 protects uninjured lung regions by promoting the isolation and compartmentalization of tissue with active inammation.
Pulmonary Hypertension

Further Reading
Belperio JA, Keane MP, Burdick MD, et al. (2001) Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. Journal of Clinical Investigation 108: 547556. Goodman RB, Strieter RM, Martin DP, et al. (1996) Inammatory cytokines in patients with persistence of the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 154: 602611. Gu L, Tseng S, Horner RM, et al. (2000) Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404: 407411. Gunn MD, Nelken NA, Liao X, and Williams LT (1997) Monocyte chemoattractant protein-1 is sufcient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inammatory activation. Journal of Immunology 158: 376383. Ikeda Y, Yonemitsu Y, Kataoka C, et al. (2002) Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats. American Journal of Physiology: Heart and Circulatory Physiology 283: H2021H2028.

Elevated levels of MCP-1 have been noted in the circulation of patients with both primary pulmonary hypertension (PPH) and chronic thromboembolic pulmonary hypertension (CTEPH). In patients with PPH, treatment with epoprostenol has been associated with signicant reductions in circulating MCP-1 levels. In patients with CTEPH, circulating MCP-1 levels were signicantly correlated with pulmonary vascular resistance. Elevated levels of MCP-1 in the circulation as well as in BAL uid have also been demonstrated in a rat model of pulmonary hypertension induced by monocrotaline. Inhibition of MCP-1 signaling in this model reduced mononuclear cell

CHEMOKINES, CC / RANTES (CCL5) 375

Katsushi H, Kazufumi N, Hideki F, et al. (2004) Epoprostenol therapy decreases elevated circulating levels of monocyte chemoattractant protein-1 in patients with primary pulmonary hypertension. Circulation Journal 68: 227231. Lu B, Rutledge BJ, Gu L, et al. (1998) Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-decient mice. Journal of Experimental Medicine 187: 601608. Miotto D, Christodoulopoulos P, Olivenstein R, et al. (2001) Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases. Journal of Allergy and Clinical Immunology 107: 664670. Moore BB, Paine R III, Christensen PJ, et al. (2001) Protection from pulmonary brosis in the absence of CCR2 signaling. Journal of Immunology 167: 43684377. Nomiyama H, Egami K, Tanase S, et al. (2003) Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters. Journal of Interferon Cytokine Research 23: 3745. Peters W, Scott HM, Chambers HF, et al. (2001) Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 98: 79587963. Reynaud-Gaubert M, Marin V, Thirion X, et al. (2002) Upregulation of chemokines in bronchoalveolar lavage uid as a predictive marker of post-transplant airway obliteration. Journal of Heart and Lung Transplantation 21: 712730. Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Openheim JJ and Feldman M (eds.) Cytokine Reference, pp. 11451160. San Diego: Academic Press. Rosseau S, Hammerl P, Maus U, et al. (2000) Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L25L35. Szalai C, Kozma GT, Nagy A, et al. (2001) Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. Journal of Allergy and Clinical Immunology 108: 375381. has the broadest range of target cells of known chemokines due to its multiple receptor usage and thus is implicated in the recruitment of memory T cells, monocytes, eosinophils, and basophils, thus making it one of the key players in respiratory disease.

Introduction
Chemokines are structurally classied into subfamilies based on their pattern of disulde bridging, which allows their division into four subclasses as dened by the distribution of the four highly conserved cysteines. Thus the a-chemokines have the rst Cys pair close to the N-terminus and separated by an amino acid (CXC chemokines) whereas the b-chemokines have the rst two Cys residues adjacent (CC chemokines). These two subclasses comprise the majority of chemokines, but there are also two minor subclasses, which each have only one or two members: CX3C chemokines have three amino acids separating the amino terminal Cys residues and C chemokines possess only one disulde bridge. Regulated upon activation normally T-cell expressed and secreted (RANTES)/CCL5 belongs to the b-chemokine or CC-chemokine subclass. RANTES was initially isolated from a T cell cDNA library although it was subsequently found to be induced in other cell types upon induction with proinammatory cytokines.

Structure
RANTES has the canonical monomeric fold of the chemokine family (Figure 1(c)) and the canonical dimeric form of the CC subfamily (Figure 1(b)) consisting of a exible N-terminal loop, followed by three antiparallel b-sheets, which are connected by short exible turns called the 30s and 50s loop and an a-helical C-terminus (Figure 1(a)). RANTES has an extremely high molecular weight, 4600 kDa, as determined by techniques such as size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). This high-molecular-weight quaternary structure is not only found in vitro, but the protein has been observed to be secreted from granules of T cells as a complex with soluble heparin, also greater than 600 kDa. Such a higher-order quaternary structure is not limited to RANTES but has also been described for MIP-1a and MIP-1b, both of which share receptor usage with RANTES. Since RANTES interacts with several receptors, mutagenesis has been used to identify the epitopes conferring receptor specicity. One common feature has emerged in that the N-terminus is critical for receptor activation. Both truncation by total protein synthesis, and extension by either retention of the initiating methionine of the recombinant protein in

RANTES (CCL5)
A E I Proudfoot, C A Power, and Z Johnson, Serono Pharmaceutical Research Institute, Geneva, Switzerland
& 2006 Elsevier Ltd. All rights reserved.

Abstract
RANTES (regulated upon activation normally T cell expressed and secreted) is a member of the subfamily of cytokines called chemokines, the name given to chemoattractant cytokines in 1994. The family consists of small basic proteins whose role is to provide the directional signal for leukocyte migration. The chemokine family are distinct from other cytokines in that they act on G-protein-coupled heptahelical transmembrane spanning receptors. Initially, chemokines were named according to their function but due to the confusion in the nomenclature which arose from the simultaneous identication by different laboratories of many chemokines in the mid to late 1990s, a systematic nomenclature was introduced in 2001. In the new nomenclature the chemokines were named according to their genomic localization, and RANTES was renamed CCL5. RANTES probably

376 CHEMOKINES, CC / RANTES (CCL5)

(a)

(b)

Arg47 Lys45

Arg44

(c)

(d)

Figure 1 Structure of RANTES. (a) Monomer; (b) dimer. (c) RANTES (red) is overlaid with six other chemokines (gray) from the CC (MCP-1 and MIP-1b), CXC (IL-8, NAP-2, and PF4) and CX3C (fractalkine) classes. (d) The BBXB motif forming the GAG binding site of RANTES on the 40s loop.

procaryotic expression systems, or by chemical coupling techniques, have shown that this signicantly affects receptor activation capacity. Truncation of the rst eight amino acids produced a protein that had acquired the ability to bind to CCR2, and extension of the N-terminal produced antagonists (see below). The one exception was the deletion of the rst two amino acids producing the variant 3-68-RANTES; this molecule had enhanced activity for CCR5 whilst inhibiting CCR1-mediated events. RANTES has a second essential interaction, which is low-afnity binding to glycosaminoglycans (GAGs), a common feature amongst the chemokine family. The binding site for GAG has been identied for RANTES as being a BBXB motif on the 40s loop (Figure 1(d)). Furthermore the variant in which the basic residues in this motif were replaced with Ala residues was shown to have lost its ability to recruit cells in vivo, thereby proving conclusively that GAG binding is essential for chemokine activity in vivo.

The RANTES gene spans 7.1 kb and comprises three exons of 133, 112, and 1075 bases respectively and two introns of approximately 1.4 and 4.4 kb. This exonintron organization is similar to that reported for other CC chemokine family members. The locus has been sequenced from nine mammalian species which code for proteins with striking homology, where the identity ranges from 71% to 100%, with similarities ranging from 79% to 100% (Figure 1).
Expression Pattern

Regulation of Production and Activity

Genomic Localization

RANTES expression was initially described as being restricted to antigen- or mitogen-activated T cells. However it turns out that RANTES is also expressed by a broad range of cell types after cellular activation by tumor necrosis factor alpha (TNF-a) and interleukin (IL)-1b including epithelial cells and broblasts. In addition RANTES is very strongly expressed by platelets. RANTES expression is a hallmark of inammatory diseases and RANTES mRNA or protein has been detected in pathological specimens (disease tissue or uids) from bronchoalveolar lavage (BAL) from asthmatics as well as many other inammatory diseases (Table 1).

The RANTES locus has been mapped to chromosome 17q11.2q12 using somatic cell hybrids and by in situ hybridization of the cDNA probe. Several other CC chemokine genes have also been mapped to this locus.

Receptors
RANTES is one of the most promiscuous chemokines in that it binds to several receptors (Figure 2). It

CHEMOKINES, CC / RANTES (CCL5) 377

binds to three CC receptors with high afnity (CCR1, CCR3, and CCR5 through which it induces cellular recruitment) and to CCR4 with low afnity. In addition, it binds to the promiscuous nonsignaling receptors DARC and D6, which are thought to act as chemokine sinks. RANTES has the highest afnity for CCR1 and CCR5, binding with low nanomolar afnity, whilst the afnity for CCR3 has been reported to range from 1 to 100 nM, and that for CCR4 is micromolar. CCR1 and CCR5 are expressed

Table 1 Disease association for RANTES receptors Receptor CCR1 Disease indication Multiple sclerosis Rheutmatoid arthritis Organ transplant Asthma, rhinitis Nephritis Asthma Allergy Multiple sclerosis Rheumatoid arthritis Transplant Asthma Nephritis IBD Acquired immune deciency syndrome (AIDS)

CCR3 CCR5

on T cells, immature dendritic cells, and monocyte/ macrophages; in the case of the latter, the expression of CCR1 is predominant on circulating monocytes, whereas CCR5 is upregulated when monocytes acquire the macrophage phenotype. Using specic small molecule inhibitors of CCR1 and CCR5, it has been shown that whilst both receptors can mediate transendothelial migration induced by RANTES, RANTES activation of CCR1 mediates cellular arrest whilst activation of CCR5 mediates cellular spreading. Its role on CCR3 and CCR4 is less clear, although high levels of RANTES expression in samples from asthmatic patients (see below) could imply that it assists in the recruitment of eosinophils through CCR3. RANTES signals through the classical chemoattractant receptor signaling pathway initiated by ligand-induced receptor activation, which results in the formation of a heterotrimeric G-protein complex at its cytoplasmic tail by recruitment of a membraneanchored Gbg heterodimer to the receptor-bound Ga subunit. The Gbg complex regulates the activation of phosphatidyl inositol-phospholipase Cb, catalyzing the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), which act synergistically in the mobilization of intracellular Ca2 . DAG and Ca2 activate various isoforms of protein kinase C, which

CXCL16 CXC13 CXCL12

CCL1

CXCR6

CCR8

CXCL20

CXCR5

CCR6 XCR1
XCL1 XCL2 CX3CL1

CCL5
CCL11

CXCR4

D6 DARC
CCR10 CCR9 CCR7

CCL5
CCL7 CCL13 CCL27 CCL28

Constitutive: basal trafficking/homing

CX3CR1 CXCR1 CXCR2 CXCR3

CXCL8 CXCL6 CXCL8 CXCL5 CXCL1 CXCL2 CXCL3 CXCL7

CCL25 CCL19 CCL21

Inducible: inflammatory

CCR5
CCL3 CCL4

CCR1 CCR3
CCL5
CCL7 CCL11 CCL13

CCR4
CCL17 CCL22

CCR2
CCL2 CCL8 CCL7 CCL13

CCL5
CCL3 CCL7

CXCL9 CXCL10 CXCL11

CCL5
CCL8

Figure 2 Chemokine receptors and their ligands. The high-afnity receptors to which RANTES/CCL5 binds are indicated as those that signal (red) and those that do not signal (black). Since RANTES has only been reported to interact with CCR4 with low (micromolar) activity it is not depicted as a CCR4 ligand in this gure.

378 CHEMOKINES, CC / RANTES (CCL5)

can phosphorylate many other downstream targets. The two major signaling pathways activated through the binding of chemokines are: (1) the phosphoinositide 3 kinase (PI3K) pathway, and studies with PI3Kg / mice have demonstrated that RANTES activity is severely hampered in the absence of this enzyme and (2) the mitogen-activated protein-kinase (MAPK) pathway, which is responsible for phosphorylating and activating downstream transcription factors that in turn regulate the synthesis of other inammatory proteins, including chemokines.

Biological Function
Since their identication, the importance of chemokines in cell migration has been demonstrated in both basal cell trafcking and in inammation. An interesting contrast in the chemokine system exists between the receptors involved in basal trafcking (controlled by homeostatic chemokines), which tend to be specic, whilst those involved in inammatory cell trafcking are shared by several chemokines (Figure 2). In inammatory situations, expression of inducible or inammatory chemokines is upregulated. Control of expression of these chemokines is under the temporal control of proinammatory cytokines. Proinammatory cytokines such as IL-1b, TNF-a, and interferon gamma (IFN-g) alone or in combination induce chemokine expression at sites of inammation in nonlymphoid tissue. RANTES is an example of an inducible/inammatory chemokine. Inammatory chemokine receptors (including those

to which RANTES binds, that is, CCR1, CCR3, and CCR5) are upregulated on inammatory cells following priming, which are then attracted to sites of inammation. RANTES may potentially mediate the activation and trafcking of a range of immune cells including T cells, monocytes, basophils, eosinophils, natural killer cells, dendritic cells, and mast cells, all of which play a role in respiratory diseases (Figure 3). RANTES is often associated with inammatory exudates and is predominantly secreted by CD8 T cells, epithelial cells, broblasts, and platelets, in which it is stored in alpha granules as densely packed aggregates. Much understanding of the chemokine system has come from studies using knockout mice. Despite its being one of the earliest identied and most widely studied chemokines, there are few reports in the literature describing the RANTES-decient mouse. Published work indicates a role for RANTES in both normal T-cell function and in monocyte and T-cell recruitment in the contact hypersensitivity response. However no data with the RANTES / mice in airway inammation are available. On the other hand there are numerous publications on studies of knockout mice for the various RANTES receptors, but again, none for CCR1 / or CCR5 / mice in airways inammation. A role for CCR1 has been demonstrated in the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE), CCR5-deleted mice are fully susceptible to EAE, surprisingly, as high levels of CCR5 expression are seen in multiple sclerosis patients. However, deletion of

Resting T cell

B cell

Activated T cell CCR1,3,(4),5

Eosinophil CCR1,3 Basophil mast CCR3,(4)

NK cell CCR5

Dendritic cell CCR1,5

Monocyte CCR1(5) Macrophage CCR5(1)

Neutrophil
Figure 3 Leukocyte subsets. Leukocytes recruited by RANTES that play a role in respiratory disease are shown in red whilst those that are not recruited by RANTES are shown in black. The RANTES receptors found on each cell type are indicated.

CHEMOKINES, CC / RANTES (CCL5) 379

CCR3, which is the predominant chemokine receptor expressed by eosinophils, results in a 5070% reduction in eosinophil accumulation in the airway lumen and the lung tissue after challenge. These data conrm the role of CCR3 in eosinophil recruitment to the lung during allergic airways inammation. However, no protection was seen in the bronchial hyperreactivity to methacholine, which could be explained by the increase in lung tissue mast cells seen in this model and highlights the controversial role of the eosinophil in respiratory disease.

Role of RANTES in Respiratory Disease

Increased RANTES expression, in combination with the presence of RANTES receptor expressing cells, has been associated with a variety of human inammatory diseases. RANTES produced by lung epithelial cells has been identied as a major eosinophil chemoattractant in the BAL uid of asthmatics. Additionally bronchial biopsies from patients with asthma show elevated RANTES mRNA expression, and levels of RANTES have been shown to reach a peak expression level approximately 4 h after allergen challenge in human subjects. Furthermore RANTES is a potent lymphocyte and monocyte chemoattractant acting via CCR1 and CCR5, and therefore contributes toward the recruitment of T cells and macrophages into the lungs of asthmatic patients. In line with its activity as a chemoattractant for T cells and eosinophils, RANTES has been observed at high levels in polyps from patients suffering from nasal polyposis in chronic hyperplastic sinusitis.
Therapeutic Potential

The therapeutic potential of RANTES as an anti-inammatory and anti-infective target has been shown by several approaches in animal models of disease. The use of neutralizing antibodies has clearly demonstrated the importance of this chemokine in several pulmonary pathologies. Airway obstruction in an experimental model of obliterative bronchiolitis and airway hyperreactivity in a respiratory syncytial viral infection model were both considerably ameliorated by treatment with neutralizing RANTES antibodies. Benecial effects were also observed with anti-RANTES antibodies in acute lung allograft rejection and allograft transplant-induced brous airway obliteration as well as in cardiac allograft rejection. As mentioned above, the N-terminal region of RANTES is critical for receptor activation. In fact modication of this region has produced several variants with receptor antagonist properties. The most

widely studied is Met-RANTES, which is produced by the extension of the N-terminus by the initiating methionine when the human protein is produced recombinantly in Escherichia coli. The protein retains high afnity for human CCR1, CCR3, and CCR5 but is unable to cause receptor activation except in the case of CCR5, on which it has partial agonist activity. Pharmacological studies on the murine receptors surprisingly showed that whilst it also retains high afnity for murine CCR1 and CCR5, it is no longer able to compete for binding of Eotaxin/ CCL11 from murine CCR3. This variant has shown the benecial effects of inhibiting RANTES receptors in many models, in the lung as well as other organs. In fact Met-RANTES was shown to be more effective than individual antichemokine antibody treatment in the ovalbumininduced airway inammation model. This was interesting since it does not bind to murine CCR3 and yet still reduces eosinophil accumulation in the lungs, indicating that blockade of CCR1 and/or CCR5 may also be potential targets for asthma. In an extensive doseresponse study in this model, the inhibition observed was inversely bell-shaped, for which a denitive explanation remains to be elucidated. However the lack of efcacy at high doses could be one of the reasons why it has not been developed for the clinic, in addition to the residual partial agonist activity, which was shown not to be the reason for the inverse bell-shaped doseresponse. CCR1 has been shown to be crucial in the inammatory response to the murine pneumonia virus, which has been used as a model for severe human respiratory syncytial virus disease. This model has highlighted the importance of inammation to the pathogenesis of chronic disease, demonstrating that the inammatory response remains active and acute even when virus replication ceases in response to appropriate antiviral therapy. Met-RANTES treatment prevented the inammatory response to the virus and resulted in reduced morbidity and mortality when administered in conjunction with the antiviral agent ribavirin. These results highlight the interesting possibility of using chemokine antagonists in dual therapies. This had been previously demonstrated by using Met-RANTES in synergy with cyclosporin to prevent organ transplant rejection, at suboptimal doses of both agents. Another N-terminally modied RANTES variant, AOP-RANTES, provided several important insights into the inhibition of human immunodeciency virus (HIV) infectivity. This variant was produced by chemical modication of the N-terminal in an attempt to improve the characteristics of Met-RANTES. Unexpectedly, this variant was even more potent on CCR5 than the parent WT-RANTES protein,

380 CHEMOKINES, CC / TARC (CCL17)

whilst retaining antagonistic properties on CCR1. It was found to be an extremely potent inhibitor of HIV infectivity, not due to its antagonistic properties on CCR5 but on the contrary due to its ability to drive internalization of CCR5, and moreover, to prevent the recycling of functional receptors to the cell surface, thereby removing the essential co-receptor. Furthermore, it provided evidence that infection of primary macrophages could be prevented, an issue that was much debated in the chemokineHIV eld since there were reports both of inhibition and enhancement of infection by R5 HIV strains (those that require CCR5 for cell entry). Despite a different mode of action, AOP-RANTES showed the same prole of inhibition of airways inammation in mice. Yet another RANTES variant has provided novel insights into strategies that can effectively inhibit inammation. As discussed above, mutation of the heparin binding site in RANTES abrogates its ability to recruit cells in vivo, but what was unexpected is that it was capable of inhibiting RANTES-mediated recruitment, even by the non-specic inammatory agent thioglycollate. This inhibitory effect translated into a potent inhibitor of clinical symptoms in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, EAE. Whilst heparin has been shown to reduce inammatory symptoms both in human and in rodent disease models, including pulmonary inammation, the effect was attributed to an effect on proinammatory cytokines. Given the fact that the GAG mutant 44AANA47RANTES had anti-inammatory properties, together with the fact that heparin could inhibit both RANTES- and thioglycollate-mediated cellular recruitment in vivo, its anti-inammatory effect could also be attributed to the interference with the establishment of chemokine gradients. Again this variant was able to reduce the cellular inltrate into inamed lungs in mice. In summary, this single chemokine provides many therapeutic targets since it acts with high afnity on three receptors, all implicated in respiratory disease. Moreover, in view of the extensive knowledge of the protein, it provides several different approaches to inhibit its activity.
See also: Chemokines. Interleukins: IL-5; IL-16. Platelets.

Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108115. Handel TM, Johnson Z, Crown SE, Lau EK, and Proudfoot AEI (2005) Regulation of protein function by glycosaminoglycans as exemplied by chemokines. Annual Review of Biochemistry 74: 385410. Hebert CA (ed.) (1999) Chemokines in Disease. Totowa, NJ: Humana Press. Johnson Z, Kosco-Vilbois MH, Herren S, et al. (2004) Interference with heparin binding and oligomerization creates a novel antiinammatory strategy targeting the chemokine system. Journal of Immunology 173: 57765785. Johnson Z, Schwarz M, Power CA, Wells TN, and Proudfoot AEI (2005) Multi-faceted strategies to combat disease by interference with the chemokine system. Trends in Immunology 26: 268274. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inammation. New England Journal of Medicine 338: 436445. Mack M, Luckow B, Nelson PJ, et al. (1998) AminooxypentaneRANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. Journal of Experimental Medicine 187: 12151224. Power CA (2002) Knock out models to dissect chemokine receptor function in vivo. Journal of Immunological Methods. Proudfoot AEI (2002) Chemokine receptors: multifaceted therapeutic targets. Nature Reviews: Immunology 2: 106115. Proudfoot AEI, Handel TM, Johnson Z, et al. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proceedings of the National Academy of Sciences, USA 100: 18851890. Rothenberg ME (ed.) (2000) Chemokines in Allergic Disease. New York: Dekker. Schall TJ, Bacon K, Toy KJ, and Goeddel DV (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347: 669671. Schall TJ, Jongstra J, Dyer BJ, et al. (1988) A human T cell-specic molecule is a member of a new gene family. Journal of Immunology 141: 10181025. Simmons G, Clapham PR, Picard L, et al. (1997) Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276: 276279.

TARC (CCL17)
T L Ness, C M Hogaboam, and S L Kunkel, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Thymus and activation-regulated chemokine (TARC) was the rst CC chemokine identied as a T-cell chemoattractant. It is known as CC chemokine ligand 17 (CCL17) according to the new nomenclature and shares most amino acid identity with CCL22. CCL17 expression is highest in the thymus, but can also be detected in other tissues such as the small intestine, colon, and lung. CCL17 is completely absent from the spleen. Dendritic cells are the predominant source of constitutive CCL17 production, whereas most immune cells and structural

Further Reading
Chvatchko Y, Proudfoot AE, Buser R, et al. (2003) Inhibition of airway inammation by amino-terminally modied RANTES/ CC chemokine ligand 5 analogues is not mediated through CCR3. Journal of Immunology 171: 54985506.

CHEMOKINES, CC / TARC (CCL17) 381

cells are capable of inducible expression. CCL17 primarily acts through its receptor CCR4 as a chemoattractant of T-helper-2 (Th2) cells. However, CCR4 is also found on Th1 cells, regulatory T cells, natural killer (NK) T cells, NK cells, platelets, and macrophages. CCL17 inhibits classical macrophage activation and type 1 responses, which further drive type 2 immune responses. CCL17 is associated with several Th2-mediated respiratory diseases such as bronchial asthma, allergic rhinitis, pulmonary brosis, and eosinophilic pneumonia, and represents a potential target for treatment of these pathologic disorders.

Introduction
Thymus and activation-regulated chemokine (TARC) was rst identied and cloned from phytohemagglutinin-stimulated peripheral blood mononuclear cells by Imai and colleagues in 1996. TARC was one of four novel sequences isolated using an Epstein-Barr virus vector signal sequence trap. As its name indicates, TARC is constitutively produced in the thymus, but can also be detected in other tissues such as the colon, small intestine, and lung. It was the rst CC chemokine identied that specically interacted with a high-afnity receptor found predominantly on T cells, to a lesser degree on monocytes, but not on granulocytes. TARC acts as a powerful T cell chemoattractant and has been implicated in the pathogenesis of several Th2-mediated diseases. TARC was renamed CC chemokine ligand 17 (CCL17) according to the new nomenclature system for chemokines and chemokine receptors.

Structure
While most CC chemokines are found on human chromosome 17 (mouse chromosome 11), CCL17 is

tightly linked to CX3CL1 and CCL22 on human chromosome 16q13 (mouse chromosome 8q). The CCL17 gene (2176 bp) comprises 4 exons and encodes a 94 amino acid preprotein with a 23 amino acid signal sequence. The CCL17 protein contains the four cysteine residues characteristic of all CC chemokines and shares the highest degree of homology with CCL22 (32% amino acid identity) (Figure 1). The secreted CCL17 protein is approximately 8 kDa and has a theoretical isoelectric point of 9.7. The tertiary monomeric structure of CCL17 is a threestranded antiparallel b-sheet anked by a C-terminal helix, like other CC chemokines, although dimerization results in a more compact molecule than other CC chemokines. CCL17 has a greater distribution of basic charge on its surface, allowing it to bind more tightly to glycosoaminoglycans than most CC chemokines. This contributes to its ability to form a gradient within the extracellular matrix of endothelial cells to attract and immobilize receptorexpressing cells. Polymorphism screening of the human CCL17 gene identied two rare single nucleotide changes within exon 3 (2134C4T and 2037G4A) and a polymorphism in the promoter region (431C4T). The promoter region contains nuclear factor kappa B (NF-kB) and signal transducer and activator of transcription 6 (STAT-6) recognition sites as well as the regulatory elements required for interleukin (IL)-4 and tumor necrosis factor alpha (TNF-a)-mediated induction. The 431C4T change alters the putative binding site for the transcription factor activator protein 4 and results in increased promoter activity in vitro. Individuals homozygous for the 431C4T

C>T

2176 bp

(a)

SP

CC

CX3CL1

CCL17

FracTARC (b)
Figure 1 Sequence structure of CCL17. (a) The genomic sequence of human CCL17 (2176 bp) is comprised of four exons. Untranslated regions are depicted as hatched boxes. The locations of the signal peptide (SP), CC motif, and 431C4T polymorphism are indicated. (b) In the mouse, tissue-specic (brain and kidney) alternative splicing results in production of fracTARC, a transcript containing the signal peptide sequence of CX3CL1/fractalkine (red) and the entire coding region of CCL17 (blue) minus the rst three N-terminal amino acids.

382 CHEMOKINES, CC / TARC (CCL17)

polymorphism have elevated systemic concentrations of CCL17 as well as an increased incidence of asthma. Murine CCL17 is unique in that its transcription is regulated not only from its own promoter, but also from the promoter of the closely linked CX3CL1 (fractalkine) gene. Alternative splicing results in expression of murine fracTARC, a protein containing the CX3CL1 signal sequence and the mature CCL17 protein. Processing of the signal sequence results in loss of the rst three amino acids from the N-terminus, normally found in CCL17. Transcriptional regulation of CCL17 is tissue specic, as CCL17 is expressed in the thymus and lung while fracTARC can only be found in the brain and kidney. fracTARC has not been detected in human tissues thus far.

Regulation of Production and Activity

Dendritic cells (DCs), specically myeloid-related DCs (CD11c CD11b ), are responsible for constitutive production of CCL17 in most peripheral lymphoid and nonlymphoid organs. Immature monocyte-derived DCs (moDCs) express high levels of CCL17 that are increased several-fold upon maturation. CCL17 is inducible in most leukocytes including DCs, macrophages, mast cells, B cells, and naive T cells after exposure to various cytokines (IL-3, IL-4, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF)), phytohemagglutinin, ovalbumin, or stimulation with Toll-like receptor agonists (i.e., lipopolysaccharide). Transforming growth factor beta (TGF-b) has been shown to act as a negative regulator of CCL17 production. Structural cells such as bronchial, epithelial, and endothelial cells, keratinocytes, broblasts, and smooth muscle cells are also a signicant source of inducible CCL17. CCL17 is not found in the spleen, even after systemic bacterial infection or in vitro stimulation of cells from the spleen. The expression pattern of this chemokine accentuates its importance in mediating the attraction of effector cells to sites commonly exposed to infection and/or stimulation by foreign antigens, i.e., mucosa and lymph nodes (LNs).

Biological Function
DCs fulll a fundamental regulatory role linking both innate and adaptive immune responses. DCs act as professional antigen-presenting cells, transporting antigens to the draining lymph nodes (LNs), where they prime T-cells and initiate antigen-specic responses. DCs produce several constitutive and inducible cytokines/chemokines that are responsible for activating and recruiting specic T-cell

populations. CCL18 and CCL19 direct naive T cells into the T-cell zone, while CCL2, CCL3, CCL17, and CCL22 attract activated and memory T cells. CCL17 is a highly selective chemoattractant for Th2 cells and CLA skin-homing memory T cells (Figure 2). CCL17 expression is upregulated in the skin of patients with psoriasis, cutaneous delayed type hypersensitivity, and atopic dermatitis (AD) as well as in the epithelium of mice with AD-like disease. Serum levels of CCL17 are increased in AD patients and correlate with disease severity, eosinophil number, and soluble E-selectin. Intradermal injection of CCL17 in mice results in dose-dependent recruitment of CD4 lymphocytes into the skin. CCL17 regulates its own production from keratinocytes and stimulates transcription of IL-4 mRNA, further highlighting its importance in driving Th2 responses. When CCL17 is neutralized (genetic deletion or antibody neutralization), mice are more resistant to allograft rejection, contact hypersensitivity, bacteriainduced fulminant hepatitis, and antigen-specic asthmatic reactions. All improvements were associated with reduced effector T-cell recruitment and decreased Th2 cytokine production. Although CCL17 is foremost recognized as a Th2 cell chemoattractant, its receptor is also found on DCs, Th1 cells, CD25 T cells, natural killer (NK) cells, NK T cells, platelets, and macrophages. Th1 cells and CD4 NK T cells migrate in response to CCL17. Receptor expression on CD25 regulatory T cells suggests that CCL17 may serve as an important immunomodulatory mediator of innate and acquired immune responses. In vitro treatment with CCL17 induces Ca2 mobilization, aggregation, and granule release from platelets. Recently, the effects of CCL17 on macrophages has been the subject of increasing interest. Macrophages undergo differentiation comparable to T-cell maturation (Th1/Tc1 vs. Th2/Tc2). Resident macrophages that are classically activated (caMf or M1 macrophages) by stimulation with microbial products exhibit strong antimicrobial innate immune responses such as production of inammatory cytokines/chemokines, expression of inducible nitric oxide synthase, efcient killing activity, and induction of a Th1 response. In contrast, IL-4 and IL-13 drive the development of alternatively activated macrophages (aaMf), characterized by expression of anti-inammatory/immunoregulatory cytokines/ chemokines, increased mannose receptor expression, arginase production, and decreased killing activity. Individuals with a predominance of aaMf are more susceptible to infection. AaMf inhibit the generation of caMf via production of IL-10 and CCL17. This

CHEMOKINES, CC / TARC (CCL17) 383

Adaptive immune response Memory T cells CD45RO Regulatory T cells Th2 cells CD25 CCR4 CCL17 Platelets Innate immune response NK cells NK T cells CCR8

CLA

Skin homing T cells

DCs

caM
/M1

Figure 2 CCL17 regulates both innate and adaptive immune responses. CCL17 is centrally involved in the adaptive immune response (shown in blue circle) as a potent chemoattractant of CD4 Th2 cells (CLA skin homing, CD25 regulatory, and CD45RO memory T cells). CCR4 is the receptor for CCL17. CCR4 is also expressed on several innate immune cells (shown in yellow circle). Natural killer (NK) cells and dendritic cells (DCs) chemotax in response to CCL17. Evidence suggests that CCL17 utilizes both CCR4 and CCR8 on NK cells. CCL17 triggers granule release and aggregation of platelets in a CCR4-dependent manner. CCL17 inhibits classical activation of macrophages (caMf), thereby suppressing type 1 innate immune responses. Natural killer T cells (NK T cells) and DCs play a fundamental role linking both innate and adaptive immune responses (shown in the overlapping green region). DCs produce several cytokines/chemokines that activate and attract innate cells while also serving as professional antigen-presenting cells priming antigenspecic responses.

has been demonstrated in murine models of thermal injury and sepsis. Thus, CCL17 functions as an effective promoter of type 2 responses primarily through its chemoattraction of Th2 cells while modulating type 1 responses by suppressing caMf.

Receptors
Initial studies demonstrated high-afnity binding of radiolabeled CCL17 to Jurkat cells; CXCL8 or other CC chemokines could not compete with this binding. CCL17-induced T-cell chemotaxis was pertussis toxin sensitive suggesting involvement of a G-protein-coupled receptor. In a series of transfection studies, CCR4 was identied as the primary receptor for CCL17. CC chemokine receptor 4 (CCR4) is found on a variety of cells (Figure 2). CCL17 and CCR4 have been implicated in the pathogenesis of many allergic diseases as they are central mediators involved in the homing of T cells to the skin. In addition to CCL17, CCL22 is the only other ligand known to bind CCR4. Despite the fact that they bind the same receptor, the different binding afnities, expression patterns, and kinetics of these two chemokines suggests important nonredundant roles in vivo. CCL22 binds CCR4 with higher afnity and is a more potent inducer of integrin-dependent

T-cell adhesion. CCR4 internalization is rapid following CCL22 binding, but delayed after exposure to CCL17. CCL22 desensitizes CCR4 T cells to CCL17, while CCL17 has no effect on CCL22 signaling. CCL22 is subject to serine protease degradation whereas CCL17 is a much more stable ligand. Considering the abundant expression of CCL17 (and not CCL22) by structural cells, it is very likely that CCL17 acts to specically arrest CCR4high cells (i.e., Th2 cells) in circulation while CCL22 subsequently directs these cells within the affected tissue. CCR8 plays a role in activation, migration, and proliferation of lymphoid cells. Early binding and chemotaxis studies also identied CCR8 as a potential receptor for CCL1, CCL4, and CCL17. However, no intracellular Ca2 changes were induced upon ligation of CCL4 or CCL17. CCL17-induced chemotaxis was attributed to low levels of CCR4 expression, as at physiologically relevant concentrations, CCL17 was unable to bind, inhibit CCL1-binding, or chemoattract CCR8high-expressing cells. However, the receptor usage of CCL17 may be cell type dependent as CCL17 is able to compete with CCL1 for binding in IL-2-activated adherent NK cells. CCL1 and CCL22 only partially inhibit CCL17-induced responses suggesting that CCL17 is able to act through both CCR4 and CCR8 in these cells.

384 CHEMOKINES, CC / TARC (CCL17)

CCL17 also interacts with both chemokine clearance receptors, the Duffy antigen/DARC and the D6 receptor. The Duffy antigen, found on red blood cells, binds both CC and CXC chemokines, while D6 is expressed on lymphatic endothelial cells and is specic for inammatory CC chemokines. While ligand binding does not produce a functional signal, it does induce rapid internalization of these ligand/receptor complexes, facilitating regulation of inammatory responses and return to homeostatic levels.

CCL17 in Th2-Type Respiratory Diseases

CD4 T cells play a fundamental role in the pathogenesis of bronchial asthma and other allergic respiratory diseases. Cytokines/chemokines are pivotal mediators of inammation, Th2 cytokine production, eosinophil and T-cell recruitment, and airway hyperresponsiveness (AHR) observed in asthmatic patients. When moDCs are treated in vitro, cells from allergic patients upregulate CCL17 expression compared to similarly treated cells from nonallergic patients. CCR4 is detected on virtually all T cells from endobronchial biopsies of asthmatic patients following allergen challenge. CCL17 concentrations are higher in the serum, airway epithelium, and bronchoalveolar lavage (BAL) uid from asthmatics after antigen challenge. Children suffering from acute and chronic asthma exhibit high levels of plasma CCL17, which directly correlate with the severity of the disease. Individuals homozygous for the 431C4T CCL17 polymorphism have more peripheral eosinophilia, are more susceptible to antigen challenge, produce higher levels of allergen-specic immunoglobulin E (IgE), and have a higher incidence of atopic asthma. Blocking CCL17 with a neutralizing antibody attenuates several aspects of pulmonary inammation (eosinophilia, T-cell recruitment, and Th2 cytokine production) and AHR in a murine model of ovalbumin-induced asthma. While mice decient for the CCL17 receptor showed no protection in this model, the CCR4 / mice were protected against allergic airway inammation initiated by chronic fungal challenge. These data clearly indicate that CCL17 plays a signicant role in the inammation and pathogenesis observed in asthmatic patients. In addition to asthma, CCL17 is centrally involved in the pathogenesis of several respiratory diseases. Allergic rhinitis is a condition characterized by eosinophil, mast cell, and Th2 cellular inltration into the nasal mucosa. The epithelium and mononuclear cells in the nasal mucosa of allergic patients express increased levels of CCL17, compared to nonallergy patients. CCL17 is signicantly increased in BAL

uid of patients with eosinophilic pneumonia, an inammatory lung disorder often caused by fungal or helminth infections or drug reactions. Concentrations of CCL17 directly parallel expression of the Th2 cytokines IL-5 and IL-13 and the number of CCR4 CD4 T cells in the BAL uid. Inammatory cell recruitment is central to the pathogenesis of interstitial lung disease. In murine bleomycin-induced pulmonary brosis and rat radiation pneumonitis models, CCL17 and CCR4 are upregulated in the lung during the disease. CCL17 was localized to epithelial cells (bleomycin model) or alveolar macrophages (radiation model) in the lung and CCR4 was predominantly found on lung macrophages as well as lymphocytes (only in the radiation model). Epithelial cells were also a prominent source of CCL17 in idiopathic pulmonary brosis patients. Neutralization of CCL17 resulted in signicantly fewer T cells, macrophages, NK cells, and neutrophils recruited into the BAL uid, as well as reduced brosis. In a murine Th2-mediated lung granuloma model, CCL17 neutralization decreased granuloma size and CCR4 cell recruitment and increased overall cytokine/ chemokine production. No CCL17- or CCR4-specic compounds have been developed for use in human trials. In Th2mediated respiratory diseases, anti-allergic and antiinammatory agents result in decreased CCL17 expression, Th2 cell recruitment, CCR4 expression, eosinophilia, IgE production, and type 2 cytokine production, further highlighting the importance of CCL17 in Th2-mediated pathogenesis.
See also: Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview; Acute Exacerbations. Bronchoalveolar Lavage. Chemokines. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibroblasts. G-Protein-Coupled Receptors. Interleukins: IL-4; IL-10; IL-13. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Platelets. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Alferink J, Lieberam I, Reindl W, et al. (2003) Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. Journal of Experimental Medicine 197(5): 585599. Belperio JA, Dy M, Murray L, et al. (2004) The role of the Th2 CC chemokine ligand CCL17 in pulmonary brosis. Journal of Immunology 173(7): 46924698. Chvatchko Y, Hoogewerf AJ, Meyer A, et al. (2000) A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. Journal of Experimental Medicine 191(10): 1755 1764.

CHEMOKINES, CC / TECK (CCL25) 385

DAmbrosio D, Albanesi C, Lang R, et al. (2002) Quantitative differences in chemokine receptor engagement generate diversity in integrin-dependent lymphocyte adhesion. Journal of Immunology 169(5): 23032312. Imai T, Yoshida T, Baba M, et al. (1996) Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using EpsteinBarr virus vector. Journal of Biological Chemistry 271(35): 2151421521. Inngjerdingen M, Damaj B, and Maghazachi AA (2000) Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309. Journal of Immunology 164(8): 40484054. Jakubzick C, Wen H, Matsukawa A, et al. (2004) Role of CCR4 ligands, CCL17 and CCL22, during Schistosoma mansoni egginduced pulmonary granuloma formation in mice. American Journal of Pathology 165(4): 12111221. Katakura T, Miyazaki M, Kobayashi M, Herndon DN, and Suzuki F (2004) CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. Journal of Immunology 172(3): 1407 1413. Katoh S, Fukushima K, Matsumoto N, et al. (2003) Accumulation of CCR4-expressing CD4 T cells and high concentration of its ligands (TARC and MDC) in bronchoalveolar lavage uid of patients with eosinophilic pneumonia. Allergy 58(6): 518523. Kawasaki S, Takizawa H, Yoneyama H, et al. (2001) Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inammation and hyperresponsiveness in mice. Journal of Immunology 166(3): 20552062. Mariani M, Lang R, Binda E, Panina-Bordignon P, and DAmbrosio D (2004) Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells. European Journal of Immunology 34(1): 231240. Schuh JM, Power CA, Proudfoot AE, et al. (2002) Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4 / mice. FASEB Journal 16(10): 13131315. Vestergaard C, Deleuran M, Gesser B, and Larsen CG (2004) Thymus- and activation-regulated chemokine (TARC/CCL17) induces a Th2-dominated inammatory reaction on intradermal injection in mice. Experimental Dermatology 13(4): 265271. that of the lymphoid chemokines, CCL17, CCL19, CCL21, and CXCL12. CCL25 mRNA was also detected in murine pro-Tcells, a population of noncommitted intrathymic T-cell progenitor cells. Detection of mCCL25 mRNA in murine fetal thymus further supported early hypotheses that the role of CCL25 was early in thymic development. This lymphoid tissue chemokine is important in the migration, localization, and maturation of immature double positive thymocytes. In addition, CCL25 participates in the development and gut-homing of T-cell subsets and localization of immunoglobulin A (IgA)-secreting plasma cells within the gut associated lymphoid tissue. CCL25 exerts these functions primarily via binding of the CC chemokine receptor CCR9.

Introduction
Thymus-expressed chemokine (TECK) is a CC chemokine that was rst identied by Vicari and colleagues in 1997. This discovery was achieved by analysis of a murine cDNA library from RAG-1 decient mice in experiments designed to identify novel genes involved in T-lymphocyte development. Initial analysis of the expression of the murine TECK transcript revealed expression predominantly in the thymus, hence thymus-expressed chemokine or TECK. It has since been designated as CCL25 according to the novel chemokine and chemokine receptor nomenclature.

Structure
Following identication of murine CCL25, homologous genes were identied in human, rat, and hamster genomes. The nucleotide homology between human (hCCL25) and murine (mCCL25) transcripts of CCL25 is 71%. The amino acid sequence of hCCL25 is described in Figure 1. The unprocessed precursor is 150 amino acids long and has a molecular weight of 16 339 Da. Alternative splicing can yield two isoforms of hCCL25. Isoform 2 is an antagonist of isoform 1. Although CCL25 contains the conserved cysteine motif, which grants membership of the CC chemokine family, the structural relationship of CCL25 to other CC chemokines is distant and its homology low. Greatest amino acid identity is shared with CCL17. The percentage protein identity shared between CCL25 and some of the most closely related chemokines is outlined in Table 1. CCL25 displays differences from other chemokines even at the gene level. The hCCL25 gene is located on chromosome 19p13.2, not chromosome 17 as would be expected. This region is syntenic with mouse chromosome 8, where the mCCL25 gene has been mapped, while the genes encoding most chemokines are clustered on mouse chromosome 11.

TECK (CCL25)
K F Buckland and C M Hogaboam, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Thymus-expressed chemokine (TECK) is a CC chemokine rst identied in 1997. The sequence of TECK/CCL25 was selected by comparison of random sequencing products to previously described molecular sequences of murine CC chemokines. CCL25 is expressed predominantly in the thymus and its function is associated with T-cell development. In addition, CCL25 is highly expressed in the small intestine and has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining of the small intestine. The function of CCL25 is comparable to

386 CHEMOKINES, CC / TECK (CCL25)

MNLWLLACLVAGFLGAWAPAVHTQGVFEDCCLAYHYPIGWAVLRRAWTY IQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHH NMQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL
Figure 1 The amino acid sequence structure of CCL25 is given using the accepted standard lettering for amino acids. The asterisk ( * ) denotes the putative cleavage site for the signal peptide. Conserved double cysteines are present at positions 30 and 31. Disulde bonds are predicted between cysteines at positions 3058 and 3175, highlighted in blue. Alternative splicing can occur at position 65 or 150, highlighted in red. Amino acids 123, highlighted in green, from a potential FT chain, a site for posttranslational modication. The sequence consisting of amino acids 24150 is known to yield small inducible protein A25, black text.

Table 1 Percentage amino acid identity between CCL25 and other chemokines mCCL25 hCCL25 hCCL17 mCCL3 mCCL4 mCCL7 mCCL12 mCCL11 49.3 28.7 23.9 23.9 22.7 23.1 22.7 hCCL25

Regulation of Production and Activity

CCL25 synthesis is highly regulated, as only a few specic cell populations are able to secrete this chemokine. Murine CCL25 mRNA is expressed at signicant levels only in the thymus and small intestine, although low levels of mRNA were also detected in liver, brain, testis, and pro-T cells. The high thymic expression was revealed to be care of major histocompatibility complex (MHC) II CD11c thymic dendritic cells (DCs). In contrast, CCL25 expression is absent from CD11c thymic DCs and neither bone marrow nor naive splenic DCs express CCL25. Thymic endothelium also expresses CCL25 at low levels, while endothelial cells are a major site of CCL25 expression in the small intestine. Both the production and the responses to CCL25 can be enhanced by antigen stimulation. Lipopolysaccharide stimulation can induce CCL25 expression in the spleen in vivo, while responsiveness of thymocytes to CCL25 is enhanced following T-cell receptor (TCR) stimulation and in CD69 thymocyte subsets. Thus costimulatory conditions may be required for CD8 T cells to retain CCL25 receptor expression and effectively migrate to the intestinal lamina propria.

Biological Function
Chemokines such as CCL25 are a key component of the system of tissue-restricted recirculation of memory and effector lymphocytes. CCL25 expression in

human and mouse thymus was localized to thymic DCs. In contrast, stimulated and unstimulated human monocyte-derived DCs and bone marrowderived cells all lacked CCL25 expression. CCL25 mRNA was also detected in murine pro-T-cells, a population of noncommitted intrathymic T-cell progenitor cells. Alternatively these cells can deviate from T-cell lineage to become natural killer (NK) cells or DCs. Detection of mCCL25 mRNA in murine fetal thymus supported early hypotheses that the role of CCL25 was early in thymic development. mCCL25 is chemotactic for murine activated monocytes, DCs, thymocytes, and a human lymphocyte cell line, THP-1, following their activation with interferon gamma (IFN-g). In contrast CCL25 was inactive on neutrophils, bone marrow cells, splenic, and peripheral blood lymphocytes or NK cell-enriched populations in vitro. Further none of these was recruited following intraperitoneal injection of CCL25 in vivo. Chemokines such as CXCL12, CCL3, CCL21, and CCL25 are involved in the selection processes allowing T-cell progenitors (pro-T cells) to progress into functional single positive T cells. In the developing thymus maturing thymocytes migrate from the cortex to the medulla. Early in embryogenesis CCL25, CXCL12, and CCL21 are expressed but not the related chemokines CCL17, CCL19, or CCL22. CCL25 expression has been localized to MHC II N418 DCs in the thymic medullary stroma. This region of the thymus is where single positive thymocytes emigrate from, into the bloodstream. CCL25 induces chemotaxis of immature CD4 CD8 double positive and mature single positive CD4 or CD8 human thymocytes. Thus CCL25 is thought to induce chemotaxis of double positive thymocytes from the cortex to the medullary region of the thymus and also to regulate localization of single positive thymocytes within the medulla. Through the chemoattraction of thymocytes and macrophages, CCL25 produced by thymic DCs may be the driving chemokine in achieving the colocalization of DCs with thymocytes and macrophages required for clonal deletion of self-reactive thymocytes. However, CCL25 is not solely responsible

CHEMOKINES, CC / TECK (CCL25) 387

for chemotaxis of thymocytes, which can also respond to other chemokines including CXCL12. The specicity of CCL25 for particular thymocyte subsets is of great interest in further elucidating its function. Lymphocyte recirculation is tightly regulated by the coordinated expression of chemoattractant receptors and adhesion molecules. This ensures that lymphocytes, which have previously encountered antigen, home to the appropriate site in which antigen exposure may reoccur. CCL25 is highly expressed in the epithelial lining of the small intestine and strongly associated with intestinal homing of particular lymphocyte populations to this antigenrich site. Importantly, CCL25 expression is weak or absent in other segments of the intestinal tract including colon and stomach, which supports the high specicity of lymphocyte trafcking. This restricted expression prole also separates the function of CCL25 from CCL17, another CC chemokine that is highly expressed in the thymus and small intestine but in contrast to CCL25, is also expressed in lung, colon, and activated mononuclear leukocytes. The CCL25 receptor is expressed only by discreet subsets of memory CD4 and CD8 lymphocytes that also express the intestinal homing adhesion molecule a4b7 but not by other systemic memory populations. In the intestine CCL25 is expressed in CD11c dendritic stromal cells, and c-kit Lin bone marrowderived cells chemotax toward CCL25. Thus CCL25 has a role in the formation of gut cryptopatches, an essential process in the generation of thymus-independent intraepithelial lymphocytes. CCL25 also has an important role in B-cell homing to the gut. CCL25 is abundantly expressed in epithelial cells lining the small intestine and is a potent and selective chemoattractant for immunoglobulin A (IgA)-secreting plasma cells. Oral antigens induce effector/memory cells that express essential receptors for intestinal homing, namely the integrin a4b7 and CCR9, the receptor for CCL25. This imprinting of gut tropism is mediated by DCs from Peyers patches in a positive feedback mechanism. Similar to T-cell development, bone marrow pre-pro B cells also migrate toward CCL25 while more mature bone marrow-derived B cells do not. Further, CCR9 / mice had a reduction in pre-pro B-cell numbers. CCR9/CCL25 interaction provides a cell survival signal in certain cell populations. CCL25 selectively rescues T cells but not thymocytes from tumor necrosis factor alpha (TNF-a)-induced apoptosis via activation of livin, a member of the inhibitor of apoptosis (IAP) family. However, antibody neutralization of CCL25 did not prevent repopulation of fetal thymus with T-cell precursors. Anti-CCL25

antibody treatment in 24-week-old mice led to a signicant reduction in the total number of CD8 gd and ab in the intraepithelial lymphocyte compartment demonstrating an important role for CCL25 in the development of these cell populations. Further, a reduction in TCRgd numbers in the intestinal mucosal epithelium correlated with an increased number present in lymph nodes and spleen in CCR9 / mice supporting the requirement for CCL25/CCL25 receptor for homing of TCRgd intraepithelial lymphocytes. Emergence of double positive thymocytes and TCRab cells during embryogenesis was also delayed by approximately 1 day and pre-pro B-lymphocyte numbers were reduced. Therefore CCL25 has differential functions across separate cell types. Immature double positive thymocytes use CCL25 for differentiation, homing, development, maturation, and selection. Mature single positive thymocytes, T and B cells, use CCL25 for localization and cell homeostasis, and to resist apoptosis. In addition malignant cells have been shown to use CCL25 for inappropriate proliferation and resistance to apoptosis as well as inltration.

Receptors
CC chemokine receptor 9 (CCR9) is a receptor for CCL25. This G-protein-coupled receptor was additionally identied and named as CCR10, D6, and GPR-9-6 but these are in fact the same receptor and are henceforth known as CCR9. CCL25 selectively and efcaciously binds CCR9 but CCR9 is not selective for CCL25 but also binds CCL25, CCL78, and CCL1213. CCR9 is highly expressed in the thymus and by T-lymphocyte subsets found in the small intestine. Expression has been localized to thymocytes and DCs in the thymus, and intraepithelial lymphocytes and lamina propria lymphocytes in the small intestine. Upregulation of CCR9 occurs during intraepithelial lymphocyte precursor differentiation allowing responsiveness to CCL25. CCR9 expression is at least several-fold greater in thymus than in spleen, bone marrow, lymph node, liver, or peripheral blood leukocytes. Activation of CCR9 leads to phosphorylation of glycogen synthase kinase 3b and forkhead transcription factor. This provides a cell survival signal. CCR9 also signals via activation of pkb/Akt, PI3-K, and mitogen-activated protein kinase (MAPK) but does not require MAPK for activation of chemotaxis. CCR9 is highly expressed by intestinal melanoma cells and abhorrent expression of CCR9 is associated with metastasis to the small intestine. CCR9/CCL25 play an important role in T-cell progenitor migration within the thymus. Immature

388 CHEMOKINES, CC / TECK (CCL25)

CCR9 = GPCR = Thymocyte DN3 DN4 pDP Subcapsular zone

DN2 DP CCL25 Thymocyte generation DN1 DC 69+ SP Medulla SP Thymus Migration/ circulation CCR11 TCR CD4 SP DC CCL17 Immunity balance & regulation CCL25 CCR9 DC Lung SP Small intestine TCR SP CCX-CKR? CCR9 SP Cortex

EC

TCR CD8 SP

Homing and expansion

CCR4

Figure 2 CCL25 is important in the migration, localization, and maturation of. immature CD4 CD8 double positive thymocytes and TCRgd T cells. In addition, CCL25/CCR9 participate in the migration and development of intraepithelial lymphocytes of the small intestine. The role of an additional receptor for CCL25, CCX-CKR, is as yet unknown but it is highly expressed in the lung. DN, double negative thymocyte; DP, double positive thymocyte; GPCR, G-protein-coupled receptor; SP, single positive thymocyte; TCR, T-cell receptor.

double negative thymocytes do not express CCR9 but CCR9 is highly expressed in double positive thymocytes. Single positive CD8 thymocytes continue to express CCR9 as they mature into naive T cells and migrate out of the thymus. CCL25 induces migration of immature double positive and mature single positive thymocytes via CCR9. CCL25 is also involved in the development of ab and gd T-cell lineages. Approximately 50% of TCRgd thymocytes express CCR9 and are responsive to CCL25 compared to 3% of TCRgd double negative cells. In TCRgd cells CCL25 response predominates over CXCL12. Circulating TCR gd T cells and CD8 T cells from lymph node, spleen, Peyers patches, and small intestine express CCR9. Conversely CD4 thymocytes have lost expression of CCR9 and this correlates with a lack of CCR9 on CD4 T cells in secondary lymphoid organs. The expression of chemokine receptors selective for intestinal trafcking is further supported by

upregulation of expression of a4b7, an intestinal homing integrin. Only Peyers patch DCs are able to induce high-level expression of a4b7 and response to CCL25 sufcient for gut homing of T cells. In correlation, CCR9 expression is maintained following activation of CD8 ab lymphocytes, with ovalbumin and lipopolysaccharide, in mesenteric lymph nodes but is rapidly downregulated on the same lymphocyte subsets if activated in the peripheral lymph nodes. Circulating CCR9 T cells that lack a4b7 are likely to have only recently exited the thymus. Surprisingly, gene deletion of CCR9 had no major effect on intrathymic T-cell development. Functional redundancy revealed by CCR9 / mice may be due to CCL25 binding by CCX-CKR.

CCL25 in Respiratory Diseases

CCL25 expression is high in the thymus where its function is associated with T-cell development, and

CHEMOKINES, CC / TECK (CCL25) 389

in the small intestine where it has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining. The functional requisite for CCL25/CCR9 in T-cell trafcking is not fully understood as mCCL25 also binds mCCX-CKR, a receptor expressed throughout the body and abundantly in the lung. The afnity of this receptor for CCL25 is high but the efcacy of this interaction is low. As in the mouse, binding of human CCL25 to CCX-CKR also fails to elicit functional responses. However signicant diversity between human and murine CCX-CKR biology has hampered investigation of the in vivo function of this ligandreceptor interaction. CCL25 mRNA has been detected in the tonsils of humans, mice, and pigs. Mucosal sites such as the intestinal and respiratory epithelium present highly specialized challenges to the immune system as they are constantly exposed to potential pathogens. The palatine and nasopharangeal tonsils produce IgA and may play a similar role in aerodigestive tract as the Peyers patches of the gastrointestinal tract. Leukocytes are central in the pathophysiology of infectious and allergic respiratory diseases. Chemokines mediate the migration and activation of lymphocytes in normal and inammatory conditions. CCL25 is known to have roles in both primary and secondary lymphoid organs, the thymus and small intestine, respectively. These different lymphoid organs and other immunological sites such as the thymus, spleen, small intestine, lymph nodes, and intestinal and respiratory epithelium can be discussed as separate compartments. Indeed, each has a specic role in lymphocyte trafcking. However, therapeutic strategies must consider that the immune system functions as a highly integrated system in vivo. Any imbalance or dysregulation, of for instance a chemokine, for example, CCL25, in one apparently isolated tissue site can be cause or effect (or exacerbation) of disease at a distant site. Despite the lack of expression of CCL25 in the lung this chemokine may still have an important contribution to the immunological component of certain respiratory diseases due to its pivotal role in thymocyte development and trafcking of these T-lymphocyte progenitor cells (Figure 2). CCL25- or CCR9-selective compounds are unlikely to be useful acute therapy in respiratory

diseases. However, CCR9/CCL25 expression or responsiveness may prove a good phenotypic marker perhaps for postimmunological therapy assessment of thymic involution and atrophy. Indeed immunodecient states caused by chemotherapy or following respiratory infection may be managed better for the long term by stimulating thymic reactivation, perhaps involving increased CCL25/CCR9 activity.
See also: Chemokines. Chemokines, CC: TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1). Dendritic Cells. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: T cells.

Further Reading
Bowman EP, Kuklin NA, Youngman KR, et al. (2002) The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. Journal of Experimental Medicine 195: 269275. Gosling J, Dairaghi DJ, Wang Y, et al. (2000) Cutting edge: identication of a novel chemokine receptor that binds dendritic celland T cell-active chemokines including ELC, SLC, and TECK. Journal of Immunology 164: 28512856. IUIS/WHO Subcommittee on Chemokine Nomenclature (2003) Chemokine/chemokine receptor nomenclature. Cytokine 21(1): 4849. Kunkel EJ, Campbell DJ, and Butcher EC (2003) Chemokines in lymphocyte trafcking and intestinal immunity. Microcirculation 10: 313323. Mora JR, Bono MR, Manjunath N, et al. (2003) Selective imprinting of gut-homing T cells by Peyers patch dendritic cells. Nature 424: 8893. Nomiyama H, Amano K, Kusuda J, et al. (1998) The human CC chemokine TECK (SCYA25) maps to chromosome 19p13.2. Genomics 51: 311312. Uehara S, Song K, Farber JM, et al. (2002) Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69 thymocytes and gammadeltaTCR thymocytes preferentially respond to CCL25. Journal of Immunology 168: 134142. Vicari AP, Figueroa DJ, Hedrick JA, et al. (1997) TECK: a novel CC chemokine specically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity 7: 291301. Youn BS, Kim CH, Smith FO, et al. (1999) TECK an efcacious chemoattractant for human thymocytes, uses GPR-9-6/CCR9 as a specic receptor. Blood 94: 25332536. Zaballos A, Gutierrez J, Varona R, et al. (1999) Cutting edge: identication of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. Journal of Immunology 162: 56715675.

390 CHEMOKINES, CXC / CXCL12 (SDF-1)

CHEMOKINES, CXC
Contents

CXCL12 (SDF-1) IL-8 CXCL9 (MIG) CXCL10 (IP-10) CXCL1 (GRO1)CXCL3 (GRO3)

CXCL12 (SDF-1)
R M Strieter and B N Gomperts, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

sequence is highly conserved among species with 99% homology between mouse and humans. This highlights the importance of this molecule in development and regeneration.

Abstract
SDF-1 (stromal cell-derived factor-1) is also known as CXCL12, and is a CXC chemokine that binds to the CXC chemokine receptor, CXCR4. It is widely expressed and highly conserved having 99% homology with lower vertebrates, suggesting that it is important in primordial development. Two splice variants, SDF-1a and SDF-1b, have been identied and determined to have different regulation and functions. The SDF-1/CXCR4 complex results in activation of a variety of signal transduction pathways. SDF-1 plays a role in several functions, including trafcking of hematopoietic and nonhematopoietic adult stem cells, development of the central nervous system, vasculature and hematopoietic cells, cell motility, chemotaxis and adhesion, and cell proliferation and survival. It also inhibits lymphotropic strains of HIV-1 to enter into CXCR4 cell lines. SDF-1 plays a critical role in lung repair after injury through recruitment of circulating mesenchymal progenitor cells such as brocytes and progenitor epithelial cells. SDF-1 is also an important molecule in invasion and organ-specic metastasis of cancer; hence, SDF-1 is one of the most important known chemokines.

Structure
Although genes encoding other members of the chemokine family are localized on chromosome 4q or 17q, the human CXCL12 gene maps to chromosome 10q. cDNA of CXCL12-a and CXCL12-b encode proteins comprised of 89 and 93 amino acids, respectively. The nucleotide sequence contains a single open reading frame of 267 nucleotides encoding the 89 amino acid polypeptide. CXCL12 contains four conserved cysteine residues of the chemokine family. It does not contain the ELR motif, although its role in angiogenesis is controversial. The genomic structure of the CXCL12 gene reveals that human CXCL12-a and CXCL12-b are encoded by a single gene that arises by alternative splicing. The strong evolutionary conservation and unique chromosomal localization of the CXCL12 gene suggest that CXCL12-a and CXCL12-b may have important functions distinct from those of other members of the chemokine family. The N-terminus appears to be critical for the activity of CXCL12, in that peptides corresponding to the N-terminal 9 residues of CXCL12 have been shown to bind to CXCR4 and to induce intracellular calcium and chemotaxis in T lymphocytes. The individual peptides have similar activities to CXCL12 but are less potent. A dimer of CXCL12 amino acid residues 19 demonstrates enhanced activity. The Nterminal peptides can act as CXCR4 agonists or antagonists. Nuclear magnetic resonance spectroscopy demonstrates that CXCL12 is a monomer with a disordered N-terminal region (residues 1 8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution (Figure 1).

Introduction
Stromal cell-derived factor-1 (SDF-1) was discovered independently by three different groups and has been known as pre-B-cell growth-stimulating factor/ stromal cell-derived factor-1 (PBSF/SDF-1) or TPA repressed gene 1 (TPAR1). SDF-1 is referred to as CXCL12 according to the consensus chemokine nomenclature. There are two alternatively spliced variants of CXCL12, CXCL12-a (68 residues) and CXCL12-b (72 residues), which have been shown to have different functions. A new isoform, CXCL12-g, was recently described. Subsequent studies show CXCL12 to be a very efcient chemoattractant for lymphocytes and monocytes. CXCL12 is constitutively expressed in a wide range of tissues and the CXCL12

CHEMOKINES, CXC / CXCL12 (SDF-1) 391

5 flanking region 5 GCGCG CXCL12(a) CXCL12-

Exon 1

Exon 2

Exon 3

Exon 4 3

89 amino acids 93 amino acids

Isoform splice site

Residues 123456789 N-terminus

Refresh motif 12-17 C-terminus

9 amino acid binding site Residues 3 N-terminus Residues 3 N-terminus (b)

Loop region docking site

Serum cleavage 67 CXCL12C-terminus

72 CXCL12C-terminus

Figure 1 (a) Schematic cartoon of the CXCL12 gene demonstrating the approximate splice site for CXCL12-a and CXCL12-b. (b) Line diagram of CXCL12 structure with binding and docking sites and sites of cleavage to create CXCL12-a and CXCL12-b isoforms. *Only Lys-1 and Pro-2 are directly involved in receptor activation.

Regulation of Production and Activity

Synthesis

CXCL12 is produced by a wide variety of cell types. mRNA for CXCL12 has been found in lung, brain, heart, liver, kidney, spleen, lymph nodes, stomach, gastrointestinal tract, muscle, lung-derived broblasts, and especially high levels are found in bone marrow stromal cells, especially those that support B cell lymphopoiesis. CXCL12 is regulated through complicated interactions among cytokines/growth factors, chemokines, and other regulatory factors. For example, CXCL12 gene expression is regulated by the transcription factor hypoxia-inducible factor1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of CXCL12 in ischemic tissues in direct proportion to reduced oxygen tension. Interferon gamma signicantly reduces CXCR4 expression and SDF-1-induced cell migration and proliferation of CXCR4-positive cells.
Processing

then at the N-terminus to produce CXCL12-a (367). By contrast, full-length CXCL12-b (172) is processed only at the N-terminus to produce CXCL12-b (372). Serum processing of CXCL12-a at the C-terminus reduces the ability of the polypeptide to bind to heparin and to stimulate B cell proliferation and chemotaxis. The additional processing at the N-terminus renders both forms of CXCL12 unable to bind to heparin and activate cells. The differential processing of CXCL12-a and CXCL12-b allows precise regulation of CXCL12s biologic activity (Figure 1). The processing of CXCL12-g has not been determined.
Activation

CXCL12-a and CXCL12-b are secreted as full-length molecules. When exposed to human serum, fulllength CXCL12-a (168) undergoes processing rst at the C-terminus to produce CXCL12-a (167) and

The binding of CXCL12 to its receptor, CXCR4, results in the activation of several signaling pathways. It has been suggested that after binding to CXCR4, CXCL12 initially triggers dimerization of the receptor. In addition, there is evidence that CXCR4 has to be included in membrane lipid rafts in order to more efciently interact with other surface receptors, downstream proteins of signal transduction pathways, and therefore to induce chemotaxis. CXCR4 activation leads to dissociation of the heterotrimeric protein complex (Gabg) to a and bg subunits that

392 CHEMOKINES, CXC / CXCL12 (SDF-1)

CXCL12 Plasma membrane CXCR4 Src ptk Gi PDK1 Ras mTOR Akt PTEN SHIP Rho Rac cdc42 PIP3 JAKS P Integrins

Gi

P13Ky

Fak src

Pax

Raf 1 B PKC mek Erk1/2 NF- B Actin polymerization Cytoskeletal rearrangements Chemotaxis

Transcriptional activation Cell growth/survival

Figure 2 Simplied version of signal transduction pathways activated by the CXCL12/CXCR4 axis that are relevant to cell growth, proliferation, and chemotaxis. Activation of these pathways varies among cell types.

results in phosphorylation of the tyrosine residues of the C-terminus via JAK2 and JAK3 kinases. The phosphorylated CXCR4/CXCL12 complex is then rapidly internalized through a mechanism involving G-protein-coupled receptor kinases followed by the binding of b-arrestin. This process terminates CXCR4 receptor signaling; and activates a number of signaling pathways, including calcium ux, and focal adhesion components, such as proline-rich kinase-2 (Pyk-2), p130Cas, focal adhesion kinase, paxilin, Crk, Crk-L, protein kinase C, phospholipase C-g, mitogen-activated protein kinase (MAPK) p42/ 44-ELK-1, PI3-kinase, AKT, and nuclear factor kappa B (NF-kB) cascades. PI3-kinase ultimately signals through Rho/Rac/cdc42 to upregulate downstream effector proteins involved in actin polymerization and cytoskeletal rearrangements resulting in chemotaxis and adhesion molecule upregulation. CXCL12 activation of CXCR4 under conditions in which the receptor is stimulated with less than saturable concentrations results in movement of CXCR4 into clathrin-coated pits and from there into endosomes, for trafcking back to the plasma membrane to be re-expressed on the cell surface. However, if CXCR4 is exposed to prolonged saturating concentrations of CXCL12 a signicant proportion of CXCR4 from endosomes moves to the lysosome for degradation.

Hematopoietic cells lacking protein tyrosine phosphatases (SHIP1 and SHIP2) have altered chemotaxis to CXCL12, implying that they are important in modulating activation by CXCR4 and the absence of membrane-expressed hematopoietic phosphatase, CD45, also results in reduced chemotaxis in lymphocytes. After CXCL12 stimulation, CD45 has also been shown to interact with CXCR4 in the membrane lipid rafts (Figure 2).

Biological Functions
Development

Targeted disruption of the CXCL12 gene in mice results in perinatal mortality at around e15.5. The numbers of B cell progenitors in mutant embryos are severely reduced in fetal liver and bone marrow, but myeloid progenitors are only reduced in the bone marrow and not in the fetal liver, indicating that CXCL12 is responsible for B cell lymphopoiesis and bone marrow myelopoiesis. Similarly, mice decient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-decient mice have severely reduced B cell lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-cell lymphopoiesis is unaffected.

CHEMOKINES, CXC / CXCL12 (SDF-1) 393

Furthermore, CXCR4 is expressed in developing vascular endothelial cells, and mice lacking CXCR4 or CXCL12 have defective formation of the large vessels supplying the gastrointestinal tract. They are also defective in cardiogenesis and have abnormal cerebellar development. The similarities between the CXCL12- and CXCR4-decient mice indicate that CXCR4 is the primary physiological receptor for CXCL12, suggesting a monogamous relationship between CXCR4 and CXCL12. This receptor ligand selectivity is unusual among chemokines and their receptors, as it is common to have several ligands that bind to one receptor.

Cell Adhesion

CXCL12 via CXCR4 increases adhesion of cells to brinogen, bronectin, stroma, and endothelial cells, through activation of adhesion molecules, such as integrins.
Cell Secretion

After stimulation by CXCL12, cells secrete more matrix metalloproteinases (e.g., MMP-9), nitric oxide, and vascular endothelial growth factor (VEGF), probably through activation of the NF-kB pathway.
Cell Proliferation and Survival

Trafcking

Varying SDF-1 expression levels in different cells and tissues create a chemokine gradient that regulates trafcking of cells that express the CXCR4 receptor namely: hematopoietic stem/progenitor cells such as CD34 hematopoietic stem cells, pre-B cells, and T cells (in particular naive T cells). In addition, more recently, CXCR4 has been found on primordial germ cells, neural stem cells, liver stem cells, muscle stem cells, retinal pigment epithelial progenitor cells, intestinal epithelial cells, brocytes/circulating mesenchymal stem cells, and circulating progenitor epithelial cells in regeneration of tissue after injury, implying that the CXCL12/CXCR4 biological axis is also important in trafcking of nonhematopoietic stem/progenitor cells.

In some experimental conditions, CXCL12 was found to stimulate proliferation and survival of hematopoietic cells, astrocytes, and some tumor cell lines through activation of the PI3-kinase-AKT and MAPK p42/44 pathways. CXCL12 has also been found to be a survival factor for glioblastoma cells and induces proliferation in a dose-dependent manner. This proliferation correlates with phosphorylation and activation of both ERK 1/2 and AKT, and these kinases are independently involved in glioblastoma cell proliferation.

Receptor
CXCR4 is a G-protein-coupled seven transmembrane receptor that was originally cloned as an orphan chemokine receptor and was known as LESTR or fusin. CXCR4 is expressed on the cell surface of most leukocytes, including all B cells, and monocytes and most T lymphocyte subsets, but just weakly on NK cells. It is also expressed on nonhematopoietic cells such as endothelial cells and epithelial cells, and adult stem cells such as brocytes and circulating progenitor epithelial cells. CXCR4 is also an essential cofactor for T-tropic HIV-1 and HIV-2 env-mediated fusion and entry into CD4 lymphocytes. Recently, a second alternatively spliced CXCR4 receptor was cloned and named CXCR4-Lo. CXCR4Lo has lower gene expression in most tissues than CXCR4 except in the spleen and lung; its function is not yet known.

Cell Motility and Chemotaxis

CXCL12 has been shown to induce cell motility through rearrangement of F-actin bundles. CXCL12 may accumulate in tissues after binding to heparin sulfate proteoglycans, which then provides a directional CXCL12 gradient for chemotaxis. CXCL12induced cell movement can be inhibited by blocking the PI3Kinase-AKT pathway in many cell lines, including A549 and H157 non-small cell lung cancer (NSCLC) cell lines. Interestingly, metastases of human NSCLC cells in severe combined immunodeciency (SCID) mice are 99% positive for CXCR4 expression, whereas signicantly fewer cells of the primary tumor express CXCR4. Under hypoxic conditions, nuclear HIF-1a induces increased levels of CXCR4 expression on these cancer cell lines, which leads to increased ability of these tumor cells to invade and metastasize. PI3-kinase inhibitors and overexpressing PTEN inhibit activation of HIF-1a and CXCR4 expression.

CXCL12 in Respiratory Diseases

Lung Injury and Repair

Circulating mesenchymal progenitor cells, brocytes, express CXCR4 and trafc to the lungs in response to CXCL12 and mediate brosis in a murine model of bleomycin-induced pulmonary brosis. Specic

394 CHEMOKINES, CXC / CXCL12 (SDF-1)

neutralizing antibodies to CXCL12 inhibit pulmonary recruitment of these circulating brocytes and attenuate lung brosis. In addition, CXCL12 expression is increased in the submucosal glands, ducts, and airway epithelium in a mouse tracheal transplant model, which provides a chemokine gradient for CXCR4 circulating progenitor epithelial cells to trafck and contribute to repair of the injured airway. The mucus in the submucosal gland lumen provides proteoglycans to depot CXCL12 and create the CXCL12 gradient for chemotaxis. The CXCL12 expression in epithelial cells moves apically to the airway surface during regeneration of the airway. Moreover, CXCL12 can induce neovascularization, which is critical in acute and chronic lung injury and repair. For example, bronchial biopsy specimens from patients with asthma show a signicant increase in the number of blood vessels and colocalization of CXCL12-positive endothelial cells together with CXCL12-positive macrophages and T lymphocytes in the submucosa compared with control subjects. These ndings suggest that CXCL12 may play a role in remodeling of asthmatic airways via angiogenesis.
Lung Cancer

Small cell lung cancer (SCLC) cells express high levels of functional CXCR4 receptors for CXCL12. CXCL12 induces integrin activation, which results in increased adhesion of SCLC cells to bronectin and collagen. This is mediated by activation of integrins and CXCR4, and is inhibited by CXCR4 antagonists. Stromal cells protect SCLC cells from chemotherapy-induced apoptosis, and this protection can also be antagonized by CXCR4 inhibitors. Renal cell carcinoma commonly metastasizes to other organs such as the adrenal gland and lung, both of which express high levels of CXCL12. Both heterotopic and orthotopic mouse models of tumors from a renal carcinoma cell line develop lung metastases, which can be abrogated by treating the mice with neutralizing antibodies to CXCL12. In summary, CXCL12 is a pleiotropic chemokine with a variety of functions beyond that of leukocyte trafcking that are relevant to the lung.
See also: Adhesion, CellCell: Epithelial. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells. Extracellular Matrix: Basement Membranes; Collagens; Matrix Proteoglycans; Surface Proteoglycans. Fibroblasts. G-Protein-Coupled Receptors. Human Immunodeciency Virus. Hypoxia and Hypoxemia. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; T cells. Matrix Metalloproteinases. Myobroblasts. Pulmonary Fibrosis. Signal Transduction. Stem Cells. Vesicular Trafcking.

CXCL12 is an important molecule in lung cancer invasion and metastasis and is also important in the development of lung metastases from tumors from other organs (e.g., breast, renal cell carcinoma, rhabdomyosarcoma, etc.). Both NSCLC tumor specimens resected from patients and NSCLC cell lines express CXCR4, but not CXCL12. NSCLC cell lines undergo chemotaxis in response to CXCL12. CXCL12CXCR4 activation of NSCLC cell lines show PI3 kinase activation, intracellular calcium mobilization and MAPK activation with enhanced ERK-1/2 phosphorylation without change in either proliferation or apoptosis. Target organs in a murine model that are the preferred destination of human NSCLC metastases produce higher levels of CXCL12 than the primary tumor, thereby creating a chemotactic gradient. The administration of specic neutralizing anti-CXCL12 antibodies to SCID mice with human NSCLC abrogates organ metastases, without affecting primary tumor-derived angiogenesis. The CXCL12CXCR4 biological axis is therefore involved in regulating the metastasis of NSCLC. In addition, the ability of NSCLC cells to metastasize correlates with their expression of CXCR4, which is upregulated under conditions of hypoxia through HIF-1a. Inhibition of PI3-kinase or overexpression of PTEN reduces CXCR4 expression and their metastatic potential.

Further Reading
Gazitt Y (2004) Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specic antibodies. Leukemia 18: 110. Kucia M, Jankowski K, Reca R, et al. (2004) CXCR4-SDF-1 signaling, locomotion, chemotaxis and adhesion. Journal of Molecular Histology 35: 233245. Luster AD (1998) Chemokineschemotactic cytokines that mediate inammation. New England Journal of Medicine 338: 436 445. Mackay CR (2001) SDF-1. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 11191127. San Diego: Academic Press. Murdoch C (2000) CXCR4: chemokine receptor extraordinaire. Immunology Reviews 177: 175184. Murphy PM (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227229. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating brocytes trafc to the lungs in response to CXCL12 and mediate brosis. Journal of Clinical Investigation 114: 438446.

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395

IL-8
R M Strieter, M P Keane, and J A Belperio, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

heparin-binding domain, which may be important in binding to glycosaminoglycans.

Regulation of Production and Activity

The gene for CXCL8/IL-8 is found on human chromosome 4, q1221, and consists of four exons and three introns. The 50 -anking region of CXCL8/IL-8 contains the usual CCAAT and TATA box-like structures. In addition, this region has a number of potential binding sites for several nuclear factors. The CXCL8/IL-8 promoter region is regulated in a cell-specic fashion requiring a nuclear factor kappa B (NF-kB) element plus either activating protein 1 (AP-1) or a C/EBP (NF-IL-6) element under conditions of transcriptional induction with tumor necrosis factor alpha (TNF-a) or interleukin-1 (IL-1). Although the CXCL8/IL-8 promoter has two C/EBP (50 and 30 ) cis-elements with NF-kB nested between them, the 50 -C/EBP element appears to be the only C/ EBP element involved in transcriptional regulation of CXCL8/IL-8. In specic cell lines, the AP-1 along with NF-kB or C/EBP and NF-kB cis-elements is sufcient for full transcriptional activation of the CXCL8/IL-8 promoter. Members of the NF-kB/rel and C/EBP families of transcriptional factors can interact on a protein:protein level via the Rel:basic leucine zipper (bZIP) domain of these proteins, respectively. In order for full transcriptional activation of the promoter, there is a cooperative interaction between all three major cis-elements (AP-1, 50 -C/EBP and NF-kB). While the promoter region of CXCL8/ IL-8 gene has been extensively studied, the mechanisms related to signal transduction and optimal transactivation of the CXCL8/IL-8 gene have been less studied. Mitogen-activated protein (MAP) kinases are necessary to optimally induce the gene expression and protein production of CXCL8/IL-8 from airway epithelial cells in response to TNF-a. TNF-a activation of the MAP kinases is able to achieve the induction of CXCL8/IL-8 gene expression and protein production by NF-kB-dependent, -independent, and posttranscriptional mechanisms. Direct inhibition of the MAP kinases (extracellular regulated kinase (ERK) and c-jun N-terminal protein kinase (JNK)) and NF-kB, but not p38, decreases TNF-a-induced transcription of the CXCL8/IL-8 promoter. Inhibition of JNK signaling reduces TNFa-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-dependent manner, whereas inhibition of ERK impairs TNF-a-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-independent and AP-1-dependent manner. Activation of the p38 MAP kinase is important for promoting TNFa-induced CXCL8/IL-8 protein production in a

Abstract
CXCL8/IL-8 is a member of the CXC chemokine family. The CXC chemokines can be further divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (GluLeuArg; ELR motif) that precede the rst cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. CXCL8/IL-8 has been shown to have an important role in the recruitment of neutrophils in bacterial pneumonia and to correlate with the development and mortality of adult respiratory distress syndrome. Furthermore, CXCL8/IL-8 also has a key role in the vascular remodeling that is seen in pulmonary brosis.

Introduction
CXCL8 is a member of the CXC chemokine family and is also known as IL-8. The CXC chemokines can further be divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (GluLeu Arg; ELR motif) that precede the rst cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors.

Structure
The precursor molecule of CXCL8/IL-8 consists of 99 amino acids with an associated 20 amino acid signal sequence. Several mature forms of CXCL8/IL8 have been identied, that are a result of repeated NH2-terminal amino acid cleavage, including the major 72 amino acid mature form. The 72 amino acid form of CXCL8/IL-8 binds to neutrophils twofold more than the 77 amino acid form, and is twoto threefold more potent in inducing cytochalasin B-treated neutrophil degranulation. Interestingly, the 77 amino acid form can induce apoptosis in leukemic cells, whereas the 72 amino acid form does not. The NH2-terminus of CXCL8/IL-8, similar to other CXC chemokines that bind and activate neutrophils, contains three highly conserved amino acid residues consisting of GluLeuArg (the ELR motif). The COOH-terminus of CXCL8/IL-8 contains a

396 CHEMOKINES, CXC / IL-8

posttranscriptional manner. These ndings would also be relevant to downstream signal transduction events related to activation of Toll-like receptors and IL-1 type I receptor for the induction of CXCL8/IL-8 gene expression and protein production.

acid residues that may be important in phosphorylation and signal coupling via G proteins.

CXCL8/IL-8 in Respiratory Disease

CXC chemokines have been found to play a significant role in mediating neutrophil inltration in the lung parenchyma and pleural space in response to endotoxin and bacterial challenge. Passive immunization with neutralizing CXCL8/IL-8 antibodies blocked 77% of endotoxin-induced neutrophil inux in the pleura of rabbits. However, in the context of microorganism invasion, depletion of a CXC chemokine and reduction of inltrating neutrophils may have a major impact on the host (Figure 2). CXCL8/IL-8 has been implicated in mediating neutrophil sequestration in the lungs of patients with pneumonia. CXCL8/IL-8 has been found in the bronchoalveolar lavage of patients with community acquired pneumonia and nosocomial pneumonia. While there is no murine structural homolog for CXCL8/IL-8, both CXCL1 and CXCL2/3 have been found in murine models of Klebsiella pneumoniae, Pseudomonas aeruginosa, Nocardia asteroides, and
Host defense/pneumonia Acute lung injury/ARDS CXCL8 Pulmonary fibrosis Angiogenesis Tumor growth
Figure 2 Role of CXCL8/IL-8 in respiratory disease.

Biological Function
CXCL8/IL-8 is a potent recruiter of neutrophils and induces angiogenesis. It is also chemotactic for eosinophils. CXCL8/IL-8 has dose-dependent effects on basophils, which can lead to either stimulation or inhibition of release of both LTB4 and histamine. CXCL8/IL-8 also leads to activation of basophils with enhanced binding of basophils to endothelial cells. CXCL8/IL-8 is not chemotactic for monocytes; however, stimulation of monocytes with CXCL8/ IL-8 leads to a rise in cytosolic calcium and generation of reactive oxygen species. CXCL8/IL-8 can selectively inhibit the IL-4-induced IgE and IgG4 production from B cells. CXCL8/IL-8 can induce migration and proliferation of keratinocytes (Figure 1).

Receptors
Chemokine activities are mediated through pertussissensitive G-protein-coupled receptors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. While the transmembrane and the second and third intracellular/cytoplasmic domains of these receptors are well conserved, the NH2- and COOH-terminal ends of these receptors are variable. The intracellular COOH-terminus of these receptors is rich in serine and threonine amino

Monocyte/macrophage Lymphocytes

Epithelial cells Endothelial cells Fibroblasts Neutrophils

CXCL8

Chemotaxis Neutrophils Eosinophils Keratinocytes Basophils

Chemotaxis

Endothelial cells

Angiogenesis
Figure 1 Source and biological functions of CXCL8/IL-8.

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397

Aspergillus fumigatus pneumonia. In a model of A. fumigatus pneumonia, neutralization of TNF resulted in marked attenuation of the expression of CXCL1 and CXCL2/3 that was paralleled by a reduction in the inltration of neutrophils and associated with increased mortality. In addition, Laichalk and associates administered a TNF agonist peptide consisting of the 11 amino acid TNF binding site (TNF7080) to animals intratracheally inoculated with K. pneumoniae and found markedly elevated levels of CXCL2/3 associated with increased neutrophil inltration. Depletion of CXCL2/3 during the pathogenesis of murine K. pneumoniae pneumonia resulted in a marked reduction in the recruitment of neutrophils to the lung that was paralleled by increased bacteremia and reduced bacterial clearance in the lung. Since ELR CXC chemokine ligands in the mouse use the CXC chemokine receptor, CXCR2, Standiford and associates used specic neutralizing antibodies to CXCR2 and demonstrated that blocking CXCR2 results in markedly reduced neutrophil inltration in response to P. aeruginosa, N. asteroides, and A. fumigatus pneumonias. The reduction in neutrophil elicitation was directly related to reduced clearance of the microorganisms and increased mortality in these model systems. These studies have established the critical importance of ELR CXC chemokines in acute inammation and the innate immune response to a variety of microorganisms. Sekido and associates demonstrated that CXCL8/ IL-8 significantly contributed to reperfusion lung injury in a rabbit model of lung ischemia-reperfusion injury. Reperfusion of the ischemic lung resulted in the production of CXCL8/IL-8, which correlated with maximal pulmonary neutrophil inltration. Passive immunization of the animals with neutralizing antibodies to CXCL8/IL-8 prior to reperfusion of the ischemic lung prevented neutrophil extravasation and tissue injury, suggesting a causal role for CXCL8/IL-8 in this model. Ventilator-induced lung injury in a murine model is associated with increased expression of CXCL1 and CXCL2 that parallels lung injury and neutrophil recruitment. Furthermore, these levels correlated with NF-kB activation. CXCR2/ mice were protected from ventilatorinduced lung injury. Several studies have demonstrated that CXCL8/ IL-8 levels correlate with the development and mortality of the acute respiratory distress syndrome. Of particular interest is the study of Donnelly and colleagues, which correlated early increases in CXCL8/IL-8 in bronchoalveolar lavage uid (BALF) with an increased risk of subsequent development of adult respiratory distress syndrome (ARDS), and also

demonstrated that alveolar macrophages were an important source of CXCL8/IL-8 prior to neutrophil inux. High concentrations of CXCL8/IL-8 were found in BALF from trauma patients, some within 1 h of injury and prior to any evidence of significant neutrophil inux. Patients who progressed to ARDS had significantly greater BALF levels of CXCL8/IL-8 than those who failed to develop this condition. Levels of CXCL8/IL-8 in plasma, as opposed to lavage, were not found to be significantly different between patients who did or did not develop ARDS. Furthermore, there is an imbalance in the expression of ELR (including CXCL8/IL-8) as compared to ELR CXC chemokines from BALF of patients with ARDS as compared to controls. This imbalance correlated with angiogenic activity and both procollagen I and procollagen III levels in BALF. These ndings suggest that CXCL8/IL-8 and other CXC chemokines have an important role in the broproliferative phase of ARDS via the regulation of angiogenesis. Idiopathic pulmonary brosis (IPF) is a disease of unknown etiology that is characterized by the accumulation of neutrophils within the airspace and mononuclear cells within the interstitium, followed by the progressive deposition of collagen within the interstitium and subsequent destruction of lung tissue. While the mechanisms of cellular injury and the role of classic inammatory cells remain unclear, increases in neutrophils in BALF and in lung tissue have been demonstrated from patients with IPF. While the number or proportion of neutrophils in BALF does not correlate with activity of alveolitis and has limited prognostic value, declines in BALF neutrophils typically occur among patients exhibiting favorable responses to therapy. Neutrophilic alveolitis has been described in humans with IPF, collagen vascular diseases with associated ILD, as well as diverse animal models of pulmonary brosis. CXCL8/IL-8 is significantly elevated in IPF, as compared to either normal or sarcoidosis patients, and correlates with BALF presence of neutrophils. The alveolar macrophage is an important cellular source of CXCL8/IL-8 in IPF. In addition, these studies have suggested that levels of CXCL8/IL-8 in IPF may correlate with a worse prognosis. While studies have suggested an important role for CXCL8/IL-8 in mediating neutrophil recruitment, CXC chemokines have been found to exert disparate effects in regulating angiogenesis. This latter issue is relevant to IPF, as the pathology of IPF demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, broproliferation, and deposition of extracellular matrix, leading to progressive brosis and loss of lung function. There have been limited investigations to delineate factors that

398 CHEMOKINES, CXC / CXCL9 (MIG)

may be involved in the regulation of this angiogenic activity during pulmonary brosis. In IPF lung tissue there is an imbalance in the presence of CXC chemokines that behave as either promoters of angiogenesis CXCL8/IL-8 or inhibitors of angiogenesis CXCL10. This imbalance favors augmented net angiogenic activity. Lung tissue from IPF patients has elevated levels of CXCL8/IL-8, as compared to control lung tissue, and demonstrates in vivo angiogenic activity that can be significantly attributed to CXCL8/IL-8. Immunolocalization of CXCL8/IL-8 demonstrated that the pulmonary broblast was the predominant interstitial cellular source of this chemokine, and areas of CXCL8/IL-8 expression were essentially devoid of neutrophil inltration. This supports an alternative biological role for CXCL8/IL-8 or other ELR CXC chemokines in the interstitium of IPF lung tissue. The pulmonary broblast is the predominant cellular source of CXCL8/IL-8 in the interstitium of IPF, supporting the notion that the pulmonary broblast has a pivotal role in mediating the angiogenic activity during the pathogenesis of IPF. Indeed, the pulmonary broblast has received increasing attention as a pivotal cell in the pathogenesis of IPF. Relative levels of CXCL8/IL-8 and CXCL10 from IPF pulmonary broblast conditioned media demonstrated a significant imbalance favoring CXCL8/IL-8-induced angiogenic activity. In contrast, normal pulmonary broblasts had greater levels of CXCL10 that favored a net inhibition of angiogenesis. The difference in expression of CXCL8/IL-8 and CXCL10 between IPF and control pulmonary broblasts lends further support to the notion of a phenotypic difference between IPF and normal pulmonary broblasts, which has been well described.
See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Chemokines, CXC: CXCL1 (GRO1)CXCL3 (GRO3). Extracellular Matrix: Basement Membranes. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells. Pneumonia: Overview and Epidemiology. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

pneumonia and adult respiratory distress syndrome. Infection and Immunity 61: 4553. Donnelly SC, Strieter RM, Kunkel SL, et al. (1993) Interleukin-8 and development of adult respiratory distress syndrome in atrisk patient groups. Lancet 341: 643647. Greenberger MJ, Strieter RM, Kunkel SL, et al. (1996) Neutralization of macrophage inammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia. Journal of Infectious Disease 173: 159. Jordana M, Schulman J, McSharry C, et al. (1988) Heterogeneous proliferative characteristics of human adult lung broblast lines and clonally derived broblasts from control and brotic tissue. American Review of Respiratory Disease 137: 579. Keane MP, Arenberg DA, Lynch JP, et al. (1997) The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary brosis. Journal of Immunology 159: 1437. Keane MP, Donnelly SC, Belperio JA, et al. (2002) Imbalance in the expression of CXC chemokines correlates with bronchoalveolar lavage uid angiogenic activity and procollagen levels in acute respiratory distress syndrome. Journal of Immunology 169: 6515. Koch AE, Polverini PJ, Kunkel SL, et al. (1992) Interleukin-8 (IL8) as a macrophage-derived mediator of angiogenesis. Science 258: 1798. Laichalk LL, Bucknell KA, Huffnagle GB, et al. (1998) Intrapulmonary delivery of tumor necrosis factor agonist peptide augments host defense in murine Gram-negative bacterial pneumonia. Infection and Immunity 66: 2822. Lynch JP, Standiford TJ, Kunkel SL, Rolfe MW, and Strieter RM (1992) Neutrophilic alveolitis in idiopathic pulmonary brosis: the role of interleukin-8. American Review of Respiratory Disease 145: 1433. Sheppard D (2001) Pulmonary brosis: a cellular overreaction or a failure of communication? Journal of Clinical Investigation 107: 1501. Southcott AM, Jones KP, Li D, et al. (1995) Interleukin-8, differential expression in lone brosing alveolitis and systemic sclerosis. American Journal of Respiratory and Critical Care Medicine 151: 1604. Strieter RM (2002) Interleukin-8: a very important chemokine of the human airway epithelium. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L688. Strieter RM, Kunkel SL, Elner VM, et al. (1992) Interleukin-8: a corneal factor that induces neovascularization. American Journal of Pathology 141: 1279. Strieter RM, Kunkel SL, Showell H, et al. (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-1b. Science 243: 1467. Strieter RM, Polverini PJ, Kunkel SL, et al. (1995) The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. Journal of Biological Chemistry 270: 27348.

CXCL9 (MIG)
Further Reading
Belperio JA, Keane MP, Burdick MD, et al. (2002) Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilatorinduced lung injury. Journal of Clinical Investigation 110: 1703. Broaddus VC, Boylan AM, Hoeffel JM, et al. (1994) Neutralization of IL-8 inhibits neutrophil inux in a rabbit model of endotoxin-induced pleurisy. Journal of Immunology 152: 2960. Chollet-Martin S, Montravers P, Gibert C, et al. (1993) High levels of interleukin-8 in the blood and alveolar spaces of patients with

P C Fulkerson and M E Rothenberg, Cincinnati Childrens Hospital, Cincinnati, OH, USA

& 2006 Elsevier Ltd. All rights reserved.

Abstract
Human CXCL9 was identied in 1993 by a differential hybridization screening technique, comparing cDNA libraries

CHEMOKINES, CXC / CXCL9 (MIG) 399

obtained from an unstimulated and interferon gamma (IFN-g)activated macrophage cell line. CXCL9 is a member of the alpha or CXC subfamily of chemokines. The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif GluLeuArg (ELR) N-terminal to the conserved CXC region. CXCL9, a non-ELR-containing CXC chemokine, mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes, preferentially of the Th1 phenotype, which express high levels of CXCR3. The expression of CXCL9 is induced primarily by the Th1-associated cytokine IFN-g and correlates with tissue inltration of T lymphocytes in a number of Th1-associated diseases, suggesting that CXCL9 plays an important role in the regulation of effector cell recruitment to sites of inammation. Remarkably, CXCL9 also regulates eosinophil accumulation in experimental asthma, a Th2-associated lung disease. In addition, CXCL9 has potent angiostatic activity, inhibiting blood vessel growth in wound repair and inhibiting tumor growth and tumor-associated vessel expansion.

preferentially of the Th1 phenotype, which express high levels of CXCR3.

Structure
CXCL9 is a member of the alpha or CXC subfamily of chemokines. CXC chemokines have a single amino acid that separates invariant cysteines one and two (of four). The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif GluLeuArg (ELR) N-terminal to the conserved CXC region. The gene coding for CXCL9, a nonELR-containing CXC chemokine, maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice (Figure 1). The 2.5 kb human mRNA transcript and 1.3 kb murine transcript is translated into a 125 and 126 amino acid protein, respectively. The human and murine CXCL9 proteins are 68% homologous. Despite differences in primary sequences, chemokines have a remarkably similar three-dimensional structure, comprised of a short N-terminal region, a large core, which is stabilized by disulde bonds and hydrophobic interactions, and a C-terminal a-helix. The most highly conserved sequence among chemokines is the four cysteine residues. The rst and third cysteines and the second and fourth cysteines are linked by conserved disulde bonds. While chemokines share four common secondary structural elements, three b-strands arranged in a single antiparallel b-sheet and one a-helix, most of the variation in chemokine amino acid sequence occurs in the Nand C-terminal regions. Structurefunction studies have shown that chemokines have two main sites of interaction with their receptors, one in the N-terminus and the other in the N-loop region after the second cysteine. Deletion of just a few of the N-terminal

Introduction
Cytokines play a central role in macrophage physiology, both as macrophage activators (e.g., interferons (IFN)) and mediators of macrophage activity (e.g., chemokines). Monokine induced by interferon gamma (IFN-g) (MIG, CXCL9) was discovered in an effort to identify genes induced during macrophage activation. A differential hybridization screening technique, comparing cDNA libraries obtained from an unstimulated and IFN-g-activated mouse macrophage cell line, was utilized to initially identify the murine CXCL9 gene. The murine CXCL9 cDNA was then used to screen a cDNA library made from an IFN-g-activated human macrophage cell line. This led to the identication of a new member of the chemokine gene family, human MIG/CXCL9, in 1993. CXCL9 mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes,
Human CXCL9/SCYB9 gene Exon 1 (a) Exon 2 Exon 3

Exon 4

Murine CXCL9/Scyb9 gene Exon 1 (b) CDS

Figure 1 Human and murine CXCL9 gene organization. The gene coding for CXCL9 maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice. CDS, coding sequence.

Exon 2

Exon 3

Exon 4

400 CHEMOKINES, CXC / CXCL9 (MIG)

residues of CXCL9 results in a loss of receptor binding capacity. In addition, the non-ELR CXC chemokines contain a C-terminal segment rich in positively charged amino acids. Notably, CXCL9 has a unique extended basic C-terminal region, which when truncated results in a dramatic decrease in its biological activity.

Regulation of CXCL9 Production and Activity

The IFNs are an important group of cytokines that mediate some of their biological effects through regulation of specic RNA and protein expression in a responding cell. The expression of CXCL9 is primarily dependent on the Th1 cytokine IFN-g, but in its absence, IFN-a or IFN-b is able to induce CXCL9 expression. The IFN-g-induced transcriptional activation of CXCL9 depends upon responsive elements in the promoter region of the gene, which are recognized by signal transducers and activators of transcription (STAT)1 or STAT1-containing factors. The promoter of CXCL9 also contains regions that mediate transcriptional activation in response to tumor necrosis factor alpha (TNF-a). IFN-g and TNF-a synergize to induce the expression of several genes, including CXCL9. In addition to inducing expression, transcription factors modulate inammatory responses by negatively regulating chemokine expression. The Th2associated transcription factor STAT6 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-g) have been shown to inhibit IFN-g-induced expression of CXCL9. The N-terminal region of most chemokines is crucial for receptor binding and signaling activities.

Amino peptidases or endopeptidases that process chemokines at the N-terminus play an important role in the regulation of chemokine activity. The membrane-associated protease dipeptidyl peptidase IV/CD26 cleaves two amino acids from polypeptides with a proline, alanine, or hydroxyproline at the second position. The removal of the N-terminal dipeptide from CXCL9 results in decreased receptor binding capacity and a dramatic decrease in chemotactic activity. In addition, matrix metalloproteinase (MMP) 9, another class of chemokine-processing proteases, cleaves CXCL9 within its extended C-terminus. C-terminal truncation of CXCL9 results in a significant decrease in its biological activity. Since the highly basic C-terminal segment of CXCL9 may associate with extracellular matrixpoteoglycans, localization and establishment of chemotactic gradients may also be affected by protease processing of CXCL9.

Biological Function
Chemokines are chemotactic cytokines that orchestrate the migration and activation of leukocyte populations under baseline (homeostatic) and inammatory conditions. The predominant function of CXCL9 is the recruitment of primed T lymphocytes to the sites of inammation (Figure 2). CXCL9 has been associated with the inltration and retention of activated T lymphocytes in inammatory diseases of the skin, joints, and central nervous system. Furthermore, studies have implicated CXCL9 as a critical mediator of primed T-lymphocyte trafcking in transplant models. In addition to promoting leukocyte recruitment, chemokines can also function as natural

Angiostasis inhibits capillary growth

CXC R3

CXCR3 Promotes T and B lymphocyte recruitment and function

CCR3 Inhibits eosinophil recruitment

Figure 2 CXCL9 regulates lymphocyte and eosinophil chemoattraction and inhibits angiogenesis. The predominant function of CXCL9 is the recruitment of primed T lymphocytes expressing high levels of CXCR3 to the sites of inammation. In addition to promoting leukocyte recruitment, chemokines can also function as natural antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. In addition, CXCL9 inhibits blood vessel growth in wound repair and tumor models.

CHEMOKINES, CXC / CXCL9 (MIG) 401

antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. Although cell trafcking is considered the central task of chemokines, there is increasing evidence that a variety of other leukocyte functions may also be modulated by chemokines. CXCL9 has also been shown to stimulate effector functions, such as cytokine production (by recruited T lymphocytes) and promote T-cell proliferation. In a host defense model using gene-targeted mice, murine CXCL9 contributed to the humoral response to a bacterial pathogen, suggesting that CXCL9 not only recruits T cells to sites of inammation, but also maximizes interactions among activated T cells, B cells, and dendritic cells within lymphoid organs. Angiogenesis, the growth of new capillaries from either pre-existing vessels or from progenitor endothelial cells, is an important biological event that is essential to several physiologic and pathologic processes, including development, wound repair, and tumor growth. CXC chemokines have pivotal, yet opposing, roles in the control of inammation and angiogenesis, as a result of the shared expression of their specic receptors by both leukocytes and endothelial cells. The ELR-containing CXC chemokines are potent promoters of angiogenesis. In contrast, members that lack the ELR motif, such as CXCL9, are potent inhibitors of angiogenesis. It is proposed that the angiostatic properties of CXCL9 are important to prevent unlimited vessel growth in wound repair.

and mouse eosinophils. This suggests that the polarization of an inammatory response may be enhanced with the expression of CXCR3 ligands, including CXCL9, that attract Th1 cells, but also concomitantly block the migration of Th2 cells in response to CCR3 ligands.

CXCL9 in Respiratory Diseases

Lung transplantation is a therapeutic option for many patients with end-stage pulmonary disorders. Acute and chronic lung rejection is a common complication. Acute rejection is characterized by perivascular and peribronchiolar leukocyte inltration and is the major risk factor for the development of chronic lung allograft rejection, or bronchiolitis obliterans syndrome (BOS), the leading cause of morbidity and mortality post-lung transplantation. BOS is a chronic inammatory process characterized by persistent peribronchiolar leukocyte inltration that eventually invades and disrupts the basement membrane, submucosa, and airway epithelium. The persistent leukocyte inltration is followed by an aberrant repair process, with increased matrix deposition and granulation tissue formation, ultimately leading to obliteration of airways. Elevated levels of CXCL9 (and the other CXCR3 ligands) have been found in the bronchoalveolar lavage (BAL) uid from patients with acute and chronic lung allograft rejection, as compared with healthy lung transplant recipients. In a murine model of BOS, neutralization of CXCL9, or its receptor CXCR3, resulted in inhibition in the recruitment of mononuclear cells and a marked reduction in extracellular matrix deposition and airway obliteration. Similarly, CXCL9 neutralization resulted in attenuation of intragraft mononuclear cells and decreased parameters in an animal model of acute lung allograft rejection. These studies demonstrate the importance of CXCL9/CXCR3 interactions during both acute and chronic allograft rejection, suggesting that CXCL9 may be an important ligand in promoting the continuum of acute to chronic rejection. Diffuse lung injury is a frequent complication of allogeneic stem cell transplantation (SCT). Idiopathic pneumonia syndrome (IPS), a noninfectious diffuse lung injury, is associated with a high degree of mortality. The pathophysiology of IPS involves toxic damage due to irradiation and chemotherapy, a donor T-cell response, and the production of inammatory cytokines. IPS is characterized by a diffuse pneumonitis involving both alveolar and interstitial space, as well as mononuclear cell inltrates around vascular and bronchial structures. These pathologic changes are further associated with decreased

Receptors
Chemokines mediate their functions through binding to seven-transmembrane-domain receptors coupled to G proteins. The receptors often recognize more than one chemokine, and alternatively, several chemokines can bind to multiple receptors. CXCL9 shares its primary receptor, CXCR3, with two other non-ELR-containing CXC chemokines, CXCL10 and CXCL11. CXCR3 is expressed on a small subset of circulating blood T cells, B cells, and natural killer (NK) cells, but T-cell activation greatly enhances CXCR3 expression. CXCR3 is preferentially expressed on Th1 lymphocytes, suggesting that CXCR3 and its ligands are more active in the setting of Th1driven inammatory responses. Besides being an agonist for CXCR3, CXCL9 has been shown to act as a natural antagonist for CCR3, a Th2-associated chemokine receptor highly expressed on eosinophils. Studies have demonstrated that CXCL9 inhibits CCR3-mediated functional responses in both human

402 CHEMOKINES, CXC / CXCL10 (IP-10)

pulmonary function. In a murine SCT model, increased BAL uid level of CXCL9 is associated with recruitment of CXCR3 expressing donor CD8 T cells to the lung after SCT. Neutralization of CXCL9 reduces the IPS severity and the recruitment of donor T cells to the lung after SCT. Together with the lung rejection studies, the significant decrease in lung injury and pathology in these models has been predominantly attributed to reduced effector cell recruitment and underscores the importance of the CXCL9/CXCR3 interaction in inammatory diseases in the lung. In addition to being associated with the chemoattraction of Th1-associated effector cells into the lung, CXCL9 has been shown to inhibit the accumulation of cells in Th2-mediated asthma models. One of the hallmarks of allergic airway disease is the accumulation of an abnormally large number of inammatory cells, such as eosinophils and T lymphocytes, into the lung. In experimental asthma models, CXCL9 has been shown to negatively regulate eosinophil recruitment into the lung. With no detectable CXCR3 on the surface of eosinophils, CXCL9 inhibited eosinophil chemoattraction in vitro. Moreover, intravenous administration of low doses of CXCL9 (B10 30 mg kg 1) induced strong and specic inhibition of eosinophil recruitment into the lung in response to a variety of stimuli. Importantly, CXCL9 also inhibited a CCR3-mediated functional response, superoxide anion formation, in eosinophils. Regulation of eosinophil accumulation and activation in the lung is important since recent studies have demonstrated dramatic protection from experimental asthma in eosinophil-decient animal models. Tumor cells have evolved the ability to regulate the angiogenic process through an imbalance in the regulatory factors resulting in an environment favoring angiogenesis. Studies have shown that the balance in expression between the angiogenic and the angiostatic CXC chemokines is correlated with the aggressiveness and metastic potential of human nonsmall cell lung carcinoma (NSCLC). Overexpression of CXCL9, an angiostatic CXC chemokine, at the tumor site resulted in inhibition of NSCLC tumor growth and metastasis via a decrease in tumor-derived vessel density. These ndings support the importance of CXCL9 in inhibiting NSCLC tumor growth by attenuation of tumor-derived angiogenesis and demonstrate the potential of delivery of potent angiostatic CXC chemokines as a therapeutic option for NSCLC.
See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: CXCL10 (IP-10). G-Protein-Coupled Receptors. Leukocytes: Eosinophils; T cells.

Further Reading
Baggiolini M (1998) Chemokines and leukocyte trafc. Nature 392: 565568. Baggiolini M, Dewald B, and Moser B (1997) Human chemokines: an update. Annual Review of Immunology 15: 675705. Cascieri MA and Springer MS (2000) The chemokine/chemokinereceptor family: potential and progress for therapeutic intervention. Current Opinion in Chemical Biology 4: 420427. Chada S, Ramesh R, and Mhashilkar AM (2003) Cytokine- and chemokine-based gene therapy for cancer. Current Opinion in Molecular Therapeutics 5: 463474. Farber JM (1997) Mig and IP-10: CXC chemokines that target lymphocytes. Journal of Leukocyte Biology 61: 246257. Liao F, Rabin RL, Yannelli JR, et al. (1995) Human mig chemokine: biochemical and functional characterization. Journal of Experimental Medicine 182: 13011314. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inammation. New England Journal of Medicine 338: 436 445. Moser B and Loetscher P (2001) Lymphocyte trafc control by chemokines. Nature Immunology 2: 123128. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 7584. Park MK, Amichay D, Love P, et al. (2002) The CXC chemokine murine monokine induced by IFN-g (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo. Journal of Immunology 169: 14331443. Romagnani P, Lasagni L, Annunziato F, Serio M, and Romagnani S (2004) CXC chemokines: the regulatory link between inammation and angiogenesis. Trends in Immunology 25: 201209. Rothenberg ME (2000) Basic science of chemokines and their receptors. In: Rothenberg ME (ed.) Chemokines in Allergic Disease, pp. 167. New York: Dekker. Schwarz MK and Wells TN (2002) New therapeutics that modulate chemokine networks. Nature Reviews: Drug Discovery 1: 347358. Zlotnik A and Yoshie O (2000) Chemokines: a new classication system and their role in immunity. Immunity 12: 121127.

CXCL10 (IP-10)
F Kheradmand and D B Corry, Baylor College of Medicine, Houston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interferon-inducible protein of 10 kDa (IP-10, CXCL10) is the tenth member of the CXC family of small chemotactic cytokines and plays an essential role in the recruitment of T-helper-1 (Th1) cells, natural killer (NK) cells, macrophages, and dendritic cells into sites of tissue inammation. In addition to leukocyte trafcking, CXCL10 binds to several G-protein-coupled receptors and induces a variety of cellular effects such as inhibition of endothelial cell proliferation, inhibition of growth factor-dependent hematopoiesis, and tumor necrosis. In humans, diverse cellular responses to CXCL10 are mediated through its two cell surface receptor isoforms that have distinct tissue expression patterns. CXCL10 acts as an antagonist of CCR3, a T-helper-2

CHEMOKINES, CXC / CXCL10 (IP-10)

(Th2) cell-specic chemokine receptor through competition with eotaxin (CCL11), a functional ligand for this receptor. Modulation of CXCL10 is implicated as a mediator of several pathological conditions of the lung such as sarcoidosis, pulmonary brosis, emphysema, and asthma. In mice, overexpression of CXCL10 results in exaggerated Th2 immune responses to antigen in a murine model of allergic lung disease, whereas deletion of this gene results in worsening of pulmonary brosis in a bleomycin-based model of acute lung injury. Excessive production of CXCL10 by human lung T cells is associated with smoking-induced emphysema. Activated human macrophages obtained from the lungs of patients with emphysema showed high expression of CXCR3, a functional CXCL10 receptor, ligation of which induced secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12), an elastolytic proteinase implicated in lung destruction in emphysema.

403

Structure
The human CXCL10 gene is located on chromosome 4 (q1221) in a cluster that contains many other members of the CXC family such as IL-8 and PF4. Mouse and human CXCL10 have 67% sequence identity while the two other mouse chemokines that bind to the same CXCR3 receptor, CXCL9, and CXCL11, are less than 30% identical to CXCL10. Nuclear magnetic resonance (NMR) spectroscopy further showed that CXCL10 and CCL11 bind in a similar manner to both CCR3 and CXCR3. A twostep model describes binding of CXCL10 to its receptor, where in the rst step, the docking domain or the region between the rst pair of conserved cysteines and the rst b-strand binds to a region of the N-terminus of CXCR3. In the second or activation step, residues near the N-terminus of CXCL10 bind to a second region on the receptor that can, only in the case of CXCL10, induce an active conformation. This second step is not fullled upon binding of CXCL10 to CCR3, providing an atomic basis for the inhibition of this receptor by CXCL10.

Introduction
As part of a directed search for cytokine-responsive genes, a gene with a predicted polypeptide size of 10 kDa (interferon-inducible protein of 10 kDa, IP10, CXCL10) was discovered. Subsequently, a variety of cells including T cells, endothelial cells, monocytes, broblasts, and keratinocytes were shown to release CXCL10 in response to interferon gamma (IFN-g) and lipopolysaccharide (LPS). CXCL10 peptide shows approximately 40% homology to the other members of the CXC family, in particular interleukin-8 (IL-8) and platelet factor 4 (PF4). Further characterization of the amino acid sequence of this protein showed that the rst two of four conserved cysteines are separated by one amino acid (CXC, where X could be any amino acid). Thus, the common name IP-10 was changed to CXCL10 using the chemokine systematic nomenclature. Consistent with its anti-angiogenic effects, this cytokine is shown to play a prominent role in T-cell-dependent tumor immunity and, most recently, upregulation of this chemokine is reported in chronic and acute inammatory diseases such as acute allograft rejection, central nervous system inammation in multiple sclerosis, psoriasis, and emphysema (see Figure 1).

Regulation of Production and Activity

Transient and rapid increase in CXCL10 mRNA and protein synthesis in response to IFN-g and LPS was rst reported in a monocytic cell line, but was later reported in a variety of primary cells. Additionally, stimulation of cells with double-stranded RNA has been shown to induce the transcription of CXCL10 in a manner dependent on intact interferon-stimulated response element (ISRE) and nuclear factor kappa B (NF-kB) binding sites within its promoter. Thus, in addition to IFN-g, multiple Toll-like receptors (TLRs) such as TLR-3 ligand or double-stranded RNAs can also induce upregulation of this chemokine. Immunohistochemical analysis has shown that bronchial epithelium is an important source of

Protective functions

Pathological diseases Emphysema Allograft rejection Sarcoidosis Asthma

Tumor immunity Lung fibrosis T-cell host defense

CXCL10

LPS
Figure 1 Biological functions of CXCL10. Many cells of the lung including epithelial cells, macrophages, and T cells express CXCL10 upon activation by IFN-g or LPS. CXCL10 plays a protective role in T-cell-dependent tumor immunity and host defense. Under pathological conditions, CXCL10 is associated with emphysema, allograft rejection, sarcoidosis, and exaggerated allergic lung inammation.

404 CHEMOKINES, CXC / CXCL10 (IP-10)

CXCL10, which serves to recruit activated T-helper1 (Th1) cells for normal host defense against intracellular pathogens of the lung. Induction of CXCL10 mRNA in response to IFN-g is seen as early as 30 min, with peak levels seen after 5 h. Signaling intermediates requisite to CXCL10 gene expression include Jak-2 and STAT1. Interestingly, prostaglandin E2, histamine, and vasoactive intestinal peptide, which all inhibit Jak-2 and STAT1, also inhibit IFNg-dependent induction of CXCL10. Peroxisome proliferator-activated receptor-g (PPAR-g), a member of the nuclear hormone receptor superfamily, originally shown to play an important role in adipocyte differentiation and glucose homeostasis, also regulates inammatory responses by inhibiting IFN-g-induced mRNA and protein expression of CXCL10 in endothelial cells, although the molecular mechanism for this inhibition is not understood.

in many pathological conditions is complicated by its dual regulatory activity since it can act as an agonist for Th1 responses by binding to its own receptor, and as an antagonist for CCR3 by competing with CCR3 ligands for binding to this receptor.

Receptors
CXCL10 binds to and activates two distinct receptors, CXCR3 and CXCR3B, as do monokine induced by IFN-g (Mig/CXCL9) and IFN-inducible T-cell alpha chemoattractant (ITAC/CXCL11). These receptors were cloned from a human CD4 T-cell library and were found to have significant sequence homology to the two IL-8 receptors. Both the human and mouse CXCR3 genes were mapped to the X chromosome. Receptor structure function analysis revealed that similar to the other chemokine receptor family members, CXCR3 is a seven transmembrane G-protein-coupled receptor, which mediates calcium mobilization and chemotaxis in response to the ligands CXCL10 and CXCL9. Initially, expression of CXCR3 was thought to be restricted to IL-2-activated T lymphocytes and not in resting T and B lymphocytes, monocytes, or granulocytes. Subsequently, CXCR3 was found on other activated cells of the immune system including natural killer (NK) cells, granulocytes, and macrophages. In addition to chemotaxis, the known CXCR3 ligands, CXCL9, CXCL10, and CXCL11, induce secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12) from activated human lung macrophages. Mutational analysis showed that binding of CXCL10 to its receptor is through the N-terminal residue Arg-8, preceding the rst cysteine. CXCR3expressing, GAG-decient Chinese hamster ovary cells remained responsive to CXCL10, suggesting that the CXCR3 and heparin binding sites of CXCL10 are only partially overlapping an interaction thought to be important for its sequestration on endothelial and other cells.

Biological Function
The most notable biological activity of CXCL10 is its ability to direct the recruitment of Th1 and related effector cells in acute and chronic inammatory conditions. Along with the other CXCL chemokine members, CXCL10 must bind to cell surface heparan sulfate proteoglycans to establish the necessary chemokine gradients required for extravasation of these critical cellular mediators of host defense and tissue transplant rejection. As with other chemokines, the functions of CXCL10 are redundant with respect to other class-specic chemokines. In experimental models of transplantation, anti-CXCL10 monoclonal antibodies prolonged allograft survival but, surprisingly, CXCL10-decient mice acutely rejected the same allografts. This can be partly explained by redundancy in the activity of two other members of the CXC family, CXCL9 (Mig) and CXCL11 (I-TAC), which share the same receptor as CXCL10. Nonetheless, in support of an important role for CXCL10 in allograft rejection, compared with wild-type donors, use of CXCL10 null donor hearts grafted into wild-type mice reduced intragraft expression of cytokines, chemokines, and their receptors, and associated leukocyte inltration, and graft injury. These ndings support the notion that tissue-specic generation of CXCL10 in response to inammation is sufcient for the progressive inltration and amplication of multiple effector pathways, and that targeting of CXCL10 can prevent acute rejection under some circumstances. Further, CXCL10 blockade impeded the expansion and migration of peripheral antigen-specic T cells in a mouse model of virus-induced organ-specic autoimmune disease. Finally, the role that CXCL10 plays

CXCL10 in Respiratory Diseases

CXCL10 has been associated with a wide spectrum of lung inammatory diseases through both pro- and antibrotic effects and its ability to recruit cells that secrete or respond to IFN-g. For example, CXCL10 mRNA and protein are markedly increased in the lungs of mice challenged with bleomycin, a potent inducer of lung brosis. However, CXCL10 is thought to be protective in this setting as mice decient in CXCL10 showed increased susceptibility to brosis while transgenic CXCL10 overexpressing mice showed reduced mortality and less severe

CHEMOKINES, CXC / CXCL10 (IP-10)

405

bleomycin-induced pulmonary brosis. The mechanism for CXCL10-dependent survival in this model was inhibition of recruitment of lung broblasts, which are largely responsible for synthesis of the matrix proteins that underlie most brosing conditions. Paradoxically, however, elevated levels of CXCL10 have been found in bronchoalveolar lavage (BAL) uids of humans who have undergone lung transplantation with acute and chronic rejection, a complication that usually leads to widespread brosis of the transplanted lung. However, in vivo neutralization of CXCR3 or CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated chronic lung rejection and bronchiolitis obliterans syndrome in mice. Furthermore, overexpression of CXCL10 was associated with worsening of lung brosis in the same transplantation model. Thus, CXCL10 appears to suppress lung brosis due to bleomycin, but is a primary mediator of lung transplant rejection. Although CXCL10 is primarily associated with Th1, or type I, immunity, its upregulation in type II inammatory conditions such as asthma has also been reported. Mice that overexpress CXCL10 in the lung were shown to exhibit significantly increased eosinophilia, IL-4 levels, and CD8 ( ) lymphocyte recruitment compared with wild-type controls that received the same allergen. In addition, there was an increase in the percentage of IL-4-secreting T lymphocytes in the lungs of CXCL10 transgenic mice. In contrast, mice decient in CXCL10 demonstrated the opposite results compared with wild-type controls, with a significant reduction in these measures of Th2-type allergic airway inammation. These ndings suggest that CXCL10, a Th1-type chemokine,
CXCR3

can contribute to Th2-type inammation in experimental asthma. Contrary to this study, adenovirusmediated overexpression of CXCL10 in the airways in a mouse model of asthma resulted in significant inhibition of eosinophils in the BAL uid accompanied by enhanced IFN-g, and reduced IL-4 secretion that was dependent on IFN-g. These ndings are more in line with the above-mentioned multifunctional activity of CXCL10 regarding its binding to CXCR3 and acting as a potent antagonist of many Th2 chemokines. Collectively, these contradictory data illustrate that local expression of the chemokine CXCL10 can exert diverse effector responses depending on the exact immunological context. Smoking-induced emphysema is associated with chronic activation of Th1 type inammation and, in particular, upregulation of CXCL9 and CXCL10, and their shared receptor, CXCR3 on lung airway epithelial cells, T cells, and macrophages. Expression of CXCL10 has also been demonstrated in the lung macrophages and in T cells isolated in the lungs of patients with smoking-induced emphysema. The accumulation of T cells and monocytes at sites of ongoing inammation represents the earliest step in a series of events that lead to granuloma formation in sarcoidosis and destruction of lung elastin bers in emphysema. High levels of a CXCL10 protein have been demonstrated in the BAL uid of patients with pulmonary sarcoidosis and lymphocytic alveolitis as compared with patients with inactive disease or control subjects. Similarly, lung CD4 T cells secreted a higher concentration of CXCL10 in emphysema subjects as compared to controls. In addition, more recently it has been shown that beyond their historic role as chemoattractants, members of the CXCL
Neutrophil Neutrophil elastase

A1AT IFNTh1 cell CXCL10 MMP-12

Elastin degradation Airway obstruction Emphysema

CXCR3 Macrophages
Figure 2 CXCL10 in emphysema. Th1 effector cells of the lung secrete CXCL10, which, through its receptor CXCR3, recruits additional lung T cells, thus amplifying the inammatory response to tobacco smoke. CXCL10 also recruits activated macrophages and neutrophils and induces macrophages to secrete MMP-12, an elastolytic enzyme that inhibits a1-antiproteinase (A1AT), which in turn is required for protection against human emphysema.

406 CHEMOKINES, CXC / CXCL10 (IP-10)

family (in particular CXCL10) actively induce production of MMP-12; this is a potent inhibitor of a1-antitrypsin, which is linked to the development of smoke-induced emphysema in humans (see Figure 2). Taken together, these data suggest that CXCL10 plays an important role in regulating recruitment of Th1 inammation and may play a role in lung destruction that is associated with the chronic inammatory lung diseases such as emphysema and sarcoidosis.
See also: Chemokines. Chemokines, CXC: CXCL9 (MIG). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General; Smoking Cessation. Drug-Induced Pulmonary Disease. Eotaxins. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. G-Protein-Coupled Receptors. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Matrix Metalloproteinases. Serine Proteinases.

Luster AD, Greenberg SM, Leder P, et al. (1985) Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315: 672676. Luster AD and Leder P (1993) IP-10, a -C-X-C- chemokine, elicits a potent thymus-dependent antitumor response in vivo. Journal of Experimental Medicine 178: 10571065. Marx N, Mach F, Sauty A, et al. (2000) Peroxisome proliferatoractivated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. Journal of Immunology 164: 65036508. Medoff BD, Sauty A, Tager AM, et al. (2002) IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inammation in a mouse model of asthma. Journal of Immunology 168: 52785286. Moser B and Willimann K (2004) Chemokines: role in inammation and immune surveillance. Annals of the Rheumatic Diseases 63: ii84ii89. Saetta M, Mariani M, Panina-Bordignon P, et al. (2002) Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 165: 14041409. Wiley R, Palmer K, Gajewska B, et al. (2001) Expression of the Th1 chemokine IFN-gamma-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. Journal of Immunology 166: 27502759.

Glossary Further Reading

Agostini C, Cassatella M, Zambello R, et al. (1998) Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Immunology 161: 64136420. Belperio JA, Keane MP, Burdick MD, et al. (2003) Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection. Journal of Immunology 171: 48444852. Campanella GS, Lee EM, Sun J, and Luster AD (2003) CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). Journal of Biological Chemistry 278: 1706617074. Dufour JH, Dziejman M, Liu MT, et al. (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-decient mice reveal a role for IP-10 in effector T cell generation and trafcking. Journal of Immunology 168: 31953204. Gasperini S, Perin A, Piazza F, et al. (1995) The IP-10 chemokine binds to a specic cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Experimental Medicine 182: 219231. Grumelli S, Corry D, Song L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. Public Library of Science Medicine 1: 7483. Hancock WW, Gao W, Csizmadia V, et al. (2001) Donor-derived IP-10 initiates development of acute allograft rejection. Journal of Experimental Medicine 193: 975980. Loetscher M, Gerber B, Loetscher P, et al. (1996) Chemokine receptor specic for IP10 and mig: structure, function, and expression in activated T-lymphocytes. Journal of Experimental Medicine 184: 963969. Loetscher P, Pellegrino A, Gong JH, et al. (2001) The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3. Journal of Biological Chemistry 276: 29862991.

Adipocytes fat cells Allograft graft of tissue from non-self donor of the same species Bleomycin a chemotheraputic agent Chemokine small chemoattractant proteins that stimulate the migration and activation of cells Cytokines proteins made by cells that affect the function of another cell Dendritic cells cells found in T-cell regions of lymph node and the most potent stimulators of T-cell responses Eotaxins CC chemokines that act predominantly on eosinophils Jak-2 a Janus-family tyrosine kinase (JAK) signaling molecules that are activated by aggregation of cytokine receptors Lipopolysaccharide a cell wall antigen present in Gram-negative bacteria Matrix metalloproteinases a large family of zincdependent endopeptidases Multiple sclerosis an autoimmune-mediated neurodegenerative disorder Proteoglycans cell surface proteins with carbohydrate side chains

CHEMOKINES, CXC / CXCL1 (GRO1)CXCL3 (GRO3) 407

Psoriasis an inammatory skin disorder STAT1 signal transducer and activator of transcription, a transcription factor, that is phosphorylated by JAKs T-helper cell type 1 a subset of CD4 T cells that are characterized by the cytokines they produce. They are mainly involved in activating macrophages Toll-like receptors cell surface receptors that are initiated by Toll pathway that activates the transcription factor NF-kB by degrading its inhibitor IkB

nuclear factor kappa B (NF-kB) transcription factor dependent. Probing against leukocyte gene library revealed two additional GRO-like molecules, designated GROb and GROg; these had respective 90% and 86% amino acid identity with GROa. Compared to GROa, there are 11 amino acid substitutions in GROb and 15 in GROg of which some confer conformational changes in protein structure. The GROb and GROg molecules were found to be identical to independently identied human MIP-2a and MIP-2b which are chemokine genes homologous to the MIP-2 genes of mice. It is now accepted that the GROa, GROb, and GROg are members of the CXCL groups of chemokines and in current standardized nomenclature are designated CXCL13.

CXCL1 (GRO1)CXCL3 (GRO3)

S W Chensue, VA Ann Arbor Healthcare System, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Structure
Genes consisting of approximately 1100 nucleotide base pairs for CXCL13 are encoded on long arm of chromosome 4 in or near the CXC chemokine locus (4q21). The protein structure of CXCL1 closely resembles other members of the CXCL group. In solution, CXCL1 is a dimeric chemokine consisting of monomers of 73 amino acid residues. The dimer contains a six-stranded antiparallel b-sheet packed against two C-terminal antiparallel a-helices (Figure 1). The CXC designation derives from characteristic conserved double-cysteine motif separated by single amino acid in the N-terminal portions of the molecule. CXCL13 are also classed as ELR chemokines based on the presence of a glutamateleucinearginine sequence adjacent and N-terminal to the CXC motif.

Abstract
CXCL13 are members of the CXCL class of chemokines with neutrophil chemotactic and angiogenic biologic properties. These molecules exist as dimers and interact principally with the guanosine nucleotide-protein-coupled, CXCR2 chemokine receptor expressed by neutrophils as well as by other cell types. CXCL13 are elicited by microbial products and cytokines and likely contribute to inammatory and repair responses as part of a broad spectrum of chemotactic mediators. Evidence to date indicates that these molecules participate in multiple pulmonary diseases including infections, adult respiratory distress syndrome, chronic obstructive pulmonary disease, hyperoxia-induced lung injury, brosis, and neoplasm.

Introduction
The GROa molecule was rst identied in the 1980s as an early response gene produced by cultured melanoma cells with autocrine growth promoting function. GROa also known as melanoma growth stimulating activity (MGSA) was expressed constitutively by serum-cultured transformed broblasts and could be induced in normal cells by the inammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor alpha (TNF-a). While initially thought to be a growth-related oncogene due to its growthpromoting effects on melanoma cells, it was quickly apparent that GROa was structurally related to the supergene family of small 815 kDa inammatory neutrophil chemotactic proteins or chemokines that included IL-8, neutrophil activating protein-2, and macrophage inammatory protein-2 (MIP-2). Like these molecules, GROa was mapped to the chemokine locus on chromosome 4 and its expression was

Figure 1 Secondary molecular structure of CXCL1 (GROa) dimer. Note six antiparallel b-pleated sheets (red) positioned against two antiparallel a-helices (blue).

408 CHEMOKINES, CXC / CXCL1 (GRO1)CXCL3 (GRO3)

Regulation of Production
While CXCL1 is produced constitutively in some transformed cell lines, under physiologic conditions it is likely induced in normal cells by exogenous stimuli such as microbial products and/or inammatory cytokines. In particular, bacterial endotoxin, IL-1, and TNF-a are potent inducers of CXCL chemokines in mononuclear phagocytes, epithelial cells, and structural mesenchymal cells. However, it should be noted that numerous infectious agents have been described as inducers of CXCL gene expression. The gene loci for CXCL13 have NF-kB binding sites and as such, the NF-kB transcription pathway is considered critical in regulating the expression of these genes. It is likely that during infection microbial products interact with Toll-like receptors on host cells to initiate phosphorylation events leading to release of active NF-kB which translocates to the nucleus to bind to gene promoter sites. The CXCL genes represent only a portion of the expressed gene prole. IL-1 can be among those induced genes and there is evidence indicating that IL-1 may regulate CXCL13 gene expressions by stabilizing transcript levels through inhibition of transcript degradation. The restoration of transcription regulatory pathways results in eventual arrest of gene expression. Neoplastic cells can display dysregulated CXCL gene expression which may allow them to function as autocrine growth or oncogenic factors.

Biological Function
Originally, CXCL1 (GROa) was considered to be an oncogenic growth factor since it appeared to have the capacity to promote growth and malignant transformation of cultured melanocytes. Since that time a number of other functions have been attributed to this group of molecules. These include regulation of tissue repair-related mesenchymal cell function. Two examples should be mentioned. First, CXCL1 caused a dose-dependent decrease in the expression of interstitial collagens by cultured rheumatoid synovial broblasts. The effect was specic, as there was no demonstrable effect on other products. Unlike its mitogenic effect on melanoma cells, it had no effect on the proliferation rate of broblasts. Thus, CXCL1 may limit or temper the scarring process following injury. Second, similar to other ELR chemokines CXCL1 is implicated as a proangiogenic factor, that is, stimulating the growth of new small vessels at sites of injury or tumor growth. This contrasts with ELR-CXCL chemokines which inhibit angiogenesis. These opposing activities suggest the presence of

carefully orchestrated signals that are required to shape events involved in the tissue repair response. The best-characterized function of CXCL13 is their chemoattractant activity. Like other ELR CXCL chemokines, these molecules have potent effects on neutrophil migration and activation. This activity appears to be further amplied by natural enzymatic truncation of the amino terminal portion of the molecules; this modication causes a 30-fold increase in biologic potency. Thus, under conditions of inammation, proteolytic enzymes may boost the chemotactic function of these molecules. Numerous studies have provided circumstantial evidence for an in vivo association of neutrophil accumulation with production of CXCL13. However, it has been difcult to provide direct evidence by neutralization approaches due to the redundancy of CXC ligands. Some direct evidence for in vivo biologic activity has been derived from CXCR2 receptor knockout mice which display impaired neutrophil recruitment and delayed wound healing. In addition, engineered chemical antagonists of the CXCR1 and CXCR2 receptors likewise cause impaired neutrophil mobilization. Taken together, the functional activities of CXCL13 would appear to be teleologically appropriate for the acute inammatory response to infection. Following epithelial injury and infection, these molecules would simultaneously promote epithelial healing, leukocyte recruitment, and neovascularization, representing a coordinated effort to prevent further infestation, initiate microbial elimination, and optimize delivery of additional leukocytes and antimicrobial humoral factors.

Receptors
The functional high-afnity receptor for CXCL13 is well characterized and is designated by standardized nomenclature as CXCR2, formerly known as IL-8B or IL-8 type 2 receptor. CXCR2 is promiscuous and binds multiple ELR CXC ligands. CXCR2 is an archetypical guanosine nucleotide-protein-coupled receptor (GPCR) with seven transmembrane hydrophobic domains having three intracellular and three extracellular hydrophilic loops. Ligation of the extracellular amino-terminal portion of the receptors triggers calcium inux and intracellular activation of transduction factors such as protein kinase C and MAP kinase. These events stimulate downstream functions such as cell movement, degranulation, adhesion, and respiratory burst. CXCR2 receptors have been detected on neutrophils, monocytes, basophils, mast cells, endothelial cells, T lymphocytes, natural killer, and neuroendocrine cells. However, neutrophils appear to be the dominant expressing cells.

CHEMOKINES, CXC / CXCL1 (GRO1)CXCL3 (GRO3) 409

Due to the promiscuity of the receptor, it is difcult to assess the contribution of individual CXC ligands in experimental animals with targeted deletion of CXCR2. Another promiscuous receptor for CXCL1 3 is the Duffy antigen/chemokine receptor. Duffy binds both CXC and CC chemokines and is expressed by erythrocytes in Duffy-positive individuals, endothelial cells of postcapillary venules, and Purkinje cells of the cerebellum. The normal physiological function of Duffy remains unclear. The transduction pathways activated by Duffy are also unknown and the receptor has not been shown to act through a G protein. Reportedly, Duffy facilitates CXCL1 distribution and enhances neutrophil movement across endothelial cell monolayers.

Role of CXCL13 in Respiratory Diseases

In addition to benecial functions, CXCL13 have been implicated to participate in multiple pulmonary diseases ranging from infectious to neoplastic and usually are part of a broader spectrum of chemokine and cytokine mediators (Figure 2). In the lung, alveolar macrophages are critical sentinel cells and are likely an important source of CXCL13 but type II alveolar epithelial cells are known to be a potential source. The roles of these chemokines in different lung diseases are discussed separately below.

significant neutrophil inux. Indeed, these chemokines are readily detectable in bronchoalveolar lavage uids of patients with infectious and inammatory lung disease but at low levels in those of normal volunteers. Experimental studies of Pseudomonas aeruginosa, an important Gram-negative bacterium causing fatal necrotizing pneumonias, have shown this agent to be capable of inducing CXCL chemokines in alveolar macrophages and epithelial cells. Depletion of alveolar macrophages impairs both the production of CXCL1 and the recruitment of neutrophils. Other Gram-negative bacterial causes of pneumonia such as Escherichia coli are similarly capable of eliciting CXCL chemokines and innate recognition of bacterial endotoxin appears to be the common stimulatory molecule. However, Grampositive bacteria and Pneumocystis are also capable of eliciting CXCL1. In general, viruses such as respiratory syncitial virus (RSV) appear to elicit strong CC chemokine responses in the lung, but epithelial damage and secondary infection will elicit CXCL chemokines.

Adult Respiratory Distress Syndrome

It is well known that critically ill patients are at increased risk for acute lung injury which is clinically manifest as adult respiratory distress syndrome (ARDS). CXCL1 is notably elevated in lung lavage uids obtained from ARDS patients. Accordingly, neutrophil inux correlates well with levels of CXCL chemokines. The alveolar epithelial damage observed in ARDS appears to be related to the inammation and as such the CXCL chemokines might be contributing to the disease. Due to extensive redundancy in neutrophil recruitment, CXCL13 likely only contribute partially and there is evidence to indicate that other chemokines such as CXCL8 may be more important.

Infections
In view of their neutrophil chemotactic properties, it might be expected that CXCL1, 2, or 3 would be detected during lung bacterial infections that elicit

Inflammation and repair Neutrophil recruitment Angiogenesis Tissue repair

Chronic Obstructive Pulmonary Disease

There is strong evidence to suggest that inammatory leukocytes including neutrophils contribute to the destruction of structural matrix observed in chronic obstructive pulmonary disease (COPD). Circumstantial evidence suggests that CXCL chemokines might contribute to neutrophil recruitment and activation during COPD. CXCL chemokines including CXCL1 are present in the sputum and lung lavage uid of COPD patients. Cigarette smoke, the major cause of COPD, has been shown to elicit CXCL1 and neutrophil inux in rat lungs. Moreover, the neutrophil mobilization could be blocked with CXCL2 receptor antagonists. This receptor also appears to show

CXCL1,2,3

ARDS Hyperoxia injury Fibrosis COPD Lung diseases

Tumor growth

Figure 2 Potential areas of functional involvement of CXCL13 in physiology and lung diseases.

410 CHEMOKINES, CXC / CXCL1 (GRO1)CXCL3 (GRO3)

enhanced expression on monocytes from smokers. These cells likewise migrate to lungs where they can potentially produce matrix destructive enzymes.

more studies would be needed to help clarify the contribution of CXCL13 in lung carcinoma.
See also: Acute Respiratory Distress Syndrome. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-1 and IL-18. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Fibrosing Alveolitis
Cryptogenic brosing alveolitis (CFA) is a progressive brotic disease of the lung characterized by significant local recruitment of neutrophils which can comprise 1020% of the cells in the lung lavage of affected patients. These cells are thought to contribute to the disease. Patients with CFA have increased levels of CXCL1 in their plasma and their neutrophils are hyperresponsive to the chemokine. Thus, chemokine-targeted therapies may have potential benet in controlling the disease.

Further Reading
Belperio JA, Keane MP, Arenberg DA, et al. (2000) CXC chemokines in angiogenesis. Journal of Leukocyte Biology 68: 18. Cao Y, Chen C, Weatherbee JA, Tsang M, and Folkman J (1995) gro-beta, a CXC chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice. Journal of Experimental Medicine 182: 20692077. Glynn PC, Henney EM, and Hall IP (2001) Peripheral blood neutrophils are hyperresponsive to IL-8 and Gro-alpha in cryptogenic brosing alveolitis. European Respiratory Journal 18: 522529. Goodman RB, Pugin J, Lee JS, and Matthay MA (2003) Cytokinemediated inammation in acute lung injury. Cytokine & Growth Factor Reviews 14: 523535. Hashimoto S, Pittet JF, Hong K, et al. (1996) Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections. American Journal of Physiology 270: L819 L828. Sager R, Haskill S, Anisowicz A, Trask D, and Pike MC (1991) GRO: a novel chemotactic cytokine. Advances in Experimental Medicine and Biology 305: 7377. Stoeckle MY (1991) Post-transcriptional regulation of gro alpha, beta, gamma, and IL-8 mRNAS by IL-1 beta. Nucleic Acids Research 19: 917920. Sue RD, Belperio JA, Burdick MD, et al. (2004) CXCR2 is critical to hyperoxia-induced lung injury. Journal of Immunology 172: 38603868. Traves SL, Smith SJ, Barnes PJ, and Donnelly LE (2004) Specic CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2. Journal of Leukocyte Biology 76: 441450. Vanderbilt JN, Mager EM, Allen L, et al. (2003) CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. American Journal of Respiratory Cell and Molecular Biology 29: 661668. Villard J, Dayer-Pastore F, Hamacher J, Aubert JD, Schlegel-Haueter S, and Nicod LP (1995) GRO alpha and interleukin-8 in pneumocystis carinii or bacterial pneumonia and adult respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 152: 15491554. Wuyts A, Govaerts C, Struyf S, et al. (1999) Isolation of the CXC chemokines ENA-78 GRO alpha and GRO gamma from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms. European Journal of Biochemistry 260: 421429.

Hyperoxia-Induced Lung Injury

Hyperoxia-induced lung injury is a potential serious side effect of oxygen therapy in hospitalized patients. The disease is characterized by inltration of neutrophils with associated endothelial and epithelial cell injury, followed by interstitial brosis. CXCL13 may be important contributors to this condition. Animal experiments demonstrate that oxygen treatment induces expression of CXCL13, which correlates with the inux of neutrophils and mortality. Genetic deletion of the CXCR2 receptor blocked neutrophil recruitment and resulted in reduced oxygen-induced mortality.

Lung Cancer
The role of CXCL13 in neoplastic disease of the lung remains controversial and studies are very limited. Due to their angiogenic and mitogenic properties, these chemokines are generally considered to support the growth of neoplastic tissue. Growth can potentially be enhanced by direct stimulation of chemokine receptors expressed by tumor cells especially melanocytic and neuroendocrine types. In addition, angiogenic activity would help to provide vascular support to growing tumors. In contrast to this notion, at least one study has provided evidence that CXCL2 is angiostatic and could inhibit the growth of Lewis lung carcinoma in mice. Clearly,

CHEMORECEPTORS / Central 411

CHEMORECEPTORS
Contents

Central Arterial

Central
N S Cherniack, UMDNJ-New Jersey Medical School, Newark, NJ, USA M D Altose, Case Western Reserve University School of Medicine, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

P aCO2

P aO2 P brainCO2

Chemoreceptors Chemical drives

Controller Ventilatory demand Respiratory muscles, lung

Abstract
The powerful stimulation of ventilation by carbon dioxide inhalation and the near-constancy of arterial PCO2 during rest and exercise indicate the importance of CO2/H receptors in the regulation of breathing. There are both peripheral and central chemoreceptors with the central ones accounting for about 70% of the CO2 effect. These sensors operate in feedback control systems that help maintain levels of CO2 in the body within narrow limits. Techniques that allow individual cells to be studied have provided much new information on central chemoreceptors. Groups of neurons that increase their activity in response to changes in acidity in their external environment are widely scattered in the medulla. It is generally believed that the proximal stimulus to these central receptors is intracellular acidity. These neuronal groups when stimulated by the changes in local hydrogen ion concentration excite breathing, but each group separately accounts for a rather small part of the increase in ventilation that can be elicited by CO2 inhalation. There are no data yet on whether the various sensory groups have different thresholds though some seem to be excited only during wakefulness, which might imply a high threshold, and others only during sleep, which is not easily explained by threshold differences. Furthermore, it is not known whether the various groups differ in their operative ranges or sensitivity. It is also possible that some of these groups are parts of other functional systems involved, for example, in producing arousal. Otherwise healthy humans who do not respond to breathing CO2 can maintain normal levels of arterial CO2 when awake. But in patients with thoracic diseases, low sensitivity to CO2 may contribute to CO2 retention. During sleep, a threshold level of CO2/H is required to prevent apnea. In the absence of a response to CO2, abnormal elevation of PCO2 occurs during sleep. Depressed chemosensitivity may be involved in the sudden infant death syndrome. In addition, depressed chemosensitivity may predispose to respiratory failure in patents with thoracic diseases.

Ventilation O2, CO2 body stores P aCO2 P aO2 P brainCO2

Figure 1 Block diagram showing components and pathways involved in the chemical control of breathing.

Introduction
Description of Central Chemoreceptor Effects on Ventilation

Respiration is regulated by a negative feedback control system that serves to hold PCO2 in the arterial

blood and in the brain within narrow limits and to maintain acidbase homeostasis (see Figure 1). This system consists of a controller made up of respiratory neurons and chemoreceptors and a plant or controlled system consisting of the body tissues in which CO2 is generated by metabolic processes. The system operates to maintain PCO2 (or H ) at some desired level called the operating point. The two components are linked via the circulation, which carries information about levels of PCO2 and PO2 in the blood. When CO2 levels in the body rise as a result of increases in metabolic CO2 production or disturbances that decrease ventilation, central chemoreceptors in the brain and peripheral chemoreceptors in the carotid and aortic bodies are stimulated. Impulses from these chemoreceptors, in turn, act on respiratory motor neurons in the medulla to produce compensatory increases in ventilation to return PCO2 to its normal level. Similarly, disturbances that increase ventilation and reduce levels of PCO2 and H ion cause compensatory decreases in breathing to help restore PCO2 /H levels. When CO2 is breathed, the chemoreceptors augment ventilation, minimizing the rise in PCO2 that would otherwise occur. The increases in ventilation

412 CHEMORECEPTORS / Central

Ventilation
Figure 2 Effect of ventilation on PCO2 . At low ventilation and high PCO2 (A, slope highlighted in red) the effect of ventilation on PCO2 is greater than at (B) where ventilation is higher and PCO2 is lower. Slope is an index of plant gain.

are proportional to the deviation in PCO2 when CO2 is inhaled but the compensation is never sufcient to restore PCO2 to its original levels. The sensitivity or gain of the chemical controller is usually expressed as the change in ventilation produced by a given change in PCO2 in the arterial blood and averages about 2 l for each mmHg change in PCO2 in adult humans. In general, the higher the gain the less is the rise in PCO2 . However, the efcacy of the control system also depends on the effect of ventilation on PCO2 . The ratio of the change in PCO2 to the change in ventilation is sometimes called plant gain. As shown in Figure 2, because the effects of ventilation on PCO2 are less at lower than at higher levels of PCO2 , plant gain increases as resting arterial PCO2 rises (when breathing CO2 free gas). Increases in plant gain and controller gain (more precisely their product or loop gain) will shorten the time needed for the control system to reach equilibrium after a disturbance. However, too high a loop gain can lead to control-system instability and oscillations in minute ventilation. All animals that extract oxygen from the atmosphere have CO2/H responsive chemoreceptors. CO2 receptors have been found on the ventral surface of the caudal and rostral medulla of the isolated bullfrog brainstem. Fish have CO2 levels of only a few mmHg and receptors for CO2/H sample the ambient water rather than changes within the body. In anuran amphibians, CO2 chemoreceptors account for 70% of the ventilatory response to CO2. In cold-blooded species the normal PCO2 varies considerably with body temperature becoming lower as temperature drops. In mammals, on the other hand, there is relatively little direct effect of temperature on PCO2 levels that roughly average 40 mmHg in the arterial blood of humans at rest. Temperature elevations, if anything, increase ventilation, particularly in panting animals.

Carbon dioxide chemoreceptors have been most extensively studied in mammals. Approximately 70% of the ventilatory response to CO2 results from the activation of central chemoreceptors in the brain. Only about 30% of the increase in ventilation following the inhalation of a CO2 enriched gas mixture is attributable to the activity of peripheral chemoreceptors, mainly the carotid body chemoreceptors. Because of the rapidity of the effects of CO2 inhalation on the arterial blood as compared to the brain, the peripheral chemoreceptors respond far more quickly to changes in CO2 than do the central ones. Both central and peripheral chemoreceptors respond to PCO2 only when it reaches a threshold value. The peripheral chemoreceptors are believed to respond to lower levels of PCO2 than the central chemoreceptors. Drives from central and peripheral chemoreceptors have an additive effect. While the peripheral chemoreceptors respond to changes in arterial PCO2 , the central chemoreceptors respond to changes in PCO2 in the brain. Levels of CO2 in the brain depend on rates of cerebral perfusion in relation to metabolism as well as ventilation. Since changes in PCO2 occurring naturally are always associated with changes in H , it is now generally considered that the proximate stimulus for central chemoreception is intra- and extracellular brain H , rather than the H concentration of the cerebrospinal uid (CSF), previously thought to be the main factor determining ventilation. The importance of chemical control in mammals in determining ventilation depends on behavioral states and higher brain center inuences may modify the characteristics of the chemical controller. It is generally agreed that chemical stimuli are the main factors regulating breathing during nonrapid eye movement (nonREM) sleep when decreases in PCO2 of only a few mmHg can produce apnea. In the waking state, neural drives emanating from sensory receptors in the body and from the environment constitute a greater portion of the ventilatory drive as shown in Figure 3. As a result breathing can be maintained at near-normal levels in the absence of any discernible response to inhaled CO2. A discernible response to CO2 is also not necessary for normal PCO2 levels to be maintained during moderate exercise, but in its absence PCO2 rises considerably rather than decreasing when the anaerobic threshold is exceeded.

PCO

Structure and Function

Location and Morphology of Central Chemoreceptors

The precise location and distribution of central respiratory-related chemoreceptors in the brain has

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Awake

Asleep

0 P aCO2

Figure 3 Effect of changes in arterial PCO2 on ventilation during sleep and wakefulness. As P aCO2 is decreased, apnea occurs during sleep but not in the awake state.

been a matter for investigation for decades and has not yet been fully established. It is generally thought that the central chemoreceptors are grouped in clusters in several different locations in the medulla and even in other areas of the brain. The characteristics of the different clusters in terms of their thresholds, sensitivities, and mechanisms of sensing CO2 seem to vary. The study of central chemoreceptors is further complicated because CO2 centrally affects more than ventilation. It increases sympathetic activity, causes the release of catecholamines, helps produce arousal, increases cerebral blood ow, increases anxiety, and can produce a sense of air hunger. Thus, it is possible that different central chemoreceptors may also differ in their functions. In studies in intact animals, central chemoreceptors were dened as cells that, when stimulated by acid or CO2, lead to an increase in ventilation or respiratory motor activity and/or when inhibited, decrease the respiratory stimulating actions of acid or CO2 without affecting respiratory activity excited by other types of respiratory stimuli. For some time it was believed that respiratory motor neurons themselves had properties of chemosensitivity. Animal studies by Loeschke and by Mitchell and others, using the direct application of acidic or basic uids, demonstrated discrete clusters of chemoresponsive neurons on the ventrolateral surface of the medulla. These include a rostral chemosensitive area (Mitchells area) and a caudal chemosensitive area (Loeschkes area). Stimulation of the Loeschke and Mitchell areas on the ventral surface of the medulla increases breathing in anesthetized animals. Correspondingly, ablation by cooling of the intermediate area between them, presumably by blocking afferent nerve bers from the rostal and caudal areas, produces apnea. It has been observed that patients with brain lesions around

Mitchells area show depressed ventilatory responses to CO2 during sleep. This supports the presumption that the ventrolateral medulla is an important site of central chemoreceptors. With time, more precise ways of stimulating or blocking specic areas of the brain using microinjection or dialysis with various agents such as carbonic anhydrase inhibitors, solutions equilibrated with CO2, neuronal stimulants and inhibitors, and neurotoxins were developed. Using these techniques, additional chemoreceptive areas were identied farther from the ventral surface and in the dorsal areas of the medulla, in the pons, in the midbrain, and even in higher brain structures. However, stimulation and ablation interventions in vivo are imperfect techniques for identifying the precise location of central chemoreceptors because the penetration of a local intervention to deeper and more distant structures cannot be totally controlled. Even quite localized stimulation and ablation can produce reactions in more distant areas through neural connections that can be either excitatory or inhibitory to breathing. These interventions can affect systems other than respiration, such as circulation, metabolism, autonomic nervous system, and arousal, which may indirectly alter breathing. Techniques that use uorescent voltage-sensitive dyes and those that measure increased activity of early immediate genes such as c-fos and c-jun to signal neural responses following exposure to high CO2 levels have identied neurons activated by CO2. However, such responses may also not be specic and lead to increases in ventilation. Studies of isolated brain and spinal cord preparations of neonatal animals and in brain slices and cell cultures have identied additional chemoreceptive areas and more importantly have elucidated critical cellular and molecular mechanisms that are involved in sensing changes in CO2/H . However, in these studies the criteria for chemoresponsiveness have been broadened to include changes in transmembrane potentials. The relationship of these membrane changes to respiratory activity is indirect. Groups of cells that depolarize and increase their excitability with hypercapnia and with local changes in acidity have also been found in nonrespiratory areas of the brain but the magnitude of the responses is less than in cells from areas involved with respiration. It is currently believed that central chemoreceptors away from sites near the ventral medullary surface (like the retrotrapezoid nucleus) are located in the dorsal medullary regions including the nucleus of the solitary tract, the locus ceruleus, the median raphe, the pre-Boetzinger complex, and the fastigial nucleus

Ventilation

414 CHEMORECEPTORS / Central

of the cerebellum. Chemosensitive cells that respond to CO2 by increasing respiratory frequency have also been found in the spinal cord of brainstemspinal cord preparations from newborn mice. Almost all of the areas in which CO2/H responsive cells have been found are concerned with other physiological functions besides respiration. For example, neurons in the raphe are also involved in blood pressure control, thermoregulation, and the sleep waking cycle making it difcult to distinguish direct from indirect effects on respiration. The effect of CO2 in producing arousal seem to be mediated in particular by receptors in the cerebellum, midbrain, posterior thalamus, and basal ganglia, in areas where chemosensitive cells have been identied. How the different chemoreceptors contribute to the overall ventilatory response to CO2/H is not clear. One possibility is that there is a hierarchical arrangement of central chemoreceptors with various groups differing in their threshold and sensitivity to CO2/H . This is suggested by the observation that direct stimulation of a single group of chemoreceptors produces only 2030% of the overall ventilatory response to the inhalation of 7% CO2. Furthermore, the threshold and sensitivity of some chemoreceptors may be inuenced by higher brain centers. For example, when articial CSF equilibrated with 25% CO2 is used to focally stimulate chemoreceptors in different locations in the rat brain, the results vary during naturally occurring cycles of sleep and wakefulness. Stimulation of the median raphe increased breathing 1520% (by increasing frequency) in sleeping rats but had no effect during wakefulness. On the other hand, stimulation of the retrotrapezoid nucleus increased tidal volume and ventilation by 24% but only during wakefulness. Stimulation with the same uid in the nucleus of the solitary tract increased breathing both during sleep and wakefulness. Other factors that can affect the responsiveness of any one group of central chemoreceptors include the blood ow to the site and excitatory or inhibitory inputs from other chemosensitive areas. Moreover, since few experiments report effects of focal stimulations on ventilation/CO2/H response slopes it is difcult to distinguish effects on the chemoreceptive process itself from effects that add to or subtract from ventilation without necessarily affecting the sensing of CO2. There are no anatomical features that distinguish chemosensitive cells that are excited by acid changes from nonchemosensitive cells. In fact, not all such cells have the same morphology. In general, they are located in close proximity to blood vessels in the brain and are situated so as to sense changes in cerebrospinal H concentration.

Chemoreceptor cells on the ventral surface of the medulla are located in the marginal glial layer and are surrounded by ne blood vessels. The technique of c-fos immunohistochemical staining has identied CO2-responsive chemoreceptor cells in both the ventral and dorsal medulla with dendrites directed toward the surface of the brain, suggesting that the dendrite may in fact be the chemosensitive portion of the cell.
Cellular Mechanisms of Chemoreception

It has been observed that for a given change in extracellular uid pH at the medullary surface, the ventilatory response is greater with CO2 inhalation than with the intravenous infusion of a xed acid. One possible explanation is that CO2 and H have separate and distinct stimulatory effects on ventilation. Alternatively, since CO2 penetrates the cell membrane much more easily than H it may be that hydrogen ion changes at some site like the cell interior, more accessible to the freely diffusible CO2 than to xed acid, is the true stimulus and that the change in internal cellular pH is the proximate signal to the central chemoreceptors. Nonchemosensitive cells rapidly restore internal pH after hypercapnic acidosis but this process is slowed in chemosensitive cells allowing them to signal H changes. Internal pH is restored through an Na /H exchange mechanism in response to acidosis and by Cl/HCO 3 exchange following an alkaline challenge. An inhibition or an absence of the Na /H subtype 3 exchanger may be important in allowing chemoreceptor cells to respond to cellular acidity. Calcium channels may also be involved in the cellular mechanisms of chemoreception. L-type calcium channels in chemosensitive cells in the locus ceruleus are activated by intracellular acidity leading to membrane depolarization. There is the possibility that some chemoreceptors are stimulated by changes in the intracellularextracellular pH gradient rather than by intracellular pH alone. Some central chemosensitive cells are also thought to contain pH-sensitive TASK-1 potassium channels as well as calcium-activated potassium channels. These potassium channels are inhibited by extracellular acidosis resulting in a reduction in the intracellularextracellular potassium gradient and a lowering of the depolarization threshold. It may be that different types of central chemoreceptors utilize different sensing processes. In ectotherms, extracellular uid H varies inversely with temperature without any effect on ventilation. This indicates that at least in cold-blooded animals H per se may not be the sole or main stimulus to the chemoreceptors. Rather, it has been

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proposed that ventilation is controlled so as to maintain a constant ratio of hydroxyl to hydrogen ion (H /OH) by an amino acid whose dissociation with temperature is like that of water. The fractional dissociation of imidazole (alpha-imidazole) like water remains constant with changes in temperature and temperature-related changes in H . However, such a protein would be affected by changes in pH under isothermal conditions. Based on these ndings, the alpha-stat theory proposes that central chemoreceptors regulate ventilation as a function of the fractional dissociation (alpha) of the imidazole group of histadine, part of a protein considered to be the pH-sensitive molecule in the central chemoreceptor. This mechanism in cold-blooded animals may in turn regulate the patency of membrane potassium or calcium channels so that intracellular pH also remains constant as the ambient temperature changes. Neurotransmitters are also considered to play a role in mediating chemoresponses. There is considerable evidence that acetylcholine through a muscarinic receptor type is involved in the transmission of both peripheral and central chemoreceptor signals. Other neurotransmitters have also been implicated in central chemoreceptor function. Suramin, a P2 purinoceptor antagonist, when injected into the retrofacial area of the medulla (Boetzinger complex) of anesthetized rats decreases phrenic nerve discharge and reduces the respiratory response to CO2. Cells in the rat medullary raphe that are stimulated by acidosis are serotonergic and contained tryptophan hydroxylase, the rate-limiting enzyme for serotonin synthesis. On the other hand, cells that are inhibited by acidosis lack this enzyme. Other studies indicate that glutamate receptors, neurokinin, and gamma-aminobutyric acid (GABA) receptors may also play a role in mediating central chemoreceptor responses. The transmission of chemoreceptor signal to respiratory motor pathways is only partially affected by synaptic blockade. This suggests the signal transmission also involves metabolic and/or electrical coupling via intracellular channels that form gap junctions. Gap junctions have been demonstrated in CO2-sensitive cells in the nucleus of the solitary tract/ dorsal motor nucleus of the vagus, the medullary raphe, and locus ceruleus. While these gap junctions seem to be involved in the sensing of CO2, how they operate to facilitate or modify chemoresponses is unknown.
Cardiovascular Effects of Central Chemoreceptors

Increased levels of CO2 heighten sympathetic discharge. About half of this increase is attributable to central and the rest to peripheral chemoreceptors. Neurons that mediate increases in sympathetic activity are found on the rostal ventrolateral medulla, in the same vicinity as respiratory chemoreceptors. However, it is not clear whether the same neurons excite ventilation and stimulate sympathetic activity. The increased sympathetic activity that is produced by both central and peripheral chemoreceptors is offset by vagal stretch receptor activity that increases as ventilation is stimulated by hypercapnia. Increases in blood pressure have been reported with cyanide injections into the ventral medulla suggesting that there are oxygen-responsive cells that can stimulate sympathetic activity. Depolarization occurs when cyanide is injected into cells cultured from the rostral ventral medullary surface but this response is absent when heme-oxygenase activity is blocked. There are also thought to be hypoxia receptors in the pre-Boetzinger complex that have a respiratory stimulating action. It has been suggested that these oxygen-sensitive neurons are involved in gasping. The effects of hypoxia on the brain are complex since hypoxia also exerts an important depressive effect on ventilation perhaps by increasing levels of GABA. Hypoxia can also increase lactate levels simulating central CO2 receptors and augment cerebral blood ow.

Central Chemoreceptors in Health and Disease

Measurement of Chemoreceptor Sensitivity

Hypercapnia in addition to its effect on respiration also stimulates the autonomic nervous system.

As CO2 increases, ventilation rises linearly until it reaches about two-thirds of the maximum breathing capacity. Tidal volume and frequency also increase linearly until tidal volumes of 1.52.0 l are attained. Tidal volumes thereafter tend to plateau and ventilation increases primarily through increases in breathing frequency. The frequency of breathing increases more as a result of a shortening of expiratory rather than inspiratory duration. CO2 sensitivity is measured by steady state and rebreathing techniques. Steady-state methods require long periods for PCO2 in both the arterial blood and the brain to reach equilibrium. In addition, hypercapnia produces a near-linear increase in cerebral blood ow so that brain PCO2 rises less than arterial PCO2 as higher concentrations of CO2 are inhaled. Consequently, changes in brain PCO2 are not simply related to arterial PCO2 . The rebreathing technique is a simpler and more rapid method for assessing chemosensitivity. After

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3045 s of rebreathing a gas mixture with an initial concentration of 7% CO2 and 30% or more oxygen, differences in alveolar, arterial, and cerebral venous PCO2 narrow greatly and with continued rebreathing increase with time at essentially the same rate. Steady-state and rebreathing methods for measuring ventilatory responses to CO2 provide comparable results in normal humans. It has been suggested that a period of hyperventilation preceding rebreathing to lower starting levels of PCO2 allows threshold levels of PCO2 to be discerned. The large contribution of neural drives from higher brain centers, independent of blood gas levels in the wakefulness state, complicates the interpretation of measurements of CO2 response in conscious subjects. Probably the most accurate measurements of CO2 sensitivity may be obtained in nonREM sleep, when breathing is almost exclusively under chemical control, provided that the test can be carried out without affecting sleep stage. The more rapid response to CO2 of peripheral versus central chemoreceptors has been used to separate peripheral from central receptor contributions. Techniques have included devolution of the steady response or by measuring the effects of a single breath of gas containing high concentrations of CO2. In animals the direct effects of CO2 or acids on peripheral chemoreceptor activity can be assessed but there is no direct way of measuring central chemoreceptor neural responses. Since diseases of the lung or thorax, by changing their mechanical properties, can alter the ventilatory response to CO2 even if chemoreceptor function is normal, more direct ways of assessing respiratory output have been proposed. These include measuring the force generated by the inspiratory muscles contracting nearly isometrically against an occluded airway (occlusion pressure) and measurement of the electrical activity of the diaphragm via surface or esophageal electrodes.
Factors Affecting CO2 Responsiveness

Ventilatory responses to CO2 are quite variable in humans. Unlike the response to hypoxia, which increases substantially soon after birth, the central response to CO2 is nearly fully developed in the newborn. The ventilatory effect of CO2 on peripheral chemoreceptors does increase somewhat in the early newborn period as evidenced by an enhanced transient response. The ventilatory responses to CO2 increase as a function of body size but otherwise do not change appreciably with advancing age. Even accounting for body size the ventilatory response to CO2 in women is less than in men. Hypercapnic

ventilatory responses in women also vary through the menstrual cycle and are greatest in the luteal phase as a result of the respiratory stimulant effect of the higher progesterone levels. High progesterone/ estrodiol levels seem to explain the hyperventilation of pregnancy. A variety of other endocrine and metabolic factors can inuence CO2 responsiveness. Hyperthyroidism increases hypercapnic ventilatory responses and the augmentation of chemosensitivity is closely linked to the increase in metabolic rate. Conversely, hypothyroidism blunts chemosensitivity and when severe, can lead to hypoventilation and hypercapnia. The acidbase status of the body also has a significant effect on CO2 responsiveness. Elevated bicarbonate ion concentration increases the buffering of H and decreases the ventilatory response to CO2. There are important behavioral inuences on breathing and on the ventilatory responses to CO2. The removal of wakefulness effects during sleep and anesthesia blunts CO2 responsiveness. Relaxation techniques such as yoga can also reduce the ventilatory response to CO2. In contrast, emotional states of fear and anxiety increase resting breathing and lower the resting PCO2 level and may also increase CO2 sensitivity. It is still not clear whether these behavioral effects originating from higher brain centers act to enhance the sensitivity of brainstem chemical control mechanisms or whether they constitute a separate parallel pathway. The hyperventilation syndrome is a condition characterized by persistent hyperventilation and hypocapnia and a variety of symptoms including shortness of breath, chest pain, palpitations, dizziness, and parasthesias. This syndrome is often associated with agoraphobia and panic attacks. The observation that panic attack can be precipitated in these patients by breathing CO2-enriched gas mixtures has led to the suggestion that the disorder is the result of chemoreceptor hyperactivity. However, ventilatory responses to CO2 are generally no greater than normal in the hyperventilation syndrome. The marked variability in respiratory chemosensitivity is due in part to genetic inuences. There is a familial correspondence in ventilatory responsiveness and the correspondence is closer in identical as compared to fraternal twins. Genetic inuences have also been shown in animal studies: mutant mice with abnormalities in RET, MASH-1, and PHOX2B genes have depressed CO2 responses.
Disorders of Central Respiratory Chemical Control

During sleep and with the loss of wakefulness drives, central chemoreceptors play a particularly important

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role in maintaining regular breathing. Normally during sleep, PCO2 rises by a few mmHg and ventilatory responses to CO2 are somewhat reduced. If CO2 sensitivity during sleep is heightened, loop gain is increased. Under these conditions a reduction in PCO2 of only a few mmHg during nonREM sleep may destabilize respiratory control and lead to the development of periodic apneas. This is the basis for CheyneStokes respiration, commonly seen in patients with heart failure, particularly during sleep. An accompanying decrease in CO2 drive to upper airway muscles can result in a loss of upper airway patency in individuals with predisposing anatomic abnormalities, setting the stage for obstructive sleep apnea. As PCO2 rises during the course of an apneic period, ventilatory drive increases or there may be a direct arousal effect so that the apnea terminates and breathing resumes only to begin the entire cycle over again. Persistent and severe periodic apneas during sleep can lead to chronic CO2 retention and hypercapnia even during wakefulness. This can be reversed by treatment of the sleep apnea with continuous positive airway pressure that serves to tether open the upper airways. Chronic hypercapnia often accompanies severe respiratory diseases that markedly impair the function of the ventilatory pump. However, not all individuals with comparable levels of ventilatory dysfunction develop hypercapnia. The propensity for hypercapnia is thought to result only when severe diseases of the ventilatory pump are associated with low CO2 responsiveness. That some individuals are intrinsically predisposed to the development of hypercapnia is suggested by the observations that normal relatives of hypercapnic patients with chronic obstructive pulmonary disease have lower CO2 responses than do relatives of normocapnic patients with chronic obstructive pulmonary disease. Other studies have failed to nd abnormal chemoresponses in patients with lung disease and hypercapnia and have attributed the elevated PCO2 levels to disordered gas exchange in the lungs and a rapid-shallow breathing pattern that minimizes alveolar ventilation. Impaired chemosensitivity is also the critical underpinning of the obesity-hypoventilation (Pickwickian) syndrome. These patients who suffer from severe obesity that imposes a mass load on the chest wall also demonstrate chronic hypercapnia and experience obstructive sleep apnea leading to severe daytime somnolence. If the impairment in chemosensitivity is sufciently severe, hypercapnia can result even in the absence of diseases of the ventilatory pump. Central alveolar hypoventilation is characterized by absent ventilatory responses to hypercapnia and hypoxia. Often

near-adequate levels of ventilation can be maintained during wakefulness but hypoventilation can become profound during sleep. It is thought that congenital central alveolar hypoventilation, also termed Ondines curse, is caused by maldevelopment of neural crest cells; it is often associated with Hirschsprungs disease in which varying lengths of the colon are aganglionic. Patients often have a family history of the disorder that is associated with several different gene mutations especially of the PHOX2B gene, the receptor tyrosine kinase, the RET gene and endothelin1 and 3 genes, and brain-derived neurotrophic factor. Articial mechanical ventilatory support is often required to maintain adequate levels of ventilation and a normal PCO2 .
See also: Hypoxia and Hypoxemia. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Other Important Neuropeptides.

Further Reading
Bradley SR, Pieribone VA, Wang W, et al. (2002) Chemosensitive serotonergic neurons are closely associated with large medullary arteries. Nature Neuroscience 5: 401402. Bruce EN and Cherniack NS (1987) Central chemoreceptors. Journal of Applied Physiology 62: 389402. Cherniack NS, Dempsey J, Fencl V, et al. (1977) Workshop on assessment of respiratory control in humans. I. Methods of measurement of ventilatory responses to hypoxia and hypercapnia. American Review of Respiratory Diseases 115: 177 181. Dean JB, Ballantyne D, Cardone DL, Erlichman JS, and Solomon IC (2002) Role of gap junctions in CO2 chemoreception and respiratory control. American Journal of Physiology. Lung Cellular and Molecular Physiology 283: L665L670. Dreshaj IA, Haxhiu MA, and Martin RJ (1998) Role of the medullary raphe nuclei in the respiratory response to CO2. Respiratory Physiology 111: 1523. Erlichman JS, Cook A, Schwab MC, Budd TW, and Leiter JC (2004) Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 286: R289R302. Kiwull-Schone H, Wiemann M, Frede S, Bingmann D, and Kiwull P (2003) Tentative role of the Na /H exchanger type 3 in central chemosensitivity of respiration. Advances in Experimental and Medical Biology 536: 415421. Morrell MJ, Heywood P, Moosavi SH, Guz A, and Stevens J (1999) Unilateral focal lesions in the rostrolateral medulla inuence chemosensitivity and breathing measured during wakefulness, sleep and exercise. Journal of Neurology, Neurosurgery and Psychiatry 67: 637645. Nattie EE (1999) CO2, brainstem chemoreceptors and breathing. Progress in Neurobiology 59: 299331. Nattie EE (2001) Central chemosensitivity, sleep, and wakefulness. Respiratory Physiology 129: 257268. Neubauer JA and Sunderram J (2004) Oxygen-sensing neurons in the central nervous system. Journal of Applied Physiology 96: 367374.

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Okada Y, Chen Z, and Kuwana S (2001) Cytoarchitecture of central chemoreceptors in the mammalian ventral medulla. Respiratory Physiology 129: 1323. Patrick W, Webster K, Puddy A, Sanii R, and Younes M (1995) Respiratory response to CO2 in the hypocapnic range in awake humans. Journal of Applied Physiology 79: 20582068. Sun MK and Spyer KM (1991) Responses of rostroventrolateral medulla spinal vasomotor neurones to chemoreceptor stimulation in rats. Journal of the Autonomic Nervous System 33: 7984. Wiemann M and Bingmann D (2001) Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity. Respiratory Physiology 129: 5770.

Arterial
F L Powell, University of California San Diego, La Jolla, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Arterial chemoreceptors include the carotid and aortic bodies, which are small organs with a very high blood ow. Glomus cells in the arterial chemoreceptors sense changes in arterial PO2 , PCO2 , and pH by mechanisms involving ion channels and hemecontaining molecules. Decreased PO2 and pH, and increased PCO2 , depolarize glomus cells and trigger the release of neurotransmitters. An unidentied neurotransmitter depolarizes sensory nerve endings in the carotid and aortic body so the intensity of the arterial stimulus is coded as the frequency of action potentials in the afferent nerves going to respiratory and cardiovascular centers in the medulla. Hypoxia and hypercapnia potentiate the effect of each other on the carotid body and the effect of PCO2 is mediated by changes in intracellular pH. The response to changes in arterial blood gases is extremely rapid (in seconds). Arterial chemoreceptors exhibit plasticity during chronic hypoxia by increasing their O2-sensitivity.

sinus at the bifurcation of the common carotid artery at the base of the skull. The aortic bodies are on the aortic arch near the aortic arch baroreceptors. Afferent nerves travel from the carotid bodies to the central nervous system (CNS) in the glossopharyngeal (IX cranial) nerve, and from the aortic bodies to the CNS in the vagus (X cranial) nerve. Efferent nerves to the carotid bodies include sympathetic and parasympathetic innervation of blood vessels, as well as sympathetic innervation of chemoreceptor cells. The carotid body is organized into lobules or glomoids that are surrounded by dense capillary networks and penetrated by the branches of the carotid sinus nerve. Blood supply to the carotid body is from the 1 2 mm long carotid body artery, which branches off the external carotid artery and continues through the carotid body to perfuse the superior cervical and nodose ganglia. Blood supply to the aortic bodies is from short vessels branching off the aortic arch. Carotid bodies have an extremely high blood ow for their size, for example, 1.5 l per 100 g per min in a cat or E15 times blood ow to the brain tissue. There may be some shunt pathways for blood ow past the capillary networks in carotid body glomoids.
Cell Types

Carotid bodies include several different types of cells (Figure 1). Glomus, or type I, cells are round or ovoid, 1012 mm in diameter, and thought to be the primary chemoreceptor cell in the carotid body. These cells develop from the neural crest and are neurosecretory. The ultrastructure of glomus cells is typical of cells that actively synthesize and secrete
O2, CO2, H+ Blood
C SN

Introduction
Arterial chemoreceptor is a generic term for both the carotid body chemoreceptors and aortic body chemoreceptors. Arterial chemoreceptors respond to changes in arterial PO2 , PCO2 , and pH and evoke negative feedback reexes in the respiratory and cardiovascular systems to maintain blood gas homeostasis. These are the most important chemoreceptors that respond to PO2 , making them essential for a normal hypoxic ventilatory response. Most of our knowledge about arterial chemoreceptors is based on studies of the carotid body, and the aortic bodies will be considered similar unless noted otherwise.

GC DA NE ACh SP

SC

Structure
The carotid bodies are small (E2 mm diameter in humans) sensory organs located near the carotid

Figure 1 Schematic of carotid body. PO2 , PCO2 , and pH in arterial blood stimulate glomus cells (GC) to release an unidentied neurotransmitter that excites the carotid sinus nerve (CSN) and sends action potentials to the medulla. Clear- and dense-cored vesicles contain dopamine (DA), norepinephrine (NE), acetylcholine (ACh), and substance P (SP). Sheath cells (SC) surround glomus cells.

CHEMORECEPTORS / Arterial

419

substances, including both clear and dense-cored vesicles. Glomus cells are connected to each other by gap junctions and make presynaptic and postsynaptic connections with afferent nerves in the carotid sinus nerve. Glomus cells contain several neurotransmitters and neuromodulators, as well as the enzymes to synthesize these substances (Figure 1). Opiates (e.g., met-enkephaline) and catecholamines are packaged in dense-cored vesicles in glomus cells. Dopamine is the most abundant catecholamine in the carotid body but norepinephrine is found as well. Clear-cored vesicles are thought to contain acetylcholine. Other neuroactive agents in glomus cells include serotonin (5-hydroxytryptamine), substance P, neurokinin A, bombesin, and atrial natriuretic peptide (ANP). Sheath cells, also known as type II or sustentacular cells, resemble glial or Schwann cells. Sheath cell bodies are located near the periphery of the carotid body and their processes reach into the organ to cover the surfaces of glomus cells that are not in contact with nerves or other glomus cells. Sheath cells generally prevent direct connections between glomus cells and capillary endothelial cells; however, they do not act as a diffusion barrier for relatively large (40 000 molecular weight) substances from the blood. Carotid bodies also include capillary endothelial cells and autonomic ganglia cells.

potassium channel conductance, which depolarizes glomus cells. Similarly, heme-oxygenase (HO) may act as an O2-sensor because hypoxia inhibits HO-2 and the production of carbon monoxide (CO); glomus cells are inhibited by low concentrations of CO. Nitric oxide synthases (NOS-1 and NOS-3) may sense O2 because hypoxia inhibits NOS and nitric oxide (NO) inhibits carotid body activity. However, NOS is only in the carotid sinus nerve afferents so it is not essential for O2-sensing by glomus cells. Finally, there is evidence for mitochondrial cytochromes with low oxygen afnity in glomus cells that may act as O2-sensors, although it is not clear how these depolarize glomus cells. Several kinds of potassium (K ) channels also contribute to O2-sensing in glomus cells. Background K channels (also known as leak currents) are sensitive to physiological levels of hypoxia, which decreases their open probability and depolarizes the glomus cell. Glomus cells also have a unique inward rectifying K channel responsible for an HERG-like K current that is sensitive to O2. However, it is not known if this channel responds to physiological levels of hypoxia. Glomus cells also have large conductance Ca2 -activated K channels (BK channels) that may be physically linked with HO-2 in the glomus cell membrane. BK channels are O2-sensitive and opened by CO so they represent a mechanism of oxygen sensing that involves oxidases and K channels.

Oxygen Sensing
Oxygen sensing in the carotid body has been localized to the glomus cells but the precise mechanism is still unknown. Decreased PaO2 depolarizes the glomus cell, which increases intracellular calcium Ca2 to cause exocytosis of a neurotransmitter and excitation of the carotid sinus nerve. In carotid bodies in vivo, PO2 at the glomus cells is less than the arterial PO2 but it is greater than tissue PO2 in most other organs. This is because blood ow is relatively high and diffusion distances are small in carotid bodies. Hence, molecular mechanisms of O2-sensing in the carotid bodies are sensitive to PO2 in the physiological range. Candidates for molecular sensors providing the initial event in oxygen sensing fall into two general classes: heme-containing enzymes and O2sensitive ion channels. Multiple O2-sensors that are sensitive to different ranges of PO2 may be involved in glomus cell chemotransduction. Heme-containing enzymes that could sense O2 include NADP(H) oxidase in glomus cells. NADP(H) oxidase generates reactive oxygen species (ROS) such as H2O2 in the presence of oxygen. ROS increases potassium channel conductance so hypoxia decreases

CO2 and H Sensitivity

The mechanism of PCO2 and pH chemoreception in glomus cells appears to be a common response to changes in intracellular pH. Intracellular acidity inhibits Ca2 -activated K currents in glomus cells, leading to depolarization and increased current through voltage-gated Ca2 channels. The increase in intracellular Ca2 promotes exocytosis of an excitatory neurotransmitter that stimulates the carotid sinus nerve. The increase in intracellular Ca2 from hypoxia is potentiated by hypercapnia, contributing to the multiplicative effect of O2 and CO2 on carotid body activity. PCO2 can diffuse into glomus cells and cause large changes in intracellular pH. Extracellular pH changes in blood cause smaller changes in intracellular pH of glomus cells. Hence, carotid body chemoreceptors are less sensitive to metabolic changes than respiratory changes in pH.

Sensory Coding
Figure 2 shows the effect of arterial stimuli on the frequency of action potentials in the carotid sinus

420 CHEMORECEPTORS / Arterial

P aCO 60 40 20

40 30 20 Action potential frequency (impulses/s) 10 0 0 (a) 25 20 15 10

50

100
2

150

200

250

P aO (mmHg)

pHa 7.25

carotid sinus nerve. Arterial chemoreceptor activity increases when PaO2 falls below 100 mmHg if PaCO2 is maintained at normal levels. However, if PaCO2 decreases, then PaO2 must decrease further to stimulate chemoreceptor activity to the same level. This latter scenario would occur, for example, when hypoxia stimulates ventilation and decreases PaCO2 . Conversely, carotid body chemoreceptors can be stimulated at higher PO2 levels if PaCO2 is increased. Hence, CO2 potentiates carotid body O2-sensitivity and vice versa. This synergistic, or multiplicative, effect of hypoxia and hypercapnia on carotid body chemoreceptors is important because it explains the multiplicative effect of PaO2 and PaCO2 on ventilation.
CO2/pH

7.45 5 0 0 (b) 20 40 60 P aCO2 (mmHg)

Figure 2 Carotid body chemoreceptor response (action potential frequency) to P aO2 and P aCO2 (a) and P aCO2 and pHa (b) for two different bers. Increasing PCO2 increases the response to PO2 , and the effect of the two stimuli together is more than the sum of the individual effects, as explained in the text. Decreasing pH increases the response at any PCO2 , but PCO2 still has an effect when pH is held constant. Adapted from Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121 and Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientic Publications.

Figure 2(b) shows the effect of pH and PCO2 on carotid body chemoreceptors. PaCO2 changes can affect chemoreceptor activity even if pHa is held constant, and vice versa. Aortic bodies in humans do not respond to pHa changes, and this is one exception to remember about the aortic bodies being qualitatively different from the carotid bodies. Therefore, the carotid bodies are the only chemoreceptors that respond to metabolic changes in pHa when PaCO2 is constant in humans. Not shown in Figure 2 is the increase of slope of the carotid body response to PCO2 caused by hypoxia. This results from the multiplicative effect of O2 and CO2 on the carotid body described above.
Pattern and Speed of Response

nerve. The response to O2 is hyperbolic while the response to CO2 is essentially linear. As explained above, hypoxia, hypercapnia, and acidity cause glomus cells to depolarize, increase intracellular Ca2 , and release an excitatory neurotransmitter. These are graded responses to stimuli so the frequency of action potentials in the carotid sinus nerve codes the stimulus intensity.
Hypoxia

The physiological stimulus for carotid bodies is O2 partial pressure; they do not respond to changes in O2 content or hemoglobin saturation if PO2 remains constant. This explains the lack of ventilatory response in clinical conditions such as anemia or carbon monoxide poisoning. There is some evidence that aortic bodies may respond to changes in O2 content. Carotid bodies are sensitive to PO2 over the entire physiological range, although they are more sensitive to hypoxia. At normal PaO2 (100 mmHg) and PaCO2 (40 mmHg), there is a low, tonic level of activity in the

Arterial chemoreceptors respond rapidly (within seconds) to changes in PaO2 , PaCO2 , and pHa. Changes in arterial blood gases that occur in phase with breathing, especially during conditions such as exercise, can be sensed by arterial chemoreceptors and may stimulate ventilation. This rapid response explains how ventilation can be altered within a single breath when arterial blood gases change. Carotid body chemoreceptors contain carbonic anhydrase, which will increase the speed of response to PaCO2 according to the intracellular pH-sensing mechanisms described above. Although different patterns of action potential frequency (e.g., bursting) can occur with different stimuli, the CNS only responds to the average discharge frequency from carotid bodies. There is no evidence that stimulating carotid body chemoreceptors with O2 versus CO2, for example, can result in different reex effects.
Neurotransmitters

Despite the identication of several neuroactive substances being released from carotid bodies during

CHEMORECEPTORS / Arterial

421

stimulation, the excitatory neurotransmitter between glomus cells and the carotid sinus nerve is not known. Acetylcholine is the most likely candidate. Acetylcholine stimulates carotid body activity via nicotinic receptors although chemoreception is not blocked by nicotinic or muscarinic receptor blockers. Substance P stimulates carotid body activity but it is not present in the glomus cells of all species. Norepinephrine and serotonin excite carotid sinus nerve activity but they do not appear to be the primary neurotransmitters from glomus cells and act as neuromodulators. Hypoxia stimulates release of dopamine from glomus cells in a graded fashion but dopamine is mainly inhibitory in the carotid body. Dopamine acts primarily on dopamine-2 autoreceptors on glomus cells to limit excitability by negative feedback. Met- and leuenkephalins also depress carotid sinus nerve activity. Multiple inhibitory neurotransmitter systems suggest that limiting and modulating chemoreceptor activity is physiologically significant.
Autonomic Modulation

during acclimatization to hypoxia at high altitude. It is reasonable to suspect that similar adaptations may take place in patients with chronic hypoxemia from respiratory disease, although arterial CO2 levels are higher than in normal subjects acclimatized to altitude. However, it has been impossible to study the arterial chemoreceptors in patients with respiratory disease to date so it is not clear if the adaptations described for normal subjects occur in patients. Similarly, it is not clear if some of the difference in the progress of respiratory diseases in different patients depends on individual differences in the premorbid arterial chemoreceptor sensitivities and reex response, or in the ability of these processes to adapt to chronic hypoxia. The effects of chronic hypoxia on arterial chemoreceptors in healthy subjects are described next.
Plasticity in Chronic Hypoxia

Stimulating the peripheral end of the transected carotid sinus nerve inhibits carotid body chemoreceptor activity in experimental animals. This involves at least two mechanisms. Parasympathetic nerves release NO to cause vasodilation, which increases local PO2 and decreases hypoxic stimulation. Sensory bers may also release NO, which increases cyclic guanosine monophosphate (GMP) in glomus cells and inhibits their activity. It is not known if the autonomic effect is physiologically significant in vivo.

Arterial Chemoreceptors and Respiratory Disease

Diseases of the arterial chemoreceptors per se are relatively rare in populations living at sea level but chemodectomas (carotid body tumors) are more common in populations found at high altitude. This may contribute to the blunted hypoxic ventilatory response observed in some high-altitude natives and people suffering from chronic mountain sickness. The carotid bodies and their innervation may also be damaged during surgical procedures in the region, such as carotid endarterectomy. However, the consequences of this for respiration are relatively unstudied. More commonly, arterial chemoreceptors are important in respiratory disease in terms of the physiological responses they elicit with, for example, hypoxemia or CO2 retention. The basic responses to changes in arterial blood gases have been described above. It is known that these responses can be altered by chronic hypoxia in healthy subjects, for example

Chronic hypoxia (days to weeks) increases O2-sensitivity of carotid body chemoreceptors. This increases afferent input from carotid bodies to respiratory centers in the brain and stimulates ventilation. This contributes to the time-dependent increase in ventilation during acclimatization to hypoxia. The effect is specic to hypoxia because it occurs even when arterial pH and PCO2 are held at normal levels during hypoxia and it does not occur with chronic application of other stimuli such as CO2. Changes in ion channels with chronic hypoxia tend to depolarize glomus cells and increase O2-sensitivity. Chronic hypoxia downregulates K conductance, increases the density of Na and Ca2 channels, and preferentially increases Ca2 inux through L-type Ca2 channels in glomus cells. Chronic hypoxia also increases the density of O2-sensitive Ca2 -activated K channels on glomus cells. Chronic hypoxia also affects neurotransmitters in the carotid body, although it does not cause a coordinated increase in excitatory, or decrease in inhibitory neurotransmitters. Substance P, an excitatory neurotransmitter in the carotid body, decreases in carotid bodies during chronic hypoxia. Dopamine, an inhibitory neurotransmitter in the carotid body, increases in carotid bodies during chronic hypoxia. Chronic hypoxia increases nicotinic receptors on afferent nerve endings in the carotid body but the effects of acetylcholine on O2-sensitivity do not change. The expression of new neuromodulators in the carotid body can increase O2-sensitivity. Endothelin1 (ET1) is not observed in carotid bodies under normoxic control conditions but it stimulates carotid bodies. Chronic hypoxia increases ET1 and blocking the ETA receptor for ET1 eliminates the increase in

422 CHEST WALL ABNORMALITIES

O2-sensitivity with chronic hypoxia in experimental animals. Chronic hypoxia also causes significant morphological changes in the carotid body but these cannot explain the functional changes. Carotid bodies show hypertrophy, hyperplasia of glomus cells, and increased capillarity after exposure to 2 or more weeks of hypoxia. These changes are reversible. Neovascularization is stimulated by endothelial growth factor (VEGF), which is controlled by hypoxic inducible factor 1a (HIF-1a), and endothelin-1 (ET1).
See also: AcidBase Balance. Arterial Blood Gases. Chemoreceptors: Central. High Altitude, Physiology and Diseases. Ventilation: Control.

Further Reading
Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121.

Dinger B, He L, Chen J, Stensaas L, and Fidone S (2003) Mechanisms of morphological and functional plasticity in the chronically hypoxic carotid body. In: Lahiri S, Semenza G, and Prabhakar N (eds.) Oxygen Sensing: Responses and Adaptation to Hypoxia, pp. 439465. New York: Dekker. Fidone S, Gonzales C, Almaraz L, and Dinger B (1997) Cellular mechanisms of peripheral chemoreceptor function. In: Crystal RG and West JB (eds.) The Lung: Scientic Foundations, 2nd edn., pp. 17251737. Philadelphia: Lippincott-Raven. Gonzalez C, Almaraz L, Obeso A, and Rigual R (1994) Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiological Reviews 74: 829898. Heath D and Williams DR (1995) High-Altitude Medicine and Pathology, 4th edn. Oxford: Oxford University Press. Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientic Publications. Prabhakar N (2000) Oxygen sensing by the carotid body chemoreceptors. Journal of Applied Physiology 88: 2287 2295. Zak FG and Lawson W (1982) The Paraganglionic Chemoreceptor System: Physiology, Pathology, and Clinical Medicine. New York: Springer.

CHEST WALL ABNORMALITIES

J L Allen and O H Mayer, The Childrens Hospital of Philadelphia, Philadelphia, PA, USA K W Gripp, A.I. DuPont Hospital for Children, Wilmington, DE, USA
& 2006 Elsevier Ltd. All rights reserved.

Introduction
The chest wall, including the ribcage and respiratory muscles, comprises the pump component of the respiratory system. Respiratory loads, primarily elastic and resistive, arise from the lung and the chest wall itself. Disorders of the thoracic cage can affect both the pump and the load components. This chapter describes some of the basic mechanisms by which congenital abnormalities of the chest wall are produced, and the evaluation and treatment of chest wall disorders.

Abstract
Chest wall abnormalities can substantially impact the quality of breathing. Chest wall dysfunction can occur as a primary disorder due to congenital (e.g., VATER syndrome), genetic (e.g., JarchoLevin syndrome), or acquired (e.g., scoliosis) causes, or can occur as a secondary disorder due to conditions such as obesity, neuromuscular disease, or chronic obstructive pulmonary disease. Recently, much has been learned about the genetic mechanisms producing chest wall disorders. These mechanisms include dominant negative action (e.g., Marfan syndrome), gain-of-function mutation (e.g., achondroplasia), haploinsufciency (e.g., Campomelic dysplasia), and loss-of-function mutations (e.g., JarchoLevin syndrome). Clinical features common to all these disorders include, in severe cases, respiratory insufciency due to pulmonary hypoplasia or respiratory pump failure. There are a variety of noninvasive interventions to help counteract the effects of chest wall dysfunction; these improve airway clearance (mechanical percussion devices and cough assist devices) and improve ventilation (noninvasive or invasive nocturnal or continuous mechanical ventilation). Surgical interventions to correct or prevent further progression of the disorder include bracing, spinal fusion, and, more recently, introduction of the vertical expandable prosthetic titanium rib (VEPTR), which has the advantage of allowing for future spinal growth.

Etiology
Chest wall abnormalities can be categorized as primary or secondary. Primary chest wall disorders can be congenital or acquired. There are two types of congenital abnormalities. Those with a genetic basis such as achondroplasia or JarchoLevin syndrome have a predictable recurrence risk, while those that arise embryologically or secondary to intrauterine environmental causes, such as VATER (vertebral, anal, tracheo-esophageal stula, radial, and renal anomalies) syndrome, are without a known genetic basis or recurrence risk. Acquired chest wall disorders, such as idiopathic scoliosis or pectus excavatum become apparent later in childhood or adolescence; while they may be familial, there is no certain genetic or environmental cause.

CHEST WALL ABNORMALITIES 423

Secondary chest wall disorders lead to dysfunction caused by other illnesses or mechanical limitations. Examples include chest wall muscle weakness due to neuromuscular disorders and malnutrition, restrictive chest wall disease due to pleural effusions or obesity, and mechanical inefciencies of the chest wall imposed by diaphragmatic attening and loss of the area of apposition seen in the pulmonary hyperination of obstructive lung diseases such as cystic brosis, asthma, and chronic obstructive pulmonary disease. Only primary chest wall disorders are discussed in this article. Secondary chest wall disorders are discussed in the articles listed in See also.

Pathology
Skeletal dysplasias may lead to severe pulmonary complications, due either to pulmonary hypoplasia or to respiratory pump failure. The underlying defect in skeletal dysplasia may affect either the extracellular matrix or the intracellular metabolism of cells forming skeletal structures. Though the ribs and cartilage provide the bulk of the support of the thoracic cavity, muscular abnormalities can also have substantial impact on thoracic mechanics and function.
Congenital Disorders, Genetic Basis Known

rhizomelic short stature and macrocephaly. Patients with achondroplasia have short ared ribs, forming a shallow thorax with decreased chest wall circumference. In addition, patients often have severe midfacial hypoplasia causing obstructive apnea, or central apnea due to brainstem compression secondary to a small foramen magnum and mild hydrocephalus. The rare patients with homozygous achondroplasia show a much more severe phenotype and die in early infancy, often from respiratory complications. In JarchoLevin syndrome, or spondylothoracic dysplasia, rib and spinal abnormalities produce a short thorax with a fan-like conguration of ribs (Figure 1) due to their fusion at the costovertebral junctions. This also presents in infancy and causes severe lung restriction and respiratory insufciency for which some patients require mechanical ventilation. The characteristic ndings of cleidocranial dysplasia are wide, late-closing fontanels, hypoplastic or absent clavicles, and supernumerary teeth. More subtle abnormalities include moderate short stature, a narrow thorax with short ribs, hypoplastic pubic bones, and brachydactyly. Respiratory distress may occur but is relatively rare. Other clinical malformation syndromes affecting the thorax and their genetic bases (where known) are listed in Table 1.
Congenital Disorders, Environmental or Genetic Basis Unknown

Intrinsic skeletal dysplasias vary greatly in severity and in their effects on the respiratory system, ranging from lethality due to pulmonary hypoplasia to respiratory pump failure and restrictive lung disease. The most common form of skeletal dysplasia (frequency 1/10 000) is achondroplasia, characterized by

Jeune syndrome, or asphyxiating thoracic dystrophy, is a disorder of rib development in which the cross-sectional area of the ribcage does not increase

Figure 1 10-year-old girl with JarchoLevin syndrome and the fan-like arrangement of ribs with bilateral vertical expandable prosthetic ribs in place.

Table 1 Malformation syndromes affecting the thorax Disorder VATER association Physical ndings Anal atresia; TE stula; radial and renal anomalies Hemifacial microsomia with microtia and small mandible; may be bilateral; preauricular skin tags; renal anomalies Aortic dilatation; lens dislocation; arachnodactyly; joint laxity Multiple fractures, joint laxity; blue sclera; dentinogenesis imperfecta Macrocephaly, short limb dwarfism; narrow foramen magnum Bowed long bones; male to female sex reversal; cleft palate Wide anterior fontanel; supernumerary teeth; short stature Vertebral and rib segmentation defects; short neck; long ngers Thoracic involvement Vertebral segmentation anomalies; structural rib anomalies; scoliosis Hemivertebrae; structural rib anomalies; scoliosis Respiratory complications Rare, due to small thoracic cage and/or scoliosis Rare, due to small thoracic cage and/or scoliosis Rarely due to abnormal thorax or pneumothorax Unstable thorax with multiple rib fractures Restrictive lung disease; central and obstructive apnea Respiratory insufciency may lead to death in infancy Rare Inheritance mode Sporadic Gene None Pathomechanism Embryologic in origin

Facioauriculovertebral spectrum; Goldenhar syndrome Marfan syndrome

Sporadic

None

Embryologic in origin

Osteogenesis imperfecta Achondroplasia

Scoliosis; pectus excavatum or carinatum; pneumothorax Rib fractures

Autosomal dominant

FBN1

Dominant negative action Dominant negative action Gain-of-function

Autosomal dominant

COLIA1 or COLIA2 FGFR3

Small thoracic cage

Autosomal dominant

Campomelic dysplasia

Small thoracic cage; kyphocoliosis

Autosomal dominant

SOX9

Haploinsufciency

Cleidocranial dysplasia Spondylothoracic dysplasia; JarchoLevin syndrome Diastrophic dysplasia

Partially or completely absent clavicle Short thorax due to vertebral and rib defects

Autosomal dominant

CBFA1

Haploinsufciency

Short limb dwarfism, clubfeet; cleft palate; cervical kyphosis

Progressive kyphoscoliosis

Respiratory insufciency due to small thoracic volume, may be lethal Respiratory compromise due to kyphosis

Autosomal recessive (most cases of Puerto Rican ancestry) Autosomal recessive

DLL3

Loss of function

DTDST

Loss of function

Category refers to the presumed genetic mechanism by which the mutations cause the phenotype (see text). Mutations in the listed genes have been identied in the respective disorders. In some disorders, there is a strict correlation between the genotype (i.e., a specic mutation or a mutation in a specic gene), while in others, heterogeneity is suspected or proven (e.g., not all patients with JarchoLevin syndrome have mutations in DLL3). The genetic basis of the disorders are discussed in detail in Pathogenesis section.

CHEST WALL ABNORMALITIES 425

Figure 2 Anterior computed tomography (CT) reconstruction of Jeune syndrome with the narrow and long thorax also showing the short ribs.

Figure 3 CT image of Jeune syndrome at the carina that demonstrates the short ribs and anterior chest wall deformity.

normally and the thorax becomes long but narrow or constricted (Figures 2 and 3). It often presents in early infancy and results in severe pulmonary compromise, often requiring invasive mechanical ventilation and aggressive airway clearance.
Acquired Disorders

The most frequent chest wall disorder in children is pectus excavatum. Though pectus excavatum and

pectus carinatum may be cosmetically disturbing, they usually have little or no impact on cardiopulmonary function. Pectus excavatum, while typically acquired as an isolated deformation, can occur with connective tissue disorders like Marfan syndrome, or in subjects with substantial respiratory muscle weakness like spinal muscular atrophy type 1. Scoliosis is most commonly idiopathic, but can also be congenital or secondary. Idiopathic scoliosis is the most common form of scoliosis, accounting for 8085% of all lateral spinal curvature. The frequency of onset increases with age, with the most common age of onset being adolescence (11 and older); idiopathic scoliosis is very uncommon under 4 years of age. Though it commonly presents with the relatively benign nding of spinal asymmetry on forward bending, if the curve is greater than 351, there is a greater than 40% chance of significant lung restriction and of moderate to severe gas trapping likely due to airway compression. The risk for progression is related to both the degree of curve and the age of the patient, with curves of 301 rarely progressing in adulthood. Scoliosis is much more likely to progress in prepubertal children. Congenital scoliosis is less common than idiopathic scoliosis and differs in having a causative spinal abnormality. Abnormalities include hemivertebrae, as in Goldenhar syndrome or the facioauriculovertebral spectrum, abnormal vertebral segmentation, as in the VATER association, and inadequate ossication. In congenital scoliosis the curve may not be clinically apparent at birth, but with longitudinal growth the spine will grow asymmetrically creating a curve. Because of the many different causes, the rate of progression and prognosis is very variable. Secondary scoliosis can occur due to inadequate truncal support, such as in neuromuscular disease like Duchenne muscular dystrophy and spinal muscular atrophy (Figure 4). The curve occurs after progressive loss of muscle tone and truncal support with the spine curving to one side and, in some situations, rotating. This can be further worsened with loss of ambulation and the different forces that act on the ribcage when a child is in the sitting position as opposed to upright. Isolated rib fusion can occur and can also lead to progressive scoliosis as the lateral edge of the thorax is xed while the spine continues to grow, leading to a poorly compliant chest wall. Conversely, absence of ribs can also cause progressive scoliosis due to inadequate support with a highly compliant or accid thoracic cavity.

426 CHEST WALL ABNORMALITIES

embryologic anomaly without a known genetic cause and no significant recurrence risk, presents with structural vertebral and rib anomalies. A second example is the facioauriculovertebral spectrum, also known as Goldenhar syndrome or hemifacial microsomia. In addition to structural vertebral and rib anomalies, eye and ear ndings and a small jaw may affect one side of the face, or rarely, both sides.
Gene Mutations: Autosomal Dominant, Dominant Negative Action

Figure 4 Chest radiograph in a 2-year-old male with spinal muscular atrophy type-1 demonstrating significant scoliosis.

Clinical Features
Though there are many different causes of chest wall abnormalities, the clinical manifestations are often quite similar. They can vary from primarily cosmetic disorders, as in pectus excavatum, carinatum, and mild idiopathic scoliosis, to severe restrictive lung disease as in the severe genetic skeletal dysplasias, progressive congenital scoliosis, and neuromuscular disease. With an increasing scoliotic curve, the lung on the concave side becomes more compressed and the range of motion of the diaphragm will decrease. This decreases vital capacity and, with further worsening, can lead to significant nocturnal hypoventilation or to more continuous hypoventilation and the need for noninvasive ventilation due to eventual respiratory muscle fatigue and failure. Pulmonary compression may also lead to poor airway clearance in the affected lung due to inability to fully expand the lung and airway narrowing that may occur from the lung compression or airway bending. In this environment, poor airway clearance can lead to retained secretions or mucus plugging and segmental atelectasis, persistent or chronic pneumonia, and bronchiectasis.

In contrast to embryologic abnormalities, disorders caused by gene mutations may have a recurrence risk, and may be present in other family members in addition to the patient. Autosomal dominant conditions include the connective tissue disorders, Marfan syndrome and osteogenesis imperfecta (Table 1). Marfan syndrome is caused by heterozygous mutations in the brillin 1 gene (FBN1) resulting in an abnormal gene product. Fibrillin is the major constitutive element of extracellular microbrils. Incorporation of a structurally abnormal protein disrupts the macromolecular structure of the extracellular microbrils, despite the fact that the patient has one wild-type allele encoding structurally and functionally normal brillin. This mechanism of action, by which the structurally abnormal gene product is disease-causing regardless of the presence of normal gene product, is called dominant negative. Another example for this mechanism is osteogenesis imperfecta, resulting from a single mutation in one of the genes encoding a procollagen chain. Mature collagen molecules consist of three procollagen chains forming a triple helical structure. Incorporation of a single structurally abnormal component disrupts this complex structure and leads to the abnormal connective tissue properties including brittle bones.
Autosomal Dominant, Gain-of-Function Mutations

Pathogenesis
Embryologic Abnormalities

Congenital anomalies of the chest wall can occur due to embryological anomalies, or due to underlying gene mutations. The VATER association, an

Achondroplasia is caused by a heterozygous point mutation in the broblast growth factor receptor 3 gene (FGFR3), resulting in a glycine to arginine amino acid substitution at position 380 of the protein product. This membrane-bound receptor has an intracellular tyrosine kinase domain that is activated upon ligand binding and ultimately regulates cell proliferation. The function of the normal receptor is negative regulation of enchondral growth, as demonstrated by the skeletal overgrowth seen in mice lacking both functional copies of the gene. The pathogenesis of the mutation involves constitutive activation of FGFR3, inhibiting proliferation of growth plate chondrocytes and thus causing short limb dwarfism. This gain-of-function mechanism is

CHEST WALL ABNORMALITIES 427

further enhanced by diversion of the abnormal gene product from the normal lysosomal degradation process to increased recycling with prolonged survival. Patients whose parents both have achondroplasia are at 25% risk for homozygosity of the mutation, resulting in extreme short limb dwarfism and lethal respiratory insufciency. A second lethal skeletal dysplasia, thanatophoric dysplasia, is also caused by mutations in FGFR3.
Autosomal Dominant, Haploinsufciency

Management and Current Therapy

Nonsurgical

A third pathogenic mechanism causing autosomal dominant skeletal disorders is haploinsufciency for the functional gene product. This mechanism most often affects protein products acting as transcription factor regulating the expression of other genes. A 50% decrease of the functional protein is diseasecausing in dosage sensitive pathways. Campomelic dysplasia and cleidocranial dysostosis are examples of skeletal dysplasias due to gene mutations resulting in haploinsufciency for the respective transcription factors (see Table 1).
Autosomal Recessive Conditions

Diastrophic dwarfism (achondrogenesis type IB) and JarchoLevin syndrome are due to mutations in both alleles of the respective gene, causing loss-of-function of protein products with enzymatic or transport function (Table 1). The pattern of malformation seen in JarchoLevin syndrome is suggestive of abnormal segmentation in early embryological development. In contrast to haploinsufciency, loss-of-function in both gene alleles is necessary to cause disease. Achondrogenesis type IB causes a clinical syndrome of extreme short stature, poor ossication of the skull and vertebral bodies, severe micromelia of the limbs, extremely short ribs, and stellate long bones. The mutation resides in the diastrophic dysplasia sulfate transporter (DTDST) gene, leading to decreased or absent sulfate transport and abnormalities in cartilage proteoglycans sulfation.
Other Disorders

For subjects with hypoventilation or respiratory failure, intermittent nocturnal noninvasive ventilation can be initiated using a nasal interface. Continuous mechanical ventilation that allows both mobility and full use of both the mouth and nose can be achieved using rhythmic abdominal compression using a Pneumo-Belts or having a tracheostomy placed to allow for invasive mechanical ventilation. In situations in which mobility is not necessary, patients may also use negative pressure ventilation, using a device that applies negative pressure directly to the thorax (cuirass or chest shell) or to the body below the neck (PortaLungs or poncho/jacket). Maximizing airway clearance with manual or mechanical chest percussion and postural drainage augmented with nebulized b2-adrenergic agonists is also very important. For subjects with segmental or lobar compression, the application of distending pressure via nasal mask or tracheostomy can help to open the airway and maintain patency during breathing and coughing. Applying cyclic positive pressure to inate the lungs rapidly followed by negative pressure using

Other disorders with structural abnormalities of the chest wall are genetic in nature but the abnormal genes have not yet been identied. Examples include Jeunes syndrome, the cerebrocostomandibular syndrome (gaps in the posterior aspects of the ribs), and the Fryns syndrome (diaphragmatic hernias in combination with multiple other anomalies). Fryns syndrome is inherited as an autosomal recessive trait, and the multiple affected organ systems are suggestive of underlying mutations in a gene widely expressed during early development.

Figure 5 Postoperative lateral view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (black arrow) and between the iliac crest and 5th rib posteriorly (white arrow). Note the expanded intercostal space between the 4th and 5th ribs.

428 CHEST WALL ABNORMALITIES

Figure 6 Postoperative posterior view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (white arrow) and between the iliac crest and 5th rib (black arrow) posteriorly.

a device such as the Cough Assists device can replace or augment coughing, especially in patients with significant muscle weakness or cognitive disability.
Surgical

Though it is not necessary to correct pectus excavatum in many situations, a ratio of the transverse to the anterioposterior diameter of greater than 3.5 is felt by many to be an indication for surgery. Severe pectus excavatum can impair cardiac output in the upright position and increase the work of breathing at high workloads. The Nuss procedure can correct an abnormality by applying internal, anterior pressure at the apex of the pectus curve. A rigid curved rod is inserted along the inner anterior chest wall and is then rotated up and sutured in place. The correction can be quite substantial and often the bar can be removed after a year without the defect returning. Correction for scoliosis has traditionally involved straightening the spine either with traction alone or permanently with posterior spinal fusion. For idiopathic scoliosis with curves of 451, surgical repair is often performed after a trial of bracing. While scoliosis surgery may not result in an immediate gain in lung volume, it helps minimize the future loss of vital capacity. However, spinal fusion reduces spinal growth in the fused segments and limits future thoracic growth. Alternatives include spinal growing

rods or the vertical expandable prosthetic titanium rib (VEPTR), both of which allow thoracic expansion. For more substantial chest wall abnormalities such as congenital scoliosis with fused ribs, absent ribs, or chest wall constriction from a variety of causes, reconstruction using the VEPTR allows for both lateral stabilization and vertical expansion of the thorax with growth (Figures 5 and 6). The devices can be arranged in a variety of orientations with expansion of the ribs laterally, or from the spine or iliac crest.
See also: Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Nutrition in Respiratory Disease. Obesity. Pectus Excavatum. Pediatric Pulmonary Diseases. Pleural Effusions: Overview. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Trauma, Chest: Bronchopleural Fistula; Postpneumonectomy Syndrome; Subcutaneous Emphysema. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading
Beiser G, Epstein S, Stampfer M, et al. (1972) Impairment of cardiac function in patients with pectus excavatum, with improvement after operative correction. New England Journal of Medicine 287(6): 267272.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 429

Boyer J, Amin N, Taddonio R, and Dozor A (1996) Evidence of airway obstruction in children with idiopathic scoliosis. Chest 109: 15321535. Byers P and Steiner R (1992) Osteogenesis imperfecta. Annual Review of Medicine 43: 269282. Campbell R and Hell-Vocke A (2003) Growth of the thoracic spine in congenital scoliosis after expansion thoracoplasty. Journal of Bone and Joint Surgery America 85-A(3): 409420. Castile R, Staats B, and Westbrook P (1982) Symptomatic pectus deformities of the chest. American Review of Respiratory Diseases 126(3): 564568. Cho J, Guo C, Torello M, et al. (2004) Defective lysosomal targeting of activated broblast growth factor receptor 3 in achondroplasia. Proceedings of the National Academy of Sciences, USA 101(2): 609614. Cornier A, Ramirez N, Arroyo S, et al. (2004) Phenotype characterization and natural history of spondylothoracic dysplasia syndrome: a series of 27 new cases. American Journal of Medical Genetics 128A: 120126. Francomano C (1995) The genetic basis of dwarfism. New England Journal of Medicine 332: 5859. Hunter A, Reid C, Pauli R, and Scott C (1996) Standard curves of chest circumference in achondroplasia and the relationship of chest circumference to respiratory problems. American Journal of Medical Genetics 62: 9197. Kose N and Campbell R (2004) Congenital scoliosis. Medical Science Monitor 10(5): RA104RA110. Lyons-Jones K (1988) Smiths Recognizable Patterns of Human Malformation. Philadelphia: Saunders. McMaster M (2001) Congenital scoliosis. In: Weinstein S (ed.) The Pediatric Spine: Principles and Practice, 2nd edn, pp. 161177. Philadelphia: Lipincott Williams & Wilkins. Park H, Lee S, Lee C, et al. (2004) The Nuss procedure for pectus excavatum: evolution of techniques and early results on 322 patients. Annals of Thoracic Surgery 77: 289295. Stacey A, Bateman J, Choi T, et al. (1988) Perinatal lethal osteogenesis imperfecta in transgenic mice bearing engineered mutant pro-alpha 1(I) collagen. Nature 332: 131136. Tosi L (1997) Osteogenesis imperfecta. Current Opinion in Pediatrics 9(1): 9499. Wong K-S, Hung I, Wang C, and Lien R (2004) Thoracic wall lesions in children. Pediatric Pulmonology 37: 257263.

Chloride Channels

see Ion Transport: Chloride Channels.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE

Contents

Overview Acute Exacerbations Emphysema, Alpha-1-Antitrypsin Deciency Emphysema, General Smoking Cessation

Overview
P J Barnes, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Chronic obstructive pulmonary disease (COPD) is characterized by progressive and largely irreversible airow limitation due to narrowing and brosis of small airways and loss of airway alveolar attachments as a result of emphysema. Cigarette smoking is the most important risk factor, but other noxious gases are important too in developing countries. Genes may determine which smokers are susceptible to the development of airow obstruction. There is a specic pattern of inammation characterized by increased numbers of macrophages, neutrophils, and T lymphocytes (particularly CD8 cells), which is an amplication of the inammation seen in normal cigarette smokers and increases with disease progression. Proteinases, particularly MMP-9, result in degradation of alveolar wall elastin resulting in emphysema. The major symptom is exertional dyspnea

caused by air trapping and hyperination as small airways close, but cough and sputum production are common due to mucus hypersecretion. Management of COPD involves smoking cessation. Bronchodilator therapy reduces hyperination; regular long-acting inhaled b2-agonists and anticholinergics are preferred to short-acting drugs. Inhaled corticosteroids do not reduce inammation but reduce exacerbations and are usually given to patients with severe disease with frequent exacerbations. Long-term oxygen is indicated for patients with respiratory failure who have evidence of pulmonary hypertension. Nonpharmacological treatments include pulmonary rehabilitation, noninvasive ventilation, and lung volume reduction.

Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by progressive development of airow limitation that is not fully reversible. The term COPD encompasses chronic obstructive bronchiolitis with obstruction of small airways and emphysema

430 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is dened by a productive cough of more than 3 months duration for more than 2 successive years; this reects mucus hypersecretion and is not necessarily associated with airow limitation. COPD is common and is increasing globally. It is now the fourth leading cause of death in the US and the only common cause of death that is increasing; this is thought to be an underestimate as COPD is likely to be contributory to other common causes of death. It is predicted to become the third commonest cause of death and the fth commonest cause of disability worldwide in the next few years. It currently affects more than 5% of the adult population and is underdiagnosed in the community. COPD consumes an increasing proportion of healthcare resources, which currently exceed those devoted to asthma by threefold.

be additional risk factors. The role of airway hyperresponsiveness and allergy as risk factors for COPD is still uncertain. Atopy, serum IgE, and blood eosinophilia are not important risk factors. However, this is not necessarily the same type of abnormal airway responsiveness that is seen in asthma. Low birth weight is also a risk factor for COPD, probably because poor nutrition in fetal life results in small lungs, so that decline in lung function with age starts from a lower peak value.
Genetic Factors

Etiology
Several environmental and endogenous factors, including genes, increase the risk of developing COPD (Table 1).
Environmental Factors

In industrialized countries, cigarette smoking accounts for most cases of COPD, but in developing countries, other environmental pollutants, such as particulates associated with cooking in conned spaces, are important causes. It is likely that there are important interactions between environmental factors and a genetic predisposition to develop the disease. Air pollution (particularly sulfur dioxide and particulates), exposure to certain occupational chemicals such as cadmium, and passive smoking may all
Table 1 Risk factors for COPD Environmental factors Cigarette smoking Active Passive Maternal Air pollution Outdoor Indoor: biomass fuels Occupational exposure Dietary factors High salt Low antioxidant vitamins Low unsaturated fatty acids Infections Endogenous (host) factors a1-Antitrypsin deciency Other genetic factors Ethnic factors Airway hyperresponsiveness? Low birth weight

Longitudinal monitoring of lung function in cigarette smokers reveals that only a minority (1540% depending on definition) develop significant airow obstruction due to an accelerated decline in lung function (two- to vefold higher than the normal decline of 1530 ml forced expiratory volume in 1s (FEV1) year 1) compared to the normal population and the remainder of smokers who have consumed an equivalent number of cigarettes (Figure 1). This strongly suggests that genetic factors may determine which smokers are susceptible and develop airow limitation. Further evidence that genetic factors are important is the familial clustering of patients with early onset COPD and the differences in COPD prevalence among different ethnic groups. Patients with a1-antitrypsin deciency (proteinase inhibitor (Pi)ZZ phenotype with a1-antitrypsin levels o10% of normal values) develop early emphysema that is exacerbated by smoking, indicating a clear genetic predisposition to COPD. However, a1-antitrypsin deciency accounts for o1% of patients with COPD and many other genetic variants of a1-antitrypsin that are associated with lower than normal serum levels of this Pi have not been clearly associated with an increased risk of COPD. This has led to a search for associations between COPD and polymorphisms of other genes that may be involved in its pathophysiology. So far, few significant associations have been detected and even those reported have not been replicated in other studies. A 10-fold increased risk of COPD in individuals who have a polymorphism in the promoter region of the gene for tumor necrosis factor alpha (TNF-a) that is associated with increased TNF-a production has been reported in a Taiwanese population but not conrmed in Caucasian populations. Several other genes have been implicated in COPD, but few have been replicated in different populations (Table 2). Techniques such as DNA microarray (gene chips) to detect single nucleotide polymorphisms, proteomics to detect novel proteins, and gene expression proling to measure which known and novel genes are expressed are now

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 431

100

FEV1 (% predicted at age 25)

Nonsmoker or nonsusceptible smoker 75 Susceptible smoker 50 Disability 25 Death 0 25 50 Age (years) 75 Stopped smoking aged 60 years Stopped smoking aged 50 years

Figure 1 Natural history of COPD. Annual decline in airway function showing accelerated decline in susceptible smokers and effects of smoking cessation. Patients with COPD usually show an accelerated annual decline in forced expiratory volume in 1 s (FEV1), often greater than 50 ml year 1, compared to the normal decline of approximately 20 ml year 1, although this is variable between patients. The classic studies of Fletcher and Peto established that 1020% of cigarette smokers are susceptible to this rapid decline. However, with longer follow-up more smokers may develop COPD. The propensity to develop COPD amongst smokers is only weakly related to the amount of cigarettes smoked and this suggests that other factors play an important role in determining susceptibility. Most evidence points towards genetic factors, although the genes determining susceptibility have not yet been determined.

Table 2 Some of the genes associated with COPD susceptibility Candidate genes a1-Antitrypsin a1-Chymptrypsin Matrix metalloproteinase-1, -2, -9, -12 Microsomal epoxide hydrolase Glutathione S-transferase Heme oxygenase-1 Vitamin D binding protein TNF-a promoter Interleukin-13 Risk ZZ genotype high risk MZ, SZ genotypes small risk Associated in some populations Associated in some studies Increased risk Increased risk Small risk Inconsistent Inconsistent Small risk

being used to study cigarette smokers who develop COPD compared with matched smokers who do not. This may identify markers of risk, but may also reveal novel molecular targets for the development of treatments of the future.

Pathology
The term COPD includes chronic obstructive bronchiolitis with brosis and obstruction of small airways and emphysema with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is dened by a productive cough of more than 3 months duration for more than 2

successive years; this reects hypersecretion of mucus and is not necessarily associated with airow limitation. Most patients with COPD have all three pathological mechanisms (chronic obstructive bronchitis, emphysema, and mucus plugging) as all are induced by smoking, but may differ in the proportion of emphysema and obstructive bronchitis (Figure 2). There has been debate about the predominant mechanism of progressive airow limitation and recent pathological studies suggest that it is closely related to the degree of inammation, narrowing, and brosis in small airways. Emphysema may contribute to the airway narrowing in the more advanced stages of the disease. There is inammation in small airways with an increase in macrophages and neutrophils in early stages of the disease indicating innate immune response, but in more advanced stages of the disease there is an increase in lymphocytes (particularly cytotoxic CD8 T cells) including lymphoid follicles, indicating acquired immunity. Emphysema describes loss of alveolar walls due to destruction of matrix proteins (predominantly elastin) and loss of type 1 pneumocytes as a result of apoptosis. Several patterns of emphysema are recognized: centriacinar emphysema radiates from the terminal bronchiole, panacinar emphysema involves more widespread destruction, and bullae are large airspaces. Emphysema results in airway obstruction by loss of elastic recoil so that intrapulmonary airways close more readily during expiration. Chronic hypoxia may lead to hypoxic vasoconstriction, with structural changes in pulmonary

432 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

Normal COPD

Disrupted alveolar attachments (emphysema) Mucosal inflammation, fibrosis Mucus hypersecretion

Emphysema + small airway obstruction Lung hyperinflation trapped gas Dyspnea Exercise tolerance Deconditioning

Mucus hypersecretion

Cough and sputum

Airway held open by alveolar attachments

Airway obstructed by Loss of attachments Mucosal inflammation + fibrosis Obstruction of lumen by mucus

Figure 2 Mechanisms of airow limitation in COPD. The airway in normal subjects is distended by alveolar attachments during expiration, allowing alveolar emptying and lung deation. In COPD, these attachments are disrupted because of emphysema thus contributing to airway closure during expiration, trapping gas in the alveoli, and resulting in hyperination. Peripheral airway are also obstructed and distorted by airway inammation and brosis (chronic obstructive bronchiolitis) and by occlusion of the airway lumen by mucus secretions which may be trapped in the airway because of poor mucociliary clearance.

Poor health status

Figure 4 Symptoms of COPD. The most prominent symptom of COPD is dyspnea, which is largely due to hyperination of the lungs as a result of small airways collapse due to emphysema and narrowing due to brosis, so that the alveoli are not able to empty. Hyperination induces an uncomfortable sensation and reduces exercise tolerance. This leads to immobility and deconditioning and results in poor health status. Other common symptoms of COPD are cough and sputum production as a result of mucus hypersecretion, but not all patients have these symptoms and many smokers with these symptoms do not have airow obstruction (simple chronic bronchitis).

Chronic hypoxia

Pulmonary vasoconstriction Muscularization Intimal hyperplasia Fibrosis Cor pulmonale Edema Death
Figure 3 Vascular changes in COPD. Chronic hypoxia results in hypoxic pulmonary vasoconstriction and leads to structural changes, resulting in secondary pulmonary hypertension. Over time, this may lead to right heart failure (cor pulmonale), which has a poor prognosis, most patients only surviving for 612 months.

Pulmonary hypertension

Obliteration

vessels that eventually lead to secondary pulmonary hypertension (Figure 3). Inammatory changes similar to those seen in small airways are also seen in pulmonary arterioles.

Clinical Features
The clinical features of COPD are usually straightforward and care should be taken to evaluate those features of the illness that are not typical.
Symptoms

symptoms of asthma. Patients have usually lost a considerable amount of their lung volume by the time they present to a doctor, with FEV1 values of o50%. There is usually a history of heavy smoking for many years, often 425 pack years (1 pack year 20 cigarettes daily for 1 year). Progressive shortness of breath on exertion is the predominant symptom and compensatory behavior of the patient may delay diagnosis. Dyspnea results from the hyperination secondary to small airway narrowing resulting in poor quality of life which is further exacerbated by the deconditioning due to reduced physical activity (Figure 4). Cough and sputum production are common symptoms but are also found in cigarette smokers without airow limitation (chronic bronchitis). A change in the character of the cough may indicate lung carcinoma. Wheezing may occur during exacerbations and periods of breathlessness. Ankle swelling may occur when there is cor pulmonale. Weight loss often occurs in advanced disease, but the mechanism is uncertain. Loss of skeletal muscle bulk may be a response to systemic inammation.
Signs

The symptoms of COPD are slowly progressive over many years, in contrast to the episodic and variable

When FEV1450% predicted, there may be no abnormal signs. The typical patient with more severe COPD shows a large, barrel-shaped chest due to hyperination, diminished breath sounds, and

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 433

distant heart sounds due to emphysema, and prolonged expiration with generalized wheezing on expiration.
Diagnosis

Diagnosis is commonly made from the history of progressive dyspnea in a chronic smoker and is conrmed by spirometry, which shows an FEV1/vital capacity (VC) ratio of o70% and FEV1 o80% predicted. Staging of severity is made on the basis of FEV1 (Table 3), but exercise capacity and the presence of systemic features may be more important determinants of clinical outcome. Measurement of lung volumes by body plethysmography shows an increase in total lung capacity, residual volume, and functional residual capacity, with consequent reduction in inspiratory capacity, representing hyperination as a result of small airway closure (Table 4). This results in dyspnea which may be measured by
Table 3 Staging of COPD by severity of airow limitation Stage 0 (at risk) Stage I (mild) Chronic symptoms (cough, sputum) Normal spirometry 7Chronic symptoms (cough, sputum) FEV1/FVC o70%, FEV1 480% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 o8050% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 45030% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1 o30% predicted or respiratory insufciency/right heart failure

dyspnea scales and reduced exercise tolerance, which may be measured by a 6 min or shuttle walking test. Carbon monoxide diffusion is reduced in proportion to the extent of emphysema. A chest X-ray is rarely useful but may show hyperination of the lungs and the presence of bullae. High-resolution computerized tomography demonstrates emphysema but is not used as a routing diagnostic test. Blood tests are rarely useful; a normocytic normochromic anemia is more commonly seen in patients with severe disease than polycythemia due to chronic hypoxia. Arterial blood gases demonstrate hypoxia and in some patients hypercapnia.
Natural History

Stage II (moderate)

Stage III (severe)

COPD is slowly progressive with an accelerated decline in FEV1, leading to slowly increasing symptoms, decreasing lung function, and eventually respiratory failure (Figure 1). The only strategy to reduce disease progression is smoking cessation, although this is relatively ineffective once FEV1 has fallen below 50% predicted. Patients with more severe COPD develop acute exacerbations, which have a prolonged effect on quality of life for many months. Most of the medical costs associated with COPD are linked to acute exacerbations that lead to hospital admission. There is still debate about the role of acute exacerbations in disease progression, but the decline in lung function may be accelerated further following an acute exacerbation.

Stage IV (very severe)

Pathogenesis
Recently, there have been important new insights into the pathogenesis of COPD.
Chronic Inammatory Mechanisms

Table 4 Lung function test abnormalities in COPD Lung function test Forced expiratory volume in 1 s (FEV1, liters) Forced vital capacity (FVC, liters) FEV1/FVC (%) Peak expiratory ow (PEF, liters/min) Total lung capacity (TLC, liters) Inspiratory capacity (IC, liters) Functional residual capacity (FRC, liters) Residual volume (RV, liters) Specic airway conductance (sGaw, cmH2O 1 s 1) Transfer factor for carbon monoxide (TLCO, ml/min/ mmHg) Transfer coefcient corrected for alveolar volume (KCO (TLCO/VA), ml/min/mmHg/l) Result k k k k m k m m k k k

There is a specic pattern of chronic inammation, particularly affecting small airways and lung parenchyma. This leads to brosis and narrowing of small airways and to destruction of the lung parenchyma, resulting in abnormalities in gas exchange and collapse of small airways on expiration. The inammation of COPD is characterized by increased numbers of neutrophils, macrophages, and T lymphocytes (particularly CD8 cytotoxic T cells) and the degree of inammation increases with disease severity (Figure 5). The pattern of inammation is markedly different from that of asthma (Table 5). Many inammatory mediators are now implicated in COPD, including lipid mediators (such as leukotriene B4), chemokines (such as interleukin (IL)-8), and proinammatory cytokines (such as tumor necrosis factor

434 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

Cigarette smoke (and other irritants) Epithelial cells TGFCTG CD 8+ lymphocyte Alveolar macrophage Chemotactic factors IL-8, CXC chemokines LTB4 Neutrophil

Fibroblast

Proteases

Fibrosis

Emphysema

Mucus hypersecretion

Figure 5 Inammatory mechanisms in COPD. Cigarette smoke (and other irritants) activate macrophages in the respiratory tract that release neutrophil chemotactic factors, including interleukin-8 (IL-8) and leukotriene B4 (LTB4). These cells then release proteases that break down connective tissue in the lung parenchyma, resulting in emphysema, and also stimulate mucus hypersecretion. These enzymes are normally counteracted by protease inhibitors, including a1-antitrypsin, secretory leukoprotease inhibitor (SLPI), and tissue inhibitor of matrix metalloproteinases (TIMP). Cytotoxic T cells (CD8 ) may also be recruited and involved in alveolar wall destruction.

Table 5 Differences between inammation in COPD and asthma Inammation Inammatory cells COPD Neutrophils CD8 T cells CD4 cells Macrophages LTB4 TNF-a IL-8, GRO-a Oxidative stress Epithelial metaplasia Fibrosis Mucus secretion AHR 7 Peripheral airways Predominantly parenchymal destruction 7 Asthma Eosinophils Mast cells CD4 T cells Macrophages LTD4, histamine IL-4, IL-5, IL-13 Eotaxin Oxidative stress Epithelial shedding Fibrosis Mucus secretion AHR All airways No parenchymal effects

Inammatory mediators

Inammatory effects

Location

Response to corticosteroids

LT, leukotriene; TNF, tumor necrosis factor; IL, interleukin; GRO, growth-related oncogene; AHR, airway hyperresponsiveness.

alpha (TNF-a) and IL-6. Oxidative stress is markedly increased in COPD and increases with disease severity; it is due to both the effects of cigarette smoke and the release of reactive oxygen species from activated inammatory cells. There is also an increase in proteases, particularly enzymes that degrade elastin, such as neutrophil elastase (derived predominantly from neutrophils) and matrix metalloproteinase-9 (MMP-9, derived predominantly from macrophages). MMP-9 may be the predominantly elastolytic enzyme causing emphysema; it also activates transforming growth factor beta (TGF-b), a cytokine that

is expressed particularly in small airways that may result in the characteristic peribronchiolar brosis (Figure 6). MMP-12 may also contribute to elastolysis and is prominent in smoke exposure models of COPD in mice, where it plays a critical role in activating TNF-a.
Amplifying Mechanisms

In normal cigarette smokers, there is a similar inammatory response to that seen in COPD patients, but COPD represents an amplication of the normal

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 435

Macrophage

Chemotactic peptides 1-AT

Neutrophils Neutrophil elastase Emphysema

Pro-MMP-9

MMP-9 Elastolysis

Latent TGF-

Active TGF-

Small airway fibrosis (chronic obstructive bronchiolitis)

Figure 6 Possible interrelationship between small airway brosis and emphysema in COPD. Transforming growth factor (TGF)-b is activated by matrix metalloproteinase-9 (MMP-9) and is in turn activated by MMP-9.

inammatory response of the respiratory tract to inhaled noxious agents. Acute exacerbations represent a further amplication of the inammatory response. The molecular and genetic mechanisms of this amplication remain to be determined. However, recently it has been shown that histone deacetylase activity (particularly HDAC2) is markedly reduced in the lungs, airways, and macrophages of COPD patients and that this is correlated with increased expression of inammatory genes, such as IL-8. This amplifying mechanism may be induced by oxidative stress, which impairs the function of HDAC2 in particular. This also accounts for the steroid resistance of COPD since HDAC2 is required for switching off of activated inammatory genes. Adenovirus infection also amplies inammation in cells in vitro and in experimental animals in vivo and its effects may also be mediated through a reduction in HDAC activity in lungs. In COPD patients, there is evidence for latent adenovirus infection in lungs of many COPD patients.
Systemic Effects

Table 6 Systemic abnormalities in COPD Systemic feature Cachexia Muscle wasting Possible mechanisms TNF-a, IL-6, leptin Apoptosis of skeletal muscle due to TNF-a? Inactivity Chronic hypoxia, erythropoetin TNF-a? TNF-a, IL-6 CRP, brinogen? Inactivity, corticosteroids, cytokines?

Polycythemia Normocytic anemia Depression Cardiovascular abnormalities Osteoporosis

TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive protein.

C-reactive protein and IL-6, are increased even in the stable state, but are further increased during exacerbations and this may be a factor predisposing to the markedly increased incidence of ischemic heart disease.

Management
COPD is managed according to the severity of the disease, with a progressive escalation of therapy as the disease progresses (Figure 7).
Antismoking Measures

There is increasing evidence for systemic (nonpulmonary) effects in patients with COPD, particularly in patients with severe disease and these might have an important negative impact on the quality of life (Table 6). The commonest systemic effect is loss of lean body mass and atrophy of skeletal muscles. This may reect poor mobility, but may also be due to systemic effects of inammatory mediators such as TNF-a and IL-6, which may induce apoptosis of skeletal muscles. Other systemic effects include osteoporosis, depression, and normocytic normochromic anemia. Circulating levels of acute phase proteins, such as

Smoking cessation is the only measure so far shown to slow the progression of COPD, but in advanced disease, stopping smoking has little effect and the chronic inammation persists. Nicotine replacement therapy (gum, transdermal patch, inhaler) helps in quitting smoking, but bupropion, a noradrenergic antidepressant, is more effective. More effective

436 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

0: at risk FEV1 80%

I: mild FEV1 80%

II: moderate FEV1 7950%

III: severe FEV1 4930%

IV: very severe FEV1 <30%

Avoidance of risk factor(s); vaccination Add short-acting bronchodilator when needed Add regular treatment with one or more long-acting bronchodilators Add rehabilitation Add inhaled corticosteroids if repeated exacerbations Add long-term O2 if chronic respiratory failure Consider surgery
Figure 7 Stepwise approach to COPD therapy. Management of COPD involves increasing the intensity of treatment depending on increasing severity of the disease. Long-acting bronchodilators are indicated in patients with moderate disease who are symptomatic, together with pulmonary rehabilitation. In severe disease an inhaled steroid may be added if there are frequent exacerbations and this is conveniently given as a combination inhaler containing a long-acting b2-agonist and corticosteroid. Long-term oxygen therapy and surgery are considerations in patients with very severe disease.

antismoking therapies, including partial nicotinic agonists and cannabinoid receptor antagonists, are currently being evaluated.
Bronchodilators

Establish diagnosis Assess symptoms

Stop smoking Healthy lifestyle Immunization

Bronchodilators are the mainstays of current drug therapy for COPD (Figure 8). The bronchodilator response measured by an increase in FEV1 is limited in COPD, but bronchodilators may improve symptoms by reducing hyperination and therefore dyspnea, and may improve exercise tolerance, despite the fact that there is little improvement in spirometric measurements. Previously short-acting bronchodilators, including b2-agonists and anticholinergics, were most widely used, but more recently, long-acting bronchodilators have been introduced. These include the long-acting inhaled b2-agonists salmeterol and formoterol and the once-daily inhaled anticholinergic tiotropium bromide. In patients with more severe disease, these therapies appear to be additive. Theophylline is also used as an add-on bronchodilator in patients with very severe disease, but systemic side effects may limit its value.
Antibiotics

Treat obstruction

Bronchodilators

Assess for hypoxia

Long-term oxygen therapy

Pulmonary rehabilitation program

Figure 8 Treatment strategy in COPD. All patients should be encouraged to stop smoking and should also receive inuenza and pneumococcal vaccination. The mainstays of treatment are bronchodilators to reduce symptoms. Bronchodilators may improve symptoms by deating the lungs, even without any significant change in spirometry. In more severe patients, pulmonary rehabilitation may be very helpful in improving health status and reducing hospitalization. For patients with respiratory failure due to right heart failure, long-term oxygen therapy is indicated.

Acute exacerbations of COPD are commonly assumed to be due to bacterial infections, since they may be associated with increased volume and purulence of the sputum. However, it is increasingly recognized that exacerbations may be due to upper respiratory tract viral infections or may be noninfective, questioning the place of antibiotic treatment in many patients. Controlled trials of antibiotics in

COPD show a relatively minor benet of antibiotics in terms of clinical outcomes and lung function. Although antibiotics are still widely used in exacerbations of COPD, methods that can reliably diagnose bacterial infection in the respiratory tract are needed so that antibiotics are not used inappropriately. There is no evidence that prophylactic antibiotics prevent acute exacerbations.
Oxygen

Home oxygen accounts for a large proportion (over 30% in the US) of healthcare spending on COPD.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 437

Long-term oxygen therapy was justied by two large trials showing reduced mortality and improvement in quality of life in patients with severe COPD and chronic hypoxemia (PaO2 o55 mmHg). More recent studies have demonstrated that patients with less severe hypoxemia do not appear to benet in terms of increased survival, so that selection of patients is important in prescribing this expensive therapy. Similarly, nocturnal treatment with oxygen does not appear to be benecial in terms of survival or delaying the prescription of long-term oxygen therapy in patients with COPD who have nocturnal hypoxemia.
Corticosteroids

chronic COPD may relate to differences in the inammatory response (increased eosinophils) or airway edema in exacerbations.
Other Drug Therapies

Inhaled corticosteroids are now the mainstay of chronic asthma therapy and the recognition that chronic inammation is also present in COPD provided a rationale for their use. Indeed, inhaled corticosteroids are now widely prescribed in the treatment of COPD. However, the inammation in COPD shows little or no suppression even by high doses of inhaled or oral corticosteroids. This may reect the fact that neutrophilic inammation is not suppressible by corticosteroids as neutrophil survival is prolonged by steroids. There is also evidence for an active cellular resistance to corticosteroids, with no evidence that even high doses of corticosteroids suppress the synthesis of inammatory mediators or enzymes. This is related to a decreased activity and expression of histone deacetylase 2, which is required for corticosteroids to switch off activated inammatory genes, such as TNF-a, IL-8, and MMP-9. Approximately 10% of patients with stable COPD show some symptomatic and objective improvement with oral corticosteroids and it is likely that these patients have concomitant asthma, as both diseases are very common. Furthermore, these patients have elevated sputum eosinophils and exhaled nitric oxide, which are features of asthmatic inammation. Long-term treatment with high doses of inhaled corticosteroids fails to reduce disease progression, even at the early stages of the disease. However, there is a small protective effect against acute exacerbations (approximately 20% reduction) in patients with severe disease. In view of the risk of systemic side effects in this susceptible population, inhaled corticosteroids are only recommended in patients with FEV1o50% predicted who have two or more severe exacerbations a year. There is a small benecial effect of systemic corticosteroids in treating acute exacerbations of COPD, with improved clinical outcome and reduced length of hospital admission. The reasons for this discrepancy between steroid responses in acute versus

Systematic reviews show that mucolytic therapies reduce exacerbation by about 20%, but most of the benet appears to derive from N-acetyl cysteine, which is also an antioxidant. This treatment should be considered in patients who have frequent exacerbations. There is no evidence that cysteinyl leukotriene antagonists, such as montelukast, are benecial in COPD, although they are commonly used. Opiates, such as codeine, may reduce dyspnea in COPD patients but the minimal benet is outweighed by side effects.
Noninvasive Ventilation

Noninvasive positive pressure ventilation, using a simple nasal mask and avoiding the need for endotracheal intubation, reduces the need for mechanical ventilation in acute exacerbations of COPD in hospital, although some studies have shown less benet. Domiciliary noninvasive positive pressure ventilation may improve oxygenation and reduce hospital admissions in patients with severe COPD with hypercapnia and may improve long-term survival, although large controlled trials are needed.
Pulmonary Rehabilitation

Pulmonary rehabilitation consists of a structured program of education, exercises, and physiotherapy and has been shown in controlled trials to improve the exercise capacity and quality of life of patients with severe COPD, with a reduction in healthcare utilization. Pulmonary rehabilitation is now an important part of the management plan in patients with severe COPD. There is debate about the duration and frequency of pulmonary rehabilitation. Most of the benets appear to relate to exercise so that modied simplied programs are now often used.
Lung Volume Reduction

Surgical removal of emphysematous lung improves ventilatory function in carefully selected patients. The reduction in hyperination improves the mechanical efciency of the inspiratory muscles. Careful patient selection after a period of pulmonary rehabilitation is essential. Patients with localized upper lobe emphysema with poor exercise capacity do best, but there is a relatively high operative mortality, particularly with patients who have a low diffusing capacity. Significant functional improvements include increased FEV1, reduced total lung capacity

438 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

and functional residual capacity, improved function of respiratory muscles, improved exercise capacity, and improved quality of life. Benets persist for at least a year in most patients, but careful long-term follow-up is needed in order to evaluate the longterm benets of this therapy. More recently, nonsurgical bronchoscopic lung volume reduction has been achieved by insertion of one-way valves by ber optic bronchoscopy. This gives significant improvement in some patients and appears to be safe, but collateral ventilation reduces the efcacy of this treatment so that significant deation of affected lung may not be achieved.
Management of Acute Exacerbations

Table 7 New treatments for COPD Mediator antagonists Leukotriene B4 antagonists (e.g., LY 29311, SB 201 146, BIIL284) 50 -lipoxygenase inhibitors (e.g., Bay x1005) Interleukin-8 antagonists (SB 225 002: CXCR2 antagonists) TNF inhibitors (monoclonal antibodies, soluble receptors, TNF convertase inhibitors) Antioxidants (stable glutathione analogs, superoxide dismutase analogs) Protease inhibitors Neutrophil elastase inhibitors (e.g., ONO-5046) Cathepsin inhibitors Matrix metalloproteinase inhibitors (marimastat, selective MMP inhibitors) a1-Antitrypsin (puried, human recombinant, gene transfer) Secretory leukoprotease inhibitor (human recombinant, gene transfer) Elan (human recombinant, gene transfer) New anti-inammatory drugs Phosphodiesterase-4 inhibitors (e.g., cilomilast, roumilast) p38 mitogen-activated protein-kinase inhibitors (e.g., SB 220 025) Nuclear factor kappa B inhibitors (IkB kinase-2 inhibitors) Adhesion molecule inhibitors (e.g., E-selectin inhibitors, glycoprotein selectin inhibitors)

Acute exacerbations of COPD should be managed by supplementary oxygen therapy, initially 24% oxygen, and checking that there is no depression of ventilation. Antibiotics should be given if the sputum is purulent or there are other signs of bacterial infection. High doses of corticosteroids reduce hospital stay and are routinely given. Chest physiotherapy is usually given, but there is little objective evidence for benet. Noninvasive ventilation is indicated for incipient respiratory failure and reduces the need for intubation.
New Treatments

Apart from quitting smoking, no other treatments, including corticosteroids, slow the progression of COPD. Yet this disease is associated with an active inammatory process and progressive proteolytic injury of lung tissues even in its most advanced stages, suggesting that pharmacological intervention may be possible. A better understanding of the cellular and molecular mechanisms involved in COPD provides new molecular targets for the development of new drugs and several classes of new drugs are now in development (Table 7). Several inammatory mediators are implicated in COPD, providing logical targets for the development of synthesis inhibitors or receptor antagonists. These include 50 -lipoxygenase inhibitors that prevent the synthesis of leukotriene B4 and specic leukotriene B4 antagonists, several of which are now being evaluated in COPD. Specic antagonists of CXCR2, one of the receptors on neutrophils that is activated by IL-8, have been developed and humanized antibodies and soluble receptors that block TNF-a have already been developed for use in other chronic inammatory diseases. More potent and stable antioxidants are also in development. However, it is not certain that antagonizing a single mediator will have a major

clinical effect, since many mediators with overlapping actions are involved in COPD. Several inhibitors of neutrophil elastase are now in clinical development. To date, only one has been reported in a clinical study and this showed no benet, but this may reect the fact that several proteases are involved or the fact that it may be difcult to monitor efcacy in such a slowly progressive disease. Several nonselective matrix metalloproteinase inhibitors have been developed and there is now a search for more selective inhibitors in order to avoid the musculoskeletal side effects that have hampered the development of this class of drug. Another approach is supplementation of endogenous antiproteinases using human recombinant a1-antitrypsin, secretory leukoprotease inhibitor or elan, or even gene therapy, although there are doubts that sufcient protein can be delivered by either approach. While corticosteroids are ineffective, other anti-inammatory treatments, particularly those that inhibit neutrophilic inammation, might be effective. The most promising amongst these are phosphodiesterase (PDE)4 inhibitors, which have an inhibitory effect on key inammatory cells involved in COPD, including macrophages, neutrophils, and cytotoxic T lymphocytes. Several PDE4 inhibitors are in development for treatment of COPD, although a limitation of drugs in this class is the common side effect of nausea and headaches, also caused by PDE4 inhibition. Other novel anti-inammatory approaches in development include inhibitors of nuclear factor kappa

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations 439

B (NF-kB) and inhibitors of p38 mitogen-activated protein-kinase, although none of these have reached clinical trials.
Drug Delivery

Rennard SI (2004) Treatment of stable chronic obstructive pulmonary disease. Lancet 364: 791802. Sutherland ER and Cherniack RM (2004) Management of chronic obstructive pulmonary disease. New England Journal of Medicine 350: 26892697. Wouters EF (2004) Management of severe COPD. Lancet 364: 883895.

Current inhaler devices have been developed to deliver drugs to the conducting airways in patients with asthma. However, COPD predominantly affects peripheral airways and lung parenchyma, which may not be optimally targeted by current inhalers. It is likely that new inhaler devices delivering aerosols of smaller particle size or systemic drugs are more likely to be useful in COPD. In the future, targeted delivery to specic cells, such as macrophages, might be possible, particularly if new treatments have systemic toxicity. There is also a rationale for giving systemic therapies to reach the lung periphery and to treat systemic features of the disease.
See also: Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General. Corticosteroids: Therapy. Leukocytes: Pulmonary Macrophages. Oxygen Therapy.

Acute Exacerbations
J A Wedzicha and J R Hurst, Royal Free and University College Medical School, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Chronic obstructive pulmonary disease (COPD) is a condition characterized by lower respiratory tract symptoms, airow obstruction, and airway inammation. The natural history of COPD is punctuated by episodes of acute deterioration in respiratory health termed exacerbations. Exacerbations are responsible for much of the morbidity, mortality, and healthcare costs associated with COPD, and also affect the rate of decline in lung function. Most exacerbations are caused by episodes of tracheobronchial infection. The clinical features comprise a worsening in symptoms beyond usual day-to-day variation. Examination may reveal tachypnea and signs of respiratory failure. Pathologically, exacerbations are associated with an increase in airway and systemic inammation which, at least in more severe disease, is predominantly neutrophilic. Simple investigations are indicated to exclude differential diagnoses and assess exacerbation severity. Treatment aims to support respiratory function whilst drugs aimed at the underlying cause are able to act. The cornerstone of therapy is inhaled bronchodilators, with oral corticosteroids in all but the mildest exacerbations. Antibiotics are indicated if there has been a change in the character of the sputum. Some patients with respiratory failure will require supplemental oxygen or assisted ventilation. Strategies to prevent exacerbations include inhaled corticosteroids, long acting bronchodilators, and inuenza vaccination.

Further Reading
Agusti AG, Noguera A, Sauleda J, et al. (2003) Systemic effects of chronic obstructive pulmonary disease. European Respiratory Journal 21: 347360. Barnes PJ (2000) Chronic obstructive pulmonary disease. New England Journal of Medicine 343: 269280. Barnes PJ (2003) New concepts in COPD. Annual Review of Medicine 54: 113129. Barnes PJ (2004) Mediators of chronic obstructive pulmonary disease. Pharmacological Reviews 56: 515548. Barnes PJ and Hansel TT (2004) Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985996. Barnes PJ, Ito K, and Adcock IM (2004) A mechanism of corticosteroid resistance in COPD: inactivation of histone deacetylase. Lancet 363: 731733. Barnes PJ, Shapiro SD, and Pauwels RA (2003) Chronic obstructive pulmonary disease: molecular and cellular mechanisms. European Respiratory Journal 22: 672688. Calverley PM and Walker P (2003) Chronic obstructive pulmonary disease. Lancet 362: 10531061. Hogg JC (2004) Pathophysiology of airow limitation in chronic obstructive pulmonary disease. Lancet 364: 709721. Hogg JC, Chu F, Utokaparch S, et al. (2004) The nature of smallairway obstruction in chronic obstructive pulmonary disease. New England Journal of Medicine 350: 26452653. Hurst JR and Wedzicha JA (2004) Chronic obstructive pulmonary disease: the clinical management of an acute exacerbation. Postgraduate Medical Journal 80: 497505. Mannino DM (2002) COPD: epidemiology, prevalence, morbidity and mortality, and disease heterogeneity. Chest 121: 121S126S. Pauwels RA and Rabe KF (2004) Burden and clinical features of chronic obstructive pulmonary disease (COPD). Lancet 364: 613620.

Introduction
It would be difcult to exaggerate the importance of chronic obstructive pulmonary disease (COPD) as a global health problem for the twenty-rst century. COPD is the fth commonest cause of death in the world and, unlike other prevalent diseases, death rates from COPD continue to rise. Much of this mortality is related to episodes of acute deterioration in respiratory health termed exacerbations. COPD is dened by the presence of lower respiratory tract symptoms, poorly reversible airow obstruction, and inammation of the lung. The natural history of COPD comprises progression of this airow obstruction, associated with worsening symptoms and increasing limitation to daily activities. However, patients are also prone to periodic

440 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations

deteriorations in respiratory health termed exacerbations. Exacerbations are important events that cause much of the morbidity, mortality, hospital admissions, and therefore healthcare costs associated with this disease. In the UK, around 10% of acute medical admissions to hospital may be attributed to exacerbations of COPD, the in-hospital mortality is 10%, and exacerbations account for 70% of the direct medical costs attributable to COPD. Exacerbations become more frequent and more severe as the severity of the underlying disease increases. In patients with moderately severe COPD, the median annual exacerbation frequency is between two and three events per year. However, this masks important differences between individual patients, some of whom are more susceptible to exacerbation than others. The factors determining a greater susceptibility to exacerbation are poorly understood but are thought to include the presence of daily bronchitic symptoms, greater severity of underlying disease, greater lower airway inammation, and the presence of lower airway bacteria in the stable state. Exacerbations are extremely heterogeneous events responsible, at one extreme, for life-threatening episodes of respiratory failure in patients with severe COPD, but to no more than a troublesome and transient increase in symptoms in patients with milder disease. This heterogeneity has caused debate about exactly how an exacerbation should be dened, but for practical purposes an exacerbation is a sustained episode of increased symptoms, acute in onset, that is beyond the patients usual day-to-day variation. This may or may not necessitate a change in therapy. In addition to direct effects on morbidity and mortality, exacerbation frequency also affects the natural history of disease. Patients susceptible to frequent exacerbations experience an accelerated decline in forced expiratory volume in 1 s (FEV1), and have a poorer quality of life. The importance of exacerbations are summarized in Figure 1.

Morbidity

Healthcare costs

Exacerbation of COPD

Mortality

Disease progression

Figure 1 The importance of exacerbations in COPD. Table 1 The most important bacterial and viral pathogens isolated from lower airway samples in patients with exacerbations of COPD Bacteria Haemophilus inuenzae Moraxella catarrhalis Streptococcus pneumoniae Pseudomonas aeruginosa Viruses Rhinovirus Coronavirus Inuenza Parainuenza Adenovirus Respiratory syncytial virus

Bold type indicates the most common isolates.

Etiology
It is generally accepted that exacerbations are caused by episodes of tracheobronchial infection, and perhaps pollution. Evidence for the former is derived from reports demonstrating a higher prevalence of pathogens in samples taken from the lower airways at the time of exacerbation than in the stable state. Evidence that pollution causes exacerbations arises from relationships between the degree of air pollution and hospital admissions for COPD. PM10

(particulate matter up to 10 mm in size), largely produced by diesel exhaust, appear particularly important. In a proportion of exacerbations, the cause remains obscure. A number of studies have investigated the isolation frequency of individual pathogens at exacerbation of COPD. The relative prevalence of specic agents varies by the methodology of the study, and the severity of both the patient and exacerbation. In patients with moderate to severe underlying disease, bacterial and viral pathogens may each be isolated from lower airway samples in approximately 50% of exacerbations. The most important bacterial and viral pathogens are listed in Table 1. The identication of organisms in lower airway samples at the time of exacerbation has been taken to imply causation, but the issue is complex because pathogenic microorganisms may also be found in the lower airways of patients with stable COPD. This is especially true for bacteria where up to 50% of patients with severe disease have the same bacterial species present in the stable state, a phenomenon

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations 441

termed lower airway bacterial colonization. More recently, it has been suggested that a change in the colonizing strain may result in exacerbation, and further evidence that bacteria do indeed cause exacerbations may be drawn from the clinical benet observed in trials of antibiotics during such events. There is ongoing controversy regarding the role of atypical organisms such as Chlamydia and Mycoplasma in the etiology of exacerbations. The most important viral pathogen is rhinovirus. Exacerbations in which a virus is isolated, or those associated with coryzal symptoms, are more severe as assessed by changes in symptoms and lung function. Reecting the circulation of respiratory viruses, exacerbations are more common in the winter than the summer months.

Pathology
COPD is dened as a disease state characterized by lower respiratory tract symptoms and airow obstruction in association with an abnormal inammatory response within the lung. A number of studies using endobronchial biopsy, bronchoalveolar lavage, sputum, and exhaled markers have demonstrated that airway inammation is increased at exacerbation compared to the baseline state. In studies examining endobronchial biopsies, exacerbations in mild COPD were associated with an inltration of eosinophils, whilst in more severe disease the inltrate was characterized by neutrophils. Regarding soluble mediators, increased concentrations of various neutrophilic indices have been reported including interleukin-8, leukotriene-B4, myeloperoxidase, and elastase.

exacerbation and that airway inammatory markers are increased at exacerbation) that exacerbations result from increased airway inammation following acquisition of a new pathogen. However, a direct relationship between the clinical severity of exacerbation and degree of airway inammation has never been demonstrated, and the mechanisms by which airway inammation results in worsening symptoms and lung function remain largely obscure. It has also been reported that systemic inammatory markers such as interleukin-6, brinogen, and C-reactive protein are higher at exacerbation than in stable COPD. This is important because of the association between systemic inammatory markers and cardiovascular events, cardiovascular disease being a common cause of death in COPD. The origin of the systemic inammatory response is unclear, though a spillover effect from the lung is thought most likely.

Animal Models
There is no animal model of exacerbation of COPD.

Management and Current Therapy

A summary of the clinical management of an exacerbation of COPD is presented in Table 2. Initial management is directed at excluding differential diagnoses and assessing exacerbation severity. The diagnosis of exacerbation is symptomatic, there is no conrmatory test, and follows exclusion of differential diagnoses such as pneumonia, pneumothorax, pleural effusion, lung carcinoma, pulmonary embolus, or cardiac disease which may also occur in a patient with COPD and mimic the presence of exacerbation. Such diagnoses may generally be excluded on the basis of a clinical history, examination, and simple investigations such as the chest radiograph and electrocardiogram. Assessment of severity is important for guiding decisions on initial treatment strategy and the need for hospital admission. However, there is no widely accepted severity scoring system. pH, more useful than PaCO2 as a marker of acute changes in alveolar ventilation, and PaO2 are important indicators. A pH o7.30 suggests a severe exacerbation. It should be remarked that measuring lung function is usually unhelpful as changes at exacerbation may be small, absolute values may be misleading in the absence of a baseline, and the tests may be difcult to perform in patients who are acutely unwell. The principles of treatment are to support respiratory function whilst therapies directed against the underlying pathophysiology, such as antimicrobial and anti-inammatory agents, are able to act.

Clinical Features
Exacerbations are dened as an increase in respiratory symptoms, acute in onset, that is beyond the patients usual day-to-day variation. These symptoms typically include increased dyspnea, sputum volume and sputum purulence, with or without increased cough, wheeze, chest tightness, coryzal symptoms, fatigue, edema, and confusion. Findings on examination are non-specic, but may include tachypnea and use of the accessory muscles of respiration, pursed-lip breathing, polyphonic expiratory wheeze, and, in severe exacerbations, signs of CO2 retention such as ap and bounding pulse.

Pathogenesis
It is generally assumed (based on observations that pathogens are more frequently isolated at the time of

442 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Acute Exacerbations

Table 2 A summary of the management of an acute exacerbation of COPD in the hospital setting Principle Assess severity Give oxygen Give bronchodilators Give corticosteroids Give antibiotics Consider need for ventilatory support Also consider Details Clinical ndings and pH/arterial blood gas tensions Controlled therapy, aiming for arterial saturations 9092%. Reassess blood gas tensions after 30 min Increased dose or frequency of b2-agonists and/or anticholinergics. Nebulised or inhaled via spacer Oral, or with initial intravenous dose When there has been a change in the character of sputum. Usually oral In respiratory failure not responding to controlled oxygen therapy Fluid balance, anticoagulation, comorbidities, and need for additional therapies, e.g., intravenous theophylline

The cornerstone of therapy, all that may be required in the most mild exacerbations, is an increase in the dose or frequency of inhaled bronchodilators. Although the airow obstruction in COPD is, by definition, largely irreversible, there may be additional bronchoconstriction at exacerbation and these drugs can achieve a dramatic symptomatic response through effects on dynamic hyperination. Shortacting b2-adrenoceptor agonists such as salbutamol have a more rapid onset of action and are preferred to anticholinergics such as ipratropium. Combinations of b2-agonists and anticholinergics are often recommended in exacerbations not responding to b2-agonists alone. Inhaled therapy via a large volume spacer is as effective as wet nebulization. Exacerbations not responding to an increase in bronchodilators alone should be treated with systemic corticosteroids. The rationale is that these drugs may act on the increased inammation observed at exacerbation, though specic evidence for this is lacking. Systemic corticosteroids result in a more rapid improvement in FEV1, though at the expense of more frequent adverse events, particularly hyperglycemia. A dose of 3040 mg prednisolone equivalent for 1014 days is appropriate. Longer courses have no additional effect and there is no benet from dose tapering. Nebulized budesonide is an alternative. The evidence of benet from antibiotics is restricted to those exacerbations characterized by a change in the character of sputum. Sputum purulence is a reliable predictor of the presence of bacteria. Parenteral antibiotics are rarely required and given that coverage of Haemophilus inuenza, Moraxella catarrhalis, and Streptococcus pneumoniae is required, an oral aminopenicillin, macrolide, or tetracycline agent is most commonly used rst line. Severe exacerbations, or exacerbations in patients with more severe underlying disease, may result in arterial hypoxemia, primarily through ventilationperfusion imbalance. Oxygen should be administered to correct this respiratory failure. The risk of

hypercapnia in a proportion of patients with COPD necessitates that oxygen be administered in a controlled manner. There is little risk of hypercapnia if oxygen is titrated to achieve saturations between 90% and 92%. Achieving adequate oxygenation only at the expense of rising PaCO2 or decreasing pH suggests the need for noninasive ventilation (NIV). NIV is clearly superior to respiratory stimulant drugs such as doxapram in the management of acute hypercapnic respiratory failure, with improvements in mortality, length of hospital stay, and the need for tracheal intubation. NIV is most commonly administered as pressure-cycled bi-level positive airway pressure (BiPAP), and is thought to be effective by off-loading fatigued respiratory muscles. Patients failing to improve on NIV, or those with a particularly severe exacerbation, may require endotracheal intubation and a period of mechanical ventilation. The decision to institute such therapy in COPD is complex and should consider the wishes and prior functional status of the patient, comorbidities, and the degree of reversibility. However, mortality in the intensive-care unit for patients with COPD, around 20%, is not greater than that seen in respiratory failure from other causes. Regarding other therapies, there is no evidence that the routine use of theophyllines, heliox, antitussives, mucolytics, or respiratory physiotherapy is benecial. Other supportive measures such as uid balance should be attended to, and comorbidities managed. Patients failing maximal therapy or in whom escalation of therapy is inappropriate should be considered for a range of palliative drugs. Given the importance of exacerbations, considerable benet may be anticipated from therapies which prevent these events. Large trials have demonstrated evidence for benet of high-dose inhaled corticosteroids (uticasone 1000 mg day 1) in patients with moderate to severe underlying disease (FEV1o50% predicted). Long-acting bronchodilators, both b2agonists and anticholinergics, have also been associated with decreased exacerbation frequency, as has

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deciency 443

inuenza vaccination. Initial trials of antibiotic prophylaxis were carried out many years ago, often in patients with simple chronic bronchitis, and current interest in this area, with more modern drugs, is high. Mucolytics may reduce exacerbation frequency in patients with milder disease. Finally, appropriate use of long-term oxygen therapy and domiciliary NIV in patients with respiratory failure is associated with reduced hospitalization, though a specic effect on exacerbations has not been demonstrated.
See also: Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Viruses of the Lung.

chronic obstructive pulmonary disease. Cochrane Database of Systemic Reviews, Issue 4.

Relevant Website
http://www.cochrane.org The Cochrane Database of Systematic Reviews.

Emphysema, Alpha-1Antitrypsin Deciency

R A Stockley, Queen Elizabeth Hospital, Birmingham, UK
& 2006 Elsevier Ltd. All rights reserved.

Further Reading
Anthonisen NR, Manfreda J, Warren CP, et al. (1987) Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease. Annals of Internal Medicine 106: 196204. Burge PS, Calverley PM, Jones PW, et al. (2000) Randomised, double blind, placebo controlled study of uticasone propionate in patients with moderate to severe chronic obstructive pulmonary disease: the ISOLDE trial. British Medical Journal 320: 12971303. Calverley PMA (2004) Reducing the frequency and severity of exacerbations of chronic obstructive pulmonary disease. Proceedings of the American Thoracic Society 1: 121124. Hurst JR and Wedzicha JA (2004) Chronic obstructive pulmonary disease: the clinical management of an acute exacerbation. Postgraduate Medical Journal 80: 497505. Lightowler JV, Wedzicha JA, Elliott MW, and Ram FSF (2003) Non-invasive positive pressure ventilation to treat respiratory failure resulting from exacerbations of chronic obstructive pulmonary disease: cochrane systematic review and meta-analysis. British Medical Journal 326: 185187. National Institute for Clinical Excellence (NICE) (2004) Chronic obstructive pulmonary disease: national clinical guideline for management of chronic obstructive pulmonary disease in adults in primary and secondary care. Thorax 59(supplement I). Pauwels R, Anthonisen N, Bailey WC, et al. (2001) Global Initiative for Chronic Obstructive Lung Disease. National Heart, Lung and Blood Institute, National Institutes of Health, Bethesday, MD. Saint S, Bent S, Vittinghoff E, and Grady D (1995) Antibiotics in chronic obstructive pulmonary disease exacerbations. A metaanalysis. Journal of the American Medical Association 273: 957960. Sethi S (2004) Bacteria in exacerbations of chronic obstructive pulmonary disease. Phenomenon or epiphenomenon? Proceedings of the American Thoracic Society 1: 109114. Siafakas NM, Anthonisen NR, and Georgopoulos D (eds.) (2003) Lung Biology in Health and Disease Volume 183: Acute Exacerbations of Chronic Obstructive Pulmonary Disease. New York: Dekker. Wedzicha JA (2004) Role of viruses in exacerbation of chronic obstructive pulmonary disease. Proceedings of the American Thoracic Society 1: 115120. Wedzicha JA and Donaldson GC (2003) Exacerbations of chronic obstructive pulmonary disease. Respiratory Care 48: 12041213. Wood-Baker RR, Gibson PG, Hannay M, Walters EH, and Walters JAE (2005) Systemic corticosteroids for acute exacerbations of

Abstract
Serum a1-antitrypsin (AAT) deciency is the most common genetic condition recognized in a Caucasian population of Northern European origin. It predisposes to several clinical entities, although the susceptibility to the development of emphysema predominates. The classical clinical presentation (although not universal) is of early onset basal panacinar emphysema associated with neutrophilic inammation in the lower airways. Proteinases released from these neutrophils remain active in the lung due to the deciency of AAT and damage many of the structural proteins (particularly elastin), leading to alveolar destruction and coalescence. The progression is generally more rapid than in nondecient subjects, leading to respiratory failure and cor pulmonale. Therapy is predominantly along conventional lines for chronic obstructive pulmonary disease, although AAT-decient patients are more likely to be accepted as potential lung transplant recipients because of their younger age and general lack of comorbidity. Augmentation therapy is available in some countries, and circumstantial evidence suggests that it is effective in disease modication. However, no suitably powered controlled clinical trial has been undertaken to prove this hypothesis.

Serum deciency of a1-antitrypsin (AAT) was rst noted and described by Laurell and Eriksson in 1963. The authors identied ve serum samples with an absence of a protein band in the a1 region on paper electrophoresis. Review of the patients indicated that three had emphysema at an early age (younger than 45 years old) and a fourth individual had a family history of emphysema. In subsequent studies, it became apparent that all individuals possessed two genes for AAT that were expressed in a codominant manner. However, some genes led to decreased serum levels, and whereas heterozygotes did not appear to be excessively at risk for developing emphysema, homozygotes did show such a risk. This led to the apparent autosomal recessive nature of inheritance of the disease trait (Figure 1).

444 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deciency

ZZ

MM

MZ

MZ

MM

ZZ

MZ

MZ

MM

MZ

MZ

MZ

MZ

ZZ

ZZ

Figure 1 Idealized ve-generation inheritance of the common Z gene. In generation 1, an affected homozygote individual marries a normal homozygote M individual. All children would be MZ heterozygotes and not at risk. In generation 2, the heterozygote marries another heterozygote, resulting in a one in four chance of a homozygote Z (at-risk) individual. In generation 3, all children are heterozygote, and at-risk individuals only recur in generation 5, if marriage to a further heterozygote occurs, thereby skipping a generation.

The patients identied initially were young, and subsequent studies suggested that the archetypal patient was an individual with early onset panacinar emphysema, predominantly basal in distribution. The highest incidence of the deciency was in Scandinavia, and the condition was primarily of Northern European origin with decreasing prevalence further away from Scandinavia. The highest incidence of serum deciency is approximately 1:1600 in Denmark, and the lowest is approximately 1:5000 in the United States.

nonsmokers with AAT deciency can develop airow obstruction, although in such individuals the onset of physiological abnormality and symptoms is generally much later than in current or ex-smokers. There have been very few other studies of factors that inuence the development of airow obstruction, although a past history of wheezing and exposure to kerosene heaters have also been implicated.

Pathology
There have been few pathological studies of the lung in patients with serum AAT. In the limited studies that have been carried out, the presence of panacinar emphysema has been well recognized. In addition, evidence of bronchitis and bronchiectasis has also been noted. Recent studies have shown that the lungs of affected individuals have evidence of neutrophilic inammation and the presence of polymerized AAT. Patients with serum deciency of AAT show accumulation of AAT in hepatocytes (Figure 2) and progressive evidence of brosis and cirrhosis. The condition often results in neonatal jaundice, which usually settles, although childhood liver failure and the development of hepatocellular carcinoma in adult patients with cirrhosis are also known to occur. Finally, other rare pathological conditions are known to occur in patients with serum deciency of AAT, including necrotizing panniculitis and vasculitis.

Etiology
Although smoking is recognized as the major etiological agent in the development of emphysema, it is recognized that not all smokers develop the condition. This suggests coenvironmental factors that amplify the detrimental effects of cigarette smoking and/ or genetic factors. Indeed, studies have indicated that emphysema runs in families, often irrespective of smoking history. Serum deciency of AAT is the major recognized genetic risk factor for the development of emphysema. Epidemiological studies have indicated that life expectancy in such individuals is reduced, particularly if they smoke, and most patients with serum deciency of AAT who present to healthcare systems have a current or past history of cigarette smoking. Despite this, it is well recognized that even

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deciency 445

Figure 2 A histological section of liver is shown demonstrating the PAS-positive inclusion bodies typical of AATD (arrow).

Clinical Features
The majority of patients with serum deciency of AAT are identied through investigation of respiratory symptoms, although approximately 20% of patients are identied through family screening. There is often a delay in the diagnosis, which may be up to 7 years from the presentation of symptoms to establishment of the cause. The typical patient often presents in the third or fourth decade with symptoms of breathlessness on exertion, which is gradually progressive. Because of the young age of onset, an initial diagnosis of asthma is often made, although lung physiology usually indicates poor reversibility and effort-dependent airways collapse. Direct questioning also indicates that the patients often have other classic symptoms of chronic obstructive pulmonary disease (COPD), including cough, sputum production, wheeze (with and without infections), and fatigue. The condition, and hence symptomatology, is progressive, especially in subjects who continue to smoke, and it generally follows a more rapid path yet broadly similar to that of patients who have normal AAT and more standard COPD. As indicated previously, nonsmokers can develop airow obstruction, although this occurs approximately 1020 years later, affecting patients in their 50s and 60s. This archetypal presentation has been shown to have some relationship to selective testing. Because the original patients who were identied in 1963 were young with extensive panlobular basal emphysema (Figure 3), there has been a general tendency to test such patients for AAT deciency and not test patients who are older with a more diffuse and centrilobular pattern of emphysema. Recent studies using high-resolution computed tomography (CT) scan have shown that approximately one-third of patients with serum deciency of AAT who are symptomatic have a more centrilobular pattern of emphysema and
Figure 3 A three-dimensional CT scan of the lungs of a patient with AATD. Emphysematous areas are dark, demonstrating the predominantly basal distribution of the disease.

typical distribution toward the apices of the lungs rather than the classical basal panacinar emphysema. In addition, approximately 30% of patients demonstrate evidence of bronchiectasis on high-resolution CT, and patients can present much later in life even with relatively mild disease when they are smokers. The average age of patients presenting to the UK ADAPT registry is 50 years (Figure 4). Patients with AAT deciency are susceptible to recurrent infections presenting as exacerbations of their condition. Studies have suggested that such episodes are associated with more inammation in the lung and a greater degree of neutrophilic recruitment than in patients who have normal serum AAT. In addition, the episodes may be longer in duration and show a relationship with the decline in lung function. This probably reects the low levels of AAT in the blood and the failure of an acute phase response during infections, leading to reduced protection of the lung and a reduced ability to dampen and switch off inammation.

Pathogenesis
AAT is a proteinase inhibitor that is the major serum inhibitor and one of the major lung inhibitors of serine proteinases. These proteinases, and particularly human neutrophil elastase, have a broad spectrum of activity and have been shown to induce many of the features of chronic lung disease in animal models, particularly the development of emphysema. Serum deciency of AAT is reected in an

446 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deciency

120 100 Number of patients 80 60 40 20 0 15 20 25 30 35 40 45 Age 50 55 60 65 70 75 n = 535

Figure 4 Frequency histogram of the age of patients referred for the rst time to the UK registry.

associated deciency of the same protein in the lung since the majority of lung AAT is derived from serum by simple passive diffusion. A variety of gene defects can result in reduced or no secretion of AAT from the hepatocytes. The most common deciency, referred to as AAT PIZ, arises from a single point mutation in the AAT gene that leads to a glutamic acid-to-lysine amino acid switch at position 342. This single amino acid change results in alteration of the tertiary structure of the protein, such that the b-sheets open and the reactive loop becomes more mobile. The net result is that the reactive loop of one protein molecule can insert into the b-sheets of a second molecule, forming a dimer. This subsequently links to a third molecule, etc., forming long polymer chains. These polymers accumulate within the hepatocyte such that less than 10% of the protein made by the gene follows the normal secretion pathway into the serum. A variety of other gene defects occur, including the complete absence of the genes, point mutations, and frame shift mutations that result in premature stop codon, and amino acid changes that markedly reduce the antiproteolytic activity of the molecule. Reduced AAT within the lung leads to an associated defect in the ability of the lung to inactivate proteinases (particularly neutrophil elastase) once they are released from the activated neutrophil. This enables the enzyme to retain its activity, causing damage to lung connective tissues and resulting in the development of the pathological changes of chronic lung disease and emphysema. The process may become self-perpetuating for several reasons. Early studies suggested that neutrophil elastase could stimulate alveolar macrophages to release neutrophil chemoattractants, particularly leukotriene B4. This would in turn lead to further neutrophil activation and recruitment into the lung,

releasing further elastase activity during the activation process and hence perpetuating the cycle of events (Figure 3). Recent studies have suggested that the AAT polymers, which are also present in the lung, may be proinammatory. The polymers have the ability to stimulate epithelial cells to release neutrophil chemoattractants, leading to an amplication of the neutrophilic inammation by an alternative route.
Animal Models

No naturally occurring animal model similar to patients with human AAT deciency exists. However, because AAT is thought to be predominantly an inhibitor of neutrophil elastase in the airways, circumstantial evidence of its role does exist. Installation of human neutrophil elastase into the airways of experimental animals reproducibly induces the development of airspace enlargement typical of emphysema. In addition, the enzyme also generates pathological changes in the airway consistent with human chronic bronchitis and mucus secretion. Similarly, strategies that lead to neutrophil accumulation in the lungs (e.g., the installation of endotoxin) also result in the development of airspace enlargement. Finally, animals exposed chronically to cigarette smoke also develop changes consistent with emphysema, and at least in the early phase of the condition, this is associated with neutrophilic inux. AAT augmentation can prevent both the early inammatory changes and the subsequent connective tissue degradation, indicating a key role for this protein. The pallid mouse has partially reduced concentrations of AAT in the plasma. This mouse is particularly susceptible to the development of emphysema. Although it is known that the mouse also has a

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, Alpha-1-Antitrypsin Deciency 447

further genetic mutation affecting the cell membrane protein called syntaxin, the mice gradually develop progressive changes consistent with emphysema. Furthermore, compared to controls, the exposure to cigarette smoke leads to the much more rapid development of emphysematous change in these mice. In addition, the pattern of emphysema in the pallid mouse is more consistent with human panlobular emphysema, whereas the control mouse tends to develop centrilobular changes. Since the classical type of emphysema in human subjects with AAT deciency is lower zone panacinar emphysema, the partially decient pallid mouse provides an animal model that may be more relevant to the human disease. The development of a knockout mouse for the AAT gene has proved to be difcult. The major reason is that the mouse has a sequential sequence of ve AAT-like genes on the same chromosome. There are many repeated sequences, making individual gene knockout a major technological problem. Recently, however, a gene disruption model of the PIZ gene was reported in the literature, but the authors did not report how this affects the response to airways inammation. Furthermore, gene disruption models have the problem of potentially reecting developmental abnormalities rather than subsequent responses to injury or insult. It is therefore imperative to develop an appropriate animal model with late (postdevelopmental) knockout of the appropriate AAT gene followed by replacement with the normal or abnormal AAT genes from the human to mimic human serum deciency. The development of such a model has major implications for future studies in AAT deciency because recurrent augmentation studies could be undertaken without concern of immune reaction to a foreign pattern.

Management
The most important management strategy is prevention of smoking or cessation of smoking as soon as the diagnosis has been made. The patient should be assessed in detail, as for all COPD individuals. Physiological testing should be carried out to determine the presence of reversible airow obstruction and response to the appropriate class of agent. In addition, subjects who have recurrent exacerbations, particularly those with more severe disease, should also be treated with inhaled corticosteroids, again according to the general guidelines of COPD patients. As indicated previously, acute exacerbations of the lung are associated with a greater degree of inammation and more rapid progression of lung disease. Thus, such episodes should be identied and treated rapidly with appropriate antibiotic therapy.

For more than 15 years, augmentation therapy with human AAT puried from plasma has been available in many countries (particularly the United States). This form of therapy seems to be logical because it increases the serum AAT and hence lung AAT levels to normal, or at least to a putative protective level. However, no sufciently powered controlled clinical trial has been undertaken to demonstrate the efcacy of such a strategy. Indirect studies have suggested that inammation in the lung can be reduced by augmentation therapy, thus modulating the potentially damaging process. In addition, cohort studies from several countries have suggested that individuals receiving augmentation therapy show a slower decline in lung function than those not receiving such therapy. These studies are clearly suggestive, but because there is a failure to match for socioeconomic status and country of origin there is a high degree of uncertainty regarding the efcacy of such therapy. The only controlled study was performed several years ago in Denmark and Holland, and it suggested that augmentation therapy may lead to a reduction in the loss of lung tissue as assessed by CT scan. The implication is that such a strategy reduces the progression of emphysema. Other conventional approaches that have been used in patients with serum deciency of AAT include lung transplantation. Patients often do well because they are younger and have less comorbidity than nondecient patients with a similar degree of physiological abnormality. Lung volume reduction surgery may be appropriate in some individuals, although the basal nature of the disease makes this less amenable to a surgical approach, and evidence suggests that any benets are relatively short lived. As our understanding of the pathogenic processes in AAT deciency and the generation of emphysema increases, exciting new strategies may be developed that may be applicable. Because neutrophilic inammation is thought to be central, strategies to reduce neutrophil trafc are therefore potentially benecial. A reduction or absence of neutrophilic trafc would reduce the proteinase burden on the lung, making the deciency in AAT less critical, as indicated previously. The chemoattractants in such patients are being characterized and leukotriene B4 may play a key role (Figure 5). Leukotriene B4 receptor antagonists have been developed and are highly effective in vitro. If leukotriene B4 is central to neutrophil trafc in AAT deciency, such a strategy may be highly effective. Patients develop emphysema, and in more than 60% of patients this tends to be panlobular and basal in origin. Studies have shown that all-trans retinoic acid can reverse emphysema in animal models in

448 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

AAT Elastase release
Dawkins PA and Stockley RA (2001) Animal models of chronic obstructive pulmonary disease. Thorax 56: 972977. Dowson LJ, Guest PJ, and Stockley RA (2001) Longitudinal changes in physiological, radiological and health status measurements in alpha-1-antitrypsin deciency and effect as associated with decline. American Journal of Respiratory and Critical Care Medicine 164: 18051809. Eriksson S (1963) Pulmonary emphysema and alpha-1-antitrypsin deciency. Acta Medica Scandinavia 175: 197205. Hill AT, Campbell EJ, Bayley DL, et al. (1999) Evidence for excessive bronchial inammation during an acute exacerbation of chronic obstructive pulmonary disease in patients with alpha-1antitrypsin deciency (piZ). American Journal of Respiratory and Critical Care Medicine 160: 19681975. Lomas DA and Parfrey H (2004) a1-Antitrypsin deciency 4: molecular pathophysiology. Thorax 59: 529535. Luisetti M and Seersholm N (2004) a1-Antitrypsin deciency 1: epidemiology of a1-antitrypsin deciency. Thorax 59: 164169. Needham M and Stockley RA (2004) a1-Antitrypsin deciency 3: clinical manifestations of natural history. Thorax 59: 441445. Piitulainen E, Tomling G, and Eriksson S (1998) Environmental correlates of impaired lung function in non-smokers with severe alpha-1-antitrypsin deciency (piZZ). Thorax 53: 939943. Sandhouse RA (2004) a1-Antitrypsin deciency 6: new and emerging treatments for a1-antitrypsin deciency. Thorax 59: 904 989. Silverman EK, Pierce JA, Province MA, et al. (1989) Variability of pulmonary function in alpha-1-antitrypsin: clinical correlates. Annals of Internal Medicine 111: 982991. Stockley RA (2002) a1-Antitrypsin deciency. In: Voelkel NF and MacNee W (eds.) Chronic Obstructive Lung Disease, pp. 8089. New York: Decker. Stoller JK and Aboussouan LS (2004) a1-Antitrypsin deciency 5: intravenous augmentation therapy: current understanding. Thorax 59: 708712.

Macrophages

Emphysema

Neutrophil recruitment

LTB4
Figure 5 The cycle of events in AATD leading to emphysema. Neutrophils in the lung release elastase. This stimulates macrophages to release the chemoattractant LTB4 because AATD results in levels of the protein too low to inactivate the enzyme. LTB4 recruits more neutrophils, causing more elastase release. Lung damage leading to emphysema is a collateral effect of the enzyme release.

which cigarette smoke or elastase have been used to induce the pathological change. It has been proved that the retinoic acid gamma receptor is the key to this repair process, and specic agonists have been developed. Preliminary studies are under way in man, specifically regarding AAT deciency. In summary, serum deciency of AAT has provided an experiment of nature in which deep understanding of the pathogenic processes in the development of emphysema has been obtained. Such patients tend to be young with little comorbidity and rapidly progressive lung disease that is associated with excessive inammation. Thus, they represent an ideal model for understanding and monitoring the pathogenic processes involved and specifically for developing and assessing new agents and strategies to retard or reverse the progressive loss of lung function.
See also: Bronchiectasis. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, General. Lipid Mediators: Platelet-Activating Factors. Retinol and Retinoic Acid. Surgery: Lung Volume Reduction Surgery.

Emphysema, General
and B G Cos o, Hospital Son Dureta, A G N Agust Palma de Mallorca, Spain
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Emphysema has been dened as abnormal permanent enlargement of airspaces distal to terminal bronchioles accompanied by destruction of their walls that lead to loss of lung elasticity and closure of small airways. Emphysema is recognized as one of the main components of chronic obstructive pulmonary disease, the second one being inammation of the airways, lung parenchyma, and pulmonary vasculature. This leads to small airway closure and air trapping with hyperination, which are responsible for the clinical features of exertional dyspnea, cough, and sputum production. Different patterns of emphysema are recognized, however, suggesting possible different pathogenic processes within the lung. The centrilobular pattern of emphysema is most closely associated to cigarette smoking, whereas the panacinar pattern, which is characterized by a more even involvement of the acinus, is often associated with a1-antitrypsin deciency. Assessment of emphysema by high-resolution computed tomography scan has been shown to correlate with histologic and functional abnormalities. Smoking cessation,

Further Reading
Alpha-1-Antitrypsin Deciency Registry Study Group (1998) Survival and FEV1 decline in individuals with severe deciency of alpha-1-antitrypsin. American Journal of Respiratory and Critical Care Medicine 158: 4959.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 449

bronchodilators, and inhaled corticosteroids are the mainstay of therapy, although pulmonary rehabilitation and, in selected cases, lung volume reduction may improve pulmonary function and quality of life of these patients.

Introduction
The Ciba Guest Symposium, later modied by Snider and others, dened emphysema as abnormal permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious brosis. This denition emphasizes the destruction of the alveolar surface with a minimal reparative response in the lung matrix and the ability of this destructive process to reduce the gas-exchanging surface of the lung (Figure 1). Pulmonary emphysema was rst described by Laennec in 1834 from observations of the cut surface of postmortem human lungs that had been xed by ination. He postulated that lesions were due to overination of the lung that compressed capillaries,

leading to atrophy of lung tissue. However, the observation more than a century later that emphysema could be produced experimentally by depositing the enzyme papain in the lung and the description of emphysema in patients with a1-antitrypsin (AAT) deciency led naturally to the hypothesis that emphysema results from a proteolytic imbalance caused by cigarette smoking. It is now recognized that emphysema is a part of a global disease known as chronic obstructive pulmonary disease (COPD). Many previous denitions of COPD emphasized the terms emphysema and chronic bronchitis, but the recognition that COPD has both airway and airspace characteristics led to the common denition of the disease. The pathological hallmarks of COPD are inammation of the large (bronchitis) and small airways (bronchiolitis), the lung parenchyma, and the pulmonary circulation, as well as the destruction of lung parenchyma (emphysema). The functional consequence of bronchiolitis and emphysema is airow limitation. These lesions are associated with a chronic inammatory response to a lifetime exposure to inhaled toxic gases and particles, mostly tobacco smoke, that involves cells of both the innate and the adaptive immune response.

Etiology
In the 1950s, air pollution and airway infections were thought to be the major etiologic factors responsible for COPD. However, the prevalence of cigarette smoking in the United States peaked by the 1960s, and by then adequate cross-sectional epidemiologic evidence had accumulated for the Surgeon Generals advisory committee to state that cigarette smoking is the most important of the causes of chronic bronchitis and increases the risk of dying from chronic bronchitis and emphysema. The relation between cigarette smoking and emphysema shows a rough dose-response curve between packs per year and the presence of emphysema, but only approximately 40% of heavy smokers develop substantial lung destruction. However, this observation should not be confused with the fact that only 1520% of people who smoke develop COPD, because emphysema is sometimes found in people who maintain normal lung function. Several endogenous (genetic background) and environmental factors increase the risk of COPD (Table 1).

Pathology
Figure 1 Giant microtome section of human lung with extensive emphysema.

Current descriptions of COPD pathology include changes in large airways, small airways (bronchiolitis), alveolar space (emphysema), and pulmonary

450 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

vasculature (Table 2). The various lesions are a result of chronic inammation in the lung, which in turn is initiated by the inhalation of noxious particles and gases such as those present in cigarette smoke.
Table 1 Risk factors for development of COPD in smokers Smoking history Quantity Age of onset of smoking Other environmental exposures Various host characteristics Impaired lung growth, shortened stable phase of lung function, or enhanced age-related decline in function Airway responsiveness Antiprotease deciency (function-modifying polymorphisms) Enhanced adaptive immunological reactivity

Table 2 Sites of inammatory cell increases in COPD Large airways Macrophages T lymphocytes (especially CD8 ) Neutrophils (severe disease only) Eosinophils (in some patients) Small airways Macrophages T lymphocytes (especially CD8 ) Eosinophils (in some patients) Parenchyma Macrophages T lymphocytes (especially CD8 ) Neutrophils Pulmonary arteries T lymphocytes (especially CD8 ) Neutrophils

Large airway changes consist of mucous gland enlargement and goblet cell hyperplasia. They are not believed to contribute directly to the airow limitation in these patients because their contribution to the increased airway resistance that occurs in COPD is marginal. In contrast, small airways (o2 mm) are the sites of increased airway resistance in COPD. Changes that predispose to narrowing of the small airways include goblet cell hyperplasia, smooth muscle hypertrophy, excess mucus, edema, and inammatory cellular inltration (Figure 2). Subepithelial brosis with collagen deposition in the small airways appears to be the most critical factor in airway narrowing. Respiratory bronchiolitis may be of particular importance since mononuclear inammatory cells may cause proteolytic destruction of elastic bers in the respiratory bronchioles and alveolar ducts. The resulting narrowing of this structure seems to be involved in the early airow obstruction in cigarette smokingrelated COPD. In addition, small airway patency is maintained by the surrounding lung parenchyma, which provides radial traction on bronchioles at points where alveolar septa attach. The loss of bronchiolar and alveolar attachments due to extracellular matrix destruction causes airway distortion and narrowing in COPD. Alveolar space changes constitute the cornerstone of the term emphysema. They are due to chronic inammation of alveolar walls and destruction with coalescence into larger alveolar spaces. Emphysema is therefore a structural abnormality of the lungs that affects the gas-exchanging airspaces (i.e., the respiratory bronchioles, alveolar ducts, and alveoli). Of note, the denition excludes enlarged airspaces caused by brosis to acknowledge that primary brotic processes can abnormally tether and enlarge airspaces.

(a)

(b)

Figure 2 (a) A normal small airway or membranous bronchiole o2 mm in diameter. (b) An abnormal membranous bronchiole in a smoker with abnormal FEV1. All the layers of the wall are thickened, there is abundant inammatory cellular inltrate, the lumen is o, McGill University, narrowed, and the epithelium is abnormal with goblet cells. Reproduced with permission of Professor M G Cos Montreal, QC, Canada.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 451

However, recent data indicate that small airway brosis may be a major manifestation of COPD. Emphysema is often classied into distinct pathological types, with centriacinar and panacinar being the most important (Figure 3). Centriacinar emphysema is characterized by enlarged airspaces found initially in association with

respiratory bronchioles, although in more severe cases virtually the whole acinar unit may be involved. In this pattern of emphysema, the focal nature of the lesions stands out against often apparently normal lung, and quite small lesions can be identied. This type of emphysema is most closely associated with tobacco smoking and is most often found

(a)

(b)

(c)
Figure 3 (a) A normal lung parenchyma macroscopic and microscopic. (b) Macroscopic and microscopic sections of centrilobular emphysema. Lobules are well dened by brous walls. By microscopy, the emphysematous area is surrounded by normal alveolar structures. (c) Macroscopic and microscopic sections of panlobular emphysema. All airspaces are enlarged in the lobule or acinus. o, McGill University, Montreal, QC, Canada. Reproduced with permission of Professor M G Cos

452 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

in upper lobes of the lung, where separate lesions may coalesce to produce larger cavities. Panacinar emphysema is characterized by abnormally large airspaces found evenly distributed across the acinar unit. Adjacent acinar units are usually involved to a similar degree, giving a conuent appearance to the cut surface of the lung, with extensive areas being involved. This type of emphysema is usually associated with AAT deciency and is more common in the lower lobes. The following are other types of emphysema:
*

Paraseptal emphysema, in which the abnormal airspaces run along the edge of the acinar unit but only where it abuts against a xed structure, such as the pleura, a vessel, or a septum. Scar or irregular emphysema, in which the emphysematous spaces are found around the margins of a scar. Because the scar may not be related to the anatomy of the acinar unit, this type of emphysema is not classied in relationship to the acinus. Bullae are areas of emphysema that locally overdistend to produce a lesion that, if supercial, protrude on the pleural surface. By convention, only lesions more than 1 cm in diameter justify the description of bullae.

Pulmonary vasculature changes in COPD include perivascular inammation, intimal thickening, muscularization of arterioles, in situ thrombosis, loss of capillaries and precapillary arterioles, and vascular congestion and stasis.

StructureFunction Correlations
Many studies have attempted to address the pathological changes of the small airways in smokers and their relationship to the ow limitation found in COPD (Table 3). Of special interest are studies comparing the airway changes in smokers with various degrees of emphysema and COPD to nonsmokers
Table 3 Causes of airow limitation in COPD Irreversible Fibrosis and narrowing of airways Loss of elastic recoil due to alveolar destruction Destruction of alveolar support that maintains patency of small airways Reversible Accumulation of inammatory cells, mucus, and plasma exudates in bronchi Smooth muscle contraction in peripheral and central airways Dynamic hyperination during exercise

since they give an indication not only of the effects of smoking and emphysema but also of the effect of aging in the airways. By studying smokers who had tests of pulmonary function done, including those reecting the small airways, before undergoing resection for lung tumors, investigators at McGill University developed a pathological score to describe the microscopic changes in the small airways that allowed them to study the correlations between morphology and function. Specically, they scored luminal occlusion, goblet cell metaplasia, squamous cell metaplasia, mucosal ulcers, muscle hypertrophy, inammatory cell inltrate, brosis, and pigment deposition of the airway wall in airways smaller than 2 mm in diameter. The rst abnormalities that could be seen in older smokers were changes in the epithelium, with squamous and goblet cell metaplasia and a chronic inammatory inltrate, and a slight increase in the connective tissue in the walls of the small airways. As the pathologic and functional abnormalities progressed, the cellular inammatory inltrate changed little, but there was a progressive increase in the connective tissue pigment and muscle in the airway wall. When the physiological measurements reecting small airway abnormalities, such as nitrogen washout test, volume of isoow, and Vmax50, and other function tests, such as the percentage of forced vital capacity in 1 s (FEV1/FVC), midow rate, and residual volume, were compared to the pathological score, all measurements showed a progressive deterioration as the score for the morphological abnormalities increased, but only the group with the most severe small airway score demonstrated a substantial amount of emphysema. The striking correlation between the progression of physiological impairment and the degree of small airway disease suggested that inammatory changes of the small airways made an important contribution to the functional deterioration seen in COPD even in the presence of emphysema. Furthermore, in subjects with a normal FEV1/ FVC ratio, two tests of small airway function, the slope of phase III of the nitrogen washout and the volume of isoow of the airhelium ow volume loops, were able to detect mild abnormalities of the small airways when spirometric tests were normal. Hogg and colleagues conrmed these ndings by studying lung tissue from patients with different degrees of airow limitation according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) classication. They showed that progression of COPD is associated with the accumulation of inammatory mucous exudates in the lumen and inltration of the wall by innate and adaptive inammatory immune cells that form lymphoid follicles.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 453

These changes are coupled to a repair or remodeling process that thickens the walls of these airways.

Clinical Features
Symptoms, signs, and the diagnosis of COPD are described elsewhere. However, the radiologic assessment of emphysema deserves special consideration. In 1978, Thurlbeck and Simon described criteria for the diagnosis of emphysema using the chest radiograph. These were based on indications of hyperination and vascular pruning and showed only a moderate correlation with macroscopic emphysema as assessed by the picture matching technique. When computed tomography (CT) scans became available, it became possible to extract quantitative data on lung density. When quantitative histologic assessment is compared with quantitative CT assessment,

a good correlation is found even within the range of those showing normal age change and early emphysema. However, attempts to use qualitative grading, scoring, or picture matching techniques have shown poorer correlation with lung function than actual quantitative measurements. The CT scan, particularly high-resolution CT scan (HRCT), can detect the extent and severity of emphysematous lung by directly visualizing areas of destroyed lung, identied as areas of low attenuation without visible walls (Figure 4). HRCT scans also allow reliable distinction of centrilobular emphysema from other causes of airow obstruction, such as bronchiectasis and constrictive bronchiolitis.

Pathogenesis
COPD is characterized by chronic inammation throughout the airways, parenchyma, and pulmonary vasculature. The intensity and cellular and molecular characteristics vary as the disease progresses. Over time, inammation damages the lungs and leads to the pathologic changes characteristic of COPD. In addition to inammation, two other processes thought to be important in the pathogenesis of COPD are an imbalance of proteinases and antiproteinases in the lung and oxidative stress (Figure 5).
Inammation

Figure 4 Specimen radiograph (left) and HRCT scan (right) show the typical, poorly dened, low-attenuation lesions of emphysema (A). Note the absence of denable walls around the areas of low attenuation.

The recognition that inammation plays a key role in the pathogenesis of COPD is now so widespread and considered so important that it has led to the inclusion of the term abnormal inammatory response in the disease denition. Thus, the GOLD guidelines dene COPD as a disease state characterized by not fully reversible airow limitation that is usually both

Noxious particles and gases Host factors Antioxidants Lung inflammation Oxidative stress Antiproteases Proteases Repair mechanisms COPD pathology
Figure 5 Pathogenesis of COPD noxious particles and gases, especially tobacco smoke, in susceptible subjects triggers an inammatory response in the lungs. In addition to inammation, two other processes thought to be important in the pathogenesis of COPD are an imbalance of proteinases and antiproteinases in the lung and oxidative stress.

454 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

progressive and associated with an abnormal inammatory response of the lungs to noxious particles or gases. It is now believed that the establishment of COPD is associated with further enhancement of the inammatory response already present in the small airways of normal smokers, which is paralleled by the development of structural abnormalities. These events are reected in the progressive deterioration of pulmonary function. The overall cellular inammatory inltrate in smokers airways was described by Finkelstein and coworkers, who carefully differentiated and quantitated the number of cells per millimeter of airway wall in smokers, categorized according to the type of emphysema, and nonsmokers lungs obtained at surgery. They found a large number and a wide variation in the number of cells between cases and also within the airways of the same patients. Patients with centrilobular emphysema tended to have more inammatory cells than patients with panlobular emphysema. It can be concluded based on their results that the peripheral airways of cigarette smokers exhibit a large number and variety of inammatory cells and that the extent of the cellular inltration shows a wide variability, indicating that inammation is unevenly distributed throughout the small airways. Other authors phenotyped the T lymphocytes found in airway biopsies and lung specimens and characterized them as predominantly CD8 T cells. Interestingly, CD8 T cells not only were increased in these subjects but also correlated negatively with the degree of airow limitation. This increase in lymphocytes is also associated with an increase in the socalled bronchial-associated lymphoid tissue (BALT), which is rarely found in healthy nonsmokers, is more frequent in cigarette smokers, and shows a further sharp increase in patients with severe (GOLD 3) and very severe (GOLD 4) COPD. This increase in lymphocyte subtypes and the appearance of BALT at this stage of COPD suggest the development of an adaptive immune response that may be driven by microbial colonization and infection, among others. The nding of increased numbers of T lymphocytes and especially CD8 T cells only in smokers who develop COPD is intriguing and supports the notion that T-cell inammation in the lungs is important and may be essential for the development of COPD, raising the hypothesis of the role of autoimmunity in COPD.
ElastaseAntielastase Imbalance

neutrophil elastase (NE). With respect to AAT, we now know that patients with the deciency have mutations in the AAT gene. The most common mutation is the Z mutation, which converts Glu to Lys. These mutations impair secretion of the protein from hepatocytes, resulting in markedly decreased circulating levels of this serine proteinase inhibitor. PiZ AAT is slightly less effective as an inhibitor due to its slower rate of association with NE than normal PiM AAT. These genetic changes allow NE to act relatively unopposed, thereby shifting the balance in favor of elastolysis. Other proteases, such as metalloproteinase-9 and -12 (MMP-9 and -12, respectively), have been shown to be related to the development of emphysema throughout transforming growth factor beta activation and brosis. Tissue inhibitor of metalloproteinase-3 (TIMP-3) deciency leads to a combination of developmental airspace enlargement and progressive destructive emphysema in adults, supporting the role of MMPs in COPD.
Oxidative Stress

In 1963, Laurell and Eriksson rst described the association between emphysema and AAT deciency, the primary inhibitor of the neutral serine proteinase

Cigarette smoke and inammatory cells have the capacity to produce reactive oxygen species, and they have been postulated to play a variety of roles in the pathogenesis of emphysema. One intriguing nding was that cigarette smoke can oxidize a methionine residue in the reactive center of a1PI, inactivating it and thus altering the elastaseantielastase balance. Oxidants cannot degrade extracellular matrix but may modify elastin, making it more susceptible to proteolytic cleavage. MacNee and others have found that intracellular oxidation of IK-kb promotes its ubiquitination and the release of free nuclear factor kappa B (NF-kB), which translocates to the nucleus, resulting in transcription of proinammatory genes such as interleukin-8 and tumor necrosis factor alpha. Barnes and colleagues found that cigarette smoke oxidizes and inactivates histone deacetylase 2 (HDAC2), which acts to counter histone acetylase. Acetylation of histone unwinds chromatin, allowing transcriptional complexes to bind to DNA. Thus, in the absence of HDAC2, RNA polymerase II and NFkB form a proinammatory transcription complex. This may explain steroid resistance in COPD because the activated glucocorticoid receptor acts to translocate HDAC2 to the nucleus, which keeps the chromatin wound inactive. Voelkel and colleagues hypothesized that oxidative stress has a central role in alveolar septal cell apoptosis and emphysema induced by vascular endothelial growth factor (VEGF) receptor blockade. Compared to control animals, rats treated with the

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General 455

VEGF receptor blocker SU5416 showed increased alveolar enlargement, alveolar septal cell apoptosis, and expression of markers of oxidative stress, all of which were prevented by the superoxide dismutase mimetic M40419.

Animal Models of Emphysema

Many investigators have tried to produce an animal model of emphysema by destroying the elastic bers in the lung of different animals using a variety of proteinases, following the rst description by Gross in 1965, who instilled papain into the lungs of rodents and found emphysema. However, none of them fully reproduce the full picture of pathologic changes seen in patients with COPD. For hypothesis testing in relation to pathogenic mechanisms of human COPD, animal models representing the various patterns of lung destruction and function observed in humans are needed. Because the major environmental factor that predisposes patients to COPD is long-term cigarette smoking, a cigarette smoke exposure model would be advantageous. A variety of animals have been exposed to cigarette smoke, including dogs, rabbits, guinea pigs, and rodents. The results in terms of lung pathology have been variable. To take advantage of the dened genome and the well-established methods of modulating gene expression, murine models of cigarette smoke exposure have been evaluated. Although airspace enlargement has been observed in these murine models, little attention has been paid to how well this simulates the varied pathologic features in smokingassociated emphysema of humans. Recent trends in molecular genetics and advances in molecular physiology make the mouse an ideal investigative model for determining genomic variants affecting susceptibility to COPD. Mice are susceptible to the development of emphysema, and several strains have been described that develop emphysema after prolonged cigarette smoke exposure. Guerassimov and colleagues characterized different phenotypes of mice in response to cigarette smoke in different strains with and without reduced AAT levels and serum elastase inhibitory capacity. Interestingly, Voelkel and colleagues described an animal model of autoimmune emphysema by injecting intraperitoneally xenogeneic endothelial cells in rats, which may help to sustain the role of autoimmunity in the pathogenesis of emphysema.

escalation of therapy as the disease progresses. Smoking cessation, pharmacological treatment with bronchodilators and inhaled corticosteroids, and oxygen therapy are the mainstay of treatment for COPD. Nonpharmacologic management of emphysema involves pulmonary rehabilitation, lung volume reduction, and, in selected severe cases, lung transplantation.
Pulmonary Rehabilitation

The goals of rehabilitation in COPD are multifactorial, including decrease and control of respiratory symptoms, increase in physical capacity, improvement in health status, reduction of the psychological inuence of physical impairment and disability, prevention of complications and exacerbations, and, ultimately, prolongation of life. There is much evidence that rehabilitation improves dyspnea on exertion and that associated with daily activities in COPD. Favorable outcomes have been reported after pulmonary rehabilitation in maximum exercise tolerance, peak oxygen uptake, endurance time during submaximal testing, functional walking distance, and strength of peripheral and respiratory muscles. Pulmonary rehabilitation also results in a clinically signicant improvement in disease-specic and general measures of quality of life. The effect size of pulmonary rehabilitation largely exceeds what can be achieved by the best pharmacological therapy. This intervention is therefore judged to be evidence based with respect to both exercise tolerance and symptoms of dyspnea and fatigue. These effects are longlasting (41 year) and not necessarily related to improvements in exercise ability.
Lung Volume Reduction

Management
COPD is managed according to the severity of the disease in a stepwise approach, with a progressive

Lung volume reduction surgery (LVRS) consists of the removal of the most damaged parts of the lungs in selected patients with emphysema, particularly those with upper lobe emphysema and poor exercise capacity after a full rehabilitation program (Table 4). Initial reports showed that LVRS appeared to produce functional benets, including increased FEV1, reduced total lung capacity and functional residual capacity, and improved function of respiratory muscles. However, uncertainty about morbidity and mortality, the occurrence, magnitude, and duration of benets, and preoperative predictors of benet led to multicenter, randomized clinical trials. The National Emphysema Treatment Trial used mortality and maximum exercise capacity 2 years after randomization as the primary outcomes. Overall mortality did not differ in this study between patients undergoing LVRS

456 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Emphysema, General

Table 4 Indications and contraindications for lung volume reduction surgery Features Clinical Indications Age o75 years Disability despite optimal medical treatment, including pulmonary rehabilitation Ex-smoker (436 months) Contraindications Age 47585 years Comorbid illness with 5-year mortality 450% Severe coronary artery disease Pulmonary hypertension Severe obesity or cachexia Surgical constraints: previous thoracic procedure, pleurodesis, chest wall deformity FEV1 post BD435%

Physiological

FEV1 post BDo3540% Hyperination Increased RV/TLC RV4200250% TLC4120% DLCOo50% CXR: hyperination CT: marked emphysema with heterogeneity (upper lobe predominance is ideal) Isotope scan: target areas for resection

RVo150% TLCo100%

Imaging

CXR: no hyperination CT: minimal emphysema; homogeneous, severe emphysema Isotope scan: absence of target zones

FEV1, forced expiratory volume in 1 s BD, bronchodilator; TLC, total lung capacity; DLCO, carbon monoxide diffusing capacity; RV, residual volume; CXR, chest X-ray; CT, computed tumography. Adapted from Flaherly KR and Martinez FJ (2000) Lung volume reduction surgery. Clinics in Chest Medicine 21(4): 835, with permission.

and those assigned medical therapy only. Exercise capacity after 24 months had improved by more than 10 W in 16% of patients in the surgery group compared to 3% of patients in the medical therapy group. Patients assigned surgery were signicantly more likely than those assigned medical therapy to have improvements in the distance walked in 6 min, the percentage predicted values for FEV1, general and health-related quality of life, and degree of dyspnea. After interim analyses, patients with FEV1 20% or less of the predicted value and either homogeneous emphysema or a diffusing capacity of carbon monoxide that was 20% or less of the predicted value had a high risk of death after LVRS, with little chance of functional benet. The National Emphysema Treatment Trial found that patients with predominantly upper lobe emphysema and low exercise capacity had lower mortality after LVRS than the corresponding medical therapy group. Cost-effectiveness analysis shows that LVRS is costly compared to medical therapy. Currently, the use of endobronchial valve placement to reduce lung volume endoscopically is being investigated. Preliminary results indicate that it can improve lung volumes and gas transfer in patients with COPD and prolong exercise time by reducing dynamic hyperination.
Lung Transplantation

smoking-related and AAT deciency emphysema have become the most common indication for pulmonary transplantation. Patients with diffuse disease, low FEV1 (o20% predicted), hypercapnia (PaCO247.3 kPa), and associated pulmonary hypertension are directed toward lung transplantation. The advantages of this approach have to be carefully balanced against the well-known disadvantages. Complete replacement of the diseased and nonfunctioning lungs can contribute to a striking improvement in pulmonary function and exercise tolerance, but the paucity of available donor lungs has created a long waiting list, the operation is associated with initial morbidity and mortality, and there is a risk of chronic allograft dysfunction or bronchiolitis obliterans syndrome. Furthermore, with strict adherence to selection criteria, very few patients with emphysema are candidates for any surgical therapy.
See also: Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deciency. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants.

Further Reading
Agusti A, MacNee W, Donaldson K, and Cosio M (2003) Hypothesis: does COPD have an autoimmune component? Thorax 58(10): 832834. Barnes PJ (2000) Chronic obstructive pulmonary disease. New England Journal of Medicine 343(4): 269280.

Pulmonary emphysema was initially thought to be a contraindication to lung transplantation, but

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 457

Barnes PJ and Stockley RA (2005) COPD: current therapeutic interventions and future approaches. European Respiratory Journal 25: 10841106. Calverley PM and Walker P (2003) Chronic obstructive pulmonary disease. Lancet 362(9389): 10531061. Cosio MG and Majo J (2002) Inammation of the airways and lung parenchyma in COPD: role of T Cells. Chest 121(supplement 5): 160S165S. Fishman AP (2005) One hundred years of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 171(9): 941948. Fishman A, Martinez F, Naunheim K, et al. (2003) A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. New England Journal of Medicine 348(21): 20592073. Flaherly KR and Martinez FJ (2000) Lung volume reduction surgery. Clinics in Chest Medicine 21(4): 835. Geddes D, Davies M, Koyama H, et al. (2000) Effect of lungvolume-reduction surgery in patients with severe emphysema. New England Journal of Medicine 343(4): 239245. Hogg JC (2004) Pathophysiology of airow limitation in chronic obstructive pulmonary disease. Lancet 364(9435): 709721. Hogg JC, Chu F, Utokaparch S, et al. (2004) The nature of smallairway obstruction in chronic obstructive pulmonary disease. New England Journal of Medicine 350(26): 26452653. Mahadeva R and Shapiro SD (2002) Chronic obstructive pulmonary disease: experimental animal models of pulmonary emphysema. Thorax 57(10): 908914. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, and Hurd SS (2001) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/ WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) workshop summary. American Journal of Respiratory Critical Care Medicine 163(5): 12561276. Shapiro SD and Ingenito EP (2005) The pathogenesis of chronic obstructive pulmonary disease: advances in the past 100 years. American Journal of Respiratory Cell and Molecular Biology 32(5): 367372. Sutherland ER and Cherniack RM (2004) Management of chronic obstructive pulmonary disease. New England Journal of Medicine 350(26): 26892697. Wouters EF (2004) Management of severe COPD. Lancet 364(9437): 883895. is unwilling to quit smoking, the 5 Rs can be employed: emphasizing relevance of cessation, identifying risks of smoking, focusing on rewards, discussing roadblocks, and using repetition. Pharmacologic therapy is recommended for most patients seriously considering smoking cessation with few exceptions. Nicotine replacement therapy and bupropion are considered rst-line agents. Combination therapy may be used in patients who are not successful in quitting while using single-drug therapy. Despite evidence that clinician advice to quit smoking increases abstinence rates, clinicians inconsistently intervene. System-based programs provide opportunities to further improve rates of smoking cessation.

Smoking and COPD

Smoking is a well-established cause of chronic lung disease and is the leading preventable behavior contributing to increased patient morbidity and mortality. In the normal aging process, the lung loses elasticity and pulmonary mechanics deteriorate with slowing of forced expiration by approximately 2030 ml per year. Smokers experience an accelerated decline in lung function, which is particularly severe in a susceptible subset of the smoking population. When forced expiratory volume in 1 s (FEV1) reaches 50% predicted, individuals become symptomatic and activity is limited by dyspnea. When FEV1 reaches 30 40% predicted, exercise limitation becomes disabling. The mean survival after the onset of disabling chronic obstructive pulmonary disease (COPD) is 5 years. Smoking cessation has a strong benecial effect on the course of COPD. In 1983, the National Heart, Lung, and Blood Institute initiated The Lung Health Study, a multicenter trial investigating the effect of smoking cessation and ipratropium bromide on the decline in lung function in adults with mild COPD. Patients who were randomized to smoking cessation received intensive counseling and pharmacotherapy. The study found that among those who quit, the accelerated decline in lung function was actually reversed during the rst year of cessation and smokers who quit had an average increase in FEV1 of 47 ml during the rst year of cessation. This was followed by a subsequent decline characterized by that of the normal aging population of 31 ml per year. This was half the average rate of decline compared to individuals who continued smoking. Among smokers who quit, improvement in FEV1 was more pronounced among heavier smokers, women, those with worse lung function, and those with greater bronchial responsiveness.

Smoking Cessation
M C McCormack and C S Rand, Johns Hopkins Univerisity, Baltimore, MD, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Smoking is a well established cause of chronic lung disease and is the leading preventable behavior contributing to patient morbidity and mortality. Smoking cessation has been shown to have a strong benecial effect on the course of chronic obstructive pulmonary disease (COPD). Research has clearly established that physician advice regarding smoking cessation will signicantly increase the likelihood that a patient will quit smoking. There are ve steps recommended as a starting point for the clinician addressing smoking cessation. The 5 As include asking the patient about smoking, advising cessation, assessing readiness to quit, assisting in the process, and arranging follow-up. If the patient

Clinical Strategies to Promote Smoking Cessation

Physician Advice

Research has clearly established that physician advice to stop smoking signicantly increases the likelihood

458 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation

that a patient will quit smoking. The Agency for Healthcare Research promotes widely used evidencebased guidelines to assist healthcare providers in brief smoking cessation counseling. These guidelines propose ve basic steps to intervention in the primary care setting, which can be summarized by the 5 As. The rst step is to ask the patient about their tobacco use. This can be consistently achieved by routinely updating smoking status while reviewing the vital signs. Next, advise the patient of the benets of smoking cessation. Here, the message should be clear, strong, and personal. This is an opportunity to relate the specic benets as they may relate to the patient. For example, As your doctor Im very concerned about the effect your smoking is having on your breathing and your overall health. If you quit smoking now, there is an excellent chance that your lungs will begin to heal and your risk of COPD, heart disease, and cancer will be signicantly reduced. For these reasons, I strongly advise you to quit smoking. Physician advice may include not only discussion of health benets but also economic factors (e.g., cost of smoking) and family concerns (e.g., passive smoking, role modeling). The third A entails assessing the patients readiness to quit smoking. This includes assessing the willingness to stop smoking in the short term (the next 30 days) as well as identifying longerterm goals. For example, one might ask, On a scale of 110 how ready are you to quit smoking in the next 30 days? Once the practitioner has ascertained that the individual is ready to quit smoking, he/she can then assist the patient in preparing for a successful quit attempt. The hallmarks for adequate preparation can be remembered by the pneumonic STAR: setting a quit date, telling family, friends, and co-workers, anticipating challenges, and removing tobacco products from the environment. The healthcare provider should also provide practical advice. This may include reviewing pitfalls in prior smoking cessation attempts and avoiding triggers. Identifying support individuals and discouraging smoking on the part of others in the home is also important. The practitioners ofce should also be identied as a source of support. Supplemental materials may be useful and pharmacotherapy should be addressed at this stage. Once a smoking cessation plan is in place, followup is essential. The nal step is to arrange a visit. The rst ofce visit should occur shortly after the quit date, preferably within 1 week, and a subsequent visit within 1 month is recommended. A more intensive, group or individual, smoking cessation program is an alternative to outpatient follow-up. If the patient is unwilling to quit after the second step assess, the clinician should employ motivation

enhancing strategies. Here, the 5 Rs may be employed. The rst is to address relevance by encouraging the patient to identify how tobacco cessation is personally relevant. Identifying risks can also be a motivating factor and the clinician can assist the patient in recognizing personal health risks. The patient should be encouraged to focus on the rewards of smoking cessation and these may include not only improved health but also economic and social benets, as well as potential improvement in personal appearance and self-esteem. The clinician should ask the patient to identify roadblocks and help troubleshoot potential barriers to successful cessation. Finally, repetition is essential in patients who are not yet ready to begin an attempt to quit as this increases the likelihood of future attempts and future success. Research suggests that the consistency of the provider message is a critical component in a patients decision to quit smoking.
Addressing Patient Barriers to Smoking Cessation

Tobacco dependence is an addictive disorder and for this reason few patients quit with their initial attempt. Tobacco dependence includes not only physical addiction (for most smokers) but also includes signicant behavioral and psychological dependence on tobacco, which can create barriers to successful cessation. For these reasons, many smokers cycle in a relapse-and-remission pattern for years before successfully quitting. According to the 2000 Surgeon Generals Report, 70% of the 50 million smokers in the US have made at least one prior quit attempt. While the tendency to relapse can be very discouraging, it is important to recognize this pattern as part of the normal process of quitting as repeated interventions are often required to attain long-term abstinence. Despite the complexities of this addiction, there has been signicant success in achieving smoking cessation at a national level over the past several decades. Between 1965 and 1993, the prevalence of adult smoking in the US decreased from 42% to 25% while quit rates increased from 25% to 50%. Although cigarettes contain as many as 600 additives, the nicotine in tobacco has been identied as the primary substance associated with addiction. Nicotine absorption is pH dependent and depends on the route of exposure. Nicotine is absorbed transdermally, through the gastrointestinal mucosa, and is rapidly absorbed across the huge surface area of the alveoli of the lungs. The half-life of nicotine is 2 h and this substance undergoes a signicant rst-pass effect in the liver, where 7080% of the metabolism occurs via the cytochrome P-450 system. The effect of nicotine is mediated by binding

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 459

to the nicotinic cholinergic receptors, a subset of acetylcholine receptors. Several additives in tobacco products are thought to inuence the metabolism of nicotine, including ammonia and menthol. While light or low-tar cigarettes have been promoted as less toxic, evidence suggests that coverage of the lter perforations with the ngertips and compensatory behavior to achieve equivalent nicotine levels, including deeper inhalation, shorter time between puffs, and smoking more cigarettes, negate the potential benet of light cigarettes over regular cigarettes. Nicotine withdrawal symptoms are an important early barrier to smoking cessation. The nicotine withdrawal syndrome is well characterized and includes craving the use of nicotine, irritability, difculty concentrating, restlessness, increased appetite, and anxiety. Some of the warning signs that a patient may have high dependence on nicotine include withdrawal symptoms during previous quit attempts, smoking more than one pack per day, smoking the rst cigarette within 30 minutes of awakening, and smoking during times of illness. Preparing the patient to deal with symptoms of nicotine withdrawal is an important aspect of counseling patients about smoking cessation. A history of withdrawal symptoms or evidence of nicotine dependence should prompt strong encouragement of nicotine replacement therapy. Social and environmental triggers comprise another potential barrier to effective long-term cessation. Smokers have generally created a world in which smoking is an important part of their social and leisure activities. Patients should be encouraged to anticipate this change by creating an alternative nonsmoking support network and avoiding activities where smoking is common (e.g., bars, clubs). Social activities and settings where alcohol is served can be risky for patients trying to quit smoking because one of the more common triggers for relapse is the use of alcohol. For this reason, abstinence from alcohol use should be encouraged during smoking cessation attempts. Weight gain after smoking cessation is a common concern among smokers considering quitting. Weight gain is likely caused by decreased metabolic activity and increased caloric intake. While women and adolescent smokers are frequently fearful of weight gain if they quit smoking, this concern is shared by many smokers and has been shown to persist in the older, medically ill population. The majority of smokers who quit will gain some weight but most will gain greater than 10 pounds (4.5 kg). AfricanAmericans, those under age 55, and heavy smokers are at greatest risk for major weight gain. There is evidence that attempts to prevent weight gain may undermine the success of cessation

attempts. However, studies suggest that increased exercise during smoking cessation can decrease and delay weight gain. Pharmacologic therapy with nicotine replacement and bupropion has also been shown to delay weight gain but not to decrease weight gain in the long run. It is important for clinicians to address the possibility of weight gain. Clinicians should stress the overall importance of quitting smoking for improved health and should help dene this as the primary goal. The clinician should also offer to help the individual address weight gain once the patient has achieved success with cessation. There is evidence that a sequential approach to cessation and weight control is superior to a simultaneous approach.
Pharmacologic Therapy

There have been many advances in the pharmacologic treatment of tobacco dependence in the recent past. It is recommended that all individuals seriously attempting smoking cessation receive pharmacologic therapy with a few exceptions. In addition to individuals with medical contraindications, special consideration of the appropriateness of pharmacotherapy should be given to individuals who smoke less than 10 cigarettes per day, pregnant women or nursing mothers, and adolescents. Pharmacologic therapy should be utilized in a coordinated approach by the healthcare provider that incorporates motivated behavioral change efforts. A list of the various smoking cessation products that are available is given in Table 1. Nicotine replacement therapy and bupropion are the recommended rst-line therapies for tobacco cessation. There is no clear distinction as to which of these therapies is superior. Therefore, the practitioner must weigh the decision and consider the circumstances specic to the patient. Bupropion and nicotine replacement therapy may be used simultaneously and there is some evidence that this may confer additional benet when compared to monotherapy. Nicotine replacement therapy Nicotine replacement therapy (NRT) is available in many forms, including the transdermal patch, chewing gum, inhaled, nasal spray, and lozenges. Nicotine substitution is effective in reducing symptoms of nicotine withdrawal. The nicotine patch is a widely used method of delivery of NRT. The patch is available as an over-thecounter or prescription medication. There is both a 24 h formulation as well as a 16 h formulation. If sleeplessness is a signicant side effect, the 24 h patch can be removed prior to bedtime or the 16 h patch can be substituted. The patch should be placed in a

460 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation

Table 1 Summary of smoking cessation products Product Over-the-counter products Nicotine gum Nicorette Dosage Package Price (US$)a Comments

2 mg 924 days 4 mg 924 days

170 pieces 110 pieces 50 pieces

59.99 49.99 31.99

Nicotine patch Nicoderm CQ

Emphasize importance of adequate use. Target is 924 per day for 46 weeks, then gradual tapering. Special instructions about chew-and-park technique Target is maintenance for 410 weeks, with gradual taper following. If sleeplessness occurs, the 24 h patch may be removed at bedtime or the 16 h patch may be used Dissolves in 1030 min. More than 9 lozenges per day recommended as initial therapy. Tapering recommended at 6 weeks

Nicotrol Generic/store brand Nicotine lozenge Nicorette

21 mg/24 h 14 mg/24 h 7 mg/24 h 15 mg/16 h 22 mg/24 h 2 mg 4 mg

14 14 14 14 14

patches patches patches patches patches

44.99 44.99 44.99 34.99 39.99 39.99

72 pieces 72 pieces

Prescription products Nicotine nasal spray Nicotrol NS

1 mg 810 mg/day 2 sprays 1 mg

100 mg

55.99

Nicotine vapor inhaler Nicotrol inhaler Bupropion SR Zyban Generic

Provides rapid onset of nicotine. Tapering recommended at 6 weeks. Nasal irritation is a frequent side effect Absorption is through the oral mucosa, not the lungs. May simulate behavioral aspect of smoking Begin treatment 1 week prior to quit date. No tapering needed. May be combined with nicotine replacement therapy

10 mg/cartridge (delivers 4 mg) 150 mg 1 per day 3 days, then 2/day 150 mg 1 per day 3 days, then 2/day

168 cartridges

153.49

60 tablets 60 tablets

136.99 105.49

Based on pricing at a large pharmacy chain in Baltimore, MD, US, 2004.

hairless area between the neck and the torso. The most common side effect is a local skin reaction, which occurs in approximately half of the patients who receive this therapy. The nicotine patch has been studied in patients with cardiovascular disease and has not been associated with an increase in cardiovascular events. While the patch offers passive dosing that provides steady-state levels of nicotine, this system does not allow the user to adjust as needed in response to craving or situational urges to smoke. Patients may benet from the addition of a shortacting nicotine replacement therapy, such as nicotine gum, if they are unable to quit with the patch alone. Nicotine chewing gum is another form of nicotine replacement therapy. This is available in a 2 mg per piece formulation, which is recommended for smokers of less than 25 cigarettes per day and a 4 mg per piece formulation for individuals who smoke 25 or more cigarettes per day. One advantage of the nicotine chewing gum is that it can be used in an ad lib fashion as cravings or symptoms of withdrawal occur. However, adherence to this therapy can be a

barrier to effectiveness. Often patients do not utilize enough of the chewing gum. This can be addressed by employing scheduled dosing intervals of 12 h. Furthermore, technique is sometimes problematic. The gum should be chewed until a mint taste is experienced. At this time, the chewing gum should be parked between the cheek and the gum to allow buccal mucosa absorption of the nicotine. This chewing and parking behavior is repeated for approximately 30 min at which time the gum is discarded. The individual should refrain from eating or drinking for 15 min before or after use to maximize absorption. While less commonly used, nicotine inhalers and nicotine nasal spray are alternative methods of nicotine delivery. They are both available by prescription only. Nicotine inhalers are dispensed as canisters containing 4 mg of nicotine delivered over 80 breaths. The recommended dose is between 6 and 16 canisters per day. The patient should refrain from eating or drinking 15 min before or after using the inhaler. The most common side effects are local irritation in the mouth and throat as well as cough and rhinitis.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Smoking Cessation 461

Nicotine nasal sprays have an aerosolized dose of 0.5 mg per nare with a combined total dose of 1.0 mg. Initial dosing should be 12 doses per hour for a total of 840 doses per day. Each bottle contains about 100 doses. The side effects of this treatment include a high frequency of nasal irritation. Nicotine nasal spray also has a greater dependence potential than other nicotine replacement therapies although less than that of cigarette smoking. Utilizing a combination of nicotine replacement strategies has been attempted. The goal is to provide a steady-state level of nicotine accompanied by ad lib use during times of cravings or abstinence symptoms. There is a suggestion that this may yield additional benet but long-term safety has not been proven. Bupropion Bupropion (Zybans) is the rst nonnicotine medication shown to be effective for smoking cessation. This is available exclusively as a prescription medication indicated for smoking cessation (Zybans) and for depression (Wellbutrins). Although the precise mechanism of action is unknown, it is presumed to be mediated by its capacity to block neural reuptake of dopamine and norepinephrine. The effectiveness of this agent for smoking cessation does not appear to be mediated through an antidepressant effect. In fact, bupropion has been found to be equally efcacious in improving smoking cessation rates in patients without depression as in those with depression. However, when choosing an agent for a depressed smoker, bupropion should be seriously considered as it may provide multiple benets. The medication should be initiated 1 week prior to quitting at a dose of 150 mg orally for 3 days, followed by 150 mg twice daily for 712 weeks. Treatment for up to 6 months can be considered for maintenance. The most commonly reported side effects are insomnia and dry mouth. Bupropion is contraindicated in individuals with a seizure disorder, with current or previous bulimia or anorexia nervosa, or in those who have taken a monoamine oxidase inhibitor within the last 14 days or are currently taking another medication containing bupropion. Other medications There are two second-line agents that have been employed for tobacco cessation when rst-line treatments are contraindicated or have failed. These include clonidine, an antihypertensive, and nortriptylene, an antidepressant. Limited research suggests that these medications may increase abstinence compared with placebo but these products are not approved by the US Food and Drug Administration for the indication of smoking cessation. The use of other antidepressants in the treatment of smoking cessation is an area of continuing

investigation as depression is more common among smokers than the general population. However, currently there are no other antidepressants recommended for smoking cessation.

Smoking Cessation Programs

Smoking cessation programs are effective in initiating and maintaining smoking cessation. In the Lung Health Study, an intensive intervention program resulted in sustained smoking cessation in 22% of patients compared to 5% of those who received usual care. A signicant doseresponse relationship has been shown between the intensity of counseling and the effectiveness in achieving cessation. Intense programs typically involve interactions that are longer than 10 min, with greater than 4 sessions, for a total of more than 30 min of counseling. These sessions might include some strategies that have been found to be particularly effective, such as practical counseling, establishing social support within the program, and helping to secure social support beyond the extent of the program. Although more intense counseling is preferable, interventions of less than 3 min have still been shown to have a positive effect. Person-to-person interaction has been found to be effective and can take the form of telephone, individual, or group counseling. Local hospitals and branches of national organizations may offer organized treatment programs. The American Lung Association offers low-cost smoking cessation programs, as well as an internet-based multistep program.

Complementary Medicine
Many patients inquire about the benets of acupuncture, hypnosis, or other complementary and alternative medicine strategies for smoking cessation. Acupuncture has been proposed to be an effective aid to cessation because it may release neurotransmitters, such as serotonin, noradrenaline, and cholecystokinin, which may affect nicotine craving and withdrawal. However, meta-analytic studies have not found acupuncture to be superior to sham acupuncture, suggesting that any potential benets are derived from positive beliefs about the utility of the procedure. Hypnotherapy has been utilized with the intent of strengthening the will to stop smoking and decreasing cravings. A meta-analysis of nine randomized trials did not conrm signicant benet from hypnotherapy compared to other interventions. While the use of complementary medicine may be helpful on an individual basis, no alternative or complementary smoking cessation techniques or products have been substantiated by evidence in the medical literature.

462 CHYMASE AND TRYPTASE

System-Based Support for Patient Smoking Cessation

Despite strong evidence that physician advice regarding smoking is an effective cessation tool, national data show that over one-third of US smokers are not advised to quit in a given ofce visit. Systemwide programs have been shown to increase the frequency with which smokers are identied. Identication of smoking status by stickers on patient charts or by recording smoking status with the vital signs are useful strategies. This also provides an opportunity for nonphysician healthcare workers to participate. The involvement of nurses, in addition to physicians, in the process of tobacco cessation intervention has been associated with increased long-term abstinence rates. System-wide programs have also been shown to increase the number of patients who receive counseling during ofce visits. Despite concerns that patients may be annoyed by increased frequency of smoking cessation interventions, follow-up studies have found enhanced patient satisfaction. Many successful system-based models have focused on removing barriers, such as access and cost. The addition of telephone-based programs has been shown to increase participation. Full coverage of the costs of patient smoking cessation services and pharmacotherapy has been associated with increased participation in cessation programs and decreased prevalence of smoking. The most successful systemwide approaches treat tobacco cessation as a key indicator of performance. These systems incorporate individuals from all levels of the organization. Successful programs solicit feedback and make interim adjustments. This results in the creation of an infrastructure capable of maintaining a smoking cessation program over time.
See also: Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations. Particle Deposition in the Lung.

Further Reading
Anthonisen NR, Connett JE, Kiley JP, et al. (1994) Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV1. Journal of American Medical Association 272: 14971505. Curry SJ, Grothaus LC, McAfee T, et al. (1998) Use and cost effectiveness of smoking cessation services under four insurance plans in a health maintenance organization. New England Journal of Medicine 339: 673679. Fiore MC, Bailey WC, Cohen SJ, et al. (2000) Treating Tobacco Use and Dependence: Clinical Practice Guideline. Rockville, MD: US Department of Health and Human Services, Public Health Service. Hollis JF, Bills R, Whitlock E, et al. (2000) Implementing tobacco interventions in the real world of managed care. Tobacco Control 9(supplement I): i18i24. Rigotti NA (2002) Treatment of tobacco use and dependence. New England Journal of Medicine 7: 506512. Scanlon PD, Connett JE, Waller LA, et al. (2000) Smoking cessation and lung function in mild-to-moderate chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 161: 381390. Wise RA (1997) Changing smoking patterns and mortality from chronic obstructive pulmonary disease. Preventive Medicine 26: 418421. Wise RA, Kanner RE, Lindgren P, et al. (2003) The effect of smoking intervention and an inhaled bronchodilator on airways reactivity in COPD: The Lung Health Study. Chest 124: 449458. Zang ZA and Wynder EL (1998) Smoking trends in the United States between 1969 and 1995 based on patients hospitalized with non-smoking-related diseases. Preventive Medicine 27: 854861.

Relevant Websites
http://www.ash.org provided by a non-prot organization, Action on Smoking and Health, and offers information about smoking risks, quitting smoking, and legal issues related to smoking. http://www.lungusa.org provided by the American Lung Association and offers an online smoking cessation program, practical smoking cessation advice, and information about smoking-related lung disease. http://www.surgeongeneral.gov/tobacco/ provided by the US Department of Health and Human Services and contains the most recent Surgeon Generals Report on the Health Consequences of Smoking, as well as information for physicians and consumers.

CHYMASE AND TRYPTASE

P Peachell, University of Shefeld, Shefeld, UK
& 2006 Elsevier Ltd. All rights reserved. synthesis of these proteases is the lung mast cell and proteases are stored within secretory granules, complexed to heparin. Activation of mast cells, by allergens and other stimuli, leads to the release of a wide variety of mediators, including proteases. The released proteases may cause bronchoconstriction, promote inammation, modulate neuronal activity, and participate in airway remodeling. These effects of proteases may be mediated by direct hydrolysis of substrates and by activation of

Abstract
The serine proteases, tryptase and chymase, contribute to the pathophysiology of respiratory disease. The principal site of

CHYMASE AND TRYPTASE 463

protease-activated receptors. These receptors are upregulated in respiratory disease. Inhibitors of these proteases, which are in development, could have a role in the therapeutic management of respiratory disease.

Introduction
The lung mast cell has long been recognized as central to the mediation of asthma, especially asthma that has an allergic basis. Activation of the mast cell, by allergens or other stimuli, leads to the generation of autacoids such as histamine, cysteinyl-leukotrienes, prostaglandin D2, cytokines, and prominent levels of proteases. These generated mediators act synergistically and can have profound effects on the airways and blood vessels causing bronchoconstriction and promoting inammation. In addition to these more immediate effects, it is likely that over a longer timeframe, mast cells contribute to airway remodeling. Mast cell-derived products can cause changes in the architecture of the airway and this could have important consequences as far as the behavior of the asthmatic airway is concerned. The importance of histamine as a mediator of allergic disorders has been known for over 70 years. Moreover, the fact that slow-reacting substance of anaphylaxis (SRS-A), later identied as a mixture of cysteinyl-leukotrienes, is a very potent bronchoconstrictor and that prostanoids can act similarly, has been known for some considerable time. However, although studies some 50 years ago demonstrated that mast cells contain proteases, a fuller appreciation of how generated proteases might inuence the development of allergic disease has only been realized more recently. A major reason for this is the identication, a little over 10 years ago, of a novel group of receptors that are activated by proteases. The widespread expression of protease-activated receptors (PARs) within the lung and the potential activation of PARs by mast cell proteases has substantially enlarged the scope of protease-mediated effects within the lung. Thus, proteases are no longer considered simply as enzymes whose egress from mast cells may cause some modest structural modications to the extracellular matrix. Indeed, a large body of evidence has now accumulated showing that mast cell proteases may contribute signicantly to the pathophysiology of asthma and other respiratory disorders. In human mast cells, tryptase is the most abundant protease and it follows that most of the available literature has focused on this protease. Other proteases variably expressed by mast cells include chymase, cathepsin, and carboxypeptidase. In fact, the expression of proteases by mast cells has led to an

important classication of mast cell populations. Mast cells that express tryptase, but not chymase, are often classied as MCT mast cells whereas those that contain tryptase and chymase (and other proteases) are referred to as MCTC mast cells. Connective tissue mast cells, such as those found in skin, are more or less exclusively MCTC mast cells. At mucosal surfaces, such as the lung, MCT mast cells appear to predominate although, within the bronchi, MCTC mast cells are more prominent. Interestingly, the heterogeneity in protease content displayed by different mast cell populations is associated with heterogeneity in function. For example, chemokine receptor expression between the two subsets differs. In addition, MCTC mast cells, but not MCT mast cells, are responsive to a wider array of activators such as neuropeptides, compound 48/80, and C5a, and the spectrum and quantities of mediators generated, quite apart from the differences in the complement of proteases that can be generated, also differ.

Structure
Mammalian mast cell chymases fall into two phylogenetic groups: alpha and beta. In primates, however, only the alpha form appears to be expressed and it is possible that beta-chymases are rodent specic. The gene for human alpha-chymase is found on chromosome 14 within a cluster encoding for a number of additional proteases. The human tryptase gene locus, found on chromosome 16, is more complex than that for alpha-chymase due to the existence of multiple tryptase genes and allelic variants. The most important mast cell tryptase is beta-tryptase, although isoforms of this type exist that may display differences in activity. In addition, alpha-tryptase has also been detected but not always, as about one-fourth of individuals lack this isoform. That interindividual differences exist in protease content could potentially have an inuence on disease susceptibility. Of particular interest are reports demonstrating that mast cell tryptase forms tetramers and that the tetrameric form is associated with optimal catalytic activity. Within the tetramer, the active sites of each of the monomers are orientated centrally creating a central pore. The geometry of this pore is such that larger substrates cannot be accommodated and, as a consequence, smaller peptidergic substrates are preferentially hydrolyzed. Although small molecule inhibitors of serine proteases are effective at attenuating the catalytic activity of tryptase, larger proteinaceous inhibitors that are effective at reducing the activity of alternative serine proteases, such as chymase, are more or less without exception ineffective. Again the reason for this resistance to inhibition

464 CHYMASE AND TRYPTASE

probably lies in the restricted access of larger moieties to the catalytic site.

capable of hydrolyzing substrates that the tetrameric form does not cleave.

Biological Function Regulation of Production and Activity

The regulation of protease synthesis by mast cells has not been clearly delineated. However, recent studies using mast cell progenitors, CD34 cells, isolated from either cord blood or peripheral blood suggest that cytokines could inuence protease expression. Culture of progenitors with the mast cell growth factor, stem cell factor (SCF), and alternative cytokines led to the development of mast cells whose tryptase content was fairly constant but where chymase levels could be detected in practically all cells but where the content was variable. The suggestion was made that MCT cells serve as precursors for MCTC cells and that exposure to appropriate stimuli can drive these cells to an MCTC phenotype. However, this concept is not supported by a more recent study in which MCT mast cells isolated from human lung were exposed to SCF and alternative cytokines, as phenotypic conversion was not observed. Data from this later study suggest parallel rather than sequential development of MCT and MCTC cells. Once synthesized, mast cell proteases are packaged within the secretory granules associated with heparin. Heparin is a heavily sulfated proteoglycan and the anionic nature serves to complex and to stabilize the proteases. Unusually, both chymase and tryptase are packaged within the granules as fully active forms and not as proforms. The mechanism of this process is unclear but the possibility that dipeptidyl peptidase 1 (DPP1), which liberates dipeptides from substrates which in turn activate proteases, might be involved has been suggested by studies utilizing DPPl-null mice. Mast cells are thus primed to liberate chymase and tryptase in active forms upon appropriate activation. Following mast cell activation by allergens and other stimuli, degranulation takes place and the granule contents are disgorged extracellularly. A wide array of granule-associated autacoids, such as histamine, is released along with proteases and heparin. Heparin appears to play a crucial role in ensuring that the active tetrameric form of tryptase is maintained. In the absence of heparin, the tetramer dissociates into monomers that are largely thought of as being relatively inactive. The tetrameric form of tryptase shows optimal catalytic activity with restricted substrate specicity governed by the size constraints of the central pore, but recent data suggest that the monomers are far from inert and may be Tryptase cleaves polypeptides on the C-side of arginine and lysine residues whereas chymase acts on the C-side of phenylalanine, tyrosine, and tryptophan. The biological effects of these proteases are a consequence of proteolysis and can be due to direct effects on substrates or those that are mediated indirectly following activation of PARs. Given the widespread distribution of PARs within the lung it is probable that PAR-mediated effects are more important and far-reaching. Potential processes that mast cell proteases may mediate in the lung are (1) bronchoconstriction, (2) inammation, (3) remodeling, and (4) neuronal effects. There appears to be some discordance in the literature as to whether mast cell proteases cause direct bronchoconstriction. Certainly, differences appear to exist among species and among the type of airway studied. Reports exist that human bronchi constrict in response to proteases, in a PAR-mediated fashion, but it is probably fair to say that alternative mast cell mediators, histamine and the cysteinyl-leukotrienes, display a greater facility to induce smooth muscle contraction than proteases. For the large part, mast cell proteases appear to display proinammatory effects. Effects on the vasculature include vasodilation and increases in permeability, these effects being PAR mediated. In addition, proteases have been shown to be chemotactic for inammatory cells, promoting cell recruitment and, additionally, a whole host of airway cells have been reported to be activated by proteases to release a variety of proinammatory autacoids. That said, conicting data have shown anti-inammatory effects of PAR activation in which agonists of PARs have been shown to attenuate inammation in various animal models. The mechanism of this effect has not been delineated but the suggestion exists that the cytoprotective and anti-inammatory prostanoid prostaglandin E2 (PGE2) may be involved. The release of proteases following mast cell activation can lead to changes in the extracellular matrix. Tryptase, for example, has been shown to hydrolyze bronectin and collagen, if presented in an appropriately denatured conformation. Alternatively, the suggestion has been made that the PARdependent release of matrix metalloproteases from airway cells may play a more prominent role in matrix protein degradation. PAR-mediated mitogenesis of cells, including airway smooth muscle cells, has also been reported. Collectively, these events are

CHYMASE AND TRYPTASE 465

likely to be important in mediating airway remodeling. Proteases may also be involved in modulating neuronal pathways. PARs have been identied on sensory nerve terminal endings and receptor activation is associated with the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP), peptides that have been implicated in the development of neurogenic inammation. That mast cells are known to be located close to nerve endings provides support for such a mechanism. Additionally, certain neuropeptides, such as vasoactive intestinal peptide (VIP), are substrates for tryptase. VIP is a bronchorelaxant and tryptase inactivates the neuropeptide suggesting a greater tendency for airway smooth muscle to constrict as a consequence of this action. Thus, proteases may play an important role in modulating levels of neuropeptides in the lung.

Receptors
Four PARs have been identied: PAR1, PAR2, PAR3, and PAR4. All are 7-transmembrane, G-proteincoupled receptors. Tryptase acts at PAR2 whereas chymase acts at PAR1. The mechanism of PAR activation is intriguing. PARs express an extended extracellular N-terminus and, following exposure to a relevant protease, the N-terminus is hydrolyzed and truncated exposing a peptide sequence (SLIGKV for PAR2 and SFLLRN for PAR1) that is still tethered to the receptor and that, autointeractively, engages the second extracellular domain of the receptor. This induces a conformational change in the receptor and activation results.

Mast Cell Proteases in Respiratory Diseases

The lung mast cell has long been recognized as central to the mediation of asthma. Mast cell numbers increase in asthma such that the potential for heightened generation of mediators, such as proteases, in response to allergens, exists. This, allied to an increase in the expression of PARs in asthma, suggests exaggerated responses to proteases in the pathophysiological context. An involvement of mast cell proteases in alternative respiratory diseases, however, is less clearly dened. For example, chronic obstructive pulmonary disease (COPD) is a disease characterized by neutrophil involvement, and the extent to which mast cells contribute is uncertain. However, since mast cells subserve a sentinel function within the lung, being found in high numbers at the mucosal surface, they are anatomically predisposed to react to

any airborne triggers. This suggests that mast cells could potentially be involved in a wide variety of respiratory disorders and, as a corollary, proteases may be important too. In asthma, the consequences of mast cell protease activity are likely to be largely proinammatory (Table 1). For example, proteases are known to cause vasodilation and increase vascular permeability as well as being chemotactic for inammatory cells, such as eosinophils. An important role for proteases in allergic inammation is supported by studies using PAR2-decient mice that demonstrated attenuated inammatory responses in a model of allergy. An additional effect of proteases in asthma is likely to involve bronchoconstriction but how important this contribution might be is uncertain. Proteases are also known to modulate levels of neuropeptides in the lung and this activity is likely to inuence the severity of both bronchoconstrictor and inammatory events in the lung. Over the longer term, mast cell proteases may be involved in airway remodeling since proteases are known to stimulate the proliferation of a variety of pulmonary cells and are also known to cause degradation of the extracellular matrix. Because proteases have the potential to mediate a very wide-ranging number of effects, there has been signicant interest in developing inhibitors of proteases for the treatment of asthma. This is especially true for tryptase and a recent study has shown that the tryptase inhibitor APC 366 partly attenuates allergen-induced late-phase airway obstruction (assessed by monitoring forced expiratory volume in 1 s) in asthma but has no effect on the early phase. Whether drugs that inhibit tryptase will show any benet in the treatment of asthma remains to be seen. However, it might be argued that strategies aimed at targeting the mast cell directly, since this would prevent the release of all mast cell mediators, might constitute a more effective approach to treat asthma.
Table 1 Main effects that the mast cell proteases, tryptase and chymase, can mediate in the lung Process Inammation Protease-mediated effect Increased vascular permeability Vasodilation Chemotaxis Airway smooth muscle contraction Increased release of neuropeptides promoting neurogenic inammation Inactivation of bronchorelaxant neuropeptides Degradation of extracellular matrix either directly or by stimulating metalloprotease release Stimulation of pulmonary cell proliferation

Bronchoconstriction Neuromodulation

Remodeling

466 CILIA AND MUCOCILIARY CLEARANCE See also: Allergy: Allergic Reactions. Asthma: Overview; Extrinsic/Intrinsic. Cysteine Proteases, Cathepsins. Extracellular Matrix: Degradation by Proteases. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide. Leukocytes: Mast Cells and Basophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids. Serine Proteinases. Smooth Muscle Cells: Airway. Systemic Disease: Sarcoidosis.
Cocks TM and Moffatt JD (2001) Protease-activated receptor-2 (PAR2) in the airways. Pulmonary Pharmacology & Therapeutics 14: 183191. Lan RS, Stewart GA, and Henry PJ (2002) Role of proteaseactivated receptors in airway function: a target for therapeutic intervention? Pharmacology & Therapeutics 95: 239 257. Levi-Schaffer F and Piliponsky AM (2003) Tryptase, a novel link between allergic inammation and brosis. Trends in Immunology 24: 158161. Reed CE and Kita H (2004) The role of protease activation of inammation in allergic respiratory diseases. Journal of Allergy and Clinical Immunology 114: 9971008. Schmidlin F and Bunnett NW (2001) Protease-activated receptors: how proteases signal to cells. Current Opinion in Pharmacology 1: 575582. Sommerhoff CP, Bode W, Matschiner G, Bergner A, and Fritz H (2000) The human mast cell tryptase tetramer: a fascinating riddle solved by structure. Biochimica et Biophysica Acta 1477: 7589.

Further Reading
Cairns JA (2005) Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inammatory disorders. Pulmonary Pharmacology & Therapeutics 18: 5566. Caughey GH (2001) New developments in the genetics and activation of mast cell proteases. Molecular Immunology 38: 1353 1357.

CILIA AND MUCOCILIARY CLEARANCE

L E Ostrowski and W D Bennett, University of North Carolina, Chapel Hill, NC, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Mucociliary clearance is an innate defense mechanism that protects the pulmonary system from the harmful consequences of inhaled agents, including those of biological, chemical, and physical nature. Ciliated cells, which line the surface epithelium of the airways, provide the force necessary for mucociliary clearance by the coordinated beating of their cilia. These highly specialized cells are therefore critical to the health and function of the pulmonary system. In this article, the basic biology of ciliated cells and the structure of the cilium are described. Their function and interaction with other components of the mucociliary system and the importance of mucociliary clearance to respiratory disease are discussed.

Introduction
Mucociliary clearance is the process whereby the pulmonary system is cleared of inhaled foreign material by the continuous beating of ciliated cells to transport mucus and any entrapped material to the pharynx, where it is subsequently removed by swallowing. This well-coordinated system consists of mucous and serous cells in the submucosal glands and secretory goblet cells in the airway epithelium that secrete water, mucus, and other proteins to produce a uid layer on the airway surface and also the ciliated cells that propel the uid out of the lung toward the mouth (Figure 1). The uid layer

consists of a low-viscosity sol phase near the cells apical surface and a more viscous gel (or mucus) phase near the tips of the cilia. Whether the two phases are entirely distinct or a viscosity gradient exists through the depth of the entire airway uid is not clear. The efciency of the mucociliary clearance (MCC) system at removing airway secretions and associated trapped substances depends on three primary factors: the beat frequency and coordination of the cilia, the quantity and rheology of airway secretions derived from surface goblet cells and submucosal glands, and the periciliary uid depth that is modulated by ion transport of the airway epithelium. In health, this system is very effective at clearing mucus and associated bacteria and toxins from the lung. However, in a variety of airway diseases (e.g., chronic bronchitis, asthma, and cystic brosis (CF)) this apparatus becomes dysfunctional, leading to further exacerbation of airway inammation and obstruction. Patients with primary ciliary dyskinesia (PCD), a genetic disease characterized by abnormal ciliary ultrastructure and function, have impaired MCC that results in chronic lung, sinus, and middle ear disease, demonstrating the critical importance of properly functioning cilia to the health of the pulmonary system.

Ciliated Cells and Cilia

Ciliated cells, which provide the force necessary for MCC, are found throughout the trachea and large

CILIA AND MUCOCILIARY CLEARANCE 467

Mucin granules

Ion channels

Cilia

Mucus

Periciliary liquid Cl Na+ Ciliated cell

Goblet cell

Basal cell Basement membrane

Figure 1 Schematic of airway epithelium illustrating the two major cell types, ciliated and goblet. The epithelium is covered by a uid layer whose depth is regulated by ion transport across the cell membranes and within which mucins are secreted to trap foreign matter for mucociliary transport out of the lung.

airways and are present distally as far as the terminal bronchioles. Figure 2 shows a cross section of a normal human large airway illustrating the heavily ciliated surface. The percentage of ciliated cells in the human airway epithelium decreases from approximately 50% in the tracheal epithelium to o20% in the bronchiolar region. Ciliated cells are also present in areas of the upper respiratory tract, including the nasal sinuses and nasopharynx. On the apical surface of each ciliated cell is an organized array of up to 200 individual cilia; the adult lung has been estimated to contain approximately 3 1012 total cilia. A typical cilium from an adult tracheal cell is approximately 7 mm long and 0.1 mm in diameter. Ciliated cells in the more peripheral airways have fewer cilia, and the cilia are shorter as well. Each cilium demonstrates the highly conserved axonemal structure, consisting of a central pair of microtubules surrounded by a ring of nine microtubule doublets (Figure 3). Each of the outer doublets consists of a complete tubule (the A tubule, containing 13 tubulin subunits) and a partial tubule (the B tubule, containing 11 subunits). Attached to each complete tubule are two rows of large multiprotein complexes, the inner and outer dynein arms. Each dynein arm contains at least one catalytic dynein heavy chain that converts the energy of ATP hydrolysis into the bending motion of the ciliary axoneme by attaching to the adjacent microtubule and undergoing a conformational change, causing the microtubules to move relative to each other. The detailed structure of the human cilium is only beginning to be unraveled, but the realization that defects in ciliary function are

Figure 2 Ciliated cells of the human airway epithelia. Photomicrograph of a large airway showing the epithelial surface covered by numerous cilia extending from the tall, columnar ciliated cells. The parafn-embedded large airway (bronchus) was stained with Richardsons to visualize the cilia.

responsible for a much wider spectrum of disease than previously thought has stimulated studies on the assembly, structure, and function of this fascinating organelle.

468 CILIA AND MUCOCILIARY CLEARANCE

Radial spoke Radial spoke Inner dynein arm Central pair microtubule A microtubule B microtubule Nexin link
Figure 3 (Left) Schematic of a prototypical axoneme illustrating some of the structural features that are highly conserved among motile 9 2 axonemes. (Right) Transmission electron micrograph showing a normal human cilium in cross section. Many of the conserved structures are easily visible.

Outer dynein arm

Outer dynein arm

Inner dynein arm

Regulation of Ciliary Beat Frequency

In the airway, cilia typically beat with a frequency of approximately 1015 Hz, although reported values vary slightly depending on the conditions used. Analysis of ciliary waveform from nasal samples by digital high-speed video has revealed a mostly planar pattern, with the cilium being fully extended in the forward power stroke. During the recovery stroke, the cilium curls backwards in the same plane. Figure 4 shows an image from a video recording of an actively beating ciliated cell. During the recovery stroke, the cilium curls backwards in the same plane. The regulation of ciliary beat frequency (CBF) has been an area of research interest for many years, and many different agents have been reported to stimulate or inhibit CBF. For example, increases in the intracellular concentration of cAMP consistently stimulate CBF in airway epithelial cells. This increase is presumed to be due to the phosphorylation of axonemal proteins by the action of cAMP-dependent protein kinase (PKA). PKA has been demonstrated to be an integral component of the ciliary axoneme; however, the regulatory protein responsible for the increased CBF has yet to be identied. Agents that increase intracellular calcium also elevate CBF in mammalian airway cells, although, again, the detailed molecular pathway has not been elucidated. Other regulatory molecules, including cGMP, PKG, protein kinase C, calmodulin, phospholipase C, and nitric oxide, have all been reported to have effects on ciliary beating, although the different experimental systems used have sometimes produced conicting results. It seems likely that the regulation of CBF occurs through the interaction of several pathways, and more research is required to determine the individual contributions of each to the overall regulation of CBF. For effective MCC to occur, ciliary beating must also be coordinated. Cilia in the airway

Figure 4 Image of an actively beating human ciliated cell. Ciliated cells isolated from human bronchial tissue were visualized using differential interference contrast optics and high-speed video images were recorded.

beat in a metachronal wave, and, again, the mechanisms that initiate and maintain the proper orientation and coordination between the ciliated cells are unknown.

Measurement of Mucociliary Clearance

MCC rates can be measured in vivo by assuming that a nonpermeating, inhaled marker depositing on the airway surface moves out of the lung at the same rate as the airway secretions in which it is immersed. The most common technique is to use inhaled, radiolabeled particles, aqueous or dry, that upon deposition in the lung can be followed by a gamma camera or scintillation detectors to determine their rate of egress from the lung. Using these techniques, scientists have been able to study MCC in healthy subjects and in patients with airways disease and to assess

CILIA AND MUCOCILIARY CLEARANCE 469

the effects of various therapies on MCC. The rate of tracheal mucus velocity in healthy controls has been estimated to be 4 or 5 mm min 1 using this technique, with slower rates reported in the more distal airways. Increases in CBF have been shown to translate into increased rates of clearance, and it has been reported that a 16% increase in CBF resulted in a 450% increase in tracheal mucus velocity. Thus, agents that increase CBF increase MCC and can provide benets to patients with a range of airway diseases.

Mucociliary Clearance in Pulmonary Disease

A number of short-acting b-adrenergic agonists (salbutamol, isoproterenol, and albuterol) intended to ease bronchoconstriction in patients with airways disease have also been shown to enhance the rate of MCC from the lung. Figure 5 illustrates this effect in a healthy subject. Figures 5(a) and 5(c) show the

gamma camera images of a healthy persons lungs immediately after inhalation of such radiolabeled particles. Figure 5(b) shows the gamma camera image 30 min after depositing the radiolabeled particles and no intervention (i.e., a control measure of MCC), illustrating a mild loss of image intensity during the 30-min period (i.e., in comparison to Figure 5(a)). On the other hand, Figure 5(d) illustrates a 30-min posttreatment gamma camera image in the same individual after inhaled albuterol treatment, showing more rapid movement of radiolabeled particles out of the lung compared to the control condition (compare Figures 5(b) and 5(d)). This enhancement in MCC is likely due to increases in CBF induced by the b-adrenergic agonist and consequent increases in intracellular cAMP discussed previously, although it is clear that these agents have pleiotropic effects. Table 1 provides a list of other pharmacological agents that can stimulate or depress in vivo MCC. Although the mucociliary apparatus in the normal lung responds quite well to pharmacological stimulation, it is less responsive in patients

Figure 5 Stimulation of mucociliary clearance by a b-adrenergic in a healthy subject. Top panels illustrate clearance associated with no intervention. (a) A gamma camera image of the lungs immediately after inhalation of radiolabeled particles (i.e., particle deposition image). (b) Thirty-minute postdeposition image. Bottom panels illustrate clearance in the same subject associated with inhaled albuterol treatment. (c) Particle deposition image that was immediately followed by inhaled albuterol treatment. (d) Thirty-minute postdeposition (and treatment) image showing increased clearance of the radiolabeled particles.

470 CILIA AND MUCOCILIARY CLEARANCE

Table 1 Factors affecting mucociliary clearance Factor Increase Decrease Anesthetics Atropine Antigen

Conclusions
MCC is a remarkably effective mechanism for maintaining pulmonary health. Impaired MCC is a common feature of many airway diseases, and agents that improve MCC are benecial in the treatment of these diseases. Many of these agents have a direct effect on ciliated cells and increase CBF. However, further research is needed to understand the complex interactions between cilia, airway surface liquid, mucus, and MCC so that more effective treatments may be developed.

Pharmacological Adrenergic agonists Cholinergic agonists Histamine ATP Amiloride Hyperosmolar solutions Pathological Pseudohypoaldosteronism Chronic cough

Cystic brosis Primary ciliary dyskinesia Chronic bronchitis Asthma Respiratory infections Chronic exposure to Sulfur dioxide Cigarette smoke Ozone NO2

Acknowledgments
The authors thank R Wonsetler, Dr P Spears, Dr M Chua, and the Hooker Microscopy Facility for preparing the ciliated cell video; K Burns and the histology core for excellent technical services; L Brown for assistance in preparing the gures; Dr S H Randell and the Cell Culture Facility for samples; and the individuals who donated tissue specimens.
See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Environmental Pollutants: Overview. Mucins. Mucus. Primary Ciliary Dyskinesia.

Environmental

Acute exposure to Sulfur dioxide Cigarette smoke Ozone

with airways disease. This may be due in part to the loss of ciliated epithelium associated with chronic inammation (e.g., patients with chronic obstructive airway disease who have smoked cigarettes most of their lives). Other pathological conditions for which MCC is known to be depressed are also listed in Table 1. In support of the need to hydrate mucus for effective MCC, patients with pseudohypoaldosteronism, a rare inherited disease characterized by poor Na absorption from the airway surface, have MCC rates three or four times higher than normal. The increased rate of clearance in these patients may be due to enhanced ciliary beating in response to an increased low-viscosity load on the cilia. In contrast, the airway surfaces of CF patients are poorly hydrated, leading to decreased MCC and promoting the formation of adherent mucus plaques on CF airway surfaces, ultimately causing airow obstruction and promoting chronic infection. Finally, a number of environmental pollutants can inuence the rate of MCC, either acutely or chronically, and are also listed in Table 1. For example, although sulfur dioxide may acutely stimulate MCC, chronic exposure to it causes a depression in MCC. This is likely due to a remodeling of the airway epithelium (i.e., a loss of ciliated cells and increase in mucus-producing cells) similar to that seen with chronic cigarette exposure.

Further Reading
Bennett WD (2002) Effect of b-adrenergic agonists on mucociliary clearance. Journal of Allergy and Clinical Immunology 110: S291S297. Bennett WD, Noone PG, Knowles MR, and RC Boucher (2001) Regulation of mucociliary clearance by purinergic receptors. In: Salathe M, et al. (eds.) Cilia and Mucus: From Development to Respiratory Defense. New York: Dekker. Brody SL (2004) Genetic regulation of cilia assembly and the relationship to human disease. American Journal of Respiratory Cell and Molecular Biology 30(4): 435437. Knowles MR and Boucher RC (2002) Mucus clearance as a primary innate defense mechanism for mammalian airways. Journal of Clinical Investigation 109(5): 571577. Noone PG, Leigh MW, Sannuti A, et al. (2004) Primary ciliary dyskinesia: diagnostic and phenotypic features. American Journal of Respiratory and Critical Care Medicine 169(4): 459467. Ostrowski LE, Blackburn K, Radde KM, et al. (2002) A proteomic analysis of human cilia: identication of novel components. Molecular & Cellular Proteomics 1(6): 451465. Robinson M and Bye PT (2002) Mucociliary clearance in cystic brosis. Pediatric Pulmonology 33(4): 293306. Snell WJ, Pan J, and Wang Q (2004) Cilia and agella revealed: from agellar assembly in chlamydomonas to human obesity disorders. Cell 117(6): 693697. Wanner A, Salathe M, and ORiordan TG (1996) Mucociliary clearance in the airways (state of the art). American Journal of Respiratory and Critical Care Medicine 154: 18681902.

CLARA CELLS 471

CLARA CELLS
B R Stripp and S D Reynolds, University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Clara cells are an abundant population of nonciliated secretory cells present within the bronchiolar epithelium of the mammalian lung. Their differentiation during lung development coincides with changes occurring within airways in preparation for adaptation to an air-breathing environment. Clara cells fulll three major functions in the adult lung. First, they represent the major secretory cell of the bronchiolar epithelium where they contribute nonmucinous secretory proteins to the extracellular lining uid. Second, Clara cells represent the principal site of xenobiotic metabolism within the lung. Finally, Clara cells represent an abundant progenitor cell pool for maintenance of the epithelium in the steady-state and for regeneration of the injured bronchiolar epithelium. Altered Clara cell function is likely to be a signicant factor contributing to declining lung function in airway disease. Remodeling of the conducting airway epithelium in the setting of chronic lung disease involves changes in Clara cell function that have been demonstrated through measurement of reduced production of the most abundant secretory product of Clara cells, CCSP. Roles for Clara cells and their secretions in airway homeostasis and responses to environmental stress have been demonstrated in studies involving genetically modied mouse models. Clara cells have been shown to play a central role in cytokine-mediated initiation of airway remodeling, and animal models of reduced Clara cell secretion have been shown to phenocopy pathophysiological outcomes observed in patients with chronic lung disease. New insights into functions for Clara cells in airway homeostasis point to central roles for this cell type in the regulation of airway inammation and protection from environmental agents.

Structurally and functionally distinct regions within the conducting airway epithelium are recognized as epithelialmesenchymal trophic units. Each of these units is composed of regionally specic epithelial (surface epithelium and submucosal glands), interstitial (basement membrane zone, broblasts, smooth muscle, cartilage and vasculature), neural (afferent and efferent nerves), and immunological (static and migratory immune system cells) tissues. Epithelial cell differentiation is likely to be inuenced by tissue interactions within the trophic unit as well as by interactions between adjacent trophic units. Therefore, the epithelium is heterogeneous in the steady state and exhibits a degree of functional plasticity in response to acute or chronic perturbation. As indicated by the title of this article, the following discussion will focus on the principal secretory cell of the bronchiolar epithelium, the Clara cell, and its role in distal airway homeostasis. Similarities and distinctions between the Clara cell and the dominant secretory cell subsets of more proximal airways will be discussed and the terminology describing the classical bronchiolar Clara cell and Clara-like cells of proximal airways will be rened.

Secretory Cells of Conducting Airways

The structural and functional distinctions that dene regions within the conducting airways are reected in the phenotypic properties of the dominant secretory cell type within each region. Accordingly, three categories of secretory cells were originally described in terms of their ultrastructural characteristics: serous cells, neuroendocrine cells (also termed dense-core granulated cells, endocrine cells, or Kultchitshy cells), and nonciliated secretory cells. Functional differences were inferred from these morphological observations and assessed in vitro using partially puried cell populations. This body of work rened suspected distinctions among the three subpopulations of secretory cells and provided support for subdivision of the nonciliated secretory cell population into bronchiolar Clara cells and tracheobronchial Clara-like and mucus cells.
Serous Cells

Introduction
Conducting airways of the lung function as the primary interface between the environment and the vast mucosal surface of the alveolus and as such are a critical component of homeostatic mechanisms aimed at maintenance of a relatively clean and sterile intrapulmonary compartment. Functions of the airway include establishment of a physical barrier to environmental agents, ltering and conditioning of inhaled air, metabolism of reactive chemicals, as well as clearance of deposited material and cellular debris. Although these activities are shared by the airway as a whole, regional specialization has evolved to account for differences in deposition of dusts and particulates, soluble and reactive oxidant gases and microorganisms, and to allow for rapid adaptation to changes in environmental conditions.

Serous cells are characterized by an electron-dense cytoplasm, discrete electron-dense granules that are 600 nm in diameter, and an abundance of both rough and smooth endoplasmic reticulum. These

472 CLARA CELLS

cells secrete neutral mucin and have been consistently described in submucosal glands, small bronchi, and bronchioles of the human airway and rat, and variably in cat and hamster. Although mice lack morphologically distinct serous cells, expression of the serous cell marker SPURT (also termed PLuNK) in tracheobronchial secretory cells suggests functional, if not phenotypic, redundancy between the human serous cell and mouse proximal secretory cell types.
Neuroendocrine Cells

conducting airway and from neuroendocrine cells. These studies indicated that the term Clara cell was a useful collective term to describe nonciliated bronchiolar secretory cells.
Tracheobronchial Clara-Like Cells

Neuroendoreine cells are an extremely rare cell type distinguished by a large number of small spherical dense core granules each of which is surrounded by an electron-lucent halo. These cells are found singly or in clusters (termed neuroepithelial bodies), are often innervated, secrete neuropeptides, and have been implicated as oxygen sensors and as a niche for maintenance of bronchiolar tissue-specic stem cells in the mouse. Hyperplasia of neuroendocrine cells and neuroepithelial bodies is a common pathological nding of neonatal and chronic lung disease and is mimicked following acute injury of mouse airways.
Bronchiolar Clara Cells

Secretory cells of the bronchiolar epithelium are referred to as Clara cells, named after Max Clara who provided a detailed description of their morphological properties in 1937. In the light of microscopic study, the Clara cell was portrayed as a cuboidal, nonciliated cell, of the human and rabbit terminal bronchiole that contained a basally situated nucleus, an apical dome that extended a variable distance into the airway lumen, and discrete, oval, densely staining granules. Subsequent ultrastructural analysis detected electrondense, 500600 nm diameter granules in humans. More detailed ultrastructural analysis of nonciliated bronchiolar epithelial cells of the mouse and rat lung detected abundant smooth endoplasmic reticulum and secretory granules located within the apical domain of the cell. Mouse Clara cells were further distinguished by their large round mitochondria that contained few poorly dened cristae and a relatively electron dense mitochondrial matrix. Comprehensive analysis of nonciliated bronchiolar cells in 15 mammals, including man, identied the presence of smooth endoplasmic reticulum (except nonhuman primates) and ovoid secretory granules (except rhesus monkey and cat) as unifying characteristics of Clara cells from multiple species. Although morphometric analysis demonstrated heterogeneity both within and among species, the organellar composition of nonciliated bronchiolar cells clearly distinguished these cells from mucus and serous cells of the more proximal

Morphological analysis of the tracheal epithelium from multiple species demonstrated that 5690% of nonciliated cells exhibited ultrastructural characteristics of Clara cells. Subsequent use of the molecular marker, Clara cell secretory protein (CCSP), to dene secretory cell populations at all airway levels led to the perception that nonciliated secretory cells of the tracheobronchial and bronchiolar epithelium were synonymous. However, the nding that CCSPexpressing cells were a subset of nonciliated/nonmucus cells in the human tracheobronchial epithelium and subsequent identication of secreted proteins unique to tracheobronchial nonciliated/nonmucus cells of both humans and mice suggested that structurally similar cells within the two compartments were functionally divergent. These molecular distinctions in combination with differential responsiveness of tracheobronchial and bronchiolar compartments to various stimuli, early segregation of the tracheobronchial and bronchiolar lineages during lung development and identication of separate hierarchies for maintenance of these two populations in the mature epithelium, led to use of the term Clara-like to describe nonciliated/nonmucus cells of the tracheobronchial epithelium. As summarized by Plopper and colleagues, the original description of the Clara cell dened a morphological entity (nonciliated cell) in a precise location (the terminal bronchiole) of two species (human and rabbit). The term Clara-like cell was developed to highlight the similarities between the bronchiolar and tracheobronchial nonciliated/nonsecretory cell and the potential for distinct functions of these populations in airway homeostasis.
Mucus Cells

Mucus cells are found primarily in the proximal airways of healthy humans and are characterized by an electron-dense cytoplasm that contains a heterogeneous population of electron-lucent conuent granules that are 800 nm in diameter, numerous Golgi complexes, and granular endoplasmic reticulum (rough endoplasmic reticulum). These cells secrete high-molecular-weight glycoprotein that is acidic in nature due to sialic acid or sulfate groups. The number of mucus cells increases with acute lung infection or injury and these cells become hyperplastic and are distributed to more distal compartments of

CLARA CELLS 473

the chronically diseased human lung. Comparative analysis of secretory cell populations in six mammalian species demonstrated that representation of mucus cells was highly variable among the species studied (020%). Few mucus cells were found in airways of rodents maintained under specic pathogen-free conditions but were a prominent component of the acute response to irritants and environmental pollutants in rats. Interestingly, mouse proximal airway secretory cells are refractory to such stimuli. However, metaplastic transition to a mucus-producing phenotype was associated with overexpression of Th2 cytokines. This analysis suggested that differential responsiveness of tracheobronchial cells to immune system factors could account for the high degree of variation in mucus cell numbers and may represent a critical adaptive response of proximal airway secretory cells to changing environmental conditions. Analysis of allergic inammation in mice also supported the idea that mucus cells were derived from a nonmucus producing cell and that expression of high-molecular-weight glycoconjugate was a gainof-function phenotype. The potential for phenotypic plasticity identied the need for molecular markers that could distinguish functionally distinct secretory cell types at the light microscopic level.

Antibodies specic for CCSP have been used to determine the number and distribution of CCSPimmunoreactive (CCSP-IR) cells in humans and commonly used animal models. Studies in humans, rats, and mice demonstrated that the majority of Clara cells expressed CCSP and that these cells were negative for periodic acid Schiff (PAS)-reactive material in normal individuals. These data suggested that CCSP was a faithful marker for human bronchiolar Clara cells and similar immunological proles of distal conducting airway secretory cells from multiple species indicated that expression of CCSP was a common molecular characteristic of secretory cells of this type. In contrast, the frequency of CCSPIR cells in the tracheobronchial epithelium of human, rat, and mouse airways was less than that of Claralike cells. Identication of subsets of tracheobronchial Clara-like cells provided further support for the notion that distinct populations of nonciliated secretory cells populate the proximal and distal airways and identied a need to further delineate these distinctions at the molecular level.

Properties of Clara Cells in the Normal Adult Lung

Early studies describing the ultrastructural properties of Clara cells provided a number of clues into their potential roles in airway biology. Abundant mitochondria and smooth endoplasmic reticulum suggested that they were a metabolically active cell, and the presence of membrane bound granules suggested a secretory function. Since then, a combination of in vivo and in vitro models has been employed to further explore these and other functions of Clara cells.
Secretion

Molecular Markers for Clara and Clara-Like Cells

Products of airway secretory cells were initially identied through immunization of rabbits with lavagable proteins from adult rat and correlation of immunoreactive antigens with cellular morphology in bronchioles and bronchi. Subsequent metabolic labeling of cell-associated and released proteins from partially puried rabbit Clara cells identied proteins of 6, 48, and 180 kDa as major products of this cell type and demonstrated that the 6 kDa protein represented 30% of total released radioactivity. A similar preparation of the low-molecular-weight protein was used to generate an afnity-puried antibody preparation and immunogold methods demonstrated that this antigen localized to secretory granules within rat Clara cells. These methods were also applied to identication of markers for human Clara cells and resulted in discovery of a homodimeric 10 kDa protein with a pI of 4.8 that was antigenically similar in human, dog, and rat. This protein, which is termed CCSP, CC10, CC16, uteroglobin, or SCGB1A1, has since been identied as the human ortholog of rabbit uteroglobin and is the founding member of the secretoglobin family of low-molecular-weight, acidic, secretory proteins.

In rodents, secretions from Clara cells account for approximately 10% of the protein content of total airway surface uid recovered by bronchoalveolar lavage and represent a much larger percentage of protein within the surface uid of conducting airways. This changes both qualitatively and quantitatively the setting of lung injury not only due, in part, to altered lung permeability resulting in the inltration of serum proteins, but also as a result of injuryrelated changes in the rate at which the secretory proteins synthesized and secreted. Even though it is clear that Clara cells constitutively secrete low-molecular-weight proteins into the lumen of airways, it is less clear whether proteins loaded into secretory granules are part, of this constitutive secretory pathway or represent a distinct pool whose secretion is regulated by extracellular cues. Evidence supporting

474 CLARA CELLS

a regulated exocytic process comes from studies investigating ultrastructural changes occurring to Clara cells following either infection with pneumotropic viruses or injury resulting from exposure to oxidant gases. In each case, the ensuing perturbations to airways result in rapid loss of Clara cell secretory granules suggesting the rapid exocytosis of stored pools of secretory proteins. Clara cells secrete a number of nonmucinous secretory proteins including surfactant proteins A and D, proteolytic enzymes, antimicrobial peptides, secretoglobins, and a variety of growth factors, chemokines and cytokines. CCSP is the most abundant secreted product of the Clara cell, accounting for up to 40% of the secreted protein product. Clara cells of CCSP-decient mice (CCSP / ) exhibit ultrastructural changes including loss of secretory granules and a dramatic reduction in the abundance of rough endoplasmic reticulum, that are associated with defects in airway secretion. The phenotype of CCSP / mice has been extensively investigated using a variety of challenge models and these studies have associated CCSP deciency with alterations in the pulmonary response to inammatory stimuli, changes in mucosal immunity, and elevated susceptibility to inhaled oxidant pollutants. The nding that the response of CCSP / mice following challenge with environmentally relevant agents phenocopies aspects of the respiratory complications seen in human patients suffering from chronic lung disease highlights a critical role for Clara cell secretion in lung homeostasis and disease.
Metabolism

generation of water-soluble glutathione adducts that can be efciently efuxed from the cell through the action of plasma-membrane-associated glutathione transporters. Clara cells are well situated to effectively clear lipophilic xenobiotic compounds delivered to the lung either through inhalation or systemically via the extensive parenchymal capillary network. The physico-chemical properties of inhaled lipid-soluble xenobiotic compounds result in deposition deep within airways of the lung. Generally, these compounds are present within inspired air at relatively low concentrations and can be effectively metabolized and cleared without overt lung injury. In contrast, systemically delivered agents, either xenobiotic pollutants or drugs, can be delivered to the lung at sufciently high concentrations to overcome the capacity of the phase II metabolizing system leading to Clara cell injury from excessive production of bioactivated products of phase I metabolism. Clara cell injury resulting from exposure to high doses of lipophilic compounds has been extensively characterized in the laboratory of Plopper and colleagues and involves dilation of the sER, formation of large cytoplasmic vacuoles and the generation of cytoplasmic blebs at the apical membrane. Differences are observed in the regio-selectivity of this injury response, with Clara cells of the bronchiolar epithelium exhibiting the greatest susceptibility. Signicant species differences in susceptibility are also observed, the principal mechanism for which is variability in the substrate specicity and expression levels of phase I metabolizing enzymes. Mechanisms of repair following Clara cell injury by lipophilic compounds will be discussed in more detail below.
Proliferation

Both the metabolic capacity of Clara cells and their location within the bronchiolar epithelium of distal conducting airways are central to their role in protection from the adverse effects of environmental lung pollutants. As is the case with hepatocytes, which are responsible for detoxication of lipophilic xenobiotic compounds including furans and aromatic hydrocarbons in the liver, Clara cells are rich in smooth endoplasmic reticulum (sER) and mitochondria and are the principal site of expression for cytochrome P450 monooxygenase family. Such enzymes are referred to as phase I metabolizing enzymes and localize to the sER fraction of intracellular membranes. Phase I metabolism of lipophilic compounds results in the generation of highly reactive intermediates that undergo secondary reactions catalyzed by the phase II class of xenobiotic metabolizing enzymes. Phase II reactions within Clara cells are principally catalyzed by enzymes belonging to the family of glutathione transferases, resulting in the

The bronchiolar epithelium is relatively quiescent in the steady state but has the capacity for rapid and complete regeneration following acute injury. Careful pulse/chase studies coupled with autoradiography and ultrastructural analysis demonstrated that normally quiescent Clara cells responded to oxidant-induced ciliated cell injury through transition to a cell type referred to as the type A Clara cell, which was distinguished from the Clara cell by virtue of its lack of secretory granules and smooth endoplasmic reticulum. These morphologically distinct cells entered the cell cycle and produced nascent Clara and ciliated cells at a ratio of 2:1. Re-differentiation of this transitional epithelium was associated with the appearance of a type B cell intermediate, distinguished from the type A cell by the presence of granules and from Clara cells by an absence of sER, which most

CLARA CELLS 475

likely represented an intermediate cell type in the reestablishment of the mature Clara cells. Subsequent pulse-labeling studies demonstrated tight correlation between DNA synthesis and expression of CCSP in cells of the steady-state and regenerating bronchiolar epithelium. Thus, mature Clara cells, which fulll a range of differentiated functions that are critical to aspects of airway homeostasis (detailed above), serve as a reservoir of abundant bipotential progenitors that mediate bronchiolar maintenance and regeneration through proliferation and differentiation.

Bronchiolar Stem Cell Hierarchy

Clara cells are part of a stem cell hierarchy that contributes to maintenance and renewal of the bronchiolar epithelium. Stem cell hierarchies have been described that contribute to maintenance of epithelia within a number of different tissues such as the small intestine and colon, skin, cornea, and hematopoietic system. Components of such hierarchies include a tissue-specic stem cell, one or more types of transitamplifying progeny, and terminally differentiated cells. Tissue-specic stem cells are a rare population of cells that are maintained within protective (either physically or biologically) microenvironments referred to as niches, and have properties of unlimited capacity for self-renewal, infrequent proliferation in the steady-state, and a relatively undifferentiated phenotype. Tissue-specic stem cells give rise to transit-amplifying progeny, frequently referred to as progenitor cells, the properties of which can vary signicantly according to tissue. Among tissues that maintain classical stem cell hierarchies, such as the intestine, transit-amplifying cells represent a continuously proliferating population that is maintained in close proximity to the stem cell niche. In such cell hierarchies, transit-amplifying cells have a nite capacity for self-renewal and eventually expend their mitotic potential. As transit-amplifying cells of classical stem cell hierarchies exit the proliferative compartment, they assume differentiated characteristics necessary for epithelial functions. Such differentiated cells have a short life span and as a result the transitamplifying cell compartment is continually activated. The existence of lung tissue-specic stem cells has been revealed using mouse models of airway injury in which proliferative cells have been selectively ablated using either chemical or conditional transgenic approaches. Parenteral administration of high does of lipophilic agents such as naphthalene causes selective injury to Clara cells as a result of xenobiotic metabolism. In contrast with epithelial regeneration following oxidant-mediated injury to ciliated cells, repair following Clara cell depletion originates from

two types of regenerative foci within bronchioles: one dened by virtue of clusters of pulmonary neuroendocrine cells that form neuroepithelial bodies (NEBs) and the other localized to the broncho-alveolar duct junction (BADJ) of terminal bronchioles. Pulse/chase studies demonstrated that a subpopulation of mitotic cells within these microenvironments exhibited characteristics of tissue stem cells, including infrequent proliferation and capacity for multipotent differentiation. The molecular phenotype of the bronchiolar tissue stem cell was revealed through conditional ablation of CCSP-expressing cells in transgenic mice. These experiments demonstrated that the CCSP-expressing component of the NEB and BADJ microenvironments, the variant CCSP-expressing cell (vCE), constituted the bronchiolar tissue-specic stem cell. A role for vCE cells in bronchiolar regeneration as well as secondary functions relating to interactions between the bronchiolar and alveolar epithelium was demonstrated by the nding that irreparable bronchiolar injury resulted in death due to compromised parenchymal function. The combination of Clara cell depletion and ablation approaches demonstrated that regeneration of the bronchiolar epithelium was entirely dependent on CCSP-expressing cells and that all proliferative cells of the bronchiolar stem cell hierarchy, including the stem cell and progenitor cell pools, share the common molecular characteristic of CCSP expression. As such, CCSP-expressing cells represent at least two functionally distinct populations within bronchioles of the mouse airway, each of which is linked as part of a common lineage. The presence of the large, uniformly distributed Clara cell population that fullls both differentiated and proliferative functions is of considerable practical importance in airways of the lung due to the need for efcient epithelial regeneration in the setting of acute airway injury. Incorporation of both maintenance and repair functions in a single cell type (the Clara cell) distinguishes the bronchiolar epithelium from other epithelia (such as the intestine and epidermis) which rely on distinct populations of cells to fulll these activities. Furthermore, the ability of the Clara cell to transition between the quiescent and proliferative states distinguished this cell from other differentiated bronchiolar cell subsets that lack this capacity. Recognition of dual roles for Clara cells in bronchiolar homeostasis and regeneration, and distinctions between the functional capacity of Clara cells and progenitor cells of other epithelial hierarchies, has led to use of the term conditionally differentiated to describe bronchiolar Clara cells. In contrast, the term terminally differentiated has been used to describe differentiated cells, such as ciliated cells, that lack

476 CLARA CELLS

mitotic potential. Mechanisms that regulate maintenance and provisional activation of the dispersed conditionally differentiated Clara cell population are likely to be distinct from those governing the activity of spatially distinct progenitor and differentiated cell pools of the gut and skin. The presence of the conditionally differentiated Clara cell population results in limited participation of the bronchiolar tissuespecic stem cell in routine epithelial maintenance.

Clara cells in Airway Disease

Histopathology and Biomarkers

Injury and remodeling of the conducting airway is a characteristic pathological feature of many types of chronic lung disease including asthma, chronic obstructive pulmonary disease (COPD), obliterative bronchiolitis, idiopathic pulmonary brosis, and cystic brosis. Changes to airways are multicompartmental, with variable contribution made by tracheobronchial and bronchiolar airways, and within these regions affecting both epithelial and mesenchymal components. Epithelial involvement has been demonstrated upon histopathological examination of lung tissue from affected patients. Contributions of the bronchiolar epithelium to the pathophysiology of airway disease are further supported by studies characterizing changes in the abundance of cell type-specic biomarkers such as CCSP within airway lining uid and serum. Due to its low molecular weight, CCSP penetrates both the epithelial and endothelial barrier of the lung resulting in serum levels that are predictive of changes to the lung. Factors that inuence serum levels of such noninvasive biomarkers include their rate of synthesis and secretion within the epithelium, integrity of both the epithelial and endothelial barrier, and the glomerular ltration rate which serves to clear and catabolize these proteins. Serum levels of CCSP increase dramatically immediately following acute lung injury due to changes in the permeability of the epithelial/endothelial barrier. However, serum CCSP content decreases in the setting of chronic lung disease due to reduced synthesis and/or secretion that results from Clara cell depletion or injury. Although changes in CCSP levels certainly reect the status of Clara cells and the bronchiolar region, changes in the integrity of more proximal airways may also inuence intrapulmonary and serum levels of this biomarker.
Functional Signicance in Disease Pathophysiology

implicated from both the histopathology and studies performed using primary cultures of airway epithelium, but have been unequivocally demonstrated in studies involving use of genetically modied mouse models. Primary cultures of human bronchial epithelium can be prepared from cells recovered by bronchial brushing. Studies comparing such cultures derived from normal subjects versus subjects with chronic lung disease have demonstrated diseaserelated differences in their functional properties that are intrinsic to the epithelium. However, even though cultures of bronchial epithelium may be reective of events occurring within bronchiolar airways, the inability to sample epithelium from smaller caliber bronchiolar airways for establishment of representative cultures makes direct analysis impossible. Direct demonstration of a role for Clara cell injury and/or loss in chronic lung disease can be demonstrated in mouse models in which Clara cells can be either selectively ablated or genetically modied. Conditional ablation of CCSP-expressing cells using a regulable transgenic mouse model results in progressive lung inammation and increased lung permeability similar to that observed with acute respiratory distress syndrome. Direct roles for altered Clara cell secretion in modulation of airway disease are suggested from studies using CCSP as a biomarker and further supported by the spectrum of pulmonary phenotypes discussed above for CCSP / mice. However, genetic manipulation of epithelial responsiveness to T-cell cytokines such as IL-13 demonstrates a direct role for CCSP-expressing cells in mediating inuences of these cytokines on outcomes such as airway hyperreactivity and mucus cell metaplasia in airways. As such, CCSP-expressing cells, of which Clara cells represent the most abundant component, are sufcient to mediate cytokine-induced airway remodeling for initiation of the asthmatic phenotype, and represent a critical cell type whose altered function contributes to perpetuation of chronic lung disease through further immunoregulatory imbalances and the altered susceptibility to environmental pathogens and pollutants.

Ontogeny of Clara Cells

Identication of specialized subsets of secretory cells in the proximal and distal airway implied segregation of the two compartments during embryonic development. Several studies have used morphological and molecular markers as well as lineage tagging methods to assess the temporal and spatial characteristics of this process. Standard embryological approaches have demonstrated that the lung is derived from the foregut endoderm 2226 days after

Roles for changes to the bronchiolar epithelium in the pathophysiology of chronic lung disease have been

CLARA CELLS 477

fertilization in humans. The trachea forms initially as a tube and is separated from the presumptive esophagus at 30 weeks gestation. In contrast, the intrapulmonary airways are formed through a budding process in which the endodermally derived epithelium invades the splanchnic mesenchyme and undergoes dichotomous branching directed by complex epithelialmesenchymal interactions involving multiple interacting signal transduction pathways. In utero lineage tagging studies in mice clearly demonstrated specication of proximal (trachea and bronchi) and distal (bronchioles, acini, and alveoli) prior to formation of the denitive lung buds on embryonic day 9.5 and tissue transplantation experiments demonstrated determination of these lineages by embryonic day 16. Within the intrapulmonary airway epithelium, cytodifferentiation occurs in a proximal to distal wave and the rst morphologically distinct phenotype to emerge is the neuroepithelial cell followed by the ciliated cell, the Clara cell, and nally the basal cell. Immunohistochemical analysis of epithelial differentiation in humans detected foci of CCSP-IR cells located predominantly at branchpoints from the 15th week of gestation onward. Localization of CCSP-IR to neuroepithelial bodies at branchpoints suggested that communication between these two cell types initiated during human lung development. Analysis of proliferation in hamster bronchiolar airways correlated regions of high epithelial proliferation with neuroepithelial bodies suggested that trophic units in the developing airway were synonymous with airway segments. Analysis of intrapulmonary airway epithelial differentiation in mice demonstrated that undifferentiated embryonic epithelial cells co-express multiple lineage markers during the embryonic stage of lung development and that this mixed pattern of gene expression resolves to the adult cell type-specic pattern as cells differentiate during the pseudograndular to canalicular transition. Studies in rabbits demonstrated reciprocal changes in cytoplasmic glycogen content (decrease) and granule content (increase) indicative of a transition from embryonic to mature secretory products. Similarly, the volume density of smooth endoplasmic reticulum and mitochondria underwent a rapid increase suggestive of maturation in metabolic capacity that reached adult levels by 10 weeks of age. Studies in rats further supported the idea that the terminal bronchiole was the last airway compartment to differentiate and that Clara cells in this region matured as a cohort. These studies support the argument that Clara cells of the adult airway are derived from immature polymorphic precursors, but did not distinguish between stochastic processes regulating differentiation of a

eld of cells and derivation from a specic progenitor cell pool.

Relationship between Clara and Clara-Like Cells

The aforementioned studies highlighted several important distinctions between the proximal and distal Clara-like and Clara cell populations. First, these data suggested that CCSP was a faithful marker for human bronchiolar Clara cells and similar immunological proles of distal conducting airway secretory cells from multiple species indicated that expression of CCSP was a common molecular characteristic of secretory cells in this region. However, representation of CCSP-immunoreactive cells in human bronchi was too low to account for all morphologically dened Clara cells in this region and led to the suggestion that proximal human Clara cells were antigenically distinct from those of the distal conducting airway. Dissimilarity between proximal and distal secretory cells was further substantiated by the identication of messenger RNAs that encoded low-molecular-weight secretory proteins specic to proximal airway secretory cells of the human and mouse. Second, early segregation of the tracheobronchial and bronchiolar domains during lung development and identication of distinct proximal and distal secretory lineages in mice suggested that the unique functional characteristics of the proximal and distal epithelialmesenchymal trophic units were based on discrete secretory cell subsets. Third, colocalization of CCSP with neutral glycoconjugate suggested a previously unrecognized precursor/progeny relationship between Clara-like and mucus cells of the upper airway. Such metaplastic transitions have been substantiated in proximal secretory cells of the mouse and have suggested that mucus production was a gain-of-function phenotype induced by proinammatory conditions. These molecular studies dovetailed nicely with ultrastructural identication of transitional cells in the upper airway. Collectively, these studies suggested that proximal secretory cell phenotype was plastic and implied that function was inuenced by both intrinsic factors and environmental conditions. Although additional studies are needed to clarify extrinsic factors regulating proximal and distal secretory cell function, existing studies suggest that the Clara-like and Clara cell populations play unique roles in airway homeostasis and that these distinctions are partially due to differences in the origin of these two cell types.
See also: Mucus. Neurophysiology: Neuroendocrine Cells. Notch. Proteinase Inhibitors: Secretory

478 COAGULATION CASCADE / Overview Leukoprotease Inhibitor and Elan. Stem Surfactant: Surfactant Protein D (SP-D). Cells.
Kuperman DA, Huang X, et al. (2002) Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nature Medicine 8(8): 885889. Plopper CG (1983) Comparative morphologic features of bronchiolar epithelial cells. The Clara cell. American Review of Respiratory Disease 128(2 Pt 2): S37S41. Plopper CG, Hill LH, et al. (1980) Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species. Experimental Lung Research 1(2): 171180. Plopper CG, Suverkropp C, et al. (1992) Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene. Journal of Pharmacology and Experimental Therapeutics 261(1): 353363. Reynolds SD, Giangreco A, et al. (2004) Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inammation and alveolar dysfunction. American Journal of Physiology: Lung Cellular and Molecular Physiology 287(6): L1256L1265.

Further Reading
Broeckaert F, Clippe A, et al. (2000) Clara cell secretory protein (CC16): features as a peripheral lung biomarker. Annals New York Academy Science 923: 6877. Bucchieri F, Puddicombe SM, et al. (2002) Asthmatic bronchial epithelium is more susceptible to oxidant-induced apoptosis. American Journal Respiratory Cell Molecular Biology 27(2): 179185. Cardoso WV (2001) Molecular regulation of lung development. Annual Review Physiology 63: 471494. Evans MJ, Cabral-Anderson LJ, et al. (1978) Role of the Clara cell in renewal of the bronchiolar epithelium. Laboratory Investigation 38(6): 648653. Hong KU, Reynolds SD, et al. (2001) Clara cell secretory proteinexpressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. American Journal of Respiratory Cell and Molecular Biology 24(6): 671681.

COAGULATION CASCADE
Contents

Overview Antithrombin III Factor V Factor VII Factor X Fibrinogen and Fibrin Intrinsic Factors iuPA, tPA, uPAR Protein C and Protein S Thrombin Tissue Factor

Overview
P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Blood coagulation is necessary for maintaining vascular integrity, and thus for maintaining life. Nature has developed an elaborate and elegant series of controlled proteolytic events that lead to the rapid formation of a stable platelet-brin plug at the site of vascular damage. Tissue factor (TF), the trigger for coagulation, is normally restricted from contact with the circulation by being expressed largely in adventitial broblasts. Once

the vessel is compromised, the exposed TF binds escaping factor VIIa (fVIIa) molecules with high afnity to form a potent procoagulant complex on local phospholipid surfaces. This enzymecofactor complex activates both fIX and fX to initiate the coagulant response, which is subsequently rapidly amplied through numerous positive feedback reactions. This burst of coagulant activity is dampened and nally quenched by specic protease inhibitors as well as the self-regulating nature of the coagulation complexes, thus preventing the potential lethal consequences of widespread thrombosis and allowing the complex process of wound healing to begin. Vascular integrity and maintenance of hemostasis is critical to normal lung function to allow efcient and unrestricted gas exchange. Lung injury or insult can lead to exposure or expression of TF in various cell types in the lung and can circumvent many of the normal coagulation control mechanisms. The resulting unregulated or

COAGULATION CASCADE / Overview 479

dysfunctional coagulation can lead to pulmonary brosis and the progression of an inammatory response such as that observed in acute lung injury and acute respiratory distress syndrome.

Extrinsic Pathway
The extrinsic pathway is the major initiator of blood coagulation in vivo. Clotting by this pathway is triggered when circulating blood comes in contact with tissue factor (TF) (Figure 2). TF is a cell-bound transmembrane glycoprotein and nonenzymatic protein cofactor that is constitutively expressed in adventitial cells of the vasculature and is not normally in contact with the circulation. Exposure of blood to TF occurs as a result of vascular damage. Circulating molecules of the plasma serine protease factor VIIa (fVIIa) (which is always present at B1% of the total fVII) then escape the circulation and bind avidly to the exposed TF generating a potent procoagulant complex. The generated fVIIaTF complex propagates and amplies coagulation via the proteolytic activation of fIX (intrinsic pathway) and fX (common pathway). Factor VII (fVII) is a member of the vitamin Kdependent coagulation protein family, which also includes fIX, fX, prothrombin, protein C, and protein S. With the exception of protein S, which is a nonenzymatic cofactor for protein C, these proteins are synthesized in an inactive zymogen form and require activation via limited proteolysis to produce an active serine protease. Each of these vitamin K-dependent proteins contains a g-carboxyglutamic acid (Gla) domain at the N-terminus followed by two epidermal growth factor-like repeats and the protease (or catalytic) domain. The Gla domain is generated through posttranslational g-carboxylation of specic glutamic acid residues by a cellular carboxylase that uses vitamin K as a cofactor. This domain is responsible for the anionic-phospholipid-binding properties of these proteins in the presence of ionic calcium. The epidermal growth factor (EGF)-like repeats are required for cofactor binding and to some extent substrate recognition, while the protease domain is responsible for the catalytic activity of the resulting enzyme.

Introduction
In 1935, W H Howell wrote: nature had a difcult problem to solve in creating a circulating medium for the animal body that would remain entirely liquid while in the blood-vessels, but would promptly set into a gel as soon as the vessels were wounded, and the blood was in danger of being lost. Indeed, while this problem alone seems quite daunting, designing a uid that can alter its response in proportion to the insult, limit the clot to the site of damage, and terminate the process once bleeding has subsided is even more remarkable. To accomplish this, nature has developed an elaborate and highly efcient sequence of enzyme-driven proteolytic reactions that utilize numerous feedback reactions, nonenzymatic cofactors, and specic inhibitors to regulate this explosive and critical, yet potentially lethal, process.

History
Perhaps one of the earliest recorded instances of research into blood coagulation is the observation in 1666 by the Italian physician Marcello Malpighi of the existence of strands of ber in the substance of a washed blood clot. Following this initial observation, more than one century passed before the next major advancement. In the mid-1800s, the concept of an enzymatic reaction that could lead to the deposition of brin was put forth, and it was not until 1904 that the rst real scientifically based theory of blood coagulation was proposed. This theory consisted of four clotting factors (tissue thromboplastin, ionic calcium, prothrombin, and brinogen) and stood for three decades. Starting in the mid-1930s, a urry of descriptions of novel clotting factors appeared in the literature, and in 1954 the International Committee for the Standardization of the Nomenclature of Blood Clotting Factors adopted the use of Roman numerals to designate the various clotting factors, with the activated form of each indicated by the addition of a lowercase a sufx. The coagulation cascade as it is known today consists of more than 10 different clotting factors organized into extrinsic, intrinsic, and common pathways (Figure 1). Although traditionally it is still referred to as a cascade, this scheme can more accurately be described as a network, as it involves numerous interlinked reactions and feedback reactions that are all intimately involved in the tight regulation of this complex physiologic process.

Intrinsic Pathway
It has long been known that plasma will spontaneously clot upon contact with a glass surface. This reaction is triggered intrinsically by contact autoactivation of fXII (Hageman factor). The generated fXIIa in complex with high-molecular-weight kininogen then activates fXI to fXIa and coagulation ensues (Figure 1). Since deciency of fXII does not result in a hemorrhagic condition the contact activation pathway is generally not considered part of the coagulation system per se but serves as a link between coagulation, inammation, and the

480 COAGULATION CASCADE / Overview

Intrinsic pathway (amplification) XI XIa TF / PL Ca2 + IX IXa VIIIa / PL Ca2 + VIIa Ca2 + TF / PL VII Extrinisic pathway (initiation)

X Xa Va / PL Ca2 +

Prothrombin (II) Thrombin (IIa) Common pathway

Fibrinogen (I) Fibrin monomer (Ia)

Fibrin clot XIII XIIIa

Stable clot

Figure 1 The modern coagulation scheme. The modern coagulation scheme is divided into the extrinsic, intrinsic, and common pathways (indicated). Although typically thought of as separate entities, these three pathways are all interlinked by numerous feedback reactions (not shown; refer to Figure 3) and cross-reactions resulting in a complex coagulation network. In this scheme, green arrows represent proteolytic processes and blue arrows represent enzymatic conversions. Required cofactors are indicated by green ovals and are juxtaposed next to their associated reaction. PL represents anionic phospholipid and the requirements for ionic calcium are indicated by Ca2 .

complement system. In contrast, deciency of fXI results in a bleeding diathesis (albeit mild), as does deciency in either fVIII or fIX (hemophilias A & B, respectively). Thus, these three proteins comprise the contemporary intrinsic pathway.

During coagulation, fIX can be activated by either of two enzymes: fXIa or the fVIIaTF complex. Since fXIa is not present initially, it is the fVIIaTF complex from the extrinsic system that is likely responsible for the initial activation of fIX. The level of fIXa

COAGULATION CASCADE / Overview 481

Figure 2 The initiation of coagulation. Blood coagulation is triggered at the site of vascular damage when escaping molecules of fVIIa bind to TF to form the procoagulant fVIIaTF complex. TF is constitutively expressed in adventitial broblasts and does not normally come in contact with the circulation. The fVIIaTF complex generated at the site of vascular injury triggers coagulation via the proteolytic activation of fX to form fXa (arrow). Platelets (small ovals) that come into contact with the exposed collagen in the basement membrane become activated (spiked ovals). These activated platelets adhere to each other to form an initial platelet plug as well as provide additional clotting factors and an effective phospholipid membrane to support coagulation reactions. This results in the formation of a stable plateletbrin plug at the site of injury to stop the outow of blood. The large curved ovals represent red blood cells, the graycolored cells lining the blood vessel represent endothelial cells, and the brown-colored cells outside the vasculature represent TF-containing adventitial broblasts.

is subsequently amplied by fXIa that is generated later via a positive feedback reaction involving thrombin (see below). fXI is one of two coagulation enzymes that is not a member of the vitamin Kdependent protein family (the other being fXIII). Although fXI is synthesized as a zymogen and requires proteolytic activation, it neither contains a Gla domain nor EGF-like repeats. As such, fXIa does not require calcium ions or a phospholipid surface for activity. fIX is a vitamin-K dependent protein with similar structure to factors VII and X. The activation of fIX requires proteolytic cleavage at two sites to produce the serine protease form of fIXa. In order to express nominal procoagulant activity fIXa must bind to its nonenzymatic protein cofactor fVIIIa, which can be considered an essential activator. It is the fIXafVIIIa complex that is the true procoagulant enzyme. The binding of fIXa to fVIIIa occurs in the presence of calcium ions and an anionic phospholipid surface. Similar to the fVIIaTF complex in the extrinsic system, the fIXafVIIIa complex also activates fX to propagate the coagulant response. FVIII is a large glycoprotein homologous to fV (the cofactor for fX; see below). FVIII is extremely labile and highly susceptible to proteolysis. It circulates in

plasma in a noncovalent complex with von Willebrands factor, which protects it from degradation and stabilizes its activity. Although not an enzyme, fVIII requires proteolytic processing at three sites to produce the active cofactor fVIIIa. The primary activator of fVIII in vivo is thrombin, although fXa can also catalyze this reaction. fVIII activation substantially reduces its size and releases it from von Willebrands factor to produce the active cofactor as a calcium-linked heterotrimer.

Common Pathway
The activation of fX can be by either the extrinsic or intrinsic pathway and produces the identical product, fXa. Once generated, fXa binds to its nonenzymatic protein cofactor (fVa) in a calciumdependent fashion on a phospholipid surface. This complex proteolytically activates prothrombin to generate a-thrombin. As with fVIII, fV requires proteolytic activation to express nominal cofactor activity. The primary activator of fV is thrombin, although fXa can also activate fV. Both fX and prothrombin are vitamin-K dependent proteins with structural characteristics similar to other members of this family. While activation of fX

482 COAGULATION CASCADE / Overview

requires a single proteolytic cleavage, activation of prothrombin requires two cleavages resulting in the release of the N-terminal Gla domain from the molecule. Thus, although prothrombin contains a Gla domain and requires phospholipid binding for activation, thrombin lacks a Gla domain and does not exhibit a phospholipid requirement. Once generated, thrombin proteolytically processes brinogen to release brinopeptides A and B and produce insoluble brin monomer, which polymerizes into a brin mesh (clot). Thrombin also proteolytically activates fXIII whose product, fXIIIa, is a calcium-dependent transglutaminase that cross-links the initially unstable brin clot to stabilize it.

XI XIa TF / PL + Ca2 + IX IXa VIII VIIIa Amplified feedbacks X PL + V Ca2 + Va Prothrombin (II) Thrombin Xa X PL + Ca2 + VIIa VII

Platelets
Blood platelets play a pivotal role in hemostasis through formation of a platelet plug at the site of vascular damage. The platelet plug is relatively friable and can be readily dislodged if left unaided. Thus, platelet activation and function are closely linked to coagulation, which welds the friable platelet plug in place. The unactivated platelet is nonthrombogenic with a neutral phospholipid surface. Upon activation, initially by exposure to collagen in the basement membrane and later by thrombin, platelet phospholipids become scrambled to expose procoagulant anionic phospholipids. Activated platelets also release fVa and expose binding sites for fIXa and fVIIIa as well as proteins involved in platelet adhesion and aggregation.
Platelet activation
Figure 3 Positive feedback reactions. There are ve major positive feedback reactions in coagulation that serve to greatly amplify the initial coagulant stimulus: the activation of fVII, fVIII, fV, fXI, and platelets. The number of feedback reactions in which an enzyme is involved increases as one progresses down the coagulation cascade (red arrows): fVIIa is involved in one feedback, fXa is involved in two, and thrombin is involved in four. Required cofactors for each feedback reaction are indicated in the gure.

Coagulation Control
Positive Feedback Reactions and Control Mechanisms

Signal amplication is an inherent characteristic of biological enzymatic cascades. The coagulation cascade builds on this using numerous positive feedback reactions. The number of feedback reactions that clotting enzymes participate in increases as one progresses down the cascade, with thrombin involved in the most (Figure 3). The result is an explosive amplication of the initial stimulus resulting in rapid formation of a clot at the site of damage. The primary feedback reaction during coagulation is the feedback activation of fVII. This occurs via feedback of fVIIaTF or fXa and rapidly amplies the number of active fVIIaTF complexes present. The activation of fVII by fVIIa is TF-dependent and can occur on any phospholipid membrane containing TF. In contrast, the activation of fVII by fXa requires an anionic phospholipid surface and does not require TF.

The feedback activation of fVIII is largely catalyzed by thrombin, although fXa can also activate fVIII in the presence of anionic phospholipid. This latter pathway may serve to generate the critical initial molecules of fVIIIa before significant thrombin is generated. Once generated, however, thrombin is a very potent feedback activator of fVIII, fV, fXI, and platelets. Thrombins feedback activation of fV serves to amplify its own formation directly, while activation of fXI serves to amplify the whole of coagulation. The former provides a smaller but rapid amplication, while the latter provides a larger and more controlled amplication due to the numerous subsequent steps encountered before clot formation. The activation of platelets by thrombin can be considered a feedback reaction due to the prior activation of platelets by extracellular matrix proteins (i.e., collagen). Thus, thrombin activation of platelets can be envisioned as a secondary activation that results in amplied platelet plug formation progressing away from the basement membrane towards the lumen of the vessel.
Cofactors and Self-Damping Reactions

One of the critical control features in coagulation is the requirement for cofactors. FVIIa, fIXa, and fXa

COAGULATION CASCADE / Overview 483

all require protein cofactors for nominal activity. These cofactors are all nonenzymatic and act strictly as regulatory molecules. Thus, the procoagulant activity of the enzyme is largely, if not entirely, dictated by the availability and activation status of the cofactor. In the case of coagulation initiation, circulating molecules of fVIIa remain essentially inert in the absence of TF. The increase in TF availability brought about by vascular damage is the triggering event, thus linking the intensity of the response to that of the insult; large ruptures expose more TF than smaller ones. Similarly, fXa has limited activity towards prothrombin in the absence of fV. Binding of fXa to fV increases its activity to a small extent, but optimal fV cofactor activity is only observed upon its proteolytic activation. This activation is largely catalyzed by thrombin. Thus, unlike fVIIa, fXa exhibits three potential levels of procoagulant activity during coagulation: fXa o fXafV o fXafVa. The activity of fIXa is also regulated by a cofactor, fVIII. Formation of the fIXafVIIIa complex on a phospholipid surface in the presence of ionic calcium increases the procoagulant activity of fIXa by a factor of B109. Thus, fIXa is an inactive catalytic subunit that becomes active only in the context of the enzymecofactor complex. FVIII does not express any measurable cofactor activity until it is proteolytically activated, unlike fV. Thus, regulation of fIXa is entirely governed by the activation status of fVIII and the level of fVIIIa. During coagulation initiation, generation of fIXa by the fVIIaTF complex will be of no immediate consequence other than to prime the system for explosive amplication immediately upon generation of small amounts of fVIIIa. This design places a critical importance on fVIII activating enzymes and their control of fVIII activation and activity. As such, nature has placed heavy restrictions on fVIII levels and fVIIIa activity: (1) fVIII levels in plasma are remarkably low (B300 pM; the lowest level of any of the clotting factors); (2) FVIII is highly susceptible to proteolytic degradation and in fact has to be protected from degradation by circulating in a noncovalent complex with a carrier protein, von Willebrands factor; (3) once proteolytically activated, fVIIIa is extremely labile and exhibits a half-life of only 1 3 min; and (4) several enzymes (largely activated protein C) can proteolytically inactivate fVIIIa.
Inhibition and Negative Control Mechanisms

1-PI XIa TF / PL AT Heparin IXa VIIIa / PL X

IX

VII VIIa TFPI TF / PL X

APC

AT Xa

APC

Va / PL Prothrombin (II)

AT

Thrombin (IIa)

HCII Heparin

Figure 4 Inhibitors of coagulation. Inhibitors of various coagulation reactions are indicated in red with associated targets encircled. a1-PI, a1-protease inhibitor; AT, antithrombin; TFPI, tissue factor pathway inhibitor; HCII, heparin cofactor II; APC, activated protein C. Heparin is required for inhibition of fIXa by AT and thrombin by HCII.

inhibitors in plasma, the ve major coagulation inhibitors are antithrombin (formerly called antithrombin III or heparin cofactor I), heparin cofactor II, a2macroglobulin, a1-protease inhibitor (formerly called a1-antitrypsin), and TF pathway inhibitor (TFPI). Antithrombin Deciency of antithrombin results in extensive venous thrombosis, suggesting that this serpin (serine protease inhibitor) is a primary coagulation inhibitor. Owing to its unfortunate designation as antithrombin, it is often thought that its target is solely thrombin. In fact, antithrombin inhibits fXa as well as thrombin. Heparin greatly augments the ability of antithrombin to inhibit both of these proteases. While antithrombin also inhibits fIXa and fVIIa, this occurs only in the presence of heparin. In the case of fVIIa, exceedingly high, and possibly not physiologically relevant, levels of heparin are required to observe this inhibition. Heparin cofactor II In contrast to antithrombin, heparin cofactor II is a specic serpin with thrombin as its sole target. The activity of this inhibitor is not only enhanced by heparin but also by other polysaccharides such as dermatan sulfate. a2-Macroglobulin Deciency of a2-macroglobulin has yet to be described, suggesting a critical function

A second critical control feature of coagulation is the presence of multiple coagulation inhibitors in plasma, which provides an additional level of safety and regulation (Figure 4). Of the numerous enzyme

484 COAGULATION CASCADE / Overview

of this inhibitor. However, only a small fraction of fXa inhibition can be attributed to a2-macroglobulin and at most 25% of thrombin inhibition under certain conditions. This, along with the propensity of this inhibitor to react with numerous other proteases, supports the notion of a2-macroglobulin being an essential general-maintenance inhibitor and protease scavenger rather than a coagulation regulatory inhibitor per se. a1-Protease inhibitor The major plasma inhibitor of fXIa is a1-protease inhibitor, which is also a weak inhibitor of fXa. Despite this, the deciency of a1proteinase inhibitor results in emphysema and not coagulopathies. Thus, the major role of this inhibitor is probably inhibition of neutrophil elastase. TF pathway inhibitor The major plasma inhibitor of fVIIa is TFPI. TFPI is a multivalent inhibitor containing three reactive sites, each of which possesses a Kunitz-type structural fold homologous to bovine pancreatic trypsin inhibitor but with very specic and distinct specicities. The rst Kunitz domain of TFPI reacts specifically with the fVIIaTF complex while the second domain reacts with fXa and reactivity of the third Kunitz domain is as yet unclear. A unique feature of TFPI is that although it is a fairly strong inhibitor of the fVIIaTF complex it does not inhibit free fVIIa. This is significant in that it allows low levels of fVIIa to circulate freely under normal conditions to serve as a primer for immediate triggering of coagulation upon vascular injury (see above). An additional unique feature of TFPI is its ability to react strongly with the direct product of fVIIaTF activity, i.e., fXa. TFPI binds to both the fVIIaTF complex and fXa to lock these proteins together in a quaternary complex and prevent further reactions from taking place. This elegant mechanism of inhibition is different from traditional modes of enzyme inhibition and allows coagulation to be triggered while subsequently rapidly shutting down the initiation complex to prevent ongoing triggering and potential excessive thrombosis. The overall afnity of TFPI for the fVIIaTFfXa tertiary complex is quite tight and in the picomolar range, thus making TFPI by far the most specic and potent coagulation inhibitor in plasma. Activated protein C (APC) Although not an enzyme inhibitor in the classical sense, APC controls coagulation by downregulating the activity of the fIXa fVIIIa and fXafVa complexes. Instead of targeting the catalytic portion of these complexes, APC regulates their activity by proteolytic inactivation of their cofactors, fVIIIa and fVa (Figures 1 and 4). This

anticoagulant pathway involves the proteolytic activation of protein C, the precursor of APC, by thrombin bound to the endothelial cell receptor thrombomodulin. Thrombomodulin serves to alter the substrate specicity of thrombin and essentially converts it from a procoagulant to an anticoagulant. In this way, thrombin can act to stop its own generation as well as that of fXa.

Coagulation in Respiratory Diseases

In normal lung function, coagulation serves the critical purpose of maintaining the hemostatic balance as well as preserving the integrity of the pulmonary vasculature to allow efcient gas exchange. Dysfunctional coagulation in the lung can lead to the onset of pulmonary brosis, which is typically followed by respiratory failure. In addition, dysfunctional coagulation within the pulmonary artery and pulmonary microvasculature can lead to thrombosis with concomitant pulmonary injury and infarction. The delicate hemostatic balance in the pulmonary vasculature can be tipped towards a more procoagulant state by a variety of acute pulmonary insults resulting from exposure to numerous toxic chemicals as well as from the development of sepsis or pneumonia. Chronic pulmonary insults such as those observed in interstitial lung diseases can also promote a procoagulant state. The elevated procoagulant state observed in acute lung injury (ALI) and interstitial lung disease is characterized by a general increase in the levels of clotting factors and decrease in the levels of anticoagulant and brinolytic factors. These factors have been measured in bronchoalveolar lavage (BAL) and/or pleural exudates from distressed lungs, indicating a concomitant increase in vascular permeability and accumulation of plasma components in the alveolar and interstitial spaces. The activation of coagulation by TF-expressing cells in these exudates ultimately results in the observed deposition of alveolar and interstitial brin. Dysfunctional activation of the coagulation cascade in ALI is spearheaded by the increased expression of TF in intravascular and extravascular cells. While TF is normally expressed in mesothelial cells and various other lung cells, alveolar macrophages, lung epithelial cells, and lung broblasts all show increased TF expression in a bleomycin-induced lung injury model. In addition, monocytes, neutrophils, and endothelial cells can all be induced to express TF under pathophysiologic conditions in response to endotoxin and various cytokines such as TNF-a and TGF-b. This increased expression of TF produces an environment conducive to constitutive lowlevel initiation of coagulation. Although the intrinsic

COAGULATION CASCADE / Overview 485

pathway of coagulation is activated to some degree, the extrinsic pathway is accepted to be the driving force in this dysfunctional clotting resulting in elevated levels of activated factor X and thrombin, and ongoing brin deposition. While extravascular brin deposition in the alveolar and interstitial spaces is a major result of dysfunctional pulmonary coagulation, several of the clotting factors are also involved in propagating an inammatory response via cell signaling through protease-activated receptors (PARs). There are currently four identied PARs, each of which is differentially expressed on various cell types in the lung: PAR-1 is expressed in platelets, mononuclear cells, and endothelial cells; PAR-2 is expressed in mononuclear cells, endothelial cells, and neutrophils; and PAR-3 and -4 are expressed in platelets. Activation of each of these receptors occurs through proteolytic cleavage of an activation site within the extracellular domain of the receptor. The newly formed N-terminus functions as a tethered ligand, which subsequently binds to an intramolecular site to induce cell signaling through coupled heterotrimeric G-proteins. Thrombin can activate cells via PAR-1, PAR-3, and PAR-4, while the fVIIaTF complex can activate cells via PAR-2 and fXa can activate cells via PAR-1, PAR2, and the nonproteolytically activated endothelial protease receptor-1 (EPR-1). As a result of cell activation and cytokine release, TF expression is further stimulated causing a proliferation of the coagulant response. Concomitant with this increase in procoagulant potential is a reduction in anticoagulant machinery: the expression of TFPI is reduced and the inhibition potential of AT is severely restricted by cleavage of heparin-like glycosaminoglycan chains from cell-surface proteoglycans (syndecans and glypicans). In addition, thrombomodulin (a cell-surface thrombin receptor required for protein C activation) is downregulated resulting in lowered APC generation. Several activated cell types also secrete elevated levels of plasminogen activator inhibitor-1 (PAI-1), thus lowering the activity of uPA and greatly attenuating brinolysis.
Anticoagulant Treatment

above placebo in overall mortality, despite a reduction in procoagulant potential. The use of APC as a potential therapy for pulmonary brosis remains encouraging since the PROWESS trials (examining the use of APC in sepsis) have demonstrated a significant reduction in mortality in cases of severe sepsis, which is a major cause of ALI and ARDS. Taken together, these studies suggest more complex interactions and place greater import on roles of clotting factors other than in coagulation per se that may involve their cell signaling functions and the linkage between coagulation and inammation.
See also: Acute Respiratory Distress Syndrome. Anticoagulants. Bronchoalveolar Lavage. Chemokines. Coagulation Cascade: Antithrombin III; Factor V; Factor VII; Factor X; Fibrinogen and Fibrin; Intrinsic Factors; iuPA, tPA, uPAR; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. Endothelins. Extracellular Matrix: Basement Membranes. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Fibroblasts. GProtein-Coupled Receptors. Interstitial Lung Disease: Overview. Leukocytes: Monocytes. Lipid Mediators: Platelet-Activating Factors. Myobroblasts. Platelets. Pleural Effusions: Pleural Fluid, Transudate and Exudate. Pneumonia: Overview and Epidemiology. Proteinase-Activated Receptors. Pulmonary Circulation. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading
Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401404. Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) (1995) Williams Hematology, 5th edn., Part X, Chs. 118147, pp. 11491591. New York: McGraw-Hill. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148157. Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) (1994) Hemostasis and Thrombosis: basic principles and clinical practice, 3rd edn., p. 1713. Philadelphia: J.B. Lippincott Company. Davie EW and Ratnoff OD (1964) Waterfall sequence for intrinsic blood clotting. Science 145: 13101312. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM (2002) Receptors of the PAR family as a link between blood coagulation and inammation. Biochemistry (Moscow) 67: 7475. Howell WH (1935) Theories of blood coagulation. Physiological Reviews 15: 435470. Idell S (2001) Anticoagulants for acute respiratory distress syndrome: can they work? American Journal of Respiratory and Critical Care Medicine 164: 517520. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and brinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238S242.

The use of various coagulation inhibitors as therapeutic treatments for pulmonary brosis has not been clearly established and remains controversial. While experiments in animal models have demonstrated benecial reductions in ALI upon treatment with anticoagulants, these results have not been borne out consistently in human trials. Although successful in phase II trials, the use of TFPI in phase III trials (OPTIMIST) failed to show any benecial effect

486 COAGULATION CASCADE / Antithrombin III

Macfarlane RG (1964) An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplier. Nature 202: 498499. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 13341349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725.

Antithrombin III
nther and C Ruppert, University of Giessen A Gu Lung Center (UGLC), Giessen, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Antithrombin, formerly referred to as antithrombin III, is a heparin-binding protein and the major plasma inhibitor of coagulation proteases, primarily thrombin and factor Xa. In addition to its function in controlling the process of coagulation, antithrombin has potent anti-inammatory properties. This, together with the observations that coagulation contributes to inammation and that antithrombin levels are significantly reduced in patients with sepsis, provided a strong rational for antithrombin replacement therapy. In numerous animal models of experimental sepsis and several small-scale studies involving patients with sepsis, administration of antithrombin has been shown to be effective. A large prospective, randomized, doubleblind, placebo-controlled phase III trial, however, failed to show a significant improvement in overall survival. More studies are needed to answer open questions, such as those that regard the concomitant use of heparin, dosing, and the patient population that may benet from antithrombin therapy.

Introduction
Antithrombin, also referred to as antithrombin III (ATIII), heparin cofactor I, and thrombin inhibitor I, is a circulating plasma protein and the major inhibitor of thrombin, FIXa, FXa, and other serine proteases, accounting for B80% of the thrombin inhibitory activity in plasma. Antithrombin functions as a key regulator of blood coagulation. It was discovered in 1905 by the German physician Paul Oskar Morawitz, who described an inhibitory substance in plasma and serum that lowered thrombin activity. In a 1918 paper, William Henry Howell and L Emmett Holt described two new factors in blood coagulation. They coined the name heparin for a new anticoagulant originally isolated from dog liver by Howells graduate student Jay McLean in 1916, and they identied an antecedent substance in plasma and serum which is converted into antithrombin by a reaction with heparin and named it pro-antithrombin. In 1939, Brinkhouse et al. showed that the anticoagulant effect of heparin was mediated by an

endogenous plasma component that they termed heparin cofactor. Fourteen years later, Monkhouse et al. isolated a protein component from plasma displaying both heparin cofactor and antithrombin activity. They suggested that both activities may be functions of the same protein. This hypothesis was substantiated in 1968, when Ulrich Abildgaard puried ATIII from plasma and showed that this protein has heparin cofactor activity and is the main thrombin inhibitor in normal plasma. It was Abildgaard who rst showed that thrombin inhibition occurs via formation of an inactive, stable complex (1969) and thus proved older theories suggesting that the inactivation occurs enzymatically (Gerendas, 1960), with antithrombin being a substrate for thrombin (Seegers et al. 1960). In 1973, Rosenberg and Damus puried a sufcient amount of ATIII for biochemical characterization. They conrmed the formation of an irreversible equimolar complex between thrombin and antithrombin and postulated that heparin specifically accelerated this complex formation by binding to ATIII and causing a conformational change in the antithrombin molecule. The term antithrombin III has largely been replaced by antithrombin alone. The designation arose from a historical classication of mechanisms responsible for thrombin inactivation made in the 1950s: ATI was dened as the ability of brin to adsorb thrombin, ATII designated heparin cofactor activity, ATIII was used to describe plasma/serum-derived inhibitors causing a slowly progressive irreversible inhibition of thrombin, ATIV designated an intermediate acting inhibitor arising during coagulation, ATV designated an inhibitor found in pathological conditions, and ATVI was dened as the anticoagulant action of elevated brin degradation products. In recent years, other functions of antithrombin independent of its effect on coagulation have become evident, including anti-inammatory and antiangiogenic activities.

Structure
Antithrombin is a single-chain glycoprotein consisting of 432 amino acids with a molecular weight of 58 kDa that belongs to the serine proteinase inhibitor (serpin) superfamily of proteins (Figure 1). Two isoforms have been isolated from normal plasma. The a form is glycosylated at four asparagine residues (Asn96, Asn135, Asn155, and Asn192) and accounts for 9095% of the circulating protein. The b form comprises a small fraction (510%) that lacks glycosylation at Asn135. This glycosylation heterogeneity is of potential functional importance since the b isoform binds heparin with B10-fold higher

COAGULATION CASCADE / Antithrombin III 487

RCL C -sheet B -sheet D -helix Heparin pentasaccharide

A -sheet

Figure 1 Structure of antithrombin and antithrombin in complex with heparin pentasaccharide. On both structures, the A b-sheet is shown in red, the B b-sheet in green, the C b-sheet in yellow, and the D a-helix in blue. The reactive center loop (RCL) is shown in purple. Reproduced from Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, and Pike RN (2004) Antithrombin: in control of coagulation. International Journal of Biochemistry and Cell Biology 36: 386389, with permission from Elsevier.

afnity. Like other serpins (antithrombin shares approximately 30% homology in amino acid sequence with other serpins), antithrombin folds into a highly conserved secondary structure with three b-sheets (AC) and nine a-helices (AI). Disulde bonds are formed between Cys8Cys128, Cys21Cys95, and Cys247Cys430, of which the rst is required for heparin cofactor activity. The reactive center loop (RCL), a exible stretch of B17 amino acids, protrudes the main body of the molecule and contains the reactive site bond Arg393Ser394 (P1P10 bond), which is cleaved by the target protease (Figure 2). A high-afnity binding site (heparin binding site) is located on the D a-helix and involves positively charged amino acids (Arg47, Lys125, Arg129, and Arg132) that are able to interact with negatively charged sulfate groups in glycosaminoglycan chains of, for example, heparin.

Cell culture experiments employing isolated rat hepatocytes showed that antithrombin synthesis is not altered in the presence of antithrombinprotease complexes but is stimulated by the supernatant medium of LPS-treated macrophages. Following reaction with the target protease and complex formation, the proteaseantithrombin complexes are rapidly cleared from the circulation t1=2 E23 min, possibly mediated by a specic receptor on hepatocytes.

Biological Function
Antithrombin is the major inhibitor of thrombin, FIXa, and FXa in plasma, but it also inactivates other serine proteases, such as FXIa, FXIIa, plasmin, kallikrein, and the complement factor C1. Inhibition by antithrombin occurs via an irreversible suicide substrate mechanism and involves the formation of a stable 1:1 stoichiometric complex between the active site of the target protease and the reactive site of antithrombin. The protease initially recognizes antithrombin as a substrate. Cleavage of the reactive site P1P10 bond (Arg393Ser394) in antithrombin triggers a striking conformational change. The RCL inserts into the A b-sheet and transports the covalently bound protease to the opposite pole of the inhibitor. This translocation results in a deformation and thus inactivation of the protease. As a result, the protease is trapped and the attempted cleavage is stopped at the stage of an acyl intermediate.

Regulation of Production and Activity

Antithrombin is synthesized in the liver and circulates in plasma at a concentration of B150 mg ml 1, with a biological half-life of 5070 h. The gene for antithrombin (SERPINC1) is located on chromosome 1 at 1q2325.1, comprises 13.37 kb, and contains seven exons and six exons. The primary translation product is a precursor protein containing a 32 amino acid signal sequence that is cleaved prior to secretion. Posttranslational modications include disulde bond formation and glycosylation. Little is known about the regulation of biosynthesis.

488 COAGULATION CASCADE / Antithrombin III

Vascular injury Coagulation Tissue factor (TF) FVII FVII TF FIX FVIIa FX Antithrombin Kinin Bradykinin Vascular permeability contraction of SMC vasodilation Immune complexes + C1 (C1q, C1r, C1s) +C4 C1qrs +C2 C4b2a C3 C3a Complement system C3bBb Plasminogen C3b C3b attachment C3a C4b2a3b (C5 convertase) C5 C5a C5b +C7 +C8 +C9 +C6 Killing of pathogens Opsonozation of pathogens Recruitment of inflammatory cells C3b C3b attachment Fibrinolysis/ MMP-system Pro-MMP MMP Degradation of ECM FVIII FIXa PL, Ca2+ FVIIIa Prothrombin FV FXa PL, Ca2+ FVa FXIIIa Fibrinogen Fibrin Crosslinked fibrin Plasmin Two chain tPA tcuPA uPAR Fibrin split products Thrombin FXIII PAR

Kallikreinkinin-system Prekallikrein FXII Kallikrein HMWkininogen FXI

Cellular effects

FXIIa

Prostacyclin release (endothelial cells) LPS-induced release of IL-6, TF Leukocyte rolling + adhesion leukocyte recruitment Platelet aggregation SMC, fibroblast proliferation ECM production Inflammatory cell recruitment Proinflammatory mediators Profibrotic growth factors

FXIa

Activating surfaces + C3b +fB +fD

C4a C2b

C3bBb3b (C5 convertase)

C5b-C9 Membrane attack complex (MAC)

Figure 2 Overview of antithrombin targets. Plasma enzyme systems and their interaction as well as the targets of antithrombin therein and on cells are shown. Components of the coagulation system are shown in black, the kallikreinkinin system is shown in green, the brinolytic and MMP system in blue, and the complement system in magenta. The cellular response is depicted in brown. Although not being the primary target, some components of plasmatic systems may not be able to interact with other systems (dotted lines). The effects of antithrombin are indicated in red by - (activation/stimulation) or (blockade). F, coagulation factor; C, complement factor; PL, phospholipids; teu-PA, two-chain urokinase; UPAR, urokinase receptor; ECM, extracellular matrix; HMW, high molecular weight; SMC, smooth muscle cell; PAR, protease activated receptor.

Although plasma concentration is quite high (23 mM), antithrombin is a relatively inefcient inhibitor in vivo. Inhibition increases B1000 times after interaction with glycosaminoglycans, such as heparin. These glycosaminoglycans bind antithrombin via its high-afnity binding site and induce a conformational change in antithrombin, making the reactive site more accessible to the protease attack. The heparin-induced allosteric activation is mediated by a unique pentasaccharide unit in the glycosaminoglycan chain. Heparin can also function as a template to which both antithrombin and the target protease bind and that brings the reactive center of antithrombin and the catalytic center of thrombin into close proximity (so-called approximation mechanism). This complex formation involves binding to

a secondary (low-afnity) heparin binding site and requires heparin molecules containing at least 18 saccharide units. The interaction between antithrombin and heparin is of high clinical and therapeutic importance, but its physiological importance is questioned since heparin is not released from mast cells into the circulation and is not detectable in plasma. Instead, a closely related glycosaminoglycan, heparan sulfate, which is located on the extracellular matrix of the endothelial lining layer of vessels, is believed to precisely control coagulation in the vasculature. In a large number of studies, anti-inammatory properties of antithrombin have been reported that are independent from its anticoagulant activity. Proposed mechanisms include the promotion of

COAGULATION CASCADE / Antithrombin III 489

prostacyclin (prostaglandin I2) production and release from endothelial cells and the inhibition of the proinammatory signaling pathway of nuclear factor-kB, thereby inhibiting tumor necrosis factor-amediated expression of cytokines, chemokines, and adhesion molecules. A modied antithrombin (inactive form with low heparin afnity in which the cleaved reactive bond loop is inserted into the A sheet in a similar manner as in the proteinase complexes) has been shown to exert potent anti-angiogenic and antitumor activity. The underlying molecular mechanism is obscure, however. Studies with a similar latent form of antithrombin (heat-denatured antithrombin with low heparin afnity) and prelatent antithrombin (i.e., another latent form with retained heparin binding) showed that the anti-angiogenic effect was mediated by induction of apoptosis by disrupting cellmatrix interactions through inhibition of focal adhesion kinase activity and focal adhesion formation. Targeted disruption of the antithrombin gene in mice resulted in an embryonic lethal phenotype. The embryos died during mid- to late embryogenesis (approximately 15.5 gestational days) and showed extensive brin(ogen) deposition in myocardium and liver as well as subcutaneous hemorrhagic lesions, possibly due to consumptive coagulopathy and/or liver dysfunction.

Receptors
There is accumulating evidence that binding of antithrombin to the cell surface is critical to exert its anti-inammatory activity. Syndecan-4, a G-proteincoupled transmembrane proteoglycan with extracellular heparan sulfate chains, may be one cellular receptor for antithrombin.

Antithrombin in Respiratory Diseases

Antithrombin deciency may be inherited or acquired due to decreased synthesis, increased loss, or increased consumption. Inherited antithrombin deciency was the rst recognized inherited thrombophilia and is heterogeneous with respect to its genetic origin and clinical manifestation. Based on results from functional and immunological assays, deciency may be type I or type II. Type I deciency is characterized by a heterozygous mutation resulting in a 50% reduction in both antigen levels and functional activity and an increased risk for venous thrombosis. In type II deciency, antigen levels are normal, but function is reduced. This group includes mostly single amino acid mutations affecting the reactive site (variant RS, with a high risk of

thrombosis) or the heparin binding site (variant HBS, with a low risk of thrombosis). A third subgroup (PE) of dysfunctional variants has pleiotropic (multiple) effects, altering both functional activity and plasma antigen levels (B70% of control). Intravascular clot formation due to the predominance of procoagulant and suppression of brinolytic factors is a key manifestation in a variety of diseases, including atherosclerosis and septic organ failure. With regard to the pulmonary vasculature, microthrombosis is a consistent nding in patients with acute lung injury (ALI), acute respiratory distress syndrome (ARDS) of both septic or nonseptic origin, and in patients with chronic pulmonary hypertension. The microvascular bed, which represents by far the largest surface in the pulmonary vasculature and plays a crucial role in dissolution of microthrombi, is particularly affected. Activation of the coagulation system with microthrombi formation, as well as uncontrolled and overwhelming inammation and damage to the microvascular bed, contributes substantially to organ dysfunction in ARDS or septic shock. Accordingly, in patients with severe sepsis, low plasma levels of antithrombin resulting from a decreased synthesis and/or enhanced clearance by thrombinantithrombin complex formation have been found. This correlates with the development of disseminated intravascular coagulation (DIC), ARDS, and poor outcome, suggesting a possible therapeutic role for antithrombin in these patients. In various animal models of ALI, sepsis, septic shock, and DIC, administration of antithrombin attenuated the inammatory response and displayed positive effects in preventing organ dysfunction and improving survival. However, the benecial effects could not be conrmed in humans. Clinical studies investigating the efcacy of antithrombin in patients with severe sepsis demonstrated a reduction in mortality and improvement of organ dysfunction. However, the ndings did not reach statistical significance due to the limited number of patients enrolled. In the large phase III multicenter KyberSept trial, which included more than 2300 patients with sepsis, no effect on 28-day mortality was achieved. Instead, evidence of an adverse interaction with heparin was observed. Patients in the antithrombin group who did not receive concomitant low-dose heparin prophylaxis exhibited a significant improvement in 90-day mortality. Possible explanations for these adverse results are the achieved antithrombin plasma levels, which were lower than expected, and the blockade of anti-inammatory actions of antithrombin by interaction with heparin (thereby preventing interaction with cellular glycosaminoglycan receptor molecules).

490 COAGULATION CASCADE / Factor V

The inuence of antithrombin on the clinical course of ARDS or ALI has not been addressed in detail. In studies investigating patients with severe sepsis, different effects on respiratory function were observed. Whereas one study showed an apparent but nonsignificant improvement in respiratory function, others showed no reduction in lung dysfunction.
See also: Acute Respiratory Distress Syndrome. Adhesion, CellMatrix: Integrins. Anticoagulants. Coagulation Cascade: Overview; Factor X; Protein C and Protein S; Thrombin. Thrombolytic Therapy.

observed in acute lung injury and acute respiratory distress syndrome.

Introduction
In 1944, the Norwegian hematologist Dr Paul Owren discovered factor V (also known as proaccelerin or labile factor) and its deciency was termed parahemophilia. In 1954, those studying coagulation adopted the Roman numeral convention and this factor was ofcially named factor V (fV) with its activated form named factor Va (fVa). Although fV was known to be important, it was not until 1959 that the role of fV as a cofactor for activated factor X (fXa) in the common pathway of blood coagulation began to emerge.

Further Reading
Bayston TA and Lane DA (1997) Antithrombin: molecular basis of deciency. Thrombosis and Haemostasis 78: 339343. Esmon CT (2001) Role of coagulation inhibitors in inammation. Thrombosis and Haemostasis 86: 5156. Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31(supplement): S329S336. Opal SM (2000) Therapeutic rationale for antithrombin III in sepsis. Critical Care Medicine 28(supplement): S34S37. Perry DJ (1994) Antithrombin and its inherited deciencies. Blood Reviews 8: 3755. Quinsey NS, Greedy AL, Bottomley SP, Whisstock JC, and Pike RN (2004) Antithrombin: in control of coagulation. International Journal of Biochemistry and Cell Biology 36: 386389. Roemisch J, Gray E, Hoffmann JN, and Wiedermann CJ (2002) Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagulation and Fibrinolysis 13: 657670. Silverman GA, Bird PI, Carrell RW, et al. (2001) The Serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Journal of Biological Chemistry 276: 3329333296.

Structure
Gene

The gene for human fV is on chromosome 1 at position q2125. It is 72.3 kb in length and contains 25 exons and 24 introns, resulting in an mRNA of 6.9 kb (Figure 1). Exons 17 encode the A1 domain while exons 812 encode the A2 domain. These are followed by the largest exon in the gene (exon 13), which encodes the entire B domain. The A3 domain is encoded by exons 1418, and exons 1922 and 2325 encode the C1 and C2 domains, respectively.
Protein

Factor V
P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Blood coagulation factor V (fV) is a large plasma glycoprotein similar in structure to fVIII. It is synthesized as a single-chain inactive precursor in the liver and present in the circulation at a concentration of roughly 10 mg ml 1. Activation of fV occurs via ordered proteolysis at three sites by thrombin or fXa. The proteolytically activated form of fV (fVa) binds tightly to fXa in the presence of ionic calcium and an anionic phospholipid surface to produce a potent procoagulant. The fully assembled complex subsequently generates a-thrombin with a catalytic efciency 300 000-fold greater than fXa alone. FV is also secreted from activated platelets, thus helping to localize fXa activity to the site of vascular damage. As with fXa, unregulated activation or activity of fV/fVa in the lung is associated with the development of pulmonary brosis and the inammatory response

fV is a very large glycoprotein (13% carbohydrate) that is the nonenzymatic cofactor for fXa. It is synthesized as a preprotein of 2224 amino acids containing 19 half cystines (Mr 249 000). Although the liver is the major source of plasma fV, endothelial cells and megakaryocytes have also been shown to synthesize fV and the latter may contribute to the presence of fV in platelets. The structure of fV is highly homologous to that of fVIII and consists of three A domains (homologous to ceruloplasmin), a single large B domain (no known homologies), and two C domains as depicted in Figure 2. fV undergoes several posttranslational modications including glycosylation at 37 potential N-linked sites. The B domain is also unique in that it contains two conserved repeats of 17 amino acids and a large region containing 35 repeats of 9 amino acids with a consensus of [Thr, Asn, Pro]LeuSerProAspLeu SerGlnThr. Despite these features, there is no known function of the B domain other than to act as a spacer (activation peptide) to separate the heavy and light chains of fVa. Other posttranslational modications of fV include the sulfation of seven

COAGULATION CASCADE / Factor V

491

4 5 6 7 8 9 10 11 12

13

14 15 16 17

18 19 20 21 22

23

24 25

A1

A2

A3

C1

C2

Figure 1 Organization of the fV gene. The organization of the fV gene depicted with corresponding encoded regions of each protein (not to scale). Exons (coding regions) are shown as black boxes and are numbered sequentially from left to right (50 to 30 ). Introns (noncoding regions) are shown as horizontal lines between exons. The region encoded by each exon is depicted below the gene as a colored bar. The fV gene is 72.3 kb in length and results in an mRNA of 6.9 kb. Exons 17 encode the A1 domain, exons 812 encode the A2 domain, exon 13 encodes the B domain, exons 1418 encode the A3 domain, and exons 1922 and 2325 encode the C1 and C2 domains, respectively.

A1 NH2-

A2

A3

C1

C2
Factor V -COOH polypeptide

Processing (a) Activation cleavages 709

SS SS SS SS

1018

1545
SS SS SS

NH2SO4 SO4 SO4 SO4 SO4 SO4 SH SH SH SO4 SH SH

-COOH

Processed factor V

(b) APC inactivation cleavages 306 NH2A1 Ca2+ COOH(c) C2 C1 A3 Factor Va -NH2 Light chain 506 679 -COOH Heavy chain

A2

Figure 2 Organization of the fV protein. (a) The immature fV polypeptide containing the prepeptide followed by the A1, A2, B, A3, C1, and C2 domains. The 37 potential N-linked glycosylation sites are indicated as tree structures. The hatched region in the B domain reects the location of the 35 repeats of 9 amino acids each and the two gray regions represent acidic regions within the B domain. The proteolytic processing site to remove the signal peptide is indicated by an arrow. (b) The fully processed and mature form of fV secreted into plasma. Predicted disulde bonds are indicated above the bar by SS and free sulfhydrils are indicated below the bar by SH. The positions of the seven potential sulfated Tyr residues are indicated below the bar (SO4) and the positions of the three phosphorylated Ser residues are indicated above the bar as solid blue ovals. The three activation cleavage sites (Arg709, Arg1018, and Arg1545) are indicated by numbered arrows. (c) Activation of fV by thrombin or fXa releases the B domain in two fragments to produce an fVa heavy chain (A1 A2 fragment) and light chain (A3C1C2 fragment) as a heterodimer held together by a single calcium atom. The APC catalyzed inactivation cleavages (Arg306, Arg506, and Arg679) are indicated by numbered arrows (see also Figure 3).

tyrosine residues (Tyr665, Tyr696, Tyr698, Tyr1494, Tyr1510, Tyr1515, Tyr1565) and the phosphorylation of three serine residues (Ser692, Ser804, Ser1506). Subsequent to fV synthesis, a 28 amino acid signal peptide is removed to produce the mature single-chain form of fV that is secreted into plasma (2196 amino acids).

Regulation of Activation and Activity

Activation

Plasma fV requires sequential proteolytic activation at three sites (Arg709, Arg1018, Arg1545) to produce the active cofactor (Figure 2). The primary activator of fV

492 COAGULATION CASCADE / Factor V

is thrombin, although fXa can also catalyze this reaction in the presence of ionic calcium and an anionic phospholipid surface. While plasmin has been reported to be capable of activating fV, this activation is transient as plasmin also proteolytically inactivates fVa (see below). The activation of fV releases the B domain as two fragments (Mr 71 000 and Mr 150 000) and produces the active fVa molecule as a noncovalent calcium-linked heterodimer composed of a heavy chain (A1A2 fragment; Mr 105 000) and a light chain (A3C1C2 fragment; Mr 74 000).
Activity

While fXa exhibits reactivity towards prothrombin in the absence of fV, this activity is very limited. The binding of unactivated fV to fXa is fairly weak and increases the activity of fXa only to a small extent. In contrast, fVa binds to fXa tightly and produces optimal fXa activity in the presence of ionic calcium and an anionic phospholipid surface. Thus, fV has the ability to directly control the activity of fXa producing three levels of procoagulant activity during coagulation: fXa with no cofactor, fXa bound to fV, and fXa bound to fVa. The latter form of the complex is the major procoagulant complex during coagulation. The membrane-binding portion of fVa resides in the light chain C1 and C2 domains (Figures 2 and 3). Although fXa can bind to anionic phospholipid in the absence of fVa, the high afnity of fVa for both phospholipid and fXa results in enhanced retention of fXa onto the lipid surface. Unlike fVIIIa, fVa is extremely stable in the presence of ionic calcium. Thus, inactivation of fVa occurs largely through proteolysis by activated protein C (APC) at Arg506 followed by Arg306 and Arg679 (Figures 2 and 3). Since both APC and fXa compete for binding to the fVa light chain, high levels of fXa can protect fVa from proteolytic inactivation by APC. However, as the ratio of fXa to APC is reduced when APC levels are increased as a result of the formation of the thrombinthrombomodulin complex, the equilibrium would be expected to shift towards fVa inactivation. Additionally, in the presence of protein S (a cofactor for APC) the protective effect of fXa is abolished. The rst cleavage by APC at Arg506 in fVa reduces the procoagulant activity of fVa by roughly 40%. Subsequent proteolysis at Arg306 (enhanced by protein S) results in complete abrogation of fVa cofactor activity. The importance of APC inactivation of fVa is evidenced in fVLeiden where Arg506 is mutated to Gln, resulting in persistent fVa activity and thrombophilia. Thrombin can partially inactivate fVa via cleavage at Arg643. Plasmin also inactivates fVa in a

phospholipid-dependent manner by proteolysis at numerous sites including Arg313 and Arg306 in the heavy chain and Arg1765 in the light chain. Since fVa is protected from inactivation by plasmin when bound to fXa, the significance of this reaction is unclear. Neutrophil elastase has also been shown to proteolytically inactivate fVa after partially activating it, similar to plasmin. It has been suggested that this may play a role during sepsis and other conditions of increased inammation when elastase levels are elevated. Interestingly, cleavage at Arg506 in unactivated fV by APC has been shown to produce an alternate form of fV (fV506) that can serve as an anticoagulant cofactor in support of the APC-dependent inactivation of fVIIIa. The in vivo significance of this reaction has been questioned. Nonetheless, in certain pathological situations, this anticoagulant role of fV506 may prove to be of import.

Biological Function
The concentration of fV in plasma is roughly 10 mg ml1 with a half-life of 12 h. Plasma fV comprises about 80% of the total fV with the remaining 20% of the fV present in platelet a granules that can be secreted upon platelet activation. Although the majority of the fV in plasma is synthesized in the liver, endothelial cells and megakaryocytes have also been shown to be capable of synthesizing fV and may be a partial source of platelet fV. Deciency of plasma fV results in the bleeding diathesis known as parahemophilia, a rare autosomal recessive trait. While causing only mild bleeding problems in humans, deletion of the fV gene in transgenic mice leads to either midembryogenic lethality or fatal perinatal hemorrhage. Deciency of platelet fV also results in a bleeding diathesis in humans (fVQuebec), although this disorder has been shown to be due to the general degradation of platelet a-granule proteins and is not solely due to deciency of platelet fV. Interestingly, while platelet fV in humans has been shown to be largely derived from uptake of plasma fV, murine transgenic models have demonstrated a distinct difference between plasma and platelet pools of fV in mice. When fV was expressed solely in murine platelets it was capable of rescuing the lethal phenotype of fV knockout mice, suggesting a critical role of platelet fV in hemostasis.

FV in Respiratory Diseases
Normal lung function requires effective hemostasis. However, if left unregulated, excessive procoagulant

COAGULATION CASCADE / Factor V

493

A2

506

A3

306 A1 C1

C2

Figure 3 Three-dimensional model of the activated fV molecule. A three-dimensional model of fVa (Protein Databank le 1FV4) depicting the relation of fVa to the phospholipid surface (along the bottom; not shown) and the organization of the fVa heavy and light chains. The various domains are labeled and color coded (A1 purple; A2 orange; A3 red; C1 cyan; C2 blue). The proposed copper-binding site is shown with a Cu2 ion as an orange sphere between the A1 and A3 domains. Two potential sites for calcium binding are possible and are indicated by a Ca2 ion as a green sphere. The free sulfhydril residues are indicated as green sticks (two in the A2 domain and one each in the C1 and C2 domains) and the two major APC inactivation sites (Arg506 and Arg306) are indicated in yellow by arrows.

activity can lead to pulmonary brosis and potentially pulmonary emboli, both of which can be followed by respiratory failure. Excessive brin deposition can occur as a direct result of septic shock as well as pneumonia and other acute pulmonary events. These situations can result in acute lung injury (ALI), which is manifested most severely in acute respiratory distress syndrome (ARDS). A hypercoagulable state is observed in these cases as increased levels of clotting factors in pulmonary uids. As with the other coagulation factors, the levels of fV are increased in bronchoalveolar lavage and pleural uids in patients with ALI. Interestingly, the fV in these uids has been reported to be largely inactive. It is unclear what this inactive fV signies, but considering the high levels of fXa and thrombin that are found in these uids and the requirement of fXa for fVa to generate a-thrombin, this may be

indicative of excessive, persistent procoagulant activity resulting in accumulation of APC-inactivated fVa. This inactive fV may also represent degradation products of plasmin and/or elastase activity. Interestingly, the therapeutic administration of APC has demonstrated benecial reductions in ALI in various animal models as well as demonstrated a significant reduction in mortality in phase III studies of sepsis (PROWESS). Thus, inactivation of fVa may be important in the control of brin deposition in cases of severe sepsis. Since sepsis is the most common cause of ARDS, this also implies an equal importance of fVa in ALI.
See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Overview; Antithrombin III; Factor VII; Factor X; Fibrinogen and Fibrin; Intrinsic Factors; Protein C

494 COAGULATION CASCADE / Factor VII and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: Monocytes. Platelets. Proteinase-Activated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Factor VII
L V M Rao, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading

Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401404. Brodsky SV (2002) Coagulation, brinolysis and angiogenesis: new insights from knockout mice. Experimental Nephrology 10(56): 299306. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148157. Duga S, Asselta R, and Tenchini ML (2004) Coagulation factor V. International Journal of Biochemistry and Cell Biology 36: 13931399. Hoyer LW, Wyshock EG, and Colman RW (1994) Coagulation factors: factors V and VIII. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 109133. Philadelphia: JB Lippincott Company. Ichinose A and Davie EW (1994) The blood coagulation factors: their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 1954. Philadelphia: JB Lippincott Company. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and brinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238 S242. Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) Williams Hematology, 5th edn., pp. 12061226. New York: McGraw-Hill. Mann KG and Kalafatis M (2003) Factor V: a combination of Dr Jekyll and Mr Hyde. Blood 101(1): 2030. Pellequer J-L, Gale AJ, Getzoff ED, and Grifn JH (2000) Three-dimensional model of coagulation factor Va bound to activated protein C. Thrombosis and Haemostasis 84: 849857. Sun H, Yang TL, Yang A, Wang X, and Ginsburg D (2003) The murine platelet and plasma factor V pools are biosynthetically distinct and sufcient for minimal hemostasis. Blood 102(8): 28562861. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334 1349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725. Yang TL, Cui J, Taylor JM, Yang A, Gruber SB, and Ginsburg D (2000) Rescue of fatal neonatal hemorrhage in factor V decient mice by low level transgene expression. Thrombosis and Haemostasis 83: 7077. Factor VII(a) is responsible for triggering the clotting cascade that eventually leads to thrombin generation, brin deposition, and platelet activation. Factor VIIa (FVIIa) is enzymatically active only after complex formation with its cellular receptor, tissue factor (TF). Factor VIIaTF activity in vivo is tightly regulated by plasma inhibitors, particularly tissue factor pathway inhibitor. In addition to maintaining hemostasis, FVIIa may also inuence various cellular processes by transmitting cellular signals via activation of G-protein-coupled proteinase-activated receptors. Since TF is constitutively expressed in the lung on airway epithelium and populations of alveolar macrophages, transudation of FVII along other plasma proteins across the injured alveolar capillary barrier leads to activation of coagulation in lung, leading to brin formation. Further, ligation of TF by FVIIa induces expression of several immunoregulatory genes in the lung. These responses play a crucial role in ordered repair and restoration of lung structure and function. However, increased production of TF in the lung following an inammatory injury would lead to an enhanced procoagulant response that is excessive relative to that needed for resolution and repair. This would lead to brin deposition in the alveoli, interstitium, and capillaries. This, coupled with increased cytokine production and cell migration and activation that may occur through FVIIaTF-induced cell signaling, independent of coagulant response, would lead to acute lung injury, such as acute respiratory distress syndrome (ARDS). Thus, blockade of FVIIa binding to TF or inhibition of FVIIaTF proteolytic activity may have therapeutic benet in patients with ARDS.

Introduction
After exposure of blood to an injured vessel wall, a native factor VII (FVII)tissue factor (TF) complex is formed as the rst step of TF-dependent blood coagulation. Tissue factor is a transmembrane cellular receptor for FVII. In health, TF is constitutively expressed in many cells, including broblasts and pericytes in and surrounding blood vessel walls, but is absent from blood cells and endothelial cells that line blood vessels. However, under certain pathophysiological conditions, monocytes and endothelial cells express TF. Thus, either vessel wall injury or a specic disease process allows FVII to come in contact with TF. Because the single-chain native FVII is a zymogen, i.e., functionally inert, there has been wide interest in elucidating how formation of the FVIITF complex triggers the coagulation process. Initially, it was believed that native FVII possessed minimal enzymatic activity, sufcient to initiate the processes. However,

COAGULATION CASCADE / Factor VII

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this was found not to be physiologically relevant. Rapid preferential activation of FVII bound to TF by trace amounts of downstream clotting proteases, factor IXa, and factor Xa, and/or autoactivation of FVII bound to TF by FVIIaTF are now thought to be responsible for the conversion of FVIITF complexes to FVIIaTF complexes. In blood, about 1% of FVII circulates as FVIIa. It is still unclear how circulating FVIIa originates. Once traces of the FVIIaTF complex are formed at the injury site, it readily activates its substrates factor X and factor IX to active serine proteases, factor Xa and factor IXa, respectively. Factor IXa generated by FVIIaTF, in the presence of its cofactor factor VIIIa, efciently activates factor X. Factor Xa, in turn, cleaves prothrombin to thrombin, which converts brinogen to brin. Factors Xa and IXa also activate FVII bound to TF to FVIIaTF, thus amplifying the initiation stimulus. Thrombin activates the cofactors, factor VIII and factor V, which serve as membrane-bound receptors/cofactors for clotting factors IXa and Xa, respectively. The need for factor IXa/factor VIIIa-induced activation of factor X for hemostasis stems from the effective inhibition of FVIIaTF complexes by a plasma inhibitor, tissue factor pathway inhibitor (TFPI) following the initial generation of factor Xa.

GLA AS EGF

EGF

Catalytic domain

Figure 1 A schematic representation of FVII structure. FVII is a multidomain protein consisting of an N-terminal domain containing 10 g-carboxy glutamic acid residues (GLA), followed by an aromatic stack (AS), two epidermal growth factor-like domains (EGF), and a catalytic domain at the C-terminal with Oglycosylation of serine 52 and 60 in the rst EGF domain and N-glycosylation of asparagines 145 and 322. b represents potential b-hydroxylation of aspartic acid 63. The arrowhead points to the activational cleavage site at Arg152-Ile153.

degree of sequence and structural homology with trypsin.

Regulation of Production and Activity

Factor VII concentration in plasma, compared to other vitamin K-dependent clotting factors, is extremely low (0.5 mg ml 1 or 10 nM). Limited steadystate FVII mRNA levels in the liver are responsible for the low mean plasma concentration of FVII. The liver is believed to be the entire source for plasma FVII as FVII mRNA expression is restricted to the liver. FVII may be synthesized locally (extrahepatically) in a limited number of cells under special circumstances, such as alveolar macrophages in interstitial lung diseases and smooth muscle cells in human atherosclerotic vessels. The human FVII gene spans 13 kb and is located in chromosome 13, just 2.8 kb 50 to the factor X gene. As in promoters of many other clotting factor genes, the FVII promoter lacks a TATA box, a sequence present in about 80% of RNA polymerase II eukaryotic promoters. A major transcription start site is identied at 51. The rst 185 bp 50 of the transcription start site are sufcient to confer maximal promoter activity. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, which are shown to bind within the rst 108 bp of the promoter region of FVII, play a critical role in FVII promoter activity. ARP1, an orphan nuclear hormone receptor, interacts with two regions of the FVII 50 anking region, the HNF-4 binding region ( 77 to 47), and the nuclear hormone response region ( 237 to 200). ARP1 binding represses the transcriptional activation of the FVII gene. Expression of FVII may also be modulated by interactions taking place outside of the HNF-4 and Sp1 binding region of the promoter. A decanucleotide insert polymorphism at 323 is shown to reduce FVII gene expression.

Structure
Human FVII is a multidomain, single-chain glycoprotein, consisting of 406 amino acid residues with a molecular weight of 50 000. FVII is synthesized primarily in the liver and is secreted into the blood as a zymogen of a serine proteinase. Prior to secretion from the liver, FVII undergoes several posttranslational modications, including g-carboxylation of 10 glutamic acid residues located in its N-terminus, incomplete b-hydroxylation of aspartic acid residue 63, N-glycosylation of two asparagine residues (residues 145 and 322), and O-glycosylation of serine 52 and serine 60 (Figure 1). The activation of FVII to FVIIa involves the hydrolysis of a single peptide bond between arginine 152 and isoleucine 153, resulting in a two-chain molecule, consisting of a light chain of 152 amino acid residues and a heavy chain of 254 amino acid residues. The light chain of FVIIa contains the g-carboxyglutamic acid (GLA) domain (located in residues 145), followed by an aromatic stack-domain and two epidermal growth factor (EGF)-like domains. The heavy chain of FVIIa contains the catalytic domain featuring the active site catalytic triad (His-193, Asp-242, and Ser344). The catalytic domain of FVIIa exhibits a high

496 COAGULATION CASCADE / Factor VII

The decanucleotide insert polymorphism correlates with both a lower FVII antigen and coagulant activity. In contrast to the decanucleotide insertion, a base substitution at 402 (G-A) is associated with the increased FVII expression. Once FVII is secreted from the liver to circulating blood, its activity is regulated by a variety of mechanisms; among them activation of FVII by cleavage of the peptide bond between Arg 152 and Ile 153, and allosteric inuences exerted by cofactor TF, substrates, and inhibitors play predominant roles. Activation of zymogen FVII to enzyme FVIIa is largely TF-dependent. Upon activation, FVII structure undergoes conformational changes that form the substrate-binding cleft. However, the conformational change in FVIIa is incomplete until it binds to TF. Thus, FVIIa alone exists only in a partially active form, and is driven to the active enzyme under the inuence of TF. Among the clotting factors, FVII has the shortest half-life approximately 23 h. In contrast to other activated clotting proteins, which are cleared rapidly from the circulation (within seconds to a few minutes), FVIIa exhibits a long circulatory half-life (t1/2 B2 h), about the same as the zymogen. Pharmacokinetic studies in rats suggested that the liver was responsible for a major proportion of the FVIIa clearance. At present, the receptors and proteins that are responsible for the clearance of FVII or FVIIa in the liver are unknown.

Biological Function
The principal biological function of FVII is to maintain hemostasis. Vascular damage that permits blood to leave the vasculature can be fatal unless the blood loss is stopped fast. FVII is responsible for initiating the cascade of highly sensitive and fast-acting proteolytic events that ultimately lead to the clot that seals the injury. Vessel wall injury disrupts the endothelial cell barrier that normally separates TFexpressing cells from the circulating blood, thus allowing circulating FVII to come in contact with TF. Complex formation with TF facilitates the cleavage of FVII zymogen to its active form, FVIIa. FVIIa, in concert with TF, then activates factor IX and X leading to localized thrombin generation, which subsequently leads to brin deposition, platelet activation, and clot formation, thereby stopping blood loss. However, FVII action can also lead to a deleterious initiation of the cascade within the vasculature in pathological conditions. For example, FVIIa-induced clotting upon the rupture of an atherosclerotic plaque would lead to a major blood clot (thrombus formation) that could become life-threatening.

Initiation of FVIIa-induced clotting in sepsis will lead to disseminated intravascular coagulation and the bleeding disorder. It is important to note that aberrant expression of TF in pathological conditions, not changes in circulating FVII, is primarily responsible for thrombotic disorders associated with the initiation of the coagulation cascade. The FVIIaTF-induced coagulation pathway not only leads to clot formation but also contributes to vascular remodeling, which is caused by growth factors secreted by activated platelets as well as the intermediary products, factor Xa and thrombin, that promote vascular smooth cell proliferation and alter endothelium. In addition, FVIIa protease may also have other biological functions that are not related to its coagulant activity. Recent studies suggest that FVIIa interaction with TF on a variety of cells, including broblasts, epithelial cells, and endothelial cells, activates multiple signaling pathways leading to altered gene expression, ultimately affecting various cellular processes, such as cell survival, proliferation, and migration. FVIIaTF is shown to activate cell signaling by activating G-protein-coupled proteinaseactivated receptors (PARs), particularly PAR2. FVIIaTF-induced cell signaling could contribute to various pathophysiological processes, such as development, inammation, angiogenesis, tumor metastasis, wound healing, and vascular remodeling. However, one should note here that, at present, the evidence for noncoagulant biological functions for FVIIa is mainly derived from data from cell model systems. This needs to be conrmed using relevant in vivo model systems.

Receptors
Tissue factor is the only known cellular receptor for FVII and FVIIa. Tissue factor is a transmembrane glycoprotein with a 219-amino acid extracellular domain, a 23-residue transmembrane region, and a 21residue intracellular domain. The lungs and central nervous system are known to contain high levels of TF activity. In the lungs, the bronchial mucosa and alveolar epithelial cells constitutively express high levels of TF. Tissue factor is constitutively expressed on the surface of many extravascular cells, such as broblasts and pericytes within the blood vessel wall, but not in cells within vasculature that contact blood, such as monocytes and endothelial cells. However, under certain pathological conditions, such as sepsis, monocytes/macrophages express TF. Although TF can be readily induced in cultured endothelial cells by a variety of stimuli, such as inammatory cytokines, bacterial lipopolysaccharides, and growth factors, its expression in vivo is somewhat

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controversial. Vessel wall injury that disrupts the endothelial cell barrier or pathological expression of TF in monocytes and endothelial cells results in circulating blood coming into contact with TF on cell surfaces, allowing FVII to interact with TF, and subsequent activation of the coagulation pathway. Recent studies suggest that, in addition to the presence of TF in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at the site of vascular injury. At present, the source of blood-borne TF and the physiological significance of blood-borne TF to hemostasis are unclear.

Factor VII in Respiratory Diseases

Fibrin is a conspicuous part of acute and chronic inammatory disorders of the lung. Extravascular brin is shown to play an important role in pathogenesis of both acute lung injury and repair. Coagulation proteases and extravascular brin may liberate cleavage products that are capable of potentiating the acute inammatory response via effects on vascular permeability, chemotaxis, and immunomodulation. Further, brin deposits inuence the reparative response to acute lung injury by promoting collagen deposition. Persistent brin deposition in areas of pulmonary scarring will lead to brosis. In addition to brin, other coagulation proteases, particularly thrombin, may also play a role in pathogenesis of lung diseases by promoting inammation, broblast proliferation, and collagen deposition through activation of proteinase-activated receptors. In acute lung injury, alveolar brin deposition is potentiated by consistent changes in endogenous coagulation and brinolytic pathways. The only known physiological pathway that leads to thrombin generation and brin formation is the FVIIaTF pathway of blood coagulation. Thus, FVII/FVIIa, through generation of thrombin and brin, plays a critical role in respiratory diseases. Consistent with this, increased FVII activity levels were observed in bronchoalveolar lavage uids of patients with a variety of lung diseases, such as sarcoidosis, idiopathic pulmonary brosis, adult respiratory distress syndrome (ARDS), and interstitial lung diseases. Acute lung injury also leads to the upregulation of TF in resident and inltrating macrophages, as well as in other constitutive cell populations within the lung. Local overexpression of TF in association with increased FVII levels and the presence of other downstream clotting proteins in alveolar lining uids predispose the patients with respiratory diseases to alveolar brin deposition. Transudation of plasma proteins across the injured alveolar capillary is

primarily responsible for the increased FVII levels in the alveolar compartment. However, alveolar macrophages were also shown to synthesize FVII locally. Although TFPI levels are also increased in alveolar lining uid in response to acute lung injury, the levels are not sufcient to block the FVIIaTF-induced coagulation. In addition to the alveolar compartment, the disordered brin is also observed in the injured pleural space. Increased coagulation and concurrently depressed brinolysis was found in the pleural uids of patients with exudative pleuritis. Increased levels of FVII and TF in the pleural uid in response to injury are responsible for the increased procoagulant activity. Although the overall pattern of increased coagulation in pleural exudates is similar to those observed in alveolar injury, the procoagulant activity expressed in exudative pleural uids was much lower than that observed in bronchoalveolar lavage (BAL). This could be due to increased concentration of coagulation inhibitors in pleural uids. Although brin deposition in the alveolar and interstitial spaces of the lung is a well-dened feature of ARDS and plays an important role in lung injury and repair, FVIIaTF complexes may also exert direct effects on the pathogenesis of lung injury (Figure 2). FVIIa binding to TF is shown to induce the expression of several immunoregulatory genes that may be involved in the pathogenesis of ARDS. FVIIa TF is shown to stimulate IL-1b, IL-8, and other chemokines and growth factors, which could regulate cell migration, activation, and collagen production. FVIIa interaction with TF on lung broblasts upregulates the expression of probrotic factors, connective tissue growth factor (CCN2), and Cyr61 (CCN1). In addition to its effects on cytokine and growth factor production, FVIIa is also shown to promote the production of reactive oxygen species by macrophages and to increase the expression of MHC class II and b integrin expression in macrophages. More importantly, serine proteases generated by FVIIaTF activation of the coagulation pathway, i.e., factor Xa and thrombin, also have independent inammatory signaling functions (Figure 2). The importance of FVII in ARDS is well illustrated in the baboon model system. Blockade of FVII interaction with TF by administering active site inactivated human FVIIa (ASIS) or by inhibiting the proteolytic function of FVIIaTF complex with TFPI, prevented respiratory failure in sepsis-induced acute lung injury. In the lung, ASIS prevented pulmonary hypertension and edema, preserved gas exchange and lung compliance, and decreased brin deposition in the alveolar space. Consistent with data obtained in experimental models of severe sepsis, phase II clinical

498 COAGULATION CASCADE / Factor VII

Coagulation proteins Signaling receptor Inflammatory and other effects IL-1, IL-6, IL-8, chemokines, growth factors, ROS, VEGF, CCN1, CCN2, collagenases, pulmonary fibrosis

TF VIIa
PAR2

Xa
PAR1

TF VIIa Xa Va Prothrombin Thrombin

PAR1 PAR2

Mostly same as above, MCP-1, edema

PAR3

Fibrinogen

Fibrin
PAR4

Platelet activation, secretion vascular tone, cell motility, cell permeability, PMN chemotaxis, inflammatory cytokines, adhesion molecules, growth factors, pulmonary fibrosis

Figure 2 Noncoagulant functions of FVIIa and downstream clotting proteases generated by FVIIa. FVIIaTF, directly or through elaboration of factor Xa and thrombin, upregulates the expression of various proinammatory cytokines and promotes cell migration and activation in lung via activation of proteinase-activated receptors. These responses contribute to ordered repair and pathogenesis of acute lung injury through multilevel interactions among them. TF, tissue factor; PAR, proteinase-activated receptor; IL, interleukin; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; MCP-1, monocyte chemoattractant protein-1; PMN, polymorphonuclear cells.

trials showed that TFPI is effective in reducing mortality in patients with severe sepsis. However, a larger phase III trial failed to show a significant difference in 28-day all-cause mortality between the TFPI treatment and placebo groups. In summary, FVIIaTF-mediated coagulation plays a major role in the development of lung injury in sepsis. FVIIa, TF, and downstream products, factor Xa, thrombin, and brin all contribute to the lung injury in ARDS. Blockade of FVIIaTF function by natural plasma inhibitor or modied FVIIa is shown to prevent lung injury, suggesting that the blockade of FVIIaTF may be a feasible therapeutic strategy in patients with ARDS.
See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Factor X; Thrombin; Tissue Factor. G-Protein-Coupled Receptors.

Further Reading
Broze GJ Jr (1995) Tissue factor pathway inhibitor and the current concept of blood coagulation. Blood Coagulation and Fibrinolysis 6: S7S13. Edgington T, Dickinson CD, and Ruf W (1997) The structural basis of function of the TF-VIIa complex in the cellular initiation of coagulation. Thrombosis and Haemostasis 78: 401405. Eigenbrot C (2002) Structure, function, and activation of coagulation factor VII. Current Protein and Peptide Science 3: 287299.

Hagen FS, Gray CL, OHara P, et al. (1986) Characterization of a cDNA coding for human factor VII. Proceedings of the National Academy of Sciences, USA 83: 24122416. Idell S (2003) Coagulation and brinolysis, and brin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213S220. Idell S, James KK, Levin EG, et al. (1989) Local abnormalities in coagulation and brinolytic pathways predispose to alveolar brin deposition in the adult respiratory distress syndrome. Journal of Clinical Investigation 84: 695705. Konigsberg W, Kirchhofer D, Riederer MA, and Nemerson Y (2001) The TF:VIIa complex: clinical significance, structure function relationships and its role in signaling and metastasis. Thrombosis and Haemostasis 86: 757771. Monroe DM, Hoffman M, and Roberts HR (2002) Platelets and thrombin generation. Arteriosclerosis, Thrombosis, and Vascular Biology 22: 13811389. Pollak ES, Hung HL, Godin W, Overton GC, and High KA (1996) Functional characterization of the human factor VII 50 -anking region. Journal of Biological Chemistry 271: 17381747. Rao LVM and Pendurthi UR (2005) Tissue factor factor VIIa signaling. Arteriosclerosis, Thrombosis, and Vascular Biology 25: 4756. Rapaport SI and Rao LVM (1995) The tissue factor pathway: how it has become a prima ballerina. Thrombosis and Haemostasis 74: 717. Thim L, Bjoern S, Christensen M, et al. (1988) Amino acid sequence and posttranslational modications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 27: 77857793. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725.

COAGULATION CASCADE / Factor X

499

Factor X
P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Blood coagulation factor X (fX) is a vitamin-K dependent serine protease zymogen synthesized in the liver and present in the circulation as a glycosylated, two-chain, disulde-linked molecule. Activation of fX occurs via limited proteolysis at a single site to release a small 52 amino acid-activation peptide. This reaction is catalyzed by either the intrinsic or extrinsic pathways of blood coagulation and requires ionic calcium as well as anionic phospholipid exposed on activated platelets or damaged cell surfaces. The activated fX that is generated, fXa, is an active serine protease and displays high homology to the trypsin family of serine proteases. FXa is involved in propagation of the coagulant response as well as in cell signaling pathways linked with the inammatory response. The procoagulant activity of fXa requires calcium and anionic phospholipid, enabling fXa to bind to its nonenzymatic protein cofactor fVa (activated fV) and activate prothrombin to generate a-thrombin. FXa can also enhance its own generation and activity via positive feedback activation of fVII and fVIII, as well as activation of fV. The cellsignaling function of fXa does not require fVa but requires a viable enzyme active site for binding to effector cell protease receptor-1 (EPR-1) or activation of protease-activated receptors PAR-1 and PAR-2. Unregulated activation or activity of fX/fXa in the lung is associated with the development of pulmonary brosis and the inammatory response observed in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).

renamed fX based on the Roman numeral naming convention adopted by the International Committee for the Standardization of the Nomenclature of Blood Clotting Factors in 1959. The function of fX in coagulation was initially unclear since fX acted as a substrate for some reactions but as an enzyme in others. The view of fX as a zymogen that is activated to an enzyme was not accepted until the early 1960s when the coagulant action of Russells viper venom was found to be directed towards fX whose product, fXa, was capable of activating prothrombin. This discovery, along with the realization that fX was activated by the intrinsic and extrinsic pathways, led in 1964 to fX being given a central position in the coagulation cascade.

Structure
Gene

Introduction
Factor X (fX), also called Stuart factor, is a vitamin-K dependent serine protease zymogen that is activated in the rst common step of the intrinsic and extrinsic pathways of blood coagulation. It was serendipitously discovered in 1956 through a patient exhibiting a hemorrhagic disorder not attributable to a deciency of any of the known clotting factors of the time. This new factor was named after the patient rst described with this deciency (Mr. Stuart). Stuart factor was

The gene for human fX is on chromosome 13 at position q32-qter. It is 27 kb long and contains eight exons (IVIII) and seven introns (AG), resulting in an mRNA of 1.5 kb (Figure 1). Exon I encodes a prepeptide and exon II encodes a propeptide and g-carboxyglutamic acid-containing domain (Gla). These are separated from each other by intron A (6.5 kb) and from the other exons by intron B (8.8 kb). Exon III is the smallest and encodes an 8 amino acid aromatic stack region separating the Gla domain from two epidermal growth factor (EGF)like repeats encoded by exons IV and V. Intron C (0.9 kb), the smallest in the gene, separates exons III and IV while intron D (1.4 kb) separates exons IV and V. Exons encoding the light chain of fX (IV) are separated from those encoding the heavy chain (VI VIII) by intron E (2.8 kb). Exon VI encodes the activation peptide region and a small portion of the protease domain. This is followed by intron F (3.2 kb), exon VII, and intron G (1.4 kb). Exon VIII

I A

II B

III IV V VI C D E F

VII VIII G

pre

pro

gla

as egf-1

egf-2

ap

Protease domain

Figure 1 Organization of the fX gene. Exons (coding regions) are shown as blue boxes and are numbered sequentially from I to VIII. Introns AG (noncoding regions) are shown as horizontal lines between exons. The regions of fX encoded by each exon is depicted below the gene as a shaded bar: pre, prepeptide coding region; pro, propeptide coding region; gla, g-carboxyglutamic acid domain coding region; as, aromatic stack coding region; egf-1 and egf-2, epidermal growth factor-like domain coding regions; ap, activation peptide coding region; protease domain, catalytic domain coding region.

500 COAGULATION CASCADE / Factor X

encodes the majority of the protease domain and at 0.6 kb is the longest uninterrupted coding sequence in the gene.
Protein

The fX protein is synthesized in the liver as a singlechain pre-proprotein of 488 amino acids with 24 half cystines (Figure 2(a)). The rst 40 amino acids of the polypeptide constitute a leader sequence containing the pre- and propeptides. The prepeptide signal sequence is cleaved off by a signal peptidase while the propeptide directs posttranslational g-carboxylation of the rst 11 Glu residues of the N-terminus (Gladomain) and is subsequently removed by a processing protease. fX also undergoes b-hydroxylation of Asp63 in the rst EGF-like domain and N-linked glycosylation at Asn157 and Asn167 within the activation peptide region. The high degree of glycosylation (15% carbohydrate) increases the relative mass

(Mr) of fX from 58 200 to 58 900. Additional processing of fX occurs by an unknown mechanism to release the basic tripeptide Arg140LysArg142 from the center of the molecule, dividing fX into two chains. The mature form of fX in plasma is a glycosylated two-chain zymogen linked by a disulde bond between Cys132 and Cys302 (Figure 2(b)). The N-terminally derived light chain (Mr 16 200) contains the Gla- and EGF-like domains, and the C-terminally derived heavy chain (Mr 42 000) contains the activation peptide and the latent serine protease domain. The activated form of fX, fXa, is an active serine protease sharing structural homology to the trypsin family and containing the canonical catalytic triad His42, Asp88, and Ser185 (His57, Asp102, and Ser195 by chymotrypsinogen numbering convention). fXa exhibits procoagulant and esterase activities. The latter is dependent exclusively on the heavy chain whereas the former requires the additional presence

Factor X polypeptide

N -linked carbohydrate

Processing NH2 COOH Pre-pro leader Gla domain sequence Processing EGF1 EGF2 Protease (catalytic) domain

(a)

Activation cleavage Activation peptide

Processed factor X
NH2 Light chain

COO NH 2

COOH Heavy chain

(b)

NH

Activated factor X (factor Xa)

NH2 Light chain

COOH

COOH Heavy chain

(c)
Figure 2 Structural elements of the fX protein. (a) The immature fX polypeptide containing the pre-propeptides, g-carboxyglutamic acid (Gla) domain, EGF-like repeats, activation peptide, and protease domain. The 11 Gla residues are indicated (g) as is the bhydroxylated Asp63 (b) and glycosylation sites in the activation peptide region. Disulde bonds are indicated by thin lines connecting looped regions of the main chain. Sites of proteolytic processing are indicated by the three arrows. (b) The fully processed and mature form of fX found in plasma. The tripeptide ArgLysArg has been removed to produce the two-chain form. The light and heavy chains (indicated) remain held together by a single disulde bond in the center of the amino acid sequence. The activation cleavage site and the glycosylated activation peptide are indicated by arrows. The activation peptide is released upon proteolysis to produce fXa (c).

COAGULATION CASCADE / Factor X

221-loop 184-loop

501

124-loop

145-loop

its activity towards fX; and (3) calcium binding at the junction of the Gla- and EGF-1 domains of the activating enzyme and fX correctly alters their conformation and topography on the phospholipid surface. The binding to phospholipid surfaces is regulated by the anionic character of the membrane on activated platelets or damaged cell surfaces and increases the local concentration of enzyme and substrate at the surface to substantially augment catalysis.
Activity

60-loop

74-loop 36-loop

243-loop

Figure 3 Insertion loops in the fXa catalytic (serine protease) domain. The crystal structure of the fXa heavy chain or protease domain (Protein Databank code 1FAX) showing the insertion regions in comparison to chymotrypsin (labeled colored regions). The canonical serine protease catalytic triad residues (Ser, His, Asp) are indicated as orange stick structures in the center of the molecule. The orange sphere in the lower left of the structure is a bound calcium ion required for the procoagulant and esterase activities of fXa.

of the light chain. Despite the high level of structural homology between fXa and the trypsin family, fXa differs from this family by the insertion of varioussized peptide sequences on the external surface of the molecule (Figure 3). These regions, or insertion loops, aid in dening the physiological behavior of fX/Xa and impart to fXa its individual specicity for substrates and inhibitors.

Regulation of Activation and Activity

Activation

fXa activates prothrombin via proteolysis of two bonds to produce a-thrombin and an activation peptide (fragment 12). While this reaction can be slowly catalyzed by fXa alone, the true procoagulant in this step is a complex of fXa and fVa, the latter being a nonenzymatic protein cofactor. The calcium- and phospholipid-dependent formation of the fXafVa complex increases the proteolysis of prothrombin roughly 3 105-fold and alters the bondcleavage order to produce a different intermediate than that observed with fXa alone: fXa produces an inactive prethrombin-2 intermediate while the fXafVa complex produces a partially active meizothrombin intermediate. FXa is inhibited in plasma principally by the serine protease inhibitor (serpin) antithrombin (antithrombin III). Heparin, or heparan sulfate on cell surfaces, mediates this inhibition by binding antithrombin and inducing a conformational change in the inhibitor. fXa can also be inhibited by tissue factor pathway inhibitor (TFPI) and, to a lesser extent, a2-macroglobulin. Attenuation of fXa activity also occurs via proteolytic inactivation of fVa by activated protein C (APC), resulting in reduced enhancement of fXa activity.

fX zymogen is activated to fXa via hydrolysis of a peptide bond in the heavy chain at Arg194. This releases the heavily glycosylated activation peptide (52 amino acids; Mr 8000) from the N-terminus of the heavy chain (Figure 2(c)). Physiologically, this reaction is catalyzed by either of two enzymecofactor complexes, fIXafVIIIa or fVIIatissue factor (TF), each of which generates the identical fXa product. Both reactions have an absolute requirement for calcium ions and anionic phospholipid. The calcium requirement is threefold: (1) calcium binding to the Gla-domains of the activating enzyme and fX refolds these domains to promote their interaction with phospholipid surfaces; (2) calcium binding to the protease domain of the activating enzyme enhances

Biological Function
Human fX zymogen exists in plasma at a concentration of 140170 nM with a half life of B1.5 days. While the major procoagulant role of fXa is to generate a-thrombin, fXa can also activate fVII in a fVaindependent positive feedback loop, as well as fV and fVIII (Figure 4). Although the latter two reactions are inefcient, they may be important in early stages of coagulation to generate initial small amounts of these active cofactors. The importance of fXa in coagulation is manifested by the bleeding diathesis observed in patients decient in fX, the severity of which is proportional to the level of deciency. Complete deletion of the fX gene in transgenic knockout mice leads to partial

502 COAGULATION CASCADE / Factor X

Intrinsic VIII VIIIa X PL, Ca2+ V IXa

Extrinsic VII VIIa TF PL, Ca2+ TFPI

Xa
Va PL, Ca2+

Antithrombin Heparin

Prothrombin Thrombin

Fibrinogen Fibrin clot

Figure 4 Biological procoagulant activities and inhibition of fXa. fX is activated to fXa by the intrinsic or extrinsic pathway. The fXa generated subsequently activates prothrombin to a-thrombin leading to brin deposition. FXa can augment its own generation by positive feedback activation of fVII and fVIII. Both of these reactions occur independent of fVa and require phospholipid (PL) and ionic calcium (Ca2 ). FXa can also enhance its own activity towards prothrombin by activation of fV to generate fVa. Solid arrows represent proteolytic conversions and dotted arrows represent enzymatic actions. Enzymes are indicated by boldface text compared to cofactors and zymogens. A lowercase a following the numerical designation designates an activated molecule. Regulation of fXa activity by the inhibitors antithrombin and TFPI are also indicated. TF, tissue factor.

integral membrane proteins containing seven membrane-spanning helices with an intracellular C-terminus and an extracellular N-terminus. The four currently identied PARs are differentially expressed on various cell types and are activated via proteolytic cleavage rather than ligand binding. PAR-1 is expressed on the surface of platelets, mononuclear cells, and endothelial cells while PAR-2 is expressed on the surface of mononuclear cells, endothelial cells, and neutrophils. Although each PAR is slightly different and exhibits a unique combination of specicities for various proteases, their general activation scheme remains the same: cleavage of an activation site within the extracellular domain of the receptor produces a new N-terminus. This new N-terminus functions as a tethered ligand that binds intramolecularly to induce an intracellular signal via activation of heterotrimeric G-proteins. Although the procoagulant action of fXa does not per se require a cellular receptor, activated platelets express the receptor EPR-1 that is involved in expression of fXa procoagulant activity. EPR-1 is also expressed in leukocyte subpopulations, human brain microvascular pericytes, smooth muscle cells, endothelial cells, and human lung broblasts. Unlike the PARs, EPR-1 does not require proteolysis to function. Although the active site of fXa is required for the cell signaling function of EPR-1, it is not required for binding to EPR-1. It is likely that the signaling function of EPR-1 on endothelial cells is mediated through PAR activation by the fXaEPR-1 complex.

embryonic lethality due to excessive bleeding as well as fatal bleeding in neonates. In addition to its procoagulant role, fXa is involved in cell signaling and induces mitosis in smooth muscle cells, nitric oxide release, and expression of adhesion molecules and TF in endothelium. Cytokine and chemokine production is also differentially affected by fXa in various cell types. FXa induces monocyte chemotactic protein-1 (MCP-1) and interleukins IL-6 and IL-8 in endothelium, and cytokines vascular endothelial growth factor (VEGF) and platelet-derived growth factor-A (PDGF-A) in lung broblasts. These signaling activities all require a functional fXa active site and either binding to effector cell protease receptor-1(EPR-1) or proteolytic activity toward protease-activated receptor-1 (PAR-1) or PAR-2.

fX in Respiratory Diseases
Effective hemostasis is paramount for the maintenance of normal lung function. Left unregulated, excessive procoagulant activity in the lung can lead to the onset of pulmonary brosis followed by respiratory failure. This circumstance can be brought about by sepsis, pneumonia, or other acute catastrophic events and is archetypical of the most severe manifestation of acute lung injury (ALI), acute respiratory distress syndrome (ARDS). The increased procoagulant state in ARDS is evidenced by increased levels of clotting factors in bronchoalveolar lavage and pleural uids of aficted patients. fXa plays a dual role in this setting by also increasing the proinammatory response via receptor-based signaling on platelets, mononuclear cells, endothelial cells, and neutrophils. Therapeutic strategies for treatment of pulmonary brosis have included the use of coagulation inhibitors such as antithrombin, heparin, TFPI, and APC to attenuate coagulation. Each of these inhibitors targets numerous enzymes including fXa: antithrombin

Receptors
The activity of fXa as a cell-signaling factor involves the cellular protease-activated receptors PAR-1 and PAR-2. These G-protein-coupled receptors are

COAGULATION CASCADE / Fibrinogen and Fibrin 503

is a broad-spectrum serine protease inhibitor whose reactivity is enhanced by heparin. TFPI targets the fVIIa-TF activation of fX, but is also a potent fXa inhibitor in the absence of fVIIa-TF. APC inactivates fVa thus attenuating the procoagulant activity of fXa. Each of these inhibitors has demonstrated benecial reductions in ALI in various animal models. Unfortunately, these results have not been reproduced in clinical studies. While TFPI was successful in phase II clinical trials, the results in phase III trials (OPTIMIST study) were disappointing and showed no benecial effect above placebo. APC, however, has demonstrated a significant reduction in mortality in cases of severe sepsis (PROWESS study), which is the most common cause of ALI and ARDS.
See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Overview; Antithrombin III; Factor V; Factor VII; Fibrinogen and Fibrin; Intrinsic Factors; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Interleukins: IL-1 and IL-18; lL-4; lL-5; lL-6; lL-7; lL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Leukocytes: Monocytes. Platelets. ProteinaseActivated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.) Williams Hematology, 5th edn., pp. 12061226. New York: McGraw-Hill. Riewald M and Ruf W (2002) Orchestration of coagulation protease signaling by tissue factor. Trends in Cardiovascular Medicine 12: 149154. Ruf W, Doreutner A, and Riewald M (2003) Specicity of coagulation factor signaling. Journal of Thrombosis and Haemostasis 1: 14951503. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 13341349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725.

Fibrinogen and Fibrin

nther and C Ruppert, University of Giessen A Gu Lung Center (UGLC), Giessen, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Fibrinogen is a complex multifunctional glycoprotein that plays a key role during blood clotting. Thrombin-mediated conversion of brinogen into brin and the subsequent cross-linking by factor XIII results in the formation of the three-dimensional framework of the thrombus. In addition, brinogen promotes platelet adhesion and aggregation via interaction with a specic integrin receptor. Next to its essential function in normal hemostasis and wound repair brin(ogen) also exerts multiple regulatory effects and is involved in pathological processes such as thrombosis and atherosclerosis. Abnormal brin turnover has also been implicated in a number of respiratory diseases including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and chronic interstitial lung diseases such as pulmonary brosis.

Further Reading
Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401404. Altieri DC (1995) Proteases and protease receptors in modulation of leukocyte effector functions. Journal of Leukocyte Biology 58: 120127. Altieri DC (1995) Xa receptor EPR-1. FASEB Journal 9: 860865. Bretschneider E and Schror K (2001) Cellular effects of factor Xa on vascular smooth muscle cells inhibition by heparins? Seminars in Thrombosis and Hemostasis. 27: 489493. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148157. Dugina TN, Kiseleva EV, Chistov IV, Umarova BA, and Strukova SM (2002) Receptors of the PAR family as a link between blood coagulation and inammation. Biochemistry (Moscow) 67: 7475. Ichinose A and Davie EW (1994) The blood coagulation factors: Their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 1954. Philadelphia: J.B. Lippincott Company. Idell S (2001) Anticoagulants for acute respiratory distress syndrome: can they work? American Journal of Respiratory and Critical Care Medicine 164: 517520. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329 S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and brinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238S242.

Introduction
In 1666 the Italian anatomist Marcello Malphigi described brin as a white brous substance in a hemostatic plug and thus conrmed the much earlier observation of the Roman physician Claudius Galenos (AD 129199) who proposed the presence of bers (Latin: brae) in circulating and clotted blood. In 1832 the German anatomist and physiologist Johannes Mu ller provided evidence that blood contains soluble brin, and in 1859 Denis de Commercy proposed the existence of a soluble brin precursor that could be salted out of plasma. In the second half of the nineteenth century Rudolf Virchow, a German pathologist, identied brinogen as being the precursor of brin, and Alexander Schmidt, a German physiologist, identied the so-called brin-ferment (i.e., thrombin) as brinogen-converting factor. At the

504 COAGULATION CASCADE / Fibrinogen and Fibrin

same time the Swedish physiologist Olof Hammarsten advanced Schmidts theory and suggested that preceding brin formation, activation of brinogen by thrombin occurred by limited proteolysis. These observations led to Paul Morawitzs classical four-factor theory of blood coagulation according to which coagulation is initiated when the clot-promoting factor thrombokinase (today referred to as thromboplastin or tissue factor) is released by destroyed tissues or liberated by platelets and leukocytes. Thrombokinase in the presence of calcium converts prothrombin into thrombin, which in turn converts brinogen into brin. In subsequent years identication of numerous clotting factors resulted in the proposal of the waterfall or cascade theory of blood clotting. In the nal step of this classical paradigm of blood coagulation soluble brinogen is cleaved by thrombin forming soluble brin monomers, which then polymerize to make an insoluble brin clot. It took several decades to elucidate the exact activation mechanism of brinogen. In the early 1950s Bailey found that thrombin removed small peptides from the N-terminus of brinogen and hypothesized that the newly exposed N-termini must be the contact sites for brin polymerization. As new analysis tools became available in the second half of the twentieth century, new insights in brin(ogen) structure were given. Fibrinogen and brin are multifunctional proteins. Fibrinogen binds via a glycoprotein receptor (GPIIb/ IIIa) to platelets mediating platelet adhesion and aggregation during hemostasis. Fibrin is an essential matrix of wound repair by stabilizing wound elds and supporting local cell proliferation and migration.

chromosome 4 (4q28). For the g-chain an elongated splice variant (427 amino acid residues) exists in human plasma accounting for B8% of the total brinogen g pool. The major form of brinogen circulating in human plasma is that of a homodimeric g/g molecule (brinogen 1), whereas heterodimeric g/g0 molecules (brinogen 2) and homodimeric g0 /g0 molecules account for B15% and less than 1%, respectively. The central E-domain contains the brinopeptides (FP)-A and -B, which are released from the N-terminus of the Aa (Aa 116) and the Bb (Bb 114) chains upon enzymatic cleavage by thrombin. The C-terminal ends of the g-chain are important for interaction with GP-IIb/IIIa receptors on platelets, polymerization of brin, and cross-linking by FXIIIa. They also contain two self-association sites (gXL and D:D, see Figure 1), which participate in the brin(ogen) assembly and cross-linking process, and a high specic binding site for tPA. The C-terminal Aa-chain protuberances contain sites for interaction with platelets (integrin binding to arginineglycineaspartic acid (RGD) sequences). Two sets of high-afnity binding sites for both tPA and plasminogen, which play an important role in the initiation of brinolysis, are located on the a-C-domain and the distal D-domain. Calcium binding sites are integral parts of the brinogen molecule. Among them there are three high-afnity binding sites, two of which are associated with the D-domain and one located at the E-domain. Ca2 is important for stabilizing brinogen against heat denaturation and proteolytic digestion by trypsin or plasmin, and for brin monomer polymerization.

Structure
Fibrinogen is a trinodular disulde-bridged molecule, composed of two symmetrical half molecules, each half consisting of three different polypeptide chains, termed Aa (610 amino acids, 67 kDa), Bb (461 amino acids, 56 kDa), and g (411 amino acids, 47 kDa) (Figure 1). The two halves are covalently joined in an antiparallel orientation in the central Edomain by ve disulde bridges. At the two ends of the cylindrical brinogen molecule, the globular Bband g-C-termini and the Aa-C-terminal random coil form the distal D-domains. The three polypetide -long coiled-coil rechains interweave forming 160 A gions, which connect the central E-domain to the outer D-domains. Inter-chain disulde bonds further stabilize the structure. The overall molecular weight of brinogen is 340 kDa and its overall length is (45 nm). B450 A Each of the polypeptide chains is a product of a single-copy gene located on the long arm of

Fibrinogen Biosynthesis, Fibrin Assembly, Cross-Linking, and Fibrin(ogen)olysis

Fibrinogen is constitutively produced in the liver and circulates in human plasma at a concentration of 150400 mg dl 1, and has a half-life of 35 days. About 75% of the total brinogen pool circulates in plasma whereas the rest distributes to tissues, interstitial uids, and lymph. Plasma brinogen levels are regulated by genetic (e.g., Bb-chain polymorphisms) as well as environmental factors. Each gene is separately transcribed and translated into a precursor polypeptide, which is then processed and assembled into the mature protein. The rate-limiting step in the brinogen biosynthesis is the synthesis of the brinogen Bb-chain. In addition to the liver, brinogen biosynthesis has been found in lung epithelial cells under inammatory conditions. The thrombin-catalyzed release of the brinopeptides FP-A and FP-B exposes two types of binding site in the N-terminal regions of the brin molecules,

COAGULATION CASCADE / Fibrinogen and Fibrin 505

C C C

28 36 65 8 9

N N N

N N N

28 36 65 9 8

C C C

A 67 kDa B 56 kDa 47 kDa

Coiled-coil region D D-domain E E-domain 450 A

Coiled-coil region D D-domain

Db D:D

c FPB

Db D:D

Da
XL

FPA
XL

Da
A

Fibrinogen Thrombin

FPA FPB

Fibrin c Db D:D EB

Db D:D

Da
XL

EA
XL

Da
A

Figure 1 Structural model and domains of brin(ogen). The major domains (E- and D-domains) of brin(ogen) and the constitutive Ddomain association sites participating in brin polymerization and cross-linking are shown. EA/EB polymerization sites that are exposed upon cleavage of the brinopeptides FPA and FPB. Da/Db association sites that interact with available EA/EB sites in brin. gA/g0 normal (gA) and elongated (g0 ) variant of the g-chains with FXIIIa-cross-linking site in the C-terminal region. gXL/D:D self-association sites. ac domain in the C-terminal region of the Aa-chain, which dissociates from its noncovalent association with the E-domain upon thrombin cleavage of FPB. Adapted from Mosesson MW (1998) Fibrinogen structure and brin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169174, with permission from Thieme New York.

termed EA and EB, which combine with constitutive complementary binding pockets in the D-domains (Da, Db) of neighboring molecules. The initial release of FP-A from the Aa-chain and (noncovalent) assembly of Da:EA associations results in the formation of double-stranded, twisted brils. The molecules within a strand are joined by longitudinal contacts between the D-domains (D:D interaction) (Figure 2). Subsequently and concomitantly, brils

undergo branching and lateral bril association resulting in a network of bers with increased thickness. The EB:Db interaction is not absolutely required for lateral bril/ber association, but it contributes to this process through cooperative interactions resulting from alignment of D-domains forming so-called trimolecular and tetramolecular branch points in the brin polymer (Figure 2). The brin clot, initially held together only by noncovalent

506 COAGULATION CASCADE / Fibrinogen and Fibrin

Fibrinogen Thrombin FPA Fibril assembly, branching and lateral fibril associations

D:E

Tetramolecular branch point Trimolecular branch point D:D

Thrombin FPB

FXIII FXIIIa

Cross-linking and fiber growth by lateral fibril associations - trimer D:D D:E c -dimer - tetramer

interactions, becomes further stabilized by the incorporation of covalent bonds between C-terminal gXL sites of brin units. FXIIIa (plasma transglutaminase) catalyzes the formation of covalent e-(g-glutamyl)lysine isopeptide bonds in the presence of Ca2 resulting in the formation of g-dimers and higherorder forms of cross-linked g-chains (g-trimers, g-tetramers). Plasmin-catalyzed degradation of brinogen and brin occurs similarly; however, degradation of cross-linked brin results in somewhat different macromolecular intermediates and end product fragments due to its g-g cross-links. When cross-linked brin is cleaved, the a-chain polymers are removed rst and degraded to low-molecular-weight fragments. Next the coiled-coil region joining the D- and E-domains are cleaved resulting in macromolecular intermediates such as fragment DD/E, fragment YD/ DY, or fragment YX/XD. Terminal split products of cross-linked brin are FsE, D-dimer, as well as D-trimer and D-tetramer derived from tri- and tetramolecular branch points (Figure 2).

Biological Function
The key role of brin(ogen) during hemostasis is to form the framework for thrombus formation. Cellular and proteinaceous components of a thrombus are stabilized by the covalent cross-linking of brin resulting in a stable hemostatic plug of the platelets. Thus, brin stabilizes damaged tissues and serves as a primary matrix of wound repair. However, this function is not solely limited to hemostasis and wound healing but may also play a role in developmental processes. Fibrin also exerts regulatory effects including the initiation of brinolysis and the regulation of thrombin activity. Fibrin, but not brinogen, accelerates the activation of plasminogen by tPA. This is mediated through formation of a tenary complex between tPA and plasminogen on the surface of brin involving the above-mentioned specic binding sites (Aa148160, g312324, Aa392610), which are cryptic in the brinogen molecule and become exposed during brin formation. Next to the initiation of brinolysis, brin is also involved in the modulation of prolonged brinolysis. The most important plasmin inhibitor a2-antiplasmin becomes cross-linked to the Aa-chain of brinogen and brin by activated factor XIII. This cross-linking process is reversible and a2-antiplasmin can also be released from degraded brin. By this, brin restricts plasmin activity to sites where it is required and protects brinogen and other plasma proteins from degradation.

Fibrinolysis
D D E D E D D E D D E D D E D D E D D E D D E D D E D D E D D E D D E D

Two-stranded cross-linked protofibril

DD/E

YD/DY

Lysis intermediate fragments

YX / XD

Lysis terminal split products

D D E D D D D D D D

FsE D-dimer

D-tetramer

D-trimer

Figure 2 Schematic model of brin assembly, cross-linking, and plasmin-catalyzed degradation. D:E noncovalent interactions between EA and Da sites that form end-to-middle staggered overlapping double-stranded brils. D:D longitudinal interaction between two D-domains within a strand. ac C-terminal a-chain protuberances, which become untethered from its noncovalent association with the E domain upon cleavage of FP-B and promote lateral bril associations by self-association with other acdomains. g-dimer, g-trimer, g-tetramer FXIIIa-mediated crosslinks between C-terminal gXL sites of D-domains. DD/E, YD/DY, YX/XD intermediate lysis fragments. FsE, D-dimer, D-trimer, Dtetramer terminal lysis fragments of plasmin-catalyzed brinolysis. Adapted from Mosesson MW (1998) Fibrinogen structure and brin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169174, with permission from Thieme New York.

COAGULATION CASCADE / Fibrinogen and Fibrin 507

Fibrin contains nonsubstrate thrombin-binding sites (two low-afnity sites in the E-domain and one high-afnity site in the g0 -chain) which mediate an antithrombin activity (referred to as antithrombin I). Experimental evidences and clinical observation suggest that antithrombin I is an important factor for downregulating thrombin activity. By this mechanism diffusion of thrombin and the extent of clot propagation is limited. Fibrinogen and brin can interact with cells via integrin binding to RGD motifs in the Aa- and g-chain. Binding of brinogen to the platelet integrin aIIbb3 (also referred to as GPIIb/IIIa-receptor, CD41a) is of central importance during primary hemostasis. Fibrinogen acts as a bridging molecule and thus mediates platelet adhesion, aggregation, and formation of platelet-rich thrombi. Recently, a regulatory role for brinogen in inammatory cell function was discovered. The nding that leukocyte engagement of brin(ogen) via the integrin receptor aMb2 is critical in the inammatory response suggested a physiologically relevant role for brin(ogen) as an inammatory mediator in innate immunity. Targeted deletion of the gene encoding the Aachain or g-chain in mice resulted in animals without detectable plasma brinogen levels. About 30% of Fbg / mice developed bleeding events shortly after birth; however, most of these animals showed a resolution of the bleeding events and survived the neonatal period. In addition, adult mice showed an increased risk for fatal abdominal hemorrhage, and pregnancy resulting in fatal uterine bleeding. A study investigating wound-healing defects in brinogennull mice showed no differences with respect to the time required to overtly heal wounds; however, a role for brin(ogen) in cellular migration and organization of wound elds could be demonstrated. Fibrinogen knockout mice crossed with atherosclerosissusceptible apolipoprotein E-decient mice did not show a decreased extent of atherosclerosis despite the absence of brinogen.

The brinogen receptor represents a target for pharmacological agents that specifically inhibit platelet aggregation. Three intravenous GPIIb/IIIa antagonists are currently marketed for the prevention of myocardial infarction in patients undergoing angioplasty or stenting: the monoclonal antibody Abciximab (mouse-human chimeric Fab fragments), and eptifibatide and tiroban, two low molecular mass inhibitors. The leukocyte integrin aMb2 (CD11b/CD18, Mac1) is a further high-afnity receptor for brinogen on stimulated macrophages and neutrophils. This receptor belongs to the b2 (CD18) subfamily of integrins, which play a pivotal role in leukocyte function. Multiple binding sites, including g190202 and g377395, are implicated in this interaction.

Fibrin(ogen) in Respiratory Diseases

A large number of congenital or acquired brin(ogen) disorders are described as being associated with a bleeding diathesis or thrombophilia. They include abrinogenemia (essentially absent brinogen), hypobrinogenemia (plasma levels less than 100 mg dl 1), or dysbrinogenemia (structurally abnormal brin molecules). Elevated brinogen plasma levels were found to be strongly and independently related to cardiovascular disease such as coronary heart disease and acute myocardial infarction. The use of brinogen plasma levels as a marker predicting the cardiovascular risk has been widely accepted. In contrast, the role of brin(ogen) in respiratory disease is still not fully settled. However, there is increasing evidence that brin(ogen) plays a central role in the pathogenesis of inammatory and chronic interstitial lung disease, including the acute respiratory distress syndrome (ARDS), severe pneumonia, and idiopathic pulmonary brosis (IPF). Hyaline membranes, that is, the accumulation of brin-rich material in the alveolar space, are commonly found in ARDS and other acute or chronic interstitial lung diseases. Such kind of extravascular brin deposition results from imbalanced coagulation and brinolysis. Analysis of bronchoalveolar lavage uids from patients with ARDS demonstrated a prominent procoagulant (tissue factor and FVII) and antibrinolytic (plasminogen activator inhibitor (PAI)-1, a2-antiplasmin) activity, while the brinolytic capacity (urokinase) was found to be markedly depressed. Thus, in concert with brinogen leakage into the alveolar space, rapid and pronounced brin formation is to be expected under these conditions (Figure 3). Furthermore, experimental data suggest a far-reaching incorporation of hydrophobic surfactant compounds into the growing brin matrix. As a

Receptors
The brinogen receptor on platelets has been identied as a membrane-bound heterodimeric glycoprotein complex, termed GPIIb/IIIa. The receptor belongs to the b3 subfamily of integrins (aIIbb3) and binds brinogen via a specic recognition site g400441. Two other potential binding sites are located at Aa9598 (RGDF) and at Aa572575 (RGDS). The latter sequence is also found in other proteins that interact with the integrin receptor such as von Willebrands factor, vitronectin and bronectin.

508 COAGULATION CASCADE / Fibrinogen and Fibrin

Phospholipids SP-B SP-C Normal

Alveolar hemostatic balance: Anticoagulation fibrinolysis Procoagulation antifibrinolysis

Inflammation fibrinogen leakage

Surfactant inhibition: Fibrinogen Fibrinogen + Fibrin oligomer + + Fibrin polymer + + + Polymerizing fibrin = Surfactant-trap Alveolar coagulation atelectasis formation Fibrin oligomer

Specialized alveolar fibrin polymer: Incorporation of pulmonary surfactant with promotion of alveolar collapse Lower susceptibility to fibrinolytic enzymes Altered mechanical properties

Fibrin oligomer

Persistent fibrin deposition Theory: Collapse induration: Persistent atelectasis/ fibrin deposition Fibroblasts Alveolar wall apposition Fibroblast activation Fibrosis Deposition of fibrous tissue Fibrosis, honeycombing
Figure 3 Diagram illustrating the potential mechanisms by which brin(ogen) contributes to respiratory disease. Under physiological conditions the phospholipid lining layer at the airwater interface reduces the surface tension and thereby promotes lung ination upon inspiration and prevents lung collapse during expiration. Under inammatory conditions, brinogen, leaking into the alveoli, is rapidly converted into brin due to a high procoagulant and antibrinolytic activity in the alveolar compartment. Surfactant function is greatly inhibited by incorporation of hydrophobic surfactant compounds (PL, SP-B/C) into polymerizing brin. Persistence or delayed clearance of this specialized brin matrix promotes broproliferative processes (collapse induration) nally resulting in a structural remodeling of the lung with loss of compliance and gas exchange properties.

result, such incorporation may yield a further increase in alveolar surface tension, alveolar instability, and nally gas exchange disturbances (Figure 3). Indeed, pronounced impairment of gas exchange could

be provoked by formation of brin in the distal lung in otherwise healthy lungs. In addition, brin clots embedding natural surfactant display markedly altered mechanical properties

COAGULATION CASCADE / Intrinsic Factors

509

and reduced susceptibility to proteolytic degradation. These properties, persistent alveolar collapse, reduced susceptibility to proteolysis, and sustained suppression of brinolytic enzymes, may prevent rapid clearance of brin from the lungs. Thus, persistent deposition of brin in the distal lung may promote broblast invasion and replacement of the primary brin matrix by a secondary collagenous matrix (Figure 3). Additionally, the brinopeptides A/B and brin(ogen) scission products have, next to thrombin itself, been shown to serve as potent broblast mitogens. However, it has to be stated that some brotic response was also encountered in brinogen null mice, suggesting that at least other extracellular matric (ECM) proteins may replace brin to some extent. Consequently, targeting abnormalities of brin turnover by anticoagulant and brinolytic interventions has been shown to protect the lung in animal models of acute lung injury and reduced the broproliferative response in bleomycin-induced lung brosis. Currently, anticoagulants such as heparin and brinolysins are being tested in ongoing experimental and clinical studies. Finally, it should be mentioned that in recent studies in a mouse model of allergic asthma evidence was provided that brin accumulation contributes to airway hyperresponsiveness.
See also: Acute Respiratory Distress Syndrome. Adhesion, CellMatrix: Integrins. Anticoagulants. Coagulation Cascade: Overview; Antithrombin III; iuPA, tPA, uPAR; Thrombin. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Interstitial Lung Disease: Overview; Alveolar Proteinosis; Amyloidosis; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Surfactant: Overview. Thrombolytic Therapy.

Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and brinolysis in endotoxemia and pneumonia. Critical Care Medicine 31(supplement): S238S242. Medved L and Nieuwenhuizen W (2003) Molecular mechanisms of initiation of brinolysis by brin. Thrombosis and Haemostasis 89: 409419. Mosesson MW (1998) Fibrinogen structure and brin clot assembly. Seminars in Thrombosis and Hemostasis 24(2): 169174. Mosesson MW (2003) Fibrinogen gamma chain functions. Journal of Thrombosis and Haemostasis 1: 231238. Ploplis VA and Castellino FJ (2002) Gene targeting of components of the brinolytic system. Thrombosis and Haemostasis 87: 2231. Wilberding JA, Ploplis VA, McLennan L, et al. (2001) Development of pulmonary brosis in brinogen-decient mice. Annals of the New York Academy of Sciences 936: 542548.

Intrinsic Factors
P F Neuenschwander, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The intrinsic coagulation pathway consists of factors XI, IX, and VIII (fXI, fIX, fVIII). FXI and fIX are serine protease zymogens requiring proteolytic activation to express protease and procoagulant activity. FVIII is a non-enzymatic cofactor that also requires proteolytic activation. FXI can be activated by either fXIIa (contact activation) or by thrombin (positive feedback). The activated fXI (fXIa) subsequently activates fIX to generate fIXa that binds to thrombin-activated fVIII (fVIIIa) to generate a potent procoagulant complex on an anionic phospholipid surface. This complex activates fX to propagate and amplify the coagulant response. The activity of the intrinsic pathway is regulated by the activation status of fVIII, dened by the spontaneous decay of fVIIIa activity. The rapid activation and decay of fVIII/fVIIIa provides a pulse of activity allowing controlled brin deposition to occur without excessive clot formation. This pathway is activated during pathological coagulation in the lung in acute lung injury models. If left unregulated it can lead to excessive generation of fXa, resulting in the deposition of extravascular alveolar brin followed by respiratory failure.

Further Reading
Bennet JS (2001) Plateletbrinogen interactions. Annals of the New York Academy of Sciences 936: 340354. Budzynski AZ (1998) Fibrinogen and brin: biochemistry and pathophysiology. Critical Reviews in Oncology/Hematology 6: 97146. Everse SJ (2002) New insights into brin(ogen) structure and function. Vox Sanguinis 83(supplement 1): 375382. Gu nther A, Ruppert C, Schmidt R, et al. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome. Respiratory Research 2: 353364. Idell S (2002) Endothelium and disordered brin turnover in the injured lung: newly recognized pathways. Critical Care Medicine 30(supplement): S274S280. Idell S (2003) Coagulation, brinolysis, and brin deposition in acute lung injury. Critical Care Medicine 31(supplement): S213S220. Koenig W (2003) Fibrin(ogen) in cardiovascular disease: an update. Thrombosis and Haemostasis 89: 601609.

Introduction
The intrinsic pathway of blood coagulation is so named due to the presence of all the required reactants in the circulation, with no external protein source required. It is evidenced most clearly in vitro by the ability of blood or plasma to spontaneously clot upon its collection into a glass vessel. The trigger for this mode of coagulation is the autoactivation of factor XII (fXII; Hageman factor), which auto-hydrolyzes on anionic surfaces (contact activation) to form fXIIa. In the presence of high-molecular-weight kininogen the fXIIa proteolytically activates fXI to generate fXIa, which subsequently activates fIX

510 COAGULATION CASCADE / Intrinsic Factors

Co nt XII activ act atio n

XIIa

XI XIa Thrombin feedback Ca2+ Ca2+ IX IXa VIIIa/PL Ca2+ X Xa

Figure 1 Intrinsic pathway of coagulation. Consisting of fXI, fIX, and fVIII, the intrinsic pathway of blood coagulation can be initiated by fXIIa via contact activation, by thrombin via feedback activation of fXI, or by the fVIIaTF complex from the extrinsic pathway via crossover activation of fIX. In all cases, fIXa is generated that can form a calcium and phospholipid dependent complex with fVIIIa. This complex subsequently activates fX to amplify and propagate the coagulant response. Green arrows indicate enzymatic actions and blue arrows indicate zymogen to enzyme conversions. Requirements for ionic calcium are indicated.

(Christmas factor). The fIXa that is formed then binds to fVIIIa and this complex activates fX to fXa as part of the common pathway (Figure 1). Deciency of fXII does not result in a hemorrhagic condition. Thus, contact activation is not considered part of the coagulation system per se but rather serves to connect coagulation with inammation and the complement system. In contrast, deciency of factors VIII or IX results in a bleeding diathesis (hemophilias A & B, respectively) as does deciency of fXI (Rosenthal syndrome or hemophilia C), albeit mildly. Thus, the formal contemporary intrinsic pathway of coagulation consists of these three factors. During in vivo coagulation, fIX can be activated by either of two enzymes: fXIa or the fVIIatissue factor (TF) complex from the extrinsic pathway. Since fXIa is not normally present in the circulation, it is the fVIIaTF complex that is likely responsible for the initial activation of fIX. This low level of fIXa is subsequently amplied by fXIa that is generated later in the pathway via a positive feedback reaction involving thrombin.

Intrinsic pathway

VIIaTF Extrinsic system

and 14 introns resulting in an mRNA transcript of 2.2 kb (Figure 2, top). Exon 1 encodes the 50 untranslated region and exon 2 encodes the prepeptide at the N-terminus of the polypeptide. The exon pairs 34, 56, 78, and 910 each encode an apple domain (Apple 14, respectively). Exon 11 encodes the activation region and the remaining exons (1215) encode the protease domain. The fIX gene is located on the X chromosome at position q26-27.3. It is 34 kb in length and consists of 8 exons and 7 introns resulting in a transcript of 2.8 kb (Figure 2, middle). The structure of the fIX gene is very similar to that of fVII, fX, and protein C. Exon 1 encodes the prepeptide, exon 2 encodes the propeptide and Gla domain, exon 3 encodes a small aromatic stack region, exons 4 and 5 each encode an epidermal growth factor (EGF)-like repeat, exon 6 encodes the activation peptide region, and exons 7 and 8 together encode the protease domain. The gene for fVIII is also located on the X chromosome in the region of q28. It is roughly 186 kb in size and consists of 26 exons and 25 introns that produce a transcript of 8.8 kb (Figure 2, bottom). Exon 1 encodes the prepeptide region while exons 2 7 encode most of the A1 domain. The small acidic a3 region is encoded by exon 8 while exons 913 encode the A2 domain. These are followed by the largest exon in the gene (exon 14; 3.1 kb), which encodes the a2, B, and a3 domains. The A3 domain is encoded by exons 1519, and exons 2023 and 2426 encode the C1 and C2 domains, respectively.
Proteins

Structure of the Intrinsic Factors

Genes

The gene for human fXI is on chromosome 4 at position q35. It is 23 kb in length and contains 15 exons

FXI is one of two coagulation enzymes that is not a member of the vitamin-K-dependent protein family. It is synthesized in the liver as a precursor that undergoes proteolytic processing to remove an N-terminal signal sequence of 18 amino acids. The domainal organization of fXI consists of four apple domains, the activation region, and the latent catalytic (protease) domain. FXI is unique among the coagulation proteases in that it exists in plasma as a homodimer linked by a disulde bond between the fourth apple domains (Cys321) of each monomer. FIX is a vitamin-K-dependent protein with a structure similar to that of fVII, fX, and protein C. The pre-pro-fIX polypeptide is synthesized in the liver and is proteolytically processed to remove the leader sequence and propeptide (46 amino acids) before secretion. FIX also undergoes several other posttranslational modications including vitamin-K-dependent g-carboxylation of the rst 12 Glu residues (Gla domain), b-hydroxylation of Asp64, partial sulfation of Tyr155, phosphorylation of Ser158, addition

COAGULATION CASCADE / Intrinsic Factors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

511

Factor XI

Pre

Apple 1

Apple 2

Apple 3

Apple 4

Ap Activation 5 6

Protease domain

Processing 1 2 3 4

Factor IX Pre Pro Gla EGF1 EGF2 AP Protease domain

Processing 1 2 34 56 7 8 9 10 12 13 14

Activation 15 19 21 22 23 24 25 26

Factor VIII

A1

A2

a1

a2

a3

A3

C1

C2

Figure 2 Gene organization of the intrinsic factors. The organization of the genes for fXI (top), fIX (middle), and fVIII (bottom) are depicted with corresponding encoded regions of each protein (not to scale). Exons (coding regions) are shown as black boxes and are numbered sequentially from left to right (50 to 30 ). Introns (noncoding regions) are shown as horizontal lines between exons. The region encoded by each exon is depicted below the gene as a colored bar. The fXI gene is 23 kb in length and encompasses 15 exons (1.5 kb) and 14 introns (21.5 kb). The gene encodes a prepeptide (Pre), four apple domains (Apple14), an activation peptide (AP) region, and the latent protease domain. The fIX gene is 34 kb in length and encompasses 8 exons (2.8 kb) and 7 introns (31.2 kb). The gene encodes a prepeptide (Pre), propeptide (Pro), g-carboxyglutamic acid domain (Gla), aromatic stack region (black), two epidermal growth factorlike domains (EGF12), an activation peptide (AP) region, and the latent protease domain. Enzymatic processing and activation sites in fXI and fIX are indicated by pointers; N-linked glycosylation sites are indicated as tree structures; b-hydroxylated Asp64 in fIX is indicated by b; and the di- and tetrasaccharide on Ser53 and Ser61 in fIX are indicated by black diamonds. The fVIII gene is the largest of the three and is 186 kb in length with 26 exons (9 kb) and 25 introns (177 kb). The gene encodes a prepeptide (red), the A1, a1, A2, a2, B, a3, A3, C1 and C2 domains. Processing and posttranslational modications of fVIII are indicated in Figure 3.

of N-linked carbohydrate at Asn157 and Asn167, and additions of disaccharide (xyloseglucose) to Ser53 and tetrasaccharide to Ser61. Thus, the mature secreted form of the fIX zymogen is a single-chain molecule of 415 amino acids (Mr 55 000) consisting of a Gla domain, two EGF-like repeats, the activation peptide region, and the latent catalytic domain. FVIII is a very large glycoprotein that is the nonenzymatic cofactor for fIXa and is synthesized in the liver. The domainal organization of fVIII (Figure 3) consists of two A domains (homologous to ceruloplasmin), a single large B domain, a third A domain, and two C domains. Each A domain is juxtaposed by a small highly acidic region designated as a1, a2, and a3. The secreted form of fVIII consists of a heavy chain (MrB200 000) and a light chain (MrB80 000) held together through a divalent metal

ion-dependent (Cu2 ) interaction. Further proteolysis within the B domain (likely by plasma proteases) produces a variably sized heavy chain. FVIII is protected from further degradation by circulating in plasma in a noncovalent complex with von Willebrands factor, which stabilizes its activity.

Regulation of the Intrinsic Factors

Activation

FXI is activated by proteolysis at a single site (Arg369) to produce fXIa and express procoagulant activity. This activation occurs via fXIIa or thrombin. Activation by fXIIa occurs when fXI zymogen binds to an anionic surface via high-molecularweight kininogen, whereas activation by thrombin does not have this requirement. In contrast, the

512 COAGULATION CASCADE / Intrinsic Factors

A1 NH2 Processing a1 A2 a2 B a3 A3 C1 C2 COOH Processing ?? SO4 SO4 SO4 SO4 SO4 SO4 SO4 SO4 Factor VIII polypeptide

(a)

s-s

s-s

s-s

s-s

s-s

s-s

s-s

s-s

s-s

s-s

NH2

COOH Processed factor VIII SH SH Heavy chain SH Light chain

(b)
Activation cleavages NH2

A1
Me2+

A2

COOH APC inactivation cleavages

Ac

COOH

C2

C1

A3

NH2
tiva

Activation cleavages

tio

Plasma factor VIII

A1 Me2+

A2

(c)

C2

C1 Factor VIIIa

A3

Figure 3 Organization of factor VIII. (a) The immature fVIII polypeptide containing the prepeptide followed by the A1, a1, A2, a2, B, a3, A3, C1, and C2 domains. The 25 potential N-linked glycosylation sites are indicated as tree structures. The two proteolytic processing sites are indicated by arrows. (b) The fully processed and mature form of fVIII, consisting of a heavy chain and a light chain. The variable size of the heavy chain is due to proteolysis of the B domain after processing (indicated by a fading color pattern). Predicted disulde bonds are indicated above the bar by SS and free sulfhydrils are indicated below the bar by -SH. The positions of the eight known/ proposed sulfated Tyr residues are also indicated (SO4). (c) The heterodimeric nature of plasma fVIII held together by a metal bridge requiring Cu2 . The three activation cleavage sites are indicated by arrows. Activation releases the B and a3 domains and separates the remaining heavy chain into A1a1 and A2a2 subunits. The resulting active fVIIIa heterotrimer is depicted in the bottom right. The A2a2 subunit is held in place solely through electrostatic interactions and readily dissociates to inactivate fVIIIa. The inactivation cleavages catalyzed by activated protein C (APC) are indicated on the fVIIIa molecule by arrows.

activation of fIX requires proteolytic cleavage at two sites (Arg145 and Arg180) to produce the serine protease. This activation is catalyzed by either fXIa or the fVIIaTF complex to produce fIXa. Plasma fVIII requires proteolytic activation at three sites (Arg372, Arg740, Arg1689) to produce the active cofactor (Figure 3). The primary activator of fVIII in vivo is thrombin, although fXa can also catalyze this reaction in the presence of anionic phospholipid and ionic calcium. The resulting active fVIIIa molecule has a substantially reduced size and is a metal-linked (Cu2 ) heterotrimer composed of the A1a1, A2a2, and A3C1C2 fragments.
Activity

Unlike the other coagulation factors, fXIa does not require a cofactor or a phospholipid surface for activity, although ionic calcium is required. This is different from fIXa, which requires binding to its nonenzymatic protein cofactor (fVIIIa) in the

presence of calcium ions and an anionic phospholipid surface to express nominal procoagulant activity. When in this complex, the activity of fIXa is increased by a factor of 109. Thus, it is only when in this complex that fIXa activates fX to propagate and amplify the coagulant response. The activity of the fIXafVIIIa complex is almost entirely regulated by fVIIIa. Once activated, fVIIIa is extremely labile and exhibits a half-life on the order of several minutes. This is largely due to the spontaneous dissociation of the A2a2 subunit domain from the activated heterotrimer (Figure 3). Although the binding of fIXa to fVIIIa somewhat stabilizes this interaction via fIXa binding sites located in the A2 domain, the dissociation of the A2a2 subunit domain remains the limiting factor in the activity of the fIXafVIIIa complex. Once dissociated, the A2a2 subunit domain is susceptible to proteolysis by activated protein C (APC) at Arg562, preventing its re-association. APC also cleaves at Arg336 in the A1a1 subunit, releasing the a1 domain and further

COAGULATION CASCADE / Intrinsic Factors

513

inactivating the cofactor. The inactivation of fVIII by APC is accelerated by protein S, a nonenzymatic cofactor for APC.

Intrinsic Factors in Respiratory Diseases

While pulmonary hemostasis is critical for maintaining lung function, it has been rigorously demonstrated that unregulated and/or ongoing procoagulant activity leads to deposition of extravascular brin. This is observed in instances of sepsis, pneumonia, and other acute events that can all lead to acute respiratory distress syndrome (ARDS). The levels of most clotting factors in these instances are increased in both the alveolar space and pleural cavity. Since the trigger for coagulation is the expression of TF, whose levels are elevated under these circumstances, the extrinsic pathway has been the major focus of investigation in lung injury models. However, the intrinsic factors have also been reported to be present in bronchoalveolar lavage of patients with ARDS, and are also likely to play a role in the propagation of pulmonary brosis. Studies investigating this inference directly have not yet been reported.
See also: Acute Respiratory Distress Syndrome. Anticoagulants. Chemokines. Coagulation Cascade: Antithrombin III; Factor VII; Factor X; Fibrinogen and Fibrin; Protein C and Protein S; Thrombin; Tissue Factor. Complement. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: Monocytes. Platelets. Proteinase-Activated Receptors. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Biological Function
The fXI and fIX zymogens exist in plasma at a level of 5 mg ml 1 with half-lives of 6080 h for fXI and 24 h for fIX. In contrast, the plasma level of fVIII is 50 times lower (B0.1 mg ml 1). The half-life of fVIII in plasma is ambiguous, but is probably around 812 h. The clearance of fVIII from plasma involves the low-density lipoprotein receptor-related protein (LRP). This is supported by a murine LRP knockout model that shows an increase in circulating fVIII levels. The major roles of the intrinsic factors are to amplify the coagulant response upon initiation by the fVIIaTF complex (Figure 1). This necessarily involves the activations of fIX by fXIa and fX by the fIXafVIIIa complex. These proteins are also all intimately involved in coagulation regulation. As centralized players in the coagulation cascade, the activity of these proteins has a major impact on the level of coagulation attained. This is due to a combination of the positive feedback activations of fXI and fVIII by thrombin, as well as the rapid decay of fVIIIa activity. The combination of these events results in an intense but brief pulse of procoagulant activity. This allows for both the rapid formation of a brin clot as well as the prevention of excessive clot formation. The importance of the intrinsic factors is evidenced in the bleeding diatheses observed in patients that are decient in these factors as well as in murine knockout models, which all show prolonged bleeding consistent with the hemophilias. Interestingly, the combined knockout of fXI and protein C partially corrects the hypercoagulable state observed in protein C knockouts, which is associated with severe protein C deciency in humans. This suggests an additional potential role of fXI (and by inference the intrinsic pathway) in certain thrombotic states.

Further Reading
Abraham E (2000) Coagulation abnormalities in acute lung injury and sepsis. American Journal of Respiratory Cell and Molecular Biology 22: 401404. Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113: 148157. Fay PJ and Jenkins PV (2005) Mutating factor VIII: lessons from structure to function. Blood Reviews 19: 1527. Gui T, Lin HF, Jin DY, et al. (2002) Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo. Blood 101(1): 153158. Ichinose A and Davie EW (1994) The blood coagulation factors: their cDNAs, genes, and expression. In: Colman RW, Hirsh J, Marder VJ, and Salzman EW (eds.) Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn., pp. 1954. Philadelphia: J.B. Lippincott Company. Idell S (2003) Coagulation, brinolysis, and brin deposition in acute lung injury. Critical Care Medicine 31: S213S220. Laterre P-F, Wittebole X, and Dhainaut J-F (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31: S329 S336. Levi M, Schultz MJ, Rijneveld AW, and van der Poll T (2003) Bronchoalveolar coagulation and brinolysis in endotoxemia and pneumonia. Critical Care Medicine 31: S238S242. Mann KG, Gaffney D, and Bovill EG (1995) Molecular biology, biochemistry, and lifespan of plasma coagulation factors. In: Beutler E, Lichtman MA, Coller BS, and Kipps TJ (eds.)

Receptors
Unlike other coagulation factors (fVIIa, fXa, and thrombin) none of the intrinsic factors possess cell signaling activity. Platelets have been shown to have specic receptors for fIXa, fVIIIa, and fXIa that may play a role in modulation of their activities. Similarly, endothelial cells and collagen IV have been reported to express fIXa receptors that may inuence fIXa plasma concentrations and/or activity. None of these receptors are currently well described.

514 COAGULATION CASCADE / iuPA, tPA, uPAR

Williams Hematology, 5th edn., pp. 12061226. New York: McGraw-Hill. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 13341349. Welty-Wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725.

iuPA, tPA, uPAR

M A Olman, University of Alabama, Birmingham, AL, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The brinolytic system is a cascade of enzymes, and their inhibitors and cell surface receptors which were initially characterized based on their capacity to proteolytically degrade brin. The effector proenzyme plasminogen is widely distributed in tissue as an inactive zymogen, and its activity is regulated largely at the level of activation by the plasminogen activators, urokinase and tissue-type plasminogen activator (tPA). Studies in vitro and in transgenic/gene deleted mice amply demonstrate that the role of this enzyme system extends far beyond the dissolution of brin, to tissue remodeling, growth factor modulation, and cell migration. Not surprisingly, plasminogen activators, their naturally occurring inhibitor, plasminogen activator inhibitor type I (PAI-1), and plasminogen activator receptors have been implicated in a number of respiratory disorders including acute lung injury, pulmonary brosis, airway remodeling in asthma and infectious pneumonia. In some disorders, the proximate consequences of the plasminogen activators/inhibitors on disease pathogenesis are independent of their known mediation of the dissolution of brin.

themselves have yielded a number of therapeutic agents that are improved over the naturally occurring molecules in their enhanced brin selectivity, prolonged half-life, and greater resistance to inhibition by PAI-1. Since then, in vitro and transgenic mice studies have extended the inuence of these serine protease/inhibitors to other biological processes including tissue repair/remodeling, development, angiogenesis, and malignancy. More recently, it has been recognized that the lung exhibits compartmentalized plasminogen activator (PA) activity, with tPA being the prime vascular compartment PA, while urokinase plasminogen activator (uPA) is the prime alveolar and interstitial PA. In contrast, plasminogen and PAI-1 are detected in all lung compartments under various circumstances.

Structure
The proteases of the brinolytic cascade are trypsinlike proteases that are produced by many cell types and tissues as inactive single-chain zymogens. They are activated through proteolytic cleavage at an Arg or Lys residue, to generate a disulde bonded, active two-chain form. The mature proteins share certain structural features including an EGF-like domain, a variable number of kringle domains, and the key serine in their protease catalytic domain in the B chain (see Table 1). The exception to this rule is tPA which is active in its single-chain form. tPA also has an N-terminal bronectin homology domain, which functions to bind to brin, while plasminogen has an N-terminal autoactivation peptide that changes its brin-binding kinetics and susceptibility to activation by uPA/tPA. A kringle-like structure, kringle 2 of tPA, binds to brin, lysine binding sites, and o amino acids (EACA). The major naturally occurring inhibitor of PA, PAI-1, is a 52 kDa glycoprotein member of the serine protease inhibitor (serpin) family that also includes the plasmin inhibitor, a-2 antiplasmin. PAI-1 is the predominant inhibitor for both tPA and uPA, and has a second order rate constant for inhibition of 107108 M 1 s 1. PAI-1 functions as a pseudosubstrate that forms sodium dodecyl sulfate (SDS)stable, PAPAI-1 complexes of 1:1 stoichiometry, followed by cleavage of the peptide bond at Arg346 Met347 (P1P10 ) bond in the PAI-1 reactive center loop. PAI-1s activity is highly dependent on the conformation of this reactive center loop. PAI-1 is secreted in an active form with an exposed reactive center loop, while spontaneous/induced insertion of the loop into the b-sheet structure results in latency (plasma half-life t1=2 90 min at 371C). In a third conformation, PAI-1 is instead cleaved by PAs at the

Introduction
The concept of brinolysis was rst proposed in the 1890s following observation of spontaneous dissolution of a blood clot, and it was recognized as a proteolytic process a decade later. Prior to World War II, it was discovered that ltrates from hemolytic streptococci (subsequently named streptokinase) required a human plasma-derived component(s) (subsequently termed plasminogen) to induce brinolysis. The recognition by Astrup and colleagues in 1947 that animal tissues contain a factor that can activate plasminogen and dissolve brin clots jump-started modern investigations in the eld. Tissue-type plasminogen activator (tPA) was puried from human uterine tissue and the general molecular mechanistic model of tPA-dependent brinolysis was developed in the late 1970s, followed by the purication and cloning of the brinolytic inhibitor, plasminogen activator inhibitor type I (PAI-1) a decade later. Structurefunction studies on the plasminogen activators

COAGULATION CASCADE / iuPA, tPA, uPAR

Table 1 Key features of brinolytic system molecules Name uPA; PLAU Chromosome 10q24 Mr (kDa) 54 AA (#) 411 Conc. 40 pM t1=2 7 min Key domains

515

tPA; PLAT

8p12-p11

68

530

70 pM

4 min

Plasminogen; PLG

6q26-q27

92

791

2 mM

50 h

PAI-1

q22.1-22.3

50

379

300 pM

10 min

uPAR; PLAUR

19q13.1-q13.2

55

313

EGFAA 4-43 binds uPAR kringle, Lys 158Ile159 activation site, B chain catalytic domain FN-repeat binds brin, EGF, kringle 1, kringle 2 binds brin, Arg275Ile276 activation site, B chain catalytic domain N-term activation peptide Lys78 enhanced PA activation and brin binding; kringle 15 bind brin, a-2-antiplasmin, extracellular matrix; Arg561Val562 activation site B chain catalytic domain AAs 55,109,110,116,123 together bind vitronectin; reactive center Arg346Met 347, reactive center loop Ser 331 Arg346 D1 binds uPA, D2/3 enhance uPA binding, C-term GPI link

Adapted from Colman RW, Hirsh J, Marder VJ, Clowes AW, and George JN (eds.) (2001) Thrombosis and Hemostasis, 4th edn. Philadelphia, PA: Lippincott Williams and Wilkins.

Arg346Met347 bond, or other nontarget proteases in the reactive center loop (i.e., elastase, thrombin). Binding of PAI-1 to vitronectin in the blood or the extracellular matrix stabilizes PAI-1 in the active conformation, alters its protease specicity, and promotes its clearance through the low-density lipoprotein receptor-related protein (LRP).

Regulation of Production and Activation

Serine proteases of the brinolytic system are synthesized in several cell types and circulate largely as inactive (zymogen) forms. The coagulation/brinolytic proteases are in the trypsin-like protease family and cleave their target proteins at arginine or lysine bonds. Key biochemical features of this cleavage include the formation of an ester bond between the Ser residue oxygen of the protease and the acyl group of the substrate, resulting in the formation of a tetrahedral complex. This is followed by the loss of the C-terminal portion of the substrate to yield an acylenzyme intermediate. The Seracyl bond is then catalytically replaced by water to release the N-terminal substrate fragment and regenerate the active site serine in the enzyme. The main site of synthesis of tPA is in endothelial cells, where tPA is stored in granules, but other mesenchymal cells can produce tPA including monocytes, smooth muscle cells, and broblasts. Regulated synthesis and release of tPA are induced by multiple stimuli including stress, adrenergic stimulation, DDAVP, histamine, and thrombin. tPA activity in plasma also exhibits diurnal variation. uPA is

present widely throughout most connective tissues, and is expressed in broblasts, epithelial, and mononuclear lineage cells. While uPA is present at high concentration in urine (50 mg ml 1) and the kidney, it is detected at low concentration (2 ng ml 1) in plasma where it is found as a free, single chain, inactive form (scuPA), with a t1=2 similar to that of tPA. Plasma levels of uPA are less volatile than those of tPA and PAI-1. ScuPA is converted into an active form through proteolytic cleavage by a number of different proteases, and through autoactivation. These include kallikrein, mast cell tryptase, cathepsins, leukocyte elastase, and some metalloproteinases. By virtue of uPA binding to its cognate receptor, uPAR, uPA-dependent plasminogen activation at the cell surface is enhanced, and uPAPAI-1 complexes bound to uPAR are internalized, with recycling of unbound uPAR to the cell surface. The serpin inhibitor, PAI-1, is produced in an active form by many cell types in a highly regulated fashion. PAI-1 expression is regulated by such varied factors and events as the cell cycle, thrombin, insulin, very-low-density lipoprotein (VLDL), brin fragments, interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF-a), and transforming growth factor beta (TGF-b). It also exhibits diurnal variation, which may account for the diurnal variation in thrombotic events. Plasma PAI-1 levels in normal subjects are highly variable ranging from 2 to 4100 ng ml 1, and it has a circulating t1=2 exponential a phase of 8 min, and b phase of 30 min. A second major pool of blood PAI-1 is contained within platelets, in an inactive form.

516 COAGULATION CASCADE / iuPA, tPA, uPAR

Biological Functions
Transgenic mice studies indicate that tPA and uPA are redundant for plasminogen activation and thrombolysis, whereas plasminogen is uniquely important for thrombolysis. tPA-dependent brinolysis is relatively brin localized, by virtue of the formation of a ternary complex of brin, plasminogen, and tPA. Mice decient in tPA exhibit a mild thrombophilic phenotype, and abnormal neurological function. The enzymatic activity of the single chain uPA is a matter of some controversy; nonetheless, in vivo, even a small amount of active plasmin or uPA can activate single chain uPA. Active uPA cleaves the Arg561Val562 bond of plasminogen to generate plasmin; and scuPA and plasminogen can undergo reciprocal activation, as well as autoactivation. Despite the absence of direct binding of scuPA to brin, it exhibits some significant brin specicity. Furthermore, the binding of scuPA to its receptor, uPAR, increases its catalytic efciency on the cell surface by two orders of magnitude. Other biologically relevant proteolytic functions of uPA are the activation of other proenzymes (i.e., MMP-2), progrowth factors (HGF, bFGF, FGF2), and cleavage of matrix bronectin (Figure 1). uPA can act as a mitogen and its N-terminal fragment binds to some integrins in a uPAR-independent manner. Numerous signaling events occur as a consequence of uPA binding to its cognate receptor (Kd 1 nM), uPAR, through uPAs

N-terminal region (see below). As with tPA deletion, mice devoid of uPA have a mild thrombophilic phenotype. In contrast to tPA deletion, uPA deletion in mice significantly affects tissue remodeling/repair processes in multiple organs including the lung, kidney, and vasculature, sometimes in a manner that is independent of uPAR. uPA knockout mice have impaired inammatory cell recruitment, and are more susceptible to infection from several Gram-positive and Gram-negative organisms, Cryptococcus neoformans, and Pneumocystis carinii, in part through mediating T-lymphocyte proliferation.

Receptors
Receptors for tPA can be divided into those that contribute to activation and those that participate in the clearance of tPA and tPA/PAI-1 complexes. The urokinase receptor (uPAR; CD-87) is a 5060 kDa heavily glycosylated cell surface receptor for the serine protease urokinase (uPA; Kd 1 nM). uPAR is linked to the exoplasmic leaflet of the plasma membrane through a C-terminal glycosylphosphatidylinositol (GPI) chain (Figure 2). Despite the absence of a transmembrane or intracytoplasmic domain, intracellular signaling through uPAR is well documented, and it is largely dependent on its ligation with uPA. Signaling through uPAR can occur directly, through poorly characterized mechanisms, or by the association of uPAR with other signaling partners such as

Prourokinase (scuPA) PAI-1 Plasminogen

tcuPA

Plasmin

-2-antiplasmin

Fibrin PAI-1 ProMMPs ECM glycoproteins Growth factors Receptor-mediated events

FDPs Active MMPs ECM fragments Activate, INH, ECM release Proliferation, adhesion/migration, survival, ECM synthesis

Figure 1 Fibrinolytic cascade in tissue repair and remodeling. The zymogen (scuPA) is activated by plasmin or auto-activated into active two-chain uPA (tcuPA). tcuPA will activate the millimolar concentrations of plasminogen present in plasma and in tissue to form the broad spectrum serine protease plasmin. The consequences of plasmin protease action include brinolysis, activation of proforms of matrix metalloproteinases (MMPs), direct cleavage of matrix glycoproteins such as laminin and helicase cleaved collagen, activation/ inactivation (INH) of growth factors and/or their release from the extracellular matrix (ECM). The serpin PAI-1 will bind and inactivate tcuPA, while a-2 antiplasmin will bind and inactivate plasmin, thereby effectively downregulating plasmin action. Through binding of scuPA and plasminogen to their cell surface receptors, plasminogen activation is more favorable, and its inhibition by a-2-antiplasmin is less favorable. Cell-localized uPA/PAI-1 may also regulate inammatory and mesenchymal cell adhesion/migration through uPARmediated events.

COAGULATION CASCADE / iuPA, tPA, uPAR

Inhibits uPA
PA I-1

517

ECM degradation Induction of cell migration Activation of growth factors Plasmin uPA

Adhesion Signal transduction ECM Vitronectin

Plasminogen uPAR Extracellular space

Integrins Cell membrane

Intracellular signaling cascade Induction of cell proliferation Src FAK

Intracellular space

Figure 2 uPAR operates independently and cooperatively as a multifunctional receptor. uPA binding to uPAR enhances its interaction with vitronectin, enhances local cell surface plasmin generation, directly initiates intracellular signal cascades, and enhances uPAR interactions with other transmembrane receptors, such as integrins. PAI-1 binds to solution phase or receptor-bound uPA, leading to internalization of PAI-1uPAuPAR complexes by LRP and recycling of uPAR to the cell surface. Under some circumstances, integrin receptors are internalized as well leading to cell detachment. uPAR is thus capable of modulating cell migration, proliferation, cell survival, as well as cell surface localized proteolysis. FAK, focal adhesion kinase. Adapted from Wilex AG and from Sperl S, Mueller MM, Wilhelm OG, et al. (2001) The uPA/uPA-receptor system as a target for tumor therapy. Drug News & Perspectives 14: 401411.

integrins and G-protein-coupled receptors. uPAR-initiated signals utilize a number of intracellular downstream pathways. uPA/uPAR may modulate cell motility/migration through protease-related and nonprotease-related mechanisms. Studies of pulmonary disease in transgenic mice support an important role for uPAR in modulating cell migration that is largely independent of its role in modulating proteolysis. For example, neutrophil recruitment in response to a murine interleukin-8 homolog is inhibited by nonproteolytically active uPA. Leukocyte recruitment in vivo is dependent on uPAR-integrin (aMb2, Mac-1) interactions as demonstrated in Pseudomonas aeruginosa infection of the lung or recruitment into the inamed peritoneum. uPAR also is important for leukocyte recruitment in the Mac-1-independent pulmonary infection due to Streptococcus pneumoniae, in a uPA-independent manner in antigen-inamed lung, or in bleomycin-induced lung injury. On balance, one can say that uPAR-b2 interactions mediate leukocyte recruitment, but other uPAR nonproteolytic functions (i.e., signaling, interactions with vitronectin, etc.) may also condition the response. The best evidence for the role of uPAR in tissue repair and

remodeling is in a ureteral obstruction model of renal brosis, where uPAR-decient mice have increased total collagen and increased brotic interstitial area.

Fibrinolytic System in Respiratory Diseases

Given the protean nature of the biological processes that depend on the brinolytic system it is not surprising that it plays a role in infectious, inammatory, allergic, brotic, and vascular diseases of the lung. The normal brinolytic activity of the lung is compartmentalized with uPA, the predominant alveolar/parenchymal PA, and tPA, the predominant endovascular PA. With an excess of PAs over PAI-1 activity, and ample tissue plasminogen, the normal lung possesses a net profibrinolytic activity.
Acute Lung Injury

Most studies of acute lung injury (ALI) in both patients and experimental animal models demonstrate an early (begins hours after injury), transient (approximately 710 days), large increase in alveolar/ parenchymal PAI-1 and a-2 antiplasmin, which is

518 COAGULATION CASCADE / iuPA, tPA, uPAR

greater than the increases observed in PAs. This incurs a net inhibition of brinolysis that, along with inux of brinogen and increased tissue factor procoagulant activity, induces an alveolar and parenchymal deposition of brin. Under some circumstances, plasminogen availability may be the rate-limiting step. Despite the inux of plasmaderived PAs and PAI-1, resident/inammatory lung cells locally produce a significant fraction of the increased PAI-1. The alveolar brin persists and contributes to the formation of hyaline membranes that are one of the hallmarks of diffuse alveolar damage, the characteristic pathology of the ALI syndrome. The increased susceptibility of mice with deletion of PAI-1 to hyperoxic lung injury supports the biological importance of this pathway. Recent data in ALI patients suggest that the level of PAI-1 in pulmonary edema uid may not only discern those with ALI from those with hydrostatic pulmonary edema, but may also predict outcome in ALI. Although PAI-1 induction appears to exert a dominant inuence over the pathobiology of ALI, uPA also plays a role. For example, uPA knockout mice are also protected from endotoxin-induced lung injury, and uPA participates in LPS-induced neutrophil activation in vitro. Hypoxia itself may contribute significantly to the induction of PAI-1 seen in ALI as hypoxic exposure of mice induces macrophage PAI-1 expression in lung. This PAI-1 increase may contribute to the excess intravascular brin observed in the hypoxia-exposed wild-type mouse as compared with the PAI-1 decient mouse.
Pulmonary Fibrosis

shares some of the histologic features of the human disease; however, it is not progressive and is highly inammatory, and when bleomycin is administered intratracheally, the brosis is bronchocentric. In the bleomycin model of broproliferative lung disease, PAI-1 is required, while plasminogen and/or tPA- or uPA-decient mice have an enhanced brotic response compared to wild-type mice, indicating a net brinolytic activity is protective against brosis in this model. Interestingly, PAI-1s effect appears to be independent of its capacity to mediate brinolysis, as mice devoid of brinogen have a brotic response to bleomycin comparable to that of wild-type mice. Other considerations may be activation and/or matrix release of growth factors by plasmin and/or uPA. Attempts at inducing a net profibrinolytic state through intratracheal exogenous urokinase, or through lung-specic or adenovirally mediated uPA overexpression, have been uniquely successful in reducing established brosis. Furthermore, aerosolized uPA can reduce both the physiological and histological and biochemical derangements seen in rabbits after bleomycin instillation, when begun during the active brotic phase. These observations provide therapeutic impetus, as patients with UIP usually present after the brotic process is well established.
Asthma

Pulmonary brosis is characterized by the deposition of disordered extracellular matrix in the alveolar and parenchymal compartments of the lung. Histologically, usual interstitial pneumonia (UIP) demonstrates a spatial heterogeneity. Areas of normalappearing tissue are adjacent to areas of relatively acellular, brin-rich, brotic tissue, which are adjacent to areas of broblast/myobroblast-rich foci, that are actively remodeling. These broblastic foci have recently been shown to correlate with prognosis in UIP. The bronchoalveolar lavage (BAL) from patients with pulmonary brosis have increased levels of PAI-1 and the procoagulant, tissue factor. Furthermore, primary broblasts isolated from patients with idiopathic pulmonary brosis express high levels of uPAR, which along with uPA stain positive in brotic lesions. Most of the observations implicating the brinolytic cascade in broproliferative lung disease have been derived from experimental animal models of bleomycin-induced brosis. This model

Mast cells activated by phorbol ester/calcium ionophore upregulate their PAI-1 expression, and lung mast cells from patients with asthma exacerbations express PAI-1. Experimental evidence for a pathogenic role for PAI-1 in airway remodeling is available. High levels of PAI-1 and TGF-b are expressed in the airway epithelium in the ovalbumin-sensitized, murine asthma model. Further, ovalbumin-sensitized PAI-1-decient mice exhibit increased airway collagen and brin deposition after ovalbumin challenge, as compared with wild-type mice, but exhibit no difference in airway inammation.
Infectious Lung Disease

In patients with moderate to severe bacterial pneumonia, excess brin degradation products and increased PAI-1 in the alveolar compartment occur, albeit less dramatically than that seen in ALI. Furthermore, these changes are anatomically restricted to the consolidated lobes. However, it is unlikely that a PAI-1-mediated inhibition of brinolysis is critical to the pathogenesis of the bacterial pneumonitis. Studies of murine pneumococcal pneumonia in PAI-1 or plasminogen-decient mice demonstrate an inammatory cell recruitment, bacterial outgrowth, and survival comparable to that in wild-type mice.

COAGULATION CASCADE / Protein C and Protein S

519

These observations support a nonbrinolytic role for uPA and uPAR in the host response to bacterial pulmonary infection, as described above. There is a role for uPA in immune-mediated lung diseases. uPA is important for T-lymphocyte proliferation, Th1-type immune response to Cryptococcus neoformans, and Th2-type cytokine response to schistosomal antigen challenge in mice. In contrast, uPAR, independent of uPA, plays a major role in pulmonary recruitment of lymphocytes after experimental intratracheal antigen challenge.
Pleural Disease

Exogenous scuPA administration has shown promise as a therapeutic agent that will prevent pleural adhesions in tetracycline-instilled rabbits. However, a recent double-blind trial of 454 patients with pleural space infection, treated with intrapleural streptokinase or placebo plus routine care, indicates no difference in the mortality rate, the rate of surgery, the radiographic outcome, or the length of hospital stay.
See also: Acute Respiratory Distress Syndrome. Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Anticoagulants. Arteries and Veins. Asthma: Overview. Caveolins. CD11/18. Coagulation Cascade: Overview. Extracellular Matrix: Degradation by Proteases. Fibrinolysis: Overview. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Matrix Metalloproteinases. Pneumonia: Overview and Epidemiology. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

matrices by inactivating integrins. Journal of Cell Biology 160: 781791. Eitzman DT, McCoy RD, Zheng X, et al. (1996) Bleomycin-induced pulmonary brosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. Journal of Clinical Investigation 1: 232237. Gunther A, Lubke N, Ermert M, et al. (2003) Prevention of bleomycin-induced lung brosis by aerosolization of heparin or urokinase in rabbits. American Journal of Respiration and Critical Care Medicine 168: 13581365. Gyetko MR, Sud S, Sonstein J, et al. (2001) Cutting edge: antigendriven lymphocyte recruitment to the lung is diminished in the absence of urokinase-type plasminogen activator (uPA) receptor, but is independent of uPA. Journal of Immunology 167: 55395542. Hattori N, Degen J, Sisson T, et al. (2000) Bleomycin-induced pulmonary brosis in brinogen-null mice. Journal of Clinical Investigation 106: 14411443. Idell S, James KK, Levin EG, et al. (1989) Local abnormalities in coagulation and brinolytic pathways predispose to alveolar brin deposition in the adult respiratory distress syndrome. Journal of Clinical Investigation 84: 695705. Maskell NA, Davies CW, Nunn AJ, et al. (2005) U.K. controlled trial of intrapleural streptokinase for pleural infection. New England Journal of Medicine 352: 865874. Olman MA (2003) Mechanisms of broproliferation in acute lung injury. In: Matthay MA and Lenfant C (eds.) Acute Respiratory Distress Syndrome, pp. 313354. New York: Dekker. Prabhakaran P, Ware LB, White KE, et al. (2003) Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema uid are associated with mortality in acute lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 285: L20L28. Sperl S, Mueller MM, Wilhelm OG, et al. (2001) The uPA/uPAreceptor system as a target for tumor therapy. Drug News & Perspectives 14: 401411.

Protein C and Protein S

Further Reading
Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890897. Blasi F and Carmeliet P (2002) UPAR: a versatile signalling orchestrator. Nature Reviews Molecular Cell Biology 3: 932 943. Blasi F, Vassalli J-D, and Dano K (1987) Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. Journal of Cell Physiology 104: 801804. Chapman HA (1997) Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Current Opinion in Cell Biology 9: 714724. Cho SH, Tam SW, Demissie-Sanders S, Filler SA, and Oh CK (2000) Production of plasminogen activator inhibitor-1 by human mast cells and its possible role in asthma. Journal of Immunology 165: 31543161. Colman RW, Hirsh J, Marder VJ, Clowes AW, and George JN, (eds.) (2001) Thrombosis and Hemostasis, 4th edn. Philadelphia, PA: Lippincott Williams and Wilkins. Czekay R-P, Aertgeerts K, Curriden S, and Loskutoff D (2003) Plasminogen activator inhibitor-1 detaches cells from extracellular

E C Gabazza, O Taguchi, and K Suzuki, Mie University Graduate School of Medicine, Mie, Japan
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Protein C and protein S are essential factors of the protein C pathway, which plays a fundamental role in the regulation of the coagulation/brinolysis system, inammation, and cell survival. The conversion of protein C to its active form, activated protein C, occurs on the phospholipid-rich surface of cell membranes in the presence of calcium, thrombomodulin, and its receptor endothelial protein C receptor. Protein S acts as a cofactor by enhancing the function of activated protein C. Activated protein C suppresses coagulation activation by inhibiting activated factors V and VIII, suppresses the inammatory response by inhibiting cytokine secretion, and improves cell survival by inhibiting apoptosis. Chronic inammatory disorders of the lung are associated with decreased activation of protein C. Treatment with activated protein C improves outcome in patients with sepsis. Activated protein C is also effective for the treatment of lung brosis and bronchial asthma in animal models. Activated protein C therefore represents a promising

520 COAGULATION CASCADE / Protein C and Protein S

therapeutic approach for incurable inammatory disorders of the lung.

Structure
PC is synthesized in the liver and endothelial and lung cells. The molecular weight of human PC is 62 kDa, and its half-life in the systemic circulation is 8 h. The gene structure of PC shows high sequence homology with other vitamin K-dependent factors. The human PC gene is located on chromosome 2, position q13q14, and spans approximately 11.2 kilobases of genomic DNA; it contains nine exons and eight introns. The molecular structure of PC consists of two polypeptide (a light and a heavy) chains of unequal length linked by a single disulde bond. The light chain contains the vitamin K-dependent g-carboxy glutamic acid (Gla) domain and two epidermal growth factor-like domains (Figure 1). Binding of the Gla domain to negatively charged phospholipids is fundamental for the anticoagulant activity of APC. The Gla domain has high afnity for calcium ions, which appear to be essential for PC binding to phospholipids on cell membrane. Calcium binding to the Gla domain also changes the conformation of the PC molecule, making its structure more suitable for interacting with the thrombinTM complex. EPCR, which inuences the rate of PC activation, also interacts with the Gla domain of PC. The two epidermal growth factor (EGF)-like domains appear to be important for changing the conformation of the PC molecule to facilitate its interaction with PS. The heavy chain of PC contains a short-activation peptide
COOH

Protein C (PC) and protein S (PS) are vitamin Kdependent glycoproteins and essential components of the PC pathway. The circulating level of PC is approximately 24 mg l 1 and that of total PS is on average 2025 mg l 1. PC is the zymogen precursor of activated PC (APC), which is the effector enzyme of the PC pathway. PC is converted to its active form, APC, by thrombin bound to thrombomodulin (TM) on the phospholipid surface of endothelial cells, epithelial cells, monocytes, and platelets. PS acts as a cofactor of APC by enhancing the proteolysis of coagulation factors Va and VIIIa. Endothelial protein C receptor (EPCR) is another factor required for optimal activation of PC. EPCR is the receptor of both PC and APC that was originally described on the cell surface of vascular endothelial cells but later found to be also present in bronchial and alveolar epithelial cells. EPCR enhances PC activation by presenting the PC substrate to the thrombinTM activation complex. The PC pathway is involved in multiple biological functions in the lung. It plays a protective role in diverse acute and chronic inammatory diseases of the lung, including acute respiratory distress syndrome, lung brosis, and bronchial inammation, by virtue of its inhibitory activity on coagulation, inammation, extracellular matrix accumulation, and its profibrinolytic effect.

Activation peptide

NH2 H

Gla domain

EGF-like domain

Catalytic domain

Figure 1 Structure of human protein C. The light chain of protein C is composed of the g-carboxy glutamic acid (Gla) domain that binds to negatively charged phospholipids, an aromatic region, and two epidermal growth factor-like domains. The heavy chain consists of the serine protease domain and the activation peptide, which is released after cleavage by the thrombin/thrombomodulin complex. Purple cycles indicate activated peptide. Red cycles indicate active residues: His211 (H), Asp257 (D), and Ser360 (S).

COAGULATION CASCADE / Protein C and Protein S

521

and a serine protease or catalytic domain. The activation peptide is released during PC cleavage by the thrombinTM complex. PS is another vitamin K-dependent anticoagulant protein whose major function is to facilitate the inhibition of factor Va and factor VIIIa by APC. PS circulates in plasma in both a free, active form and an inactive form bound to C-4b binding protein (C4BP) of the complement system. Expression of PS has been described in the liver, lung, lymphocytes, brain, megakaryocytic cell lines, and osteoblasts. Two genes of PS have been identied on chromosome 3, in position p11.111.2; one is the active gene that contains 15 exons and 14 introns, and the other (pseudogene) has high sequence homology with the active gene but lacks exon 1. The molecular weight of PS is 69 kDa, and its half-life in circulation is 30 h. The mature PS is a multidomain glycoprotein containing a phospholipid-binding Gla domain, a thrombin-sensitive region, four EGF-like domains, and a sex hormone-binding globulin-like domain region containing two laminin G-type repeats (Figure 2). As in other vitamin K-dependent proteins, the Gla domain is necessary for PS binding to negatively charged phospholipid and for supporting APC binding to the membrane. The thrombin-sensitive region and the rst EGF-like domain appear to help in the orientation and localization of APC during its interaction with factor Va and factor VIIIa. The second

EGF-like domain and the laminin G-type domains are also indispensable for full expression of APC cofactor function of PS. The laminin G-type domain contains the binding site for C4BP.

Activation of PC
Several factors, including negatively charged phospholipids, calcium ions, thrombin, the two cell membrane-bound proteins TM and EPCR, and platelet factor-4, are required for optimal generation of APC. PC activation occurs in a stepwise manner. In the rst step, thrombin binds to TM, and this binding alters the substrate specicity of thrombin so that it is no longer able to convert brinogen to brin, activate factor V, or induce aggregation of platelets. In the second step, PC interacts with both components of the complex through calcium bridges. The thrombinTM complex then activates PC by the cleavage of an internal peptide bond and releases an activation peptide. EPCR favors the interaction of PC with the thrombinTM complex and thereby enhances generation of APC (Figure 3). Platelet factor-4 may modulate PC and TM by interacting with the Gla domain of PC and with the O-linked carbohydrate region of TM, respectively, resulting in enhanced APC generation. PC inhibitor (PCI) may also regulate the activation of PC by the thrombinTM

NH2

COOH

Gla Thrombindomain sensitive domain

EGF-like domains

Sex hormone-binding globulin-like domain

Figure 2 Structure of human protein S. The mature protein S contains a g-carboxy glutamic acid (Gla) domain, an aromatic region, a thrombin-sensitive region, four epidermal growth factor-like domains, and the sex hormone-binding globulin-like domain.

522 COAGULATION CASCADE / Protein C and Protein S

TF/VIIa IX IXa PL, Ca2+, Mg2+, VIIIa PS APC X Xa PL, Ca2+, Va PAR-1 EPCR PS-C4BP APC-PCI PCI

Prothrombin

Thrombin

Thrombin TM

EPCR

Fibrinogen

Fibrin

PC

Figure 3 Coagulation cascade and anticoagulant protein C pathway (dotted arrows indicate inhibition). PC, protein C; TM, thrombomodulin; EPCR, endothelial protein C receptor; APC, activated protein C; PS, protein S; PCI, protein C inhibitor; C4BP, C4b binding protein; PAR-1, protease-activated receptor-1; TF, tissue factor; PL, phospholipids.

complex on negatively charged proteoglycan and phosphotidylethanolamine of cell membrane.

Biological Functions of APC

Inhibition of Coagulation

thrombin, which is necessary for activation of the zymogen, thrombin-activatable brinolysis inhibitor. Vitronectin accelerates the inhibition of PAI-1 by APC. APC may also suppress the action of other brinolytic inhibitors, such as PCI and a1 antitrypsin.
Inhibition of Inammation

APC inhibits activation of the coagulation cascade by proteolytic degradation of factor Va and factor VIIIa in the presence of phospholipids, calcium, and PS. Factor V and factor VIII circulate in plasma in inactive forms and are converted into their active forms, factor Va and factor VIIIa, by activated factor X (factor Xa) and thrombin during the initiation of the coagulation cascade (Figure 3). The clinical importance of the PC pathway in the inhibition of coagulation is illustrated by the frequent occurrence of thromboembolic complications in subjects with deciency of PC or resistance to APC. The phospholipid surface of various cells, including vascular endothelial cells, platelets, monocytes, alveolar macrophages, and bronchoalveolar epithelial cells, may support activation of PC. Thus, generation and function of APC may take place in the intravascular and extravascular spaces of a wide variety of organs, including those of the respiratory system (Figure 4).
Stimulation of Fibrinolytic Activity

APC may inhibit the inammatory response by blocking the cellular secretion of inammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, monocyte chemoattractant protein-1, and macrophage inammatory protein 1-a, and by downregulating the cell-surface expression of adhesion molecules (e.g., intercellular adhesion molecule-1) required for transendothelial migration of inammatory cells to the sites of tissue injury. APC may also protect against inammation by decreasing endothelial cell permeability and migration of inammatory cells. APC enhances lung endothelial barrier by binding to EPCR and transactivation of the sphingosine 1-phosphate receptor. APC also suppresses the nuclear translocation of transcription factors, including those from the nuclear factor kappa B (NF-kB) family. The cellular effect of APC appears to be mediated by EPCR and, interestingly, by protease-activated receptor-1 (PAR-1), the thrombin receptor.
Regulation of Tissue Remodeling

APC may directly stimulate brinolytic activity by inactivating plasminogen activator inhibitor-1 (PAI1) or PCI, another inhibitor of plasminogen activator, and indirectly by decreasing the formation of

APC may suppress excessive extracellular matrix deposition by stimulating the expression and activation of premetalloproteinases and by blocking the

Inhibition of coagulation

Stimulation of fibrinolysis

Regulation of remodeling

Inhibition of inflammation

Inhibition of apoptosis

TB

TAFI

TAFIa APC PCI APC PAI-1 Pro-MMP

MMP PDGF

Permeability Cytokines

P53 Bax Bcl-2

PTB Prothrombinase

C4BP

PS

APC C4BP APC FX Tenase Va APC PS TM VIIIa APC PS TB PC EPCR PAR-1 EPCR ? SP1R

Cell membrane of endothelial cells, monocytes/macrophages, platelets or epithelial cells

Figure 4 Activation and biological functions of the protein C pathway. Protein C (PC) is converted to activated protein C (APC) on the phospholipid-rich surface of endothelial cells, monocytes/macrophages, platelets, and epithelial cells by the thrombin (TB)thrombomodulin (TM) complex in the presence of calcium. The endothelial protein C receptor (EPCR) enhances PC activation. APC blocks prothrombinase formation and activation of coagulation by inactivating activated factor V (Va) and activated factor VIII (VIIIa). APC stimulates brinolysis directly by forming a complex with plasminogen activator inhibitor-1 (PAI-1) and protein C inhibitor (PCI) or indirectly by preventing thrombin-induced activation of thrombin-activatable brinolysis inhibitor. APC exerts anti-inammatory activity by reducing endothelial permeability and by suppressing the secretion of cytokines. APC affects cell survival by regulating the expression of pro- and antiapoptotic factors. The anti-inammatory and antiapoptotic activity of APC appears to be mediated by EPCR, the protease-activated receptor-1 (PAR-1), or the sphingosine 1phosphate receptor (SP1R).

524 COAGULATION CASCADE / Protein C and Protein S

secretion of platelet-derived growth factor from a variety of cells, including monocytes/macrophages and endothelial and epithelial cells. APC may also affect tissue repair by promoting angiogenesis.
Regulation of Cell Survival

free PS level induced by injection of C4BP, which blocks the APC cofactor activity of PS, exacerbates the systemic levels of cytokines induced by Escherichia coli infection, with this response being suppressed by PS supplementation.
Miscellaneous Functions

APC may promote cell survival by upregulating the expression of antiapoptotic factors. APC protects from neural and brain endothelium apoptosis by inhibiting the expression of the tumor suppressor protein p53 and the proapoptotic protein Bax and by stimulating the expression of Bcl-4, an antiapoptotic protein. This protective action of APC is also mediated by both EPCR and PAR-1 receptors.

PS is also a mitogen of smooth muscle cells and thus it may play a role in vascular repair. The receptor that mediates this action is still elusive, but it may be similar to a tyrosine kinase receptor of the Tyro3/Axl family. PS is synthesized by osteoblasts and thus it may participate in bone formation.

Biological Functions of PS
Inhibition of Coagulation

PC and PS in Respiratory Diseases

Thromboembolism with secondary pulmonary hypertension may occur in patients with hereditary deciency of PC or PS, suggesting the clinical importance of the anticoagulant PC pathway. Impairment of PC function may also occur in acquired conditions. Activation of PC is impaired in the lungs of patients with several inammatory diseases, such as sarcoidosis and bronchial asthma, and of patients with idiopathic pulmonary diseases and collagen vascular disease-associated pulmonary brosis. Impairment of APC generation has also been demonstrated in experimental animal models of lung injury and bronchial allergic inammation. The mechanism of this low APC generation is not completely understood. There is increased concentration of PAI-1 and PCI in the intracellular space in patients with inammatory lung disorders, and several inammatory mediators such as TNF-a may decrease in vitro the expression of PC, TM, and EPCR in endothelial cells or bronchial epithelial cells. Proteolytic cleavage of membrane-bound TM and EPCR may also occur during lung injury. Thus, the subnormal level of APC in the lung may result in part from its accelerated inhibition by the serpin family of proteases, by excessive consumption or downregulation of PC and PS, or by low availability of cell surface TM and EPCR. Dysfunction of the PC pathway may perpetuate the activation of mechanisms known to play crucial roles in the pathogenesis of lung injury, pulmonary brosis, and bronchial asthma. In experimental animal models, administration of exogenous APC protects from acute lung injury by limiting excessive activation of these host defense mechanisms. Therapy with APC also improves clinical outcomes in patients with sepsis, some of them also with acute respiratory distress syndrome. Administration of exogenous APC blocks development of pulmonary

PS exerts anticoagulant activity mainly by acting as a cofactor to APC in the inactivation of factors Va and VIIIa. The high afnity of PS for negatively charged phospholipids helps in APC binding to the membrane. This function is lost when PS binds to the b chain of C4BP; thus, only free PS supports the anticoagulant activity of APC in vivo. This function has clinical importance because a deciency of PS is associated with severe thrombotic complications. PS also exhibits in vitro APC-independent anticoagulant activity by directly inhibiting the prothrombinase complex and the factor X activating complex. PS blocks the binding of prothrombin to factor Va and the amidolytic activity of factor Xa. Both the free form and PS in complex with C4BP can display this direct anticoagulant property.
Stimulation of Fibrinolysis

PS may accelerate brinolysis by suppressing activation of thrombin-activatable brinolysis inhibitor or by supporting the APC-mediated inhibition of PAI-1. However, the physiological significance of this profibrinolytic function of PS is unknown.
Regulation of the Inammatory Response

Removal of dying cells is crucial for avoiding inammation induced by autoantigens. PS may regulate the inammatory response by helping in the clearance of apoptotic cells. PS binds to negatively charged phospholipids exposed on apoptotic cells and enhances severalfold their phagocytosis by macrophages. PS also serves to localize C4BP to cell membrane to inhibit activation of the complement system. Indirect observations also indicate that PS supports host defense mechanisms. For example, a decreased

COAGULATION CASCADE / Thrombin 525

brosis in the mouse. The benecial effect of APC may depend on its ability to inhibit generation and the probrotic activity of thrombin and to decrease the secretion of the potent broblast mitogen platelet-derived growth factor and the expression of various cytokines with brogenetic activity. The stimulatory activity of APC on brinolysis and prometalloproteinases may also explain its antibrotic effect. APC has the potential to inhibit allergic bronchial inammation, as demonstrated in animal models of bronchial asthma. Th2 cytokines, including IL-4, IL-5, and IL-13, play a key role in the pathogenesis of asthmatic inammation. APC may block Th2 cytokine expression from lymphocytes, leading to restoration of Th2/Th1 balance, less eosinophil inltration and IgE concentration in the lung, and reduced airway hyperresponsiveness. APC appears to inhibit Th2 cytokine expression by decreasing nuclear translocation of NF-kB and the signal transducer and activator of transcription. APC may also block allergic inammation in the lung by inhibiting recruitment of eosinophils.

Future Perspectives
Studies have demonstrated that dysfunction of the PC pathway due to low availability of some of its components plays a pivotal role in the pathogenesis of lung injury and bronchial inammation. Correction of this dysfunction by exogenous administration of APC may help cure these disorders. The success of APC in the treatment of patients with sepsis supports this assumption. APC also holds promise for the treatment of chronic inammatory diseases of the lung. Studies using appropriate delivery systems such as inhalation therapy are needed to prove its effectiveness.
See also: Coagulation Cascade: Overview; Factor V; Factor X; Fibrinogen and Fibrin; Thrombin; Tissue Factor. Complement. Fibrinolysis: Overview; Plasminogen Activator and Plasmin. Interstitial Lung Disease: Overview. Serine Proteinases.

Gabazza EC, Taguchi O, Kamada H, et al. (2004) Progress in the understanding of protease-activated receptors. International Journal of Hematology 79: 117122. Kobayashi H, Gabazza EC, Taguchi O, et al. (1998) Protein C anticoagulant system in patients with interstitial lung disease. American Journal of Respiratory and Critical Care Medicine 157: 18501854. Rezende SM, Simmonds RE, and Lane DA (2004) Coagulation, inammation, and apoptosis: different roles for protein S and the protein SC4b binding protein complex. Blood 103: 11921201. Shimizu S, Gabazza EC, Taguchi O, et al. (2003) Activated protein C inhibits the expression of platelet-derived growth factor in the lung. American Journal of Respiratory and Critical Care Medicine 167: 14161426. Suzuki K (2005) Protein C. In: High KA and Roberts HR (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 393423. New York: Dekker. Suzuki K (2005) Protein S. In: High KA and Roberts HR (eds.) Molecular Basis of Thrombosis and Hemostasis, pp. 459478. New York: Dekker. Suzuki K, Gabazza EC, Hayashi T, et al. (2004) Protective role of activated protein C in lung and airway remodeling. Critical Care Medicine 32: S262S265. Van de Wouwer M, Collen D, and Conway EM (2004) Thrombomodulinprotein CEPCR system: integrated to regulate coagulation and inammation. Arteriosclerosis, Thrombosis and Vascular Biology 24: 13741383. Yasui H, Gabazza EC, Tamaki S, et al. (2001) Intratracheal administration of activated protein C inhibits bleomycin-induced lung brosis in the mouse. American Journal of Respiratory and Critical Care Medicine 163: 16601668. Yuda H, Adachi Y, Taguchi O, et al. (2004) Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice. Blood 103: 21962204.

Thrombin
R C Chambers, University College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Thrombin is a serine protease that plays a central role in hemostasis by promoting blood coagulation. It is formed from its precursor, prothrombin, following tissue injury and converts brinogen to brin in the nal step of the coagulation cascade. Thrombin also exerts numerous cellular effects, including promoting platelet aggregation, inammatory cell recruitment, endothelial cell activation, and proliferation of mesenchymal cells. These effects are mediated via the proteolytic cleavage and activation of a novel family of cell-surface receptors known as proteinase-activated receptors. Thrombin activity is under tight regulatory control, and excessive or persistent generation of thrombin has been implicated in a number of respiratory diseases, including acute lung injury, interstitial lung disease, asthma, and chronic lung disease of prematurity.

Further Reading
Bernard GR (2003) Drotrecogin alfa (activated) (recombinant human activated protein C) for the treatment of severe sepsis. Critical Care Medicine 31: S85S93. Castellino FJ (2001) Gene targeting in hemostasis: protein C. Frontiers in Bioscience 6: D807D819. Dahlback B and Villoutreix BO (2003) Molecular recognition in the protein C anticoagulant pathway. Journal of Thrombosis and Haemostasis 1: 15251534. Esmon CT (2003) The protein C pathway. Chest 124: 26S32S. Esmon CT, Fukudome K, Mather T, et al. (1999) Inammation, sepsis, and coagulation. Haematologica 84: 254259.

Introduction
At the end of the nineteenth century, Buchanan, a Scottish physiologist, suggested the presence of a

526 COAGULATION CASCADE / Thrombin

blood-borne clotting agent, which he named thrombin. In 1951, the protein was puried, and since then there have been rapid developments in our understanding of the structurefunction relationships of this proteinase. Thrombin is activated during the nal stages of the coagulation cascade and promotes the cleavage of soluble brinogen to form an insoluble brin clot. Besides its critical role in brin formation, thrombin exerts numerous cellular effects via the proteolytic activation of a novel family of cell surface receptors, the proteinase-activated receptors (PARs).

Arg/Ser (where / represents the cleavage site). The high specicity of thrombin toward its substrates and receptors is conferred by the depth and narrowness of the active site cleft, as well as by its unique anionbinding exosite (basic patch). In addition, thrombin possesses three specicity pockets that interact with inhibitors.

Regulation of Production and Activity

Thrombin is the major effector of the coagulation cascade and is generated following the stepwise activation of coagulation proteinases (Figure 1). In vivo, the activation of the coagulation cascade is initiated via the extrinsic pathway. Upon tissue injury, plasma is exposed to tissue factor expressed on nonvascular cells or activated endothelial cells, allowing a complex between factor VII and tissue factor to form. Factor VII is then activated to factor VIIa. The tissue factorfactor VIIa complex subsequently catalyzes the initial activation of factor IX (to factor IXa) and factor X (to factor Xa). Factor Xa in association with activated factor V catalyzes the conversion of prothrombin to thrombin, which in turn catalyzes the conversion of brinogen to brin, the main constituent of a clot. This mechanism is believed to generate only limited amounts of thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial factor VIItissue factor complex pathway catalyzes the activation of factors XI, IX, VIII, and X. In this manner, the intrinsic pathway is activated, leading to the sustained generation of thrombin and blood coagulation. The extrinsic pathway is therefore paramount in initiating
Intrinsic pathway Thrombin

Structure
The prothrombin gene is located on chromosome 11 (cytoband p11q12) and contains 14 exons and 13 introns. Prothrombin is synthesized in the liver as a pre-propeptide that undergoes extensive posttranslational modication prior to secretion. This includes the carboxylation of glutamyl residues by the microsomal enzymes, the vitamin K-dependent carboxylases. The resultant g-carboxy glutamyl residues are necessary for calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin by the prothrombinase complex. The resultant 39 kDa globular protein comprises two chains cross-linked by four disulde bonds that house a deep, narrow groove that contains the catalytic triad consisting of His43, Asp99, and Ser205. This groove is hydrophobic and exhibits a preference for apolar amino acids preceding arginine at a thrombin susceptible bond, such as Leu Asp Pro
Extrinsic pathway Tissue factor

Factor VII

Factor VIIa

Factor XIa

Factor XI

Factor IXa

Factor IX

Factor X

Factor Xa

Factor X

Prothrombin

Thrombin

Fibrinogen
Figure 1 The coagulation cascade.

Fibrin

COAGULATION CASCADE / Thrombin 527

coagulation via the activation of a limited amount of thrombin, whereas the intrinsic pathway maintains coagulation by the dramatic amplication of the initial signal. The coagulation cascade is tightly controlled by both negative feedback mechanisms, as well as by circulating and locally produced inhibitors. The extrinsic pathway is mainly controlled by tissue factor pathway inhibitor (TFPI). This protein forms a complex with factor Xa generated at the initiation of coagulation. This complex then inactivates factor VIIatissue factor complexes, thus inhibiting further synthesis of factors IXa and Xa. The intrinsic pathway is controlled by antithrombin III, which inhibits thrombin and other serine proteases of the coagulation cascade in the presence of heparin. Other important physiological inhibitors include heparin cofactor II and protease nexin-1, which inhibit thrombin, and a2-macroglobulin and a1-antitrypsin (also known as a1 proteinase inhibitor), which inhibit thrombin and factors IXa, Xa, and XIa. Finally, blood coagulation is also regulated when thrombin binds to the endothelial cell surface receptor, thrombomodulin, and is converted from a procoagulant to an anticoagulant by activating protein C. Protein C, in conjunction with protein S, inactivates factors Va and VIIIa and thereby prevents further thrombin generation.

inuences inammatory cell trafcking by inducing the expression of cell surface adhesion molecules, such as P-selectin and intercellular adhesion molecule-1. Finally, thrombin is a potent broblast and smooth muscle cell mitogen and induces extracellular matrix synthesis via the production of brogenic mediators, including PDGF, TGF-b, and connective tissue growth factor, which act via both autocrine and paracrine mechanisms.

Receptors
Most of thrombins cellular effects are mediated via the proteolytic activation of ubiquitously expressed cell surface receptors, termed PARs (Figure 2). These receptors belong to the family of G-protein-coupled seven-transmembrane domain receptors and currently comprise four members, of which three (PAR1, PAR3, and PAR4) are thrombin receptors. These receptors display a unique mechanism of activation involving limited proteolysis of the N-terminus, resulting in the unmasking of a cryptic ligand. This ligand binds to the second extracellular loop of the receptor and thereby triggers signaling via heterotrimeric G-proteins. Synthetic peptides corresponding to the tethered ligand sequences of PAR1 and PAR4 are capable of activating these receptors independently of receptor cleavage and are widely used as experimental tools to invoke the involvement of these receptors in mediating thrombin cellular responses. In contrast, PAR3 is not capable of initiating cell signaling by thrombin but, rather, is thought to act as an accessory receptor in concert with other PARs.

Biological Function
Thrombin plays a critical role in blood coagulation by converting brinogen to brin and by promoting platelet aggregation. In addition, thrombin exerts a range of other cellular effects that may play important roles in subsequent inammatory and tissue repair responses, as part of the normal response to injury, but that may also lead to lung pathology. In addition to being one of the most potent physiological agonists of platelet aggregation, thrombin also promotes the release of thromboxane A2 and platelet factor-4 and the brogenic mediators, platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-b), involved in early tissue repair responses. Thrombin exerts dramatic effects on vascular tone and permeability via both direct effects on endothelial cell contraction and indirect mechanisms involving the release of nitric oxide and serotonin from endothelial cells and histamine from mast cells. In terms of inuencing inammatory responses, thrombin is a chemoattractant for inammatory cells and stimulates the release of a host of proinammatory cytokines, including monocyte chemotactic protein-1, interleukin-6 (IL-6), and IL-8, by a number of cell types. Thrombin further

Thrombin in Respiratory Diseases

Activation of the coagulation cascade is a feature of a number of respiratory conditions associated with inammation and excessive deposition of connective tissue proteins. Intra-alveolar accumulation of brin occurs in the lungs of patients with pulmonary brosis, in acute lung injury (ALI), and in acute respiratory distress syndrome (ARDS), in which rapid broproliferation and matrix synthesis can lead to the development of extensive brotic lesions. Bronchoalveolar lavage uid obtained from patients with ARDS has also been reported to contain tissue factorfactor VII/VIIa complexes that can trigger activation of the extrinsic coagulation cascade. Levels of active thrombin are increased in the lungs of patients with pulmonary brosis associated with systemic sclerosis, in pulmonary brosis associated with chronic lung disease of prematurity, and in asthma. Increased procoagulant activity and/or tissue factor

528 COAGULATION CASCADE / Thrombin

Thrombin

Fibrin deposition

PAR1 activation

Inhibition of surfactant function Fibroblast & inflammatory cell recruitment

Microvascular permeability Production of proinflammatory cytokines Inflammatory cell trafficking and mitogenesis

Production of fibrogenic mediators Fibroblast chemotaxis & proliferation Fibroblast to myofibroblast transformation and ECM synthesis

Lung inflammation and fibrosis

Figure 2 Proposed mechanisms for the role of thrombin in lung inammation and brosis.

levels have also been identied in patients with idiopathic bronchiolitis obliterans-organizing pneumonia, sarcoidosis, hypersensitivity pneumonitis, and pleural disease. The pathological importance of excessive or smoldering activation of the coagulation cascade in both acute and chronic lung injury has been demonstrated in extensive studies employing experimental animal models using direct coagulation proteinase inhibitors and genetically modied mice. Evidence suggests that both brin persistence and thrombin signaling contribute to the pathogenesis of ALI/ARDS and pulmonary brosis. In terms of the receptor by which thrombin acts in these disease settings, studies of PAR1-decient mice suggest that PAR1 plays an important role in both the acute inammatory phase (inammatory cell recruitment and microvascular injury) and the chronic brotic phase (lung collagen accumulation) in experimentally induced lung injury. Several agents aimed at targeting the coagulation cascade, including antithrombin III and TFPI, have undergone trials in humans with ALI/ARDS, but the results have been largely disappointing. The results of the phase III, randomized, double-blind, placebocontrolled multicenter PROWESS trial of intravenous infusion of the anticoagulant activated protein C in severe sepsis (which included patients with ARDS) and subsequent reduction in mortality resulted in approval of the use of this drug in severe sepsis. ARDS subgroup analysis was not presented so

that the ability of this agent to protect the lung in ARDS remains to be determined. Future studies of this agent in this and other respiratory disease settings associated with uncontrolled thrombin generation are eagerly awaited.
See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Asthma: Overview. Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12). Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-ProteinCoupled Receptors. Interstitial Lung Disease: Overview. Pulmonary Fibrosis. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
Bernard GR, Vincent JL, Laterre PF, et al. (2001) Recombinant Human Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efcacy and safety of recombinant human activated protein C for severe sepsis. New England Journal of Medicine 344: 699709. Chambers RC (2003) Proteinase-activated receptors and the pathophysiology of pulmonary brosis. Drug Development Research 60: 2935. Cocks TM and Moffatt JD (2000) Protease-activated receptors: sentries for inammation? Trends in Pharmacological Science 21: 103108. Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407: 258264. Dahlback B (2000) Blood coagulation. Lancet 355: 16271632. Gunther A, Lubke N, Ermert M, et al. (2003) Prevention of bleomycin-induced lung brosis by aerosolization of heparin or

COAGULATION CASCADE / Tissue Factor 529

urokinase in rabbits. American Journal of Respiratory and Critical Care Medicine 168: 13581365. Howell DCJ, Goldsack NR, Marshall RP, et al. (2001) Direct thrombin inhibition reduces lung collagen accumulation and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary brosis. American Journal of Pathology 159: 13831395. Howell DC, Johns RH, Lasky JA, et al. (2005) Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inammation and brosis. American Journal of Pathology 166: 13531365. Idell S (2003) Coagulation, brinolysis, and brin deposition in acute lung injury. Critical Care Medicine 31: S213S220. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R (2001) Proteinase-activated receptors. Pharmacology Reviews 53: 245282. Ploplis VA, Wilberding J, McLennan L, et al. (2000) A total brinogen deciency is compatible with the development of pulmonary brosis in mice. American Journal of Pathology 157: 703708. Ruf W (2004) Protease-activated receptor signaling in the regulation of inammation. Critical Care Medicine 32: S287S292. Suzuki K, Gabazza EC, Hayashi T, et al. (2004) Protective role of activated protein C in lung and airway remodeling. Critical Care Medicine 32(5 supplement): S262S265. van der Poll T, Levi M, Nick JA and Abraham E (2005) Activated protein C inhibits local coagulation after intrapulmonary delivery of endotoxin in humans. American Journal of Respiratory and Critical Care Medicine 171: 11251128. Wagers SS, Norton RJ, Rinaldi LM, et al. (2004) Extravascular brin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness. Journal of Clinical Investigation 114: 104111. organ dysfunction. Blockade of TF attenuates acute lung injury associated with Gram-negative sepsis in experimental animal model systems.

Introduction
The main function of tissue factor (TF) in vivo is to trigger the blood coagulation process. After vascular injury, circulating plasma clotting factor VII (FVII) comes in contact with TF. Tissue factor binds and activates FVII. The resultant TFFVIIa complexes initiate coagulation cascade by activating both factors IX and X, which ultimately leads to thrombin generation, platelet activation, and brin deposition. The primary role of TF in the complex is to induce allosteric changes in FVIIa, which dramatically increase FVIIa activity, and thus making it an efcient catalyst of factor IX and factor X activation. Tissue factor is constitutively expressed in mesenchymal cells residing in the adventitial lining of blood vessels but not in intravascular cells. Bacterial infection or certain other pathological conditions induce TF expression in monocytes, and probably in endothelial cells. Thus, FVII comes in contact with TF only upon disruption of blood vessel barriers or in a disease state. However, recent studies suggest TF presence in plasma, and the blood-borne TF may contribute to thrombosis. At present, the source of blood-borne TF and its pathophysiological significance are unclear. In addition to its role in coagulation, TF may also have a nonhemostatic function. Tissue factor-FVIIa, and the coagulation proteases generated by TFFVIIa, i.e., factor Xa and thrombin, are shown to activate proteinase-activated receptors (PARs). The TF-dependent signaling pathways contribute to a variety of pathophysiological processes, including pathogenesis of acute lung injury (ALI) and organ dysfunction.

Tissue Factor
L V M Rao and U Pendurthi, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Tissue factor (TF) is a cellular receptor for coagulation factor VII (FVII), and its activated form, factor VIIa (FVIIa). Exposure of TF to blood upon vascular injury leads to TF-FVIIa complex formation, which triggers blood coagulation cascade. In health, TF is constitutively expressed in many extravascular cells, including broblasts and pericytes in and surrounding blood vessel walls and lung epithelial cells, but absent from blood cells and endothelial cells that line blood vessel wall. Certain pathological conditions, such as sepsis, induce TF expression in monocytes, and increase the expression in lung, kidney, and other tissues. Such increased TF activity contributes to the procoagulant environment in both intravascular and extravascular compartments. Increased TF expression in parenchymal and recruited lung cells, coupled with transendothelial leakage of plasma proteins, leads to alveolar brin deposition. In addition, TF-FVIIa, and downstream coagulant proteases generated by TF-FVIIa amplify multiple steps of the inammatory response through activation of proteinase-activated receptors (PARs). Both the TF pathway-induced brin deposition and cell signaling contribute to the pathogenesis of acute lung injury and

Structure
Tissue factor is an integral membrane glycoprotein. The mature protein, following cleavage of a 32-residue leader sequence from the primary translational product, consists of a single polypeptide of 263 amino acids, with the predicted mol wt. of 29 593. The glycosylated TF migrates as a B45 000 Da protein on SDS polyacrylamide gels. The majority of the molecule resides on the cell surface except for 23 residues in the transmembrane region and a 21-residue C-terminal region in the cytoplasm. The extracellular domain of TF contains three potential N-glycosylation sites (NXT/S) at residues Asn11, Asn124, and Asn135. However, the carbohydrate chains are not required for the procoagulant function of TF. There are ve half-cystine residues; four

530 COAGULATION CASCADE / Tissue Factor

half-cystines in the extracellular domain of TF form two disulde bonds and the half-cystine in the cytoplasmic region (Cys245) is acylated by palmitic acid and stearic acid. Analysis of the human TF protein sequence revealed the presence of two putative phosphorylation sites (Ser253 and Ser258) in the cytoplasmic domain. Protein kinase C-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a Pro-directed kinase. Palmitoylation of the cytoplasmic Cys245 negatively regulates Ser258 phosphorylation. Tissue factor exhibits a distant sequence homology with members of the class 2 cytokine receptor superfamily. Similar to cytokine receptors, the extracellular domain of TF is composed of two immunoglobulin-like domains. The crystal structure of TF revealed that the two Ig-like domains are tightly packed with loops from both modules interdigitating to make a dovetailed joint at the interface (Figure 1). The binding site for factor VII lies at the interface region and involves residues from both the domains. TF does not undergo major structural changes upon FVIIa binding. In contrast, FVIIa loses its segmental exibility upon binding to TF. This positions the FVIIa active site at an appropriate distance above the membrane surface for efcient cleavage of its substrates, factors IX and X.

Domain 1

Regulation of Production and Activity

Tissue factor is expressed in a cell-specic manner. The TF gene is constitutively expressed in many extravascular cell types, such as broblasts in adventitia, epithelium of skin and lung, astrocytes in brain, Bowmans capsule of glomeruli in the kidney, and stromal cells in endometrium. Although the TF gene is not expressed in luminal vascular cells in health, it is inducibly expressed in several vascular cell types, including monocytes, vascular endothelial cells, and smooth muscle cells. Detailed studies on the regulation of the TF promoter in various cell types indicate that Sp1 controls the basal (constitutive) TF gene expression whereas a distal enhancer ( 227 to 172) containing two AP-1 sites and a nuclear factor kappa B (NF-kB) site mediates the induction of the human TF gene in monocytes and endothelial cells (Figure 2). Proximal enhancers ( 109 to 59) containing overlapping Egr-1 and Sp1 sites mediate TF induction in epithelial-like cells and vascular smooth muscle cells, respectively. Various exogenous and physiological agents can induce TF expression in vascular endothelial cells, epithelial cells and cells of the monocyte/macrophage lineage. The agents that induce TF synthesis that are relevant to pathophysiology include inammatory

Domain 2

Figure 1 Structure of tissue factor extracellular domain. Factor VII binding sites are shown in yellow and the region important for factor X activation is shown in red. Reproduced with minor modications from Harlos K, Martin DMA, OBrien DP, et al. (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370: 662666, with permission from Nature Publishing Group.

cytokines (interleukin-1 a and b, interleukin-2, and tumor necrosis factor alpha (TNF-a)), growth factors (broblast growth factor, platelet-derived growth factor, and vascular endothelial cell growth factor), thrombin, C-reactive protein, endotoxins, virus infections, and immune complexes. The level of TF induction and expression varies significantly from one stimulus to another, and from one cell type to another. Functional expression of TF is tightly regulated at the cell surface by multiple mechanisms. Cellular localization of TF dictates its functional activity. TF residing in the anionic phospholipid milieu of the plasma membrane is more active than the TF present within neutral phospholipids. Similarly, TF association with caveolae limits its activity, and the disruption of caveolae leads to increased TF functional

COAGULATION CASCADE / Tissue Factor 531

250 AP-1 Inducible 200 NF- B c-rel/p65 150 Sp1 100 Sp1/Egr-1 Egr-1 50 TATA +1 bp

c-fos/c-jun

Sp1

Sp1

Constitutive

Figure 2 The promoter region of tissue factor gene. Transcription factor binding sites in the human TF promoter are shown by boxes. The binding sites consist of (left to right): two activator protein-1 (AP-1, beige), one nuclear factor kappa B (NF-kB, red), two Sp1 (turquoise), and three overlapping Sp1 (turquoise)/early growth response gene product (Egr-1, magenta). A summary of constitutive (c-fos/c-jun (oblong) and Sp1 (round))and inducible transcription factors (c-Rel/P65 (diamond) and Erg-1 (pointed cylinder)) that bind to the promoter of TF gene is also shown.

activity. Although it is not well established, transformation of TF from dimers to monomers is also shown to regulate TF activity. Tissue factor pathway inhibitor-1 (TFPI-1) regulates TFFVIIa activity by dual mechanisms, forming the inactive ternary complex TFFVIIaFXaTFPI and promoting the endocytosis (and subsequent degradation) of TFFVIIa complexes.

Biological Function
The absence of TF-decient humans and the embryonic lethality of TF / mice indicate that TF is essential for life. The primary function of TF is to initiate blood coagulation cascade following the vascular injury that ultimately leads to platelet activation and brin formation, which are vital in sealing the injured vessels to protect against hemorrhage after tissue injury. Aberrant TF expression in the vessel wall and/or circulating cells initiates life-threatening intravascular thrombosis in various diseases, such as atherosclerosis, cancer, and sepsis. In addition, TF plays a nonhemostatic role in many biological processes, such as vascular development, inammation, angiogenesis, and tumor metastasis.

PAR-1 and PAR-2, whereas thrombin activates PAR1, PAR-3, and PAR-4. PARs are members of a large family of seven transmembrane domain cell surface receptors that transmit extracellular signals through G-proteins. Activation of PARs involves proteolytic cleavage of a specic susceptible bond in the extracellular N-terminal portion of the receptors, which uncovers a new N-terminus that acts as a tethered ligand capable of activating the self or an adjacent PAR. PAR signaling inuences cytoskeletal functions and gene expression patterns that regulate diverse cellular responses including cell migration, proliferation, and apoptosis. TF-dependent PAR-mediated cell signaling is thought to be in part responsible for pathogenesis of ALI and brotic lung disease.

Tissue Factor in Respiratory Diseases

Tissue factor plays a critical role in intra-alveolar and intravascular brin deposition, which is frequently associated with ALI or ARDS (Figure 3). In lung, the microvascular endothelium and the alveolar epithelium form the alveolar-capillary barrier. The disruption of the alveolar-capillary barrier, which often occurs in acute injury or ARDS, results in leakage of plasma into the alveolus. This allows FVII to come in contact with TF on alveolar macrophages and lung epithelial cells. TFFVIIa complexes thus formed trigger the coagulation cascade, with subsequent thrombin formation and brin deposition. Systemic or pulmonary bacterial infections activate inammation, which in turn increases TF expression in alveolar macrophages and lung epithelial cells. Clinical studies in patients with ARDS or evolving pneumonia show an increased TF and FVIIa activity in bronchoalveolar lavage uid (BALF). Although TFPI-1, the inhibitor of TFFVIIa, also increases in

Receptors
Tissue factor acts as a cellular receptor for FVIIa. The resultant TFFVIIa complexes not only activate the coagulation pathway but also activate, either directly or through generation of factor Xa and thrombin, PAR-mediated cell signaling. The TFFVIIa complex activates PAR-2, whereas the TFFVIIa FXa complex activates both PAR-1 and PAR-2. Factor Xa, although not efciently, also activates both

532 COAGULATION CASCADE / Tissue Factor

Microorganisms endotoxins

Alveolar space TF Macrophages epithelial cells fibroblasts

Plasma exudation TF FXa

PAR-1/PAR-2

TFFVIIa TFFVIIaFXa

Thrombin Inflammatory cytokines Fibrin

PAR-2, PAR-1

FXa

DIC, Microthrombosis

IL-1, IL-6, IL-8

Thrombin

Fibrin Cell recruitment, surfactant dysfunction etc.

PAR-1 ALI Vascular space

CTGF/Procollagen Fibrosis

Figure 3 Role of tissue factor in acute lung injury. Microorganisms/endotoxin induce TF expression in monocytes and endothelial cells in vascular space and upregulate TF expression in alveolar macrophages and lung epithelial cells in the alveolar compartment. Damage to alveolar capillary barrier allows plasma-clotting factors to come in contact with TF expressed macrophages, alveolar epithelial cells, or broblasts in the alveolar compartment. Formation of TFFVIIa complex on these cells triggers the activation of coagulation cascade leading to thrombin generation and brin formation. TFFVIIa, either directly or through generation of thrombin, activates PAR-mediated cell signaling that could promote lung brosis through elaboration of connective tissue growth factor (CTGF) and procollagen synthesis in broblasts. PAR signaling upregulates cytokine production and TF in endothelial cells and macrophages. All these processes contribute to acute lung injury (ALI) and lung brosis. DIC, disseminated intravascular coagulation.

BALF in ARDS patients, the increase is much smaller than the increase in TF expression. Thus, TFPI-1 levels are not adequate to inhibit TF-induced coagulation in the alveolar space. The imbalance between TF and TFPI-1, coupled with impaired brin removal caused by depression of brinolysis in ARDS patients, leads to intrapulmonary brin deposition. Although brin provides the critical matrix for cell migration and collagen deposition that are necessary for wound healing and repair mechanisms, excessive brin accumulation in the lung contribute to injury in multiple ways. Fibrin deposits enhance inammatory response by increasing vascular permeability, activating endothelial cells to produce proinammatory cytokines and chemokines, inducing the accumulation of activated neutrophils, and inactivating lung surfactant function. Excess brin deposition in the lung can also be probrotic. Tissue factor and proteases generated by the TF pathway of coagulation can also exert direct effects on both intravascular and extravascular cells in amplifying the inammatory cascade. In lethal Escherichia coli sepsis animal models, inhibition of the

TFFVIIa complex significantly reduced the inammatory response, as measured by a decrease in the plasma levels of IL-6 and IL-8, and protected the animals against the lethal effects of septic shock. Blockade of TFFVIIa assembly or its function prevented sepsis-induced hypoxemia, pulmonary hypertension, lung edema, and loss of pulmonary system compliance. In contrast, efcient inhibition of brin formation by blocking the coagulation cascade at the downstream of TFFVIIa neither attenuated IL-6 and IL-8 increase nor reversed the lethal outcome of sepsis. Genetically modied mice expressing low levels of TF exhibited reduced IL-6 expression and increased survival in a mouse model of endotoxemia. These data suggest that TFFVIIa contributes directly to ALI in sepsis in part through selective stimulation of proinammatory cytokines. Recent in vitro cell studies have established that TF-dependent initiation of coagulation provides an efcient signaling mechanism via PARs. TFFVIIa stimulates a number of proinammatory cytokines and chemokines that are relevant to ALI through PAR2-mediated cell signaling. Nascent factor Xa in the transient

COAGULATION CASCADE / Tissue Factor 533

ternary TFFVIIaFXa complex also efciently activates both PAR-1 and PAR-2. PAR signaling in endothelial cells and respiratory epithelial cells induces proinammatory cytokines, such as IL-6 and IL-8. TFFVIIa, FXa, and thrombin activation of PARs also upregulate the expression of connective tissue growth factor (CTGF) in lung broblasts. CTGF is a potent broblast mitogen and chemoattractant, and plays an important role in promoting connective tissue formation after tissue injury. However, overexpression of CTGF, which may occur in excessive and/ or persistent activation of the TF pathway of coagulation, would lead to pulmonary brosis. The role of TF in ALI is not limited to the alveolar compartment or lung interstitium. Aberrant expression of TF may promote thrombi in the pulmonary artery, resulting in pulmonary dysfunction. Tissue factor-induced diffused intravascular thrombosis and disseminated intravascular coagulation could also cause acute edematous pulmonary injury. Further, activation PAR signaling in endothelial cells upregulates IL-6 and TF, which provides a self-amplifying loop to sustain the inammation. The coagulation abnormalities observed in ALI are similar to those present in sepsis, which is a major risk factor for ALI. Therefore, selective anticoagulants used as adjunctive therapy for the treatment of sepsis are expected to inuence the clinical course of ALI. Although animals studies have demonstrated a consistent reduction in lung injury with the administration of anticoagulants that selectively block TF FVIIa function, the information on their potential benets in humans are either lacking or, where available, less convincing. A phase II study evaluating TFPI-1 in the treatment of severe sepsis suggested that lung function in ARDS patients was improved. However, a phase III trial failed to demonstrate a survival benet. Active site-inhibited FVIIa (ASIS), which blocks TFFVIIa complex formation by competing with endogenously generated FVIIa for limited TF sites, prevented lung injury after E. coli sepsis in baboons. The outcome of phase II clinical trails with ASIS in ARDS is yet to be reported.

brin deposition and mediating cell signaling via PARs. Selective blockade of TFFVIIa complex formation or its activity may have therapeutic benets in patients with ARDS.
See also: Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-Protein-Coupled Receptors. ProteinaseActivated Receptors. Pulmonary Fibrosis. Signal Transduction.

Further Reading
Camerer E, Kolsto AB, and Prydz H (1996) Cell biology of tissue factor, the principal initiator of blood coagulation. Thrombosis Research 81: 141. Eilertsen KE and Osterud B (2004) Tissue factor: (patho)physiology and cellular biology. Blood Coagulation and Fibrinolysis 15: 521538. Giesen P, Rauch U, Bohrmann B, et al. (1999) Bloodborne tissue factor: another view of thrombosis. Proceedings of the National Academy of Sciences, USA 96: 23112315. Harlos K, Martin DMA, OBrien DP, et al. (1994) Crystal structure of the extracellular region of human tissue factor. Nature 370: 662666. Idell S (2003) Coagulation and brinolysis, and brin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213S220. Idell S, Gonzalez K, Bradford H, et al. (1987) Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome contribution of tissue factor associated with factor VII. American Review of Respiratory Diseases 136: 14661474. Konigsberg W, Kirchhofer D, Riederer MA, and Nemerson Y (2001) The TF: VIIa complex: clinical significance, structurefunction relationships and its role in signaling and metastasis. Thrombosis and Haemostasis 86: 757771. Mackman N (1997) Regulation of the tissue factor gene. Thrombosis and Haemostasis 78: 747754. Mackman N (2004) Role of tissue factor in hemostasis, thrombosis, and vascular development. Arteriosclerosis Thrombosis and Vascular Biology 24: 10151022. Morrissey JH (2001) Tissue factor: an enzyme cofactor and a true receptor. Thrombosis and Haemostasis 86: 6674. Rao LVM and Pendurthi UR (2003) Regulation of tissue factorfactor VIIa expression on cell surfaces: a role for tissue factorfactor VIIa endocytosis. Molecular and Cellular Biochemistry 253: 131140. Rao LVM and Pendurthi UR (2005) Tissue factor-factor VIIa signaling. Arteriosclerosis Thrombosis and Vascular Biology. 25: 4756. Rapaport SI and Rao LVM (1995) The tissue factor pathway: how it has become a prima ballerina. Thrombosis and Haemostasis 74: 717. Ruf W (2004) Protease-activated receptor signaling in the regulation of inammation. Critical Care Medicine 32: S287S292. Welty-wolf KE, Carraway MS, Ortel TL, and Piantadosi CA (2002) Coagulation and inammation in acute lung injury. Thrombosis and Haemostasis 88: 1725.

Summary
Tissue factor, a cellular receptor that occupies a central position in hemostasis and thrombosis, plays a key role in ALI. Tissue factor contributes to the pathogenesis of ALI by generating extravascular

Collagens

see Extracellular Matrix: Collagens.

534 COLLECTINS

COLLECTINS
J A Whitsett and P S Kingma, Cincinnati Childrens Hospital, Cincinnati, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Collectin Family of Polypeptides

The collectins are a family of collagenous proteins that belong to the C-type lectin superfamily. The collectin family includes the pulmonary collectins, SP-A and SP-D, as well as six other collectins that were identied in mammals: mannose-binding protein (MBP), collectin liver-1 (CL-1), collectin placenta 1, conglutinin, collectin of 43 kDa (CL-43), and collectin of 46 kDa (CL-46). Collectins are dened by four structural domains shared by all family members: a short amino-terminal cross-linking domain, a triple-helical collagenous domain, a neck domain, and a carbohydrate recognition domain (CRD) (Figure 1). Three neck domains combine to form a triple coiled-coil structure and subsequently orchestrate the zipper-like assembly of the remaining domains into a trimer that serves as the minimal functional unit of each collectin. Trimers further combine into multimeric proteins through disulde bonds among the cysteine-rich amino-terminal domain. The collagen domain consists of repeating triplets of GlyXY, where X and Y can be any amino acid but are frequently proline or hydroxyproline. In SP-D, conglutinin, CL-1, CL-46, and CL-43, the collagen domain preferentially associates to form cruciform tetramers of 12 monomeric proteins. In contrast, the collagenous sequences of both SP-A and MBP are relatively short and interrupted in the Gly XY repeat, which inserts a bend in the collagenous domain that produces regions of exibility that enable the collagen domains of SP-A and MBP to angle away from the protein core and form an octameric bouquet-like structure of 18 monomers. Whether in cruciform or bouquet forms, the collagenous domains provide proper spacing between the separate trimeric subunits, which may allow crosslinking of carbohydrate structures on the surface of microorganisms facilitating their aggregation and neutralization. The CRD of all known collectins is composed of 115130 amino acid residues. Comparisons of multiple collectins reveal that 22 amino acids of the CRDs are highly conserved. The majority of these residues preserve the C-type lectin fold within the CRD; however, the remaining sequences diverge significantly and probably dictate saccharide binding specicity. Although several calcium-binding sites have been identied, crystallographic analysis of the CRD of MBP and SP-D showed the presence of

Abstract
Surfactant protein A (SP-A) and surfactant protein D (SP-D) are members of the collectin family of mammalian lectins that are expressed in the lung. Although SP-A and SP-D inuence pulmonary surfactant structure and homeostasis in the alveoli, both are essential components of the innate immune system that recognize, bind, and clear invading microbes from the lung. SP-A and SP-D bind polysaccharides, phospholipids, and glycolipids expressed on the surface of viral, fungal, and bacterial pathogens. Both proteins directly kill pathogens or enhance their uptake by phagocytes. As a consequence of their physiologic function, SP-A and SP-D are targets for developing clinical therapies designed to limit the proliferation of microorganisms and the resulting inammatory damage from persistent infection.

Each day, the human lung exchanges approximately 15 000 liters of air that contains a multitude of microorganisms and ne particles. Despite the absence of a rigid protective physical barrier at the airtissue interface, the lung is infrequently subjected to infection or injury by toxicants. Pulmonary homeostasis is maintained in large part by multilayered innate and acquired immune systems that simultaneously defend B150 m2 of lung surface area from microbial invasion and maintain the integrity of the alveoli. Most infectious agents that enter the respiratory system are trapped in nasal passages and proximal conducting airways and removed by mucociliary clearance. Microbes that evade this rst level of defense are deposited in the distal airways or alveolar compartments, where they interact with host defense proteins, respiratory epithelial cells, and marrowderived cells, including macrophages, leukocytes, and lymphocytes. Whereas resident macrophages respond rapidly, subsequent activation of the acquired immune system and recruitment of other immune cells takes time, during which pathogen invasion and proliferation may occur. The collectin family of host defense proteins has evolved to quickly recognize pathogens and instruct innate inammatory responses to clear invading pathogens from the lung. This article focuses on the role of the pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), which are recognized as critical components of the innate host defense of the lung that link innate and acquired immunity.

COLLECTINS 535
SP-A CRD Neck domain Collagenous domain cysN-terminal domain Monomer Trimer Octadecamer

CHO-

SP- D

CRD CHONeck domain Collagenous domain N-terminal domain Trimer Dodecamer

cys-

Monomer

Figure 1 Structure of pulmonary collectins. Each pulmonary collectin contains a carbohydrate recognition domain (CRD), a neck domain, a collagenous domain, and a cysteine (cys)-rich amino-terminal cross-linking domain. The primary sites of N-linked glycosylation (CHO) are shown. Three neck domains combine to form a triple coiled-coil structure that leads to the formation of a collagen-like triple helix and a functional trimer. Trimeric subunits further combine into higher oligomeric forms containing 12 (SP-D) or 18 (SP-A) monomers.

between two and four calcium ions in most collectins. One calcium ion is known to be part of a troughlike pocket that facilitates saccharide binding. Within this pocket, binding occurs through direct coordination of the calcium ion, as well as hydrogen bond interactions with amino acid side chains. Another calcium ion, which is found only in SP-D, is located close to the neck domain and may explain why the neck domain of SP-D is able to bind lipopolysaccharide (LPS) and phospholipids. The role of the remaining calcium ions and the exact number of calcium ions required for collectin function remain unclear.

Synthesis of Pulmonary Collectins

Surfactant Protein A

SP-A was the rst pulmonary collectin identied and was named a surfactant protein on the basis of its close association with lipids in pulmonary surfactant. SP-A is encoded by two genes located within a collectin locus on the long arm of chromosome 10 that also includes the genes for SP-D and MBP.

In humans, SP-A synthesis begins during the second trimester of gestation and occurs primarily in the alveolar type II epithelial cells, Clara cells, and in cells of tracheobronchial glands. Although the SP-A protein was initially recognized in the lung, it also has been detected in extrapulmonary tissues; however, the reported tissue distribution of SP-A in the literature is inconsistent. This is due primarily to non-specic binding of anti-SP-A antibodies as well as tissue-specic differences in mRNA versus protein expression. For example, reverse transcriptase polymerase chain reaction analysis has shown low amounts of SP-A mRNA in human prostate, pancreas, thymus, colon, and salivary gland. This expression pattern, however, varies among species, and when tested by either immunohistochemistry or Western analysis, the SP-A protein was not detected in extrapulmonary tissues in humans. Therefore, whereas SP-A gene expression may occur at low levels in multiple tissues, the SP-A protein seems to be primarily restricted to the respiratory system. Expression of SP-A increases with advancing gestation and in response to multiple forms of lung injury. Increased protein levels can be detected within

536 COLLECTINS

hours of exposure to bacterial endotoxin or after challenging mice with inhaled Pseudomonas aeruginosa. In addition, SP-A content and mRNA expression increase following toxin-mediated lung injury including hyperoxia. The mechanisms that control SP-A gene expression are complex and occur at the levels of gene transcription and mRNA stability. In studies using alveolar type II cells, as little as 400 bp of the 50 -anking region of the SP-A gene was required for appropriate cell-specic, developmental, and inammatory expression. Comparisons across multiple species revealed that several response elements are present within this region. These include a cAMP response element, an E box that binds upstream stimulatory factor-1 and -2, a GT box that binds Sp1, a thyroid transcription factor-1 (TTF-1) binding element, a GATA binding site, and a reversed nuclear factor kappa B (NF-kB) binding site. Transcription factor activation and binding to these response elements was increased in human fetal lung following exposure to epidermal growth factor, cAMP, g-interferon, and interleukin (IL)-1b and inhibited by glucocorticoid. cAMP, which increases in response to hyperoxia, activates protein kinase A, which in turn causes increased phosphorylation and activation of TTF-1. IL-1, which is increased as part of the response to infection, activates NF-kB. TTF-1 and NFkB subsequently act in an additive manner to bind the SP-A promoter and initiate transcription.
Surfactant Protein D

Unlike SP-A, the tissue distribution of SP-D requires that the SP-D promoter respond to relatively ubiquitous transcription factors. To facilitate this, the mouse SP-D promoter contains consensus transcription factor-binding sequences for the AP-1 family, forkhead transcription factors FoxA1 and FoxA2, TTF-1, nuclear factor of activated T cells (NFAT), and multiple sites for CCAAT enhancer-binding proteins (C/EBPs). NFATs, C/EBPs, and members of the AP-1 family activate the transcription of immune response genes in several organs. Interestingly, members of the C/EBP family are also prominent factors in the regulation of the hepatic acute phase response to systemic injury, suggesting that the regulation of SP-D by C/EBP may mediate an analogous pulmonary acute phase response to microbial- or toxinmediated damage within the lung. Developmentally regulated expression of SP-D is increased by glucocorticoid treatment in utero. Steroids likely increase SP-D mRNA levels through activation of C/EBP binding. In addition, as part of the innate immune system, SP-D protein levels may increase up to 70-fold following exposure to IL-4 or IL-13 in vivo. IL-4 and IL-13 induce calcineurin-mediated dephosphorylation of NFAT. Once activated, NFAT works cooperatively with TTF-1, AP-1 family members, and C/EBPs to form a multiprotein transcriptional complex that induces SP-D expression.

Relationship between Collectin Structure and Function

Pulmonary collectins are pattern-recognition molecules that bind to polysaccharides, phospholipids, and glycolipids on the surface of a variety of microorganisms (Table 1). In order to recognize the large variety of microorganisms that enter the lung, SP-A and SP-D must have a broad binding specicity. This is achieved by an open through-like pocket that facilitates binding interactions between the terminal carbohydrate of polysaccharides on the surface of microbes and the amino acid side chains within the CRD. Despite the required broad binding specicity, SP-A and SP-D display a monosaccharide binding preference for mannose or glucose over galactose. C-type lectins can be divided into two distinct subtypes with monosaccharide binding preference for either mannose/glucose or galactose. Oligosaccharide binding preference is determined by a GluProAsn or GluProArg motif that is present within the binding pocket of SP-D and SP-A, respectively. Computational docking studies have shown that additional binding specicity arises through hydrogen binding between saccharide residues anking the terminal

Human SP-D was named on the basis of sequence similarity to SP-A, expression in alveolar epithelial cells, and association with pulmonary surfactant. SP-D is encoded by a single gene located in close proximity to the SP-A gene on human chromosome 10. Although SP-D was rst recognized in the lung and is expressed primarily by type II and other nonciliated bronchiolar respiratory epithelial cells, SP-D mRNA and protein were detected in many nonpulmonary tissues. SP-D immunostaining was detected in the epithelial cells of parotid glands, sweat glands, lachrymal glands, skin, gall bladder, bile ducts, pancreas, stomach, esophagus, small intestine, kidney, adrenal cortex, anterior pituitary, endocervical glands, seminal vesicles, and urinary tract. Extrapulmonary levels of SP-D mRNA and protein, however, are severalfold lower than those detected in the lung. SP-D mRNA is rst detected in the mouse or rat lung at midgestation and increases prior to birth and during the neonatal period. SP-D mRNA increases following lung injury caused by bacterial endotoxin, inhaled microorganisms, and hyperoxia.

COLLECTINS 537
Table 1 Binding interactions of SP-A and SP-Da Organism Gram-negative bacteria Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli 0111 Escherichia coli K12 Escherichia coli J5 Hemophilus inuenzae, type A Hemophilus inuenzae, type B Hemophilus inuenzae, nontyped Salmonella minnesota Gram-positive bacteria Staphylococcus aureus Streptococcus pneumonieae Group B streptococci Fungi Aspergillus fumigatus Cryptococcus neoformans Candida albicans Pneumocystis carinii Viruses Inuenza A Respiratory syncytial virus Herpes simplex type I Cytomegalovirus Rotavirus Other Mycobacterium tuberculosis Mycoplasma pneumoniae Lipopolysaccharide Rough Smooth Lipid A
a

SP-A

SP-D

than 10 000-fold when all CRDs are simultaneously bound to an organism. The collagenous domain of collectins is thought to have functional roles beyond the positioning of the CRD. For example, a specic GEKGEP motif within the collagen domain of MBP is involved in binding to the C1q receptor. This same sequence is present in SP-A but absent in SP-D, which does not interact with the C1q receptor. In addition, the collagen domain of MBP binds two serum proteases (MBP-associated serum protease-1 and -2) and consequently activates the complement cascade. Collectin function is also inuenced by the distribution of attached carbohydrates. In particular, collectins vary in the location and number of asparagine-linked glycosylation sites across species. Examination of SP-A and SP-D across multiple species reveals at least one conserved site of glycosylation in each protein. The conserved sugar in SP-A is linked to the CRD, whereas the conserved sugar in SP-D is linked to the collagen domain. Mutant SP-A proteins lacking the glycosylation sequence are defective in interaction with the inuenza A virus. However, functional differences between wild-type SP-D and a SP-D mutant lacking the conserved glycosylation site were not detected.

Collectins Modulate the Inammatory Response in the Lung

SP-A and SP-D modulate the host defense response to a variety of pulmonary pathogens (Figure 2). Several studies support both anti- and proinammatory mechanisms for collectin-mediated control of immune cells. SP-A and SP-D indirectly decrease inammatory cell activation by neutralizing noxious stimuli within the respiratory environment. When in the fully assembled multimeric form, a single collectin binds the saccharide ligands present on multiple organisms and consequently forms protein bridges between microbes. These bridges induce extensive microbial aggregation, which enhances clearance of organisms by mucociliary cells lining the respiratory tract. In addition, microbial binding of both proteins increases cell wall permeability and inhibits growth of both Gram-negative bacteria and fungal pathogens. Finally, studies of aerosolized LPS and SP-D demonstrated that SP-D scavenges inhaled LPS and limits the inammatory effects of this molecule. The pulmonary collectins stimulate immune cellmediated recognition and clearance of pulmonary pathogens through three mechanisms. First, SP-A and SP-D act as chemoattractants and recruit alveolar neutrophils and macrophages to sites of infection. Second, collectins coat the surface of microorganisms

The presence ( ) or absence ( ) of binding is shown. Where no symbols are given, no consistent data are available.

carbohydrate and amino acid side chains outside the CRD binding pocket. The majority of collectinpolysaccharide binding interactions occur within the CRD; however, the amino terminus, collagenous domain, and neck domain determine the degree of multimerization and spacing of the CRD, which in turn inuences collectin function. For example, dodecameric SPD can bind, aggregate, and enhance neutrophil inactivation of inuenza A virus, whereas trimeric SPD binds and weakly aggregates the virus. The monomeric CRD is defective in polysaccharide binding. This cooperative nature of polysaccharide binding by the CRD allows multiple CRDs from a single fully assembled collectin to bind clusters of glycoproteins or glycolipids on the surface of a microorganism. The interaction of multiple CRDs can increase binding afnity more

538 COLLECTINS
Alveolar macrophage Phagocytosis Oxygen radicals Cytokines Phagocytosis Oxygen radicals Cytokines

SP-A

SP-D

Bacteria, viral, and fungal pathogens Aggregation Clearance Aggregation Clearance

Figure 2 Pulmonary collectins in host defense. In vitro and genetically engineered animal models suggest that SP-A and SP-D play multiple roles in pulmonary host defense. The pulmonary collectins bind and aggregate viral, bacterial, and fungal pathogens. SP-A and SP-D enhance phagocytosis of microorganisms and regulate the inammatory response of alveolar macrophages.

and act as opsonins. With the CRD bound to the pathogen, the exposed domain of the collectin interacts with specic receptors on the surface of phagocytic cells and induces uptake and killing of the microorganism. Third, SP-A and SP-D have a direct, nonopsonic stimulatory effect on phagocytosis by immune cells. This is likely due to binding and upregulation of the mannose receptor, a pattern-recognition receptor involved in phagocytosis. The most compelling evidence for the role of SP-A and SP-D in modulating the inammatory response comes from animal models of collectin deciency, which show that the anti-inammatory effects of SP-A and SP-D predominate in vivo. Mice with targeted deletions of the surfactant protein A (Sftp-a) gene (Sftp-a / ) survived normally and had no differences in pulmonary surfactant composition, secretion, clearance, or pool size. However, Sftp-a / mice had defects in pulmonary host defense. Clearance of group B Streptococcus, Hemophilus inuenza, respiratory syncytial virus (RSV) and P. aeruginosa was delayed in Spa / mice. Decreased bacterial clearance in Sftp-a / mice was associated with defects in alveolar macrophage-mediated recognition and uptake of bacteria. In addition, oxygen radical production and killing of engulfed microorganisms by Sftp-a / macrophages was markedly reduced. Although alveolar macrophage

phagocytosis and oxygen radical production were decreased, markers of lung inammation were increased in Sftp-a / mice. Sftp-a / mice had an exaggerated inammatory response and produced increased levels of proinammatory cytokines TNFa, IL-6, and MIP2 when challenged with an infectious organism. Unlike Sftp-a / mice, which had relatively normal surfactant function, deletion of the Sftp-d gene generated defects in both pulmonary host defense and surfactant homeostasis. Sftp-d / mice survived normally but developed a gradually worsening pulmonary inammation and surfactant lipid accumulations. Inammatory lesions consisted of increased numbers of enlarged, foamy alveolar macrophages. Progressive emphysema developed in Sftp-d / mice, probably as a result of the reactive oxygen species and metalloproteinases that were released by the abnormally activated alveolar macrophages. Uptake and clearance of viral pathogens, including inuenza A and RSV, were decient in Sftp-d / mice. In contrast, clearance of group B Streptococcus and H. inuenza was unchanged. When exposed to various infectious pathogens, oxygen radical release and production of the proinammatory mediators, TNF-a, IL-1, and IL-6, were increased in Sftp-d / mice. SP-A and SP-D play both anti- and proinammatory roles in pulmonary host defense. Evidence

COLLECTINS 539

suggests a mechanism by which SP-A and SP-D can simultaneously mediate anti- and proinammatory processes in the lung. In the unbound state, the CRDs of SP-A or SP-D bind to signal-regulating protein-a (SIRPa), a transmembrane protein involved in signal transduction, which initiates a signaling cascade that inhibits proinammatory mediator production by alveolar macrophages. In contrast, if the CRDs of SP-A or SP-D are occupied by a microbial ligand, the collectin reverses orientation and binds to calreticulin/ CD91 via the collagenous tail. Calreticulin/CD91 subsequently initiates a pathway that releases proinammatory mediators and stimulates phagocytosis. Therefore, depending on the presence of infectious particles and subsequent binding orientation of the CRD, SP-A and SP-D can either enhance or suppress immune cell function.

Collectins in Clinical Disease

Human disease resulting from a mutation or deletion in SP-A or SP-D has not been identied. However, the apparent immune modulatory activities of these proteins, combined with the results of microbial challenge experiments in SP-A- and SP-D-decient mice, suggest that altered function or decreased levels of either protein would result in clinical disease that would reect a loss of either the pro- or anti-inammatory properties of each protein. A variety of clinical conditions characterized by increased inammation or lung parenchyma damage, including adult respiratory distress syndrome, bronchopulmonary dysplasia, asthma, chronic obstructive pulmonary disease, and cystic brosis, are associated with decreased levels of SP-A or SP-D in broncheolar lavage uid. Altered collectin function is also associated with increased susceptibility to infection. The severity of RSV infections correlates with genetic polymorphism in SP-D. Specifically, infants hospitalized with RSV were more likely than controls to be homozygous for the allele coding methionine at amino acid 11 of SP-D. Furthermore, in a population of children who have increased susceptibility to infection, mutations were identied in the serum collectin, MBP, that interfered with appropriate protein folding and secretion.

SP-A and SP-D Receptors

SP-A and SP-D interact with several receptors or binding molecules on host cells. These include glycoproteins and glycolipids, which bind the CRD, as well as membrane proteins, which interact with other regions of the collectin. Both SP-A and SP-D bind the LPS receptor CD14; however, the mechanism of binding differs between each protein. SP-D binds CD14 via interactions between the CRD and N-linked oligosaccharides on CD14, whereas SP-A binds directly to the protein backbone. As mentioned previously, SP-A and SP-D bind the LPS signaling protein SIRPa. SP-A also inhibits peptidoglycan-induced cytokine release by the Toll-like receptor-2. The fact that both collectins bind LPS and multiple proteins involved in LPS signaling suggests a possible mechanism for controlling the cellular response to LPS; however, the precise steps of this pathway remain to be determined. SPR-210 is a receptor found on alveolar type II cells, macrophages, and lymphocytes. SP-A binding to SPR-210 inhibits lipid secretion by type II cells, suppresses T lymphocyte proliferation, and enhances uptake of mycobacterium by macrophages. Evidence indicates that SPR-210, through the activation of cellular myosins, mediates high-afnity binding and uptake specifically of SP-A-coated particles and bacteria. SP-A and SP-D show calcium-dependent but CRDindependent binding to gp340, a member of the scavenger receptor superfamily. Although gp340 is located on alveolar macrophages and colocalizes with SP-D in multiple tissues, there is no evidence that gp340 represents a physiologically relevant receptor for either protein.

Summary
Collectins are an essential component of the innate immune system and modulate the inammatory response of immune cells. In the absence of infection, SP-A and SP-D can inhibit inammatory responses within the lung. However, in the presence of a microbial challenge, SP-A and SP-D recognize invading pathogens and orchestrate immune cell activation and phagocytosis of microbes. Therefore, the pulmonary collectins can provide a critical dual-role mechanism for initiating the critical host defense response to infection while limiting the potential damaging effects of inammation and microbial invasion on the lung parenchyma.
See also: Clara Cells. Endotoxins. Epithelial Cells: Type II Cells. Leukocytes: Pulmonary Macrophages. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Protein D (SP-D). Toll-Like Receptors. Transcription Factors: Overview.

Further Reading
Clark H and Reid KB (2002) Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology 205: 619631.

540 COLONY STIMULATING FACTORS

Crouch E and Whitsett JA (2003) Diverse roles of lung collectins in pulmonary innate immunity. In: Ezekowitz RAB and Hoffman JA (eds.) Innate Immunity. Totowa, NJ: Humana Press. Crouch E and Wright JR (2001) Surfactant proteins A and D and pulmonary host defense. Annual Review of Physiology 63: 521 524. Gardai SJ, Xiao YQ, Dickinson M, et al. (2003) By binding SIRPa or clareticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inammation. Cell 115: 1323. Hawgood S and Poulain FR (2004) The pulmonary collectins and surfactant metabolism. Annual Review of Physiology 63: 495 519. Hickman-Davis JM, Fang FC, Nathan C, et al. (2001) Lung surfactant and reactive oxygennitrogen species: antimicrobial activity and hostpathogen interactions. American Journal of Physiology: Lung Cellular and Molecular Physiology 281: L517L523. Korfhagen TR, Bruno MD, Ross GF, et al. (1996) Altered surfactant function and structure in SP-A gene targeted mice. Proceedings of the National Academy of Sciences, USA 93: 95949599. Korfhagen TR, Sheftelyevich V, Burhans MS, et al. (1998) Surfactant protein D regulates surfactant phospholipid homeostasis in vivo. Journal of Biological Chemistry 273: 2843828443. Kuroki Y and Sano H (1999) Functional roles and structural analysis of lung collectins SP-A and SP-D. Biology of the Neonate 76(supplement 1): 1921. Kuroki Y, Takahashi H, Chiba H, and Akino T (1998) Surfactant proteins A and D: disease markers. Biochimica Biophysica Acta 19: 334345. Palaniyar N, Nadesalingam J, and Reid KB (2002) Pulmonary innate immune proteins and receptors that interact with Grampositive bacterial ligands. Immunobiology 205: 575594. Van de Wetering JK, van Golde LMG, and Bateenburg JJ (2004) Collectins: players of the innate immune system. European Journal of Biochemistry 271: 12291249.

COLONY STIMULATING FACTORS

B C Trapnell and S Abe, Cincinnati Childrens Hospital, Cincinnati, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The colony stimulating factors (CSFs) comprise a small family of homodimeric cytokines central to blood formation, leukocyte function, and overall immune competence. Family members include macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and interleukin-3 (IL-3). M-CSF and G-CSF are relatively lineage-specic and affect the survival, growth, differentiation, and function of mononuclear phagocytes (monocytes/macrophages) and neutrophils, respectively. GM-CSF is less restricted and stimulates growth of myeloid progenitors as well as the survival and function of both macrophages and neutrophils. IL-3 is pluripotent and affects all hematopoietic lineages. CSF production and clearance are strictly regulated to maintain homeostasis and are modulated during conditions of stress such as the increased turnover of leukocytes in intensely inammatory lung diseases like cystic brosis. The CSFs function within a complex yet precisely regulated signaling network involving local, paracrine, and endocrine pathways, the effects of which extend beyond hematopoiesis. For example, a critical role has been established for GM-CSF in the regulation of pulmonary surfactant homeostasis and innate immunity in the lung. Deciency of endogenous GMCSF bioactivity due to neutralizing anti-GM-CSF antibodies is central to the pathogenesis of idiopathic pulmonary alveolar proteinosis.

family members were identied, puried, and studied intensely. The genes for each CSF have been isolated from multiple species, cloned, and characterized in detail, and both human and murine recombinant CSF molecules are commercially available. Their respective receptors have also been identied, cloned, and characterized. M-CSF and G-CSF are relatively lineage-specic and stimulate formation of macrophage and granulocyte (neutrophil) colonies from bone marrow precursors, respectively. GM-CSF gives rise to both macrophage and neutrophils and IL-3 (also called multi-CSF) is a pluripotent factor stimulating formation of macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes, and erythroid cells.

M-CSF
Structure

Introduction
In the mid-1960s, Donald Metcalf identied colony stimulating factor (CSF) in mice and several years later in humans. Over the next decade, four CSF

Human M-CSF (also known as CSF-1) is a complex cytokine that exists in three isoforms, each of which is composed of disulde-linked homodimers derived from alternatively spliced mRNA transcripts. M-CSFb is comprised of two 522 amino acid (AA) polypeptides representing the full-length M-CSF coding sequence. M-CSFa is composed of two 224 AA polypeptides that lack residues 150447. M-CSFg is composed of two 406 AA polypeptides and lacks residues 332447. The molecular weight of M-CSFb (4590 kDa) exceeds the predicted value (60 kDa) due to glycosylation. The crystallographic structure resolution of M-CSF has been determined at 2.5 A

COLONY STIMULATING FACTORS 541

S C S S N C N M-CSF G-CSF C S S GM-CSF N IL-3 N

Figure 1 Crystal structures of cytokine growth factor monomers. All structures are depicted in worm format using the Cn3D viewer from the National Center for Biotechnology Informations Molecular Modeling DataBase (MMDB) structural database. The structures were derived from the following NCBI Protein DataBase (PDB) les: Human MCSFaPDB 1HMC A; Human G-CSFPDB 1PGR A; Human GM-CSFPDB 1CSG; Human IL-3PDB IJLI. The amino terminal (N) and carboxy terminal (C) end of each polypeptide is indicated. Disulde bonds are marked (S).

and M-CSF shares structural similarity to GM-CSF and G-CSF (Figure 1). Mature murine and human M-CSF are similar except that the former is 519 AA residues in length.
Production and Activity

Human M-CSF is derived from a single gene at 1p13 21; its predicted length is 554 AA including a 32 AA signal peptide. M-CSF mRNA is alternatively spliced within exon 6 resulting in three major transcripts encoding the three protein isoforms. All isoforms contain the 149 N-terminal residues required for biological activity. M-CSF is present as an integral cellsurface protein and several soluble forms. An active soluble form is released from the cell surface by proteolytic cleavage. Murine and human M-CSF are highly homologous within the N-terminal (bioactive) but not the C-terminal region of the molecule. M-CSF is produced in a wide variety of cells and normal blood levels (38 ng ml 1) are tightly controlled. More than 95% of M-CSF present in the blood is cleared by intravascular monocytes and macrophages, which internalize, degrade M-CSF, and proliferate in response. M-CSF pools in blood and tissues appear to be compartmentalized. Superimposed upon this coarse control of M-CSF levels is a ne control which can be recruited under stress conditions such as infection. At such times, local M-CSF release further stimulates local mononuclear phagocytes. Other factors stimulating M-CSF production include lipopolysaccharide (LPS), IL-2, phorbol esters, tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-g).
Receptors

140150 kDa (972 AA) polypeptide with extracellular, membrane-spanning, and cytoplasmic domains. The cytoplasmic region includes an autophosphorylation domain that associates with phosphoinositol-3 OH kinase (PI3K) and is required for signaling. The M-CSF receptor gene is located at 5q33-3. It is normally expressed on macrophage lineage cells, osteoclasts, and placental trophoblasts. It is also expressed on mammary epithelial cells during lactogenic differentiation and other cell types in certain pathological conditions. The half-life is short (23 h) in the absence of ligand and M-CSF receptors are internalized and rapidly degraded upon ligand binding. Receptor expression is modulated by endotoxin and various cytokines. Ligand binding causes receptor dimerization, auto- and intermolecular phosphorylation of intracytoplasmic residues, activation of PI3K, Srcfamily kinases, and Raf-1 and other downstream signaling events. Human M-CSF is able to activate the murine M-CSF receptor but not vice versa.
Biological Functions

M-CSF stimulates bone marrow progenitors to form macrophage colonies in soft agar as well as promoting the survival, growth, differentiation, and activation of macrophage lineage cells. In mature macrophages it stimulates production of cytokines (IL-1b, TNF-a, IFN, and G-CSF), plasminogen activator, prostaglandin E, and reactive oxygen species and regulates other metabolic processes. M-CSF stimulates the antimicrobial activity of macrophages and is important in bone metabolism, atherogenesis, lipoprotein clearance, and uterine function.

G-CSF
Structure

The M-CSF receptor (CD115) belongs to the protein tyrosine kinase receptor superfamily and is identical to the cfms proto-oncogene. It consists of one

Human G-CSF (also known as CSFb and pluripoietin) is expressed in two isoforms (177 and 174 AA)

542 COLONY STIMULATING FACTORS

due to alternative splicing. Its molecular weight (21 kDa) is larger than predicted (19 kDa) due to Olinked glycosylation at Thr133. There are two interchain disulde bonds (Cys36Cys42 and Cys64 Cys74) important for solubility and stability. The crystallographic structure of G-CSF has been deter resolution (Figure 1). Mature murine mined at 2.5 A and human G-CSF are similar except that the former is 178 AA residues in length.
Production and Activity

Human G-CSF is derived from a single gene at 17q2122; its predicted length is 207 AA including a 30 AA signal peptide. G-CSF mRNA is polyadenylated and alternatively spliced in humans (but not mice); the 174 AA isoform is the dominant variant and is 20-fold more active than the 177 AA isoform. It is biologically active as a soluble homodimer. G-CSF is produced by activated macrophages/ monocytes as well as various other cells; its production is under tight control and blood levels are normally very low. G-CSF is cleared by granulocytes, which bind, internalize, degrade, and respond to it. G-CSF production is under both coarse and ne regulatory control. Factors stimulating its production include other CSFs (IL-3, GM-CSF, M-CSF), IL-1, IL-4, TNF-a, IFN-g, LPS, and phorbol esters. G-CSF expression is regulated at transcriptional and posttranscriptional levels.
Receptors

the survival, proliferation, and differentiation of neutrophil lineage cells. G-CSF stimulation of mature neutrophils results in increased production of reactive oxygen intermediates, expression of C3b receptors, and release of arachidonic acid and myeloperoxidase. Neutrophil functions stimulated by G-CSF include phagocytosis, chemotaxis, and antibody-dependent cellular cytotoxicity (ADCC) against tumor cells. G-CSF promotes the proliferation and migration of endothelial cells in vitro, the secondary platelet aggregation induced by adenosine diphosphate, and angiogenic activity in rabbit cornea, and stimulates growth of some small cell lung cancer cell lines. It also stimulates production of acute phase response proteins.

GM-CSF
Structure

The G-CSF receptor (CD114) belongs to the hematopoietic receptor superfamily and comprises a single 120150 kDa polypeptide with extracellular, membrane-spanning, and cytoplasmic domains. It does not have intrinsic tyrosine kinase activity but is thought to bind and mediate phosphorylation of the Jak2 kinase upon ligand-mediated dimerization. The human G-CSF receptor gene is located at 1p3534.3 and expression is strictly regulated transcriptionally. It is normally expressed on neutrophils, neutrophil progenitors, and platelets, but can also be found on endothelial and other cells. Expression of the G-CSF receptor is stimulated by all-trans retinoic acid and downregulated by GM-CSF, TNF, LPS, C5a, formylMet-Leu-Phe (fMLP), and phorbol esters. Ligand binding causes receptor dimerization, phosphorylation by Jak2 kinase, and downstream signaling events. Human G-CSF binds and stimulates the murine G-CSF receptor and vice versa.
Biological Functions

GM-CSF (also known as CSF-2, CSFa or pluripoietin-a) is a 22 kDa homodimeric polypeptide cytokine in humans and mice that exists as a single soluble isoform. The mature human polypeptide is 127 AA in length (124 AA for mouse) and contains two intrachain disulde bonds (Cys54Cys96 and Cys88 Cys121 in human, Cys51Cys93 and Cys85Cys118 in mouse) and two potential N-linked glycosylation sites (Asn27 and Asn37 in human, Asn66 and Asn75 in mouse). The crystallographic structure of resolution GM-CSF has been determined at 2.5 A (Figure 1).
Production and Activity

G-CSF stimulates bone marrow progenitors to form neutrophil colonies in soft agar as well as promoting

Human GM-CSF is derived from a single gene at 5q2331; its predicted length is 144 AA including a 17 AA signal peptide. Glycosylation is not required for biological activity, but nonglycosylated GM-CSF is 10-fold more active than the glycosylated form. Murine and human GM-CSF share approximately 56% homology but do not cross-react immunologically or functionally. GM-CSF is produced by T lymphocytes, macrophages, epithelial cells, endothelial cells, and various other cells. Production is tightly regulated. GM-CSF levels are normally only B545 pg ml 1 in serum but increase during infection. The levels are maintained by dual coarse and ne regulatory control. Factors stimulating GM-CSF production include LPS, IL-2, IL-1, and TNF-a. Production by macrophages is also stimulated by adherence and by phagocytosis. GM-CSF pools in the lungs and blood are compartmentalized and expression is regulated transcriptionally and posttranscriptionally.

COLONY STIMULATING FACTORS 543 Receptors

The GM-CSF receptor belongs to the hematopoietic receptor superfamily and is composed of heterodimeric a and b chains. The human a chain (CDw116) is an 80 kDa transmembrane protein with an extracellular region, a membrane-spanning region, and a short cytoplasmic tail and has 11 potential N-linked glycosylation sites. The human b chain (KH97) is a 130 kDa transmembrane protein with an extracellular domain, a membrane-spanning region, and a large cytoplasmic domain, and three potential N-linked glycosylation sites. The b chain is shared with the receptors for IL-3 and IL-5. The GM-CSF receptor is normally expressed on macrophages, neutrophils, and eosinophil lineage cells, and has been also demonstrated on CD34 progenitor cells, erythrocyte and megakaryocyte precursors, B and T fetal lymphocytes, vascular endothelial cells, broblasts, osteoblasts, and uterine cells. Expression of the GM-CSF receptor is stimulated by IFN-g, TNF-a, IL-3, erythropoietin (EPO), and stem cell factor (SCF). The a chain binds GM-CSF with low afnity (3.23 10 9 M). The b chain does not bind GMCSF, but associates with the ligand-bound a chain increasing the ligand-binding afnity (1.203 10 10 M). The b chain constitutively associates with Jak2 kinase and association with the ligand-bound a chain results in tyrosine phosphorylation of the receptor and Jak2, and signaling through multiple pathways.
Biological Functions

surfactant homeostasis and innate immunity in the lung. Many of the effects of GM-CSF on alveolar macrophage innate immune and other functions are mediated by the transcription factor PU.1 (Figure 2).

IL-3
Structure

lnterleukin-3 (IL-3) (also known as multi-CSF, hematopoietic cell growth factor, burst-promoting activity, P-cell stimulating factor activity, thy-1 inducing factor) is a 1430 kDa homodimeric polypeptide cytokine in humans (28 kDa in mice) that exists as a single isoform. Mature human IL-3 is 133 AA in length and contains one intrachain disulde bond (Cys16Cys84) and two potential N-linked glycosylation sites (Asn15 and Asn70). Mature mouse IL-3 is 140 AA in length and contains two disulde bonds (Cys17Cys80 and Cys89Cys140). It has four potential N-linked glycosylation sites, but is glycosylated only at Asn16 and Asn86. The crystallographic structure of a variant of human IL-3 has been determined (Figure 1).
Production and Activity

GM-CSF stimulates bone marrow progenitors to form neutrophil and macrophage colonies in soft agar and has pleiotropic effects on hematopoietic and other cell types. It is a survival and growth factor for hematopoietic progenitors and stimulates differentiation and activation of granulocytes and monocytic cells, and serves as a growth factor for endothelial and epithelial cells, erythroid cells megakaryocytes, and T cells. GM-CSF exhibits overlapping activities on hematopoietic progenitors with other cytokines including M-CSF, G-CSF, IL-3, IL-6, and SCF. GM-CSF stimulates a broad range of innate immune functions in macrophage and neutrophil lineage cells including phagocytosis, chemotaxis, cytokine release, superoxide radical production, antigen presentation, and others, and contributes to host defense against bacterial, viral, fungal, and parasitic infections and to antitumor surveillance. GM-CSF has critical roles in the lung in mice and humans where it stimulates terminal differentiation of alveolar macrophages and is required for

Human IL-3 is derived from a single gene at 5q23; its predicted length is 152 AA including a 19 AA signal peptide. Glycosylation is not required for biological activity. Human and murine IL-3 share only 29% homology and do not cross-react immunologically or functionally. IL-3 is produced by activated T lymphocytes, and various other cells. IL-3 gene expression is regulated transcriptionally and posttranscriptionally.
Receptors

The IL-3 receptor belongs to the hematopoietic receptor superfamily and is composed of heterodimeric a and b chains. The human a chain (CD123) is a 70 kDa transmembrane protein with an extracellular region, a membrane-spanning region, and a short cytoplasmic tail with six potential N-linked glycosylation sites. The extracellular domain of the a chain shares tertiary structural similarity with the GM-CSF receptor a chain. The human b chain (KH97) is shared with the receptors for GM-CSF and IL-5 and has been described above. Mice have two b chains: AIC2B, which corresponds to the human b chain (KH97), and another, AIC2A, which associates only with the IL-3a chain. The IL-3 receptor is normally expressed on hematopoietic progenitor cells, monocytes, and B lymphocytes. Expression of the a chain is tightly regulated at the level of transcription.

544 COLONY STIMULATING FACTORS

Surfactant film Alveolar stabilization

Large surfactant aggregates Tubular myelin

SP-A SP-D

Host defense Surfactant pool size

Small surfactant aggregates

Secretion ABCA3 transporter Lamellar body SP-B SP-C Lipids Secretory vesicle SP-A SP-D SP-A Pro-SP-B Pro-SP-C SP-D Recycling Uptake

Alveolar macrophage

L PU.1 GM-CSF Catabolism

G Processing ER N

Type II alveolar epithelial cell

Type I alveolar epithelial cell

Figure 2 Regulation of surfactant homeostasis by GM-CSF. Surfactant proteins (SP)(SP-A, SP-B, SP-C, and SP-D) and surfactant lipids are produced in pulmonary type II alveolar epithelial cells and released as large aggregates into the alveolar space (green arrows). A thin surfactant lm stabilizes the alveolus by reducing the tremendous force of surface tension at the airliquidtissue surface. Small surfactant aggregates derived from this lm are either recycled in type II cells (yellow arrow) or taken up into alveolar macrophages. GMCSF, also produced by type II cells, stimulates increased levels of the transcription factor PU.1 in alveolar macrophages. PU.1, in turn, is required to stimulate catabolism of surfactant lipids and proteins in these cells (white arrows in macrophage). In the absence of GM-CSF signaling (human idiopathic pulmonary alveolar proteinosis, GM-CSF or GM-CSF receptor knockout mice), surfactant production proceeds as usual, but catabolism in alveolar macrophages is impaired resulting in intra-alveolar and intracellular surfactant accumulation. GM-CSF, via PU.1, stimulates alveolar macrophage terminal differentiation. ER, endoplasmic reticulum; G, Golgi body; N, nucleus.

In humans, only the a chain binds IL-3 and does so with low afnity (10 7 M). Association of the ligand-bound a chain with the b chain, which does not bind IL-3, converts ligand binding to high afnity (10 10 M). In mice, the same pattern is observed for binding to the a chain (53 10 8 M) and conversion to high-afnity binding by association with AIC2B, which increases a chain binding afnity (33 10 10 M). The mouse a/b (AIC2B) receptor binds IL-3 with high afnity (43 10 10 M). Ligand binding results in phosphorylation of the b chain and multiple other cellular proteins. Signal transduction requires the presence of the b subunit, which apparently does not have intrinsic kinase activity.
Biological Function

monocytes and may have an immunomodulatory role.

CSF in Respiratory Diseases

Each of the CSFs discussed above have immunomodulatory properties on mature myeloid cells in mice and humans. Targeted gene ablation studies in mice have been particularly helpful in delineating the roles of CSFs in the lung.
CSF in Murine Lung Diseases

IL-3 stimulates bone marrow progenitors to form erythroid, granulocyte, and macrophage colonies in soft agar. It serves as a growth factor stimulating progenitor to form erythroid, megakaryocyte, neutrophil, eosinophil, basophil, mast cell, and monocytic lineages. IL-3 is capable of activating

Naturally occurring osteopetrotic (Op/Op) mice are decient in M-CSF due to a null-mutation in the M-CSF gene. At birth, there are reduced numbers of macrophage lineage cells including pulmonary alveolar macrophages. Alveolar macrophage numbers correct with age in association with increased pulmonary IL-3 levels, alveolar macrophage metalloprotease release, and development of emphysema. G-CSF knockout mice have neutropenia and do not develop neutrophilia during infection. They have

COLONY STIMULATING FACTORS 545

increased incidence of spontaneous lung infections, which contribute significantly to increased mortality. Recently, a homeostatic mechanism regulating blood neutrophil counts has been identied involving IL-23, IL-17, and G-CSF. GM-CSF knockout mice do not have gross hematological abnormalities other than reduced eosinophil counts, but develop a lung phenotype characterized by the accumulation of surfactant lipids and surfactant proteins. The lung abnormality is due to decreased surfactant catabolism in alveolar macrophages. GM-CSF, via PU.1, regulates surfactant catabolism and a number of innate immune functions in macrophages. GM-CSF also regulates innate immune functions in mature neutrophils. GMCSF receptor b chain (AIC2B) knockout mice have a pulmonary phenotype similar to GM-CSF knockout mice. IL-3 knockout mice have impaired delayed-type hypersensitivity. In contrast, gene targeting of either AIC2B or A1C2A did not result in hematologic abnormalities (other than decreased numbers of eosinophils in AIC2B-decient mice), apparently due to the presence of redundant IL-3 receptors in mice.
CSF in Human Lung Diseases

Proteinosis. Leukocytes: Neutrophils; Pulmonary Macrophages. Stem Cells. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D). TollLike Receptors. Transcription Factors: PU.1. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Dranoff G, Crawford AD, Sadelain M, et al. (1994) Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264(5159): 713716. Fitzgerald KA, ONeill LAJ, Gearing AJH, and Callard RE (2001) The Cytokine Facts Book, 2nd edn. San Diego: Academic Press. Gaviria JM, Garrido SM, and Root RK (2001) Clinical use of granulocyte colony-stimulating factor in infectious diseases. Current Clinical Topics in Infectious Diseases 21: 302322. Hubel K, Dale DC, and Liles WC (2002) Therapeutic use of cytokines to modulate phagocyte function for the treatment of infectious diseases: current status of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and interferon-gamma. Journal of Infectious Diseases 185: 14901501. Kitamura T, Tanaka N, Watanabe J, et al. (1999) Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colonystimulating factor. Journal of Experimental Medicine 190(6): 875880. LeVine AM, Reed JA, Kurak KE, Cianciolo E, and Whitsett JA (1999) GM-CSF-decient mice are susceptible to pulmonary group B streptococcal infection. Journal of Clinical Investigation 103(4): 563569. Lieschke GJ and Burgess AW (1992) Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (1). New England Journal of Medicine 327: 2835. Lieschke GJ and Burgess AW (1992) Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (2). New England Journal of Medicine 327: 99106. Nishinakamura R, Wiler R, Dirksen U, et al. (1996) The pulmonary alveolar proteinosis in granulocyte macrophage colonystimulating factor/interleukins 3/5 beta c receptor-decient mice is reversed by bone marrow transplantation. Journal of Experimental Medicine 183(6): 26572662. Roilides E and Farmaki E (2001) Granulocyte colony-stimulating factor and other cytokines in antifungal therapy. Clinical Microbiology and Infection 7(supplement 2): 6267. Roilides E, Lyman CA, Mertins SD, et al. (1996) Ex vivo effects of macrophage colony-stimulating factor on human monocyte activity against fungal and bacterial pathogens. Cytokine 8: 4248. Rosen SG, Castleman B, and Liebow AA (1958) Pulmonary alveolar proteinosis. New England Journal of Medicine 258: 11231142. Seymour JF, Presneill JJ, Schoch OD, et al. (2001) Therapeutic efcacy of granulocyte-macrophage colony-stimulating factor in patients with idiopathic acquired alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 163(2): 524 531. Shibata Y, Berclaz P-Y, Chroneos Z, et al. (2001) GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU. 1. Immunity 15: 557567. Shibata Y, Otake K, Zsengeller Z, Plalaniyar N, and Trapnell BC (2001) Alveolar macrophage deciency in M-CSF-decient,

All of the CSFs are capable of modulating functions of myeloid lineage cells in humans and pulmonary CSF levels are increased during lung inammation. For example, GM-CSF and IL-3 levels are elevated in asthma; GM-CSF is elevated in cystic brosis; and GM-CSF and MCSF are elevated in smokers. CSF levels may participate in the pathogenesis of these and other lung disorders by modulating macrophage and neutrophil functions and the inammatory response. Genetic disorders of CSF deciency corresponding to each of the CSF knockout models discussed above have not been identied in humans. However, idiopathic pulmonary alveolar proteinosis is specifically associated with high levels of neutralizing, anti-GMCSF autoantibodies, which completely eliminate GM-CSF bioactivity and result in a functional deciency of GM-CSF. Clinically, this disease is characterized by surfactant accumulation in the lungs and an increased rate of secondary infections, similar to that in GM-CSF-decient mice. Together, data in mice and humans strongly suggest that GM-CSF, via PU.1, regulates pulmonary surfactant homeostasis and innate immunity in the lung by modulating alveolar macrophage function.
See also: CD14. Croup. Hypoxia and Hypoxemia. Inhibition of Differentiation (ID) Proteins. Interleukins: IL-12. Interstitial Lung Disease: Alveolar

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osteopetrotic mice spontaneously corrects with age and is associated with matrix metalloproteinase expression and emphysema. Blood 98: 28452852. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 25272539.

Relevant Websites
http://www.ncbi.nlm.nih.gov National Center for Biotechnology Information: Molecular Modeling DataBase. http://rarediseasesnetwork.epi.usf.edu Rare Lung Diseases Consortium.

COMPLEMENT
N Rawal, University of Texas, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Complement is an important part of the hosts immune system that ghts infections. The complement system consists of about 35 different proteins; most of these are soluble plasma proteins, but some are membrane bound. The complement system recognizes, opsonizes, kills, and clears bacteria, parasites, and other pathogens. It also aids in the clearance of immune complexes and altered host cells, for example, apoptotic or necrotic cells. Bacteria or immune complexes activate complement to initiate a cascade in which one complement component activates another. Products generated during the activation process elicit cellular responses such as phagocytosis and inammation that involve innate and adaptive immune systems. Complement activation ultimately results in assembly of the cytolytic membrane attack complex, which then lyses targeted bacteria. Activation of complement is under the strict control of complement regulatory proteins, which include about one third of the total complement proteins. Excessive or inappropriate activation of complement contributes to the pathogenesis of several immuno-inammatory diseases and conditions, including glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, hemolyticuremic syndrome, multiple sclerosis, tissue graft rejection, and reperfusion injury. Complement activation products are implicated in lung inammation and injury from various causes, and respiratory diseases, such as pneumonia, acute respiratory distress syndrome, pulmonary brosis, smoke-induced damage and asthma, may involve complement activation.

of complement activation was the rst to be discovered, components involved in its activation were puried and characterized only in the 1960s and 1970s from work done in the laboratories of Nelson and Mu eller-Eberhard. In the 1950s, Pillemer suggested an alternative pathway to the classical pathway. This second pathway of complement activation did not require an antibody, and its existence was not accepted until later in the 1970s. More recently (1996), Matsushita and Fujita discovered a third pathway of complement activation. The lectin pathway involves recognition of sugar residues present on the cell surface of microbes by mannan-binding lectin (MBL) and colins.

Structure
Complement proteins can be divided into two general groups based on their structure. Components C3, C4, C5, C1 inhibitor, C3a receptor (C3aR), and C5a receptor (C5aR) form one group, whose primary structure resembles members of other protein families almost across their entire length. The second group includes most of the remaining complement components. These are modular proteins with just short sequences of amino acids in common with other proteins. C3, C4, and C5 belong to the same family as a2macroglobulin. Except for C5, these proteins have an internal thioester bond, which becomes exposed when activated by proteolysis. Then the metastable thioester can be hydrolyzed, or it can react with hydroxyl or amino groups on cells or proteins to form ester (Figure 2) or amide bonds. C1 inhibitor belongs to the serpin superfamily of serine protease inhibitors, while C3aR and C5aR belong to the family of 7 membrane domain G-protein-coupled receptors. C1r, C1s, factor D, factor B, mannan-binding lectin-associated serine proteases (MASPs), C2, and factor I belong to the second group. These are enzymes of the complement system that generate biologically active complement fragments during

Introduction
Complement was rst described in the 1890s as a heat-labile bactericidal activity in the bloodstream that complemented heat-stable antibodies in the killing of microorganisms. Bordet was awarded the Nobel Prize in 1919 for discovering this factor and dening its actions. He showed that the bactericidal factor present in normal serum had no activity by itself, but that it became lytic in the presence of the heat-stable antibody (i.e., antibody-mediated immunity). Since then, three distinct pathways of complement activation have been recognized (Figure 1). Although the antibody-dependent classical pathway

COMPLEMENT 547

Classical pathway (IgM- or IgG-containing immune complexes) C1 C4 C2

Lectin pathway (carbohydrates) MBL/ficolins MASPs C4 C2 C3 P

Alternative pathway (bacteria, viruses, yeast) C3(H2O) B D

Opsonization

C3 convertase C3a C5 C5 convertase C5a C5b C6 C7 C8 C9 Membrane attack complex Lysis Terminal pathway Inflammation

Figure 1 The complement system. Activation of complement occurs via three pathways: the classical pathway, the lectin pathway, and the alternative pathway. The established nomenclature and symbols for complement proteins have been used. C1, C4, C2, C3, C5, C6, C7, C8, C9 are complement components that directly participate in the activation process. C3(H2O), an inactive form of C3; MBL, mannan-binding lectin; MASPs, mannan-binding lectin-associated serine proteases; B, complement factor B; D, complement factor D; P, complement protein, properdin; C3a, one of the cleavage products of C3; C5b and C5a, the cleavage products of C5; C3 convertase and C5 convertase, serine proteases that are transiently generated up on the activation of complement and cleave C3 and C5, respectively.

C3a or C4a C3 or C4 O C SH O OH C+ H O H C3 convertase C3b or C4b S C3b or C4b

O O

SH

have similar structures, and they interact with C5b and each other in a sequential manner to form the cytolytic membrane attack complex that lyses bacteria. Complement receptors, CR1 and CR2, and complement regulatory proteins, C4b-binding protein (C4bp), factor H, decay accelerating factor (DAF), and membrane cofactor protein (MCP) also belong to the second group. These proteins are composed of repeats of complement control protein (CCP) modules.

Cell surface

Surface-bound C3b or C4b

Figure 2 Activation and deposition of C3 and C4. C3 and C4 have an internal thioester bond that is exposed when they are cleaved. The reactive thioester bond reacts with hydroxyl groups (shown in the gure) or amino groups found on a wide range of proteins and cell membranes of the target surface via an ester or an amide bond.

Functions of Complement
Complement proteins circulate in blood as inactive molecules. When activated, complement components are cleaved into two products that have biological activity (Figure 3). The larger fragment called b usually remains attached to the surface of the microorganism while the smaller fragment called a may diffuse away. Activation of the complement via any of the three pathways has two phases: opsonization and formation of membrane attack complex

complement activation. All have a catalytic domain that is structurally similar to that found in serine proteases of the trypsin subfamily. C2 and factor B are structurally very similar. C6, C7, C8, and C9

548 COMPLEMENT

(Figure 1). In the rst phase, when an activator is recognized, the complement system is activated, and a series of proteinprotein interactions and proteolytic cleavages culminate in the assembly of transient complexes with protease activity. These complexes are called C3 convertases. Although the three pathways are activated by different mechanisms, formation of C3 convertase results in cleavage of the same peptide bond in C3. Cleavage of C3 into C3b and C3a is central to the process of complement activation. The larger fragment, C3b, covalently attaches to (Figure 2) and coats (opsonizes) the cell surface so that phagocytic cells recognize the particle, while the smaller C3a fragment promotes an inammatory response by attracting eosinophils and phagocytes to the site. The second part of complement activation is the effector phase. C3b attached to a foreign surface is cleaved again forming smaller fragments iC3b and C3d. These fragments are ligands that present the particle to the adaptive immune system through the CD21/CD19 receptor system; this antigen presentation then prompts production of antibodies by the CD58 B cell population. In this phase of complement activation, cleavage of C5 by C5 convertase initiates the formation of a membrane attack complex (Figure 1). This last enzymatic step in the complement cascade constitutes the terminal activation common to all three pathways. The products of C5 cleavage, C5b and C5a, play an important role in killing microorganisms (Figure 3). The larger fragment, C5b, binds with other complement proteins C6, C7, C8, and C9 to form the membrane attack complex and lyses cells to which the complex is

attached. The smaller fragment, C5a, is one of the most potent proinammatory mediators known. It has many diverse activities, including the release of mediators from mast cells, chemoattraction of neutrophils and eosinophils, upregulation of b2 integrins, and it causes L-selectin shedding from eosinophils along with release of granules and an oxidative burst.

Alternative Pathway of Complement

The alternative pathway is one of the hosts innate immune responses to infection. Six complement proteins, C3, properdin (P), and factors B, D, H and I are involved in activation, amplication, and regulation of this pathway. The alternative pathway can operate in the absence of antibodies. It is activated in response to the activating surfaces of certain bacteria, viruses, fungi, and yeast within few minutes after they come in contact with plasma. It is also spontaneously activated in the uid phase at a very low rate. Initiation of complement activation via the alternative pathway involves spontaneous activation of C3 in the uid phase. C3 has a thioester bond that reacts with any exposed hydroxyl or amino group. The slow hydrolysis of the thioester converts inactive C3 to C3(H2O) (Figure 4). C3(H2O) is like a functionally active C3b-like molecule; it binds factor B in a Mg2 dependent fashion to form the complex C3(H2O),B. Factor B in complex with C3(H2O) is then cleaved by factor D to form a C3 convertase, C3(H2O),Bb. The C3 convertase cleaves C3 to generate C3b and C3a. C3b generated by C3(H2O),Bb has an active thioester through which it can bind to

Activation products C3b, C4b, iC3b C3b

Activity Opsonization of bacteria and immune complexes Solubilization and clearance of circulating immune complexes Activation of lymphocytes and phagocytes Increased vascular permeability smooth muscle contraction Activation and degranulation of mast cells and basophils Activation and chemotaxis of neutrophils Lysis of bacteria and foreign cells

Function Phagocytosis Lymphocytosis and phagocytosis

Complement activation

C3a, C5a

Inflammation and degranulation Chemotaxis

C5a C5b-9 complex

Lysis

Figure 3 Functions of complement. Products generated during complement activation possess biological functions, which elicit cellular responses of the innate and adaptive immune systems to help ght infections. C3b and C3a, cleavage products of C3; C5b and C5a, the cleavage products of C5; C4b, one of the cleavage products of C4; iC3b, inactivated C3b; C5b-9 complex, the membrane attack complex.

COMPLEMENT 549
C3

Initiation

C3 convertase (fluid phase)

Metastable C3b Deposition of C3b Surface-bound C3b

C3

Amplification Recognition and regulation Nonactivator Activator

iC3b

C3 convertase C5 convertase Lysis Membrane attack complex

Figure 4 Activation of the alternative pathway of complement. Activation of the alternative pathway of complement involves initiation, deposition of C3b, recognition of an activator, amplication, regulation, and lysis. C3, the third component of complement that directly participates in the activation process; C3b, the proteolytically activated form of C3; iC3b, inactivated C3b; C3 convertase and C5 convertase, serine proteases that are transiently generated up on the activation of complement and cleave C3 and C5, respectively. The dashed lines represent the amplication loop.

a surface. Deposition of C3b is continuous and occurs on host cells (self) as well as foreign particles (nonself). The regulatory proteins, factors H and I, control discrimination between self and nonself surfaces. They readily inactivate C3b that deposits on host cells, while they are much less effective on C3b deposited on an activating surface such as bacteria, fungi, and viruses (Figure 4). Other complement regulatory proteins, such as DAF and MCP, are found on host cell surfaces where they can inactivate the C3 convertase, thereby preventing autologous complement activation on host tissue. A special feature of the alternative pathway is the amplication of the number of C3b molecules deposited on the surface (Figure 4). This occurs by C3b-dependent positive feedback. C3b is the product of C3 cleavage catalyzed by C3 convertase. It forms the noncatalytic subunit of the C3 convertase (C3b,Bb). Thus, every C3b deposited can form a new C3 convertase with factors B and D in the presence of Mg2 . This mechanism produces more C3b and consequently more enzymes. Factors H and I control the amplication process. Factor H enhances the dissociation of C3b,Bb, while C3b in complex with factor H is inactivated to iC3b by factor I. Properdin is a positive regulator that enhances amplication of surface-bound C3b by binding to C3b,Bb and stabilizing it. The C3 convertase, C3b,Bb, also cleaves C5, but does so inefciently. However, as C3b density

increases due to amplication of the number of C3b molecules deposited by C3b,Bb, C3b complexes form. These C3b complexes form C3 convertases that exhibit higher afnities for C5 and cleave C5 at a velocity approaching Vmax thereby switching the enzyme from C3 to C5 cleavage.

Classical Pathway of Complement

Seven proteins, C1, C2, C4, C3, C4b-binding protein, C1 inhibitor and factor I, are responsible for recognition, activation, and regulation of the classical pathway of the complement. The pathway is primarily activated by the interaction of C1 with immune complexes or aggregates containing IgG or IgM. The C1 complex consists of three components: C1q, proenzyme C1r, and proenzyme C1s in the ratio 1:2:2. C1q is the recognition unit of the C1 complex and binds to the Fc region of the antibody. The C1q molecule consists of six globular heads, each connected by a central bril region. When two or more heads of C1q interact with an immune complex or an activator, a conformational change occurs that results in the autoactivation of proenzyme C1r, thereby converting it to the active protease C1r. Activated C1r in turn cleaves proenzyme C1s converting it to the active protease C1s. C1 inhibitor is the only plasma inhibitor that inactivates C1r and C1s enzymes. The mechanism of inactivation involves C1 inhibitor binding to active sites on activated C1s and C1r.

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Activated C1s cleaves C4 into two products, C4a and C4b. The smaller fragment, C4a, is released into the uid phase, while the larger fragment, C4b, may attach to the surface of an activator. Freshly activated C4b has a reactive acyl group through which it binds covalently to hydroxyl (Figure 2) or amino groups close to the C1 molecule. C2 forms an Mg-dependent complex with C4b. If binding of C2 to C4b occurs close to activated C1s, then C2 is cleaved by C1s to C2b and C2a. The larger fragment C2a remains attached to C4b to form a bimolecular complex (C4b,C2a), which is the C3 convertase of the classical pathway of the complement. The C3 convertase, C4b,C2a, essentially cleaves C3 to C3b and C3a. Attachment of C3b results in opsonization of the target surface for phagocytosis. C3b can also attach to C4b, the noncatalytic unit of the C3 convertase enzyme, to form the complex, C3bC4b,C2a. This complex is also a C3 convertase, but because it has a much higher afnity for C5 than for C3, the enzyme primarily cleaves C5 and is referred to as a C3/C5 convertase. C3/C5 convertases of the classical pathway are inactivated in three ways. First, the soluble plasma complement regulatory protein, C4b-binding protein, binds to C4b and prevents assembly of C3/C5 convertase. Second, C4b-binding protein and DAF dissociate the catalytic subunit, C2a, from bound C4b causing the enzyme to lose activity. Third, C4b is inactivated to iC4b by the proteolytic activity of factor I, for which C4b-binding protein and the red cell complement receptor, CR1, act as cofactors.

(MBLMASP1MASP2) appears to be analogous in overall organization and function to the C1 complex (C1qC1r2C1s2) of the classical pathway. The proenzyme forms of MASPs are activated when MBL binds to the cell-surface carbohydrates of microbes. Activated MASP-1 and MASP-2 are regulated by C1 inhibitor in a manner similar to that observed for C1s and C1r enzymes. Activated MASP-2 cleaves C4 and C2 to form the classical pathway C3/C5 convertases (C4b,C2a), which then generate the membrane attack complex.

Complement Receptors
There are four complement receptors for C3 fragments: CR1, CR2, CR3, and CR4. CR1 (CD35) is found on erythrocytes, lymphocytes, neutrophils, dendritic cells, and others. The main functions of CR1 are phagocytosis and clearance of immune complexes. Erythrocytes bearing CR1 bind to circulating immune complexes or particles that have complement ligands C3b, iC3b, and C4b deposited on them. The erythrocytes then transport the complexes to liver and splenic macrophages for destruction. CR1 also regulates complement activation. It acts as a cofactor for factor I to cleave surface-bound iC3b or iC4b to smaller fragments: surface-bound components C3dg or C4d and soluble components C3c or C4c. Immune complexes with xed iC3b attach to neutrophils via CR1 and CR3. Subsequent release of cytotoxic enzymes, oxygen metabolites, and leukotrines by the activated neutrophils kills the bacteria. However, in autoimmune diseases (e.g., systemic lupus erythematosus) where immune complexes with xed iC3b are trapped in normal tissue, the same mechanism turns against the host to cause neutrophil-mediated tissue damage. CR2 (CD21) is a receptor for C3 cleavage fragments C3b, iC3b, C3d, and C3dg. CR2 enhances recognition of antigens by B cell activation and proliferation. CR3 (CD11b/CD18) is expressed mainly on mononuclear phagocytes and natural killer cells; it helps to clear opsonized bacteria. This complex is a receptor for iC3b and is used by macrophages to adhere to an extracellular matrix. CR3, along with CR4, mediates adhesion of neutrophils to endothelial cells at inammation sites via ICAM-1. CR4 (CD11c/CD18) is primarily expressed on myeloid cells, dendritic cells, natural killer cells, platelets, B lymphocytes, and alveolar macrophages. Binding of CR4 to iC3b initiates ingestion of particles that are deposited on the membrane via CR3. The C5a (CD88) and C3a receptors (C3aR) are G-protein-coupled receptors. Both C3a and C5a

Lectin Pathway of Complement

The lectin pathway of the complement is activated when MBL binds to hexoses with carbon 3 and 4 OH groups such as N-acetyl-D-glucosamine, glucose, fucose, and mannose. These sugars are expressed as repetitive O-polysaccharide structures on surfaces of bacteria, yeast, parasites, mycobacteria, and certain viruses. Because MBL does not recognize the OH conguration present in galactose and sialic acid, the sugars commonly found on mammalian glycoproteins, the bodys innate immune system can readily distinguish nonself structures from self. The lectin pathway plays a major protective role during the vulnerability window experienced by infants between decay of maternal antibody and establishment of an effective adaptive immune system. Activation of the complement via the lectin pathway occurs when the proenzyme forms of MASPs are activated. MASPs resemble the proteolytic enzymes, C1r and C1s, of the classical pathway. A complex of MBL, MASP-1, and MASP-2

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receptors are found on circulating white cells such as neutrophils, monocytes, basophils, and eosinophils. In addition, they are also found on endothelial cells, lung epithelial cells, hepatocytes, astrocytes, and microglial cells. Binding of C3a or C5a to their respective receptors invokes functions such as increased vascular permeability, edema, and degranulation of mast cells. C1qRp, C1qRo2, cC1qR, and CR1 are thought to be C1q receptors. Binding of C1q to these receptors on various cells initiates cellular responses such as phagocytosis, chemotaxis, clearance of apoptotic cells, and increase in superoxide metabolism by neutrophils.

damage. Complement activation also contributes to transfusion-related acute lung injury, which has features similar to early ARDS. The development of transfusion-related acute lung injury in most cases is attributed to the presence of antigranulocyte antibodies and other substances in blood-derived products, which activate granulocytes and the complement. Complement activation may also cause vascular endothelium damage during reperfusion of ischemic tissues. Specic inhibition of C5a or inhibition of the complement cascade by general depletion of the complement confers protection in animal models with acute lung injury.

Immune Complex Disease Complement in Respiratory Diseases

Various cells of the lung, including alveolar macrophages, lung broblasts, and type II alveolar cells, produce most of the complement components. Thus, although the complement system in the lung is well poised for killing and clearance of pulmonary infections, unwanted activation of the complement system can lead to lung injury. Immune injury to the lung is classied according to type of hypersensitivity (I-IV), depending upon the type of immunological reaction responsible for injury. Complement is involved in types II and III hypersensitivity reactions, which include certain drug allergies and immune complex diseases, but the complement and its products may also contribute to anaphylaxis and asthma, which are type I hypersensitivities. Immune complexes form in the body when an antibody recognizes a specic antigen. The complement system prevents the immune complex from precipitating in the serum by making it more soluble and then removing it through mechanisms involving IgG, C3b, iC3b, and complement receptors CR1 and CR3. Various insults may lead to immune complex diseases. For example, immune complexes can form the following: persistent infection (e.g., streptococcus infection); production of antibody to self-antigen (e.g., systemic lupus erythematosus); or inhalation of antigenic materials from molds, plants, or animals (e.g., Farmers lung and Pigeon fanciers lung). The continued production of antibodies leads to extended formation of immune complexes that can overwhelm the complement system. Because the soluble immune complexes are poorly cleared from circulation, they often become embedded in various tissues where they trigger inammatory reactions. Complement activation in the lung induced by immune complexes leads to neutrophil accumulation and lung injury. The role of the complement was dened in experiments with mice where treatment with a C5a blocking antibody attenuated the immune complex-induced permeability and neutrophil inltration into the lung. In addition, mice lacking the C5aR were completely protected from immune complex alveolitis, and treatment with a C5aR antagonist reduced edema formation in normal mice. These studies suggest C5a and C5aR are important mediators of lung injury in immune complex disease.

Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a pulmonary disorder resulting from a variety of severe injuries. The injury may be directed at the lungs, as with pulmonary infection, aspiration of gastric contents, severe thoracic trauma, toxic gas (smoke) inhalation, or it may be indirect as in the acute systemic inammatory response to severe sepsis, acute pancreatitis, drug overdose, reperfusion injury, and others. Although the pathogenesis of ARDS is not yet fully dened, it is clear that activation of the complement cascade and proinammatory cells injures the lung. The inadvertent activation of the complement system results in C5a generation, which increases capillary permeability. Clinical studies correlate the extent of complement activation (indicated by increased plasma levels of activation products, C3a and C5a) with the development of ARDS in patients. C5a attracts neutrophils and macrophages to the lungs where they release oxygen free radicals to cause inammation and alveolar epithelial

Asthma
The role of the complement in asthma is controversial, but recent studies of allergen-induced asthma in C3- or C3aR-decient mice showed that lack of these complement components significantly reduced airway hyperresponsiveness, lung eosinophilia, and

552 COMPLEMENT

IL-4-producing cells. In addition, attenuation of Agspecic IgE and IgG1 responses was also observed. These ndings suggest that the complement may modulate susceptibility to asthma. Interaction of C3a with epithelial cells can regulate lung inammatory responses such as synthesis of mucus, while increased production of C3a in asthmatic patients during exercise implies a role for complement in exercise-induced asthma. In addition, complement depletion by cobra venom factor, a protein found in snake venom that consumes C3 and C5, blunted the asthmatic response in a guinea pig model of occupational asthma, indicating a role for complement in cellular inltration and lung injury.

Therapeutic Considerations
There is no single inhibitor of all three pathways of complement activation. Since activation products C3a and C5a mediate inammatory reactions leading to tissue damage, attempts at inhibition focused on blocking complement activation at the C3/C5 level. Studies with mice either decient in C3/C5 or transgenically lacking C3aR/C5aR indicate that selective control of complement activation products reduces lung injury, yet maintains opsonization and lysis of bacteria. Small antagonist molecules that inhibit C5a generation or its binding to C5aR can reduce proinammatory activities of C5a in rats, while recombinant sCR1 not only reduces inammation and tissue injury by injected immune complexes but also protects against lung injury in different models of complement-dependent acute inammation. These animal studies, together with data from ongoing human clinical trials of small molecular antagonists for C3a and C5a receptors, will facilitate development of clinically useful inhibitors to control complement-mediated inammation.
See also: Acute Respiratory Distress Syndrome. Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; ExerciseInduced; Extrinsic/Intrinsic. G-Protein-Coupled Receptors. Pleural Effusions: Overview.

Anaphylaxis
Complement activation does not occur in systemic anaphylaxis because IgE immune complexes do not activate complement. In this immediate hypersensitivity reaction, the antigen binds to an IgE antibody on mast cells or basophils, causing release of mediators that produce life-threatening symptoms. However, studies with mice that lack IgE suggest that generation of C3a and C5a via complement activation contributes to the bronchoconstriction and hypotension of anaphylaxis. Indeed, additional animal studies implicate C3a and C5a as putative mediators of anaphylactic shock. Both C3a and C5a peptides contract airway smooth muscle in the guinea pig independent of histamine, and treatment with soluble CR1 (sCR1), which inhibits complement activation, eliminates the antigen-induced hypotensive response in these animals.

Further Reading
Czermak BJ, Lentsch AB, Bless NM, et al. (1998) Role of complement in in vitro and in vivo lung inammatory reactions. Journal of Leukocyte Biology 64: 4048. Davies KA and Walport MJ (1998) Processing and clearance of immune complexes by complement and the role of complement in immune complex diseases. In: Volanakis JE and Frank MM (eds.) The Human Complement System in Health and Disease, pp. 423454. New York: Dekker. Guo RF and Ward PA (2002) Mediators and regulation of neutrophil accumulation in inammatory responses in lung: Insights from the IgG immune complex model. Development of C5a receptor antagonists. Free Radical Biology and Medicine 33: 303310. Joseph J and Sahn S (1993) Connective tissue diseases and the pleura. Chest 104: 262270. Ko hl J (2001) Anaphylatoxins and infectious and non-infectious inammatory diseases. Molecular Immunology 38: 175187. Prince JE, Kheradmand F, and Corry DB (2003) Immunologic lung disease. Journal of Allergy and Clinical Immunology 111: S613 S623. Regal JF (1997) Role of the complement system in pulmonary disorders. Anaphylatoxins and infectious and non-infectious inammatory diseases. Immunopharmacology 38: 1725. Tsokos GC and Tolnay M (2004) Complement and autoimmunity. In: Szebeni J (ed.) The Complement System: Novel Roles in Health and Disease, pp. 307314. New York: Kluwer Academic Publishers.

Pleural Disease
Pleura effusions accompany lung injury resulting from various insults, including immune mechanisms that occur in diseases such as rheumatoid arthritis and lupus erythematosus. Circulating immune complexes localize in the pleural capillaries and activate the complement, thereby causing endothelial injury. Also, immune complexes and complement can be generated locally, further adding to the injury. Measurement of the complement in posttraumatic pleural effusions shows that levels of C3 and C4 are decreased relative to normal serum, suggesting local consumption of the complement via the classical pathway. The fact that levels of SC5b-9 are higher in pleural effusions from patients with tuberculosis than in those with malignant effusions suggests that measuring SC5b-9 levels might differentiate the mechanism and origin of the effusion.

CONNECTIVE TISSUE GROWTH FACTOR 553

CONNECTIVE TISSUE GROWTH FACTOR

J A Lasky, Tulane University Health Science Center, New Orleans, LA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Connective tissue growth factor (CTGF) is a peptide growth factor that is upregulated during brogenesis. CTGF was originally found to promote broblast proliferation, migration, adhesion, and extracellular matrix formation, and subsequently has been implicated in brogenesis, wound healing, chondrogenesis, angiogenesis, transdifferentiation, and tumor growth. CTGF has numerous cellular probrotic activities, which include increasing synthesis of brillar collagen and bronectin, enhancing secretion of factors that impede matrix degradation, and stimulating the transformation of broblasts into the more brogenic myobroblast phenotype. Increased CTGF expression is observed during lung brogenesis in animal models, as well as in diseases that cause lung brosis in humans. Overexpression of CTGF has been reported to cause lung brosis in rats, as well as to render lung brosis-resistant mouse strains more susceptible to brogenic agents. These observations have raised interest in developing CTGF inhibitors for employment in trials to arrest lung brogenesis in humans.

Introduction
Connective tissue growth factor (CTGF), also termed sp12, bIG-M2, and IGF-BP8, is a secreted cysteinerich growth factor that belongs to the CCN (cyr61, CTGF, nov) family. It was rst isolated in 1991 from serum-stimulated NIH3T3 cells and named sp12 (broblast-inducible secreted protein-12). The term connective tissue growth factor was introduced the same year by others to describe a cysteine-rich mitogen secreted by human vascular endothelial cells.

Since then the CTGF cDNAs for pig, rat, cow, and frog have been sequenced. CTGF is a highly conserved protein. Compared to human CTGF, there is 94% homology displayed in murine CTGF, 96% homology in rat CTGF, and 92% homology in pig CTGF. CTGF was originally found to promote broblast proliferation, migration, adhesion, and extracellular matrix formation as its name suggested. It has also been reported that CTGF is transcriptionally activated by transforming growth factor beta (TGF-b) and is an important downstream mediator of TGFbs probrotic activities. In light of these ndings, CTGF is proposed as a probrotic factor that plays an important role in the pathways leading to brosis. However, a host of evidence has shown that the physiological functions of CTGF are far more than what its name suggests. Other than broblasts, expression of CTGF has been reported in diverse cell types such as epithelial cells, neuronal cells, endothelial cells, smooth muscle cells, and chondrocytes. In contrast, under certain circumstances CTGF has been shown to act as an antimitogenic factor or apoptosis inducer instead of as a mitogen. CTGF has thus been implicated in brogenesis, wound healing, chondrogenesis, angiogenesis, transdifferentiation, and tumor growth.

Gene and Protein Structure

Both human and murine CTGF genes span about 3 kb of DNA and are composed of ve exons and four introns (Figure 1). The hCTGF gene resides on

CTGF gene structure

lntron 1 Exon 1
5

Intron 2 Exon 2 Exon 3

Intron 3
Exon 4

Intron 4 Exon 5
3

1 N

21 SP

27 I IGFBP

97

101

167

199

243

256

330

3498

II
VWC

III TSP1

IV
CT

CTGF protein structure

Figure 1 Gene and protein structure of CTGF. The CTGF gene is composed of ve exons and four introns. The dark blue boxes in the gene structure map represent open reading frames. CTGF protein consists of a signal peptide (SP) at the N-terminal and four modules, shown as light blue boxes. The numbers shown on top of each region are the amino acid numbers at the beginning or end of the region. IGFBP, I-insulin growth factor binding protein; VWC, von Willebrand factor type C repeat; TSP1, thrombospondin type 1; CT, C-terminal module.

554 CONNECTIVE TISSUE GROWTH FACTOR

chromosome 6q23.1 and the mCTGF gene maps to the (10A310B1) region of the murine genome. The 50 untranslated region of the CTGF gene contains a variety of transcriptional regulatory elements such as AP-1, TATA, M-CAT, and SP-1 as well as a unique TGF-b response element (TbRE), which accounts for induction of CTGF expression by TGF-b. hCTGF also contains a cis-acting repressive element in the 30 untranslated region. A single 2.4 kb mRNA transcript of CTGF has been reported in most studies and encodes for a 349 amino acid hCTGF peptide (348 amino acids for mCTGF). Two larger CTGF transcripts (3.5 and 7 kb) are found in glioblastoma cells, and the functional significance of these elongated transcripts is presently unclear. CTGF is a 3638 kDa peptide that is highly conserved among species. The most frequent variations occur in the N-terminal signal peptide region. The secreted form of the protein consists of 323 amino acids containing 38 conserved cysteine residues that form disulde bridges within the peptide. As a member of the CCN family, CTGF protein has four modules that are shared with all other CCN family members. The four domains are: an I-insulin growth factor binding module (module I, IGFBP), a von Willebrand factor type C repeat (module II, VWC), a thrombospondin type 1 module (module III, TSP1), and a C-terminal module (module IV, CT), which contains a cystine knot (Figure 1). Interestingly, each module is encoded by one exon and is separated by an intron; however, the exact biological function of these four modules remains unknown. Module 3

of CTGF has been shown recently to bind to lowdensity lipoprotein receptor-related protein (LRP). Several lines of evidence suggest that module 4, which harbors the cystine knot of CTGF, might be the functional domain for the majority of CTGFs activities. For example, module 4 accounts for the heparin-binding capacity of CTGF. More compelling evidence arises from studies on some smaller CTGF variants existing in various cell types and body uids. These 1020 kDa proteolytic fragments are composed of either module III and IV or module IV only. Similar to the full-length CTGF, these CTGF fragments are able to bind to heparin as well as to stimulate mitosis and cell adhesion.

CTGF Receptors
There is very limited information regarding the CTGF receptor/s (Figure 2). CTGF binding was evaluated in a human chondrocytic cell line using radiolabeled CTGF. Scatchard analysis demonstrated two classes of CTGF binding sites, but only one band at approximately 280 kDa was detected using a crosslinking study. As of yet, the bona de CTGF receptor/s has not been isolated, and the receptor gene has not been cloned or sequenced. A study using afnity cross-linking and SDS-polyacrylamide gel electrophoresis analysis has revealed CTGF binding to a membrane protein of a mass of approximately 620 kDa which is characterized as the multiligand receptor LRP. Consistent with this observation, CTGF induces tryrosine phosphorylation of the cytoplasmic

Fibroblasts Epithelial cells Endothelial cells Airway smooth muscle cells Tumor cells LRP

CTGF

Integrin

v 3

Receptor (?)

FAK(?) (?) CRMP-1 (inhibit metastasis)

(?)

ERK1/2

Target gene

Gene

Bcl-2 (apoptosis)

5 Integrin (cell adhesion) Extracellular matrix

1-Type I collagen Fibronectin (fibrosis and airway remodeling)

Figure 2 CTGF receptor binding, signal transduction, and biological activities. CRMP-1, collapsin response mediator protein 1; ERK, extracellular signal regulated kinase; FAK, focal adhesion kinase; LRP, low-density lipoprotein receptor-associated protein.

CONNECTIVE TISSUE GROWTH FACTOR 555

domain of LRP in broblasts, and the LRP antagonist, receptor-associated protein (RAP) inhibited CTGF-induced tryrosine phosphorylation of LRP. The biological significance of CTGF binding to LRP is highlighted by the fact that inhibition of LRP signaling reduced CTGF- mediated synergistic induction of alpha-smooth muscle actin (SMA) protein. Another CTGF receptor candidate is integrin avb3 which functions as a co-receptor with heparan sulfate proteoglycans (HSPG) for CTGF-mediated hepatic stellate cell (HSC) adhesion. Integrin avb3 is also involved in the angiogenic activity of CTGF.

Modulators of CTGF Expression

There are multiple factors and stimuli that regulate the expression of CTGF. TGF-b1 is perhaps the bestcharacterized factor capable of increasing CTGF expression. CTGF expression is upregulated in response to TGF-b1 through a novel TGF-b1 response element in the CTGF promoter. Inhibition of extracellular signal regulated kinase (ERK) and c-jun N-terminal kinase (JNK), but not of p38 mitogen-activated protein kinase (MAPK) and phosphoinositol-3 kinase (PI3 K), blocks the effect of TGF-b1 on CTGF mRNA and protein expression. Although IL-4 was found to have no effect on basal CTGF expression in cultured human lung broblasts, it decreases TGF-b-induced CTGF expression through a transcriptional mechanism that involves modulation of Smad and ERK signaling. Notably, IL-4 did not affect bronectin or alpha1(I) collagen mRNA expression induced by TGF-b in human lung broblasts. There are other peptide factors that have the capacity to induce CTGF expression, such as VEGF and thrombin. Similarly, angiotensin II, alpha-tocopherol, shear stress, cell stretch and static pressure, H2O2, and O2 increase the expression of CTGF. The potent vasodilator NO decreases CTGF expression. Thus, CTGF is thought to play a role in vascular wall remodeling. Conversely, there are several proinammatory cytokines, for example, tumor necrosis factor alpha (TNF-a), IL-1, and IL-4, which decrease CTGF expression. The effect of any given factor on CTGF expression under the complex milieu that occurs in injury and repair may not correlate closely with the in vitro ndings. For example, TNF decreases CTGF mRNA and peptide expression in cultured bovine artery endothelial cells, 3T3 broblasts, and smooth muscle cells. TNF and IL-1b reduce TGF-b1-stimulated CTGF mRNA expression, but unlike IL-4, do not inhibit TGF-b-induced Smad2/3 phosphorylation. In comparison, CTGF expression is actually upregulated at times when TNF expression is increased in the bleomycin model of murine pulmonary

brosis. In addition, dexamethasone has been reported to increase CTGF expression in BALB/c 3T3 broblasts in vitro, which is of concern since corticosteroids are frequently employed in an attempt to arrest inammation in subjects aficted with brotic diseases. However, dexamethasone did not increase the expression of CTGF in a murine model of wound healing. In yet another example, the 3-hydroxy3-methylglutaryl (HMG) CoA reductase inhibitor, simvastatin, inhibits TGF-b-induced CTGF mRNA and protein expression in lung broblasts isolated from patients with interstitial pulmonary brosis (IPF). In addition, simvastatin was found to reduce TGF-b-induced alpha smooth muscle action and collagen gel contraction through a Rho-dependent signaling mechanism. A large retrospective review did not demonstrate any survival advantage between IPF patients that were taking HMG CoA reductase inhibitors and those patients that were not. However, by nature of the retrospective design, it is not known whether the IPF patients taking statins had lower expression of CTGF compared with the IPF population who were not taking statins. These examples demonstrate the necessity of evaluating factors that regulate CTGF expression in vivo as well as in vitro.

CTGF and Fibrogenesis

There are now numerous publications showing that CTGF expression is enhanced during brogenesis in multiple organs, including lung, skin, kidney, liver, bowel, and heart, as well as within desmoplasia surrounding tumors. This is not unexpected since TGFb1 is increased in broproliferative pathology, and because TGF-b1, -b2, and -b3 upregulate CTGF expression in vitro. Importantly, subcutaneous injection of TGF-b1 results in increased expression of CTGF within dermal broblasts and subsequent formation of a brotic lesion. A rat skin wound model has revealed an upregulation of CTGF mRNA expression that follows an increase in TGF-b1 mRNA expression, which suggests that CTGF is induced by the increased expression of TGF-b1 in vivo. Because dermal broblasts synthesize CTGF and proliferate in response to CTGF, investigators have proposed that CTGF may act as an autocrine mitogen induced by TGF-b1. In addition, many manuscripts demonstrate that CTGF expression is reduced in mice that are protected from brosis. For example, proteinaseactivated receptor 1 (PAR-1) knockout mice are protected from bleomycin-induced lung injury and express less TGF-b1 and CTGF mRNA compared with wild-type littermates. Urinary CTGF excretion is reduced in patients with diabetic nephropathy

556 CONNECTIVE TISSUE GROWTH FACTOR

treated with Losartan, an angiotensin converting enzyme inhibitor. In sum, these studies clearly show that there is a correlation between CTGF expression and brogenesis, but do not prove a cause-and-effect relationship between CTGF and brosis. The majority of publications regarding CTGF expression and brogenesis have been correlative; however, there are a few manuscripts that attempt to dene the role of CTGF in brogenesis. A study using a model of carbon tetrachloride-induced liver brosis demonstrated that CTGF antisense oligonucleotides (ODN) decreased liver CTGF mRNA expression as well as that of type I collagen mRNA, but did not affect tissue inhibitor of metalloproteinase-1 (TIMP-1) expression or the degree of brosis. In contrast, two models of renal brosis demonstrate that treatment with a CTGF antisense ODN attenuates the induction of CTGF, bronectin, alpha1(I) collagen, plasminogen activator inhibitor 1, and TIMP-1, and suppresses renal interstitial brogenesis, without affecting TGF-b mRNA expression. Repair of cartilage is another area strongly supporting a healing role for CTGF. A rat model of osteoarthritis revealed CTGF expression at the sites of wound repair, and the administration of rCTGF enhances chondrogenesis in vivo. It is difcult to reconcile these disparate ndings regarding the importance of CTGF in brogenesis, although species variances and magnitude of CTGF inhibition could be entertained as possible explanations.
CTGF and Dermal Fibrogenesis

also depends upon factors that degrade the matrix. Administration of recombinant CTGF reduced matrix degradation in human mesangial cells exposed to high concentrations of glucose. Furthermore, a neutralizing anti-CTGF antibody reduced TGF-b1mediated inhibition of matrix degradation. CTGF is heavily implicated in the pathogenesis of scleroderma. Indeed, serum CTGF levels correlate with the extent of skin and lung brosis in patients aficted with scleroderma. Normal unperturbed skin broblasts do not express CTGF, whereas broblasts in areas of wound repair or isolated from the skin of people aficted with scleroderma express abundant CTGF. The previously mentioned probrotic activities of CTGF in vitro, namely broblast mitogen, stimulator of collagen expression, and enhancer of cell adhesion, are considered as support for the role of CTGF in the pathobiology of scleroderma. Interestingly, in contrast to normal wound repair wherein CTGF promoter activity is under control of a TGF-b response element, scleroderma broblast CTGF expression is resistant to SMAD3 inhibition of TGF-b signal transduction, but rather is dependent upon Sp1.
CTGF and Lung Fibrogenesis

Dermal brogenesis is addressed here because of the high frequency of pulmonary brosis in association with scleroderma, and because there is increasing evidence that CTGF is involved in the pathogenesis of scleroderma. This is not unexpected since TGF-b1 is increased in broproliferative dermal pathology, such as scleroderma, and because TGF-b1, -b2 and -b3 upregulate CTGF expression in vitro. Importantly, subcutaneous injection of TGF-b1 results in increased expression of CTGF within dermal broblasts and subsequent formation of a brotic lesion. In addition, a positive correlation between CTGF expression and skin sclerosis has been reported. A rat skin wound model has revealed an upregulation of CTGF mRNA expression that follows an increase in TGF-b1 mRNA expression, which suggests that CTGF is induced by the increased expression of TGF-b1 in vivo. Administration of rCTGF alone did not result in dermal brosis; however, rCTGF did prolong the stability of brosis caused by administration of rTGF-b. An explanation for these ndings may be related to the fact that collagen accumulation

Considerable attention has also been given to dening the role of CTGF in lung brogenesis. CTGF mRNA expression is increased in the lungs of mice that develop pulmonary brosis following administration of bleomycin as well as mice treated with an adenoviral vector expressing TGF-b1. Moreover, CTGF expression is elevated in lavage specimens derived from brotic human lung. The cellular immunoreactivity for CTGF was markedly increased in the lung tissue of patients with IPF compared to normal lungs, and immunolocalization of CTGF was conned predominantly to proliferating type II alveolar epithelial cells and activated broblasts. Interestingly, TGF-b mRNA expression decreased in a population of patients with IPF who improved clinically following treatment with interferon gamma (IFN-g), but changes in CTGF expression were not reported in a larger study of IPF patients who underwent lung biopsy before and 6 months after administration of IFN-g versus placebo. The ambiguity regarding the biological signicance of enhanced CTGF expression in lung brogenesis mirrors that discussed above for renal and liver brosis. Others have clearly demonstrated that adenoviral gene transfer of CTGF into rat lung induces transient brosis. Moreover, administration of a hCTGF adenoviral vector intranasally 3 days prior to intratracheal administration of bleomycin caused

CONNECTIVE TISSUE GROWTH FACTOR 557

the normally bleomycin-resistant strain of BALB/c mice to develop lung brosis. In contrast, another study found normal lung histopathology in three lines of transgenic mice that overexpress CTGF using the lung-specic surfactant protein-C promoter. Moreover, the CTGF transgenic mice did not demonstrate a significant change in the severity of bleomycin-induced lung brosis in comparison to their wild-type littermates. In another experiment, the degree of bleomycin-induced lung brosis in mice that were heterozygous for a CTGF null allele was evaluated. Although lung broblasts from heterozygous CTGF knockout lungs express 50% less CTGF mRNA compared with those from wild-type littermates (pre or post in vitro treatment with TGFb1), there was no difference observed in the severity of bleomycin-induced lung brosis in heterozygous CTGF knockout mice compared with their wild-type littermates. In total, these ndings suggest that small variances in CTGF expression have at best small and transient effects on the severity of lung brosis. The data also indicate that if anti-CTGF therapy is to be effective in limiting the progression of lung brosis, then the degree of inhibition will need to be much greater than 50%.

secreted by endothelial cells. The release of CTGF from tumor cells appears to be essential for the angiogenesis in chicken chorioallantoic membrane angiogenesis assays. In summary, CTGF is a peptide growth factor that affects an array of biological processes, and is clearly necessary for normal development. CTGF expression is mediated by factors implicated in brogenesis and CTGF has profibrogenic activity in vitro and in vivo. It remains to be seen whether or not inhibition of CTGF expression will result in a significant improvement in the course of patients suffering from broproliferative diseases.
See also: Interleukins: IL-1 and IL-18; IL-4. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
Blom IE, Goldschmeding R, and Leask A (2002) Gene regulation of connective tissue growth factor: new targets for antibrotic therapy? Matrix Biology 21(6): 473482. Bonniaud P, Martin G, Margetts PJ, et al. (2004) Connective tissue growth factor is crucial to inducing a probrotic environment in brosis-resistant BALB/c mouse lungs. American Journal of Respiratory Cell and Molecular Biology 31(5): 510516. Chang CC, Shih JY, Jeng YM, et al. (2004) Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis. Journal of the National Cancer Institute 96(5): 364 375. Grotendorst GR, Okochi H, and Hayashi N (1996) A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differentiation 7: 469480. Ivkovic S, Yoon BS, Popoff SN, et al. (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130(12): 27792791. Leask A and Abraham DJ (2003) The role of connective tissue growth factor, a multifunctional matricellular protein, in broblast biology. Biochemistry and Cell Biology 81(6): 355363. Nishida T, Kubota S, Kojima S, et al. (2004) Regeneration of defects in articular cartilage in rat knee joints by CCN2 (connective tissue growth factor). Journal of Bone and Mineral Research 19(8): 13081319. Okada H, Kikuta T, Kobayashi T, et al. (2005) Connective tissue growth factor expressed in tubular epithelium plays a pivotal role in renal brogenesis. Journal of the American Society of Nephrology 16(1): 133143. Uchio K, Graham M, Dean NM, Rosenbaum J, and Desmouliere A (2004) Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver brosis. Wound Repair and Regeneration 12(1): 6066. Yokoi H, Mukoyama M, Nagae T, et al. (2004) Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial brosis. Journal of the American Society of Nephrology 15(6): 14301440.

Modulation of Cancer Biology

Early studies demonstrated significant associations between CTGF expression and advanced features of breast cancer, which implicated CTGF in the progression of breast cancer. CTGF functions as a survival and differentiation factor in human rhabdomyosarcoma. However, conicting results arose from studies pertaining to the role of CTGF with other types of carcinomas. CTGF impedes the growth of oral squamous cell carcinoma cells and inhibits metastasis and invasion of human lung adenocarcinoma in a collapsin response mediator protein (CRMP)1-dependent mechanism. CTGF regulates normal endothelial homeostasis, as well as the angiogenic process during embryonic development, placentation, tumor formation, brosis, and wound healing. CTGF knockout mice exhibit vascular defects during embryogenesis and fetal development. CTGF modulates tumor-associated angiogenesis through both intrinsic angiogenic activity and induction of other angiogenic molecules (e.g., broblast growth factor (bFGF), vascular endothetial growth factor (VEGF)). CTGF released by human breast cancer cells exposed to hypoxia promotes angiogenesis through modulating the balance of extracellular matrix synthesis and degradation via matrix metalloproteinases (MMPs)

558 CONNEXINS, TISSUE EXPRESSION

CONNEXINS, TISSUE EXPRESSION

M Koval, Emory University School of Medicine, Atlanta, GA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Connexins are transmembrane proteins that form gap junction channels. Gap junctions interconnect cells to allow the diffusion of signaling molecules and metabolites between cells. Cells in the respiratory system exhibit unique patterns of connexin expression which determine whether they are interconnected with neighboring cells and the types of signals transmitted between them. Connexin expression in the lung is altered in response to injury as a compensatory mechanism to alter the extent of cell cell coupling. Gap junctional communication between type II and type I cells plays a critical role in integrating the alveolus to enable type I cells to act as mechanical sensors to regulate pulmonary surfactant secretion by type II cells. By regulating intercellular signaling, controlling the ow of metabolites, and restricting the ow of toxic agents, connexins enable the cells of the lung to act as an integrated system.

in close contact were separated by a uniform 23 nm gap, hence the term gap junction. Perfection of techniques to isolate junctions from rat liver and heart enabled the identication and subsequent cloning of the channel forming proteins of gap junctions, known as connexins.

Structure
Connexins are transmembrane proteins that span the membrane bilayer four times with both the N- and C-terminus oriented towards the cytoplasm (Figure 1). Unlike most transmembrane proteins, connexins are not glycosylated. Based on the human and murine genomes, there are almost two dozen mammalian connexins which show cell- and tissue-specic expression patterns. By protein homology, connexins fall into two subgroups, alpha and beta, which are dened by characteristic amino acids in the rst and third transmembrane domains and second extracellular loop. The cytoplasmic loop and C-terminal tail domains show the greatest level of sequence divergence. A functional gap junction channel is composed of two connexin hexamers (or hemichannels) in two adjacent cells which dock to form a complete channel. Gap junction channels are typically arranged in semicrystalline arrays, known as plaques, at sites of cellcell contact where intercellular communication occurs. However, free connexin hemichannels dispersed throughout the plasma membrane can also act

Introduction
Gap junction channels interconnect cells by forming a direct link to enable the diffusion of small aqueous molecules and ions from one cell to its nearest neighbor. This enables both the ow of specic intercellular signals and metabolic cooperation between communicating cells in a tissue. Intercellular communication was initially correlated with sites identied by electron microscopy where two adjacent cells

Lumenal/extracellular

N C

Cytosolic (a)

Cx37 Cx43 Cx46 Cx40 Cx50 Cx59 Cx62 Cx40.1 Cx31.9 Cx45 Cx47 Cx36 (b)

Cx25 Cx26 Cx30 Cx32 Cx31.1 Cx30.3 Cx31 (c) Connexin hexamer

Figure 1 Connexins. (a) Connexins are transmembrane proteins that span the bilayer four times, with N- and C-terminus oriented towards the cytoplasm. (b) Dendrogram showing connexins aligned by protein sequence homology. Based on sequence homology, connexins form two subgroups, alpha and beta. (c) Gap junction channels are composed of two hexamers, one in each cell, that dock to form a complete channel. Dashed lines indicate the path of diffusion for aqueous molecules and ions through gap junctions from one cell to another.

CONNEXINS, TISSUE EXPRESSION 559

as bona de plasma membrane channels, enabling the exchange of aqueous molecules between the cytoplasm to the extracellular environment.

Regulation of Production and Activity

Connexins have a relatively rapid half-life of 15 h, suggesting that gap junction turnover is a constant process. Connexins are cotranslationally inserted into the endoplasmic reticulum membrane and then assembled into hemichannels. The site of hemichannel assembly depends on connexin subtype, where beta connexins form hemichannels in the endoplasmic reticulum/Golgi while alpha connexins form hemichannels in intermediate compartment aspect of the Golgi apparatus. Hemichannels are then transported to the plasma membrane where they dock with hemichannels on neighboring cells to form complete gap junction channels. Gap junctions are turned over by a unique endocytic mechanism shared by tight junctions where one cell engulfs a portion of the other cell to internalize a piece of a gap junction plaque. Internalized plaques are degraded by a combination of proteosomal and lysosomal hydrolysis. Cells that express multiple connexins have the potential to form heteromeric or mixed gap junction channels. The rules that govern connexin intermixing are a combination of innate incompatibility and regulated assembly. For instance, alpha and beta connexins are innately incompatible; an alpha connexin such as Cx43 cannot form a mixed gap junction channel with a beta connexin such as Cx32. However, cells also regulate the formation of mixed gap junctions by compatible connexins. For instance, two of the compatible connexins expressed by alveolar epithelial cells are Cx43 and Cx46. Type I alveolar epithelial cells form mixed gap junctions composed of Cx43 and Cx46, whereas type II cells prevent Cx43 and Cx46 from intermixing. Other examples of cells which regulate connexin assembly include endothelial cells which restrict formation of mixed gap junctions containing Cx37 and Cx43. The mechanisms that regulate connexin intermixing are not known at present. However, one ramication of differential connexin expression and assembly is that cells within a given tissue can create distinct cellcell interfaces to preferentially allow or restrict intercellular communication. Connexin expression is regulated at the transcriptional level. In the normal lung, most epithelial cells express Cx32 and Cx43, whereas endothelial cells express predominantly Cx37, Cx40, and Cx43. Type I and type II alveolar epithelial cells have been studied in considerable detail. The major connexins expressed by alveolar epithelial cells are Cx26, Cx32,

Cx43, and Cx46. Others are expressed at low levels, such as Cx30.3, Cx37, and Cx40. Based on immunohistochemistry of whole lung sections, Cx32 is expressed exclusively by type II alveolar epithelial cells in normal adult rat lung. Cx43 is fairly ubiquitous and is the major connexin functionally interconnecting type II and type I cells. Expression of Cx37 by alveolar epithelial cells in situ is low, but consistently detectable by immunohistochemistry. Considerably more Cx37 is expressed by bronchiolar epithelium, but this is still less than the level observed for pulmonary endothelial cells.

Biological Function
The primary function of gap junction channels is to interconnect cells. Gap junction channels have relative selectivity, that is, gap junction channels composed of different connexins show different rates of diffusion for a given molecule. For instance, diffusion of ATP is B10-fold faster through Cx43 channels as compared to Cx32. Conversely, glutathione ux through Cx32 and Cx43 channels is roughly comparable. One consequence of gap junction formation by two or more connexins is that cells form gap junction channels with unique permeability characteristics. For instance, Cx26 has a fairly restrictive permeability and forms heteromeric gap junction channels with Cx32. Cx32 is fully permeable to cAMP and cGMP. However, channels containing both Cx26 and Cx32 are preferentially permeable to cGMP as compared to cAMP. Other examples of mixed gap junction channels which have unique permeability include Cx37/Cx43, Cx40/Cx43, and Cx43/Cx45. In addition to metabolic coupling, gap junction channels also transmit signals from one cell to another. Most typically, this is observed by the transmission of transient increases in cytosolic calcium (calcium waves) throughout a tissue. For instance, transmission of calcium signals through gap junctions regulates vascular tone, particularly in small vessels where gap junctions interconnect endothelial cells to smooth muscle cells. In airway epithelium, intercellular transmission of inositol trisphosphate creates oscillatory waves that synchronize ciliary beat frequencies in order to coordinate the outward ow of mucus. In the alveolus, gap junctions between type I and type II cells enable these two classes of alveolar epithelial cells to act in concert to regulate surfactant secretion. During normal breathing the changes in lung volume generally correspond to relatively small changes (B5%) in alveolar surface area, although during sighs or yawns, alveolar surface area may

560 CONNEXINS, TISSUE EXPRESSION

increase by about 1015%. Stress analysis of alveoli indicates that type I cells stretch to a level equivalent to the overall level of alveolar stretch. However, type II cells are more or less shielded from stretch. In response to stretch, type I cells increase cytosolic calcium, which is subsequently transmitted to type II cells through gap junctions. The rise in type II cell cytosolic calcium is sufcient to trigger lamellar body fusion with the plasma membrane and stimulates surfactant secretion. Thus, gap junctions enable type I cells to act as mechanosensors and to actively contribute to the regulation of pulmonary surfactant release. Interestingly, a role for disrupted cellcell communication in regulating surfactant release is potentially underscored by surfactant abnormalities associated with diseases such as acute respiratory distress syndrome (ARDS) and ventilator-induced injury, where cellcell contacts are disrupted. To date, there are few direct data from transgenic mouse models related to respiratory function. This is due, in part, to the difculty of working with some connexin-decient models. For instance, Cx43-decient mice have a neonatal lethal phenotype due to cardiac malformations of the outow tract which, in turn, lead to pulmonary edema. Whether Cx43 has a role in control of lung uid balance through regulation of epithelial barrier function is not known at present. However, mice doubly decient in Cx37 and Cx40, two prominent blood vessel connexins, show permeability defects and localized hemorrhages, consistent with a role for endothelial gap junctions in regulating vessel barrier function. Note, however, that any role for connexins in altering barrier function will be regulatory rather than structural, since the structural component of endothelial or epithelial barrier function is due to claudin-family tight junction proteins.

mean time that channels are open. For instance, calmodulin binds to Cx32-containing gap junction channels and increases their sensitivity to calciuminduced closure. Connexins are also regulated by phosphorylation on the C-terminus, which typically decreases connexin open probability. Phosphorylation of plasma membrane connexins also acts as a signal for gap junction disassembly. Several kinases are known to phosphorylate connexins, including sarcoma (src) kinase and mitogen-activated protein kinases, which help decrease the extent of gap junctional communication during cell division and proliferation.

Connexins in Respiratory Diseases

Human diseases directly attributable to mutant connexins include peripheral neuropathy (Charcot MarieTooth disease; Cx32), premature cataract (Cx46, Cx50), and musculoskeletal disorders (oculodentodigital dysplasia; Cx43). Interestingly, different mutations in the same connexin can cause different diseases. For instance, distinct mutations in Cx26 are associated with either deafness or skin disease (keratoderma). To date, no human respiratory disease has been directly attributable to a connexin deciency or mutation; however, potential roles for connexins in lung disease can be deduced from animal and in vitro models. In the injured lung, type II cell hyperplasia increases the frequency of type II cells in direct contact with other type II cells, both of which express Cx32. Since type Itype II cell communication is mediated through Cx43-compatible connexins and is not mediated by Cx32, this provides type II cells with an independent pathway for communication that does not involve type I cells. During the acute phase of acute lung injury, connexin expression in the alveolus is altered, where both Cx43 and Cx46 expression is elevated. Cx46expressing alveolar epithelial cells do not express typical type II cell markers and thus may represent a distinct subtype of cells proliferating in response to injury. Interestingly, Cx46 has relatively limited permeability as compared to Cx32 and Cx43, suggesting a possible role for Cx46 in limiting metabolic depletion or intercellular transmission of toxic agents. Transmission of toxic agents through gap junctions is very efcient, a phenomenon known as the bystander effect. Even radiation levels on the order of one alpha particle per ve cells can create cytotoxic substances transmitted through Cx43 gap junction channels. Although deleterious in the setting of lung injury, bystander effects have been explored as a

Connexin Binding Proteins

The extracellular portion of connexins interacts exclusively with other connexins. However, the cytoplasmic C-terminus interacts with a number of different protein cofactors, which interlink them with the cytoskeleton and regulate connexin function. Since there is considerable variability in the C-terminus, different connexins interact with different proteins. For instance, the zona occludens-1 (ZO-1) scaffold protein, which links junction proteins to the actin cytoskeleton, binds to Cx43, Cx45, and Cx46, but not Cx32. As another example of a connexincytoskeleton interaction, tubulin binds to Cx43 and provides a direct link to microtubules. In addition, connexin binding proteins can regulate connexin open probability, which relates to the

CONTRACTILE PROTEINS 561

chemotherapeutic approach, most notably in the use of viruses expressing thymidine kinase combined with ganciclovir as a potential treatment of asbestosinduced mesothelioma. A limitation in the use of bystander effects as an anticancer therapy is that tumor cells typically have relatively low levels of connexin expression and gap junctional communication, which limit their susceptibility to cellcell transfer of toxic agents. However, this underscores the notion that gap junctional communication plays a role in the control of cell growth. Although the precise role for gap junctions in regulating cell growth is largely unknown, mice decient in Cx32 exhibit increased lung tumor load in response to carcinogens such as urethane and radiation, suggesting that Cx32 might be a lung tumor suppressor. There are also clues that connexins may play a role in the pathology of cystic brosis (CF). Cells expressing the DF508 mutant of the cystic brosis transmembrane conductance regulator (CFTR) associated with CF lack efcient connexin assembly and show abnormally low levels of gap junctional communication. Further work is required to dene a functional link between CFTR and connexin function.
See also: Acute Respiratory Distress Syndrome. Adhesion, CellCell: Vascular; Epithelial. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene.

Further Reading
Abraham V, Chou ML, George P, et al. (2001) Heterocellular gap junctional communication between alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L1085L1093. Ashino Y, Ying X, Dobbs LG, and Bhattacharya J (2000) [Ca2 ]i oscillations regulate type II cell exocytosis in the pulmonary alveolus. American Journal of Physiology 279: L5L13. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cellcell interactions in regulating lung function. American Journal of Physiology: Lung Cellular and Molecular Physiology 287: L455L459. Chanson M, Berclaz PY, Scerri I, et al. (2001) Regulation of gap junctional communication by a pro-inammatory cytokine in cystic brosis transmembrane conductance regulator-expressing but not cystic brosis airway cells. American Journal of Pathology 158: 17751784. Chipman JK, Mally A, and Edwards GO (2003) Disruption of gap junctions in toxicity and carcinogenicity. Toxicological Sciences 71: 146153. Goldberg GS, Valiunas V, and Brink PR (2004) Selective permeability of gap junction channels. Biochima et Biophysica Acta 1662: 96101. Goodenough DA and Paul DL (2003) Beyond the gap: functions of unpaired connexon channels. Nature Reviews: Molecular Cell Biology 4: 285294. Harris AL (2001) Emerging issues of connexin channels: biophysics lls the gap. Quarterly Reviews of Biophysics 34: 325472. Kelsell DP, Dunlop J, and Hodgins MB (2001) Human diseases: clues to cracking the connexin code? Trends in Cell Biology 11: 26. Koval M (2002) Sharing signals: connecting lung epithelial cells with gap junction channels. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L875L893. Saez JC, Berthoud VM, Branes MC, Martinez AD, and Beyer EC (2003) Plasma membrane channels formed by connexins: their regulation and functions. Physiological Reviews 83: 13591400.

CONTRACTILE PROTEINS
J R Sellers, National Institutes of Health, Bethesda, MD, USA
Published by Elsevier Ltd. discussed. During many types of respiratory disease smooth muscle proliferates, hypertrophies, and/or upregulates its contractile protein complement in response to the stress.

Abstract
All cells must have machinery in them to move vesicles, proteins, and mRNA, to carry out membrane trafcking of receptors and synthesized proteins, and to promote adhesion with other cells or their substrate. In addition, most cells undergo cell division in which a single cell is divided in half by a cytokinetic ring. Many types of cells can secrete vesicularized contents or undergo endocytosis or phagocytosis. The machinery responsible for this plethora of functions is loosely termed contractile proteins and consists of actin, myosin, and associated regulatory or structural proteins. While the function of these proteins is well documented in smooth and striated muscle, less is known about their roles in nonmuscle cells. In this article the function of these proteins in both muscle and nonmuscle cells will be

Introduction
The respiratory system contains a multitude of different cell types. Smooth muscle cells line the trachea, the bronchi, and the bronchioles and are present in the vascular system. Ciliated columnar epithelium, cuboidal epithelium, and Clara cells are present at different locations. Endothelial cells are present in the vasculature and elsewhere. Mast cells are involved in the immunologic response and motile macrophages phagocytose extracellular debris. A common feature of all these cells is that they contain the cellular machinery to move intracellular vesicles that

562 CONTRACTILE PROTEINS

maintain a cytoskeleton. The term contractile proteins will be used to dene the proteins used in cell motility, cytokinesis, intracellular vesicular trafcking, and maintaining cell morphology and cell contacts.

Regulation of Striated Muscle Contraction

In resting muscle (in the absence of calcium), troponin holds tropomyosin in a position on the actin lament that blocks myosin from binding in a productive manner. Contraction is initiated following nerve stimulation, which releases calcium from internal stores called the sarcoplasmic reticulum. Calcium binds to the TN-C subunit, which induces a rearrangement of the other troponin subunits, resulting in a movement of tropomyosin from its blocking position to one that allows myosin to interect with actin laments and pull them toward the center of the thick lament, thus shortening the sarcomere and the muscle. When calcium is sequestered in relaxation, troponin and tropomyosin move back to their inhibitory positions, myosin no longer interacts productively with actin, and the giant elastic protein titin restores the sarcomere to its resting length. Titin, the largest protein in the genome, stretches from the Z-line to the center of the myosin thick lament. Portions of titin consist of repeating elastic elements that can be unfolded to allow for lengthening during muscle contraction. Upon relaxation of the muscle ber, the titin elastic elements begin to refold, thus shortening the molecule and aiding return of the muscle ber to resting length. The fact that muscle shortens without either of its two lament systems undergoing a decrease in length is due to the fact that these two laments slide past one another during contraction. The function and localization of nebulin on the thin lament is not well known. It may be template to determine the length of the actin lament and/or may play a role in the calcium-dependent regulation. The individual myosin molecules in a thick lament interact asynchronously with actin in a manner

Role of Muscle in Respiration

The diaphragm muscle is of the skeletal or striated type and is the major muscle of ventilation. Accessory muscles of ventilation include the scalene, the sternocleidomastoid, the pectoralis major, the trapezius, and the external intercostals. Smooth muscle is found in the trachea and in the pulmonary arteries and smaller vessels. Striated muscles contain regular arrays of thick and thin laments that make up the sarcomere, the basic contractile unit (Figure 1). Bipolar thick laments containing the motor protein myosin lie at the center of the sarcomere. Thin laments lie on either side of the thick lament and are attached to Z-lines that are built of many proteins including a-actinin, an important structural protein, and CapZ which binds to the end of an actin lament and helps anchor it to the Z-line. The thin lament is composed of polymerized G-actin subunits and contain the regulatory proteins troponin and tropomyosin as well as the large protein nebulin. Troponin consists of three subunits: TN-C, a small calmodulin-like calcium-binding protein; TN-I, an inhibitory subunit; and TN-T, a tropomyosin-binding subunit. Troponin and tropomyosin are each present in a stoichiometry of one per seven actin molecules (Figure 1). Tropomyosin is an elongated coiled-coil a-helical protein whose length matches the distance spanned by seven actin monomers. It lies in the groove of the thin lament.

Titin Thin filament Nebulin

Thick filament Z-lines CapZ

Figure 1 Schematic of a striated muscle sarcomere. The schematic shows the manner in which the thin laments and thick laments interdigitate. When the muscle shortens, the myosin molecules in the thick lament actively pull the thin laments toward the center of the thick lament. Other important proteins are shown as described. An expanded view of a thin lament is given showing the positions of tropomyosin and troponin. The position of nebulin on the thin lament is not shown since its localization has not been rmly established.

CONTRACTILE PROTEINS 563

that is tightly coupled to the hydrolysis of ATP. To illustrate the coupling of this process to ATP, a single powerstroke of myosin is shown in Figure 2. We start with myosin at the end of its powerstroke where the products of ATP hydrolysis have just been released.

Myosin head AM ATP Actin

In this state (often referred to as the rigor state), the myosin head is bound to actin at a 451 angle with respect to actin. Upon binding ATP the myosin head dissociates from actin, and hydrolyzes ATP to give the products ADP and Pi which are still bound to the active site. While dissociated from actin, the myosin head recocks to assume the prepowerstroke conformation. Myosin then rebinds to actin at a 901 angle and subsequently undergoes a tilting conformational change which moves the actin lament incrementally toward the center of the thick lament. This process is associated with the ordered release of Pi and ADP from the myosin active site to restore the starting rigor conformation.

ATP (A)M.ATP

Smooth Muscle Contractility

Smooth muscle is found in tissues such as the stomach, the intestines, the bladder, arteries, the trachea, and the uterus. Smooth muscle contains interdigitating thick and thin laments, but their order is not nearly as precise as exists in striated muscle, giving rise to a smooth versus striated appearance in the light microscope. The difference in microscopic appearance between smooth muscle and skeletal muscle is but one of the profound differences between the two systems. The activation of skeletal muscle tissue is directly driven by electrical activity at the neuromuscular junction. Smooth muscle on the other hand can also be activated by adrenergic and cholinergic receptors on the cell surface. In general, smooth muscles contract more slowly than do skeletal muscle bers and maintain the contraction for longer periods of time. Smooth muscles can be divided into phasic muscles which contract more rapidly and tonic muscles which contract slowly and maintain tension for long periods of time. At least part of the difference in contractile speed of phasic and tonic smooth muscles derives from the expression of different, alternatively spliced isoforms of the smooth muscle myosin gene. The mechanism of calcium-dependent regulation of smooth muscle is profoundly different from that of skeletal muscle. The dominant site of regulation of smooth muscle is at the thick lament. The enzymatic activity of smooth muscle myosin is greatly activated by phosphorylation of its regulatory light chain subunit at Ser19 by myosin light chain kinase (MLCK) which, in turn, is active when bound to the calcium-bound form of calmodulin (Figure 3). Myosin is inactivated by dephosphorylation of Ser19 by a myosin phosphatase (MYPT1). The dephosphorylated myosin is essentially completely inactive. Signal transduction pathways impinge on both MLCK and MYPT1 affecting their respective

M.ATP

ADP.Pi M.ADP.Pi

(A)M.ADP.Pi

ADP.Pi

Pi

AM.ADP

ADP

AM
Figure 2 Myosin cross-bridge cycle. See text for description of events. A, actin; M, myosin; AM, myosin bound strongly to actin; (A)M, myosin bound weakly to actin.

564 CONTRACTILE PROTEINS

RhoA signaling pathway Other signaling pathways ??? ROK active Myosin-P active P-MYPT1 less active MYPT1 fully active Myosin inactive MLCK.Ca.CaM active Ca2+ MLCK inactive CaM kinase II +Ca2+

Phosphatase
Figure 3 Regulation of smooth and nonmuscle myosin.

activities. Since the steady state level of myosin phosphorylation and, thus its activity, is primarily a function of the ratio of the activity of these two important regulatory proteins, pathways such as the RhoA pathway or those regulated by heterotrimeric G-proteins can profoundly affect the contractile state of smooth muscle and also the activity of myosin in nonmuscle cells. RhoA signaling results in the activation of Rho kinase (ROK) which phosphorylates MYPT1 and inhibits its activity. MLCK can be phosphorylated by protein kinase A and CaM kinase II resulting in a reduction of its activity. Some kinases in various signal transduction pathways can directly phosphorylate myosin at Ser19 of the regulatory light chain. Smooth muscle contains tropomyosin, but lacks troponin on its thin laments. Instead, smooth muscle thin laments bind another potential regulatory protein, termed caldesmon. Caldesmon is an elongated protein whose C-terminal end binds to actin and the small calcium-binding protein calmodulin, while its N-terminal end binds to myosin. In vitro experiments have shown that in the absence of calcium, caldesmon can inhibit the enzymatic and mechanical activities of myosin and that this inhibition can be reversed by the addition of calcium and calmodulin. Caldesmon might also play a structural role by tethering thick and thin laments via its spatially separated binding sites for actin and myosin. Many of the striated muscle contractile proteins have homologous isoforms that are expressed in smooth muscle tissue. There is a smooth muscle-specic myosin heavy chain gene and the genes for the light chains of smooth muscle myosin differ from those of skeletal muscles. In place of well-dened Zlines, smooth muscles have dense bodies which contain a smooth muscle-specic isoform of a-actinin.

review. Nevertheless, these cells contain contractile proteins, including actin, myosin, tropomyosin, caldesmon, MLCK, and a myosin-specic phosphatase that are very similar to those found in smooth muscles. These proteins are used in cells for a variety of purposes including maintaining cellcell and cell substrate adhesion, cell division, cell motility, and cell morphology. The clearest role for contractile proteins in nonmuscle cells is the constriction of the cytokinetic ring during cell division. In another example, a dense cortical bundle of actin and myosin laments is found in the periphery of resting basophil and mast cells. Upon stimulation this cortical network is disassembled to allow the secretory vesicles access to the plasma membrane. In addition, directed cell motility requires the coordinated action of both the actin lament network and myosin. The contractile states of various cells are similarly affected by the RhoA signaling pathway described above and also by other signal transduction networks, such as the Rac pathway. Here, one of the downstream kinases from these pathways, such as ROK and, possibly PAK, can directly phosphorylate Ser19 of the nonmuscle myosin regulatory light chain. Discussed below are more detailed descriptions of actin and myosin and roles that they play.
Actin

Contractile Systems in Nonmuscle Cells

Most of the cells of the respiratory tissue are not muscle and will be called nonmuscle cells for this

Actin is often the most abundant protein in cells. G-actin, the globular monomeric form of actin, has a molecular weight of about 42 kDa. Many G-actin subunits polymerize to create a lamentous form of actin, F-actin, which has the appearance of a twisted string of pearls (Figure 2). The pseudo repeat of the F-actin helix is about 37 nm and contains 14 actin monomers. Actin monomers preferentially bind at the barbed end of the actin lament and preferentially dissociate from the pointed end giving the structure polarity. Unlike in striated muscle tissue where actin plays predominantly a single role as the

CONTRACTILE PROTEINS 565

structural element upon which myosin pulls, in nonmuscle cells actin does many chores. It forms a structural cytoskeleton which plays important roles in cellcell and cellsubstrate adhesion. Motile cells have an actin-rich lamillipodial region at the leading edge. Many proteins are involved in this process including a complex called Arp2/3 which binds to the side of an existing actin lament and nucleates the polymerization of another branching actin lament at a 701 angle to create a dendritic network. Since most actin laments have their barbed ends at the plasma membrane, the polymerization of actin becomes the driving force for membrane protrusion. In cells with sterocelia or microvilli, multiple actin laments are held in closely spaced, parallel bundles via cross-linking proteins such as mbrin and villin to create membrane-bound spikes which effectively increase the surface area. Dynamic lopodial extensions of the cell membrane are formed when fascin bundles actin laments that are actively polymerizing. Actin-binding proteins such as vinculin, a-actinin, and talin link actin to focal adhesions that promote cellsubstrate adhesions. There are many actin-binding proteins that control the polymerization state of actin laments. Some proteins bind to and sequester G-actin subunits to prevent polymerization, whereas other proteins facilitate polymerization by increasing the rate of addition of actin to the barbed end of the actin laments. There are capping proteins such as CapZ that bind to the barbed end of the actin lament and prevent actin monomer addition. Proteins such as gelsolin or colin sever F-action laments and then remain bound as a cap to the barbed end to expedite depolymerization from the pointed ends of laments. In some cases, actin polymerization can drive the movement of vesicles inside the cell and some bacterial pathogens such as Listeria and viruses such as Vaccinia hijack this process by recruiting polymerizing actin networks to drive their movements within the cytoplasm of infected cells. Infection to other cells occurs when the pathogen drives into the cell membrane of one cell and is engulfed by a neighboring cell.
Myosins

Myosin was rst isolated by Kuhne from skeletal muscle tissue in the nineteenth century. In addition to the striated and smooth muscle myosins already discussed which function in muscle contraction, it is now clear that there is a large superfamily of myosins that perform a variety of other functions within cells. These include functions such as phagocyotis, endocytosis, providing structural stability for stereocelia

and microvilli, and movement of intracellular cargo such as vesicles, melanosomes and mRNA. There at least 19 classes of myosins based on the sequence of the heavy chain gene, but no species contains representatives of each class. The human genome contains 3940 possible myosin heavy chain genes representing 11 of the different classes of myosins (Figure 4). The myosins discussed in muscle cells and the nonmuscle myosin isoforms described above are all class II myosins which are also called conventional myosins since these were the rst myosins to be discovered and studied. Myosins from the other classes are commonly referred to as unconventional myosins. Myosins are typically composed of one or two heavy chains and one to twelve associated light chain subunits. There are three major functional domains in myosins, a motor, a neck, and a tail. The motor domain, which is usually located at or near the N-terminus of the protein, is about 700 amino acids in length. It interacts with actin and contains the nucleotide-binding site. Some myosins have N-terminal extensions that range in size from a few amino acids to over 1000 amino acids in length although their functions have not been clearly elucidated. The neck domain which follows the motor is composed of an a-helical sequence of the heavy chain which interacts with lower-molecular-weight proteins termed light chains. The light chains are either calmodulin or calmodulin-family member proteins. The light chains provide rigid support for the extended heavy chain helix. There is evidence that the neck region may function as a lever arm during actin translocation. Light chains are critical for regulation of the enzymatic activity of the smooth and nonmuscle myosin II, as seen above, but may also play regulatory roles in other myosin classes as well. Together the motor domain and neck region are sometimes termed the head. The third functional domain of myosin is the tail, located at the C-terminus of the molecule. The tail is at least partially responsible for the targeting of myosin to its proper cellular localization or cargo and may be involved in the regulation of enzymatic activity. The tail regions of different myosin classes are very diverse and may contain different functional domains such as FERM (band 4.1, ezrin, radixin, moesin), PH (pleckstrin homology), MyTH4 (myosin tail homology-4), SH3 (src homology-3), or zincbinding domains. Myosin IX molecules have a functional GTPase activating (GAP) domain in their tails. The tails of many classes of myosins have coiled-coil forming domains that may allow two heavy chains to dimerize, creating molecules with two heads. Others have phosphorylation sites regulating their self-association or their association with cargo.

566 CONTRACTILE PROTEINS

XVI III XVIII MYO18B MYO18A MYO3A MYO3B MYO16 VII X

MYO 7A MY O 7B

MY

O1

XV

Y0

15

) 5B ene 1 YO og (M eud ps

Nonmuscle myosin-IIc MYH14 Smooth muscle myosin-II MYH11 Nonmuscle myosin-IIb MYH10 Nonmuscle myosin-IIa MYH 9

MYO9A MYO9B MYO1A MYO1B

IX

[KIAA1000] MYH15
[KIAA1512] {MYH ?}

MYO1D MYO1G MYO1C

II

Cardiac- MYH7 Cardiac- MYH6 Cmbryonic MYH3 Extraocular MYH13 IIx/d MYH1 IIb MYH4 Perinatal MYH8 lla MYH2 MYO5A MYO5B MYO5C

MYO1H MYO1E MYO1F

MYO6 VI MYO19 XIX

V
Figure 4 Human myosin family tree. The various classes of myosin present in humans are shown. Myosin 15B is a pseudogene and is thus indicated with a dashed line.

Myosin classes Some of the interesting myosin classes that exist in vertebrates will be discussed next, starting with the class II, conventional myosins. Myosin II molecules have long C-terminal coiledcoil regions which dimerize to form two-headed structures. Filaments are formed by a self-association of the tail regions. The structure of laments formed by different isoforms of myosin II vary in both their packing and in their size. Vertebrate striated muscle myosins form bipolar thick laments of around 1.2 mm in length. The bipolar nature allows a single thick lament to pull actin laments from adjacent Z-lines toward its center. Analysis of the human genome predicts 14 myosin II heavy chain genes. Eight of these are found in either cardiac or skeletal muscle tissue where they are expressed in a muscle type and, sometimes, developmentally controlled manner. For example, some myosin heavy chain genes are expressed in fast versus slow muscle or in the cardiac ventricle as opposed to the atria. Others are found in fetal muscle, but not

in adult. There are three genes for nonmuscle myosin II (also called cytoplasmic myosins II) and one for smooth muscle myosin II. Most cells contain more than one nonmuscle myosin II isoform, but the individual functions of these myosins are still not well known. Humans have eight genes for myosin I. These proteins are characterized by a motor domain with little or no N-terminal extension. The neck region contains one or more IQ motifs. Their short tails do not have any coiled-coil forming sequences and do not appear to dimerize. The study of myosin I in various organisms has been complicated by the number of isoforms present. Some myosin I isoforms are expressed fairly ubiquitously in many tissues while others are more cell or tissue specic. For example, myosin Ie is abundant in the enterocyte brush border epithelium where it is associated with the actin bundle via its motor domain and a protein constituent of a lipid raft through its tail domain. In contrast myosin Ic is

CONTRACTILE PROTEINS 567

expressed in most tissues examined. It is found in the stereocilia of the hair cells of the inner ear and may play a role in sensory adaptation in hearing. In other cells, it likely plays a more generalized function. Other myosin I isoforms play roles in vesicle transport, endocytosis, and recycling of endosomes. The human genome has three myosin V heavy chain genes. These myosins have a long neck region with six IQ motifs that bind calmodulin and a coiledcoil segment of the tail that dimerizes to produce a two-headed structure. Myosin Va is a cargo transporting protein. Its role in transporting the pigmented melansome granules in melanocytes is well studied. It has also been implicated in the transport of vesicles in neuronal cells. The two other mammalian myosin V isoforms, Vb and Vc, also participate in intracellular vesicle trafcking. Two problems must be overcome to efciently move cargo along cytoskeletal actin laments in cells. First, it is necessary that the cargo remain attached to the actin lament at all times via the myosinactin interaction. Otherwise, the cargo would rapidly diffuse away from actin lament. To overcome this, myosin V has evolved its kinetics such that a single molecule can move processively on actin laments. In other words, the two motor domains of a single myosin V molecule are capable of taking many alternating steps in which one of the two heads remains bound to actin while the other head is moving forward searching for a new binding site. The second problem is related to the helical structure of an Factin lament. If the myosin merely stepped from one actin monomer to the next closest actin monomer, it would have to follow the helical pitch of the actin lament which would have the effect of running the cargo into the cytoskeleton after taking a few steps. To overcome this, myosin V has evolved a long neck which allows the molecule to take 36 nm steps (the distance of about 13 actin monomers) coinciding with the half helical pitch of the actin laments. Thus, myosin V is able to literally step across the top on an actin lament while keeping its cargo positioned above the cytoskeleton. Myosins Va, Vb, and Vc associate either directly or indirectly with Rab proteins which are G-protein family members associated with membrane vesicles. Myosin Va has been shown to indirectly interact with Rab27a via a linker protein, termed melanophilin. Myosin Vb interacts with Rab11a on perinuclear vesicles and myosin Vc with Rab8. Most myosins move toward the barbed end of actin laments, but myosin VI moves in the opposite direction. Mutations in the single myosin VI gene result in deafness in humans. However, the protein has a broad expression distribution in other cells and

tissues. Mice lacking myosin VI are deaf and show circling behavior, but appear to be otherwise healthy, although there are defects in membrane trafcking. Myosin VI is found on clathrin-coated vesicles in polarized cells and is thought to play a role in endocytosis. In some cells, it is also found associated with the Golgi complex and myosin VI mutant mice have smaller Golgi complexes than do wild-type mice. A single myosin X gene is found in humans. The tail contains coiled-coil, PH, FERM, and MyTH4 domains. It is widely distributed in cells and tissues, but present in low abundance mainly at the tips of lopodia. In macrophages, it localizes to areas of phagocytosis. In frog oocytes, myosin X interacts with microtubules and is involved in spindle assembly and nuclear anchoring. For more details on the other classes of myosin, the reader is directed toward the Further reading section.

Role of Contractile Proteins in Respiratory Disease

No mutations involving contractile proteins have been shown to be responsible for hereditary respiratory diseases, but they play important roles in the motility of macrophages, mast cells, and broblasts that lead to invasion of these cells during lung injury and disease states. The role of airway smooth muscle in the pathophysiology of asthma has been well studied, but there is considerable disagreement as to whether smooth muscles increase in size, number, or content of contractile proteins in asthma.
See also: Cytoskeletal Proteins. Leukocytes: Mast Cells and Basophils; Pulmonary Macrophages. Platelets. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signal Transduction. Smooth Muscle Cells: Airway. Vesicular Trafcking.

Further Reading
Barany M (ed.) (1996) Biochemistry of Smooth Muscle Contraction. New York: Academic Press. Bresnick AR (1999) Current Opinion in Cell Biology 11: 2633. Buss F, Luzio JP, and Kendrick-Jones J (2002) Trafc 3: 851858. Pollard TD (2003) Nature 422: 741745. Pollard TD and Borisy GG (2003) Cell 112: 453465. Reck-Peterson SL, Provance DW, Mooseker MS, and Mercer JA (2000) Biochimica et Biophysica Acta 1496: 3651. Ridley AJ, Schwartz MA, Burridge K, et al. (2003) Science 302: 17041709. Schiwa M (ed.) (2003) Molecular Motors. New York: Wiley-VCH. Sellers JR (1999) Myosins, 2nd edn. Oxford: Oxford University Press. Sellers JR (2000) Biochimica et Biophysica Acta 1496: 322. Somlyo AP and Somlyo AV (2003) Physiological Reviews 83: 13251358. Stephens NL (2001) Lung 179: 333373.

568 CORTICOSTEROIDS / Glucocorticoid Receptors

CORTICOSTEROIDS
Contents

Glucocorticoid Receptors Therapy

Glucocorticoid Receptors
I M Adcock, R Hayashi, and K Ito, Imperial College London, London, UK G Caramori, University of Ferrara, Ferrara, Italy
& 2006 Elsevier Ltd. All rights reserved.

led to debilitating side effects, much work has been performed in an attempt to understand the mechanism of corticosteroid action. It is hoped that by understanding how corticosteroids function at the cell and molecular level it will be possible to develop new safer drugs in the future.

Abstract
Corticosteroids bind to and activate a cytoplasmic glucocorticoid receptor (GR) which exists as several isoforms derived from a single gene product by alternative splicing. The activated glucocorticoid receptor translocates into the nucleus and binds to specic response elements in the promoter regions of antiinammatory genes such as lipocortin-1 and secretory leukocyte protease inhibitor. However, the major anti-inammatory effects of glucocorticoids appear to be due largely to interaction between the activated glucocorticoid receptor and transcription factors, notably nuclear factor kappa B (NF-kB) and activator protein-1, that mediate the expression of inammatory genes. NF-kB switches on inammatory genes via a process involving recruitment of transcriptional coactivator proteins and changes in chromatin modications such as histone acetylation. The interactions between NF-kB and the glucocorticoid receptor result in differing effects on histone modications and subsequent chromatin remodeling. GR is subjected to posttranslational modications and these may inuence hormone binding and nuclear translocation, alter glucocorticoid receptor interactions and protein half-life. Therapeutically, drugs that enhance glucocorticoid receptor nuclear translocation (long-acting b-agonists) and GR-associated histone deacetylase activity (theophylline) have been shown to be effective add-on therapies. In addition, dissociated glucocorticoids that target NF-kB preferentially have also been successful in the treatment of allergic disease in the skin.

The Glucocorticoid Receptor

Classically, corticosteroids exert their effects by binding to a single 777 amino acid glucocorticoid receptor (GR) that is localized to the cytoplasm of target cells. GRs are expressed in almost all cell types and their density varies from 200 to 30 000 per cell, with an afnity for cortisol of B30 nM, which falls within the normal range for plasma concentrations of free hormone. GR has several functional domains (Figure 1). The corticosteroid ligand-binding domain (LBD) is at the C-terminus of the molecule and is separated from the DNA-binding domain (DBD) by a hinge region. There is an N-terminal transactivation domain that is involved in activation of genes once binding to DNA has occurred. This region may also be involved in binding to other transcription factors. The inactive GR is part of a large protein complex (B300 kDa) that includes two subunits of the heat shock protein hsp90, which blocks the nuclear localization of GR and one molecule of the immunophilin p59 (FKBP4). In 1985, GR was the rst transcription factor to be cloned and two isoforms were described: the classical 94 kDa a isoform and a C-terminal truncated isoform, GRb, which lacked the N-terminal 9b exon and consequently the LBD. GRb is localized to the nucleus and due to its inability to bind ligands it does not switch on gene expression. However, enhanced expression of GRb can have a dominant negative effect on GRa via the formation of GRa/GRb heterodimers. The physiological role of abnormal GRb expression has been suggested to be important in reducing corticosteroid responses in severe asthma but this is controversial. Interestingly, the highest expression of GRb is seen in neutrophils, which are relatively corticosteroid insensitive. The human GR gene covers a region more than 80 kb in length within chromosome 5 and contains 8

Introduction
Corticosteroids are vitally important steroid hormones that control a number of key physiological functions in man including glucose homeostasis, after which they are named, and the regulation of metabolism, cell survival/death, development, and neurological function. Corticosteroids are also one of the most effective anti-inammatory agents available and are used to treat a number of chronic diseases including inammatory bowel disease, psoriasis, rheumatoid arthritis, and asthma. Since the elucidation of their clinical effectiveness in the early 1950s and the realization that high doses of corticosteroids

CORTICOSTEROIDS / Glucocorticoid Receptors 569

3 4 5 6 78

34 5 6 78

GR Transactivation/repression
0

GR Dominant negative

Figure 1 The GR is comprised of nine exons. Alternative splicing of exon 9 at the 5 end of the coding region leads to the formation of the classic GRa isoform and the dominant negative GRb isoform.

coding exons (exons 29) and alternative 50 -noncoding exon 1s. The promoter region does not contain either a TATA or a CCAAT box but is characterized by the presence of multiple GC boxes. The promoter contains DNA-binding sites for a number of transcription factors including AP-1, NF-kB, Sp1, and YY1 and also GR itself, although the regulation of GR expression in response to physiological and inammatory stimuli is unclear. There are at least three distinct promoters for the human GR, although correlation with the rat GR suggests that more may exist. It is clear that each GR initiation site has its own promoter region and this leads to the idea that multiple independent promoter regions producing the same protein by splicing upstream of the translation initiation site may partially explain tissue-specic GR expression (Figure 2). Furthermore, a single GR mRNA species can produce up to eight functional N-terminal GR isoforms via alternative translation initiation mechanisms including leaky ribosomal scanning and ribosomal shunting. Importantly, these GR isoforms can display diverse cytoplasm-to-nucleus trafcking patterns and distinct transcriptional activities and each GR isoform regulates both a common and a unique set of genes. The levels of these GR isoforms differ significantly among tissues and it is possible that cell-specic GR isoforms can generate specicity in glucocorticoid control of transcription in different tissues.

GR combines with another GR to form a dimer at consensus DNA sites termed glucocorticoid response elements (GREs, GGTACAnnnTGTTCT) in the regulating regions of corticosteroid-responsive genes. This interaction allows GR to associate with a complex of DNAprotein modifying and remodeling proteins, including steroid receptor coactivator-1 (SRC-1) and cAMP response element binding protein (CREB) binding protein (CBP), which produce a DNAprotein structure that allows enhanced gene transcription. The particular ligand, the number of GREs, and the position of the GREs relative to the transcriptional start site may be important determinants of the magnitude and direction of the transcriptional response to corticosteroids.

Nuclear Localization of GR
Control of nuclear protein import allows regulation of transcription factor activity and gene regulation. Nuclear importation of proteins is an active process for proteins 440 kDa that contain a nonconsensus basic targeting sequence or nuclear localization sequence (NLS). GR contains a classical basic NLS (NLS1, residing in the hinge region) and a second poorly characterized NLS (NLS2, residing across the LBD); nuclear import via NLS1 proceeds more rapidly than that seen under NLS2 control. Recent studies have shown that nuclear import of GR is mediated through its NLS and interaction with importins, with importin-a selectively binding to NLS1 and importins 7 and 8 binding to both NLS1 and NLS2. GR nuclear export is also tightly regulated; however, the role of the exportin-1 (CRM1) pathway is currently unclear. Importantly, the NLSimportin-a interaction is often inuenced directly by the phosphorylation status of the imported proteins.

Mechanism of GR Action
Corticosteroids are thought to freely diffuse from the circulation into cells across the cell membrane and bind to cytoplasmic GR (Figure 3). Once the corticosteroid binds to GR, hsp90 dissociates, allowing the nuclear localization of the activated GRcorticosteroid complex and its binding to DNA.

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1 421 486 777 1 421 486 742

GR

DBD

LBD

GR

DBD

1 27 86 90 98 316 331 336 A B C1 C2 C3 D1 D2 D3

1 27 86 90 98 316 331 336 A B C1 C2 C3 D1 D2 D3

GR -A

DBD

LBD

Possible phosphorylation/ nitration sites

GR -A

DBD

Figure 2 The GR can produce eight distinct products (A, B, C1, C2, C3, D1, D2, and D3) by use of alternative translation initiation sites corresponding to methionine residues at 1, 27, 86, 90, 98, 316, 331, and 336. These alternative proteins may be produced from both GRa and GRb transcripts. Posttranslational modication of GR particularly by phosphorylation and nitration alters GR function and contributes to the potential for diverse function in distinct tissues.

Histone Modications and Chromatin Remodeling

GR, like other transcription factors, increases gene transcription through its action on histone modications, chromatin remodeling, and recruitment of RNA polymerase II to the site of local DNA unwinding. DNA is tightly compacted around a protein core. This chromatin structure is composed of nucleosomes, consisting of an octamer of two molecules each of core histone proteins (H2A, H2B, H3, and H4) surrounded by B146 bp DNA. Expression and repression of genes is associated with alterations in chromatin structure by enzymatic modication of core histones. Specic residues (lysines, arginines, and serines) within the N-terminal tails of core histones are capable of being posttranslationally modied by acetylation, methylation, ubiquitination, or phosphorylation, all of which have been implicated in the regulation of gene expression. The histone code refers to these modications, which are set and maintained by histone-modifying enzymes and contribute to coactivator recruitment and subsequent increases in transcription. Transcriptional coactivators such as CBP have intrinsic histone acetyltransferase (HAT) activity. This activity is recruited to the site of active gene transcription by the binding of transcription factors to DNA, thus suggesting an amplifying mechanism for histone acetylation. Increased gene transcription is

therefore associated with an increase in histone acetylation, whereas hypoacetylation induced by histone deacetylases (HDACs) is correlated with reduced transcription or gene silencing. Histone acetylation is an active process whereby small changes in the activity of HATs or HDACs can markedly affect the overall histone acetylase activity associated with inammatory genes. Importantly, these changes in histone acetylation appear to be targeted toward regions of DNA associated with specic activator sites within the regulatory regions of induced inammatory genes, although a global loosening of histone structure has also been proposed. However, this regulatory system is more complex. Under resting conditions, just under half of the potential lysine residues available for acetylation are in fact acetylated, and these residues have a rapid turnover. Upon stimulation small increases in the total number of acetylated lysines occur at specic loci. This situation suggests that even small changes above or below the resting level are enough to lead to an activated chromatin state. Acetylation of lysines has two effects: (1) a loss of the electrostatic attraction between charged histones and DNA; and (2) formation of bromodomains by the acetylated lysine residues. This allows loosening of the chromatin structure, recruitment of chromatin remodeling complexes such as switch/sucrose nonfermentable (SWI/SNF), and subsequent recruitment of further large transcriptional coactivator complexes and RNA polymerase II.

CORTICOSTEROIDS / Glucocorticoid Receptors 571

Corticosteroid

GR

Cofactors SRC-1 CBP GR GR GRE Ac Ac Ac Histone acetylation RNAP II

SWI/SNF

SLPI, MKP-1, -receptor, CD163

Figure 3 Mechanism of gene activation by the GR. Corticosteroids can freely diffuse across the plasma membrane where it associates with the inactive cytosolic GR. Upon ligand binding GR is activated and can translocate to the nucleus where it binds to a glucocorticoid response element (GRE) within the controlling region for glucocorticoid-responsive target genes. These GREs may be 50 or 30 to the start site for transcription. Once bound to DNA, GR recruits a complex containing basal transcription factors, coactivators such as CBP and SRC-1, chromatin modiers such as SWI/SNF and RNA polymerase II (RNAP II), which together induce histone modications including acetylation (Ac) and chromatin remodeling, and subsequent production of mRNAs encoding various genes including SLPI, MKP-1, CD163, and the b2-adrenoceptor.

Recruitment of Transcriptional Coactivators by GR

GR interacts with CBP and other transcriptional coactivator proteins, including SRC-1 and glucocorticoid receptor interacting protein-1 (GRIP-1), that enhance local HAT activity. In addition, correct association of GR with DNA and other proteins determines the assembly of coactivator complexes on GR target promoters, resulting in differential acetylation of histones on distinct lysine residues. Steroid receptor coactivators recruited by GR can, in turn, recruit other coactivators and remodeling complexes including SWI/SNF and mediator complexes in a coordinated manner through bromodomain interactions. Histone H1 phosphorylation may also play a role in gene expression activated by the GR. Only the phosphorylated form of histone H1 can be displaced from the mouse mammary tumor virus (MMTV) promoter by the GR. Furthermore, long exposure to corticosteroids leads to H1 dephosphorylation. This

may explain the previously puzzling refractory state of the MMTV promoter obtained on long exposure to corticosteroids. In vitro, GR has a hit-and-run mechanism of action rather than a stable association with the GRE. GR resides on DNA for less than 10 s before being ejected and replaced by another GR. This ejection may allow binding of additional regulatory factors that enhance gene transcription, such as HAT-containing complexes, and may also play a role in feedback regulation. Interestingly, in the absence of adenosine triphosphate and chromatin remodeling factors, the GR stably interacts with the corticosteroid-responsive MMTV long terminal repeat promoter.

Gene Repression by GR
In spite of the ability of corticosteroids to induce gene transcription, the major anti-inammatory effects of corticosteroids are through repression of inammatory and immune genes. GR binding to a more variable negative GRE (nGRE, ATYACnnTnTGATCn)

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was originally proposed as a possible mechanism for corticosteroid-mediated gene repression; however, most glucocorticoid repressible inammatory genes do not generally possess nGREs in their promoters, suggesting that other mechanisms of inhibition are more important. The inhibitory effect of corticosteroids appears to be due largely to interaction between the activated GR and the transcription factors, such as NF-kB and AP-1, which mediate the expression of inammatory genes. Full inammatory gene expression probably requires that a number of transcription factors act together in a coordinated manner, and repression of a single transcription factor may only partially modify the full response. Glucocorticoids may be able to reduce inammatory gene expression by repressing downstream targets of transcription factor activation, irrespective of the precise activated transcription factors involved. GR, acting as a monomer, binds only to specic coactivator/modulator complexes that are activated by proinammatory transcription factors (Figure 4). The GR complex can regulate gene products in at least ve other ways (Figure 4). Firstly, GR acting as a monomer can bind directly or indirectly with the transcription factors AP-1 and NF-kB, which are upregulated during inammation, thereby inhibiting the proinammatory effects of a variety of cytokines.

The interaction between proinammatory transcription factors and the GR may result in differing effects on histone modications such as acetylation/deacetylation through a number of different mechanisms including GR binding to, or recruiting, nuclear receptor co-repressors such as NCoR and HDACs or possibly by direct repression of NF-kB-associated HAT activity. Secondly, the GR dimer can bind to a GRE that overlaps the DNA-binding site for a proinammatory transcription factor or the start site of transcription, thus blocking gene expression. Thirdly, the GR dimer can induce the expression of the NF-kB inhibitor IkB-a in certain cell types. Similarly, induction of GILZ (glucocorticoid inducible leucine zipper) can prevent AP-1 DNA binding and activity in some cells. Fourthly, corticosteroids can increase the levels of cell ribonucleases and mRNA destabilizing proteins, thereby reducing the levels of mRNA (Figure 3). Finally, GR may affect RNA polymerase II phosphorylation status (see below). As mentioned above, the context in which the GRE and kB sites are found within a promoter of specic genes may drastically affect the nal effect on gene expression. For example, dexamethasone can enhance cytokine-inducible expression of TLR2 via a GR-p65 association on the promoter where the GRE overlaps with the kB site. This does not absolutely

X
GR GR

Nongenomic effect on membrane or kinases (ERK, Akt, PI3K) GR GR 6

1 GRGR 2

GR ? 5

I B , GILZ

GR GR

GRGR GR

mRNA stability

GR

3 GR 4

AP-1/ NF- B

GR

RNA pol II phosphorylation RNAP II

X
P

Figure 4 Mechanisms of gene repression by the GR. Activated GR acting as a homodimer can bind to a GRE that overlaps the DNAbinding site for a proinammatory transcription factor or the start site of transcription to prevent inammatory gene expression 1 . Activated GR can repress AP-1/NF-kB-mediated gene expression by either inducing the expression of the NF-kB inhibitor IkB-a or the AP-1 inhibitor GILZ via classic gene induction mechanisms 2 . Acting as a monomer, GR can directly or indirectly suppress AP-1/NF-kB activity by 3 altering cofactor activity or by 4 dephosphorylating RNA polymerase II (RNA pol II). GR can also reduce the levels of mRNA by either inducing the dual specicity MAPK phosphatase-1 (MPK-1) that in turn regulates p38 MAPK-mediated mRNA stability or increasing the levels of cell ribonucleases and mRNA destabilizing proteins 5 . Finally, GR can induce rapid nongenomic effects either through a membrane receptor or affecting activation of kinases such as ERK, Akt, or PI3K 6 . Mechanisms that have been reported to be reduced under conditions of oxidative stress are shown by #.

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require GRE and NF-kB linear association as similar effects of p65 and GR can be seen during the induction of stem cell factor (SCF) in human broblasts despite the GRE and kB-RE being separated by B1700 bp implying a more complex interaction between GR and NF-kB, for example, than previously thought. An alternative mechanism for GR-p65 cross-talk has been proposed by Yamamoto who clearly showed that 100 nM dexamethasone acts by repressing the phosphorylation of RNA polymerase II CTD induced by tumor necrosis factor alpha (TNF-a), thereby altering the ability of RNA polymerase II to elongate. Explanations for the differences between the models may involve concentration and timing of dexamethasone treatment of cells along with the precise inammatory stimulus used. Evidence for cross-talk between GR and AP-1/NFkB has been described in a series of elegant experiments using mice expressing mutated GRs that are unable to dimerize and subsequently bind to DNA. In this model, Schutz and colleagues have conrmed a role for GR DNA binding as a dimer in the control of pro-opiomelanocortin expression and T-cell apoptosis but not in that of inammatory genes regulated by AP-1 or NF-kB. This suggests that it will be possible to develop corticosteroids with a greater therapeutic window. In addition, corticosteroids may play a role in repressing the action of mitogen-activated proteinkinases (MAPKs), such as the extracellular signalregulated kinase (ERK) and c-jun N-terminal kinase (JNK). More recently, it has been shown that dexamethasone can rapidly induce the dual specicity MAPK phosphatase-1 (MKP-1) and thereby attenuate p38 MAPK activation. Rogatsky and colleagues have in turn shown reciprocal inhibition of rat GR reporter gene activity by JNKs by a direct phosphorylation of Ser-246, whereas ERK can inhibit GR action by an indirect effect, possibly through phosphorylation of a cofactor. In addition, we and others have shown that enhanced MAPK activity is associated with attenuated GR function in severe asthma.

(TTP), which promotes mRNA decay, and HuR family members, which are associated with mRNA stability. Importantly, HuR binding to AREs is dependent upon p38 MAPK. In several cell types dexamethasone has been reported to alter the expression of either TTP and/or HuR, thereby affecting cyclooxygenase-2 (COX-2) and CC chemokine ligand 11 (CCL11) mRNA half-life possibly through induction of MKP-1. However, this does not appear to be the case for dexamethasone actions on granulocytemacrophage colony-stimulating factor mRNA stability in lung epithelial cells.

Nongenomic Actions of Glucocorticoids

The traditional genomic theory of glucocorticoid action, whether directly interacting with DNA or involving cross-talk with other transcription factors, does not fully explain the rapid effects of glucocorticoids, and it is thought that some nongenomic actions are mediated by a distinct membrane receptor. Initially described in amphibians this receptor has been described in mammalian cells and has distinctive hormone-binding properties compared to the well-characterized cytoplasmic receptor and is probably linked to a number of intracellular signaling pathways acting through G-protein-coupled receptors and a number of kinase pathways including MAPKs, Akt, and PI3K. There are a number of reviews in the literature providing a summary of the evidence for the rapid effects seen and, of these, one important in respiratory disease may include changes in bronchial blood ow induced by inhaled glucocorticoids during treatment of asthmatic exacerbations. In addition, the classical receptor is associated with a number of kinases and phosphatases within the inactive GR/hsp90 complex. These enzymes are released upon hormone binding and may also account for the rapid induction of tyrosine kinases by glucocorticoids seen in some cell types.

Posttranslational Modications of GR
GR is a phosphoprotein containing numerous potential phosphorylation sites, including those for ERK, p38 MAPK, protein kinase C, and protein kinase A. Evidence obtained during the past 10 years clearly suggests that altered GR phosphorylation status can affect GR-ligand binding, hsp90 interactions, subcellular localization, nuclear-cytoplasmic shuttling, and transactivation potential, possibly through association with coactivator molecules. Ligand binding induces GR hyperphosphorylation at seven sites, which regulates transactivation and reduces nonspecic DNA binding, although this response varies

Regulation of mRNA Stability

Adenylate-uridylate-rich elements (AREs) are found within the 30 -UTR of many inammatory genes and control the stability of mRNA. These sequences are very heterogeneous and include both AUUUA pentamers and AT-rich stretches. Binding of mRNA to ARE-binding proteins results in the formation of messenger ribonucleoprotein (mRNP) complexes, which control mRNA decay. Several ARE-binding proteins have been reported and include tristetrapolin

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during the cell cycle, with cells being less sensitive to corticosteroids during G2/M. Thus, global changes in GR charge may affect its function as well as specic phosphorylation events. MAPK activation or overexpression can also target specic serine/threonine residues in GR, decreasing GR-mediated transactivation (Figure 2). In addition, recent evidence suggests that GR phosphorylation is involved in receptor turnover and that phosphorylation can target the receptor for hormone-mediated degradation. As such, phosphorylation-induced targeting of GR for ubiquitination and proteosomal degradation may play an important role in overall GR responsiveness. GR sumoylation appears to have the opposite effect of ubiquitination and results in increased GR activity. In addition to phosphorylation, nitrosylation of GR at an hsp90 interaction site induced by the nitric oxide (NO) donor S-nitroso-DL-penicillamine has also been shown to prevent GR dissociation from the hsp90 complex and a reduction in ligand binding. It has become clear that histones are not the only targets for histone acetylases and recent evidence has suggested that acetylation of transcription factors can modify their activity. For example, the p65 component of NF-kB can also be acetylated thus modifying its transcriptional activity. Recent evidence suggests that GR is also acetylated upon ligand binding and that deacetylation is critical for interaction with p65 at least at low dexamethasone concentrations.

transrepression properties of GR ligands may result from glucocorticoid side chains lling this pocket. The crystal structure of GR antagonists and dissociated ligands in this model will conrm this view.

Therapeutic Implications
Inhaled glucocorticoids are now used as rst-line therapy for the treatment of persistent asthma in adults and children in many countries, as they are the most effective treatments for asthma currently available. However, at high doses systemic absorption of inhaled glucocorticoids may have deleterious effects, so there has been a search for safer steroids for inhalation and even for oral administration.

Effects of Long-Acting b-Agonists on GR Function

Long-acting b2-adrenoceptor agonists (LABAs) and glucocorticoids are the two most effective treatments for asthma and are more potent in combination than either drug alone. Whereas glucocorticoids are used to treat airway inammation, LABAs are used as bronchodilatory agents to bring rapid relief of airway bronchoconstriction. LABAs do not have major anti-inammatory actions in themselves in vivo as opposed to their potent effects in vitro, suggesting that the added benet of combination therapy probably relates to cross-talk between the two drugs. In several lung cell types salmeterol and formoterol can induce GR nuclear translocation and enhance GR glucocorticoid response element binding in the absence of ligand. Translocation of GR by b2-agonists was less effective than that seen with dexamethasone and was protein kinase A (PKA) dependent. This study has since been conrmed in vivo, which has indicated that salmeterol and formoterol can induce GR nuclear translocation and that this may prime the receptor to be more responsive to a concomitant or subsequent challenge with glucocorticoid.

GR LBD Crystal Structure

The crystal structure of the GR LBD has been determined in a ternary complex with dexamethasone and a TIF2 coactivator peptide following point mutation of Phe602. The overall structure is similar to that of other nuclear hormone receptor (NHR) LBDs but contains a unique dimerization interface and a second charge clamp that may be important for cofactor selectivity. Unlike other NHR LBDs, the GR LBD also has a distinct binding pocket that may explain ligand selectivity and lead to rational-based design of selective dissociated GR agonists. Modication of the dimerization interface by mutation of I628A resulted in reduced transactivation ability without affecting the ability to repress an NF-kB-driven reporter gene. The contrasting effects of this mutant suggests that the monomer and dimer forms of GR may regulate distinct signaling pathways conrming data obtained from the GRdim( / ) mouse. The most intriguing aspect of the ligand-binding pocket compared to other NHRs is the presence of an additional branch extending from the center. This pocket is opposite C17 of dexamethasone and it is possible to speculate that altered transactivation or

Dissociated Glucocorticoids
Whilst the major anti-inammatory effects of corticosteroids are almost certainly due to transrepression, the underlying molecular mechanisms for the side effects of glucocorticoids are complex and not fully understood. Certain side effects such as diabetes and glaucoma are due to transactivation events whilst others are due to transrepression (hypothalamic-preoptic area-adrenal (HPA) suppression). In addition, the precise molecular events underlying glucocorticoid induction of osteoporosis is unclear but probably requires both gene induction and gene repression. In

CORTICOSTEROIDS / Glucocorticoid Receptors 575

mice with GRs that do not dimerize there is no transactivation, but transrepression appears to be normal. Despite this uncertainty there has been a search for dissociated glucocorticoids that selectively transrepress without significant transactivation, thus potentially reducing the risk of systemic side effects. Several nonsteroidal selective glucocorticoid receptor agonists (SEGRA) have recently been reported that show dissociated properties in human cells. These are now in clinical development where they show good separation between transrepression and transactivation actions in the skin. This suggests that the development of glucocorticoids and SEGRA with a greater margin of safety is possible and may even lead to the development of oral compounds that have reduced adverse effects. Furthermore, the newer topical glucocorticoids used today, such as uticasone, mometasone and budesonide, appear to have more potent transrepression than transactivation effects, which may account, at least in part, for their selection as potent anti-inammatory agents. This suggests that the development of glucocorticoids with a greater margin of safety is possible and may even lead to the development of oral glucocorticoids that do not have significant adverse effects. The recent resolution of the crystal structure of the glucocorticoid receptor may help in better design of dissociated glucocorticoids.

glucocorticoids have additional effects, so it is uncertain whether IKK-2 inhibitors will parallel the clinical effectiveness of glucocorticoids. They may have side effects, such as increased susceptibility to infections; however, as a corollary to this, if glucocorticoids were discovered today, it is unlikely that they would be used in humans because of the low therapeutic ratio and their side effect prole (Figure 4).

Conclusion
Advances in delineating the fundamental mechanisms of gene transcription, especially recruitment of histone-modifying cofactors, have resulted in better understanding of the molecular mechanisms whereby glucocorticoids suppress inammation. The challenge is to see if these mechanisms hold true in primary cells in vivo. This will undoubtedly lead to the development of drugs that target novel aspects of GR function and potentially restore glucocorticoid sensitivity to diseases that are unresponsive to current therapeutic strategies.

Acknowledgments
We would like to thank other members of the Cell & Molecular Biology Group for their helpful discussions. Work in our groups is supported by Associazione per la Ricerca e la Cura dellAsma (ARCA, Padua, Italy), Asthma UK, The British Lung Foundation, The Clinical Research Committee (Brompton Hospital), The Medical Research Council (UK), The National Institutes of Health (USA), The Wellcome Trust, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline (UK), Mitsubishi Pharma (Japan), Novartis, and Pzer.
See also: Allergy: Overview. Corticosteroids: Therapy. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Lipid Mediators: Prostanoids. Transcription Factors: Overview. Vasculitis: Microscopic Polyangiitis.

Other Approaches to Anti-Inammatory Therapy

The elucidation of the molecular mechanisms of glucocorticoids raises the possibility that novel nonsteroidal anti-inammatory treatments might be developed that mimic the actions of glucocorticoids on inammatory gene regulation. Inhibition of specic HATs activated by NF-kB may prove to be useful targets, especially if they also repress the action of other proinammatory transcription factors. Alternatively, activation of HDACs may have therapeutic potential, and theophylline has been shown to have this property, resulting in marked potentiation of the anti-inammatory effects of glucocorticoids both in vitro and in vivo. This action of theophylline is not mediated via phosphodiesterase inhibition or adenosine receptor antagonism and, therefore, appears to be a novel action of theophylline. It may be possible to discover similar drugs that could form the basis of a new class of anti-inammatory drugs without the side effects that limit the use of theophylline. Many of the anti-inammatory effects of glucocorticoids appear to be mediated via inhibition of the transcriptional effects of NF-kB, and small-molecule inhibitors of kappa B kinase 2 (IKK-2), which activate NF-kB, are in development. However,

Further Reading
Barnes PJ (2002) Scientic rationale for inhaled combination therapy with long-acting beta2-agonists and corticosteroids. European Respiratory Journal 19: 182191. Barnes PJ and Adcock IM (2003) How do corticosteroids work in asthma? Annals of Internal Medicine 139: 359370. Bledsoe RK, Montana VG, Stanley TB, et al. (2002) Crystal structure of the glucocorticoid receptor ligand binding domain reveals a novel mode of receptor dimerization and coactivator recognition. Cell 110: 93105. Dean JL, Sully G, Clark AR, and Saklatvala J (2004) The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal 16: 11131121.

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De Ruijter AJ, Van Gennip AH, Caron HN, Kemp S, and Van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochemical Journal 370: 737749. Hermanson O, Glass CK, and Rosenfeld MG (2002) Nuclear receptor coregulators: multiple modes of modication. Trends in Endocrinology and Metabolism 13: 5560. McNally JG, Muller WG, Walker D, Wolford R, and Hager GL (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287: 12621265. Norman AW, Mizwicki MJ, and Norman DP (2004) Steroid hormone rapid actions, membrane receptors and a conformational ensemble model. Nature Reviews: Drug Discovery 3: 2741. Reichardt HM, Kaestner KH, Tuckermann J, et al. (1998) DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93: 531541. Schacke H, Schottelius A, Docke WD, et al. (2004) Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proceedings of the National Academy of Sciences, USA 101: 227232. Urnov FD and Wolffe AP (2001) Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 20: 29913006. Yudt MR and Cidlowski JA (2002) The glucocorticoid receptor: coding a diversity of proteins and responses through a single gene. Molecular Endocrinology 16: 17191726.

1950s and remain the most effective therapy available for this condition. The introduction of inhaled corticosteroids, initially as a way of reducing the requirement for oral steroids, has revolutionized the treatment of chronic asthma, and they are now used as rst-line therapy for all patients with persistent symptoms. In contrast, inhaled corticosteroids are much less effective in chronic obstructive pulmonary disease (COPD) and should only be considered in patients with severe disease. Oral steroids are indicated in the treatment of several other pulmonary diseases, such as sarcoidosis, interstitial lung diseases, and pulmonary eosinophilic syndromes.

Chemical Structures
The adrenal cortex secretes cortisol (hydrocortisone) and, by modication of its structure, it was possible to develop derivatives, such as prednisolone and dexamethasone, with enhanced corticosteroid effects but with reduced minerallocorticoid activity. These derivatives with potent glucocorticoid actions were effective in asthma when given systemically but had no antiasthmatic activity when given by inhalation. Further substitution in the 17a ester position resulted in steroids with high topical activity, such as beclomethasone dipropionate (BDP), triamcinolone, unisolide, budesonide, and uticasone propionate, which are potent in the skin (dermal blanching test) and were later found to have significant antiasthma effects when given by inhalation (Figure 1).

Therapy
P J Barnes, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Corticosteroids are by far the most effective anti-inammatory treatment for asthma and switch off multiple activated inammatory genes through reversing histone acetylation. They suppress inammation in the airways and reduce airway hyperresponsiveness. Inhaled corticosteroids are now rst-line therapy for all patients with persistent asthma, controlling asthma symptoms, and preventing exacerbations. Long-acting inhaled b2-agonists improve control and are commonly given as combination inhalers with the corticosteroid, which improves compliance and controls asthma at a lower dose of corticosteroid. Oral steroids have numerous metabolic and endocrine side effects, and the lowest dose needed to control the disease should be used. Inhaled corticosteroids, which are absorbed from the lungs into the systemic circulation, have negligible systemic side effects at the doses most patients require. In contrast, inammation in chronic obstructive pulmonary disease is corticosteroid resistant due to inactivation of histone deacetylase, and steroids are only indicated in reducing exacerbations in patients with severe disease. Systemic corticosteroids are also used as antiinammatory treatments in pulmonary eosinophilic syndromes and pulmonary vasculitides and in interstitial lung disease.

Mode of Action
Corticosteroids enter target cells and bind to glucocorticoid receptors in the cytoplasm. The steroidreceptor complex is transported to the nucleus, where it binds to specic sequences on the upstream regulatory element of certain target genes, resulting in increased (or rarely decreased) transcription of the gene that leads to increased (or decreased) protein synthesis. Glucocorticoid receptors may also interact with transcription factors and coactivator molecules in the nucleus and thereby inuence the synthesis of certain proteins independently of any direct interaction with DNA. The repression of transcription factors, such as activator protein-1 and nuclear factor kappa B (NF-kB), is likely to account for many of the anti-inammatory effects of steroids in asthma. In particular, corticosteroids reverse the activating effect of these proinammatory transcription factors on histone acetylation by recruiting histone deacetylase-2 (HDAC2), which reverses the acetylation of activated inammatory genes (Figure 2).

Corticosteroids are used in the treatment of several lung diseases. They were introduced for the treatment of asthma soon after their discovery in the

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CH2 OH HO H3C Cl O Hydrocortisone O Beclomethasone dipropionate O Budesonide H3C C O 17 OH HO

CH2OCOC2H5 C O OCOC2H5 CH3 HO

CH2 OH C O O O C H CH2CH2CH3

SCH2F C O HO OCOC2H5 CH3 F O F Fluticasone propionate O F Flunisolide HO

CH2 OH C O O O C CH3 CH3 HO

CH2 OH C O O O C CH3 CH3

F O Triamcinolone acetonide

Figure 1 Chemical structures of inhaled corticosteroids.

Inflammatory stimuli e.g., IL-1 , TNF-

Corticosteroids (low dose)

IKK-2 p65 NF- B p50

GR

pC
p65 B Inflammatory genes cytokines, chemokines adhesion molecules inflammatory receptors, enzymes, proteins p50 Acetylation

AF
CBP HDAC2 HAT Deacetylation

Gene transcription

Gene repression

Figure 2 Corticosteroids suppression of activated inammatory genes. Inammatory genes are activated by inammatory stimuli, such as interleukin-1b (IL-1b) or tumor necrosis factor alpha (TNF-a), resulting in activation of IKK-2 (inhibitor of IkB kinase-2), which activates the transcription factor NF-kB. A dimer of p50 and p65 NF-kB proteins translocates to the nucleus and binds to specic kB recognition sites and also to coactivators, such as CREB-binding protein (CBP) or p300/CBP-activating factor (pCAF), which have intrinsic histone acetyltransferase (HAT) activity. This results in acetylation of core histone H4, resulting in increased expression of genes encoding multiple inammatory proteins. Glucocorticoid receptors (GR) after activation by corticosteroids translocate to the nucleus and bind to coactivators to inhibit HAT activity directly and recruiting HDAC2, which reverses histone acetylation leading to suppression of these activated inammatory genes.

The mechanisms of the action of corticosteroids in asthma are poorly understood but are most likely related to their anti-inammatory properties. Corticosteroids have widespread effects on gene transcription,

increasing the transcription of anti-inammatory genes and suppressing transcription of inammatory genes (Table 1). Steroids have inhibitory effects on many inammatory and structural cells that are

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activated in asthma (Figure 3). Inhaled steroids reduce the number and activation of inammatory cells in the epithelium and submucosa, with a healing of the damaged epithelium. Steroids potently inhibit the formation of proinammatory cytokines and decrease eosinophil survival by inducing apoptosis. They also inhibit the expression of multiple inammatory genes in airway epithelial cells; indeed, this may be the most important action of inhaled corticosteroids in suppressing asthmatic inammation (Figure 4). Inhaled steroids reduce airway hyperresponsiveness, but this effect may take several weeks or months and

presumably reects the slow healing of the damaged inamed airway. It is important to recognize that steroids suppress inammation in the airways but do not cure the underlying disease. When steroids are withdrawn, there is a recurrence of the same degree of airway hyperresponsiveness, although in patients with mild asthma it may take several months to return. Steroids increase the expression of b2-adrenoceptors in the lung, and this may prevent the loss of nonbronchodilator response to b2-agonists that occurs after regular b2-agonist use.

Table 1 Effect of corticosteroids on gene transcription Increased transcription Lipocortin-1 b2-Adrenoceptor Secretory leukocyte inhibitory protein IkB-a (inhibitor of NF-kB) Anti-inammatory or inhibitory cytokines IL-10, IL-12, IL-1 receptor antagonist Mitogen-activated protein kinase phosphatase-1 (inhibits MAP kinase pathways) Decreased transcription Inammatory cytokines IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, IL-13, IL-15, TNF-a, GM-CSF, SCF Chemokines IL-8, RANTES, MIP-1a, eotaxin Inducible nitric oxide synthase (iNOS) Inducible cyclooxygenase (COX-2) Inducible phospholipase A2 (cPLA2) Endothelin-1 NK1 receptors Adhesion molecules (ICAM-1 and VCAM-1)

Pharmacokinetics
Prednisolone is readily and consistently absorbed after oral administration with little interindividual variation. Enteric coatings to reduce the incidence of dyspepsia delay absorption but not the total amount of drug absorbed. Prednisolone is metabolized in the liver, and drugs such as rifampicin, phenobarbitone, or phenytoin, which induce P-450 enzymes, lower the plasma half-life of prednisolone. The plasma half-life is 2 or 3 h, although its biological half-life is approximately 24 h, so it is suitable for daily dosing. There is no evidence that previous exposure to steroids changes their subsequent metabolism. Prednisolone is approximately 92% protein bound, the majority to a specic protein transcortin and the remainder to albumin; it is the unbound fraction that is biologically active. The pharmacokinetics of inhaled corticosteroids is important in relation to systemic effects. The fraction of steroid that is inhaled into the lungs acts locally on

Inflammatory cells Eosinophil Numbers (apoptosis) T lymphocyte Cytokines Mast cell Numbers Macrophage Cytokines Dendritic cell Numbers Corticosteroids

Structural cells

Epithelial cell Cytokines mediators

Endothelial cell Leak Airway smooth muscle 2-Receptors Cytokines Mucus gland Mucus secretion

Figure 3 Effect of corticosteroids on inammatory and structural cells in the airways.

CORTICOSTEROIDS / Therapy 579

the airway mucosa but may be absorbed from the airway and alveolar surface. This fraction therefore reaches the systemic circulation. The fraction of inhaled steroid that is deposited in the oropharynx is swallowed and absorbed from the gut. The absorbed fraction may be metabolized in the liver before reaching the systemic circulation (rst-pass metabolism) (Figure 5). Budesonide and uticasone propionate have a greater rst-pass metabolism than BDP and are therefore less likely to produce systemic effects at high inhaled doses. The use of a large-volume spacer chamber reduces oropharyngeal deposition and therefore reduces systemic absorption of corticosteroids, although this effect is minimal in corticosteroids with a high rst-pass metabolism.
Inhaled corticosteroids

Mouth rinsing and discarding the rinse has a similar effect, and this procedure should be used with highdose dry powder steroid inhalers since spacer chambers cannot be used with these devices.

Role of Corticosteroids in Respiratory Medicine

Acute Severe Asthma

Epithelial cells Cytokines IL-1 IL-6 GM-CSF RANTES Eotaxin MIP-1

Although the value of corticosteroids in acute severe asthma has been questioned, others have found that they speed the resolution of attacks. Hydrocortisone is given intravenously in acute severe asthma. There is no apparent advantage in giving very high doses of intravenous steroids (e.g., 1 g methylprednisolone). Intravenous steroids are indicated in acute asthma if lung function is o30% predicted and for patients in whom there is no significant improvement with nebulized b2-agonist. Oral prednisolone (4060 mg) has a similar effect as intravenous hydrocortisone and is easier to administer.
Asthma

Enzymes iNOS COX-2 cPLA2

Peptides ET-1

Adhesion molecules ICAM-1

Inflammation

Figure 4 Inhaled corticosteroids may inhibit the transcription of several inammatory genes in airway epithelial cells and thus reduce inammation in the airway wall. NF-kB, nuclear factor kB; AP-1, activator protein-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; NO, nitric oxide; COX-2, inducible cyclooxygenase; cPLA2, cytoplasmic phospholipase A2; PG, prostaglandin; ET, endothelin; ICAM, intercellular adhesion molecule.

Inhaled steroids are recommended as rst-line therapy for all patients with persistent asthma. Inhaled steroids should be started in any patient who needs to use a b2-agonist inhaler for symptom control more than three times a week. They are effective in mild, moderate, and severe asthma in children as well as adults. Although it was recommended that inhaled corticosteroids should be initiated at a relatively high dose and then the dose reduced once control was achieved, there is no evidence that this is more effective than starting with the maintenance dose. Doseresponse

MDI ~1020% inhaled Mouth and pharynx Lungs ~8090% swallowed ( spacer/mouthwash) Absorption from GI tract Liver GI tract Systemic circulation

Inactivation in liver 'first pass'

Systemic side effects

Figure 5 Pharmacokinetics of inhaled corticosteroids. GI, gastrointestinal; MDI, metered dose inhaler.

580 CORTICOSTEROIDS / Therapy

studies for inhaled corticosteroids are relatively at, with most of the benet derived from doses o400 mg BDP or equivalent. Oral steroids are reserved for patients who cannot be controlled on other therapy, with the dose being titrated to the lowest dose that provides acceptable control of symptoms. Short courses of oral steroids (3040 mg prednisolone daily for 1 or 2 weeks) are indicated for exacerbations of asthma, and the dose may be tailed off over 1 week once the exacerbation is resolved; although the tail-off period is not strictly necessary, patients nd it reassuring. For most patients, inhaled steroids should be used twice daily, which improves compliance once control of asthma has been achieved (which may require 4-times daily dosing initially or a course of oral steroids if symptoms are severe). If a dose of more than 800 mg via a metered dose inhaler is used daily, a spacer device should be used because this reduces the risk of orpharyngeal side effects. Inhaled steroids may be used in children in the same way as in adults, and at doses of 400 mg daily or less there is no evidence of growth suppression. The dose of inhaled corticosteroid should be the minimal dose that controls asthma, and once control is achieved the dose should be slowly reduced. There is convincing evidence that addition of an inhaled long-acting b2-agonist (salmeterol or formoterol) improves asthma control and compliance with therapy, leading to the widespread use of xed combination inhalers. Combination inhalers provide better control of asthma at lower doses of inhaled corticosteroids.
COPD

Other Diseases

Systemic corticosteroids are indicated in selected patients with severe interstitial lung disease, sarcoidosis, and pulmonary vasculitis. They are not effective in bronchiectasis or cystic brosis, although inhaled corticosteroids are commonly used in these diseases.

Adverse Effects and Contraindications

Corticosteroids inhibit adrenocorticotrophic hormone (ACTH) and cortisol secretion by a negative feedback effect on the pituitary gland. Hypothalamopituitary-adrenal (HPA) axis suppression is dependent on dose and usually only occurs when a dose of prednisolone 47.510 mg daily is used. Significant suppression after short courses of steroid therapy is not usually a problem, but prolonged suppression may occur after several months or years. Steroid doses after prolonged oral therapy must therefore be reduced slowly. Symptoms of steroid withdrawal syndrome include lassitude, musculoskeletal pains, and, occasionally, fever. HPA suppression with inhaled steroids is seen only when the daily inhaled dose exceeds 2000 mg BDP or equivalent daily. Side effects of long-term oral corticosteroid therapy are well described and include uid retention, increased appetite, weight gain, osteoporosis, capillary fragility, hypertension, peptic ulceration, diabetes, cataracts, and psychosis. Their frequency tends to increase with age. The incidence of systemic side effects after inhaled steroids is an important consideration (Table 2). Initial studies suggested that adrenal suppression only occurred when inhaled doses of more than 15002000 mg daily were used. More sensitive measurements of systemic effects include indices of bone metabolism, such as serum osteocalcin and urinary pyridinium cross-links, and, in children, knemometry, which may be increased with
Table 2 Side effects of inhaled corticosteroids Local side effects Dysphonia Oropharyngeal candidiasis Cough Systemic side effects Adrenal suppression and insufciency Growth suppression Bruising Osteoporosis Cataracts Glaucoma Metabolic abnormalities (glucose, insulin, and triglycerides) Psychiatric disturbances (euphoria and depression)

COPD patients occasionally respond to steroids, and these patients are likely to also have asthma. Steroids have no objective short-term benet of airway function in patients with true COPD, although they may often produce subjective benet because of their euphoric effect. Corticosteroids do not appear to have any significant anti-inammatory effect in COPD, and there appears to be an active resistance mechanism that may be explained by impaired activity of HDAC2. Inhaled corticosteroids have no effect on the progression of COPD, even when given to patients with presymptomatic disease. Inhaled corticosteroids reduce the number of exacerbations in patients with severe COPD (FEV1 o50% predicted) who have frequent exacerbations, and they are recommended in these patients. Oral corticosteroids are used to treat acute exacerbations of COPD and reduce the rate of recovery, although the effect is small.

CORTICOSTEROIDS / Therapy 581

inhaled doses as low as 400 mg in some patients.The clinical relevance of these measurements is not clear, however. Nevertheless, it is important to reduce the likelihood of systemic effects by using the lowest dose of inhaled steroid needed to control the asthma, by using add-on therapies such as long-acting b2-agonists, and by using a large-volume spacer to reduce oropharyngeal deposition. Several systemic effects of inhaled steroids have been described and include dermal thinning and skin capillary fragility, which is relatively common in elderly patients after high-dose inhaled steroids. Other side effects, such as cataract formation and osteoporosis, are reported, but often in patients who are also receiving courses of oral steroids. There has been particular concern about the use of inhaled steroids in children because of growth suppression. Most studies have been reassuring in that doses of 400 mg or less have not been associated with impaired growth, and there may even be a growth spurt as asthma is better controlled. There is evidence that use of high-dose inhaled corticosteroids is associated with cataract and glaucoma, but it is difcult to dissociate the effects of inhaled corticosteroids from the effects of courses of oral steroids that these patients usually require. Inhaled steroids may have local side effects due to the deposition of inhaled steroid in the oropharynx. The most common problem is hoarseness and weakness of the voice (dysphonia), which is due to laryngeal deposition and may be due to atrophy of the vocal cords. Oropharyngeal candidiasis occurs in 5% of patients. The incidence of local side effects may be related to the local concentrations of steroid deposited and may be reduced by the use of largevolume spacers, which markedly reduce oropharyngeal deposition. Local side effects are also less likely when inhaled steroids are used twice daily rather than four times daily. There is no evidence of atrophy of the lining of the airway or of an increase in lung infections (including tuberculosis) after inhaled steroids. It is difcult to extrapolate systemic side effects of corticosteroids from studies in normal subjects. In asthmatic patients, systemic absorption from the lung is reduced, presumably because of the reduced and more central deposit of the inhaled drug,

particularly in more severe cases. Another important issue is the distribution of inhaled corticosteroids in patients with asthma since most of the drug deposits in larger airways. Because inammation is also found in small airways, particularly in severe cases, the inhaled corticosteroids may not adequately suppress inammation in these airways.
See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Corticosteroids: Glucocorticoid Receptors. Gene Regulation. Systemic Disease: Sarcoidosis.

Further Reading
Adcock IM and Lane SJ (2003) Corticosteroid-insensitive asthma: molecular mechanisms. Journal of Endocrinology 178: 347 355. Allen DB (2004) Systemic effects of inhaled corticosteroids in children. Current Opinion in Pediatrics 16: 440444. Barnes PJ (2004) Corticosteroid resistance in airway disease. Proceedings of the American Thoracic Society 1: 264268. Barnes PJ and Adcock IM (2003) How do corticosteroids work in asthma? Annals of Internal Medicine 139: 359370. Barnes PJ, Ito K, and Adcock IM (2004) A mechanism of corticosteroid resistance in COPD: inactivation of histone deacetylase. Lancet 363: 731733. Barnes PJ, Pedersen S, and Busse WW (1998) Efcacy and safety of inhaled corticosteroids: an update. American Journal of Respiratory and Critical Care Medicine 157: S1S53. Efthimou J and Barnes PJ (1998) Effect of inhaled corticosteroids on bone and growth. European Respiratory Journal 11: 1167 1177. Kankaanranta H, Lahdensuo A, Moilanen E, and Barnes PJ (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respiratory Research 5: 17. Leone FT, Fish JE, Szeer SJ, and West SL (2003) Systematic review of the evidence regarding potential complications of inhaled corticosteroid use in asthma. Chest 124: 23292340. Leung DY and Bloom JW (2003) Update on glucocorticoid action and resistance. Journal of Allergy and Clinical Immunology 111: 322. Lipworth BJ (1999) Systemic adverse effects of inhaled corticosteroid therapy: a systematic review and meta-analysis. Archives of Internal Medicine 159: 941955. Pedersen S (2001) Do inhaled corticosteroids inhibit growth in children? American Journal of Respiratory and Critical Care Medicine 164: 521535. Rowe BH, Edmonds ML, Spooner CH, Diner B, and Camargo CA Jr (2004) Corticosteroid therapy for acute asthma. Respiratory Medicine 98: 275284.

Cough

see Symptoms of Respiratory Disease: Cough and Other Symptoms.

582 CROUP

CROUP
G Geelhoed, Princess Margaret Hospital, Perth, WA, Australia
& 2006 Elsevier Ltd. All rights reserved.

in children under 8 years of age. Any swelling in this area due to inammation results in significant impingement on the airway. The younger the child is, the greater the need to monitor them closely.

Abstract
Croup is a common childhood problem with a peak incidence of 60 per 1000 child years in those aged between 1 and 2 years. All children who present to emergency departments with croup should be treated with steroids, oral dexamethasone 0.15 mg kg 1 or prednisolone 1 mg kg 1 being the drugs of choice. A compromised but functioning airway should never be made worse by upsetting the child. Children who require epinephrine may be sent home safely provided they have also received steroids and have improved sufciently to have no stridor at rest over a number of hours.

History
The typical presentation of croup is in a preschool child with a history of a recently acquired upper respiratory tract infection. The child develops a barking or seal-like cough, a hoarse voice, and, if obstruction is severe enough, stridor. If stridor is inspiratory this indicates obstruction at the laryngeal level or above while expiratory stridor or biphasic stridor indicates problems in and around the trachea and more severe obstruction. Less commonly, older children may present with recurrent croup with no viral prodrome. They and their families tend to be atopic and suffer from asthma more than the general population. They should, however, be treated in the same manner as those with viral croup. In the smaller child especially, problems with feeding, swallowing difculties, and whether the child has been cyanosed should be ascertained. It is important to know whether or not the child has had croup in the past and specifically whether the child has had mild stridor in between acute attacks. This is important as any child who has a pre-existing narrowing of the airway is much more likely to proceed to dangerous obstruction with an acute obstruction superimposed on it. Immunization history is important to obtain to exclude the diagnosis of diphtheria, which is very rare, and to check whether the child has been given the Hib vaccine should epiglottitis be suspected.

Introduction
The term croup describes an acute clinical syndrome of hoarse voice, barking cough, and stridor; it is usually seen in young children. Croup results from swelling of the upper airway, in and around the larynx, usually as a result of a viral infection. Parainuenza virus type 1 accounts for around half the cases during winter, and parainuenza type 2, inuenza type A, adenoviruses, respiratory syncytial virus, enteroviruses, and possibly mycoplasma pneumonia cause most of the other cases. Croup is a common childhood problem with a peak incidence of 60 per 1000 child years in those aged between 1 and 2 years, although it may be seen up to the teenage years. As such, it is by far the most common cause of acute upper airways obstruction likely to present to emergency departments.

Pathogenesis/Pathology
Unless otherwise qualied, the term croup in this article refers to viral laryngotracheobronchitis. In viral laryngotracheobronchitis the causative virus is transmitted via the respiratory route with the port of entry being the nose and nasopharynx. Viral replication occurs, and as the infection spreads, the walls of the larynx and trachea become edematous with a brinous exudate partially occluding the lumen of the trachea. In addition to luminal narrowing, edema of the vocal cords and subglottic larynx leads to stridor, hoarseness, and a characteristic bark-like cough. Distress caused by obstruction tends to be most marked in younger children due to the small size of their larynx, the presence of loose submucous tissues, and the tight encirclement of the subglottic area by the cricoid cartilage. This is the narrowest airway point

Examination
Most children with croup are not distressed and have only a barking cough with no stridor at rest or stridor that is audible only with physical activity. Signs due to viral illness such as mild fever and nasal discharge are often present. In more severe cases, the child may have a more pronounced stridor at rest. If obstruction progresses, the child may exhibit increasing substernal, intercostal, and subcostal retractions. A worrying sign is that of altered consciousness reected as anxiety, restlessness, and obvious fatigue. Decreased air entry and respiratory effort, extreme pallor, and cyanosis require immediate intervention. Patients with mild croup should have their throats examined but

CROUP 583

this should be deferred in more severe cases. A compromised but functioning airway should never be made worse or compromised by upsetting the child. The childs preferred position may also give clues as to the severity of obstruction and to a diagnosis other than croup. Hyperextension or other abnormal positioning of the neck may suggest epiglottitis or a retropharyngeal abscess.

Treatment and Disposition

All children who present to emergency departments with croup should be treated with steroids. Prior to the regular use of steroids a general rule of thumb was to admit children with stridor at rest to hospital for observation while allowing those with occasional stridor and barking cough only to be managed at home. As many children will improve within a few hours of taking steroids they may be discharged home after a short stay in an emergency department or an observation ward. Other factors such as the distance of home from medical care, the availability of transport, the childs past history with regard to severe airway obstruction, and parental concern and attitude all need to be taken into account when making the decision to admit. Oral dexamethasone in a one-off dose of 0.15 mg kg 1 (or an equivalent dose of prednisolone of 0.75 mg kg 1) is recommended. Most children with croup will require only one dose but if croup (as opposed to viral) symptoms persist, a further dose may be given 24 h later. It is often more convenient to use prednisolone (rounded off to a simple 1 mg kg 1) in the community as it is more readily available. Unpublished work suggests that children treated with prednisolone may present more commonly than those treated with dexamethasone and would benet from a second dose. Steroids may be administered intramuscularly or intravenously (in injectable form) in the rare case of severe obstruction where there is concern that the child may aspirate given their respiratory difculties. Oral dexamethasone has been found to be as effective as inhaled steroids such as budesonide and to work as fast at a fraction of the cost. Combining dexamethasone and budesonide is no more effective than dexamethasone alone. There is no place for antibiotics in a typical case of croup. The effectiveness of steam or humidied air is largely unproven despite its once common usage. Where obstruction is judged to be severe, the use of nebulized epinephrine should be considered. It is generally considered that epinephrine does not change the natural history of croup, such as length of stay in hospital or need to intubate, due to its short-lasting effects. However, it will buy time while waiting for steroids to take effect or waiting for an anaesthesiologist to arrive in a worst case scenario. Five milliliters of 1 in a 1000 epinephrine nebulized with oxygen can be used for all children and may be repeated after 10 min if needed. While in the past it was recommended that any child who received epinephrine for croup should be admitted, a number of studies have now shown that children may be sent home safely provided they have also received steroids

Diagnostic Tests
Oximetry is of limited value as children may maintain oxygen saturations in the high nineties even when badly obstructed. In difcult diagnostic cases, a lateral soft tissue X-ray of the neck may help; however, the possible benets should be weighed against the risks of moving or disturbing the child if obstruction is more than mild and expert advice should be sought. Croup, however, is usually an easy spot diagnosis requiring no diagnostic tests.

Differential Diagnosis
Although rare, it is important to establish that other more sinister causes of acute upper respiratory tract obstruction masquerading as croup are not present. Especially in the younger child one should inquire regarding long-term symptoms preceding the present episode, such as low-grade stridor. This might suggest underlying congenital airway or a vascular anomaly (e.g., tracheomalacia, congenital subglottic stenosis, congenital bilateral cord paralysis, laryngeal web, or vascular ring compression of the trachea). One should also inquire as to possible trauma, toxic ingestions, dysphagia, and drooling. Drooling may suggest epiglottitis, peritonsil abscess, or foreign body in the airway or esophagus. Classic croup and epiglottitis are hard to confuse as the latter usually presents as a pale, toxic, drooling child with a short history who does not cough much. Early on, however, the distinction may be more difcult to make. A child with severe croup with a high fever who does not respond to epinephrine and steroids may have tracheitis and will need a more aggressive approach. The possibility of a foreign body should be kept in mind for children who do not respond to treatment or have a prolonged course. While usually parents will volunteer a history of an acute obstruction or a sudden coughing t the history may not always be known to them. A denitive diagnosis may be made by directly viewing the upper airway but this should only be done by an experienced pediatric anaesthesiologist, intensivist, or emergency physician in an appropriate clinical setting.

584 CRYOGLOBULINEMIA

and have improved sufciently to have no stridor at rest over a number of hours. The universal use of steroids for croup in emergency departments has resulted in a fall in the relapse rate of those sent home, a fall in the average length of stay in hospital, and a dramatic reduction in the number of children needing intensive care and intubation.

with croup, a second dose of prednisolone 24 h later may be needed in some cases.
See also: Corticosteroids: Therapy. Laryngitis and Pharyngitis. Lung Anatomy (Including the Aging Lung). Lung Development: Congenital Parenchymal Disorders. Pediatric Pulmonary Diseases. Signs of Respiratory Disease: Breathing Patterns; General Examination. Upper Airway Obstruction. Vaccinations: Bacterial, for Pneumonia.

Prognosis
Most children with croup have mild symptoms and do not need hospitalization, and will recover within a few days. Their symptoms will be shortened even further with the use of steroids. Despite the substantial impact of steroids an occasional child will still follow a prolonged course with cough and marked stridor for many days. While other diagnoses such as foreign body need to be considered, most children will settle with time.

Further Reading
Bouchier D, Dawson KP, and Fergusson DM (1984) Humidication in viral croup: a controlled trial. Australian Paediatric Journal 20: 289291. Denny FW and Clyde WA Jr (1986) Acute lower respiratory tract infections in nonhospitalized children. Journal of Pediatrics 108: 635645. Geelhoed GC (1996) Sixteen years of croup in a Western Australian Teaching Hospital: the impact of routine steroid treatment. Annals of Emergency Medicine 621626. Geelhoed GC (1997) Croup. Pediatric Pulmonology 23(5): 370 374. Geelhoed GC and Macdonald WGB (1995) Oral dexamethasone in the treatment of croup: 0.15 mg/kg is as effective as 0.3 mg/kg or 0.6 mg/kg. Pediatric Pulmonology 20: 362367. Geelhoed GC, Turner J, and Macdonald WB (1996) Efcacy of a small single dose of oral dexamethasone for outpatient croup: a double blind placebo controlled clinical trial. British Medical Journal 313(7050): 140142. Kelley PB and Simon JE (1992) Racemic epinephrine use in croup and disposition. American Journal of Emergency Medicine 10(3): 181183. Klassen TP, Craig WR, Moher D, et al. (1998) Nebulized budesonide and oral dexamethasone for treatment of croup: a randomized controlled trial. JAMA 279(20): 16291632. Neto GM, Kentab O, Klassen TP, and Osmond MH (2002) A randomized controlled trial of mist in the acute treatment of moderate croup. Academic Emergency Medicine 9: 873879. Prendergast M, Jones JS, and Hartman D (1994) Racemic epinephrine in the treatment of laryngotracheitis: can we identify children for outpatient therapy. American Journal of Emergency Medicine 12(6): 613616. Stoney PJ and Chakrabarti MK (1991) Experience of pulse oximetry in children presenting with croup. Journal of Laryngology and Otology 105: 295298.

Prevention
For most children, croup is a one-off episode and well tolerated especially if steroids are used. Children who suffer repeated episodes of recurrent croup as described above may benet from steroid use at home at the rst hint of croup symptoms. Although no trials have evaluated this approach, anecdotal evidence suggests this practice appears to have benet.

Controversies/Future Research
Although once controversial, use of steroids is now generally accepted for all children who present to emergency departments with croup. While it is clear that both prednisolone and dexamethasone are effective in the treatment of croup, direct comparisons have not been published as yet. Unpublished work suggests the shorter half-life of prednisolone may result in a greater number of children returning to medical care. While a once-only dose of dexamethasone is sufcient for the vast majority of children

CRYOGLOBULINEMIA
` degli Studi di Parma, Parma, Italy A Pesci, Universita P Manganelli, Azienda Ospedaliero Universitaria di Parma, Parma, Italy
& 2006 Elsevier Ltd. All rights reserved. associated with the presence of circulating cryoglobulins. It may be secondary to infectious, neoplastic, and autoimmune diseases. A striking association between hepatitis C virus (HCV) infection and mixed cryoglobulinemia has been documented in the majority of patients (7595%). In mixed cryoglobulinemia, mild to moderate lung involvement may be shown, but severe lung involvement is very rare. Chest X-rays and high-resolution computed tomography may show interstitial lung involvement ranging from mild ground glass and/or consolidation appearance to honeycombing pattern. Most frequently, lung involvement is

Abstract
Mixed cryoglobulinemia is a disease characterized by the clinical triad of palpable purpura, arthralgia, and weakness. It is

CRYOGLOBULINEMIA 585
subclinical. In nonsmoking patients with HCV-associated mixed cryoglobulinemia, free of pulmonary symptoms and with normal chest radiographs, bronchoalveolar lavage demonstrated a T lymphocyte alveolitis not predictive of a significant later deterioration in lung function. Patients without respiratory symptoms do not usually need treatment, even in the presence of high levels of cryoglobulins, whereas in those with progressive pulmonary disease, anti-inammatory/immunosuppressive treatments should be considered.

Mixed cryoglobulinemia is a distinct clinical entity that belongs to the small vessel systemic vasculitides, according to the Chapel Hill Consensus Conference nomenclature. It is a systemic inammatory syndrome due to deposition of both soluble and cryoprecitable circulating immune complexes within the wall of small-sized vessels (i.e., capillaries, venules, and arterioles), resulting in activation of complement and vascular injury. Cryoglobulinemia refers to the presence in serum of one or more immunoglobulins (Igs) that precipitate at temperatures below 371C and redissolve on rewarming. Cryoglobulins are usually classied into three subgroups according to the Ig composition: (1) single or type I, composed of single monoclonal Ig, usually IgM or, less frequently, IgG; (2) mixed, type II; and (3) mixed, type III. Mixed cryoglobulins are immune complexes composed of monoclonal IgM (type II) or polyclonal IgM (type III) that have rheumatoid factor (RF) activity and bind to polyclonal IgG. The analysis of cryoprecipitates is generally performed by means of immunoelectrophoresis or immunoxation. However, using more sensitive methodologies, such as immunoblotting or two-dimensional polyacrylamide gel electrophoresis, type II mixed cryoglobulins may result composed of oligoclonal IgM or a mixture of polyclonal and monoclonal IgM associated with polyclonal IgG (type IIIII mixed cryoglobulins), indicating a possible transition from type III to type II mixed cryoglobulins. Type I cryoglobulins are nearly always found in patients with myelo- and lymphoproliferative diseases. Type II and III mixed cryoglobulins can be associated with various infectious, neoplastic, or autoimmune diseases.

as the major etiological agent of non-A/non-B chronic hepatitis, many epidemiological studies have suggested an important role for HCV in the pathogenesis of mixed cryoglobulinemia. Indeed, 7595% of patients with mixed cryoglobulinemia have signs of HCV infection, and examination of the soluble and cryoprecipitating immune complexes suggests that the IgG component, to which the IgMRF fraction binds, is directed against the HCV proteins. Indeed, the cryoprecipitate contains most of the known HCV antigens (core, E1, E2, NS3, NS4, and NS5) and their corresponding antibodies. Moreover, the concentration of HCV RNA in the cryoprecipitate is up to 1000-fold greater than that in the supernatant. Finally, a direct involvement of HCV in immune complex-mediated cryoglobulinemic vasculitis has been suggested by immunohistochemical and molecular biology studies, including HCV RNA detection by in situ hybridization. Therefore, because of the striking association between HCV infection and mixed cryoglobulinemia, the term essential mixed cryoglobulinemia should be reserved for only a small number of patients without a known cause of the disease.

Pathology
The information on the lung pathology in mixed cryoglobulinemia is scanty. Cellular inammation and brosis are the most common lung manifestations. Widespread vasculitis involving small and medium-sized vessels in the lung, as well as in other organs, has been reported in postmortem examination of cases of mixed cryoglobulinemia. The pattern of organizing pneumonia has also been reported from surgical lung biopsies in patients with cryoglobulinemia.

Clinical Features
The major clinical manifestations of mixed cryoglobulinemia include palpable purpura (which primarily appears on the lower limbs but less frequently on the buttocks and trunk), arthralgia, and weakness (Meltzers triad). Cryoglobulinemic purpura is typically intermittent. Other clinical manifestations are due to the involvement of the liver (mild to moderate chronic hepatitis and, less frequently, overt cirrhosis), kidneys (type I membranoproliferative glomerulonephritis), and the peripheral nervous system (sensory or sensorimotor peripheral neuropathy and, less frequently, multiplex mononeuropathy). The clinical picture of mixed cryoglobulinemia may also include arthritis (generally mild and nonerosive oligoarthritis), leg ulcers, Raynauds phenomenon, sicca syndrome,

Etiology
Because chronic hepatitis is frequently observed during the clinical course of mixed cryoglobulinemia, a possible role for hepatotropic viruses in the pathogenesis of the disease has long been suggested. It is estimated that hepatitis B virus is a causative factor in mixed cryoglobulinemia in less than 5% of patients. Since the discovery of hepatitis C virus (HCV)

586 CRYOGLOBULINEMIA

and, less frequently, the central nervous system, cardiac and gastrointestinal involvement, and hyperviscosity syndrome. It should be emphasized that low levels of circulating mixed cryoglobulins can be detected in more than 50% of HCV-infected individuals, whereas overt cryoglobulinemic vasculitis develops in approximately 5%.
Pulmonary Manifestations

Pulmonary symptoms are infrequent. However, mixed cryoglobulinemia patients may complain of

exertional dyspnea, wheezing, and pleuritic pain. Severe lung involvement due to alveolar hemorrhage, adult respiratory distress syndrome, or acute lung injury is a very rare complication of mixed cryoglobulinemia. Chest X-rays and high-resolution computed tomography (HRCT) may show interstitial lung involvement ranging from mild ground glass (Figure 1) and/or consolidation appearance to a honeycombing pattern (Figure 2). In a large series of mixed cryoglobulinemia patients, clinically evident interstitial lung disorder was observed in only four of 206 patients (2%) during a long-term follow-up.

Figure 1 High-resolution computed tomography demonstrating bilateral ground glass appearance in a patient with mixed cryoglobulinemia.

Figure 2 High-resolution computed tomography demonstrating bilateral honeycombing pattern in a patient with mixed cryoglobulinemia.

CRYOGLOBULINEMIA 587
p <0.001 100

75 p <0.001 % 50

25

N.S.

0 Macrophages Lymphocytes Neutrophils

Figure 3 Bronchoalveolar lavage cell count of 16 patients with mixed cryoglobulinemia (solid triangles) and 13 healthy control subjects (open triangles). Lines are means. Data from Manganelli P, Salaf F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978982, by permission of Oxford University Press.
p <0.004 100

N.S.

75 N.S. 50

25

subclinical T lymphocyte alveolitis) (Figure 3). The percentage of CD3 lymphocytes was significantly higher in mixed cryoglobulinemia patients than in controls, whereas no significant differences were found in the percentage of CD4 , CD8 , and CD19 lymphocytes, neutrophils, and eosinophils (Figure 4). The 5-year follow-up of these patients did not demonstrate deterioration in lung function. Interestingly, treatment of ve patients with recombinant interferon-a for a mean period of 12 months resulted in a significant decrease in the percentage of BAL lymphocytes without changes in the T cell subsets.

0 CD3+ CD4+ CD8+

Figure 4 T cell subsets in bronchoalveolar lavage of 16 patients with mixed cryoglobulinemia (solid triangles) and 13 healthy control subjects (open triangles). Data from Manganelli P, Salaf F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978982, by permission of Oxford University Press.

Diagnosis

Chest radiographs may also demonstrate pulmonary inltrates, cavitary lesions, and pleural thickening. Lung function tests often reveal airow obstruction and impairment of gas exchange with reduced DLCO. 67-Gallium scintigraphy may demonstrate hilar and/or parenchymal uptake of the radionuclide.
Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) performed on nonsmoking patients with HCV-associated mixed cryoglobulinemia, free of pulmonary symptoms and with normal chest radiographs, demonstrated a lower percentage of alveolar macrophages and higher percentage of lymphocytes than in healthy controls (i.e.,

There are no available classication schemes or diagnostic criteria for mixed cryoglobulinemia. In clinical practice, the main diagnostic parameters are serum mixed cryoglobulins with RF activity, low C4 serum levels, skin purpura, and leukocytoclastic vasculitis of small-sized blood vessels due to the deposition of circulating immune complexes and activation of complement. Diagnostic features may also include the involvement of one or more organs (i.e., peripheral nerves, renal, and/or liver). Although cryoglobulin detection and immunologic characterization are necessary for a correct classication and diagnosis, the amount of serum cryoglobulins does not generally correlate with the severity, activity, and prognosis of the disease. When pulmonary involvement is suspected, complete lung function testing, resting room air, arterial blood gases, and chest X-rays should be obtained. If any abnormality is observed, HRCT of the thorax should be performed. Fiberoptic bronchoscopy with transbronchial biopsy

588 CRYOGLOBULINEMIA

and BAL should be reserved for patients with dubious diagnosis or severe pulmonary involvement.
Outcome

The most common clinical pattern in patients with mixed cryoglobulinemia is a mild, slowly progressive disorder with good prognosis and survival. However, in one-third of the cases, a moderate to severe clinical course is observed; in these patients, the prognosis can be affected by renal failure. Mixed cryoglobulinemia may also be complicated, generally after a long-lasting benign clinical course, by malignancies such as B cell non-Hodgkins lymphoma and hepatocellular carcinoma. Lung involvement is usually mild and does not affect survival, but it may predispose the lung to infectious complications and rarely lead to respiratory insufciency from clinically evident pulmonary brosis.

Pathogenesis
The mechanism(s) of cryoprecipitation remains unknown. However, different hypotheses have been suggested to explain this in vitro phenomenon, namely (1) structural modication of the variable proportions of Ig heavy and light chains, (2) a reduced concentration of sialic acid, (3) reduced amounts of galactose in the Fc portion of the Ig, and (4) the presence of N-linked glycosylation sites in the CH3 domains as a result of somatic Ig mutations during autoimmune responses. Moreover, this phenomenon can be caused by specic interactions among single components of the cryoprecipitate, such as the IgM cryoprecipitable RF and the Fc portion of IgG, the corresponding autoantigen.
Relationship to HCV Infection and Lung Diseases

HCV RNA has been detected in pulmonary tissue and BAL uid from HCV patients with diffuse parenchymal lung disease. Furthermore, BAL from patients with both chronic hepatitis C and HCV-associated mixed cryoglobulinemia, without clinical symptoms and radiological ndings of diffuse parenchymal lung disease, demonstrated a neutrophilic or lymphocyte alveolitis. Unfortunately, it is not clear if this nding may contribute to the process that leads to pulmonary brosis seen in a minority of cases of chronic hepatitis C with or without mixed cryoglobulinemia. Finally, HCV may affect lung function in patients with obstructive lung diseases. Indeed, it has been found that chronic HCV infection is associated with an accelerated decline in lung function in both patients with chronic obstructive pulmonary disease and patients with bronchial asthma. Chronic HCV infection also impairs responses to inhaled corticosteroid therapy, as well as reversibility with salbutamol in bronchial asthmatic patients who do not respond to interferon-a therapy.

Management and Current Therapy

Since HCV plays a crucial role in the etiology/pathogenesis of mixed cryoglobulinemia, HCV eradication with interferon-a and ribavirin should be attempted in the majority of patients. In other patients, or in those who have failed to respond to the antiviral therapy, anti-inammatory/immunosuppressive treatments, including corticosteroids, low antigen content diet, plasma exchange, and cytotoxic drugs (mainly cyclophosphamide), should be considered. Patients without respiratory symptoms do not usually need treatment, even in the presence of high levels of cryoglobulins, whereas in symptomatic patients with progressive pulmonary disease, anti-inammatory/immunosuppressive treatment should be taken into account.
See also: Bronchoalveolar Lavage. Interferons. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Fibrosis. Vasculitis: Overview.

Since HCV is the main etiological agent suspected in mixed cryoglobulinemia, the possible role of this viral agent in some lung diseases deserves consideration. Indeed, it has been suggested that HCV may play a role in the pathogenesis of pulmonary brosis due to an increased incidence of serologic markers of HCV infection in Japanese and Italian patients with lung brosis compared to healthy controls. This observation, however, was not conrmed in a British series of patients with pulmonary brosis. The discrepancy among these results may depend on differences in both the geographical prevalence of HCV infection and serological assays for its detection. However, lung function tests and HRCT may show ndings compatible with diffuse parenchymal lung disease in patients with chronic HCV infection, even in the absence of respiratory symptoms. In addition,

Further Reading
Bombardieri S, Paoletti P, Ferri C, et al. (1979) Lung involvement in essential mixed cryoglobulinemia. American Journal of Medicine 66: 748756. Dammacco F, Sansonno D, Piccoli C, Tucci FA, and Ravanelli V (2001) The cryoglobulins: an overview. European Journal of Clinical Investigation 31: 628638. Ferri C, Giaggioli D, Cazzato M, et al. (2003) HCV-related cryoglobulinemic vasculitis: an update on its etiopathogenesis and therapeutic strategies (review). Clinical and Experimental Rheumatology 21(supplement 31): S78S84.

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 589

Ferri C, La Civita L, Fazzi P, et al. (1997) Interstitial lung brosis and rheumatic disorders in patients with hepatitis C virus infection. British Journal of Rheumatology 36: 360365. Ferri C, Sebastiani M, Giuggioli D, et al. (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Seminars in Arthritis and Rheumatism 33: 355374. Irving WL, Day S, and Johnston ID (1993) Idiopathic pulmonary brosis and hepatitis C virus infection. American Review of Respiratory Disease 148: 16831684. Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis & Rheumatism 37: 187192. Lamprecht P, Gause A, and Gross W (1999) Cryoglobulinemic vasculitis (review). Arthritis & Rheumatism 42: 25072516. Manganelli P, Salaf F, and Pesci A (1997) Clinical and subclinical alveolitis in connective tissue diseases assessed by bronchoalveolar lavage. Seminars in Arthritis and Rheumatism 26: 740754. Manganelli P, Salaf F, Subiaco S, et al. (1996) Bronchoalveolar lavage in mixed cryoglobulinemia associated with hepatitis C virus. British Journal of Rheumatology 35: 978982. Meliconi R, Andreone P, Fasano L, et al. (1996) Incidence of hepatitis C virus infection in Italian patients with idiopathic pulmonary brosis. Thorax 51: 315317. Okutan O, Kartaloglu Z, Ilvan A, et al. (2004) Evaluation of highresolution computed tomography and pulmonary function tests in patients with chronic hepatitis C virus infection. World Journal of Gastroenterology 10: 381384. Salaf F, Manganelli P, Carotti M, et al. (1996) Mixed cryoglobulinemia: effect of alpha-interferon on subclinical lymphocyte alveolitis. Clinical and Experimental Rheumatology 14: 219220. Ueda T, Ohta K, Suzuki N, et al. (1992) Idiopathic pulmonary brosis and high prevalence of serum antibodies to hepatitis C virus. American Review of Respiratory Disease 146: 266268. Viegi G, Fornai E, Ferri C, et al. (1989) Lung function in essential mixed cryoglobulinemia: a short-term follow-up. Clinical Rheumatology 8: 331338.

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

V C Manganiello and J Fontana, National Institutes of Health, Bethesda, MD, USA E Degerman, Lund University, Lund, Sweden F Ahmad, National Institutes of Health, Bethesda, MD, USA
Published by Elsevier Ltd. only known association of PDE mutations with human disease, for example, certain subclasses of retinitis pigmentosa. The mammalian PDE superfamily is a major potential target for drug discovery, that is, PDE3 inhibitors for certain types of cardiac failure and peripheral vascular disease, PDE4 inhibitors for inammatory disorders such as chronic obstructive pulmonary disease and asthma, and PDE5 inhibitors for erectile dysfunction and, more recently, for pulmonary hypertension.

Abstract
The cyclic nucleotides, cAMP and cGMP, are important intracellular messengers that regulate multiple critical signaling pathways important in normal physiology, as well as pathogenesis of disease. Their intracellular concentrations are regulated by two enzyme systems: cyclases, which catalyze formation of cAMP and cGMP from ATP and GTP, respectively, and cyclic nucleotide phosphodiesterases (PDEs), which cleave the high-energy 30 50 cyclic phosphate bond of cAMP and cGMP to yield 50 AMP and 50 GMP, respectively. PDEs are divided into two major classes: I and II. Class I PDEs contain a conserved catalytic core (B250300 amino acids) and include the majority of presently identied PDEs. The 11 structurally related, highly regulated, and functionally distinct mammalian PDE gene families (PDEs 111) are class I PDEs, as are PDEs found in Drosophilia, nematodes, yeast, sponges, and parasites such as Trypanasoma brucei and T. cruzei. Class II PDEs are fewer in number and structurally unrelated to class I PDEs; no mammalian class II PDEs have been identied. Mammalian PDEs possess a conserved modular molecular architecture, with a conserved catalytic domain containing a signature sequence motif proximal to the C-terminal tail and downstream from a regulatory region in the N-terminal portion of the molecule. It is becoming clear that PDEs are important components of signaling modules and microdomains that are critical to establishing and maintaining intracellular cyclic nucleotide gradients, and compartmentalization of cyclic nucleotide pathways and responses. At present, mutations in the PDE6 gene represent the

Introduction
After Sutherland and co-workers discovered cAMP and cGMP in the late 1950s, these intracellular second messengers became rapidly recognized as important regulators of myriad critical cellular processes, many of which are central to pulmonary physiology in health and disease, that is, cell proliferation and differentiation, metabolism, secretion, vascular and airway smooth muscle relaxation, production of inammatory mediators, cytokines, chemokines, adhesion molecules, extracellular matrix, collagen, etc. Although signaling induced by cAMP and cGMP primarily involves their activation of cAMP- and cGMP-dependent protein kinases (PKAs and PKGs), with subsequent phosphorylation of critical effectors, direct interactions of cyclic nucleotides with binding proteins reect alternative mechanisms for transduction of their signals. These binding proteins include cyclic nucleotide-gated channels, cAMPactivated guanine nucleotide exchange factors (GEFs) that regulate Rap1, and several cyclic nucleotide phosphodiesterases (PDEs), which contain allosteric, noncatalytic cyclic nucleotide binding sites (Figure 1).

590 CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

Rap1

PDEs PDE1 PDE2 PDE3 PDE10 PDE11 5AMP 5GMP

cAMP GEF

PDE4 PDE7 PDE8

PDE5 PDE6 PDE9

PDEs 2,5,6

PKA

cAMP

cGMP

PKG

CNG channels

CNG channels ATP Cyclases GTP

Figure 1 Role of cyclic nucleotide phosphodiesterases (PDEs) in regulation of signaling by cyclic nucleotides. GEF, guanine nucleotide exchange factor; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; CNG channels, cyclic nucleotide-gated channels.

The Sutherland group discovered PDEs almost simultaneously with cAMP and cGMP. PDEs catalyze hydrolysis of cAMP and cGMP, and thereby regulate their intracellular concentrations, and, consequently, their signaling pathways and effects. In tissue extracts and cells, maximal rates of cyclic nucleotide hydrolysis (via PDEs) exceed rates of synthesis, and PDE inhibitors are required to produce measurable increases in cyclic nucleotides during incubations of cells and tissue preparations with hormones and agents that increase cyclic nucleotide synthesis. Molecular genetics has occasioned a virtual explosion of information concerning diversity and complexity of the mammalian PDEs, and the organization of the large mammalian PDE superfamily into 11 structurally related, highly regulated, and functionally distinct gene families (PDE111). We are only beginning to understand the extent and complexity of regulation of PDE expression and activity, as well as the complexity of regulation of cyclic nucleotide signaling by PDEs. Not only are PDEs important regulators of the generation, amplitude, and duration of intracellular cyclic nucleotide signals, but their specic subcellular locations, their association with molecular scaffolds and interacting partners, and their contribution to regulation of signaling compartmentalization, specicity, and functions within signaling microdomains are becoming a common theme in cell biology.

PDE Structure
Catalytic Domain

The 11 PDE gene families differ in their primary amino acid sequences, substrate specicities, kinetic

characteristics, sensitivities to effectors and pharmacological agents, physiological roles, and mechanisms by which they are regulated. Most families contain more than one gene; within the same family and subfamily, multiple homologous isoforms are generated from the same or different genes via utilization of different promoters/transcriptional initiation sites, alternative mRNA splicing, or regulation of translation. Twenty PDE genes encode more than 50 proteins. PDE nomenclature includes, in order, two letters to indicate species, followed by a number identifying the specic gene family and letters that represent individual subfamilies and splice variants. For example, the PDE4 family is comprised of four subfamilies, PDE4AD: HSPDE4D3 indicates the human PDE4 gene family, PDE4D gene, splice variant 3. PDEs share a common structural organization, with a conserved catalytic domain (B250300 amino acids) in the C-terminal portion, followed by a short hydrophilic C-terminal tail and preceded by an N-terminal regulatory region (Figure 2). The conserved catalytic core contains histidine-containing signature motifs, HDX2HX4N, and consensus metal-binding domains. Catalytic domains contain family-specic sequences that determine differences in substrate afnities, catalytic properties, and sensitivities to specic effectors and inhibitors, as well as common structural determinants involved in cleavage of cyclic phosphate bonds. Some families are relatively specic for cAMP as substrate (PDEs 4, 7, 8); others, cGMP (PDEs 5, 6, 9); some exhibit high afnities for both cAMP and cGMP (PDEs 1, 2, 3, 10, 11) (Figure 1). Some of the latter hydrolyze cAMP and cGMP in a mutually competitive manner. Although methylxanthines inhibit almost all PDEs,

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 591

Regulatory domain Conserved catalytic domain C-terminal domain

Targeting domains Autoinhibitory domains Phosphorylation sites Allosteric ligand-binding sites, i.e., GAF domains Effector interaction sites, i.e., calmodulin-binding, PAS domains Dimerization domains

PDE signature motif Substrate specificity Inhibitor sensitivity -Arrestin interaction sites ERK phosphorylation sites

Figure 2 Common structural pattern for different PDE gene families.

family-selective inhibitors, that is, pharmacological agents that inhibit specic families with 10100-fold greater potency than other families, have been discovered for several families, for example, PDEs 1, 2, 3, 4, 5, 6, 7. The crystal structure of the catalytic cores of several PDEs has been solved. This information should aid in the design of drugs with greater selectivity, specicity, and potency for individual PDE isoforms.
Regulatory Domain

Although cGMP binding is not the primary function of GAF domains, PDEs 2, 5, and 6 contain homologous GAF domains that bind cGMP with high afnity and selectivity, but with different functional sequellae.

PDE Functions
PDEs: Critical Components of Spatially Organized Signaling Modules and Microdomains

PDEs are modular enzymes with differences in cyclic nucleotide substrate specicities and inhibitor sensitivities, and with isoform-specic regulatory domains/cassettes (Figure 2). These characteristics allow cells to utilize PDEs with subtle differences in their catalytic and regulatory properties at different subcellular locations to achieve specicity of cAMP signaling and function. Divergent N-terminal portions of PDE molecules contain structural determinants involved in selective responses of individual PDEs to specic signals that regulate catalytic activity, proteinprotein interactions, or targeting to specic subcellular locations. These regulatory domains contain autoinhibitory modules, dimerization domains, GAF domains (an acronym for proteins, i.e., cGMP-binding PDEs, Arabena adenylyl cylase, and fhlA (an Escherichia coli transcriptional activator) that contain these regions), subcellular localization signals, sites for phosphorylation by PKA, PKG, Ca2 /calmodulindependent sensitive protein kinases, protein kinase B (PKB/Akt), and protein kinase C, etc. PDE1 proteins are activated by binding of calmodulin to Ca2 /calmodulin-binding regions; PDE8 proteins are activated by interactions of regulatory PAS (acronym for Per, ARNT, and Sim proteins) domains with inhibitor kappa B (IkB). PDEs 2, 5, 6, 10, and 11 contain N-terminal region GAF domains; they function as conserved nucleotide switches that regulate adjacent catalytic domains.

Regulation of signaling specicity and specic functional readouts in intracellular microdomains is achieved by using small, discrete subsets of related proteins with subtle differences in their structural, regulatory, and catalytic/functional properties. From a traditional perspective, PDEs are important for stringent regulation of the generation, amplitude, duration, and termination of intracellular cAMP and cGMP signals and their physiological effects. Newer techniques, for example, use of uorescence-resonance energy transfer (FRET) and cyclic nucleotide biosensors (cyclic nucleotide-gated channels, whole cell patch clamps, etc.), indicate that, in different types of cells, localized activation of cyclases produces localized increases in cyclic nucleotides, and that these intracellular pools of cAMP and cGMP are temporally, spatially, and functionally compartmentalized. Specic PDE isoforms clearly play important roles in these spatially constricted and temporally regulated microdomains, especially in establishing cyclic nucleotide gradients and regulating their turnover and diffusion. Inhibition of specic PDEs allows diffusion of cAMP from localized microdomains to different subcellular compartments. Although differential localization of specic PDE isoforms supports the concept that localization is related to function, the most salient example of a specifically localized PDE serving as a critical effector and regulator of a unique cellular function is the

592 CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

photoreceptor PDE6. Light-induced activation of PDE6, whose expression is almost virtually conned to photoreceptor rod and cone cells, results in cGMP hydrolysis, closure of cGMP-gated cation channels, hyperpolarization of cell membranes, and initiation of visual signal transduction. Recent studies, including RNA silencing of specic PDE4 variants or PDE4B and D knockout (KO) mice, as well as real-time dynamic imaging and FRET analysis of cAMP gradients, indicate that PDE4D, not PDE3, is primarily responsible for terminating the increase in cAMP induced by activation of the b-receptor in rodent cardiomyocytes. Conversely, accumulation of cAMP in response to forskolin, which activated the entire pool of myocyte adenylyl cyclase, was markedly potentiated by specic PDE3, not PDE4, inhibitors. Confocal microscopy suggested localization of PDE3 to internal membranes, and PDE4 isoforms, along with PKA, to sarcomeric M and Z lines. Different targeting domains in PDE4 variants allow them to be spatially compartmentalized in different subcellular regions. In Calu-3 airway epithelial cells, PDE4D may be localized in a microdomain of the apical membrane, in proximity to adenosine A2B receptors and the cystic brosis transmembrane conductance regulator (CFTR). Thus, PDE4D may form a barrier to diffusion of cAMP and regulation of CFTR by PKA at the apical membrane. Historically, in cells containing multiple PDEs, family-specic PDE inhibitors have been utilized to pharmacologically link specic PDEs to regulation of specic intracellular cyclic nucleotide pools, their specic signaling pathways, and physiological effects. For example, in cultured mammalian oocytes, inhibitory effects of cAMP on meiotic progression and oocyte maturation are enhanced or mimicked by PDE3, not PDE4 or 5, inhibitors, suggesting that PDE3 regulates a cAMP pool that controls maturation. (In fact, female PDE3A-decient mice are sterile.) In addition, PDE KO mice exhibit distinct phenotypes, with disruption of specic signaling pathways, indicating that other PDEs cannot compensate for targeted PDE isoforms. Production of tumor necrosis factor alpha (TNF-a) in response to lipopolysaccharide (LPS) is dramatically inhibited in PDE4B KO mice. Studies with PDE4B and 4D KO mice indicate that they play complimentary, but not redundant, roles in control of neutrophil function and that one gene cannot completely compensate for the other. PDE4D KO mice do not exhibit acetylcholine-induced contraction and hyperreactivity of airway smooth muscle. The molecular basis for compartmentalization of cAMP signaling involves, in large part, the targeting/

anchoring of PKA isoforms at specic subcellular locations via A-kinase anchoring proteins (AKAPs). PKA catalytic subunits bind to AKAPs via PKA regulatory subunits. AKAPs contain targeting domains, which specify their intracellular addresses, and scaffolding domains, which organize formation of localized signaling modules/microdomains consisting of PKA, other kinases, kinase substrates, phosphatases, PDEs (especially PDE4 isoforms), and other signaling molecules. Different AKAPs are associated with different constellations of signaling effectors. These spatially segregated modules sense intracellular cAMP gradients and effectively compartmentalize activation of effectors, transduction of signals, and propagation of specic physiological responses. In some functional microdomains, cAMP activation of PKA results in phosphorylation of substrates as well as phosphorylation/activation of PDE4, resulting in modulation and termination of cAMP signals and return of PKA to its basal state. PDE4D isoforms, together with PKA and other signaling effectors, interact/associate with b-arrestins, which are scaffolding proteins that interact with activated b-receptors and regulate b-receptor signaling, trafcking, and internalization. Internalization of b-receptor/b-arrestin complexes reduces generation of cAMP by adenylyl cyclase, and targeting of PKA and PDE4D to activated b-receptors increases localized degradation of cAMP, thus desensitizing the complex to future signaling via cAMP. In T lymphocytes, recruitment of PDE4 (in complex with b-arrestin) to lipid rafts potentiates T-cell receptor (TCR) signaling during T-cell activation induced by ligation of the TCR and co-receptor CD28.
PDEs: Signal Integrators

Because of their intrinsic regulatory properties, different responses to regulatory signals, and different interacting partners, PDEs serve as a locus for crosstalk between different signaling pathways. For example, PDE2, which is allosterically activated by binding of cGMP to noncatalytic sites in GAF domains, is highly concentrated in adrenal glomerulosa cells. In these cells, atrial natriuretic factor activates guanylyl cyclase and increases cGMP, which in turn activates PDE2, reduces cAMP, and inhibits aldosterone production. On the other hand, in other cells, nitric oxide (NO) activates soluble guanylyl cyclase and increases cGMP, which inhibits PDE3 and increases cAMP, resulting, for example, in smooth muscle relaxation, inhibition of platelet aggregation, or stimulation of renin secretion from the renal juxtaglomerular apparatus. In cultured vascular smooth muscle cells, agents that increase cGMP

CYCLIC NUCLEOTIDE PHOSPHODIESTERASES 593

inhibit PDE3, resulting in activation of PKA and inhibition of nuclear factor kappa B (NF-kB)-dependent regulation of chemokine and adhesion molecule expression. In platelets, effects of NO, cAMP, and cGMP seem to be regulated by the interplay of PDEs 2, 3, and 5.
PDEs: Homeostatic Regulators

afnity of the catalytic site for cGMP and catalytic activity.

Clinical Applications: Specic PDE Inhibitors

Nonselective PDE inhibitors such as theophylline are used under certain circumstances to treat asthma. Despite considerable effort to develop family-specic inhibitors to replace theophylline, no PDE inhibitors are currently used as therapy for major pulmonary diseases. PDE3 inhibitors, which inhibit platelet activation/aggregation, relax airway and vascular smooth muscle, and enhance myocardial contractility, failed in long-term trials of chronic cardiac failure. One PDE3 inhibitor, milrinone, is used for acute treatment of adults with decompensated and refractory cardiac failure, while another, cilostazol (Pletaal), is used for treatment of intermittent claudication. PDE4 enzymes are relatively highly expressed in inammatory/immune cells, and specic PDE4 inhibitors exhibit potent anti-inammatory actions in preclinical studies and model systems. Roumilast, a PDE4 inhibitor, is currently in phase 3 trials for treatment of chronic obstructive pulmonary disease (COPD) and asthma. On the other hand, the PDE5 inhibitors Viagra, Cialis, and Levitra have achieved remarkable success, with very favorable side-effect proles, in the treatment of erectile dysfunction. The success of these drugs may be related to their inhibition of PDE5, at a time and locus where NO, produced in response to sexual arousal, activates guanylyl cyclase. PDE5 inhibitors protect cGMP from hydrolytic destruction; cGMP activates

To ensure tight regulation of intracellular concentration of cAMP and cGMP, cyclic nucleotide signaling pathways contain intrinsic mechanisms for negative feedback control. cAMP-induced activation of PKA can result in phosphorylation/activation of PDE3 and PDE4 isoforms, enhanced destruction of cAMP, and termination or dampening of cAMP signals. Chronic, long-term elevation of cAMP also initiates negative feedback control, by increasing transcription of PDE3 and PDE4 genes, resulting in increased enzyme protein and activities. In some cells, this phenomenon upregulates cAMP hydrolysis and is related to ligand-induced downregulation of cAMP signaling and cellular responses, such as tachyphylaxis or desensitization. In cultured rat and human vascular myocytes, cAMP increases expression of membrane-associated PDE3B, without any effect on PDE3A. Thus, cell-specic adaptive mechanisms to prolonged elevation of cAMP have specic functional consequences. Negative feedback regulation by increased intracellular cGMP involves binding of cGMP to GAF domains, resulting in allosterically induced conformational changes that facilitate PKG-induced phosphorylation of the N-terminal domain and increases
Neuronal (n)NOS Endothelial (e)NOS GTP Soluble guanylyl cyclase cGMP

Proteins ATP PKG ADP Ca2+

NO

PDE5 inhibitors

PDE5

Protein-P

5 GMP

Lower Ca2+

Ca2+

Ca2+

Relax vascular smooth muscle

Penile erection

Pulmonary vasodilation

Figure 3 Mechanism of action of PDE5 inhibitors. NOS, nitric oxide synthase.

594 CYSTEINE PROTEASES, CATHEPSINS

PKG, which phosphorylates critical effectors that lower intracellular Ca2 and induce vascular relaxation, leading to penile erection (Figure 3). These drugs are beginning to show progress as potential therapy for pulmonary hypertension (PHT); whether their effectiveness in PHT is related to NO physiology in the pulmonary vascular bed, as in the corpus cavernosum, is not certain.

Further Reading
Beavo JA and Brunton LL (2002) Cyclic nucleotide research still expanding after half a century. Nature Reviews, Molecular and Cellular Biology 3: 710717. Conti M (2004) A view into the catalytic pocket of cyclic nucleotide phosphodiesterases. Nature Structural and Molecular Biology 11: 809811. Corbin JD and Francis SH (1999) Cyclic GMP phosphodiesterase 5: target of sildenal and related compounds. Journal of Biological Chemistry 274: 1372913733. Degerman E, Rahn Landstrom T, Stenson Holst L, et al. (2004) Role for phosphodiesterase 3B in regulation of lipolysis and insulin secretion. In: LeRoth D, Taylor SI, Olefsky JM (eds.) Diabetes Mellitus: A Fundamental and Clinical Text, 3rd edn., pp. 373383. Philadelphia: Lippincott Raven. Francis SH, Turko IV, and Corbin JD (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Progress in Nucleic Acid Research and Molecular Biology 65: 152. Houslay MD and Adams DR (2003) PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signaling cross-talk desensitization, and compartmentalization. Biochemical Journal 370: 118. Jin SL, Lan L, Zoudilova M, et al. (2005) Specic role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages. Journal of Immunology 175(3): 1523 1531. Masciarelli S, Horner K, Liu C, et al. (2004) Cyclic nucleotide phosphodiesterase 3A-decient mice as a model of female infertility. Journal of Clinical Investigations 114(2): 196205. Mongillo M, McSorley T, Evellin S, et al. (2004) Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. American Heart Association Journal 95(1): 6775. Rich T and Karpen JW (2002) Cyclic AMP sensors in living cells: what signals can they actually measure? Annals of Biomedical Engineering 30: 10881099. Spina D (2004) The potential of PDE4 inhibitors in respiratory disease. Current Drug Targets Inammation and Allergy 3: 221226. Torphy T (1998) Phosphodiesterase isoenzymes: molecular targets for novel antiasthma agents. American Journal of Respiratory and Critical Care Medicine 157: 351370. Wong W and Scott JD (2004) AKAP signaling complexes: focal points in space and time. Nature Reviews, Molecular and Cellular Biology 5: 959970.

Conclusions
Compartmentalization of cyclic nucleotide signaling requires the successful conuence of multiple factors. Scaffolding molecules, at different intracellular addresses, spatially organize different subsets of signaling molecules, including PDEs, kinases, phosphatases, substrates, and other signaling and effector molecules, and thus generate discrete functional microdomains. By virtue of their distinct intrinsic characteristics, their differential expression and regulation, and their targeting to different subcellular locations and microdomains where they interact with cellular structural elements, regulatory partners and molecular scaffolds such as AKAPs and b-arrestin, different PDEs respond to and regulate multiple inputs, and modulate the intracellular diffusion of cyclic nucleotide gradients and functional compartmentalization of cyclic nucleotide signals. Integration of the myriad combinations of signaling molecules provides a variety of networks involved in generation, transduction, modulation, and termination of cyclic nucleotide-gated signals and actions, and establishes unique cyclic nucleotide phenotypes that characterize individual cells.
See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Transcription Factors: NF-kB and Ikb.

CYSTEINE PROTEASES, CATHEPSINS

hling, Otto-von-Guericke University of F Bu Magdeburg, Magdeburg, Germany
& 2006 Elsevier Ltd. All rights reserved. mice, and selective protease inhibitors have added new insight into the specic functions of single proteases. This article discusses some of these functions in relation to the lung, especially the role of lysosomal cysteine proteases in matrix remodeling, immunoregulation, surfactant protein processing, and apoptosis.

Abstract
Lysosomal cysteine proteases or cathepsins are a family of more than 10 enzymes. The description of new lysosomal cysteine proteases with restricted expression and outstanding enzymatic activity and also the use of molecular biologic methods, knockout

Introduction
The term cathepsin was introduced in 1920 and it stands for lysosomal proteolytic enzyme, regardless

CYSTEINE PROTEASES, CATHEPSINS 595

of the enzyme class. Therefore, in addition to cysteine proteases, this term also includes enzymes using other catalytic mechanisms, such as serine proteases (cathepsins A and G) and aspartic proteases (cathepsins D and E). This article focuses on cysteine proteases, which are part of the papain family. These proteases are localized predominantly in the lysosomes of cells. Other nonlysosomal cysteine proteases are caspases, calpains, and hemoglobinases. Since the discovery of cathepsin B in 1941, more than 12 cathepsins belonging to the cysteine proteases have been described. Until the 1980s, classical protein biochemical methods were used to characterize cathepsins B, C, H, L, and S. The importance of these enzymes became clear when it was shown that up to 40% of the cellular protein turnover could be suppressed by cysteine protease inhibitors. Later, molecular biology was used to discover, for example, cathepsins F, K, O, V, W, and X. Sequence homologies of the cysteine proteases cathepsins B, C, H, L, and S indicate that these enzymes diverged early during eukaryotic evolution. Some other genes encoding novel enzymes, such as cathepsins F, K, V, W, and X, may be the result of relatively recent gene duplication events. In addition, rodents differ from humans with respect to cathepsin expression; that is, in contrast to humans, rodents do not express cathepsin V, but they do express a group of homologous cathepsins in the placenta.

distinguished. Some of the genes may be the result of relatively recent gene duplication events, as suggested by three gene pairs with common chromosomal localization and high sequence homologies: cathepsins S and K, F and W, and L and V. Active cathepsins are mostly monomeric proteins. Several three-dimensional structures of cysteine proteases are available, and they demonstrate that enzymes of the papain family share a common fold. The main structural features of a papain-like twodomain catalytic platform are depicted in Figure 1 using a model of mature cathepsin H; the active site is located at the interface between the two domains. It contains cysteine and histidine, which form the nucleophile thiolateimidazole ionic pair. Most cysteine proteases of the papain family are endopeptidases by design. Exopeptidases, such as cathepsins B, C, H, and X, are likely to have evolved from the endopeptidase template by addition of structural elements capable of interacting with the C or N terminus of a substrate.

Structure
Cathepsin genes are localized on different chromosomes (Table 1). Based on their amino acid sequence homology and specic sequence motifs, cathepsin Blike (B, O, C, and Z), cathepsin L-like (L, V, K, S, and H), and cathepsin F-like (W and F) enzymes may be
Table 1 General characteristics of cathepsins Cathepsin (synonyms) B Chromosomal localization (human/mouse) 8/14 Expression in respiratory system Ubiquitous macrophages Comments; disease association Carboxypeptidase; aberrant expression in lung tumors and numerous inammatory diseases Dipeptidyl peptidase I; Papillon Lefevre syndrome Aminopeptidase, surfactant processing Collagenase, elastase; pyknodysostosis, lung brosis Pulmonary emphysema Elastase, stable at neutral pH Elastase Activity unknown Carboxypeptidase

Figure 1 Structural model of mature cathepsin H. Courtesy of Dr W Brand, Martin-Luther-University Halle, Halle, Germany.

C (DPPI) F H K (O2) L O S V (L2, U) W (lymphopain) X (Z, B2)

11/7 11/19 15/9 1/3 9/13 4/3 1/3 9/ 11/19 20/2

Mast cells Ubiquitous Ubiquitous, type II pneumocytes Bronchial epithelial cells, activated macrophages and broblasts Ubiquitous, macrophages Macrophages ? Cytotoxic lymphocytes Ubiquitous, granulocytes

596 CYSTEINE PROTEASES, CATHEPSINS

Regulation of Production and Activity

Cathepsins are synthesized as inactive pre-proenzymes and undergo posttranslational glycosylation. The prepart of the molecule is necessary for intracellular trafcking toward the lysosomal compartment, which is realized using mannose 6-phosphate receptors. Truncated forms lacking the pre-protein are misdirected and therefore secreted or accumulated in the cytoplasm of cells; these forms have been found in some tumor cells. The pro-parts of the cathepsins serve as competitive inhibitors by occupying the active center of the enzymes. They are cleaved under acid conditions within the endosomal/lysosomal compartment. The activity of cathepsins is regulated at different levels. First, the transcription of cathepsin genes is controlled by cell-type-specic mechanisms and exogenous factors. Thus, different cell types do express different cathepsins. Some cathepsins, such as cathepsins B, H, F, L, and X, are ubiquitously expressed in almost all cells but differ in their expression level. These cathepsins are found in high concentrations in alveolar macrophages and at lower levels in almost all other cell types. Cathepsin H is increased in type II pneumocytes. Other cathepsins show a more specic expression level, such as cathepsins K and S. Cathepsin S is predominantly expressed in macrophages, whereas cathepsin K has been found in bronchial epithelial cells, activated macrophages (epithelioid cells and multinucleated giant cells), and activated lung broblasts. In addition, cytokines may regulate cathepsin expression in a cell-type-specic way. Proinammatory cytokines (i.e., interleukin (IL)-6 and IL-1b) induce cathepsin expression. Transforming growth factor beta may suppress the expression. Microbial components also increase cathepsin expression in several cells. Second, the maturation and posttranslational processing is regulated by other proteases and the pH in the endosomal/lysosomal compartment. Intracellular microorganisms, which are able to prevent the acidication of the lysosomal compartment, prevent the maturation and activation of cathepsins and, thus, their proteolytic degradation. Third, the activity of mature enzymes depends on the pH of the environment. Neutral or alkaline pH induces the irreversible inactivation of most cathepsins except cathepsins S and K. Furthermore, because of the exceptional proteolytic activity and the high concentration of cathepsins (up to 1 mM in lysosomes), their activity is controlled by a network of endogenous inhibitors. These inhibitors are cystatins, which are expressed intracellularly (cystatins A and B) or extracellularly (cystatins C, D, and F), and other endogenous cathepsin inhibitors, including kininogens, serpins, and a2-macroglobulin.

Cathepsins cleave a variety of proteins and polypeptides. On the one hand, they are involved in lysosomal bulk protein degradation. On the other hand, single cathepsins specifically degrade certain proteins. For example, cathepsins S, L, and V are involved in the processing of the invariant chain of MHC class II molecules, cathepsin K degrades collagen with a unique specicity, and cathepsins K and V are very efcient elastases. Other proteins that are cleaved by cathepsins include surfactant proteins; kinins; thyroid hormones; other enzymes or their precursors, such as granzymes, cathepsin D, neutrophil elastase, cathepsin G, and proteinase 3; trypsinogen; neurotransmitters; and amyloid proteins.

Biological Functions Related to the Respiratory System

During recent years, several knockout and transgenic mouse models have been generated. The published phenotypes and the functional associations found in these animals are summarized in Table 2. The biological functions directly linked to the respiratory system are antigen presentation, matrix remodeling, surfactant processing, and apoptosis (Figure 2).
Antigen Presentation

The process depends on proteolytic processing at different levels. First, extracellular antigens, which are taken up by antigen presenting cells, are mostly degraded in the endosomal/lysosomal compartment. The resulting peptides are displayed on the cell surface after formation of MHC class II peptide complexes. Studies have shown that the pattern of peptides to be presented depends on distinct proteases. For example, positive selection in the thymus depends on the antigen peptides presented on epithelial cells and negative selection of peptides on dendritic cells; in mice, the former depends on cathepsin L and the latter on cathepsin S. Removal of one of these two enzymes by generating chimeric knockout mice leads to a failure of T cell maturation. Second, during the early stages of biosynthesis, MHC class II ab heterodimers associate with a type II membrane protein, the invariant chain (Ii), to form a nonameric complex of abIi. Degradation of Ii is a precondition for binding of antigenic peptides to class II molecules. It has been shown that the cathepsins F, L, S, and V are involved in Ii processing. In thymic epithelial cells of mice, cathepsin L is critical for Ii processing. In human thymus, this function is likely performed by cathepsin V. Cathepsin S may be involved in Ii processing in other human epithelial cells. Cathepsin S is also the critical enzyme in dendritic

CYSTEINE PROTEASES, CATHEPSINS 597

Table 2 Characteristics of cathepsin-decient and -overexpressing mice Cathepsin B C H K Generated by Peters C et al. Ley TJ et al. Peters C et al. Saftig P et al. Kola I et al. Kiviranta R et al. (transgen) Peters C et al. Chapman HA et al. Peters C et al. Functional association Thyroglobulin processing, trypsinogen processing, apoptosis Proteolytoic activation of granzyme A.B, cathepsin G, proteinase 3, neutrophil elastase, mast cell chymase Bone resorption, lung matrix degradation brosis

L S W

Antigen presentation, hair growth, myocardial remodeling, neurotransmitter processing, cathepsin D processing apoptosis Antigen presentation, angiogenesis, atherogenesis

Antigen processing

Lysosomal protein breakdown

Matrix remodeling

Invariant chain processing

Surfactant protein processing

Cathepsins Prohormone processing

Protease activation/inactivation

Regulation of apopt osis

Defensin inactivation

Protease inhibitor cleavage

Figure 2 Biological functions of cathepsins. Functions that are discussed in this article are shown in dark blue.

cells and B lymphocytes. Mouse macrophages express large amounts of cathepsin F, which seems to be responsible for Ii processing in these cells. The suppression of cathepsin F in mice prevents eosinophilia and IgE response after ovalbumin challenge, which illustrates the potential role of dened cathepsins as targets for new immunomodulatory drugs. Further studies should characterize the expression and function of additional cysteine proteases (e.g., cathepsins B, K, and L) in antigen presenting cells of the lung and also explore in more detail the effects of selective inhibition of these enzymes on the development of allergic reactions.
Matrix Remodeling

Cathepsins are involved in intracellular and extracellular matrix (ECM) degradation. Within the lysosomes of macrophages and broblasts, cathepsins

degrade phagocytozed fragments of ECM proteins. It has been shown that macrophages, mast cells, smooth muscle cells, broblasts, and various tumor cells release cathepsins. Macrophages can acidify their environment upon release of lysosomal proteases and thus contain their activity. In addition, cathepsins C, K, S, and V are characterized by relatively high pH stability, and they retain some proteolytic activity at neutral pH. Cathepsin K is characterized by a unique collagenolytic activity. The enzyme cleaves collagen bers at multiple sites, whereas mammalian proteases, such as collagenases, gelatinases, and stromelysin, cleave collagen helices at a dened site, leaving 2/3 and 1/3 fragments. Other cathepsins remove only teleopeptide fragments. The enzyme seems to be important in the development of lung brosis. In a mouse model of bleomycin-induced lung brosis, cathepsin K plays

598 CYSTEINE PROTEASES, CATHEPSINS

a critical role in the degradation of surplus ECM proteins; therefore, cathepsin K-decient mice develop more lung brosis than do wild-type mice. Cathepsins V, K, and S are the most efcient elastases described so far. These enzymes are coexpressed in activated macrophages of artherosclerotic plaques, in which 60% of the elastinolytic activity may be attributed to cysteine proteases. Therefore, it has been suggested that they play a crucial role in degradation of the elastin matrix. Further studies will show whether the same enzymes are involved in regulation of elastin turnover in the lung. In transgenic murine models employing the inducible expression of cytokines along the alveolar surface, the overproduction of IL-13 or interferon gamma caused a phenotype that mirrors human chronic obstructive pulmonary disease, with emphysema, enlarged lungs, and enhanced pulmonary compliance. These changes were accompanied by increased expression of matrix metalloproteinases and cathepsins in the lung. When mice were given the cysteine protease inhibitor E64, they showed markedly attenuated emphysematous changes, implying that cysteine proteases are important in the development of cytokine-induced emphysema. Lung cancer cells can secrete cathepsins L and B in vitro, and anticathepsin L antibodies protect against the formation of solid tumors by implanted myeloma cells in vivo. Therefore, it has been suggested that cathepsins may play a critical role in tumor invasion and progression. In addition, in a number of in vitro models using cells of different origin, it was shown that inhibition of cathepsin B or cathepsin L by specic inhibitors, or suppression of protease expression using antisense technologies, decreases the invasion of tumor cells into the extracellular matrix.
Processing of Surfactant Proteins by Cysteine Proteases

undiscovered, cause of surfactant dysfunction in pulmonary diseases in infants, children, and adults. The primary translation products of SP-B and SP-C (i.e., proproteins (proSP-B and proSP-C) with molecular masses of 42 and 21 kDa, respectively) undergo extensive C- and N-terminal processing in multivesicular bodies and lamellar bodies. The lysosomal protease cathepsin H is localized in lamellar bodies of type II pneumocytes. Immunoelectron microscopic investigations have shown that cathepsin H, proSP-B, and proSP-C are colocalized in multivesicular and lamellar bodies. In human fetal type II pneumocytes, the expression and enzymatic cathepsin H activity is upregulated during in vitro differentiation in parallel with surfactant protein secretion. Incubation of differentiated type II pneumocytes with E64, a potent inhibitor of cysteine proteases, interrupts SP-B processing and leads to additional features of SP-B deciency, including abnormal SP-C processing and aberrant lamellar bodies. In addition, experiments using recombinant proSP-B have shown that cathepsin H may be involved in N- and C-terminal proSP-B processing. The SP-C processing by cathepsin H has been investigated in more detail in vitro, and it has been suggested that cathepsin H is involved in the N-terminal processing of proSP-C in electron-dense multivesicular bodies. In general, published results indicate that in vitro processing of proSP-B and proSP-C by cathepsin H alone does not result in the mature surfactant proteins; therefore, additional enzymes also seem to be involved in this complex processing cascade. Studies using knockout mice for different combinations of lysosomal proteases have been initiated to determine the in vivo relevance of cathepsin H and to characterize other enzymes involved in surfactant protein processing.
Apoptosis

The surfactant proteins SP-B and SP-C play an essential role in the metabolism and dynamics of the pulmonary surfactant lipids. Hereditary SP-B deciency in infants or mice leads to respiratory failure at birth. Mutations in the SP-C gene and SP-C deciencies are associated with interstitial lung disease. In addition, both hereditary alveolar proteinosis in infants without detectable mutations in the SP-B or the SP-C gene and acquired pulmonary alveolar proteinosis in children and adults are characterized by intraalveolar accumulation of mature surfactant proteins and abnormal SP-B precursors. Therefore, insufcient processing of proSP-B or proSP-C due to a lack or dysfunction of one or more proteases involved in surfactant protein processing may be another,

Evidence has accumulated that shows that lysosomal proteases play an important role in the regulation of apoptosis. Destabilization of the lysosomal membrane and release of proteases (cathepsins) trigger or support the process of apoptosis in various systems. The process was described to be the third pathway of apoptosis, in addition to the Fas/CD95- and the mitochondrial pathways. Cysteine proteases and cathepsin D play the most important roles in apoptosis. For example, it was shown in vitro and in vivo that cathepsin B is an important execution protease involved in apoptosis of tumor cells and hepatocytes. Cathepsin B/L double-knockout mice died from apoptotic neuronal loss.

CYSTIC FIBROSIS / Overview 599

Cathepsins in Respiratory Disease

Cathepsins are specifically involved in processing and degradation of a variety of protein substrates and therefore regulate different cellular functions. Future studies involving genetically modied animals and specic inhibitors will determine more specically the function of dened enzymes in vivo. Data suggest that cathepsins contribute to the pathogenesis of the following respiratory diseases, which may be inuenced by the modication of cathepsin activity:
*

Proteasomes and Ubiquitin. Proteinase Inhibitors: Alpha-2 Antiplasmin; Antichymotrypsin; Cystatins; Secretory Leukoprotease Inhibitor and Elan. ProteinaseActivated Receptors. Pulmonary Fibrosis. Serine Proteinases. Surfactant: Overview; Surfactant Proteins B and C (SP-B and SP-C). Transgenic Models.

Further Reading
Barrett AJ, Rawlings ND, and Woessner JF (1998) Handbook of Proteolytic Enzymes. New York: Academic Press. Bromme D and Kaleta J (2002) Thiol-dependent cathepsins: pathophysiological implications and recent advances in inhibitor design. Current Pharmaceutical Design 8: 16391658. Bu cken C, Brasch F, et al. (2004) Pivotal role of ca hling F, Ro thepsin K in lung brosis. American Journal of Pathology 164: 22032216. Bu hling F, Waldburg N, Reisenauer A, et al. (2004) Lysosomal cysteine proteases in the lung. Role in protein processing and immunoregulation. European Respiratory Journal 23: 620628. Chapman HA, Riese RJ, and Shi GP (1997) Emerging roles for cysteine proteases in human biology. Annual Review of Physiology 59: 6388. Lah TT and Kos J (1998) Cysteine proteinases in cancer progression and their clinical relevance for prognosis. Biological Chemistry HoppeSeyler 379: 125130. Riese RJ and Chapman HA (2000) Cathepsins and compartmentalization in antigen presentation. Current Opinion in Immunology 12: 107113. Turk V, Turk B, and Turk D (2001) Lysosomal cysteine proteases: facts and opportunities. EMBO Journal 20: 46294633. Wolters PJ and Chapman HA (2000) Importance of lysosomal cysteine proteases in lung disease. Respiratory Research 1: 170177. Yan SQ, Sameni M, and Sloane BF (1998) Cathepsin B and human tumor progression. Biological Chemistry HoppeSeyler 379: 113123.

Lung brosis and emphysema have been shown to be associated with insufcient or excessive cathepsin activity, respectively. Adequate regulation of cathepsin expression or activity may prevent tissue remodeling via the regulation of matrix remodeling and apoptosis of broblasts. Allergic immunoreactions may be suppressed by the selective inhibition of cathepsin activity via the suppression of antigen presentation and/or the shift of the antigenic epitopes that are presented by the accessory cells. Alveolar proteinosis and similar diseases may in part be caused by insufcient surfactant protein processing due to cathepsin insufciency that leads to accumulation of surfactant proteins in the alveolar space.

See also: ADAMs and ADAMTSs. Apoptosis. Chronic Obstructive Pulmonary Disease: Emphysema, General. Interstitial Lung Disease: Alveolar Proteinosis; Amyloidosis; Idiopathic Pulmonary Fibrosis.

CYSTIC FIBROSIS
Contents

Overview Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Overview
+ and J F Collawn, University of Alabama, Z Bebok Birmingham, AL, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Cystic brosis (CF) or mucoviscidosis is the most frequent autosomal recessive genetic disorder in people with European ancestry. The classic form of CF results from loss of function

mutations in the cystic brosis transmembrane conductance regulator (CFTR) gene. The gene is localized to the long arm of chromosome 7 and encodes a protein that acts both as a chloride channel and as a regulator of other transporters. Loss of CFTR activity results in a plethora of pathological consequences, suggesting a central role for CFTR in epithelial physiology. The disease is characterized by viscous secretions of the exocrine glands in multiple organs and elevated levels of sweat chloride. The most life-threatening clinical features of CF include chronic bacterial infections of the upper and lower airways, airway obstruction, bronchiectasis, respiratory failure, and cor pulmonale. The gastrointestinal manifestations are

600 CYSTIC FIBROSIS / Overview

primarily caused by inefcient exocrine pancreatic function and include meconium ileus, maldigestion, and failure to thrive. Male infertility and reduced fertility in females are also important symptoms in adults with CF. Current therapy focuses on ameliorating the symptoms of malnutrition, airway obstruction, and inammation. As a result of these symptomatic treatments, the survival improved dramatically. The new therapeutic approaches fall into two broad categories, gene replacement and targeted drug development.

Introduction
Cystic brosis (CF) is a monogenic disorder with a complex phenotype and clinical variability. It is observed in approximately 1 in 2500 births among Caucasians, and has a carrier frequency of 1 in 25 individuals, making it the most common lethal monogenetic disease within this demographic group. CF is caused by mutations in a 180 kb gene localized to the long arm of chromosome 7. The gene encodes the 1480 amino acid cystic brosis transmembrane conductance regulator (CFTR) protein. CF cases present with complex phenotypes and clinical variability. Defects in CFTR lead to altered exocrine secretion and pathological changes in multiple organ systems. Therefore, CF provides an excellent example of the ways in which a single gene defect confers a wide variety of clinical symptoms. The most commonly affected organs include the airways, gastrointestinal (GI) tract, pancreas, sweat glands, hepatobiliary system, and genital tract. The most life-threatening clinical features of CF are recurrent and persistent airway infections leading to bronchiectasis, respiratory failure, and cor pulmonale. Pancreatic insufciency is present in 90% of patients due to obstruction of the pancreatic ducts and subsequent brosis. Exocrine pancreatic dysfunction begins in utero and causes postnatal steatorrhea and failure to thrive. Meconium ileus is present in B10% of newborns with CF. CF patients also present abnormally high levels of chloride (Cl ) and sodium (Na ) in the sweat, which is considered as a diagnostic standard of the disease before genetic testing. In 95% of adult CF males, infertility is caused by azoospermia as a result of the complete bilateral absence of vas deferens (CBAVD). In females, reduced fertility may result from abnormally viscous intrauterine mucus. Other manifestations of the disease include liver cirrhosis and diabetes mellitus. Early references to CF describe a serious infant disorder characterized by unusual saltiness of skin. Detailed studies have been performed only since the beginning of the twentieth century, prior to which most infants died soon after birth. Landsteiner, who also dened the basic human blood groups, described cases of meconium ileus associated with defective

pancreas function. However, CF was rst recognized as a distinct pathological entity in the 1930s by pathologist Dorothy Anderson, who dened one of the most important signs of the disease, the cystic brosis of the pancreas. After this report, Farber described CF as generalized state of thickened mucus and named it mucoviscidosis, a term that is still widely used in Europe. Anderson and Hodges discovered the autosomal recessive inheritance pattern of the disease in 1946, and suggested that CF was caused by a single gene defect. In the 1950s, Di SantAgnese and colleagues observed that levels of Cl and Na were increased in the sweat of children with CF. This led to the development (in 1959) of the rst accurate diagnostic test, the sweat test for cystic brosis. In the 1980s, two critical observations led to a better understanding of the basic pathophysiology of the disease. First, Knowles and colleagues found that respiratory epithelium in CF has defective Na and Cl transport. Second, Paul Quinton described the sweat ducts of CF patients as impermeable to Cl . The gene responsible for the disease was identied in 1989. This opened exciting possibilities for understanding the molecular basis of CF and provided new hope for potential therapies. Fifteen years later, this advance was further reinforced by the successful completion of the lowresolution structure of the CFTR protein, along with the high-resolution structures of mouse and human wild-type and DF508 NBD1 domains. Although there is no cure for CF to date, the life expectancy of CF patients has increased significantly. In the 1950s, few children with CF lived to school age; whereas today, patient survival extends to the mid-30s.

Etiology
CF is caused by mutations of the CFTR gene encoding the CFTR protein. Mutations result in decreased levels or inefcient function of CFTR and subsequent pathological changes (Figure 1). CFTR is a multidomain, integral membrane glycoprotein consisting of two homologous halves. Each half contains six predicted transmembrane segments (TMD1 and TMD2) and a nucleotide-binding domain (NBD1 and NBD2). A regulatory domain (R domain) is interposed between the two halves of the protein. CFTR is part of a multiprotein assembly in the apical plasma membrane of epithelial cells (Figure 2). Although more than 1400 mutations (listed in the CFTR Mutation Database, see Relevant websites) are known in CFTR, deletion of phenylalaline at the 508 position (DF508) is the most frequent cause of the disease and contributes to over 90% of CF cases. Other mutations are often linked to certain

CYSTIC FIBROSIS / Overview 601

geographical regions or are unique. Only four of them (G542X, N1303K, G551D, and W1282X) have a frequency above 1%. CFTR gene mutations are classied into six broad groups based on their impact on CFTR synthesis and

CFTR gene mutations

Absent or defective CFTR protein

Altered secretion in exocrine glands

Impaired mucosal defense

Infections
Figure 1 Etiology and pathogenesis of cystic brosis. Summary of how mutations in the CFTR gene lead to infections.

intracellular trafcking (Figures 3(a) and 3(b)). Class I, nonsense, and frameshift mutations abolish CFTR synthesis. Examples include the G542X and R1162X nonsense mutations that encode premature stopcodons. These mutations result in truncated mRNA transcripts that are unstable. Mutations that result in low CFTR levels (Class V) include missense defects such as A455E, and promoter and splicing abnormalities. Class I and V mutations result in little or no protein product. Class II mutations include the most frequent mutation, DF508. Deletion of phenylalanine at the 508 position leads to the synthesis of a protein that is recognized by the ER quality control system as misfolded and degraded by the proteasome after retrotranslocation from the ER. Class III mutations lead to the production of CFTR that reaches the plasma membrane, but has defective regulation and function. G551D, a missense mutation within NBD1, is an example of this class and the third most common disease-associated mutation. The G551D mutant is unable to conduct chloride in response to cAMP. Other mutants in this category exhibit ineffective nucleotide binding or hydrolysis (S1255P, G551S, G1244E, and G1349D). Class IV mutants, such as the R117H and R347P mutants, result in the synthesis of

Glycan chains

COOH EC Syntaxin 1A

TMD1

TMD2

Cell membrane

IC H3 H2 H1 COPII NH2 Blocked by Munc-18 R domain Syntaxin 8 NH 2 NBD1 NBD2 YSDI AP-2 E3KARP CAL CAP70 EBP50 Ezrin PKA Actin DTRL

Figure 2 Model of CFTR and interacting proteins. The CFTR molecule is an integral membrane glycoprotein composed of 12 membrane-spanning segments forming two transmembrane domains (TMD1 and TMD2), two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain (R domain). Syntaxin 1A binds to the N-terminal tail and inhibits CFTR function. This interaction can be blocked by Munc-18. N -linked glycosylation sites are located on the fourth extracellular loop. CFTR C-terminal tail associates with PDZ domain containing proteins (ezrin-radixin-moesin (ERM)-binding phosphoprotein, EBP50; Na /H exchanger 3 kinase A regulatory protein, E3KARP; CFTR-associated ligand, CAL; CFTR-associated protein of 70 kDa, CAP70) through the DTRL sequence. These interactions inuence function or trafcking, or both. Sequential interactions through EBP50, ezrin, and protein kinase A (PKA) anchor CFTR to the actin cytoskeleton. The adaptor complex II (AP-2) interacts with CFTRs internalization signal, YSDI, to promote CFTR endocytosis. COPII binds to NBD1 and facilitates CFTR exit from the ER. Syntaxin-8, a t-SNARE protein that associates with CFTR through the R-domain, facilitates CFTR recycling. EC, extracellular space; IC, intracellular space.

602 CYSTIC FIBROSIS / Overview

Cl
TJ

Cl

6 4 Carrier vesicle 8 Golgi LS Lysosome 5 ES 7 Endosome

ER

Proteasome

AA

Nucleus (a)

Cl

Class IV: altered conductance

TJ

Class III: altered regulation

Class VI: inefficient recycling

Golgi

ER

Class II: block in processing

Class V: reduced synthesis Class I: no synthesis Nucleus (b)

Figure 3 (a) Model for CFTR synthesis, trafcking, and degradation. 1, Synthesis and co-translational insertion of CFTR into the endoplasmic reticulum (ER) membrane; 2, proteasomal degradation of misfolded CFTR; 3, ERGolgi processing, glycosylation; 4, vesicular trafcking to the cell surface; 5, endocytosis through clathrin-coated pits; 6, recycling to the cell surface; 7, lysosomal; and 8, proteasomal degradation. (b) Classes of CFTR mutations. Class I: defective protein production (G542X and R1162X); Class II: defective protein processing (DF508); Class III: defective regulation (G551D); Class IV: defective conduction (R117H, R347P); Class V: decreased levels of CFTR (A455E); and Class VI: unstable CFTR. TJ, tight junction; ES, endosome.

CYSTIC FIBROSIS / Overview 603

a CFTR protein with defective chloride conductance. The most recently identied class of CFTR mutants (Class VI) maintain normal biosynthetic processing and chloride channel function of the truncated CFTR, but the biological stability of the fully processed protein is dramatically reduced.

Pathology
Respiratory Tract

Histological signs of CF lung disease have been observed as early as the third trimester, with the obstruction of submucosal gland ducts by thick mucus. Recurring pulmonary exacerbations, due to repeated cycles of infection and inammation, result in lung damage and compromise pulmonary function. Inammation is present at an early age and remains active even in clinically stable patients. As a result, pulmonary brosis, bronchiectasis, respiratory insufciency, and cor pulmonale remain the hallmarks of CF pathology and the primary cause of morbidity.
Gastrointestinal Tract

before adolescence, but some remain asymptomatic until adult age. In 10% of newborns with CF, meconium ileus is the rst diagnostic sign. Later, the symptoms are related mainly to respiratory infections and intestinal malabsorption. The classical diagnostic sweat test for CF involves the biochemical detection of high levels (4100 mg ml 1) of sodium and chloride in a sweat sample collected following pilocarpine iontophoresis. The excessive loss of electrolytes may result in heat exhaustion in hot climates. Diagnosis can be conrmed by genetic testing of blood or buccal cells. The classic clinical symptoms of CF are summarized in Table 1.
Airway Infection and Inammation

The pathological features of the gastrointestinal tract are linked to reduced exocrine pancreatic functions and inadequate intestinal absorption. Neonatal meconium ileus, a diagnostic feature of CF, occurs only in 10% of cases. Distal intestinal obstruction syndrome (DIOS), constipation, megacolon, rectal prolapse, and pancreatic brosis develop later during adolescence. Pancreatic insufciency is present in approximately 90% of CF subjects. Pancreatic brosis leads to an increased incidence of diabetes mellitus. Diabetes affects B1% of children and 15% of adults with CF.
Hepatobiliary System

At birth, typically there are no significant signs of lung disorder in infants with CF. During early childhood, however, cough and thick mucus production, impaired mucociliary clearance, and high susceptibility to infections begin to manifest themselves. Chronic upper airway infections and the production of thick, viscous mucus result in sinus obstruction and nasal polyposis. Pulmonary infection and inammation in the CF lung are caused by surprisingly few bacterial pathogens. Pseudomonas aeruginosa is the most common isolate (80%), followed by Staphylococcus aureus (51%), Haemophilus inuenzae (17%), Methicillin-resistant Staphylococcusaureus (MRSA) (12%), and Stenotrophomonas maltophilia (11%). Viral infections with respiratory syncytial virus (RSV),
Table 1 Clinical features of cystic brosis Respiratory tract Chronic respiratory tract infection Recurrent wheeze Bronchiectasis Nasal polyposis Chronic sinusitis Cor pulmonale Hepatobiliary system Liver cirrhosis Genital tract Male infertility (90%, CBAVD, azoospermia) Reduced fertility in females (20%) Gastrointestinal tract Meconium ileus Steatorrhea Distal intestinal obstruction syndrome Vitamin deciency Failure to thrive Rectal prolapse Diabetes mellitus Electrolyte imbalance High sweat sodium and chloride Excessive salt loss in sweat Systemic electrolyte imbalance

Hepatic dysfunction increases with age and 1015% of adolescents and adults with CF have liver cirrhosis.
Genital Tract

Azoospermia is present in 95% of adult CF males as a result of CBAVD and consequential dilated or absent seminal vesicles. In women, there is reduced fertility that is caused by abnormally viscous intrauterine mucus.

Clinical Features
Diagnosis

CF cases represent a great variety of clinical symptoms that vary from patient to patient and by age groups. In the majority of cases, CF is diagnosed

604 CYSTIC FIBROSIS / Overview

inuenza, and adenovirus are also frequent in children with CF. Therefore, CF should be considered in any young child with persistent symptoms following infection with these pathogens. Chronic lower airway infections leading to brosis, bronchiectasis, respiratory insufciency, and cor pulmonale remain the hallmarks of morbidity in CF.
Gastrointestinal and Pancreatic Manifestations

CF patients suffer from gastrointestinal problems related to inadequately controlled intestinal absorption and pancreatic insufciency. These include meconium ileus in newborns, steatorrhea, DIOS, constipation and acquired megacolon, rectal prolapse, pancreatitis, and failure to thrive during adolescence.
Genital Manifestations, Fertility

depends on the function of the epithelia in which CFTR is expressed. In the apical surface of the secretory coils of sweat glands, CFTR controls Cl efux. Movement of Cl promotes H2O movement and hydration of the secretum. In contrast, in the ducts of the sweat glands, Cl is reabsorbed through CFTR. Because the secretory coil cells express either CFTR or other (non-CFTR, Ca2 -activated) anion channels, in the absence of CFTR, Cl can still be secreted in the secretory coils. However, Cl (and consequently Na ) reabsorption are decient because in the duct cells, CFTR is the only chloride channel able to reabsorb Cl (Figure 4). Pathogenesis of airway abnormalities in CF Because of the multiple physiological functions of CFTR, several models have been proposed to link CFTR mutations to the development of airway infection. Results from CF mice, raised under germfree conditions and from infants with CF studied by polymerase chain reaction (PCR) or other sensitive pathogen detection methods, suggest that inammation precedes infection. Not all studies, however, are in agreement with this hypothesis and support the idea that infection precedes inammation. Alterations in salt and uid secretion The development of infection in the CF lung may result from alterations in salt and uid secretion. Two models have been put forward to explain how uid and electrolyte secretion alterations may contribute to lung infections. Under physiological conditions, the function of CFTR in the apical membrane of airway epithelial cells and serous secretory cells is to secrete or reabsorb Cl (Figure 5(a)). Intracellular ion conditions are maintained mainly through the action of the basolateral sodiumpotassium pump and other ion transporters. Under basal conditions, both serous acinus cells and surface epithelial cells secrete Cl , Na , and H2O to hydrate the airway surface liquid (ASL). Physiological ASL volume and salt concentration is maintained through regulated Na and Cl reabsorption through ENaC and CFTR. The rst hypothesis, referred to as the low-volume hypothesis, suggests that in the absence of CFTR, ENaC is hyperactive, resulting in increased Na absorption from the periciliary uid layer. This causes increased water absorption from the airways and leads to isotonic, but diminished airway surface uid. As a result, the airway uid is poorly hydrated (Figure 5(b)). Infections follow because the bacteria are not cleared and become trapped in the viscous surface uid. A second model termed the high-salt hypothesis is based on the assumption that under normal

Ninety-ve per cent of adult CF males are infertile as a result of CBAVD and consequential dilated or absent seminal vesicles. Although B80% of adult female CF patients are fertile, reduced fertility may result from abnormally viscous intrauterine mucus, that is, o80% water in contrast to the normal 93 96% hydration that appears to be necessary for sperm migration.

Pathogenesis
Ion channels selectively expressed in the apical or basolateral membrane domains of epithelial cells regulate ion composition and hydration of secreted material. Discrepancies in the expression of these ion transporters engender impaired salt composition and aberrant hydration of the glandular uid. CFTR is a cAMP-regulated, bidirectional Cl channel primarily expressed in the apical membranes of epithelial cells in a variety of organs including the respiratory tract, gastrointestinal tract, exocrine secretory glands, kidneys, and bile ducts. Tissue specicity is known to be acquired both at the level of transcription and by expression of CFTR splice variants. Interestingly, CFTR mRNA, protein, and functional activity have also been demonstrated in T and B lymphocytes, red blood cells, and cardiomyocytes. Based on its broad expression pattern, it is apparent that CFTR has a central role in electrolyte transport regulation in a number of tissues and cell types. The current concepts regarding CF pathogenesis can be summarized based on the primary functions of CFTR.
CFTR as Ion Transporter

Pathogenesis of sweat abnormalities in CF The direction of chloride movement through CFTR

CYSTIC FIBROSIS / Overview 605

Skin surface TJ Duct (absorption) Cl Cl Na+ Na+ cAMP Cl

Na+ K+

K+

TJ Duct cell electrolyte reabsorption Secretory unit Cl Na+ TJ Cl Cl Na+ K+ K+ cAMP Na+ TJ TJ Secretory cells fluid and electrolyte secretion H2O TJ Cl Ca2+ Na+ CFTR

Na+

Cl Na+ K+ K+

Figure 4 CFTR function in sweat glands. A predominant function of CFTR in the secretory coils of sweat glands is to secrete Cl . In contrast, CFTR in the apical membrane of the sweat gland ducts primarily reabsorbs Cl . The secretory coils of sweat glands secrete electrolytes and uid that is regulated mainly through the action of basolateral (sodiumpotassium pump, Cl and K transporters) and apical (ENaC, CFTR, Ca2 activated) ion channels. Na and Cl are reabsorbed from the ducts of the sweat glands. Because the secretory coil cells express either CFTR or other (non-CFTR, Ca2 -activated) anion channels, in the absence of CFTR, Cl can still be secreted in the secretory coils. However, Cl (and consequently Na ) reabsorption is decient because in the duct cells, CFTR would be the only chloride channel able to reabsorb Cl . TJ, tight junction.

conditions, airway and submucosal gland epithelial cells behave in a similar way to sweat glands and absorb more Cl and Na than water, resulting in hypotonic airway surface liquid. In CF, CFTR is missing or functionally defective, and therefore Cl absorption is decreased, resulting in higher luminal Cl and Na concentrations than in healthy individuals (Figure 5(c)). This model is supported by the nding that defensins are inactivated under high salt conditions, and loss of defensin activity may predispose patients to bacterial infections. Distinguishing between these two possible models continues as an ongoing debate.

CFTR as Regulator of Other Transporters

CFTR as Receptor

Loss of the CFTR protein has also been linked to altered cell surface protein sialysation, resulting in increased asialo-GM1 molecule expression. This result has significance because asialo-GM1 is a receptor for a number of respiratory pathogens including P. aeruginosa and S. aureus. CFTR itself has been proposed as receptor for P. aeruginosa, suggesting that epithelial cells without CFTR are unable to bind, internalize, and clear this pathogen.

CFTR plays a central role not only in secretion of chloride and bicarbonate ions, but also regulates a number of other ion channels. These regulatory functions appear to be tissue-and cell type-specic, and reect the ion channel expression proles of the specic cells. CFTR has been shown to regulate Na reabsorption by inhibiting the activity of the epithelial Na channel, ENaC, suggesting that CFTR defects may lead to disturbances of both Na secretion and absorption. It has been proposed that the lung pathology in CF is primarily due to disregulation of sodium transport through ENaC. Supporting this hypothesis, transgenic mice with airway-specic overexpression of ENaC (i.e., increased Na reabsorption), but normal CFTR levels, have recently been shown to develop mucus obstruction, goblet cell metaplasia, neutrophilic inltration, and poor bacterial clearance, that are the hallmarks of CF lung disease. In addition to the evidence that CFTR conducts HCO3 , it also regulates HCO3 transport through the HCO3 /Cl exchanger. In the intestinal lumen and pancreatic ducts, secretion of uid containing high concentrations (100140 mM) of HCO3 is

606 CYSTIC FIBROSIS / Overview

Cl

Na + Normal airway submucosal gland ASL

H2O Na + Cl

Cl cAMP Ciliated, surface cells Mucussecreting cells Na+ 2Cl K + Na + Serous secretory cells CFTR

Ca 2 + K+

(a)

Na + CF airway submucosal gland and surface epithelial cells

Na +

X
Low ASL Cl cAMP Na+ 2Cl K + Na +

Ciliated, surface cells Mucussecreting cells (b)

Serous secretory cells

Na + CF airway submucosal gland and surface epithelial cells NaCl NaCl NaCl NaCl ASL

Na +

X
Cl cAMP Na+

NaCl Mucussecreting cells (c)

Ciliated, surface cells 2Cl K + Na +

Serous secretory cells

Figure 5 (a) CFTR function in the airways. The function of CFTR in the apical membrane of airway epithelial cells and serous secretory cells is to secrete or reabsorb Cl . Intracellular ion conditions are maintained mainly through the action of the basolateral sodium potassium pump and other ion transporters. Under basal conditions, both serous acinus cells and surface epithelial cells secrete Cl , Na , and H2O to hydrate the airway surface liquid (ASL). Physiological ASL volume and salt concentration is maintained through regulated Na and Cl reabsorption through ENaC and CFTR. The ASL is cleared by mucociliary clearance and evaporated. (b) Lowvolume hypothesis. The low-volume airway surface liquid (ASL) hypothesis argues that both the airway surface cells and serous secretory cells of the acini secrete Cl . In the absence of CFTR, Cl secretion is diminished; ENaC is hyperactive and absorbs more Na than under physiological conditions. This results in increased salt and water reabsorption and loss of ASL uid volume. In CF, the surface epithelial cells are attened and the ducts of the submucosal glands are plugged with viscous mucus. (c) High-salt hypothesis. The highsalt hypothesis argues that CFTR is the predominant pathway for Cl reabsorption from the airways and in the absence of CFTR, Cl cannot be absorbed from the airways. However, Cl can be secreted through alternative Cl channels resulting in hyperpolarization of the luminal surface. As a consequence, Na reabsorption is inefcient without a counter ion and NaCl accumulates in the ASL.

S
K+ H2O H2O Cl Ca 2 + K+ K+ Cl H2O Cl Ca 2 + K+

S
K+

CYSTIC FIBROSIS / Overview 607

necessary to maintain the solubility of mucins and the inactive state of digestive enzymes. The majority of bicarbonate transport in these organs is achieved through an epithelial Cl /HCO3 exchanger, regulated by CFTR. This particular CFTR function is therefore likely to have important physiological implications, and may help explain the severely im paired HCO3 secretion in CF. Chloride channels other than CFTR, including Ca2 , activated and outwardly rectied anion channels require the presence of CFTR in the plasma membrane. Loss of CFTR has profound effects on chloride transport both directly and indirectly through these pathways, compounding the defects in epithelial chloride transport that result from loss of CFTR. Another important cellular effect of CFTR is volume regulation through the Ca2 -dependent potassium channel, KCNN4. During regulatory volume decrease (RVD), anion and cation channels are activated, permitting the passive loss of inorganic ions and osmotically obliged water. In a CFTR knockout mouse model, cell volume regulation in jejunal crypts is decient as a consequence of dysfunctional KCNN4. The activity of other K channels such as ROMK2 and KvLQT1 have also been shown to be modulated by CFTR expression. To make matters worse, proper cell volume through aquaporin 3 also appears to be dependent on CFTR. Finally, the activity of gap junction channels is affected by functional CFTR expression, indicating that CFTR plays a significant role in maintaining electrolyte transport not only across individual cells, but also across the epithelial monolayer as a whole.
CFTR and Gene Expression Regulation

observed in CF patients. Although the complete list of inammatory genes affected by CFTR expression in different tissues remains incomplete, this area of research may explain the multifaceted nature of CF lung disease and will remains an important aspect for future studies.

Animal Models
Important structural and functional characteristics of CFTR have been discovered using cell culture models. Despite the progress made, animal models are necessary to investigate the pathogenesis of CF and evaluate novel therapeutic strategies. After cloning the CF gene in 1989, a novel approach was applied to generate mouse strains with mutations in the mouse Cftr gene. Mutations to specic sites in the mouse genome were targeted using homologous recombination in mouse embryonic stem cells. The mutant embryonic stem cells were then injected into blastocysts to create chimeric mice. Chimeric mice were bred to obtain individuals homozygous for the null mutation. Several different CF mouse strains were developed using this approach. A comprehensive list of models can be found in the Virtual Repository of Cystic Fibrosis European Network.
Gene Targeted (Knockout or KO) and Residual Function CF Mouse Models

Because of the difculties that have been experienced in attempting to correlate CF genotype and pulmonary disease severity, alternative functions of CFTR remain of considerable interest. One of the hallmarks of CF is enhanced expression of proinammatory mediators in CF lungs. For example, reduced Smad3 expression has been reported to selectively alter TGFb1-mediated signaling in CF epithelium, and CF epithelial cells may be functionally decient in IL10, a key anti-inammatory cytokine that controls expression of the inhibitory subunit, IkB. The presence of cell surface CFTR has also been shown to be necessary for RANTES (regulated upon activation, normal T-cell expressed, and presumably secreted) expression, a chemokine that selectively inuences the migration of monocytes, CD45 RO memory T lymphocytes, and eosinophils. Furthermore, CF airway epithelial cells fail to express RANTES in response to P. aeruginosa, the predominant bacterium

Gene targeted (KO) mouse models were created using a method called replacement strategy. An interruption that was introduced to the mouse Cftr gene resulted in complete loss of CFTR mRNA and protein. Residual function models were created using the insertion strategy. These models, despite the interruption in the Cftr genome may produce some CFTR mRNA and demonstrate minimal residual CFTR activity. Although these models represented an important step in CF research, it was apparent that the articial mutations would not accurately model naturally occurring mutations.
Mouse Models with Naturally Occurring, DiseaseCausing Mutations in the Mouse Cftr Genome

New mouse strains with naturally occurring mutations, most importantly those with DF508, G480C, (Class II), and G551D (Class III) mutations, have been developed. These models were created either by the exon 10 replacement strategy or by the hit and run procedure. The exon replacement strategy uses a selection marker inserted into one of the introns that regulates the activity of the transcription of the mutant gene. The hit and run method results in a

608 CYSTIC FIBROSIS / Overview

mutant exon only and the transcription and activity of the mutant allele is therefore identical to the normal allele. It has been shown that the DF508 mutation in the mouse Cftr sequence caused a similar processing defect as in the human protein.
Transgenic Mouse Models Expressing Human CFTR

have already been used to test new therapeutic interventions.

Other Animal Models

Transgenic mouse models were constructed on a knockout background with a null mutation in the endogenous CFTR locus (Cftr / ). First, the human wild-type CFTR transgene was expressed in the intestinal tract under the control of the fatty-acidbinding protein (FABP) promoter from rat. These mice have an improved survival rate compared to the null mutants. Using a similar approach, a second transgenic mouse model was developed expressing human CFTR carrying the G542X mutation. These mice have been used to study the effects of different compounds on the suppression of this premature stop mutation.
Differences in the Phenotypic Properties of CF Mouse Models and Human CF

Sheep, pig, and ferret models are being considered because the structure of their lungs resembles the human lung more. These models may become more important in the future for testing new therapeutic interventions.

Therapy
Traditionally, CF treatments have focused on ameliorating symptoms of intestinal obstruction, malnutrition, airway obstruction, infection, and inammation. Pancreatic enzyme replacement decreased the severity of these symptoms significantly, resulting in improved survival, and increased the importance of ambulatory care. Increased understanding of CF pathophysiology and genetic defects provide a conceptual basis for targeted drug development. New therapeutic approaches fall into two broad categories: gene replacement and drug development (CFTR repair). In theory, the gene replacement therapies could be applied to all mutations, whereas drugs target specic mutations. Compounds intended for CFTR repair are being screened to rescue DF508 from the ER quality control (Class II mutants), to stimulate defective channels at the cell surface such as G551D (Class III mutants), or to promote readthrough of premature stopcodons for mutants such as W1282X (Class I mutants).

Because a number of laboratories have created CF mouse models, it is important to note that although the phenotypic hallmarks are the same, important differences between models with the same mutation have been observed. These variations relate to the genetic background and a number of other factors. In general, intestinal disease is the most prominent feature of CF mice and the symptoms are comparable to CF in humans. Because of the severity of intestinal obstruction, CF mice are kept on a liquid diet to improve survival. The pancreatic involvement in CF mice is less severe than in humans. The most striking differences regarding the respiratory defect between the mouse models and human CF were described in studies. Initial characterization of CF mice showed no significant lung disease. Later, pulmonary abnormalities were described in mice bred on a different genetic background, suggesting the presence of genetic modiers. However, despite extensive studies, an appropriate model for CF lung disease in CF mice has proved challenging. Pathological changes in the lungs of CF mice resemble pulmonary brosis rather than abnormalities seen in human patients with CF. It appears that there are additional Cl transporters in mouse lungs that make modeling of the human disease incomplete. Interestingly, infection of CF mice with Pseudomonas promotes some changes consistent with CF. Although the suitability of mouse models for CF lung disease remains controversial, some of the models

Preventive and Symptomatic Treatments

The increased life expectancy of CF patients is a direct result of the improved preventive care and antibiotic treatments against respiratory pathogens. A variety of general and transmission-based, CFspecic infection control guidelines are now being implemented. General precautions focus on the potentially infectious nature of body uids, particularly respiratory tract secretions. Transmission-based precautions, on the other hand, prevent transmission of multidrug-resistant organisms such as Burkholderia cepacia, P. aeruginosa, or viruses such as RSV, inuenza, and adenovirus by employing procedures such as maintaining sterilized respiratory equipment, segregating patients, and vaccinating young CF patients. These measures have decreased the severity of lung disease and resulted in a dramatic improvement in the patients health status.
Antibiotic Treatments

S. aureus and H. inuenzae are often the rst agents to infect patients with CF. In the short term,

CYSTIC FIBROSIS / Overview 609

treatment of these pathogens with oral antibiotics is typically effective. Later in the course of the disease, P. aeruginosa (particularly the mucoid strains) become predominant. Mucoid P. aeruginosa is remarkably resistant to even intensive antibiotic regimens in part because antibiotics fail to penetrate bacterial biolms. A specifically formulated tobramycin (TOBI) solution developed for use in inhalers provides a high-dose aminoglycoside antibiotic in the lungs that is effective against P. aeruginosa. TOBI has been shown to increase lung function in CF patients, with less toxicity compared to systemic aminoglycoside treatments. Macrolide antibiotics are well tolerated by CF patients. One member of this class, azithromycin, exhibits broad antibacterial activity and has also been shown to decrease neutrophil chemotaxis, inhibit the expression of proinammatory cytokines, and interfere with biolm formation by P. aeruginosa. It is clear that the use of combined antibiotic therapy during acute exacerbations significantly reduces the development of resistant organisms and chronic airway infections. To date, however, no consensus has been reached regarding the possible benet of periodic antibiotic administration in the absence of overt pulmonary exacerbation. No significant advantage of this type of treatment (compared to symptomatic antibiotic therapy) has been shown for this type of therapy.
Anti-Inammatory Therapy

intrapulmonary percussive ventilator (IPV) to help loosen thick, mucous plugs within the airways. Similarly, aerosolized medicines such as albuterol, ipratropium bromide, and Pulmozymes (dornase alfa) are used to solubilize and break up CF airway secretions.
Lung Transplantation

Lung or heartlung transplantation is an important option for CF patients with severe lung disease. Based on present criteria, patients are considered for transplant waiting lists when the forced expiratory volume in s (forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC)) falls below 30%. While precedence should be given to patients with the most severe symptoms, due to the long waiting period for a transplant, patients should be evaluated well before FEV1/FVC falls below 30%. Contraindications for transplantation include ventilator dependence, drugresistant bacterial infection, and poor overall health/ nutritional status. Based on recent reports, this treatment increases the 5-year survival rate from o30% to 66% and perhaps more importantly, dramatically improves quality of life.

Treating the Fundamental CF Defect Prospects for CF Interventions

Gene Therapy

Both steroid and nonsteroid anti-inammatory drugs have been used in CF treatments with limited benets. Although treatment with steroids may improve function, they also have a number of deleterious side effects (growth retardation, glucose intolerance, and cataract development). High doses of nonsteroidal anti-inammatory drugs such as ibuprofen have also been used to treat CF lung disease and have been shown to reduce the decline in lung function. Based on altered pharmacokinetics of ibuprofen in CF patients and the potential adverse effects, however, the widespread use of anti-inammatory agents of this sort has been limited.
Physiotherapy and Mucolytic Treatment

Since the discovery of the CFTR gene in 1989, gene replacement strategies have been sought using both nonviral and viral vectors. Proof-of-principle in the lung has been established and progress achieved in understanding the barriers preventing efcient gene transfer to surface airway epithelium. A number of gene therapy clinical trials have been completed, although none with positive, long-term effects. Newly developed adeno-associated and lenti-viral vectors provide continuing hope for gene therapy-based CF interventions in the future.
Translational Readthrough, Rescue Agents, and Channel Modulators

A major goal regarding CF therapy is to prevent the development of pulmonary hypertension and consequent cor pulmonale. Daily chest physiotherapy and aerosol inhalation are often used to improve oxygen delivery. A typical physiotherapy session involves either manual chest percussion (pounding), or use of a device such as the ThAIRapy vest or the

Treatment of premature stop mutations with aminoglycoside antibiotics is based on the nding that these agents promote translational readthrough. Although clinical results from using this strategy have been promising, mutations that are treatable with this approach (such as W1284X) are rare. Agents that rescue the common DF508 mutant from the ER quality control are of particular interest. A number of laboratories and pharmaceutical and academic centers

610 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

have launched high throughput screening programs to identify compounds that correct CFTR folding. Similar studies concentrating on modulators of dysfunctional CFTR mutants such as G551D are being tested in preclinical and clinical trials. It is likely that a combination of several of these approaches will be required to overcome the CFTR defects. For example, treatment of patients with DF508 CFTR may require both, rescue of the protein from ERAD and methods or agents that activate the channel and stabilize the protein at the apical surface. The past few years have provided significant advancements in our understanding of the CFTR protein, and future prospects for alleviating many of the symptoms of CF are at hand. Establishing new interventions based on molecular understanding of CF defects will require further insights regarding CFTR regulation, processing, and proteinprotein interactions. Having said this, the rapid progress over the past few years regarding CFTR structure and function suggests the possibility of a number of promising therapies in the near future.
See also: Bronchiectasis. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defensins. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Mucins. Mucus. Oxygen Therapy.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

R G Crystal and R G Pergolizzi, Weill Medical College of Cornell University, New York, NY, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Cystic brosis (CF), the most common autosomal recessive disorder in Caucasians, primarily manifests in epithelial cells in the gastrointestinal system, airways, reproductive system, and sweat glands. CF is caused by mutations in the cystic brosis transmembrane conductance regulator (CFTR) gene, a 230 kb, 29 exon gene on chromosome 7q31.2. The 480 amino acid singlechain CFTR protein functions as a cyclic AMP-regulated chloride channel on the surface epithelium. Over 1000 different CFTR mutations have been identied and although there has been some success correlating CFTR genotype and the severity of CF disease, especially with regard to pancreatic disease, many questions remain unresolved. CF pulmonary disease is highly variable among individuals with the same genotype, most likely arising from the presence of modier genes and environmental factors. This article summarizes the structure and function of the CFTR gene and the protein, the current classication of CFTR mutations, and the relationship of those classes to CF disease phenotype.

Introduction
Cystic brosis (CF) is the most common autosomal recessive disorder in Caucasians, with a frequency of about 1 in 2500 live births. The disease is caused by mutations in the cystic brosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel in the apical membrane of epithelial cells. The CF phenotype is characterized by pulmonary, pancreatic, gastrointestinal, and reproductive abnormalities. The greatest impact of mutations of the CFTR gene is on pulmonary disease, accounting for most morbidity and deaths. The relationship between specic mutations in CFTR and severity of CF is very complex, leading to broad variability in symptoms of disease. Improved treatment modalities for CF have improved life expectancy for patients over the last two decades. Data from the US registry show that median age at death in patients with CF has doubled in the last 20 years to mid-30s in 2005. The major clinical phenotype of CF is poor mucociliary clearance with chronic airway bacterial infection, excessive mucus secretion, and obstructive lung disease. Most affected individuals (495%) have pancreatic insufciency, and a high level of male infertility results from the absence or obstruction of the vas deferens. Infants with CF are likely to present with meconium ileus, obstruction of the bowel with inspissated secretions.

Further Reading
Kirk KL and Dawson DC (2003) The Cystic Fibrosis Transmembrane Conductance Regulator. New York: Kluwer Academic/ Plenum. Riordan JR (2005) Assembly of functional CFTR chloride channels. Annual Review of Physiology 67: 29.129.18. Welsh MJ, Ramsey B, Accurso FJ, and Cutting GR (2001) Cystic brosis. In: Scriver CR, Beaudet AL, Sly WS, et al. (eds.) The Metabolic and Molecular Bases of Inherited Disease, 8th edn., pp. 51215188. New York: McGraw-Hill. Yankaskas JR, Marshall BC, Suan B, Simon RH, and Rodman D (2004) Cystic Fibrosis Adult Care, Consensus Conference Report. Chest 125: 1S39S.

Relevant Websites
http://www.genet.sickkids.on.ca This is a database of mutations in the CFTR gene and is currently maintained by the laboratory of Lap-Chee Tsui. http://central.igc.gulbenkian.pt This is a database maintained by the laboratory of Dr Margarida Amaral that provides information on CFTR expression, function, and detection. http://www.cff.org This is a patient registry that was started by the CF Foundation to track the condition of patients with CF in the US. The registry is updated yearly by the CF Foundation. http://www.sciencedirect.com The cited issue of the Journal of Cystic Fibrosis summarizes the consensus work of the European Cystic Fibrosis Network and provides detailed information about CFTR structure, function, genetics, and disease models.

CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene 611

In this article we summarize the available information regarding the advances in understanding the CFTR gene and its mutations, and the effects these have on CFTR function and the clinical CF phenotype.

CFTR Structure and Organization

The human CFTR gene occupies 230 kb on the long (q) arm of human chromosome 7 at region q31.2, encoding an mRNA transcript of 6129 bp and a single-chain 480 amino acid polypeptide, which functions as a chloride channel in membranes of epithelial cells of the lung, liver, pancreas, intestines, reproductive tract, and sweat glands. The CFTR protein is made up of ve domains: two transmembrane domains (MSD1 and MSD2) that form the chloride ion channel, two nucleotide-binding domains containing several phosphorylation sites (NBD1 and NBD2) that bind and hydrolyze ATP, and a regulatory (R) domain (Figure 1). Expression of the CFTR protein confers cyclic adenosine monophosphate (cAMP)-regulated chloride conductance in nonepithelial cells, and reconstitution of puried CFTR protein into lipid bilayers generates cAMPactivated chloride channels with properties similar to those in CFTR-expressing cells.

of CFTR channels is linked to ATP hydrolysis. CFTR gating activity is acutely modied by phosphorylation. Modulation of gating activity of the CFTR chloride channel is regulated by kinases, especially protein kinase A (PKA), and phosphatases. However, the impact of PKA on CFTR gating activity is not an allor-nothing process, since biochemical measurements show that PKA readily phosphorylates the CFTR to a stoichiometry of at least ve ATPs per CFTR molecule, and incremental changes in gating appear with each additional event. Most of the residues in CFTR that become phosphorylated are located in the large cytoplasmic R domain. Deletion of the R domain renders CFTR constitutively active in the absence of phosphorylation. Therefore, it is likely that the R domain plays a role in regulating the gating activity of CFTR and that this regulation depends on the phosphorylation state of this domain. Potential phosphorylation sites outside the R domain have also been identied, and one in NBD1 at G551 has been linked to particularly severe symptoms of CF. Adenine nucleotides, magnesium ion concentration, and inorganic phosphate also impact the rate and duration of opening of the chloride channel.

CFTR Mutations
Over 1000 mutations in the CFTR gene have been described. There is variability in why these mutations cause CF, but all result in a loss of chloride ion transport, which upsets the sodium and chloride ion

CFTR Activity and Regulation

The CFTR protein itself comprises a metabolically gated chloride ion pore, and the opening and closing

MSD1

MSD2

Cell membrane

N P

P P NBD2

R domain P

NBD1 ATP ATP ADP

Figure 1 Schematic of the CFTR protein with respect to the epithelial cell membrane. The major domains of CFTR are shown, including the membrane-spanning domains (MSD1 and 2), nucleotide-binding domains 1 and 2 (NBD1 and 2), and the regulatory (R) domain. The N-terminal and C-terminal regions are indicated. MSD2 has asparagine N-glycosylation sites. The NBDs utilize adenosine triphosphate (ATP), converting it to adenosine diphosphate (ADP). The R domain has several phosphorylation sites. The membranespanning domains form the chloride channel in the cell membrane.

ADP

612 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene
Table 1 Cellular phenotype of CFTR mutations Class I II III IV V VI Affected Biosynthesis Maturation Cl gating Cl conductance Splicing Protein stability Location in CFTR protein Various Various NBDs Intramembrane domains Exons 8 and 12 C-terminus Examples G542X, R553X F508del, S945L, H949Y, D979A G551D R117H, G622D, M1137V, I1139V, DM1140, D1152H, and D1154G A455E, Exon 8 Tn, 1717-9T, C-D565G Q1412X, 4326delTC, 4279insA, 4271delC

balance needed to maintain the normal mucus layer lining the lung and other organs. In the lung, the ion imbalance leads to the formation of a thick mucus layer that traps bacteria, resulting in chronic infections. In the gastrointestinal and reproductive tracts, the thick mucus blocks organ function, while in the skin, the sweat glands produce sweat with high sodium ion concentrations. The mutations of the CFTR gene are grouped into functional classes; ve classes are universally agreed upon, and a proposed sixth class relating to the possible impact of the mutation on biosynthesis is controversial (Table 1). Class I relates to protein synthesis, class II to protein maturation, class III to chloride channel regulation and gating, class IV to chloride conductance, class V to CFTR mRNA splicing, class VI to protein stability, and class VII to effects on the regulation of other channels. Although attempts to correlate specic CFTR mutations with severity of CF disease have not been completely successful, mutants dened as classes I to III tend to have severe CF symptoms, whereas classes IV and V are linked to milder manifestations of CF disease. The most common CFTR mutation worldwide is a deletion of phenylalanine at position 508 (F508del) causing problems in maturation and trafcking of the protein to the cell surface. There is variability in the occurrence of this mutation among ethnic groups. F508del occurs in the DNA sequence that codes for the rst nucleotide-binding domain (NBD1). Homozygotes for F508del tend to have the most severe symptoms of CF. Other mutations are found throughout the entire promoter and coding regions of the CFTR gene. Mutations are particularly common in the NBD1, NBD2, and R domains, which is probably related to the importance of proper function of these domains. Twelve CFTR mutations, including 17171G4A, G542X, W1282X, N1303K, F508del, 3849 10kbC4T, 621 1G4T, R553X, G551D, R117H, R1162X, and R334W account for over 90% of all CF patients. There is also a genetic element modulating posttranscriptional processing of CFTR mRNA

transcripts. There is a polymorphic string of thymidines at the end of intron 8 of the CFTR gene, with three different alleles identied based on the number of thymidines (T5, T7, or T9) present at this locus. It has been shown that the shorter the poly(T) tract, the greater the relative amount of exon 9-CFTR mRNA transcripts present in respiratory epithelium. Since exon 9 encodes part of the important rst nucleotidebinding domain, the resulting CFTR protein is inactive. A polymorphic TG repeat adjacent to the thymidine repeat region also inuences the CF phenotype, with individuals carrying 12 or 13 TG repeats being more likely to exhibit an abnormal phenotype than those with 11 repeats.
Class I Mutations Affecting Biosynthesis (No CFTR Synthesis)

Class I mutations lead to reduced production of CFTR mRNA. This class includes the most severe CF phenotypes due to the total lack of CFTR protein. In those cases where truncated proteins are produced, they are usually unstable, and are rapidly degraded in the endoplasmic reticulum (ER). The most common examples of class I mutations are G542X and R553X.
Class II Mutations Affecting Protein Maturation (Block in CFTR Processing)

In class II mutations, such as F508del, CFTR is trapped in the endoplasmic reticulum and subsequently proteolytically degraded. Small amounts of F508del CFTR might reach the apical domain of respiratory epithelial cells in patients with cystic brosis, suggesting that defective CFTR maturation might still be rescued after passage through the endoplasmic reticulum. CFTR chaperones such as phenylbutyrate, 8-cyclopentyl-1,3-dipropylxanthine (CPX), and genistein that can force functional protein to the cell membrane, increase the efciency of protein folding, or suppress protein degradation could provide therapy for class II mutants. Other examples of class II mutations are S945L, H949Y, and D979A.

CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene 613 Class III Mutations Affecting Chloride Channel Regulation/Gating (Block in Regulation)

Class III mutants produce a CFTR protein that gets transported to the cell membrane but does not respond to cAMP stimulation. All mutations so far attributed to this class are located within the NBDs, and probably act by preventing conformational change. Examples of class III mutations include G551D.
Class IV Mutations Affecting Chloride Conductance (Altered Conductance)

CFTR-associated ligand, CAP70, and Shank2. These interactions regulate CFTR maturation, oligomeric state, channel gating, and phosphorylation state. Phosphorylation of the R domain stimulates CFTR channel activity and is regulated by protein kinases A and C (PKA and PKC).

Genotype/Phenotype Correlation
The presence of particular CFTR genotypes (classes IIII) has been shown to correlate with pancreatic insufciency, liver disease, early onset, high sweat chloride levels, and meconium ileus. However, it has not been possible to closely correlate specic CFTR mutations with the severity of lung disease, although the fraction of normal CFTR mRNA detected in splicing mutants Exon 8-5T and 3849 10kbC4T correlates directly with the penetrance and severity of lung disease. Unlike lung disease, the severity of pancreatic insufciency in CF can be correlated with specic CFTR mutations. R117H, R334W, and R347P are associated with mild pancreatic disease, and individuals carrying two severe alleles will present with pancreatic insufciency. The environment and modier genes may significantly alter the progression and severity of CF. Secretion of electrolytes and water by the epididymal epithelium is critical for sperm maturation and transport. Only 23% of male CF patients are fertile, with the absence of vas deferens occurring in essentially all patients. There is also a reduction in fertility in female CF patients, although not to the extent seen in males. Menstrual irregularity and oligomenorrhea coupled with the presence of a plug of thick mucus in the cervix contribute to reduced fertility in female CF patients. Congenital bilateral absence of the vas deferens (CBAVD) patients may carry CFTR mutations without exhibiting other aspects of the CF phenotype. CBAVD prevents transport of spermatozoa from the testicles or epididymis to the vas deferens, resulting in infertility. It has been suggested that a combination of particular alleles at several polymorphic loci affects the quantity/quality of CFTR, resulting in a partial phenotype like CBAVD in which 70% of patients carry one CFTR mutation and 10% carry two. A mild CFTR mutation may then become a diseasecausing mutation in the vas deferens of CBAVD patients due to the overall decrease in full-length CFTR mRNA. However, CBAVD patients show no evidence of pulmonary disease, suggesting a possible CFTR mRNA transcript threshold level for appearance of pulmonary manifestations of CF. It is clear that CFTR genotype alone does not account for the diverse spectrum of disease observed

This group includes cases where the CFTR protein is correctly trafcked to the cell membrane and responds to cAMP stimulation but the resulting chloride ion current is reduced. Examples of class IV mutations are R117H, M1137V, I1139V, M1140del, D1152H, and D1154G. Many of the class IV mutations are located within the intramembrane regions of the CFTR protein, particularly within transmembrane helix 12. Class IV mutants tend to have milder forms of CF.
Class V Mutations Affecting CFTR Splicing

Class V mutations affect splicing of the mRNA resulting in exon skipping or extra-cryptic exons. Both mRNA and protein stability may be affected by these mutations. Examples of this class include the exon 8 thymidine polymorphism (discussed above) and 1717-9 T C-D565G in exon 12.
Other CFTR Mutations

It has been suggested that mutations affecting protein stability that result in a short lifetime for the CFTR protein should be placed in class VI. Examples of class VI mutants involve frameshifts leading to terminal truncations, including Q1412X 4326delTC, 4279insA, and 4271delC. Although the CFTR C-terminal domain is not critical for essential CFTR functions, it appears to be required for maintaining the stability of the protein.

Regulation of CFTR by Other Proteins

CFTR is associated with other membrane proteins in a regulatory complex, which includes other transport proteins such as the epithelial sodium channel. The N-terminus of CFTR binds to the N-terminus of syntaxin 1, a vesicle fusion protein found at neuronal synapses and airway epithelium. Binding of CFTR with syntaxin 1 interferes with CFTR channel activity. The DTRL motif at the C-terminus of CFTR binds to scaffolding/adaptor proteins such as EBP50 (a sodium hydrogen exchange regulatory factor),

614 CYSTIC FIBROSIS / Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

for the CF pulmonary phenotype. Secondary genetic factors distinct from CFTR have been identied that may inuence the severity of CF lung symptoms. These proposed modier genes include inammatory and anti-inammatory molecules, antioxidants, molecules that inuence airway reactivity, molecules capable of inducing alterations in CFTR trafcking, and alternative ion channels. It has been suggested that particular alleles at several polymorphic loci might cooperate, resulting in an overall decrease in the function of the CFTR protein. The subtle contribution of polymorphic alleles could explain why apparently individually normal CFTR alleles cause disease and might be responsible for variation in the phenotypic expression of CFTR. An example of this can be seen in the cooperation of two mild mutations, R347H and D979A. R347H alone leads to somewhat decreased Cl channel activity, and D979A alone causes misprocessing of CFTR. However, an individual carrying both mutations showed a severe decrease in Cl channel activity.

circumvent the abnormalities caused by mutations in the CFTR gene promise to yield major improvements in the treatment of CF. One of the most attractive approaches to CF is the augmentation of the existing genetic complement of airway epithelial cells with a normal CFTR transgene. The possibility for CFTR gene transfer was quickly established in vitro and in animal models after cloning of the gene in 1989. The rst gene therapy clinical trials in CF patients were carried out in 1993, and 29 clinical trials have been published as of 2004. Repeat administration of CFTR adenoviruses, adeno-associated virus, and nonviral strategies have been shown to be safe to humans. Progress has been made in overcoming the problems associated with gene transfer to airway epithelial cells but difculties remain in the establishment of long-term expression of CFTR in vivo.

Acknowledgments
We thank N Mohamed for help in preparing this manuscript. These studies were supported in part by NIH P01 HL51746 and the Will Rogers Memorial Fund, Los Angeles, CA, USA.
See also: Bronchiectasis. Cystic Fibrosis: Overview. Ion Transport: Calcium Channels; Chloride Channels.

Associated Disorders
Disseminated bronchiectasis is an obstructive pulmonary disorder involving structural abnormalities often associated with childhood lung infections and immunodeciencies. CFTR mutations may contribute to the phenotype in non-CF obstructive pulmonary diseases such as disseminated bronchiectasis. In two studies of 16 and 32 patients, respectively, the frequency of the T5 allele was significantly increased over controls and 13 CFTR mutations were detected. Two recent studies of 39 and 20 idiopathic chronic pancreatitis patients showed that at least 30% of patients carried CFTR mutations, demonstrating that CFTR mutations are associated with this disorder. Chronic rhinosinusitis, a chronic inammation of the sinus epithelium, is also associated with CF. Individuals with chronic rhinosinusitis are three times more likely to carry CFTR mutations than controls. Asthma has been suggested to be more common in association with CFTR mutations, but these observations have not been found to be statistically significant. Pancreatitis and atrophy of pancreatic acinar tissue has also been correlated with an increased incidence of CFTR mutations.

Further Reading
Chiba-Falek O, Kerem E, Shoshani T, et al. (1998) The molecular basis of disease variability among cystic brosis patients carrying the 3849 10kb c4 T mutation. Genomics 53: 276283. Chu CS, Trapnell BC, Curristin S, Cutting GR, and Crystal RG (1993) Genetic basis of variable exon 9 skipping in cystic brosis transmembrane conductance regulator mRNA. Nature Genetics 3: 151156. Crystal RG, McElvaney NG, Rosenfeld MA, et al. (1994) Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic brosis. Nature Genetics 8: 4251. Dalemans W, Barbry P, Champigny G, et al. (1991) Altered C1channel kinetics associated with the major (F508) cystic brosis mutation. Nature 354: 526528. Dorwart M, Thibodeau P, and Thomas P (2004) Cystic brosis: recent structural insights. Journal of Cystic Fibrosis 3(supplement 2): 9194. Harvey BG, Leopold PL, Hackett NR, et al. (1999) Airway epithelial CFTR mRNA expression in cystic brosis patients after repetitive administration of a recombinant adenovirus. Journal of Clinical Investigation 104(9): 12451255. Kerem B and Kerem E (1996) The molecular basis for disease variability in cystic brosis. European Journal of Human Genetics 4: 6573. McKone EF, Emerson SS, Edwards KL, and Aitken ML (2003) Effect of genotype on phenotype and mortality in cystic brosis: a retrospective cohort study. The Lancet 361(17): 16711676. Ratjen F and Do ring G (2003) Cystic brosis. The Lancet 361: 681689.

New Therapies
Although there have been improvements in the understanding of the genetic basis and management of CF in the last decade, there is as yet no cure. However, additional study of the structure and function of CFTR and the development of new drugs to

CYTOSKELETAL PROTEINS 615

Rave-Harel N, Kerem E, Nissim-Rafinia M, et al. (1997) The molecular basis of partial penetrance of splicing mutations in cystic brosis. American Journal of Human Genetics 60: 8794. Riordan JR, Rommens JM, Kerem B, et al. (1989) Identication of the cystic brosis gene: cloning and characterization of complementary DNA. Science 245: 10661073. Vankeerberghen A, Cuppens H, and Cassiman JJ (2002) The cystic brosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions. Journal of Cystic Fibrosis 1: 1329. Welsh MJ, Ramrey BW, Accurso F, et al. (2001) Cystic brosis. In: Scriver CR, Sly WS, Childs B, et al. (eds.) The Metabolic and Molecular Bases of Inherited Disease, 8th edn, pp. 51215189. New York: McGraw Hill.

CYTOSKELETAL PROTEINS
A D Verin, Johns Hopkins University School of Medicine, Baltimore, MD, USA N V Bogatcheva, Baylor College of Medicine, Houston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The cytoskeleton represents the intricate inner carcass that stabilizes the cell by a network of three major components: microlaments, microtubules, and intermediate laments. This network is anchored to the membrane by highly specialized protein complexes, providing both cell attachment to other cells or extracellular matrices, as well as stabilization and dynamic rearrangement of cell-surface structures. Not only does the cytoskeleton maintain cell shape but it also manages compartmentalization and proper orientation of many biochemical processes including signal transduction. Cytoskeletal remodeling is critical for cell contraction, motility, and morphogenesis; in addition, the cytoskeleton serves as a cellular mechanosensor and mechanotransducer. Signals originating from cytoskeletal elements control gene expression, cell differentiation, proliferation, and survival. The vast majority of physiological responses, such as leukocyte migration and phagocytic activity, the ability of broblasts to heal wounds, angiogenesis, control over vascular/bronchial tone, and regulation of endothelial/epithelial barrier function depend upon cytoskeletal proteins, involved in the organization of intercellular contacts, and contractile proteins, inducing tension. Maintenance of barrier function is of particular importance in lung physiology, since an increase in alveolar capillary permeability and resultant edema represent the underlying causes of lung damage characterized as acute lung injury or acute respiratory distress syndrome. Different therapeutic strategies counteracting hyperpermeability are currently under development; some of which target signaling pathways, leading to cytoskeletal rearrangement.

providing bonds between laments of the same or different types and attachment to the nuclear or plasma membranes and cell organelles. The current hypothesis holds that living cells organize their cytoskeleton by the principle of tensional integrity architecture. Such structure gains stability from continuous inward-directed tension counterbalanced by the resistance of elements, which either provide peripheral attachment or cannot be compressed. Thus, cell shape is determined by the net effect of the pull of contractile microlaments and the adhesive tethering and rigidity of microtubules. The lattice of intermediate laments stiffens the whole cell including the cell nuclear compartment to render protection from both centrifugal and centripetal forces. This interplay between cytoskeletal elements may result in cell spreading or contraction, development of asymmetrical shape, or cell migration. Organelle and vesicle intracellular motility is also governed by cytoskeletal rearrangement. Remodeling of specic cytoskeletal structures, especially those associated with the membrane, initiates a series of signaling events, consequently the cytoskeleton provides not only the structural basis for cell shape maintenance and movement, but also mediates cell physiological responses serving as an important signal transducer.

Microlaments (Actin Cytoskeleton)

The actin cytoskeleton plays a central role in cell contractility, motility, vesicle and organelle movement, and overall organization of cell architecture. Two processes are of particular importance for these cell functions: actin polymerization and the interaction of actin with molecular motor myosin. In the cell, the complex actin network is assembled from linear protein complexes called microlaments, or thin laments (Figure 1). The microlament core consists of brillar actin (F-actin), formed from globular actin (G-actin) in the process of polymerization. Actin monomers and

Introduction
The dynamic cytoskeletal network composed of microlaments, microtubules, and intermediate laments is arranged into a functional system with the help of multiple associated proteins. These proteins regulate lament polymerization/depolymerization processes and dene overall cytoskeleton organization,

Vessel lumen

Centrosome

ZO proteins

ZO proteins

Centrosome

Tight junctions Catenins Catenins

Nucleus
Adherent junctions Complexus adherent junctions

Nucleus

FAK

FAK

FAK

FAK

Focal adhesion

Extracellular matrix Focal adhesions Hemidesmosomes

Extracellular matrix

Hemidesmosome

Figure 1 Schematic representation of the cytoskeletal organization of endothelial cells. In a functional monolayer, endothelial cells are attached to each other via several junctional complexes (tight junctions, adherent junctions, complexus adherent junctions). Cell adhesion to the extracellular matrix is provided by integrins (shown in green), the transmembrane elements of focal adhesions and hemidesmosomes. All junctional adhesion complexes are linked to microlaments (red), microtubules (black), or intermediate laments (purple) via numerous specic proteins. An important regulatory role is attributed to focal adhesion kinase (FAK), which controls signaling cascades, governing overall cytoskeletal organization. In an agonist-challenged monolayer, the development of the actomyosin-driven inward-directed tension, along with the disruption of interendothelial cell contacts, results in endothelial barrier dysfunction, a prerequisite of pulmonary edema.

CYTOSKELETAL PROTEINS 617

newly formed actin nuclei are known to possess structural polarity; it is not surprising then, that the polymerization rates at the two ends of microlaments are different. Polymerization and depolymerization processes are regulated by numerous actin-binding proteins, including severing and capping proteins (ADF/colin, gelsolin, severin, fragmin), and proteins controlling actin nucleation and elongation (ARP2/3, profilin). Some of these proteins associate with the lament in a way that may enable lament branching (colin, ARP2/3). Single actin laments may be organized into bundles or gels by the numerous bundling or gelating proteins (a-actinin, villin, synapsin, mbrin, lamin). In nonmuscle cells, actin structures are very dynamic, being in constant transition between polymerized actin and monomers. In muscle cells, the equilibrium is shifted toward the microlament formation; nevertheless, relaxed smooth muscle cells still retain detectable amounts of nonpolymerized actin. In the quiescent cell, the actin cytoskeleton might consist of peripheral bundles (so-called cortical actin) or cell-crossing bundles (stress bers). In spreading or migrating cells, actin polymerization accounts for the appearance of surface protrusions such as lamellipodia and lopodia. Several proteins help to organize cortical actin and actin of cellsurface structures. Peripheral actin bundles contain the actin-binding protein cortactin, known to regulate both actin nucleation and actin crosslinking. Branching activity of ARP2/3 and anticapping, antibranching, and profilin-recruiting activities of the actin-binding protein VASP seem to control the dynamics and architecture of the orthogonal (lamellipodia) and parallel (lopodia) actin network. Depending on the cell type, actin laments may be aligned with different structural and regulatory proteins (troponins, tropomyosin, caldesmon, calponin), affecting F-actin stability and interaction with myosins. Myosins are molecular motors, with diverse structure and function; however, all of them are able to move along actin laments in an ATP-consuming fashion. Cell contractile activity depends mainly on the unique capacity of conventional myosin II to form laments (thick laments). Myosin II laments bundle actin threads into stress bers or tension bers; tension is generated by the sliding of actin and myosin laments along each other. Whereas most myosin molecules move toward faster growing (plus) ends of actin laments, some nonconventional myosins (myosin VI and IX) are able to move toward the minus ends of actin laments, a unique function of these cellular motors. So far, nonconventional myosins have been shown to be important primarily for the vesicular transport

and maintenance of specic structures on the cell surface. The force generated by actin polymerization or actomyosin interaction is usually directed towards the membrane, providing either vesicle movement or cell shape change. Actin association with the membrane might be rendered by several proteins or protein complexes, dictating the specic contacts within the membrane compartment. For instance, myosin V, important for vesicular transport, is recruited to different kinds of cellular vesicles by specic proteins of the Rab family. Cleavage furrows, microvilli, lopodia, and lamellipodia contain proteins of the ezrinradixin-moesin (ERM) family, linking the actin cytoskeleton to the plasma membrane. Cortical actin bers are attached to the membrane via cell junction complexes, whereas tensile bers associate with the membrane through the focal contacts, integrincontaining complexes anchoring the cell surface to the substrate (Figure 1). The temporal and spatial organization of the actin cytoskeleton and the contractile state of the actomyosin system depends upon several factors, which include intracellular calcium levels, activity of small GTPases of the Rho family (Rho, Rac, and Cdc42), and phosphatidylinositol (4,5) bisphosphate (PIP2) concentration. In smooth muscle and nonmuscle cells, the calciumcalmodulin complex activates myosin light chain kinase, which phosphorylates myosin light chains (MLC). MLC phosphorylation induces myosin ATPase activity, myosin polymerization, and stress ber formation. Rho, via activation of its downstream effector Rho kinase, also increases MLC phosphorylation levels and induces stress ber formation. In addition, Rho signaling elevates the level of PIP2, which affects actin polymerization via the regulation of ARP2/3, gelsolin and colin/ADF activity, and actin linkage to the membrane through a-actinin, lamin, ERM, talin, and vinculin. Other GTPases of the Rho family, namely Rac and Cdc42, induce formation of lopodia and lamellipodia, respectively. Rac has been shown to be involved in cortical actin formation and endothelial barrier enhancement via p21-activated protein kinase (PAK)/LIM kinase/colin phosphorylation pathway.

Microtubules (Tubulin Cytoskeleton)

The tubulin cytoskeleton is critical for cell migration, chromosome, and vesicle movement and the organization of some cell-surface structures such as agella and cilia. Tubulin-associated motor activity as well as tensile force produced by microtubule

618 CYTOSKELETAL PROTEINS

depolymerization is involved in diverse cellular events including cilia and agella movement, organelle transport, and mitotic and premeiotic cell rearrangement. Within the cell, heterodimers of a- and b-tubulin are polymerized in a head-to-tail manner to form a linear protolament. Protolaments are subsequently joined through lateral interactions to form the wall of the cylindrical microtubule. Microtubules are polymerized from microtubule organizing centers, such as centrosomes (Figure 1) or agella basal bodies. In some cell types, including epithelium, microtubules are able to disconnect and move from the centrosome after nucleation. Microtubules are highly dynamic, constantly switching between phases of growth and shrinkage mediated by the addition or loss of tubulin dimers from the ends of the microtubule. This property of microtubules is known as dynamic instability and is dependent on the binding and hydrolysis of GTP by tubulin subunits. Rapidly growing and shrinking ( ) ends of microtubules are oriented toward the cell periphery while the slower () ends are closer to the nucleus. The assembly and stability of microtubules is regulated not only by the nucleotide state of tubulin, but also by their interaction with cellular factors like microtubule-associated proteins (MAPs). The most studied MAPs are tau protein, dynein, and kinesin. As is the case with many other MAPs, tau controls the assembly of microtubules in a phosphorylation-dependent manner: in the unphosphorylated form tau promotes assembly of microtubules and inhibits the rate of depolymerization. Similar to myosin, dynein and kinesin function as ATP-consuming microtubule motors. The role of dynein is to transport cargo towards the minus end of microtubules. Dynein motor domains (heads) have microtubule-activated ATPase activity. The tail of dynein connects to the large dynactin protein complex, which targets and tethers dynein to diverse intracellular structures such as Golgi membranes, chromosomes, and microtubules. In contrast to dynein, most kinesins (N-kinesins) move towards the plus end of microtubules. Remaining kinesins (C-kinesins) move in the opposite direction. Similar to dynein, kinesin has an N-terminal motor domain (head) and C-terminal tail, which determines the cargo specicity for each individual kinesin. The interaction between the catalytic domain (head) and the adjacent linker region determines the direction of movement for each kinesin isoform. There has been accumulating evidence of coordination and interaction between microlaments and microtubules. For instance, myosin V, involved in vesicular transport, is known to associate with kinesin. The myosin V/kinesin heteromotor complex allows long-range movement of vesicles along

microtubules, followed by the short-range movement on actin laments. Microtubules have also been shown to be essential for the regulation of cell migration. Whereas this process is largely driven by actin polymerization and subsequent actomyosin contraction, the interaction between microtubules and microlaments is necessary to guide the cell. In migrating broblasts, the growing ends of microtubules were shown to be targeted and attached to newly formed cortical actin foci. The actin-binding proteins capturing microtubules remain unknown, but several proteins have been shown to bind both polymerized actin and tubulin (ezrin, Mip-90, plakins, mDia, caldesmon, tau) or MAP (cortactin). Microtubules are also known to suppress cell contractility, although the mechanism of such suppression is not completely understood. According to the tensegrity model, microtubules mechanically resist actomyosin-driven tension; however, there is evidence that some microtubule-associated molecules may regulate the state of contractile proteins. For example, the disruption of microtubules by microtubule inhibitors, like vinblastine or nocodazole, activates the Rho/Rho kinase complex, inhibits MLC phosphatase activity thereby increasing MLC phosphorylation and induces stress ber formation.

Intermediate Filaments
The function of intermediate laments (IFs) is less well understood than that of microlaments and microtubules. The prevailing hypothesis holds that IFs provide stability to individual cells and cell monolayers, helping to organize the latter into an integral system. In some cell types, IFs have been shown to be important for contractile and motile activity. Several studies have demonstrated interdependence between the IF network organization and the state of microlaments and microtubules in the cell. IFs are formed by groups of proteins exhibiting characteristic cell-expression proles (e.g., keratins are expressed in epithelia, neurolament proteins in neuronal cells, desmin in myogenic cells, and vimentin in mesenchymal cells) and ubiquitous nuclear lamins. Whereas keratins, desmin, and vimentin form the cytoplasmic IF network, nuclear lamins assemble the nuclear envelope. IFs represent dynamic polymers maintaining an equilibrium with the IF protein tetramers. IF organization is likely controlled by phosphorylation and by the association with proteins affecting IF assembly, such as a-crystallin. IF-associated proteins help to organize IFs into a network by linking them either together or to other cytoskeletal elements. For example, plectin has been shown to cross-link IFs to microlaments,

CYTOSKELETAL PROTEINS 619

microtubules, and membrane adhesions. IF attachment to the plasma and nuclear membranes might be provided by integral or membrane-associated proteins (including ankyrin and spectrin).

Integrins and Focal Adhesions

Cell attachment to the surrounding environment is provided by several surface proteins, with the most studied group consisting of transmembrane proteins called integrins. Some integrins act as substrate adhesion molecules, whereas others mediate cellcell interactions. The establishment of integrin-containing contacts is critical for the processes of cell spreading, migration, contractility, and barrier function maintenance. Integrins are composed of separate a and b subunits, noncovalently linked through their extracellular regions. Subunit combination determines integrin specicity toward the ligands. Integrins, which are involved in extracellular matrix binding, are usually organized into complexes called focal adhesions (Figure 1). These complexes consist of an intricate combination of cytoskeletal proteins, linking the cytoplasmic domain of b integrin to actin laments through a-actinin, vinculin, talin, and tensin. Some types of integrins may interact with intermediate laments, creating basal membrane hemidesmosomes (Figure 1). While integrins on migrating cells are dispersed and form small adhesion sites, clusters of integrins forming larger focal adhesion plaques may be found at sites of cell attachment to the extracellular matrix. Initial adhesion sites and mature adhesion plaques appear to be associated with different pools of actin laments, since protruding lamellipodia establish contacts via ARP2/3-branched actin meshwork, whereas mature adhesion plaques are linked to the parallel actin lament bundles, forming stress bers. Integrin binding to ligands is known to promote the attachment of integrins to the actin cytoskeleton while the size of focal adhesion sites depends on the actomyosin-driven tension applied to the point of contact. Additionally, the tubulin network regulates focal adhesion dynamics. Extensions of microtubules targeted to newly formed adhesion sites arrest focal adhesion growth and trigger their disassembly. Such control may be important for the coordination of the process of cell migration, since stabilization of cell substrate contacts in the cells leading edge would interfere with polarized movement. Several signaling molecules regulate the establishment of integrin-containing contacts. Rho GTPase affects integrin association with the cytoskeleton via phosphatydylinositol 4-phosphate 5-kinase/PIP2/

vinculin and talin conformational changes. Focal adhesion-specic protease, calpain, controls both turnover and activation of focal adhesion components. Small GTPases of the Ras family have been shown to alter integrin clustering or change integrin afnity to ligands. Focal adhesions are known to serve as sensors and external signal transducers as they coordinate several signaling cascades involving numerous proteins including tyrosine kinases (focal adhesion kinase (FAK), members of Src family, and C-terminal Src kinase (CSK)), serine/threonine kinases (PKC, PAK, and ILK), tyrosine phosphatases, and modulators of small G proteins. A predominant role in the assembling of focal adhesion protein complex belongs to paxillin and zyxin, which function as adapters for an array of signaling and cytoskeletal molecules. The phosphorylation of FAK and paxilin appears to be a prerequisite of external stimuli-induced cytoskeletal reorganization. One molecule that couples to phosphorylated paxillin is adapter protein CrkII, and is implicated in actin cytoskeleton remodeling. An emerging body of evidence indicates that, upon dissociation from focal adhesions, paxillin and zixin may be shuttled to the nucleus to regulate gene transcription.

Cell Adhesion Molecules

Several types of molecule are specifically expressed on the cell surface to provide cellcell recognition and cell adhesion to surrounding cells. Under quiescent conditions, adhesion molecules participate in maintenance of endothelial barrier function, enforcing contacts between cells in a monolayer. Inammation changes the pattern of adhesion molecules exposed on the cell surface, inducing the interaction of activated mobile cells from the blood vessel lumen with activated endothelial cells. Adhesion molecules are structurally diverse and include selectins, selectin ligands, integrins, and adhesion molecules of the IgG superfamily, such as intercellular adhesion molecules (ICAMs), vascular adhesion molecule (VCAM), platelet-endothelial adhesion molecule (PECAM), and junction adhesion molecule (JAM). These last two proteins are involved in homophilic contact (JAMJAM and PECAMPECAM) organization, whereas ICAMs and VCAM serve as ligands for the specic integrins. Adhesion proteins can associate with the actin cytoskeleton through actin-binding proteins such as cortactin or ERM, or comprise parts of large junctional protein complexes, characterized as tight or adherent junctions. In addition to their cytoskeletal functions, some adhesion molecules (for instance,

620 CYTOSKELETAL PROTEINS

PECAM-1) might serve as scaffolding molecules in several signaling pathways. As mentioned before, control over cell adhesion mostly occurs via alteration of adhesion protein expression on the cell surface. Function of some adhesion proteins, in turn, is regulated by their phosphorylation.

Tight Junctions
Tight junctions, or zonula occludens (ZO), are characteristic of epithelial and endothelial cells (Figure 1). Located at the border between apical and lateral membranes, tight junctions regulate the passage of proteins and liquids across the cell monolayer. Tight junctions include occludin, claudin family members, JAMs 13, cingulin, and linker proteins from the ZO family, which serve to bind the former proteins to each other or to the actin cytoskeleton. In both endothelial and epithelial cells, VASP has been found in the complex with zonula occludens-1 (ZO-1) protein. The regulation of tight junction assembly is achieved via phosphorylation by several protein kinases. Thus, activation of the MAP kinase pathways controls ZO-1-occludin interaction with plasma membrane and VASP association with tight junctions is regulated by PKA-dependent phosphorylation.

Junctional organization and stability of junctional proteins within cells is tightly controlled by PKA-, PKC-, and tyrosine kinase-dependent phosphorylation as well as the balance between Rho and Rac activity. Moreover, several signaling proteins associate with adherent junctions, including receptor tyrosine kinase VEGFR-2, Src homology phosphatase 2 (SHP2), protein tyrosine phosphatase m (PTPm), and IQ GTPase activating protein (IQGAP). The establishment or disruption of cell junctions is now thought to affect gene expression patterns and overall cell fate via nuclear translocation of bcatenin. g- and p120 catenins, members of the same armadillo family of transcriptional regulators, may also be involved in regulating gene expression.

Desmosomes
Desmosomes are basolaterally located junctions restricted mostly to epithelial and myocardial cells. They are critical for tissue integrity, play an important role in epithelial morphogenesis and may act as signaling centers. Desmosomes are formed by the transmembrane glycoproteins of the cadherin superfamily (desmogleins and desmocollins), associated with placoglobin and desmoplakins, which, in turn, anchor keratin- or desmin-containing IFs. Endothelial cells do not have desmosomes but they assemble unique structures called complexus adherence junctions (Figure 1), in which a network of vimentin is linked to classical cadherin via the desmosomal proteins plakoglobin and desmoplakin. These latter proteins may recruit PECAM-1, driving its interaction with intermediate laments.

Adherent Junctions
Adherent junctions, or zonula adherens, are also widely represented in both endothelial and epithelial cells although located more basolaterally relative to tight junctions (Figure 1). Adherence junctions are known to play a role in monolayer barrier maintenance, vascular morphogenesis, and cell migration. The core of adherent junctions is formed by cellspecic transmembrane proteins called cadherins. Extracellular domains of cadherins from adjacent cells undergo calcium-dependent homophilic binding, while cytoplasmic domains associate with b-catenin or g-catenin (placoglobin), linking the complex via a-catenin/vinculin to the actin cytoskeleton. p120 catenin binds the juxtamembrane domain of cadherins and controls their lateral dimerization. Newly formed cadherin-containing contacts have been found to contain VASP. This protein appears to be important in the establishment of monolayers, since the disruption of VASP function interferes with epithelial sheet formation. PECAM-1 is also found within adherent junctions, but is not exclusive to them. PECAM-1 is able to act as a scaffold to recruit b- and g-catenins to cell contacts in a phosphorylation-dependent manner.

Gap Junctions
Gap junctions are membrane structures, serving to provide electrical and metabolic coupling of adjacent cells, such as capillary endothelial cells and alveolar epithelial cells, or type I (gas exchanging) and type II (surfactant-producing) epithelial cells. Cell interconnection is thought to coordinate microcompartment responses to the external stimuli; however, it could also result in the transmission of toxins, for instance, in the setting of oxidative stress, and therefore affect cell survival. Adjacent cells form gap junctions by the association of two hemichannels, each consisting of six tissue-specic connexins. On the cytoplasmic side, connexins may associate with ZO-1 and tubulin, which have the potential to modulate their function. The regulation of gap junctions can also be achieved by their interaction with the calcium-binding protein

CYTOSKELETAL PROTEINS 621

calmodulin, and PKA-, PKC-, or Src-dependent phosphorylation.

Cytoskeleton in Mechanotransduction
Airway smooth muscles, airway and alveolar epithelial cells, lung broblasts, and adherent inammatory cells upon inltration into the lung are continuously subjected to mechanical forces caused by changes in lung volume during the respiratory cycle. While endothelial cells also experience such forces, endothelial cells of larger vessels are subjected to constant changes in blood ow or shear stress. Cells are known to sense their mechanical environment and respond to it with cytoskeletal rearrangement and alterations in gene expression. Changes in local tension, generated during exercise, and in diseases such as asthma, emphysema, and cystic brosis activate several signaling pathways affecting cell contractility, migration, proliferation, monolayer integrity, synthesis of proinammatory mediators, and/or surfactant exocytosis. Excessive forces, like those applied during mechanical ventilation, may trigger mechanosensitive signaling cascades leading to catastrophic consequences manifested as ventilator-induced lung injury. While the cytoskeleton represents the target of the mechanical stress-induced cell response; mechanotransduction per se relies upon cytoskeletal structures associated with plasma membrane anchors. Mechanical strain, administered to extracellular domains of integrins, results in increased tyrosine phosphorylation of proteins, associated with focal adhesion sites. This event activates multiple signaling cascades, leading to alterations in cell metabolism relative to the changing environment. Propagation of mechanical deformation-induced signals also depends upon cytoskeletal structures. Calcium elevation waves are known to diffuse via gap junctions from the stretchinjured alveolar epithelial cells to affect uninjured cells in monolayer. Further research will be needed to understand the major pathways responsible for mechanical deformation-triggered alterations in cell metabolism, in order ultimately to prevent the detrimental consequences of such alterations.

vascular permeability and marked interstitial edema formation, implicating the direct involvement of microlaments in the regulation of permeability in vivo. While the importance of the microtubule component in the maintenance of endothelial cell barrier function remains largely unexplored, intravenous administration of anticancer drugs, which specifically target microtubules, can lead to the sudden development of pulmonary edema in breast cancer patients suggesting the potential importance of the microtubule network for the regulation of lung permeability. Proinammatory mediators, released by activated cells at the site of inammation (reactive oxygen species, tumor necrosis factor (TNF-a), interleukins, platelet activator factor (PAF), endothelin, prostanoids and leukotrienes), may significantly affect endothelial barrier function. Two parameters are of particular importance for the maintenance of barrier function: the contractile state of the actin cytoskeleton and intercellular junctional integrity. Edemagenic factors are known to induce stress ber formation and increase endothelial cell contractility, while altering tight junction and adherent junction organization. Notably, those oxidants and other inammatory mediators, while increasing endothelial permeability, enhance cellsubstrate adhesion. Such strengthening of focal contacts is likely to be a prerequisite of activation of integrin-mediated signal transduction pathways but also can serve to prevent detachment of endothelial cells from the monolayer. Because several proinammatory mediators lead to cytoskeletal changes via the same signal transduction pathways, direct interference of intracellular signaling may have profound therapeutic implications. One potential strategy would be to block key signaling events leading to cytoskeletal rearrangement, namely calcium entry, Rho kinase, PKC or Src family kinase activation. The clinical significance and safety of several pharmacological inhibitors, including PKC inhibitor LY333531 and Rho kinase inhibitor Y27632, are currently under investigation.
See also: Adhesion, CellMatrix: Integrins.

Cytoskeleton in Lung Hyperpermeability

Reorganization of the endothelial cytoskeleton leads to alteration in cell shape and provides a structural basis for increase of vascular permeability, which has been implicated in the pathogenesis of many diseases including asthma, sepsis, and acute lung injury. In perfused rabbit lungs, selective disruption of actin microlaments leads to a significant increase in

Further Reading
Alexander JS and Elrod JW (2002) Extracellular matrix, junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation. Journal of Anatomy 200: 561574. Bershadsky AD, Balaban NQ, and Geiger B (2003) Adhesion-dependent cell mechanosensitivity. Annual Review of Cell and Developmental Biology 19: 677695. Chidgey M (2002) Desmosomes and disease: an update. Histology and Histopathology 17: 11791192.

622 CYTOSKELETAL PROTEINS

Dos Santos CC and Slutsky AS (2000) Mechanism of ventilatorinduced lung injury: a perspective. Journal of Applied Physiology 89: 16451655. Dudek SM and Garcia JGN (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 14871500. Ilan N and Madri JA (2003) PECAM-1: old friend, new partners. Current Opinion in Cell Biology 15: 515524. Johnson-Leger C, Aurrand-Lions M, and Imhof BA (2000) The parting of endothelium: miracle, or simply a junctional affair? Journal of Cell Science 113: 921933. Koval M (2002) Sharing signals: connecting lung epithelial cells with gap junction channels. American Journal of Physiology 283: L875L893. Krause M, Dent EW, Bear JE, Loureiro JJ, and Gertler FB (2003) Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annual Review of Cell and Developmental Biology 19: 541564. Lee TJ and Gotlieb AI (2003) Microlaments and microtubules maintain endothelial integrity. Microscopy Research and Technique 60: 115127. Sellers JR (2000) Myosins: a diverse superfamily. Biochimica et Biophysica Acta 1496: 322. Van Nieuw Amerongen GP and van Hinsbergh VWM (2003) Targets for pharmacological intervention of endothelial hyperpermeability and barrier function. Vascular Pharmacology 39: 257272. Wang Y and Gilmore TD (2003) Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear. Biochimica et Biophysica Acta 1593: 115120. Wojciak-Stothard B and Ridley AJ (2003) Rho GTPases and the regulation of endothelial permeability. Vascular Pharmacology 39: 187199. Yin HL and Janmey PA (2003) Phosphoinositide regulation of the actin cytoskeleton. Annual Review in Physiology 65: 761789.

D
Deep Venous Thrombosis (DVT)
see Pulmonary Thromboembolism: Deep Venous Thrombosis.

DEFENSE SYSTEMS
R Boyton, Imperial College London, London, UK R Wilson, Royal Brompton and Hareeld NHS Trust, London, UK D M Altmann, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The lung is the anatomical site not only for gaseous exchange, but also for high-turnover sampling of pathogens and other antigenic and allergenic material. It constitutes a vast mucosal surface area, constantly exposed to inhaled substances that, allowed free ingress, would cause substantial damage or death. Any defense mechanism must accommodate the fact that the respiratory tract is made up of delicate tissues required for gas exchange. The lung, therefore, requires a multilayered system of protection that allows a fast, efcient, and effective response able to deal with infectious pathogens and other foreign particles without damaging the lung itself in the process. This encompasses physical barriers, innate immune mechanisms, and the cellular and antibody adaptive immune response. The price of excessive or inappropriate inammatory responses can be high and may lead to lung damage. The rst line of defense against entry to the lung is from physical barriers such as mucus and cilia, followed by the innate immune response that includes a battery of mediators such as lactoferrin, lysozyme, collectins, and defensins. Release of these molecules leads directly to lysis of pathogens, or destruction through opsonization or the recruitment of inammatory cells. Rapid activation of the innate immune response also comes through the triggering of Toll-like receptors (TLRs) and the chemotactic recruitment and activation of neutrophils. Natural killer (NK) cells provide a fast and effective immune surveillance response against infectious pathogens. Type I interferons exert a direct antiviral effect. The adaptive immune response contributes by making neutralizing antibodies and T lymphocytes orchestrate antigen-specific immune responses through their ability to differentiate self from nonself and provide memory. T lymphocyte populations of different cytokine phenotypes so-called T-helper-1 (Th1) and T-helper-2 (Th2) dramatically alter the balance between pathogen clearance and tissue damage.

Introduction
Most pathogens gain entry to the body through mucosal surfaces. The lung, being the largest epithelial

surface in the body, constitutes the major site of entry. The airways are continually challenged by airborne microorganisms and ingress of environmental particles and employ robust mechanisms to combat infection, starting with physical barriers to entry, and failing this, mounting innate or specific adaptive immune responses. Lung diseases such as cystic brosis (CF) and the acquired immunodeciency syndrome (AIDS) constitute illustrative examples of how certain types of respiratory infections thrive when elements of the innate response in the rst case, or the specific immune response in the second, are impaired. The specific part of the protective response that is missing or suboptimal dictates, both qualitatively and quantitatively, the nature of the infecting organism and lung damage. It is possible that some chronic lung diseases such as bronchiectasis or chronic obstructive pulmonary disease (COPD) may result from a dysregulated immune response to infectious pathogens or inhaled foreign particles driven in part by underlying differences in the individuals immune response repertoire and also by levels of exposure. The specific prole of cytokines made by activated T lymphocytes must be highly regulated and appropriate; the appropriateness of T-helper-1 (Th1) and T-helper-2 (Th2) cytokine proles in the respiratory immune response is critical to maintaining the balance between protective immunity and excessive inammation. The balance between cytokines that promote, for example, bronchial inltrates (primarily either neutrophilic or eosinophilic) will have a major impact on the type of disease. Immunity in the lung faces the particular problem that, at the same time as requiring the capacity for rapid and potent clearance of infection, an overzealous or inappropriate cellular or cytokine response, either due to immune dysregulation or failure to clear antigen, may cause inammatory changes that can permanently alter the function and architecture of the airways.

2 DEFENSE SYSTEMS

Immune mechanisms in the lung must deal with a wide range of pathogens, including bacteria, mycobacteria, fungi, and viruses. While specific mechanisms for dealing with pathogens may differ to some extent (e.g., the importance of type I interferons and cytotoxic T cells for antiviral responses), there are features common to the immune mechanisms. These consist of a multilayered defense that may involve several modes of innate mediators, Tolllike receptor (TLR) activation, leukocyte inltration, antibodies, natural killer (NK) cells, and activation of specific T lymphocytes.

Physical Barriers and Soluble Mediators

Mechanical barriers offer the rst line of defense to microorganisms entering the respiratory tract. Mucins of the mucociliary blanket lining the surface of the airways trap microorganisms which can then be cleared by ciliary movement. Cells of the respiratory tract produce a range of soluble mediators, either constitutively or following activation in response to those particles that pass this barrier. Airway surface uid (ASF) contains several proteins with antimicrobial activity. These mechanisms are sensitive to local salt concentrations, and are therefore believed to be impaired in CF. Furthermore, the CF transmembrane conductance regulator (CFTR), mutation of which is the cause of the disease, has itself been shown to act as a receptor for internalization and clearance of some microorganisms, particularly pseudomonas. Airway epithelial secretory proteins can be divided into mucins and nonmucins. The nonmucin secretory proteins include lactoferrin, peroxidase, lysozyme, and antileukoprotease as well as type I interferons. Most of the nonmucin secretory proteins are secreted by the serous cells contained within submucosal glands. Among proteins known to bind bacteria in the host innate response are lipopolysaccharide (LPS)binding protein (LBP) and bactericidal/permeabilityincreasing protein (BPI). Genes with sequence homology to LBP and BPI have been identied and shown to be expressed in the upper respiratory tract. The genes include plunc (palate, lung, and nasal epithelial clone), lunx, and spurt. The role of these related gene family members in innate immunity is largely inferred from their relationship to the LBP and BPI proteins, their distribution in the respiratory tract, and ndings in disease, such as the observation that SPLUNC1 is increased in the sputum of patients with COPD. There are at least 12 mucin genes. The mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC5B are abundantly expressed in the respiratory

tract. Their gene products contribute directly or indirectly to the pulmonary defense mechanism against infectious pathogens. Respiratory tract mucus is not only essential for lubricating the tissue surface and protecting from injury but also has the function of trapping inhaled particles. These can then be excluded from entry to the airways by the beating action of the cilia. Goblet cells in the airway epithelium and secretory cells in the submucosal glands are the main contributors to mucus secretion. However, overproduction of mucus, as seen in inammatory contexts such as asthma, chronic bronchitis, bronchiectasis, and CF, causes airway narrowing or obstruction, impeding airow. Lysozyme made by glandular serous cells, surface epithelial cells, and macrophages is a major constituent of lavage uid and sputum. It represents an important antimicrobial defense, particularly against Gram-positive bacteria. In transgenic mice overexpressing lysozyme in the lung, elimination of pulmonary pseudomonas infection as well as group B streptococci is dramatically enhanced, leading to a decrease in systemic dissemination, enhanced recruitment of neutrophils and protection from death. Furthermore, clinical study of susceptibility and resistance to acute bronchitis show a correlation of protection with levels of macrophage-derived lysozyme. Lactoferrin, another arm of the constitutive defense in the airways, is produced by serous cells and neutrophils. It is able to kill and agglutinate bacteria which it recognizes on the basis of carbohydrate motifs, and also stimulates superoxide production by neutrophils. As with lysozyme, the concentration of lactoferrin is markedly increased in the lower respiratory tract in subjects with chronic bronchitis. The a- and b-defensins have broad microbicidal activity against mycobacteria, Gram-negative and -positive bacteria, fungi, and some viruses. They act by inducing permeabilization and show upregulation in the lung in response to interleukin-1 (IL-1). Binding of defensins to complement components also leads to triggering of the alternative complement cascade. The function of the defensins is highly dependent on salt concentration and likely to be impaired in CF. One of the members of the b-defensin family is tracheal antimicrobial peptide (TAP), which is strongly expressed in the ciliated airway epithelium and upregulated in response to bacterial LPS. The collectins are a family of pre-immune opsonins, able to recognize carbohydrates on the surface of a broad range of pathogens, including bacteria and viruses. This, in turn, leads to recruitment of other cells and defense mechanisms, including macrophage chemotaxis, phagocytosis, immune cell proliferation,

DEFENSE SYSTEMS 3

and activation of the alternate complement cascade. Members of the collectin family include surfactant proteins A and D (SP-A and SP-D), conglutinin, collectin-43, and mannose-binding lectin (MBL). Collectins can directly affect activation of inammatory cells, including macrophages and lymphocytes. In SP-A knockout mice there is enhanced susceptibility to group B streptococcal pneumonia and sepsis after intratracheal infection. Also, following infection with pseudomonas there is an inability to destroy bacteria and decreased phagocytosis by alveolar macrophages. SP-A function is dependent on binding to the TLR recognition system (see below) as a functional TLR4 molecule is required for induction of leucocyte activation. Human defense pathways share the use of many highly conserved genes and functions with invertebrate species that use these mechanisms in the absence of any adaptive immune response. A feature common to induction of many of the protective factors that contribute to the rapid, innate response is that these mechanisms are often evolved to recognize and respond to conserved structures of microorganisms. The family of Toll receptors were initially described in fruit ies as transmembrane receptors, stimulated by the secreted Spatzle factor, and responsible for the developmental establishment of dorsoventral polarity. However, using mutant strains for several molecules in this pathway, it was subsequently appreciated that there was a functional role also in innate immunity, through activation of the antifungal peptide drosomycin, to fungal infection and of a response to Gram-positive bacterial infection. Furthermore, a role in a conserved pathway of innate immunity was suggested by homology to the mammalian IL-1 receptor gene. Mammalian TLRs were subsequently cloned and shown to constitute the receptors for a large, evolutionarily conserved system of pattern recognition, leading both to innate immune mechanisms and to priming for antigen presentation to the adaptive immune system. Analysis of the natural agonists of TLRs is an area of intense research activity. Generic structures for recognition can include bacterial carbohydrates, LPSs, or forms of bacterial DNA (unmethylated cysteine guanosine-rich areas known as CpG sequences) that are not seen in human genes. Known ligands for TLR1 include triacyl lipopeptides of bacteria and mycobacteria. TLR2 is activated by diverse ligands (including fungal zymosan and bacterial peptidoglycan), TLR3 by double-stranded RNA during viral infection, TLR4 by several ligands (including LPS), and TLR9 by bacterial CpG DNA sequences. Singlestranded RNA viruses are recognized by TLR8 in humans and TLR7 in mice.

The downstream inammatory consequences of TLR binding and activation are multiple and diverse. A common downstream signaling pathway acts through activation of nuclear factor kappa B (NFkB). The impact of this activation can encompass release of cytokines, chemotaxis for inammatory cell types, and priming of the adaptive immune response through the activation of dendritic cells. TLRs are widely expressed and are further inducible during inammation. TLR expression in the lung encompasses both the expression patterns on inammatory cells of hematopoietic origin and the expression by lung tissue itself. Alveolar epithelial cells express TLR2 and TLR3, as do alveolar macrophages. TLR4 and TLR6 expression is also found in the lung. Information on TLR expression and susceptibility to lung infection has come mainly from studies of mice carrying null mutations for TLR genes or common TLR signaling molecules such as MyD88. From such studies TLR4 is important for the control of chronic Mycobacterium tuberculosis infection. Granulomas from patients with active tuberculosis show expression of TLR15 and TLR9. TLR4 is also implicated in defense against respiratory syncytial virus (RSV) from studies in knockout mice. TLR4null mice infected with RSV show impaired NK cell trafcking, reduced cytokine expression, and reduced virus clearance. For virus recognition, there also exist other non-TLR pathways such as PKR and RIG1. Immunoglobulin A (IgA) is considered the most important immunoglobulin for defense in the respiratory tract. This is borne out by the susceptibility to chronic bronchial infections in patients with IgA immunodeciencies. Although, like other immunoglobulins, it is secreted by B lymphocytes, unlike the other immunoglobulins such as IgG and IgE found in the lung, synthesis is not dependent on specific T lymphocyte help. Thus, CD4 T cell knockout mice have normal IgA responses. It is thought that IgAsecreting B cells are stimulated locally in the lung. The IgA response is an important component of rapid, local lung immune responses to infection by viruses including inuenza and RSV. An important component of the direct response of cells in the respiratory tract, including epithelial cells and macrophages, to viral infection comprises the type I interferons, a and b. Following type I interferon release, several factors are released with the ability to interfere with viral replication. Knockout mice carrying null mutations for the receptor to these interferons show enhanced viral replication in the lung. Interestingly, protection may nevertheless be maintained through an augmented neutralizing antibody response. Furthermore, resolution of infection in

4 DEFENSE SYSTEMS

these circumstances can be associated with an altered inammatory response, with the appearance of granulocytic pulmonary inammatory cells.

Cells of the Innate Immune Response

Activation of many of the pathways described above can, in turn, lead to release of mediators that increase neutrophil migration to the lung. Within hours of experimental infection or LPS injection, there is massive neutrophil recruitment until these cells constitute 6080% of bronchial alveolar lavage (BAL) cells. This can occur through the stimulation of factors including IL-8, complement activation, or the release of chemokines. Activated neutrophils have considerable capacity to phagocytose and neutralize bacteria, and can also secrete various factors including defensins, tumor necrosis factor alpha (TNF-a), IL-1b, and IL-6. For example, experimental infection with Haemophilus inuenzae causes respiratory epithelial cells to both release IL-8 and upregulate expression of a molecule called intercellular adhesion molecule-1 (ICAM-1). The IL-8 leads to neutrophil recruitment and the enhanced ICAM-1 to increased neutrophil adherence, both of these contributing to clearance of the infection. Furthermore, neutrophils utilize a range of microbicidal products. These include oxidants, microbicidal peptides, and lytic enzymes. Neutrophil generation of microbicidal oxidants results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, responsible for transferring electrons from NADPH to O2, and thus leading to formation of superoxide anion. Indeed, a wide array of functionally overlapping cytotoxic pathways has so far been elucidated in neutrophils, and it remains uncertain which are the most significant in host defense. Knockout mouse studies have tended to place more emphasis on the pathways associated with the neutrophil granule proteins than the direct effect of free radical reactions. Evidence also comes from the analysis of patients with chronic granulomatous disease (CGD), an immunodeciency most commonly associated with a mutation in the gene encoding the b subunit of cytochrome b558 or gp91, called CYBB on the X chromosome and associated with defects in the neutrophil oxidative respiratory burst. While CGD is a profound immunodeciency in terms of susceptibility to bacterial and fungal infections, the patients show considerably greater resistance to infection than individuals with severe neutropenia (and so decient in the full gamut of neutrophil-dependent host defense mechanisms).

Alveolar macrophages respond to signals from microbes both through TLR recognition and through a number of other specific, cell-surface receptors. The repertoire of receptors expressed on the alveolar macrophage surface is vital for binding, uptake, and response to microbes and inhaled particles. An important family of receptors is the class A macrophage scavenger receptors (SRAs), a group of pattern recognition receptors comprising three members: SRA-I/ II, macrophage receptor with collagenous structure (MARCO), and scavenger receptor with C-type lectin (SRCL). In a murine model of pneumococcal pneumonia, MARCO knockout mice show impaired clearance of bacteria from the lungs, increased pulmonary inammation and cytokine release, and diminished survival. NK cells pose a further barrier to infectious agents entering the lung. NK cells derive from the same hematopoietic lineage as T lymphocytes, but, unlike classical T cells, do not mature in the thymus and do not express rearranged antigen receptors. In view of the relationship between the role of receptors expressed by NK cells in recognition of alterations in self-proteins and that of T cells, NK cells may be regarded as occupying the boundary between evolution of the innate and adaptive immune systems. NK cells use what is now appreciated to be a large number of families of cellular receptors to survey the body most notably the large, polymorphic family of killer cell immunoglobulin-like receptors (KIRs) to screen for cells that, for reasons of infection or transformation, have altered expression of human leukocyte antigen (HLA) class I tissue antigens. The rules governing NK cell activation are complex, not the least because different members of the receptor families can be either inhibitory with respect to the intracellular signal they transmit on ligand engagement, or activatory. Thus, while NK cells were until recently dened simply as cells that patrol for HLA class I expression that has been diminished as a consequence of infection or cellular transformation, the missing self hypothesis, they are now considered cells that survey for targets that either lack proper class I expression or overexpress ligands for activatory NK cell receptors. The KIR family of receptors is particularly focused on recognition of alterations in HLA-C expression. If the NK cells fail to receive a cellular signal that normal HLA class I has been recognized, they can enter a program of activation leading to lysis of the infected cell and release of interferon gamma (IFN-g). This can, in turn, lead to recruitment of other cells. In experimental RSV infection, there is an extremely rapid antiviral NK cell IFN-g response that precedes and leads to recruitment of virus-specific, cytotoxic T lymphocytes.

DEFENSE SYSTEMS 5

Local release of IL-12 is an important early event leading to stimulation of NK cells for rapid antiviral immunity in the lung. Recent research in sarcoidosis has focused on a specific population of NK cells, termed NKT cells, which are considered to have regulatory properties in many diseases. It has been observed that patients with sarcoidosis show a significantly reduced population of CD1d-restricted NKT cells, a nding that is considered consistent with the excessive Th1 inammatory activation associated with the disease.

Adaptive Immunity
The specific, adaptive immune response to infection in the lung depends on the specific recognition of foreign, microbial antigens by the clonotypic receptors of B and T lymphocytes. The antibodies against a given pathogen may be directed against several different binding sites, termed epitopes. Some antibodies are able to neutralize infections through interference with a vital, structural component of the microbe. Others may clear infections through opsonization for phagocytosis and through activation of the complement system. After initial exposure, a state of B cell memory is established, leading to the ability to mount a more rapid IgG antibody response on any subsequent re-exposure. Furthermore, the B-lymphocyte-derived IgG response is unique in the immune response in showing afnity maturation. This is the ability to introduce small numbers of random mutations into the genes for the antibody, somatically mutating the sequence so that receptors with better afnity for the epitope are selected at each generation of cell division. This antibody response and its afnity maturation are absolutely dependent on cytokine-mediated help ensuing from antigen recognition by CD4 T cells. The antibody response to infection mounted by B lymphocytes acts alongside the T lymphocyte response. T lymphocytes carry the surface markers CD3 along with either CD4 or CD8, mature in the thymus, and express an antigen receptor that must recognize peptide fragments of microbial antigen presented by host HLA molecules. T lymphocytes mediate their effector function either through the release of a wide range of soluble mediators called cytokines or, in the case of CD8 cytotoxic T cells, by direct killing of target cells. Early studies aimed at cloning the genes for the antigen receptor used by T lymphocytes identied two separate systems of receptors now known to delineate separate lineages of thymus-differentiated T cells. One receptor heterodimer was the ab receptor, the other the gd receptor. The cells expressing the ab T cell receptor are the

conventional CD4 and CD8 populations with the properties of mounting cytolytic responses to infected cells, making cytokines, and stimulating B cells. This involves recognition of peptide fragments of micorobial antigens loaded through the biosynthetic pathway of classical HLA class I or class II molecules into the peptide-binding groove. The T cells expressing the gd receptor are particularly associated with defense at epithelial surfaces, have a different mode of recognizing foreign antigens, but are also involved in lung immunity. They do not need antigen processing or major histocompatibility complex (MHC) restriction for antigen recognition. The types of antigen they recognize can be categorized into those that are widely distributed and are constitutively expressed in host cells and by microbial pathogens, and those that are inducibly expressed and might be restricted to specific cell types. Presentation may be by classical MHC molecules or by related molecules such as CD1d. Examples of the families of recognized antigens include the nonprotein pyrophosphates and alkylamines that are found in bacteria, plant or animal cells, and bacterial and mammalian homologs of the stress protein Hsp60/GroEL. CD4-positive, TCR ab expressing T lymphocytes are termed Th cells. Their effector function is through the release of cytokines that stimulate or activate some other cell type: for example, IL-4, which stimulates B cells and attracts eosinophils; IL-2, which drives other T lymphocytes, including CD8 cells to divide; and IFN-g, which causes other T lymphocytes to differentiate at the same time as it activates macrophages and dendritic cells. CD4 cells can themselves kill target cells through the release of TNF-a. CD8-positive, TCR ab T lymphocytes are termed cytotoxic cells (or sometimes, killer cells). On recognition of microbial peptides presented by HLA class I molecules, they function by lysing infected cells, particularly by release of granules of perforin.

Responses by T-Cell Subpopulations

The functional distinction between Th1 and Th2 subpopulations was initially made in experimental mouse models in which antigens were given under rather polarizing conditions, although the categorization, while often less clear-cut in naturally induced T cell immune responses of humans, appears to have meaning in this context also. Th1 cells are driven to differentiate primarily by the presence of the cytokines IL-12 and IL-18, and, in response to antigen, can make IFN-g as well as IL-2 and TNF-a. These are the cells historically described as those mediating delayed hypersensitivity responses to local viral or

6 DEFENSE SYSTEMS

bacterial infection, stimulating local macrophage activation, neutrophil recruitment, and specific cytotoxic T-cell responses. In the respiratory immunology sphere, sarcoidosis represents an overtly Th1 environment in which the majority of CD4 T cells express receptors for IL-12 and make Th1 cytokines. Th2 cells are driven to differentiate primarily by the presence of the cytokine, IL-4. They can respond to antigen challenge by making more IL-4 as well as IL-5 and IL-13. IL-10 is considered a regulatory cytokine that may be associated with Th1, Th2, or regulatory T cell (T reg) responses (see below). The Th2 cytokine response prole is important for driving B lymphocytes to make antibody responses and in classswitching of antibody production to make IgG1a and IgE. Th2 cytokines can also lead to recruitment and activation of basophils and eosinophils. Thus, in the respiratory context, a prototypic Th2 response is that seen in atopic asthma or in aspergillosis. The choice of differentiation pathway from the Th0 precursor to Th1 to Th2 is made by mature, post-thymic T cells in the peripheral immune system and is inuenced by many variables including the local cytokine milieu, the nature of the antigen-presenting cell and available co-stimulatory molecules, the avidity of the interaction, and the nature of the antigen. Cytolytic CD8 cells can similarly be divided into Tc1, and Tc2. This distinction follows the same split as for T-helper cells: Tc1 cells are primarily characterized by release of IFN-g, and Tc2 cells primarily by release of IL-4. The various T cell subsets described here Th1, Th2, Tc1, and Tc2 can each be associated either with protection from pathogens or (in the case of either an overzealous, dysregulated immune response or the need to mount a continued response in the face of an inability to cope with antigen load) with damaging immune pathology. A number of specific observations have been made about mechanisms regulating Th1/Th2 balance in the lung. It remains to be seen whether these observations are unique to pulmonary immune responses or also have relevance to other sites. Pulmonary dendritic cells (DCs) from mice exposed to respiratory antigen transiently produced IL-10. These pulmonary DCs can stimulate the development of CD4 Treg cells that also produce high amounts of IL-10 and have the ability to transfer specific immunological tolerance. Many observations have been made about the possible role of Treg cells in modulating potentially immunopathogenic immune responses in the lung, particularly in asthma. A particularly novel Treg population has been described in the studies of pulmonary immunity. A population of Treg cells develops in vivo from naive CD4 CD25 T cells during

Th1-polarized responses, distinct from CD25 TR cells. In terms of transcription factor expression, they are positive both for Foxp3 and T-bet, thus showing features intermediate between Th1 and Treg cells. These antigen-specific Treg cells are induced by CD8 DCs, produce IL-10 and INF-g, and inhibit development of airway hyperreactivity. The contributions of different T cell populations to protection and tissue damage during lung infection have been modeled in a wide variety of transgenic and knockout mouse strains. Protection of mice from experimental inuenza infection is particularly related to the transfer of IFN-g secreting CD8 cells, although CD4 cells also offer protection. While Th1 CD4 cells protect against lethal challenge, transfer of Th2 cells exacerbates disease. Absolute numbers of systemic antiviral T cells can be enumerated using uorescent peptide/MHC tetramers as a probe. From these studies, it is clear that there exists robust, long-lasting CD8 immunity following viral infection, with cytotoxic precursor cells detectable at more than 20-fold increased levels for life. Thus, immune individuals have potent memory such that virus can be cleared considerably faster than in immunologically naive individuals. The ability of CD8 cells to clear a pulmonary viral infection is not simply related either to the number of cells or to their cytolytic capacity, as Tc1 cells are able to isolate and clear inuenza infections far more rapidly than equally cytolytic Tc2 cells. Thus, besides the cytolytic activity, the ability to migrate appropriately to the site of infection and attract other appropriate cell types is also crucial. Mice expressing transgenic IL-4 targeted to the lung show delayed clearance of virus, although this, in turn, leads to an enhanced neutralizing antibody response. This is in line with the view that there is considerable redundancy in mechanisms for clearing infection from the lung, although these may be associated with different inammatory consequences. In experimental infection with RSV, Th2-dependent eosinophilia can be reversed by treatment with the Th1-polarizing cytokine IL-12. However, despite reversing the eosinophilia, the clinical outcome is relatively unchanged. A number of studies show inability to clear infection in knockout mice lacking IFN-g or IFN-g receptors. The role of gd T cells in protection from respiratory pathogens is less well characterized than TCR ab CD4 and CD8 cells. Experiments have compared susceptibility to infection in knockout mice lacking TCR ab cells compared with those lacking TCR gd cells. The TCR gd knockout mice are extremely susceptible to mycobacterial and nocardia infections as well as to lethal bacterial pneumonia following infection with Klebsiella.

DEFENSINS 7 See also: Cilia and Mucociliary Clearance. Collectins. Complement. Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defensins. Immunoglobulins. Interferons. Interleukins: IL-1 and IL-18; IL-4; IL-5; IL-6; IL-7; IL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Mucins. Mucus. Surfactant: Surfactant Protein A (SPA); Surfactant Protein D (SP-D). Toll-Like Receptors.
Moore TA, Moore BB, Newstead MW, and Standiford TJ (2000) Gamma delta T cells are critical for survival and early proinammatory cytokine gene expression during murine Klebsiella pneumonia. Journal of Immunology 165: 26432650. Rogge L, Papi A, Presky DH, et al. (1999) Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo. Journal of Immunology 162: 39263932. Rowe SM, Miller S, and Sorscher EJ (2005) Cystic brosis. New England Journal of Medicine 352: 19922001. Sallusto F, Geginat J, and Lanzavecchia A (2004) Central memory and effector memory T cell subsets: function, generation, and maintenance. Annual Review of Immunology 22: 745763. Segal A (2005) How neutrophils kill microbes. Annual Review of Immunology 23: 197224. Takeda K, Kaisho T, and Akira S (2003) Toll-like receptors. Annual Review of Immunology 21: 335376. Turner SJ, Kedzierska K, La Gruta NL, Webby R, and Doherty P (2004) Characterization of CD8 T cell repertoire diversity and persistence in the inuenza A virus model of localized, transient infection. Seminars in Immunology 16: 179184. Underhill DM (2004) Toll-like receptors and microbes take aim at each other. Current Opinion in Immunology 16: 483487. Woodland DL (2003) Cell-mediated immunity to respiratory virus infections. Current Opinion in Immunology 15: 430435. Zangh P, Summer WR, Bagby GJ, and Nelson S (2000) Innate immunity and pulmonary host defence. Immunological Reviews 173: 3951.

Further Reading
Basu S and Fenton MJ (2003) Toll-like recpeptors: function and role in lung disease. American Journal of Physiology. Lung Cellular and Molecular Physiology 286: L887L892. Crouch E, Hartshorn K, and Ofek I (2000) Collectins and pulmonary innate immunity. Immunological Reviews 173: 5265. Diamond G, Legarda D, and Ryan LK (2000) The innate response of the respiratory epithelium. Immunological Reviews 173: 2738. Doherty PC, Hou S, and Tripp RA (1994) CD8 T cell memory to viruses. Current Opinion in Immunology 6: 545552. Kronenberg M (2005) Toward an understanding of NKT cell biology: progress and paradoxes. Annual Review of Immunology 23: 877900. Lanier L (2005) NK cell recognition. Annual Review of Immunology 23: 225274.

DEFENSINS
P S Hiemstra, Leiden University Medical Center, Leiden, The Netherlands
& 2006 Elsevier Ltd. All rights reserved.

Introduction
The host is equipped with a variety of antimicrobial mechanisms, including the action of antimicrobial reactive oxygen and nitrogen intermediates, and antimicrobial peptides and proteins. These antimicrobial polypeptides have been the subject of intense research in the last decade and are considered interesting templates for the pharmaceutical development of efcient broad-spectrum antimicrobial peptides. Mammalian defensins are small (35 kDa) cationic peptides that have been identied in humans, monkeys, cows, and rodents. These peptides kill a wide range of Gram-negative and Gram-positive bacteria, fungi, and viruses. Following the discovery of arginine-rich antimicrobial components in rabbit and guinea pig neutrophils in the 1960s and 1970s, the rst human defensins were described and named in 1985. Three neutrophil defensins were isolated from human neutrophils and named human neutrophil peptides 13 (HNP13). Soon afterwards, the fourth much less abundant neutrophil defensin, HNP-4, was identied. Neutrophil defensins are highly abundant in neutrophils: they make up 5% of the total protein

Abstract
Defensins are members of a large family of cationic antimicrobial peptides that form an essential element of innate immunity. They are key effector molecules in host defense against infection due to their broad-spectrum antimicrobial activity. Defensins are small, cationic peptides that contribute to the antimicrobial activity of phagocytes, the skin, and the mucosa (including that of the lung). Neutrophils and epithelial cells are the main cellular sources of these peptides, but monocytes, macrophages, dendritic cells, and lymphocytes also produce defensins. The two families of antimicrobial defensins expressed in the human lung are the a- and the b-defensins, mainly produced by neutrophils and epithelial cells, respectively. The members of these families are structurally different, but display partly overlapping activities. Recent studies show that defensins not only act as endogenous antibiotics, but also display a range of activities involved in inammation, immunity, and wound repair. Studies in animal models and human disease strongly suggest an important role of these molecules in vivo. In addition, genetic studies show associations between polymorphisms in defensin genes and inammatory lung disease.

8 DEFENSINS

content of the neutrophil, and are the predominant component of myeloperoxidase-containing neutrophil azurophilic granules. Like all defensins that have been characterized at the peptide level to date, neutrophil defensins display broad-spectrum antimicrobial activity against a range of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and (enveloped) viruses. Based on their structure, neutrophil defensins belong to the a-family of defensins. The Paneth cells in the human small intestine express other a-defensins, named human defensin (HD) 1 and 2. The rst mammalian b-defensin was discovered in the bovine respiratory tract in 1991. The rst human b-defensin (human b-defensin 1; hBD-1) was isolated from hemoltrate and characterized in 1995. Subsequently, several human b-defensins were characterized at the protein and gene level. j-Defensins are the last family of defensins that have been identied. These circular mini-defensins were isolated from rhesus monkeys in 1999. They appeared to have evolved in primates, but the human y-defensin genes are pseudogenes that are not expressed.

Table 1 Human defensin families Peptide family a-Defensins Neutrophil defensins HNP-1, HNP-2, HNP-3, HNP-4 Paneth cell defensins HD-5, HD-6 Distribution Neutrophils; also NK-cells and CD8 cells Paneth cells of the small intestine; also nasal and bronchial epithelial cells, female reproductive tract

b-Defensins Epithelial defensins hBD-1, hBD-2, hBD-3, hBD-4

Epithelial cells; also various leukocyte subsets

In addition to this cluster on chromosome 8, four additional clusters have been identied encoding defensin genes that are transcribed. However, the gene products of these defensin genes have not yet been explored. The gene for HNP-2 has not been found; for this reason and based on the amino acid sequence of the peptide, it is likely that HNP-2 is generated from HNP-1 and/or HNP-3 by proteolytic processing (Table 1).

Structure
Defensins are small (35 kDa), nonglycosylated cationic peptides with a b-sheet structure that contain six cysteine residues that form three intramolecular disulde bridges. Defensins are divided into the three families (a, b, and y) based on the spacing and connectivity of the cysteines in the disulde bridges, and on the overall molecular structure. a-Defensins are arginine-rich peptides containing 2935 amino acid residues, with the following disulde alignment: 16, 24, and 35. b-Defensins are 3642 amino acids in length and are characterized by an 15, 24, 36 disulde alignment. y-Defensins are circular minidefensins that arise from two a-defensin mRNA precursors, and are expressed in rhesus monkeys. The genes encoding these defensins are present in humans, but not expressed due to the presence of a premature stopcodon. The genes encoding the human a-defensins and the human b-defensins (hBD) 16 are located in a cluster on the short arm of chromosome 8. The HE2 (human epididimys secretory protein) gene is located in close proximity of the hBD-3 gene. A splice variant of this HE2 gene, HE2b1, is expressed in bronchial epithelium and encodes a cationic peptide containing the six-cysteine-motif that is characteristic of b-defensins. Multiple copies and polymorphisms for several a- and b-defensin genes have been demonstrated, and complicate the ne mapping of this region.

Regulation of Defensin Production

Defensins are produced as precursor polypeptides containing a signal peptide, a propeptide, and the mature peptide. The propiece is removed by proteolytic processing, resulting in the release of the mature peptide. For neutrophil a-defensins, this processing occurs in the developing neutrophils in the bone marrow, and the mature peptide is stored in the azurophilic granules of neutrophils. However, circulating prodefensin levels are increased in patients with inammatory disorders. Paneth cell a-defensins are stored in the secretory granules of these cells, and following secretion into the intestinal lumen processing occurs extracellularly. Paneth cell trypsin has been identied as the major processing enzyme for HD-5. Phagocytosis of microorganisms by neutrophils leads to release of the defensins in the phagolysosome, where they contribute to intracellular killing. Neutrophil stimulation may also lead to release of the granular content into the environment of the cell, thus explaining the high amounts of neutrophil defensins that are observed in inammatory exudates. HD-5 and HD-6 are primarily expressed in the Paneth cells. All human b-defensins that have been identied in some detail (hBD1-4; HE2b1) are expressed in human lung tissue. These b-defensins are mainly

DEFENSINS 9

produced by epithelial cells, but hBD-1 and hBD-2 are also expressed in monocytes, macrophages, dendritic cells, and lymphocytes. Inamed epithelia are a rich source of antimicrobial peptides such as b-defensins. Indeed, production of b-defensins by epithelial cells is mainly inducible under inammatory conditions, although production of hBD-1 is constitutive. Expression of b-defensins is increased by a variety of stimuli. Microbial products increase b-defensin expression through the action of Toll-like receptors (TLRs) such as TLR2, TLR4, and TLR9. TLRs are pattern-recognition receptors for conserved molecular patterns present on microbial products that allow the innate immune system to recognize microbial presence and respond accordingly. In addition, proinammatory cytokines (such as IL-1b, TNF-a, and IL-17), growth factors (such as TGF-a), and activators of protease-activated receptors increase b-defensin expression. Selected microorganisms have been shown to downregulate expression of b-defensins, thus increasing susceptibility of the host to infection.

-Defensins (HNP1 4)

-Defensins (hBD16)

Activation of epithelial cells and fibroblasts

Antimicrobial activity Chemoattractant for T cells, dendritic cells, monocytes, and macrophages Mast cell activation

Biological Function
Most defensins that have been characterized at the peptide level were identied based on their antimicrobial activity. Defensins exert this activity by interfering with the integrity of the microbial membrane. Defensins kill a range of Gram-positive and Gram-negative bacteria, fungi, and viruses. Selected defensins display anti-HIV activity. Differences exist with respect to the selective activity against specific microorganisms for the different defensins. The main sites of antimicrobial action of defensins appear to be the phagolysosome of neutrophils, where ingested microorganisms are killed, and the surface of the skin and mucosa. Defensins in these compartments form part of a cocktail of antimicrobial molecules, and may act in synergy with these molecules. Pathogens and commensals have developed a variety of resistance mechanisms that serve to protect them against the action of antimicrobial peptides such as defensins. This bacterial resistance is determined by a variety of mechanisms, including changes in the charge or structure of the bacterial cell membrane. Also the ability of certain bacteria to decrease epithelial bdefensin expression may contribute to pathogen survival. In addition to their antimicrobial activity, defensins display a range of other activities (Figure 1). Defensins display chemotactic activity for a range of cell types, are involved in adaptive immunity through their effect on T cells and dendritic cells, and activate mast cells, epithelial cells, and broblasts leading to

Figure 1 Defensin production and activities in the lung. Neutrophils inltrating the airways release neutrophil a-defensins (HNP14), whereas airway epithelial cells are the main cell type producing b-defensins. However, other cell types such as lymphocytes, macrophages, and dendritic cells also contribute to b-defensin production in human lung tissue. a- and b-defensins display a range of overlapping and distinct activities. See text for details.

release of proinammatory mediators, proteinase inhibitors, and extracellular matrix components. They also modulate broblast and epithelial cell proliferation and aid in mucin production and wound repair in airway epithelium. The role of human defensins in animal models has been demonstrated in a number of studies. Intratracheal instillation of neutrophil defensins into mice induced inammation, and increased lung permeability and dysfunction. HD-5 transgenic mice that express the human transgene in the intestinal Paneth cells were found to be markedly resistant to oral challenge with Salmonella typhimurium. Further support for the in vivo contribution of defensins to host defense against infection comes from studies in knockout mice lacking murine defensins or the metalloprotease involved in processing of Paneth cell a-defensins. The observation that mice decient in mouse b-defensin 1 (mBD-1) clear Haemophilus inuenzae inefciently from their lungs further supports a role for defensins in defense against respiratory infection. Although various in vivo studies have shown that defensins contribute to host defense against infection, it is likely that many of these observations are not solely explained by direct antimicrobial activity of defensins. The ability of defensins to promote chemotaxis, cause mast cell degranulation, and their other activities (Figure 1) likely contributes markedly to their in vivo antimicrobial action.

10

DENDRITIC CELLS

Receptors
The ability of defensins to activate eukaryotic cells has prompted the search for cellular receptors that bind defensins. This led to the discovery that b-defensins bind to the chemokine receptor CCR6 on immature dendritic cells, and murine b-defensin-2 (mBD-2) activates cells via TLR4. The receptor involved in recognition of a-defensins has not yet been identied.

cause inactivation of defensins. However, these increased salt concentrations in epithelial lining uid of the airways in cystic brosis were not conrmed in all studies. Conversely, because a1-antitrypsin is an inhibitor of the proinammatory activity of neutrophil a-defensins, its deciency may facilitate this activity. Taken together, these data demonstrate that defensins play a role in a variety of lung diseases.
See also: Cystic Fibrosis: Overview. Defense Systems. Leukocytes: Neutrophils. Matrix Metalloproteinases. Mucus. Panbronchiolitis. Pneumonia: Overview and Epidemiology. Proteinase Inhibitors: Secretory Leukoprotease Inhibitor and Elan. Thrombolytic Therapy.

Defensins in Respiratory Disease

Variations in gene-copy numbers and single nucleotide polymorphisms have been described for various a- and b-defensin genes. These polymorphisms may contribute to inammatory lung disease, as demonstrated by the association of single nucleotide polymorphisms in b-defensins with chronic obstructive pulmonary disease (COPD). The contribution of variable copy numbers of defensin genes to disease susceptibility or severity has not yet been established. An increasing number of studies have explored the concentrations of defensins in lung diseases. Defensin levels are increased in airway secretions and plasma in a range of respiratory disorders, which is probably explained by the fact that lung inammation is often characterized by neutrophil accumulation and epithelial cell activation. Lung disorders, in which increased defensin levels are found, include cystic brosis, diffuse panbronchiolitis, acute respiratory distress syndrome, a1-antitrypsin deciency, mycobacterial infections, pneumonia, pulmonary brosis, and sarcoidosis. Whereas increased levels may be a mere reection of inammation or may contribute to disease, functional impairment of defensin activity may also play a role in inammatory lung disease. Defensins show optimal activity at the low-salt conditions that appear to be characteristic for mucosal secretions of the lung. It has been suggested that these salt concentrations are higher in the epithelial lining uid of patients with cystic brosis, and that this may

Further Reading
Bals R and Hiemstra PS (2004) Innate immunity in the lung: how epithelial cells ght against respiratory pathogens. European Respiratory Journal 23: 327333. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews: Microbiology 3(3): 238250. Devine DA (2003) Antimicrobial peptides in defence of the oral and respiratory tracts. Molecular Immunology 40(7): 431443. Devine DA and Hancock REW (eds.) (2005) Mammalian Host Defense Peptides. Cambridge: Cambridge University Press. Gallo RL (ed.) (2005) Antimicrobial Peptides in Human Health and Disease. Norfolk, UK: Horizon Bioscience. Ganz T (2002) Antimicrobial polypeptides in host defense of the respiratory tract. Journal of Clinical Investigation 6: 693697. Ganz T (2003) Defensins: antimicrobial peptides of innate immunity. Nature Reviews: Immunology 3(9): 710720. Schutte BC and McCray PB Jr (2002) b-Defensins in lung host defense. Annual Review of Physiology 64: 709748. Van Wetering S, Tjabringa GS, and Hiemstra PS (2005) Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells. Journal of Leukocyte Biology 77: 444450. Yang D, Biragyn A, Hoover DM, Lubkowski J, and Oppenheim JJ (2004) Multiple roles of antimicrobial defensins, cathelicidins, and eosinophil-derived neurotoxin in host defense. Annual Review of Immunology 22(1): 181215. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415(6870): 389395.

DENDRITIC CELLS
J W Upham and T L Hughes, Telethon Institute for Child Health Research, Perth, WA, Australia
& 2006 Elsevier Ltd. All rights reserved. are especially sensitive to signals derived from microbes, allergens, and the airway tissue microenvironment. They can polarize na ve T cells into either Th1 and Th2 effector cells, and are increasingly recognized as having a central role in the establishment of long-lasting adaptive immunity as well as the maintenance of tolerance to inhaled antigens. DCs form a closely meshed network within the respiratory mucosa, and are rapidly recruited from the circulation in response to a variety of proinammatory stimuli.

Abstract
Dendritic cells (DCs) are specialized, migratory antigen presenting cells which are able to regulate immune reactions. DCs

DENDRITIC CELLS 11
DCs also have a crucial role in the initiation and maintenance of the dysregulated immune responses seen in many respiratory diseases. In allergic asthma, for example, allergens are captured by DCs in the airways, subsequently priming a Th2 immune response, with the subsequent development of allergic airway inammation. Increased numbers of DCs also inltrate the airways and lung parenchyma in a variety of other inammatory diseases. This chapter summarizes the current literature on the origin and function of DCs, as well as their contribution to the pathology seen in respiratory disease. The future is likely to witness a concerted effort to develop a range of new therapies targeted at DCs, including DC-based vaccines and DC-based immunomodulation.

the respiratory intraepithelial tissue, at a density of 5001000 cells mm 2. Under experimental conditions, they can be derived from either lymphoid or myeloid progenitor cells; however, the differentiation pathways that generate DCs in vivo are largely unknown. Over 30 years ago, Ralph Steinman discovered DCs in murine lymphoid tissues. He described them as phagocytic cells with unusual shapes that were distinct from macrophages and had a highly potent stimulatory role in immune function.

Dendritic Cell Subpopulations Introduction

The lung mucosa together with the skin and gastrointestinal tract comprise the extensive interface that exists between the host and the external environment. This faces a continuous threat from foreign proteins and pathogens and thus requires a highly procient immune surveillance system. Dendritic cells (DCs) provide a crucial role in this process. Their migratory nature allows them to sample environmental proteins, process them, and subsequently present them in regional lymph nodes to T lymphocytes, initiating the adaptive immune response. The term dendritic cell arises from their characteristic morphology (Figure 1). These cells extend long, irregular dendrites in order to maximize their surface area and thus their exposure to environmental antigens. They exist as a high-density network in Dendritic cells can be divided into subsets, partly determined by their origin and function but also by their location. Myeloid DCs can be recognized by the expression of the myeloid markers CD11c, CD33, and CD13. They have a lineage relationship with macrophages and monocytes and require granulocyte-macrophage colony-stimulating factor (GM-CSF) for survival. Myeloid DCs can also be differentiated from monocytes in vitro using cytokines such as GM-CSF and interleukin-4 (IL-4) or IL-13. Differentiation of monocytes into DCs is likely to be a normal phenomenon. There seem to be some monocytes (identied by CD16 expression) that are committed to differentiating into DCs. However, in the presence of inammatory signals all monocytes are thought to be able to differentiate into either macrophages or DCs.

Figure 1 (a) Confocal microscopic image of DCs within the epithelium of a normal mouse trachea. Longitudinal section, magnication 40. The extensive branching processes are thought to facilitate antigen capture. These processes retract when DCs migrate to regional lymph nodes. Photograph courtesy of Phil Stumbles and Roz Wealthall. (b) Transmission electron micrograph of myeloid DCs isolated by enzymatic digestion from normal mouse lung. Scale 2 mm. Image courtesy of Phil Stumbles, Christoph von Garnier, and Luis Filgueira.

12

DENDRITIC CELLS

The origin of the plasmacytoid subset of DCs has not been established. They are long-lived cells, which morphologically resemble plasma cells and do not express myeloid markers. They express the IL-3 receptor a chain CD123, and respond to IL-3 for their growth and survival. Producing large amounts of ainterferon, it is thought that their primary role is in the host defense against viral infections and in the development of peripheral tolerance. Langerhans cells (LCs) represent one subpopulation of DCs that are located in epithelial tissues. These cells have been most extensively characterized in the epidermis of the skin where they express a distinctive array of molecules including CD1a, Ecadherin, Langerin, and Birbeck granules. Less is known about LCs in the lung, though it appears that many DCs in the airway epithelium express LC-specic markers.
Dendritic Cells in the Lung

specifically target foreign proteins. These receptors include C-type lectin receptors, such as the mannose receptor, Langerin, and DEC205, as well as receptors such as Fcg and complement receptors, which recognize opsonized bacteria. Specic receptors for the uptake of apoptotic bodies are also expressed. The high expression of the high-afnity IgE receptor FceRI by human asthmatic airway DCs indicates a role for these receptors in the uptake of the inhaled allergens.
Migration and Maturation

Dendritic cells form a tightly meshed network within antigen-exposed tissues of the upper airway, bronchial mucosa, lung interstitium, and pleura. The density of DCs in the airways is related to the extent of antigen exposure, being greatest in the proximal airways, and diminishing towards the periphery. A variety of proinammatory stimuli induce a marked increase in the density of lung DCs, including exposure to bacterial and viral infection.

Dendritic Cell Function in the Normal Lung

Dendritic cells at mucosal surfaces display a distinct phenotype in the steady state, being adept at antigen uptake, and having a poor capacity to present antigen to T cells. They are able to respond immediately and efciently to many microbial and inammatory stimuli via Toll-like and cytokine receptors. Subsequent to antigen uptake and environmental signals, the DC morphology and cell surface marker expression is altered. This facilitates migration to regional lymph nodes, where antigens are presented and T cell responses effected (see Figure 2).
Antigen Uptake

In response to pathogens, tissue damage, or proinammatory cytokines, DCs migrate to regional lymph nodes and acquire the ability to prime na ve T cells. The process of DC maturation involves changes in the expression of specic chemokine receptors, with concomitant downregulation of tissue-homing receptors and upregulation of lymph node-homing chemokine receptors. In addition to chemokines, other mechanisms are likely to regulate DC migration, including integrin and cadherin expression, and extracellular matrix-degrading enzymes, although these have not been well characterized in relation to respiratory tract DCs. Spontaneous migration of DCs to draining lymph nodes is also observed under healthy steady-state conditions in the absence of tissue inammation, although the mechanisms involved are not well characterized. These spontaneously migrating DCs retain a somewhat immature phenotype, even after moving to the lymph nodes, and this is likely to be critical for the development of immune tolerance. A number of mechanisms exist to regulate DC function within the lungs. Freshly isolated lung DCs exhibit a poor capacity for antigen presentation and express only low levels of costimulatory molecules. However, culturing puried lung DCs for short periods in the absence of exogenous stimuli is sufcient to elicit spontaneous DC activation, suggesting that the tissue environment in the lung provides inhibitory signals that counteract DC activation in vivo. Alveolar macrophages are known to maintain lung DCs in an immature state through the release of nitric oxide. Furthermore, airway epithelial cells may also have the ability to regulate DC function.
Antigen Presentation

Antigen uptake occurs via several mechanisms. Constitutive, low-level uptake of small soluble particles is mediated by micropinocytosis, with larger particles also being taken up in the uid phase by the cytoskeleton-dependent process of macropinocytosis. Receptor-mediated endocytosis, however, is 100-fold more efcient at antigen uptake. There are various specialized receptors on the dendritic cell surface that

Dendritic cells not only contribute to the expansion and differentiation of T lymphocytes, but also the qualitative nature of the T cell response. The role of DCs in shaping the differentiation pathway of a na ve T cell is potentially powerful because DCs provide the precursor T cell with its rst activation

DENDRITIC CELLS 13
Antigen capture Dendritic cells at the environmental interface. Antigen uptake is facilitated by receptors specific for opsonins, pathogen-associated molecular patterns and apoptotic bodies

Migration Following antigen uptake and environmental signals, dendritic cells migrate through the lymphatics to regional lymph nodes

Antigen presentation Dendritic cells present the processed antigenic peptideMHC complex. Surface costimulatory molecules facilitate the dendritic cell-T cell interaction

T cell response In response to antigen presentation, costimulatory molecule ligation and cytokine production by the DCs, T cells proliferate and differentiate along one of several pathways

Th1

Th2

Regulatory

Figure 2 The key concepts of DC function. Upon capturing antigen at the environmental interface, DCs migrate via lymphatics to the secondary lymphoid organs. Here they act as potent antigen-presenting cells, inuencing subsequent T lymphocyte responses.

signals. The mechanisms by which DCs differentially regulate Th1 and Th2 immune responses is incompletely understood, but is likely to be due to several factors, such as antigen dose, the spectrum of costimulatory molecule expression, and the inuence of microbial stimuli that induce Th1-polarizing cytokines (IL-12, IL-23, IFN-a). Airway DCs are also able to inuence the induction of peripheral T cell tolerance. Exposure to inhaled innocuous antigen under normal conditions induces a state of immunological tolerance. In this regard, it has been well established that soluble antigen delivered via the respiratory mucosa, in the absence of inammatory signals or sensitized T cells, induces a profound state of T cell tolerance

following systemic antigen challenge. Recent studies have conrmed a role for airway DCs in mediating this tolerogenic process. This mechanism is governed by the expression of IL-10 by pulmonary DCs that drives the generation of a population of IL-10-producing regulatory T cells (T reg) in draining lymph nodes capable of suppressing subsequent responses to antigenic challenge. T reg induction is critically dependent on inducible costimulatory (ICOS)inducible costimulatory ligand (ICOSL) interactions, suggesting the requirement for specialized costimulatory pathways in this process. Thus, although the mechanisms governing the development of T cell tolerance or sensitization are not entirely clear, it seems likely that this will involve the coordinated

14

DENDRITIC CELLS

regulation of DC maturational state together with DC-derived cytokines.

fungi, the extent to which deciencies in lung DC function contributes to particular human pulmonary infections has not been well dened.
Therapeutic Considerations

Dendritic Cells in Respiratory Diseases

Dendritic Cells in Asthma and Allergic Rhinitis

In both asthma and allergic rhinitis, increased numbers of dendritic cells are present within the airway mucosa of the lung and nose, respectively. Many of these cells express the high-afnity IgE receptor, and treatment with inhaled corticosteroids leads to a decrease in the numbers of DCs within the airway mucosa. Following allergen inhalation challenge in allergic asthma there is a rapid reduction in the numbers of myeloid DCs within the circulation as myeloid DCs are recruited to the lung under the inuence of chemokines and leukotrienes. In contrast, while short-term allergen inhalation does not appear to alter plasmacytoid DC numbers in the airway, repeated low-dose allergen exposure over several days induces the accumulation of plasmacytoid DCs in the nasal mucosa. Increasing evidence points to changes in the status of blood DCs in asthma. Alterations in the relative proportions of plasmacytoid versus myeloid DC have been reported in asthma. Various allergens and microbial stimuli have the ability to directly alter the function of DCs, inducing both DC activation and secretion of inammatory mediators such as IL-1b, IL-6, TNF-a, IL-10, IL-12, and PGE2. Importantly, the pattern of mediator release differs between atopic and nonatopic individuals, and this may be one mechanism by which DCs contribute to Th1/Th2 polarization and the pathogenesis of atopy and allergic airway inammation.
Dendritic Cells in Other Respiratory Diseases

Various other diseases of the respiratory tract are associated with changes in the number of dendritic cells in the airway mucosa or lung parenchyma, including idiopathic non-specic interstitial pneumonia, hypersensitivity pneumonitis, obliterative bronchiolitis following lung transplantation, diffuse panbronchiolitis, Langerhans cell histiocytosis, and cigarette smoking. In each of these conditions it is not clear whether the observed accumulation of DCs in the lung plays a role in the pathogenesis of the disease, or has developed as a non-specic or even protective response to lung injury or inammation, so there is a clear need for further longitudinal studies of DC function in each of these conditions. While it is clear from various animal models that DCs play a key role in the development of protective immunity to viruses, bacteria (including Mycobacteria), and

Because DCs are extremely responsive to external stimuli, and because they are thought to play a role in many disorders of the respiratory tract, there is considerable interest in the development of new therapeutic strategies targeted at DCs. In inammatory lung diseases such as asthma, the aim is to inhibit DC function in order to reduce inammation. By contrast, in pulmonary infection and malignancy, the aim is to augment DC function in order to enhance adaptive immunity directed against microbial pathogens or tumor antigens. Many well-known anti-inammatory drugs are known to inhibit DC activation, including corticosteroids, cyclosporin, and tacrolimus, though these agents clearly affect other cells such as lymphocytes and other molecules involved in the inammatory cascade. Corticosteroids inhibit DC maturation and antigen uptake and induce DC apoptosis, while agents such as vitamin D analogs and N-acetylcysteine block DC maturation via inhibition of the transcription factor (i.e., nuclear factor kappa B (NFkB)). Imidazoquinolines (e.g., imiquimod and resiquimod) and specic immunostimulatory sequences derived from bacterial DNA (e.g., CpG motifs) enhance the ability of DCs to release Th1-polarizing cytokines such as IL-12 and IFN-a. In the search for new vaccines against pulmonary infections, researchers are increasingly seeking to target DCs in order to develop protective immune memory. DC-based therapies for lung cancer have been tested in phase 1 and II clinical trials, using the patients own DCs pulsed with tumor antigens. As more is learnt about DC biology and its role in a variety of lung diseases, it is likely that new molecules will be developed that specifically target DCs.
See also: Allergy: Overview. Asthma: Overview. Chemokines. Dust Mite. Epithelial Cells: Type I Cells; Type II Cells. Interferons. Interleukins: IL-1 and IL-18; IL-4; IL-5; IL-6; IL-7; IL-9; IL-10; IL-12; IL-13; IL-15; IL-16; IL-17; IL-23 and IL-27. Interstitial Lung Disease: Overview. Leukocytes: Monocytes; Pulmonary Macrophages.

Further Reading
Akbari O, Freeman GJ, Meyer EH, et al. (2002) Antigen-specic regulatory T cells develop via ICOS-ICOS ligand pathway and inhibit allergen-induced airway hyper reactivity. Nature Medicine 8: 10241032. Banchereau J and Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673): 245252.

DIFFUSION OF GASES 15
Cochand L, Isler P, Songeon F, and Nicod LP (1999) Human lung dendritic cells have an immature phenotype with efcient mannose receptors. American Journal of Respiratory Cell and Molecular Biology 21: 547554. Engleman EG (2003) Dendritic cell-based cancer immunotherapy. Seminars in Oncology 30(3 supplement 8): 2329. Hammad H, Charbonnier AS, Duez C, et al. (2001) Th2 polarization by Der p 1-pulsed monocyte-derived dendritic cells is due to the allergic status of the donors. Blood 98: 11351141. Holt PG and Upham JW (2004) The role of dendritic cells in asthma. Current Opinion in Allergy and Clinical Immunology 4: 3944. Lambrecht BN, De Veerman M, Coyle AJ, et al. (2000) Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inammation. Journal of Clinical Investigation 106: 551559. Lambrecht BN and Hammad H (2003) Taking our breath away: dendritic cells in the pathogenesis of asthma. Nature Reviews: Immunology 3(12): 9941003. Long J, Fogel M, Thompson PJ, and Upham JW (2004) Higher prostaglandin E2 production by dentritic cells from subjects with asthma compared with normal subjects. American Journal of Respiratory and Critical Care Medicine 170: 485491. Mellman I and Steinman RM (2001) Dendritic cells: specialized and regulated antigen processing machines. Cell 106(3): 255258. Moser M and Murphy KM (2000) Dendritic cell regulation of Th1Th2 development. Nature Immunology 1(3): 199205. Tailleux L, Schwartz O, Herrmann JL, et al. (2003) DC-SIGN is the major Mycobacterium tuberculosis receptor on human dendritic cells. Journal of Experimental Medicine 197: 121127. Todate A, Chida K, Suda T, et al. (2000) Increased numbers of dendritic cells in the bronchiolar tissues of diffuse panbronchiolitis. American Journal of Respiratory and Critical Care Medicine 162: 148153. Tunon-de-lara JM, Redington AE, Bradding P, et al. (1996) Dendritic cells in normal and asthmatic airways: expression of the a subunit of the high afnity immunoglobulin E receptor (FceRIa). Clinical and Experimental Allergy 26: 648655. Upham JW, Denburg JA, and OByrne PM (2002) Rapid response of circulating myeloid dendritic cells to inhaled allergen in asthmatic subjects. Clinical and Experimental Allergy 32: 818823. Upham JW and Stumbles PA (2003) Why are dendritic cells important in allergic diseases of the respiratory tract? Pharmacology and Therapeutics 100: 7587.

DIFFUSION OF GASES
R E Forster, University of Pennsylvania, Philadelphia, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
This article summarizes different diffusion processes that transport respiratory gases in the body, their theoretical bases and pertinent equations. Gases move within cells by diffusion alone, so it is essential to maintain life. Gases that are neither metabolized nor produced (inert gases), such as nitrogen and anesthetic gases, equilibrate between capillary blood and alveolar gas or peripheral tissues in one capillary transmit time. Metabolism of O2 in a cell simultaneously with diffusion causes a marked drop in PO2 with distance from a capillary which limits the size of a cell, even under steady state conditions. Chemical reaction simultaneous with diffusion also severely reduces the transient rate of uptake of O2, CO, and NO by red cells; the more rapid the chemical reaction, the greater the relative reduction. The rate of CO uptake by red cells limits the rate of its uptake in the lungs. Gas diffusion can be facilitated by reacting reversibly with a carrier molecule which also diffuses, adding to the net ux. Cell membranes have been considered highly permeable to gases, because the gases dissolve in membrane lipid but recently some have been found to be relatively impermeable and some to contain membrane proteins which transport gas.

produced by the random thermal motion of the molecules. Diffusion can be considered a mixing process in which a particular molecular species moves under its concentration gradient independent of other molecules. It is the ultimate transport mechanism for respiratory gases as it alone carries them between capillary blood and cell cytoplasm. The rate of diffusion (dq/dt in mol s 1) in a steady state is proportional to the concentration gradient (@ c/ @ x in mol cm 3 cm 1) times the area (A in cm2), times a constant, the diffusion coefcient (d in cm2 s 1): dq=dt Ad@ c=@ x 1

Basic Concepts
Diffusion is the statistical movement of molecules from a region of higher to one of lower concentration

This equation is known as Ficks rst law of diffusion. (Note: respiratory physiologists and clinicians have been measuring partial pressure in mmHg with mercury monometers for centuries and most workers in the eld today are most familiar with these units, at least in the USA, so these will be used rather than units derived from the cgs system. Cubic centimeters (cc) and milliliter is the volume of 1 g water at 41C and is equal to 1.0000 27 cc, a discrepancy immeasurable by physiological or clinical techniques. Cubic centimeters will be used in this article as the measure of volume primarily to balance the units in several equations.) The rate of diffusion of a molecular species is proportional to the mean linear velocity

16

DIFFUSION OF GASES

(v in cm s 1) of its molecules. The kinetic energy of the gas, (1/2) mv2, where m is the molecular weight in g mol 1, approximately equals its thermal energy, RT. Therefore RT 1=2 mv2 2

where R perfect gas constant in ergs mol 1 1C 1 and T absolute temperature in kelvin. Therefore, the rate of diffusion of a molecular species is proportional to (T/m)1/2. A lighter gas diffuses more rapidly and all diffuse faster at higher temperatures. Diffusion in a gas phase, as in alveoli, is about a million times faster than in watery solution; it is difcult to separate from convection and will not be discussed here. Gas diffusion in the airways can be calculated from changes in volume of the dead space. In the body, gases exist in gas phases and liquid phases. At chemical equilibrium the concentration of a gas in the liquid equals its partial pressure, p (in mmHg), in the gas to which it is exposed, times its solubility in the liquid, a (in mol cm 3 mmHg 1), a relation which is known as Henrys law: c ap 3

volume (lefthand side or LHS) equals the change in concentration of the gas in that volume. Solutions for this differential equation for various geometries are available in the literature and show that inert gases equilibrate between alveolar gas and capillary blood early in its transit. This conclusion is supported by the nding that calculations of pulmonary blood by inert gas uptake in the lungs, which assume such equilibria, agree with measurements by other techniques. Similarly, the halftime computed for inert gas to equilibrate between blood in a 8 mm diameter capillary and a surrounding peripheral tissue cylinder of 36 mm diameter is less than 0.1 s. With a capillary transit time of 1 s the gas would be completely equilibrated in the tissue cylinder. This means that inert gas exchange in body tissues is not rate limited by diffusion but by capillary blood/tissue volume and relative gas solubility. This is pertinent to exchange of anesthetic gases and decompression disease (bends).

Diffusion and Simultaneous Chemical Reaction: Steady State

The rate of diffusion of gases that react with the cytoplasm is determined as much or more by the rate of the chemical reaction as by diffusion itself. Some gases are consumed (O2), bound (CO, NO), or produced (CO2) as they diffuse through a tissue. Under steady-state conditions this is described as follows, using O2 as a model: d@ 2 O2 =@ x2 m 6

A corollary to this is that a dissolved gas in a liquid exerts a measurable partial pressure or escaping tendency, even though there is no gas phase present. Substituting eqn [3] in eqn [1] we obtain dq=dt Aad@ p=@ x 4

Much of gas transport in the body takes place between a gas phase and a liquid phase or between two liquid phases with different solubilities. At the boundary there is a discontinuity in gas concentration but not in p, in which case eqn [4] should be used.

The term on the left-hand side is the difference between the rate of diffusion of O2 into and out of a differential volume, and m is the rate of consumption of O2 in that volume, measured in mol s 1. The solution of this equation for a spherical cell with p as the driving force is
3 PO2 ;o PO2 m=6adr2 or

Inert Gases
Gases that do not react chemically with cell cytoplasm are called inert gases; these include nitrogen, helium, and anesthetic gases. They diffuse so rapidly that their rate of equilibration with tissues cannot be measured directly by experiment. By default, reliance has been on theoretical calculations using solutions of the following equation (Ficks second law of diffusion) for nonsteady, that is, transient conditions: d@ 2 c=@ x2 dc=dt 5

This equation states that the difference between the diffusion of a gas into and out of a differential

where PO2 ;o oxygen partial pressure in mmHg at the surface of the cell, PO2 oxygen partial pressure in mmHg at the radial distance r (cm) from the surface of the cell, d diffusion coefcient of O2 in 7.6 10 6 cm2 s 1, and m is the rate of oxygen metabolism in mol cm 3 s 1 and is considered uniform with r and with PO2 above 2 mmHg. PO2 ;o PO2 is the partial pressure drop at a distance r from the center of the cell. The greater the metabolic rate, m, and the lower the diffusion coefcient, d, the greater the drop. The maximum size of the cell is reached when PO2 in the center (r 0) becomes less

DIFFUSION OF GASES 17

than approximately 2 mmHg. For resting skeletal muscle and cardiac muscle, with m equal to 0.00196 and 0.08 mM s 1, respectively, the maximal cell radii are 354 and 50 mm, respectively. In order to get around this limitation on size, multicellular organisms have developed circulatory systems to convect a uid that binds oxygen between an exchanger that equilibrates with the ambient medium (lungs, gills) and one that exchanges with peripheral cells (tissue capillaries). Peripheral capillaries deliver O2 to only one surface of a cell, but do not surround a cell as is implied in eqn [7]. A more realistic model of mammalian skeletal muscle is a capillary as a tube surrounded by a concentric cylinder of muscle cells for which the decrease in PO2 with radial distance is described by this solution of eqn [6]:
2 PO2 PO2 ;o M=2ar2 r2 c =2 rt ln r=rc

60

PO 2 difference (mmHg)

Hear t mus cle

1 5 10 Tissue cylinder radius (m)

Figure 1 The log10 of the PO2 difference between the surface of a skeletal muscle capillary and the perimeter of a surrounding concentric cylinder of cells is plotted against the radius of the cylinder for heart muscle (open circles) and resting skeletal muscle (solid circles) with O2 consumption of 0.08 and 0.002 mM s 1, respectively. Data points were computed using eqn [8]. After Landis and Pappenheimer.

Res ting

10

ske

leta

l mu

scle
100

where PO2 the oxygen partial pressure at any radial point in the tissue cylinder, PO2 ;o is the oxygen partial pressure at the junction of the surface of the capillary and the tissue, r is the radial distance, rc is the radius of the capillary, the inner cylinder, and rt is the radius of the tissue cylinder of the muscle ber. This is known as the KroghErlang equation because it was developed for August Krogh in his original explanation of how tissue capillaries delivered oxygen to cells. The decrease in PO2 with radial distance from the capillary surface as calculated using eqn [8] is plotted in Figure 1 for resting skeletal muscle cells and cardiac cells. The important point is that these graphs demonstrate the maximum distance a muscle ber of a given O2 consumption can be from a capillary and still be supplied with O2. Resting striated muscle can survive at a greater distance from a capillary than cardiac muscle because of its lower metabolic rate. During exercise additional capillaries open up, effectively reducing the diffusion distance to the perimeter of the model cylinder. In the early part of the twentieth century it was known that blood entered the lung with HbO2 about 75%, corresponding to 60 mmHg PO2 , and left the lung nearly 100% saturated with O2 corresponding to 100 mmHg. The mechanism by which O2 was transported from alveolar gas to the capillary blood hemoglobin was in dispute. Thus, it became important to determine the ability of the lung to equilibrate mixed venous blood with alveolar gas. In the lung, blood capillaries with the internal diameter of a red blood cell (8 mm) are separated from gas in the alveoli only by the alveolar membrane, consisting of single layers of capillary endothelium

200

and alveolar epithelium, a diffusion distance of only several micrometers. O2 was considered to diffuse from alveolar gas into the capillary blood where it bound reversibly and instantaneously with hemoglobin in the erythrocytes. Any O2 gradients in capillary blood were neglected in comparison to the diffusion resistance of the alveolar membrane. O2 uptake in this model could be calculated by eqn [1] as follows, with units changed to those commonly using clinically: dVO2 =dt DL PA;O2 average PC;O2 9

where VO2 is the amount of oxygen diffusing across the capillary wall (alveolar membrane) in cc, t time in min, DL was originally called the diffusing capacity of the lung, a phenomenological transfer factor in cc mmHg 1 min 1, PA;O2 alveolar PO2 in mmHg, and PC;O2 capillary blood PO2 in mmHg. VO2 can be measured as the volume of inspired minus expired O2, or the rate of change of alveolar O2 concentration alveolar volume. Alveolar PO2 varies with time during the respiratory cycle and amongst alveoli, depending on the ventilation/capillary blood ow. While a reasonable average value

18

DIFFUSION OF GASES

can be obtained for the alveolar PO2, this is not true for the capillary blood PO2 . As an increment of O2 is taken up by the blood and combines with hemoglobin, the blood PO2 rises by an amount determined by the S shaped oxygenhemoglobin equilibrium curve, for which there is no useful analytical expression. Equation [9] can be solved by a numerical integration (Bohr integration), but the difference between alveolar and end capillary blood PO2 must be known and is critical. First, a sample of this blood cannot be obtained; only arterial blood is available, and this is a mixture from all the alveoli and is contaminated by small amounts of blood that bypass the alveoli. Second, although it was possible to measure blood O2 content and HbO2 saturation, prior to the 1950s it was not possible to measure PO2 accurately. Thus, it was not certain whether end capillary blood PO2 equilibrated with alveolar gas or not. J S Haldane in fact argued for a long time that the lung could secrete O2 actively when necessary, for example, at high altitude or during heavy exercise, raising arterial PO2 higher than alveolar gas. A great advance in studies on gas diffusion in the lung was made by Christian Bohr and August and Marie Krogh who used CO exchange instead of O2. CO, like O2, binds reversibly with hemoglobin but with 225 times the afnity. The Pco for 50% saturation of HbCO is about 0.1 mmHg, while that of O2 is 28 mmHg. Therefore, with a low and safe, for a short period, alveolar Pco, the increase in blood HbCO during capillary transit is negligible. Thus, DL,CO can be calculated from eqn [9] substituting dVCO/dt and PA,CO for those terms in O2 and neglecting blood HbCO. DL;O2 can then be calculated from DL,CO on the assumption that diffusion through a watery alveolar membrane between alveolar gas and capillary blood is rate limiting. In this case the rate of diffusion is proportional to the solubility of the gas in water and inversely proportional to the square root of its molecular weight (Grahams law): DL;O2 DL;CO mCO =mO2 1=2 aO2 =aCO 10

It was next found that DL,CO decreases when alveolar PO2 increases, which is explained by the competition of O2 and CO for hemoglobin. The rate of formation of HbCO in hemoglobin solution is given by the bimolecular reaction: dHbCO=dt k1 COHb k1 HbCO 11

where k1 is the reaction velocity constant for the binding of the rst CO on tetrameric hemoglobin in mM 1 s 1, k 1 is the reaction velocity constant for the dissociation of HbCO in s 1, and CO, Hb and HbCO are the concentrations of carbon monoxide, unliganded (reduced) hemoglobin and carbonmonoxyhemoglobin in mM. k1 and k 1 can be measured in a solution of hemoglobin by several types of rapid reaction apparatus. When PO2 is present, as it in this case, [Hb] decreases slowing the rate of formation of HbCO by mass action.

Diffusion and Simultaneous Chemical Reaction: Transient

The rate of uptake of CO by red blood cells can be measured best in a continuous-ow rapidmixing apparatus and at a normal alveolar PO2 of 100 mmHg is found to be 33% of that in solution. This reduction in CO uptake rate is caused by the interaction of diffusion and chemical reaction, an important phenomenon in reactive gas exchange. Theoretically this combined process is described by the following equation: @ 2 CO=@ x2 @ CO=@ t k1 COHb k1 HbCO 12

giving a value for the ratio of 1.20. A normal value for DL,CO is 31 cm3 mmHg 1 min 1 (single breath technique) and doubles with heavy exercise, increasing the uptake of O2 for the same mean O2 gradient from alveolar gas to capillary blood. This explains how arterial blood PO2 is maintained during exercise, obviating the need to propose O2 secretion. From the O2 consumption and DL;O2 , using the Bohr integration, one can show that end capillary PO2 is almost completely equilibrated with alveolar gas.

which states that the difference between the rate of CO diffusing into and out of an innitesimal volume equals the rate of change of CO less the net consumption of CO to form HbCO in that volume. It is not possible to obtain an analytical solution of eqn [12] because of the nonlinear term k 1 [CO][Hb]. However, representing the red blood cell as a semiinnite layer of 30% hemoglobin 0.7 mm thick, it can be shown that upon suddenly exposing the surface to CO, a prole of [CO] in the layer will be established before a significant increase in Hb occurs at any point. The average of this prole equals the mean CO concentration in the layer, which multiplied by the initial concentration of Hb, obtained from the hemoglobin equilibrium curve, gives an initial value of d[HbCO]/dt from eqn [11], HbCO remaining constant. This corresponds to the initial slope of the experimentally determined rate of [HbCO] increase in a red blood cell suspension. It

DIFFUSION OF GASES 19
1.00

Measurement of Capillary Blood Volume In Vivo

CO + 598 mmHg O2

Fraction of surface partial pressure

0.80

0.60 CO + 100 mmHg O2 0.40

The decrease in DL,CO as alveolar PO2 increases proves that the uptake of CO in the lungs is rate limited by the rate at which red blood cells in the alveolar capillaries can take up CO. Considering CO uptake in the alveoli as passing through two resistances in series, that of the alveolar membrane and that of the red blood cells in the capillaries, we can state that Total lung resistance membrane resistance red blood cell resistance

13

0.20 NO + 100 mmHg O2 0.00 0.00 0.14 0.28 0.42 0.56 0.70

Distance from surface into hemoglobin layer (m)

Figure 2 Initial partial pressure of a ligand gas in a 0.7 mm layer of 30% hemoglobin as a fraction of its value at the surface calculated from eqn [12] and plotted against distance into the layer. Unliganded Hb was considered constant, at pH 7.4 and 371C. PCO and PNO were 75 mmHg.

By analogy with Ohms law, resistance equals (partial pressure difference across the path)/(rate of gas ow). The total resistance is thus the reciprocal of the diffusion capacity. In succinct form: 1=DL;CO 1=Dm 1=DRBC 14

was found experimentally that the rate of uptake of a ligand gas by red blood cells did not increase indefinitely with the reaction rate in hemoglobin solution, but approached a limit. This is illustrated in Figure 2, which presents the initial prole of the partial pressure of ligand gas in the hemoglobin layer as a fraction of its value at the surface for three cases with increasing reaction velocity constants. These are CO in the presence of 585 mmHg PO2 and CO and NO in the presence of 100 mmHg with reaction velocity constants of 137 525, and 25 000 mM 1 s 1, respectively. The mean ligand gas partial pressure in the layer as a fraction of its value at the surface is 0.85, 0.52, and 0.02, respectively. Therefore, the velocity constants for the uptake of ligand in the layer would be 116, 273, and 500 mM 1 s 1 and experimental measurements on red blood cells are in agreement. The uptake of NO by red blood cells approximates the conditions for an innite reaction velocity, the so-called advancing front process, in which the ligand reacts instantaneously with the hemoglobin in the rst differential volume, but requires time to diffuse through it to the next step, and so on. Red blood cells cannot take up a ligand any faster than this limit.

where Dm diffusion capacity of the membrane alone in cc min 1 mmHg 1 and DRBC so-called CO diffusion capacity of the red blood cells in the capillary in cc min 1 mmHg 1. This can be broken down into YCO VC, where YCO is the rate of uptake of CO by a cubic centimeter of blood in the alveolar capillaries in cc min 1 mm Hg 1 and VC is the volume of blood in the capillaries in cc: YCO k1;c aCO blood CO capacity 60 s per min 15

k1,c is a pseudo bimolecular reaction velocity constant calculated from eqn [11] with: (1) d[HbCO]/dt taken from the initial slope of a graph of [HbCO] versus time measured with a continuous-ow rapid reaction apparatus; (2) [CO] considered that at the surface of the cells; (3) [Hb] obtained from the PO2 and an HbO2 equilibrium curve; and (4) [HbCO] neglected. Blood CO capacity in cubic centimeters of gas per cubic centimeter of blood is that found in the capillary, which is considered the same as that in peripheral blood. Normally this is 0.2 cc cc 1 but is decreased in anemia, as is DL,CO. By measuring DL,CO at different alveolar PO2 greater than 100 mmHg, eqn [14] can be solved for VC and Dm. Representative values are about 100 cc for VC, and about 65 cc min 1 mmHg 1 for Dm.

20

DIFFUSION OF GASES See also: Arterial Blood Gases. Carbon Dioxide. Carbon Monoxide. Diving. Extracorporeal Membrane-Gas Exchange. Hemoglobin. Myoglobin. Nitric Oxide and Nitrogen Oxides. Oxygen Hemoglobin Dissociation Curve. Peripheral Gas Exchange. Pleural Effusions: Overview. Pulmonary Function Testing in Infants.

Facilitated Diffusion
Facilitation of gas diffusion occurs when the gas combines rapidly and reversibly with another molecule, a carrier, which also diffuses, adding to the total transport. The carrier molecule is generally much larger than the gas molecule but is in greater concentration. Examples are O2 and CO reacting with hemoglobin in red blood cells and myoglobin in muscle, and CO2 forming H and HCO3 . The pair of equations describing steadystate-facilitated transport, using O2 and hemoglobin as an example, are as follows: dO2 @ 2O2 =@ x2 dHbO2 @ HbO2 =@ x2 k1 HbO2 k1 HbO2 16

Further Reading
Cotes JE (1979) Lung Function, 4th edn., chs. 8 and 9, pp. 203250. Oxford: Blackwell. Crank J (1956) Mathematics of Diffusion. Oxford: Clarendon. Donaldson TL and Quinn JA (1974) Kinetic constants determined from membrane transport measurements: carbonic anhydrase activity at high concentrations. Proceedings of the National Academy of Sciences, USA 71: 49954999. Forster RE, II, Dubois AB, Briscoe WA, and Fischer AB (1986) The Lung, 3rd edn., ch. 8 and appendices, pp. 190222. Chicago, IL: Year Book Medical Publishers. Forster RE (1987) Diffusion of gas across the alveolar membrane. In: Fishman AP, Farhi L, Tenney SM, and Geiger SR (eds.) The Respiratory System, Handbook of Physiology, vol. IV, ch. 5, sect. 3, pp. 7188. Bethesda, MD: American Physiological Society. Forster RE (1987) Rate of gas uptake by red cells. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 33, sect. 3, pp. 827838. Bethesda, MD: American Physiological Society. Forster RE (1987) Diffusion of gases. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 32, sect. 3, pp. 839872. Bethesda, MD: American Physiological Society. Forster RE, Gros G, Lin L, Ono Y, and Wunder M (1998) The effect of 4,40 -isostilbene-2-20 -disulfonate on CO2 permeability of the red blood cell membrane. Proceedings of the National Academy of Sciences USA 95: 1581515820. Kety SS (1951) The theory and application of the exchange of inert gas at the lungs and tissues. Pharmacological Reviews 3: 140. Landis EM and Pappenheimer JR (1963) Exchange of substances through capillary walls. In: Hamilton WF (ed.) Circulation, Handbook of Physiology, vol. II, ch. 29, sect. 2, pp. 9611034. Bethesda, MD: American Physiological Society. Nicholson P and Roughton FJW (1951) A theoretical study of the inuence of diffusion and chemical reaction velocity on the rate of exchange of carbon monoxide and oxygen between the red cell corpuscle and surrounding uid. Proceedings of the Royal Society of London, Series B 138: 241264. Roughton FJW (1987) Transport of oxygen and carbon dioxide. In: Fenn WO and Rahn H (eds.) Respiration, Handbook of Physiology, vol. I, ch. 32, sect. 3, pp. 767826. Bethesda, MD: American Physiological Society.

Analogous sets of equations describe other facilitated pairs. These equations cannot be solved analytically because of the nonlinear (bimolecular) reaction term but have been approximated with numerical integration. The expression for facilitation of isotopically labeled 13CO2 transport at chemical equilibrium can be solved analytically because H in the nonlinear term in the equation is constant. Facilitation has been measured experimentally in articial layers, but is more difcult to prove in vivo.

Diffusion through Cell Membranes

Until recently it has been assumed that gases pass easily through cell membranes by dissolving in them. It is technically difcult to substantiate this experimentally because exchanges across the membrane are so rapid and the membranes so permeable that diffusion gradients outside the cells, stagnant layers, and diffusion within the cells cannot be differentiated from gradients across the membrane itself. However, the apical membranes of some epithelial cells of the gastrointestinal tract have recently been found experimentally to be relatively impermeable to CO2, permitting measurements. There is some evidence that gases such as CO2 and NH3 may pass through channels or membrane proteins.

Dilators

see Bronchodilators: Anticholinergic Agents; Beta Agonists; Theophylline.

DIVING 21

DIVING
R E Moon and J P Longphre, Duke University Medical Center, Durham, NC, USA
& 2006 Elsevier Ltd. All rights reserved.

Immersion Effects
In the upright position on dry land gravity tends to cause blood to pool in the legs. During water immersion the hydrostatic pressure of the water counteracts the venous pressure, reducing venodilatation and causing blood in the legs to redistribute into the large veins and pulmonary vessels. The resulting increase in cardiac preload is equal to a transfusion of 500800 ml of blood. The reduction in lung volumes (e.g., vital capacity) is augmented by cold water, in which active vasoconstriction increases the volume of redistributed blood, and a negative static lung load when the diver is in the head-up position (Figures 1 and 7). Lung engorgement also reduces lung compliance (Figure 2).

Abstract
Humans are poorly adapted to the underwater environment. Breath-hold diving is limited by limits on the capacity of the thorax to compress during descent, and ability to withstand hyperoxia and hypercapnia. Using compressed gas technology humans can breathe underwater, although with effects on pulmonary statics, dynamics, and gas exchange. Breathing compressed gas causes an increase in density, leading to impairment of both inspiratory and expiratory ow, and hence a reduction in maximum exercise ventilation. In addition, there is an increased Bohr dead space, which can further lower alveolar ventilation. Hypercapnia is common during diving. Immersion causes blood redistribution from the periphery into the lung, and a decrease in compliance. Gaseous compounds exert their physiological effects in proportion to their partial pressures. Thus, gases that are in concentrations that are safe to breathe at 1 ATA can become toxic during diving, including oxygen. Pulmonary O2 toxicity can occur with prolonged breathing of gas with a PO2 that exceeds 0.50.6 atmospheres (ATA). Other diving hazards include pulmonary barotrauma, and pulmonary edema due to venous gas embolism or the hemodynamics of immersed exercise. Mild long-term reductions in pulmonary function have been observed in professional divers and may be due to effects other than diving. Some lung conditions preclude safe diving.

Breath-Hold Diving
The world record for a breath-hold dive is currently 171 m (18 atm absolute, ATA, Figure 3). In order for a select group of humans to reach this depth and return alive, several physiological challenges must be met. During a breath-hold dive lung volume decreases with the increase in hydrostatic pressure according to Boyles law. During descent, lung volume will eventually reach residual volume (RV). Total lung capacity (TLC)/RV ratio is a predictor of maximum achievable. In order to allow for further lung volume reduction, continued descent will cause stretching of the diaphragm. Additional lung volume reductions can be accommodated by translocation of blood into pulmonary vessels to a limit determined by the allowable vascular transmural pressure. When this limit is exceeded, pulmonary capillary rupture can occur, causing intrapulmonary hemorrhage (referred to as lung squeeze). Adaptations that facilitate deep breath-hold diving include compliant chest wall, high TLC/RV ratio, and high pulmonary vascular compliance. During the descent, alveolar gas tensions rise in direct proportion to the ambient pressure. For instance, if alveolar PO2 is 100 mmHg at the surface, then an instantaneous compression to 10 m (2 ATA) would cause the alveolar PO2 to rise to 200 mmHg. At depth, O2 is continuously taken up by the pulmonary blood and CO2 is released. The breath-hold break point is determined mainly by the rising PCO2 , thus the diver begins ascending while the PO2 is still high. However, during ascent the alveolar gas partial pressure falls due to the reduced ambient pressure.

Introduction
Humans are poorly adapted to the underwater environment, and breath-hold dives are limited to only a few minutes duration. Early attempts to make use of compressed gas technology were impaired by physiological problems such as hypothermia and decompression sickness. The latter occurs during decompression, when the reduction in ambient pressure occurs at a rate such that the inert gas (e.g., N2) in tissues supersaturates and forms bubbles. The practical realization of commercial, military, and recreational diving has occurred through technological advances in compressed gas delivery systems, thermal protection, and an understanding of the physiology of inert gas elimination. Divers breathing compressed gas mixtures can stay underwater for prolonged periods, even up to weeks at a time. Nevertheless, both breath-hold and compressed gas diving induce physiological changes in respiratory function that can limit diver performance and cause morbidity.

22

DIVING

RV VT

Standing dry

Immersed in water

ERV FRC

25 mmHg

RV

70 mmHg

140 mmHg

Figure 1 During immersion there is a central redistribution of blood due to the hydrostatic effect of the water column. In addition, depending upon the divers position, there is a static lung load, which in the upright position amounts to approximately 25 mmHg. Redrawn from Moon RE and Stolp BW (1997) The lung during anesthesia and in adverse environments. In: Schwinn D (ed.) Scientic Principles of Anesthesia, Atlas of Anesthesia (Miller RD, series ed.) vol. 2, pp. 6.26.15. Philadelphia, PA: Current Medicine, with permission.

As the diver nears the surface he may become hypoxic and lose consciousness (see Figure 4). Breath-hold time can be increased by at least two mechanisms. The rst is a low respiratory exchange ratio CO =V O : This can occur if carbohydrates have V 2 2 not been consumed for several hours, when basal metabolism is fueled to a greater extent by lipid. This slows the rise in PCO2 during a breath-hold, and thus postpones the break point. The second mechanism by which breath-hold time can be prolonged is prior hyperventilation. This reduces the PCO2 at the start, but increases O2 tension minimally. Both of these factors increase the likelihood of hypoxia. Other

mechanisms are probably in play for maintaining brain PO2 . Arterial gas embolism has been observed in individuals who repetitively breath-hold dive. The mechanism may involve air entering pulmonary blood vessels due to pulmonary stretch injury or vascular rupture as described above.

Compressed Gas Diving

Anyone attempting to breathe from a snorkel at a depth exceeding a few centimeters will realize the impossibility of inhaling against the high hydrostatic

DIVING 23

pressure (see Figure 5). Thus, underwater breathing can only occur if the breathing gas is delivered at an ambient pressure close to that of the diver. Each 10 m (33 feet) of depth is equivalent to 1 atm of pressure. Thus, the ambient pressure at 30 m depth is 4 ATA (1 ATA at the surface plus an additional 3 ATA). Air is the most commonly used breathing gas. The maximum working depth breathing air is limited by

1000 Lung volume (ml)

Submersion to xiphoid

Submersion to neck

750

500

250

5 10 15 Pressure (cmH2O)

20

Figure 2 Pressurevolume relationships in the lung during immersion. Redistribution of blood into the thorax during immersion to the neck reduces lung compliance. Reproduced from Hong SK, Cerretelli P, Cruz JC, and Rahn H (1969) Mechanics of respiration during submersion in water. Journal of Applied Physiology 27: 535538, used with permission from The American Physiological Society.

the narcotic properties of high pressure nitrogen (nitrogen narcosis), which significantly affects psychomotor function at depths exceeding around 50 m. Helium is not narcotic, and thus can be used for much deeper dives. However, another clinical entity impairs human performance at depths: high pressure nervous syndrome. Its manifestations, including tremor, ataxia, nausea and vomiting, can be ameliorated to a large extent either by slow compression or addition of a small amount of narcotic gas to a heliumoxygen mixture. The record for human exposure during an experimental dive in a hyperbaric chamber is 701 m equivalent depth, using a mixture of helium, hydrogen, and oxygen. Typically for a self-contained underwater breathing apparatus (scuba) the gas is delivered to the diver via a regulator close to the mouthpiece (Figure 6). Depending upon the divers angle in the water, this may induce a small negative or positive static lung load (see Figure 7). Commercial divers do not use scuba, but instead gas is delivered from the surface via an umbilical hose (Figure 6), which runs in parallel with communication cables and sometimes a hot water source in order to maintain thermal balance. Saturation diving refers to prolonged exposure to increased depth for a period of time sufcient to allow tissues to be saturated with inert gas (i.e., tissue and ambient inert gas partial pressures are equal). In saturation diving the divers are usually compressed to bottom depth in a hyperbaric chamber. Entry into

Year 1940 0 20 40 60 Depth (m) 80 100 120 140 160 180

Figure 3 Breath-hold diving records (from various sources).

1950

1960

1970

1980

1990

2000

Women Men

24

DIVING
340 320 300 280 260 240 220 Partial pressure (mmHg) 200 180 160 140 120 100 Surface Dive O2 CO2

80 60 55 50 45 40 35 30
0 0 5 Descent 10 15 20 25 20 m 30 35 40 Time (s) 45 50 55 Ascent 60 65 70 75

Figure 4 Partial pressures of O2 and CO2 in the lungs during submersed breath-holds at the surface and during simulated dives to 20 m in a hyperbaric chamber, both under resting conditions. During descent, both PO2 and PCO2 increase. During the time at maximum depth PO2 drops faster than it does at the surface because of the increased blood content of O2 due to its higher partial pressure and a higher cardiac output. At the end of the dive, alveolar PO2 is lower after a dive than after a breath-hold at the surface of similar duration. During exercise, PO2 would drop even faster at depth, thus increasing the likelihood of hypoxemia and loss of consciousness when the diver approaches the surface. Redrawn from Liner MH, Ferrigno M, and Lundgren CE (1993) Alveolar gas exchange during simulated breath-hold diving to 20 m. Undersea Hyperbaric Medicine 20: 2738, with permission.

1 ATA

1 ATA 1 ATA + water pressure

Figure 5 Breathing underwater via a snorkel longer than a few inches is impossible because the inspiratory muscles cannot overcome a pressure greater than around 100 mmHg. Adapted from Lundgren CEG and Miller JN (eds.) (1999) The Lung at Depth. New York: Dekker.

DIVING 25

Figure 6 (a) Scuba diver. The diver is wearing a wetsuit for thermal protection. The gas delivery hose is seen winding around the right side of her head. The red bib-like device is a buoyancy compensator. Photographer Reese. (b) Tethered diver. Breathing gas is supplied from the surface. Behind the divers head can be seen the tip of a tank containing an emergency gas supply (bailout bottle). Photos courtesy of National Oceanographic and Atmospheric Administration OAR/National Undersea Research Program (NURP).

SLL = 20 cmH2O

SLL = +25 cmH2O

Figure 7 Static lung load is the difference in pressure between the breathing regulator at the mouth and the lung centroid pressure. In the head-up position the inspiratory muscles experience an additional static load; in the head-down position the diver experiences positive pressure (CPAP). Reproduced from Lundgren CE (1999) Immersion effects. In: Lundgren CE and Miller JN (eds.) The Lung at Depth, pp. 91128. New York: Dekker, with permission.

the water can be accomplished via a closed diving bell lowered into the water. When the water pressure is equal to the internal bell pressure, the hatch can be opened and the divers can exit, connected to the bell and receiving breathing gas by an umbilical. At the end of a diving shift the divers re-enter the bell, which is then closed, lifted to the surface, and mated with a living chamber where the divers can relax, eat, and sleep. Provided appropriate environmental conditions are maintained, divers can remain at pressure indefinitely. The advantage for commercial diving operations is the ability to perform multiple days of work with the requirement for only a single decompression, which may take several days. The increased ambient pressure requires that the physical characteristics of the gas will change. Air at 1 ATA has a density of around 1.1 g l 1 at 371C. At 50 m depth, compressed air has a density of 6.6 g l 1. In direct proportion to the ambient pressure, the density and partial pressures of the component gases increase. Gas viscosity is unchanged by the increased pressure. The increase in gas density has effects on both ow resistances and pulmonary gas exchange. Although vital capacity is not affected by gas density, all ow-related spirometric indices (e.g., forced expiratory volume in 1 s (FEV1), FEF2575) are reduced. From studies in humans during dry experimental dives in hyperbaric chambers while breathing gas up to 25 times as dense as air at 1 ATA, it can be deduced that airways resistance increases approximately in proportion to the square root of gas density. Expiratory ow limitation occurs at higher lung volume and lower ow rates (Figures 8 and 9).

26

DIVING
10 8 MEF (l s1) 6 4 2 0 100 10 8 6 4 2 80 60 40 20 Volume (% VC) 0 0 2 4 6 8 Pressure (atm) 10

Figure 8 Expiratory ow-volume relationships breathing air at increased ambient pressure in a dry hyperbaric chamber. VC, vital capacity. Reproduced from Miller JN, Lanphier EH, and Wangensteen OD (1971) Ventilatory limitations on exertion at depth. In: Lambertsen CJ (ed.) Underwater Physiology IV. Proceedings of the Fourth Symposium on Underwater Physiology, pp. 317323. New York: Academic Press.

75% VC

1 ATA

5 V (l s1)

4 ATA

3 10 ATA

0 20 10

0+

10

20

30

40

50

Ppl (cmH2O)
Figure 9 Isovolume pressureow curves at 75% vital capacity at increased ambient pressure breathing air. Reproduced from Wood LD and Bryan AC (1978) Exercise ventilatory mechanics at increased ambient pressure. Journal of Applied Physiology 44: 231237, used with permission from The American Physiological Society.

Inspiratory ow limitation in the upper airway has also been observed. The functional result is a lower maximum breathing capacity (maximal voluntary ventilation, MVV) (see Figure 10). MVV at any gas density r can be estimated from the baseline MVV measurement (MVV0 obtained at r0) according to the following equation:  0:4 r MVV MVV0 1 r0

MVV while immersed is 510% lower than in the dry. External breathing resistance in the breathing apparatus can induce a further reduction in MVV. At 1 ATA maximum exercise ventilation is generally no higher than 70% of MVV. However, during a dive, the exercise ventilation required for normocarbia may exceed MVV, leading to CO2 retention. In addition to the pulmonary mechanical changes induced by breathing dense gas, there is impaired alveolarcapillary gas exchange. Oxygen exchange

DIVING 27
150 MVV (l min1) 130 110 90 70 0 200 400 600 800 1000 Dry Immersed VD (l, BTPS) Depth 1

Depth (fsw)
Figure 10 Effect of depth and immersion on maximum voluntary ventilation (MMV) in divers breathing heliumoxygen. fsw, feet of sea water (33 fsw is equivalent to 1 atmosphere pressure (760 mmHg)). Redrawn from Wright WB and Crothers JR (1973) Effects of immersion and high pressures on pulmonary mechanical functions in man. Aerospace Medical Association Annual Scientic Meeting, p. 39.

0.5 Surface

2 VT (l, BTPS)

does not appear to be affected. Additionally, the PO2 in the breathing mix is usually increased above normal. For instance, if the breathing gas is air, the ambient PO2 at 1 ATA is 159 mmHg, while at 10 m (2 ATA) it is 318 mmHg. However, CO2 exchange, as assessed by the physiological (Bohr) dead space, is impaired. Studies in experimental dives (ranging from 18 to 650 m equivalent depth, breathing gas densities 3.117.1 g l 1) during rest and exercise have revealed that physiological dead space is approximately twice normal (Figure 11). The most likely reasons for this are: (1) a higher anatomic dead space due to increased lung volume (to overcome the added breathing resistance induced by a greater gas density); and (2) nonuniformity of ventilation induced by altered gas density, thus creating gas exchange units with high ventilation/perfusion ratios. The human lung appears to have evolved to work most efciently when the breathing gas density is around 1 g l 1, not higher as is more common in diving. The net effect of this dead space increase is to reduce alveolar ventilation, which in divers is not compensated by higher total ventilation, thus PCO2 increases. In the depth range of most scuba diving, during mild exertion, significant hypercapnia is unlikely, although respiratory compensation for exercise-induced metabolic acidosis is impaired. During heavy exercise in deep diving, arterial PCO2 above 60 mmHg has been observed.

Figure 11 Dead space as a function of tidal volume during experimental deep dives (equivalent depths 18650 m, breathing gas densities 7.917.1 g l 1). The data points correspond to exercise levels of rest: 360, 720, 960, and 1080 kp m min 1. Reproduced from Salzano JV, Camporesi EM, Stolp BW, and Moon RE (1984) Physiological responses to exercise at 47 and 66 ATA. Journal of Applied Physiology 57: 10551068, used with permission from The American Physiological Society.

Table 1 Clinical problems of diving related to the lung Pulmonary oxygen toxicity Pulmonary barotrauma Pneumomediastinum Pneumothorax Arterial gas embolism Effects of venous gas embolism Cardiorespiratory decompression sickness (chokes) Chronic effects of saturation diving Immersion pulmonary edema

Oxygen Toxicity

Health Consequences
Clinical problems associated with diving, pertinent to the lung, are listed in Table 1.

Gaseous compounds exert their physiological effects in proportion to their partial pressures. Thus, gases that are in concentrations that are safe to breathe at 1 ATA can become toxic during diving. This includes oxygen, for which pulmonary toxicity can occur when the breathing gas PO2 exceeds 0.50.6 ATA (380450 mmHg) for a prolonged time. This most commonly occurs during treatment for decompression sickness or arterial gas embolism, when the diver is administered 100% O2 at 2.8 ATA (breathing gas PO2 2143 mmHg). However, pulmonary O2 toxicity can also occur when lower O2 percentages are breathed, especially during saturation diving. For example, breathing air for a prolonged time would produce pulmonary O2 toxicity at ambient pressures

28

DIVING

exceeding 3 ATA (20 m). Therefore, for long, deep dives the O2 percentage in the breathing gas must be adjusted to achieve an appropriate PO2 (for instance, during an experimental deep dive to 686 m, the oxygen concentration was 0.6%).
Pulmonary Barotrauma

capillary network, and generally no symptoms occur. However, high-grade VGE can cause cough, dyspnea, and pulmonary edema (cardiorespiratory decompression sickness or chokes), for which the initial treatment is high concentration O2, appropriate support of cardiovascular parameters. The denitive treatment is hyperbaric O2 in a recompression chamber.
Immersion Pulmonary Edema

During ascent intrapulmonary gas expands and must be exhaled. If the diver holds his breath or regional airway obstruction causes gas trapping, alveolar rupture can occur (pulmonary barotrauma, PBT), causing pneumomediastinum, pneumothorax, or arterial gas embolism (AGE). Pneumomediastinum often presents as altered voice or subcutaneous emphysema. Pneumothorax is the least common manifestation of PBT. AGE typically presents with impaired consciousness and cortical signs such as hemiparesis, within minutes of surfacing. Risk factors for PBT include rapid ascent, as well as obstructive diseases, bullae, blebs, and interstitial restrictive lung diseases. Immediate treatment for AGE includes O2 administration; the denitive treatment is hyperbaric oxygen treatment in a recompression chamber.
Venous Gas Embolism

Immersion pulmonary edema is a condition in which the diver develops a cough with pink frothy sputum, usually shortly after entering the water. Chest radiography reveals pulmonary edema indistinguishable from other forms (Figure 12). The condition is believed to be more common in individuals who subsequently develop hypertension or after diving in cold water, although it has occurred in tropical water. Immersion pulmonary edema has been fatal. The pathophysiology of immersion pulmonary edema is believed to be high pulmonary capillary pressure due to the normal pulmonary vascular response to immersed exercise, augmented by transient cardiac failure due to the afterload induced by inspiring against high intrapulmonary and extrapulmonary resistance.
Long-Term Effects of Diving on Pulmonary Function

Venous gas embolism (VGE) occurs during and after ascent as a result of inert gas supersaturation. VGE usually consists of low levels of bubbles, which can only be detected by ultrasound. During a week of diving 80% of recreational divers will have detectable VGE. VGE also commonly occurs during decompression from saturation dives. In the absence of an intracardiac or intrapulmonary shunt, VGE are cleared from the circulation by the pulmonary

Following deep saturation dives increases have been observed in dead space and reductions in FEV1, CO transfer factor, and exercise capacity. These have been attributed variously to deconditioning, persistent low levels of otherwise asymptomatic VGE during decompression, high oxygen partial pressures, and breathing gas contaminants.

Figure 12 Immersion pulmonary edema. (a) A 55-year-old male who dived to 20 m for 24 min in 171C water. He experienced respiratory distress at depth and surfaced with dyspnea, chest tightness, and cough productive of pink frothy sputum. Chest CT shows patchy densities in both lungs. (b) A male of the same age with a history of hypertension, who also experienced respiratory distress after diving to 24 m for 7 min.He surfaced with dyspnea, cough, and hemoptysis. Plain chest radiograph was normal. CT shows densities in the right middle lobe. From Cochard G, Arvieux J, Lacour JM, et al. (2005) Pulmonary edema in scuba divers: recurrence and fatal outcome. Undersea Hyperbaric Medicine 32: 3944, with permission.

DIVING 29

Fitness to Dive Evaluation

Pulmonary evaluation for tness to dive with compressed gas consists of two aspects: pulmonary function and susceptibility to pulmonary barotrauma.
Pulmonary Function

One way of considering this issue is as follows. According to eqn [1], a normal divers predicted MVV is at 70% of his or her surface value when he or she reaches a depth of 10 m. An exercise ventilation of 70% of MVV may be required to maintain a normal arterial PCO2 . Therefore, if the diver has a low MVV due to respiratory disease, he or she will not be able to achieve adequate exercise ventilation even at this shallow depth. Thus, abnormal 1 ATA spirometry would be disqualifying.
Susceptibility to Pulmonary Barotrauma

Diseases in which air trapping is likely are disqualifying for diving. This includes a history of spontaneous pneumothorax, blebs, bullae or other lung cysts, or interstitial diseases. Asthmatics may also be at increased risk. However, the risk appears to be similar to the general population if there are no active symptoms of asthma (e.g., dyspnea, cough, or wheezing), spirometry is normal, and a provocative test, such as exercise while breathing dry air, does not produce a decrement in spirometry (see American Thoracic Societys Guidelines for Methacholine and Exercise Challenge Testing, 1999).
See also: Arterial Blood Gases. Diffusion of Gases. Exercise Physiology. Hyperbaric Oxygen Therapy. Mediastinitis, Acute and Chronic. Oxygen Toxicity. Pneumothorax. Space, Respiratory System. Hypoxia and Hypoxemia.

Further Reading
American Thoracic Society (2000) Guidelines for Methacholine and Exercise Challenge Testing, 1999: ofcial statement of the American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 161: 309329. Bert P (1978) Barometric Pressure (La Pression Barome trique) (Translated by Hitchcock MA and Hitchcock FA). Bethesda, MD: Undersea Medical Society. Bove AA (ed.) (2004) Diving Medicine. Philadelphia, PA: Saunders. Brubakk AO and Neuman TS (2003) The Physiology and Medicine of Diving. New York: Elsevier.

Camporesi EM and Bosco G (2003) Ventilation, gas exchange and exercise under pressure. In: Brubakk AO and Neuman TS (eds.) The Physiology and Medicine of Diving, pp. 77114. New York: Elsevier. Cochard G, Arvieux J, Lacour JM, et al. (2005) Pulmonary edema in scuba divers: recurrence and fatal outcome. Undersea Hyperbaric Medicine 32: 3944. Flook V and Fraser IM (1989) Inspiratory ow limitation in divers. Undersea Biomedical Research 16: 305311. Hong SK, Cerretelli P, Cruz JC, and Rahn H (1969) Mechanics of respiration during submersion in water. Journal of Applied Physiology 27: 535538. Kohshi K, Wong RM, Abe H, et al. (2005) Neurological manifestations in Japanese Ama divers. Undersea Hyperbaric Medicine 32: 1120. Liner MH, Ferrigno M, and Lundgren CE (1993) Alveolar gas exchange during simulated breath-hold diving to 20m. Undersea Hyperbaric Medicine 20: 2738. Lundgren CEG and Miller JN (eds.) (1999) The Lung at Depth. New York: Dekker. Miller JN, Lanphier EH, and Wangensteen OD (1971) Ventilatory limitations on exertion at depth. In: Lambertsen CJ (ed.) Underwater Physiology IV. Proceedings of the Fourth Symposium on Underwater Physiology, pp. 317323. New York: Academic Press. Moon RE and Stolp BW (1997) The lung during anesthesia and in adverse environments. In: Schwinn D (ed.) Scientic Principles of Anesthesia, Atlas of Anesthesia (Miller RD, series ed.), vol. 2, pp. 6.26.15. Philadelphia, PA: Current Medicine. Mummery HJ, Stolp BW, Dear GdL, et al. (2003) Effects of age and exercise on physiological dead space during simulated dives at 2.8 ATA. Journal of Applied Physiology 94: 507517. Saltzman HA, Salzano JV, Blenkarn GD, and Kylstra JA (1971) Effects of pressure on ventilation and gas exchange in man. Journal of Applied Physiology 30: 443449. Salzano J, Rausch DC, and Saltzman HA (1970) Cardiorespiratory responses to exercise at a simulated seawater depth of 1,000 feet. Journal of Applied Physiology 28: 3441. Salzano JV, Camporesi EM, Stolp BW, and Moon RE (1984) Physiological responses to exercise at 47 and 66 ATA. Journal of Applied Physiology 57: 10551068. Skogstad M, Thorsen E, Haldorsen T, and Kjuus H (2002) Lung function over six years among professional divers. Occupational and Environmental Medicine 59: 629633. Thorsen E, Hjelle J, Segadal K, and Gulsvik A (1990) Exercise tolerance and pulmonary gas exchange after deep saturation dives. Journal of Applied Physiology 68: 18091814. Thorsen E, Risberg J, Segadal K, and Hope A (1995) Effects of venous gas microemboli on pulmonary gas transfer function. Undersea Hyperbaric Medicine 22: 347353. Wood LD and Bryan AC (1969) Effect of increased ambient pressure on owvolume curve of the lung. Journal of Applied Physiology 27: 48. Wood LD and Bryan AC (1978) Exercise ventilatory mechanics at increased ambient pressure. Journal of Applied Physiology 44: 231237. Wright WB and Croltiers JR (1973) Effects of immersion and high pressures on pulmonary mechanical functions in man. Aerospace Medical Association Annual Scientic Meeting, p. 39.

30

DNA / Repair

DNA
Contents

Repair Structure and Function

Repair
D M Wilson III, H-K Wong, D R McNeill and J Fan, National Institutes of Health, Baltimore, MD, USA
Published by Elsevier Ltd.

Abstract
Our genetic material, DNA, can be damaged by environmental (including sunlight and ionizing radiation and manmade products) and intracellular (e.g., reactive oxygen species) chemical and physical agents. These agents create lesions that if unrepaired can lead to genetic change (mutations) and accompanying cellular dysfunction. Thus, to avert or limit cell death and the mutagenic consequences of DNA damage, organisms are equipped with an array of DNA repair systems. Such systems function to recognize DNA damage, remove it, and restore genome integrity. Signicantly, defects in these repair responses are associated with human disease, most notably cancer and tissue (namely neuronal) degeneration. In this article, we discuss the general mechanisms of the major DNA repair systems and highlight their relationship to specic human disorders, and we also present what little is known about their connection to respiratory disease.

DNA encodes the blueprint of the organism. In brief, genes (located within the DNA) are expressed in a complex, orchestrated manner to generate proteins that comprise the structural architecture and carry out the functional operations of the cell. The gene expression pattern, which is unique for each cell type, produces specic and distinctive cellular characteristics.

If this highly regulated network is upset or disrupted, either by dysregulation of gene expression or by the production of mutant (dysfunctional) proteins, cells can become atypical, creating problems for the organism as a whole. DNA is subject to constant molecular change (Figure 1) resulting from reactions with DNA-damaging agents that exist both within the environment and as natural byproducts within the cell. In particular, extracellular physical and chemical agents, such as ultraviolet light (sunlight), ionizing radiation, aromatic and heterocyclic amines, asbestos, vinyl chloride, and certain heavy metals can generate, directly or indirectly, harmful DNA damage. Additionally, aerobic organisms (e.g., humans) metabolize oxygen to generate energy, and during this process they form chemicals known as free radicals (or reactive oxygen species) that can react with DNA to produce dangerous intermediates. Unrepaired DNA damage can disrupt gene expression or promote permanent genetic mutations and, if excessive, can drive programmed cell death (apoptosis) or irreversible proliferation arrest (senescence). Dysregulation of or mutations in genes that control cell growth and function can promote disease development. To avert the deleterious effects of DNA damage and to maintain the delity of the genome, organisms have evolved a complex array of systems, known collectively as DNA repair, that recognize specic forms of DNA damage, remove the damage from DNA, and ultimately restore genome integrity.

Base modification: spontaneous deamination, alkylation, oxidation

Thymine dimer Abasic site

Mismatched nucleotides

G T

Double-strand break Single-strand break

Figure 1 Several major forms of DNA damage. See Table 1 for associated repair pathways. Note that the alkylation damage referred to here is distinct from the active methylation used to regulate promoter activity (i.e., enzyme-directed methylation of cytosine).

DNA / Repair 31
Table 1 Summary of the major human DNA repair pathways Major damage substrates Nucleotide mismatches (e.g., G:T) and short nucleotide loop segments Spontaneous and oxidative forms of DNA damage (e.g., base modications, abasic sites, and single-strand breaks) Helix-distorting bulky DNA lesions (e.g., T-T photodimers) Alkylation base damage (e.g., O6alkylguanine) Double-strand breaks and unresolved, complex DNA intermediates
a

DNA repair pathway Mismatch repair Base excision repair

Relationship to disease a Colon and uterus cancer predominantly, but several sporadic tumors as well, including lung Colon cancer and potentially other forms of cancer (e.g., lung and B cell); indirect connection to spinocerebellar ataxias Skin cancer predominates; some instances of neurological degeneration; clinical features vary depending on gene mutated Likely role in preventing alkylating agent-induced carcinogenesis; role in inuencing the therapeutic effectiveness of alkylating agents Leukemia, lymphoma, brain, skin, breast, ovary, and bone tumors

Nucleotide excision repair

Direct reversal

Recombinational repair

This is a broad representation. The precise clinical features will be determined by the specic gene mutated.

We briey discuss aspects of the ve major mammalian DNA repair pathways (summarized in Table 1) and report known associations to human disease.

Mismatch Repair
The eld of DNA repair remained relatively obscure until the discovery that mutations in components of mismatch repair (MMR) are genetically linked to forms of hereditary nonpolyposis colorectal cancer (HNPCC). This repair process is largely responsible for correcting replication errors, most notably mismatched nucleotides or small insertion/deletion loops, which arise during copying of the doublestranded genome. Defects in MMR lead to genomewide instability, particularly in simple repetitive sequences (known as microsatellite instability). The current understanding of the molecular details of this pathway is illustrated in Figure 2, yet is limited, particularly following the recognition step. Since the association of MMR with HNPCC was established, it has been found that defects in MMR are linked to some types of sporadic tumors as well. Many of these fall into the HNPCC spectrum that is, colon, rectum, uterine endometrium, stomach, and ovarian cancer. Recently, it has been realized that nonHNPCC-related sporadic cancers may also result from defects in MMR, including breast, prostate, and lung cancer. However, the mutational proles of these cancers differ from those found in the HNPCC-related tumors, implying different molecular mechanisms. In addition to its role in processing DNA replication intermediates (Figure 2), the MMR system contributes to genomic stability by mediating programmed cell death in response to certain DNA-damaging agents. In this process, MMR operates to recognize specic DNA lesions and initiate an apoptotic response to eliminate severely damaged cells

from the organism, thereby preventing tumorigenesis. In fact, in the absence of MMR, cells display increased resistance to certain therapeutic drugs and a concomitant propensity for mutagenesis. These observations have prompted investigators to further examine the role of MMR in determining responsiveness to cell killing by certain anticancer agents, namely alkylating and DNA cross-linking compounds.

Base Excision Repair

Base excision repair (BER) is the major pathway for dealing with spontaneous, nonbulky forms of DNA damage, including simple oxidative and alkylation damage. These include base modications (e.g., the common mutagenic 8-oxoguanine lesion), sites of base loss, and single-strand breaks in DNA. The fundamental molecular details of this workhorse pathway are well understood and are detailed in Figure 3. Note that although multiple subpathways exist in BER, the general biochemical steps remain the same. Until recently, no direct link of BER with human disease had been demonstrated. However, mutations in the human MutY homolog (MYH) DNA glyco sylase, which recognizes and initiates repair of certain base abnormalities, were linked to cases of HNPCC. Genetic analysis has also revealed that B30% of human tumors express a DNA polymerase b (POLb) variant, with one of these mutant proteins (Lys289Met) exhibiting reduced polymerase delity. Moreover, several epidemiological association studies have suggested correlations of specic BER gene variants with certain cancer types, although direct links have not been established. Last, it is worth noting that a central component of BER, X-ray cross-complementing 1 (XRCC1) (Figure 3), has been connected to two proteins tyrosyl DNA phosphodiesterase 1 (TDP1)

32

DNA / Repair

G T Large insertion/deletion loops DNA damage recognition MSH2 MSH6 PCNA MSH2 MSH3

G MSH2 T Incision, excision + ATP EXO1 RFC PCNA RPA A G MSH2 T PCNA Gap-filling MSH6 MSH6

MLH1 MLH1 MLH1

PMS2

MLH3

MLH1 PMS1

RFC

T LIG1 Ligation

Figure 2 Mismatch repair (MMR). MMR is responsible for correcting mismatched nucleotides or insertion/deletion loops that inadvertently arise during copying of the genome. MSH2MSH6 is primarily involved in targeting basebase (e.g., G:T) and small insertion/ deletion loop mismatches, whereas MSH2MSH3 acts predominantly on insertion/deletion loops up to 12 nucleotides in length (details not shown). The MSH proteins have been argued to interact with the replication fork through associations with the replication processivity factor, proliferating cell nuclear antigen (PCNA). This interaction is thought to be important for determining which strand is the newly replicated DNA (i.e., the target strand) during the so-called strand discrimination step of MMR. ATP binding and hydrolysis are likely critical for the early steps of MMR, including MSH-mediated mismatch recognition and subsequent MSHMLH interactions. The MLH1PMS2 complex (or one of the putative backup complexes shown to the right) is hypothesized to signal mismatch recognition to downstream effectors, which are largely unknown. MLH1 has been shown to physically interact with EXO1, a 50 to 30 double-stranded DNA exonuclease implicated in both 50 and 30 nick-mediated MMR. EXO1 operates, likely in cooperation with PCNA and replication factor C (RFC), to remove the mismatched nucleotide or loop region, with the generated single-stranded portion (hundreds of nucleotides; not drawn to scale here) being stabilized by replication protein A (RPA). The replicative DNA polymerases e and d and DNA ligase 1 (LIG1) complete the process by lling the gap and sealing the nal nick, respectively.

and aprataxin (APTX) in which defects have been associated with spinocerebellar ataxias.

Nucleotide Excision Repair

The connection of DNA repair to cancer susceptibility was rst recognized upon the discovery that the rare human disorder xeroderma pigmentosum (XP), which is characterized by severe sensitivity to ultraviolet light and a 1000-fold increase in skin cancer susceptibility, is defective in components of the nucleotide excision repair (NER) response. In general,

NER copes with helix-distorting base lesions, which arise from sunlight or bulky chemicals such as benzopyrene. In Figure 4, the well-understood molecular mechanics of NER are shown. It is important to emphasize that NER has two functionally distinct branches one that operates to repair lesions in the bulk inactive genome and one that works in cooperation with the transcription machinery to remove damage from actively transcribed regions. Defects in NER are connected to at least three human genetic disorders: XP, Cockaynes syndrome (CS), and trichothiodystrophy. Seven complementation

DNA / Repair 33
IR OH PARP1 XRCC1 PNK AP site incision OH dRP APE1 APE1 End processing

PP, P, or PG NEIL 1 Base excision UNG

Strand displacement synthesis Gap-filling POL PCNA,RFC,RPA, and POL / POL

dRP excision

POL FEN1

SP-BER Nick ligation XRCC1 LIG3 LIG1

Flap excision LP-BER

Nick ligation

Figure 3 Base excision repair (BER). BER is typically initiated by recognition and excision of a base damage (indicated as a black residue) by a DNA glycosylase. Such enzymes include uracil DNA glycosylase (UNG) and endonuclease VIII-like 1 protein (NEIL1). Monofunctional DNA glycosylases (e.g., UNG) generate an abasic (AP) site product, whereas bifunctional DNA glycosylases (e.g., NEIL1) create an AP site and then nick the DNA backbone. In the former case (pathway to the left), the AP site is cleaved by AP endonuclease 1 (APE1), creating a strand break with a normal 30 -hydroxyl (OH) group and a nonconventional 50 -terminus harboring a deoxyribose phosphate moiety (dRP). The one nucleotide gap and 50 -dRP residue are lled and excised, respectively, by DNA POLb. The remaining nick is sealed by a protein complex containing XRCC1 and DNA ligase 3 (LIG3), which together complete the process of short-patch BER (SP-BER). In the case of a multifunctional DNA glycosylase, or situations in which a strand break arises from the direct action of ionizing radiation (IR) or free radical attack (pathway to the right), the DNA ends are processed by a series of proteins, including poly(ADP-ribose) polymerase 1 (PARP1), XRCC1, polynucleotide kinase/phosphatase (PNKP), and APE1. Once the termini have been converted to normal 30 -OH ends by the appropriate phosphodiesterase and 50 -phosphate (P) groups by the PNKP kinase, the gap is lled by a DNA polymerase. In certain situations (not fully understood), the replicative polymerases (namely, d and e) operate to perform repair synthesis in cooperation with their accessory factors PCNA, RFC, and RPA. At times (again, not fully understood), gap-lling involves strand displacement synthesis, which generates a 50 -ap structure that requires subsequent removal by the ap endonuclease 1 (FEN1). In cases of long-patch BER (LP-BER), DNA ligase 1 (LIG1) functions to seal the nal nick. 4-Hydroxypentenal phosphate (PP) is the 30 -terminal strand break product of some bifunctional DNA glycosylases. Phosphate (P) and phosphoglycolate (PP) are products of IR and reactive oxygen species attack. NEIL1 also generates 30 -P termini.

groups have been associated with XP (AG), whereas two groups have been linked to CS (A and B), with each group representing a different repair gene. Although clinical manifestations differ considerably, varying from high cancer predisposition to aging symptoms, the common feature of the three syndromes is photosensitivity, manifested at different levels. Evidence has also pointed toward a role for NER in the repair of oxidative DNA damage that may be related to the neurodegenerative and premature

aging phenotypes associated with certain NERdefective individuals.

Direct Reversal
Unlike the other repair processes that entail multiprotein complexes, direct reversal involves a single protein that transfers the modied portion of DNA to a residue within the proteins active site. This action restores DNA back to its natural state but leads

34

DNA / Repair

DNA damage recognition

HR23B XPC GGR

CSA CSB TCR

RNA Pol

DNA unwinding XPA RPA XPD XPB TFB5

TFIIH

Incision of damaged DNA XPF ERCC1 XPA RPA XPD Fragment release, repair synthesis, and DNA ligation XPB TFB5 TFIIH XPG

RFC

LIG1 DNA Pol PCNA RPA

Repaired double stranded DNA

Figure 4 Nucleotide excision repair (NER). NER is subdivided into two major, distinct pathways. In global genome repair (GGR), the protein complex XPC/HR23B initially recognizes and binds to the helix-distorting damage site (shown as a thymine dimer photoproduct). In contrast, transcription-coupled repair (TCR) begins when an RNA polymerase (RNA Pol) stalls at the site of the lesion during transcription, followed by recruitment of CSA and CSB. Either of these events causes the main repair apparatus to be recruited in a sequential manner to the scene of the damage. The site of the lesion is rst opened by the action of the transcription factor II H (TFIIH) complex (which contains two bidirectional helicase subunits, XPD and XPB (which unwind DNA in an ATP-dependent manner), and the recruitment subunit TFB5) and stabilized by XPA and RPA. Next, the ERCC1/XPF complex is directed to cut the damaged region on the 50 side of the lesion, while XPG is positioned to cleave 30 to the damage (resulting in an approximately 20-nucleotide incised fragment). Excision of the damage occurs due to the interaction between RPA, RFC, DNA polymerases (DNA Pol, likely d and e), and PCNA. At the end of the synthesis stage, the resulting nick is sealed by the action of DNA LIG1, producing a repaired double-stranded DNA molecule.

to irreversible inactivation of the covalently modied protein. Such a repair response has been termed suicidal because the protein is no longer functional. In mammals, the repair protein O6-methylguanineDNA-methyltransferase (MGMT) transfers alkylation damage at the O6 position of guanine, and to a lesser extent the O4 position of thymine, to an active site cysteine residue. This repair step leads to the production of normal guanine or cytosine while leading to protein degradation. If unrepaired, O6alkylguanine lesions can promote mutagenesis upon replication of the strand containing damage.

MGMT is of particular interest because of its role in both cancer prevention and treatment. First, MGMT repair of O6-alkylguanine, a damage that promotes G to A transition, suppresses the mutagenic effects of exogenous carcinogens. For instance, transgenic mouse models indicate that MGMT expression levels are inversely related to the frequency of alkylating agent-induced tumorigenesis. Second, many anticancer agents, such as the alkylating compounds temozolomide, streptozotocin, procarbazine, dacarbazine, and the nitrosoureas, kill actively dividing cells by introducing cytotoxic DNA intermediates.

DNA / Repair 35

Thus, strategic manipulation of MGMT activity can have a profound impact on the efcacy of certain anticancer treatments for cancers. In particular, normal cells can be protected from the cytotoxicity of alkylating agents by increasing MGMT activity via gene therapy techniques, whereas cancerous cells (e.g., gliomas, brain tumors, lymphomas, and Hodgkins disease) can be killed more effectively by selectively inhibiting MGMT function. Current work on MGMT (as well as other DNA repair responses) focuses largely on maximizing its potential as a target for improved anticancer treatment strategies.

Recombinational Repair
DNA double-strand breaks (DSBs) are direct products of ionizing radiation but are also formed during DNA replication when the progressing polymerase is disrupted by a DNA damage (e.g., a single-strand break) and the fork collapses. DNA DSBs are potent cytotoxic lesions and are capable of promoting gross chromosomal rearrangements (e.g., translocations). To repair DSBs, organisms are equipped with two corrective processes: homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR occurs in S and G2 phase cells and relies on the extensive nucleotide sequence identity between the damaged chromatid and the intact homologous chromatid as the basis for strand exchange and repair. Since it involves an undamaged complementary template for repair, this process is error-free and restores the genome to its natural state; repair by HR is therefore conservative. That is, in the repaired chromosome, both strands are restored to the original template. NHEJ requires little or no sequence homology and is mediated by direct end-to-end ligation. However, depending on the terminal processing that occurs, NHEJ can result in the loss or gain of unwanted DNA nucleotides. The current understanding of these complex processes is depicted in Figure 5. Besides operating as key pathways for repair of DSBs, it is important to keep in mind that HR and NHEJ also function in essential cellular processes, such as meiotic recombination, which creates genetic diversity among the reproductive cell population, and V(D)J recombination, which generates diversity in the antigen receptor genes during lymphocyte development. Defects in HR or NHEJ are associated with chromosome instability, DNA-damaging agent hypersensitivity, cancer susceptibility (particularly hematological cancers), and immunodeciency. This fact is perhaps best exemplied by the human diseases ataxia-like disorder and Nijmegen breakage syndrome (NBS), which are defective in MRE11 and

NBS1, respectively. Evidence also indicates that BRCA1 and BRCA2, originally identied as the breast cancer genes, and components of the autosomal recessive disorder, Fanconi anemia, contribute to the HR process. WRN (Werners syndrome) and BLM (Blooms syndrome), proteins associated with premature aging and cancer diseases, have been shown to help resolve or process complex replication/recombination DNA intermediates. Signicantly, a network linking the various DSB repair participants to cell-cycle checkpoint responses has emerged. Defects in components of cell-cycle regulation (most notably, ATM, p53, and CHEK2), which operate to arrest cell-cycle progression to allow chromatin remodeling, repair of DNA damage, and avoidance of error-prone replication across a damaged template, are commonly associated with cancer or specic cancer-prone syndromes (i.e., ataxia telangiectasia and LiFraumeni). In cases of extensive damage, these processes also function to activate cell death and remove such precarious cells from the organism.

Associations with Lung Cancer and Respiratory Disease

As outlined previously, evidence clearly links defects in DNA repair to cancer predisposition and neurological abnormalities. Not surprisingly, both epidemiological and molecular biology studies have indicated an inverse correlation between DNA repair capacity and lung cancer risk. Although associations have been reported, studies examining the direct relationship of DNA repair defects and lung cancer susceptibility are limited and still in the early stages. A hypothetical model outlining the potential relationship of DNA repair with respiratory disease is presented in Figure 6. The major risk factor for lung cancer the leading cause of cancer-related death in the world is exposure to tobacco smoke. In vivo experiments in rodents have shown that exposure to tobacco smoke causes bulky aromatic DNA adducts, oxidative damage (including single-strand breaks), and chromosome aberrations. Human biomarker studies conducted on smokers and nonsmokers have uncovered similar results, corroborating that tobacco smoke (and its constituents) is a potent genotoxin/mutagen. As alluded to previously, initial studies suggest that defects in components of BER, NER, and the cellcycle response are most prominently associated with increased risk of lung cancer. For instance, experiments indicate that deciencies or genetic polymorphisms in the oxidative DNA damage repair

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DNA / Repair

Recognition and end binding DNAPKcs

Ku70/80 5 3 RPA RAD52 5 RAD51 3 5 5 End trimming 5 Polymerization, ligation 5 3 3 3 MRN 3

Recruitment of proteins for end processing

Artemis

End Recognition, processing,singlestrand binding RAD51-Mediated homology search, strand invasion

Recruitment of proteins for ligation

XRCC4/LIG4

5 3 Polymerization, branch migration 5 3

3 5

End ligation

HJ resolution, end ligation 5 3 (b)

3 5

(a)

Figure 5 Double-strand break (DSB) repair pathways. (a) NHEJ. A DSB is recognized and bound by the Ku70/80 heterodimer, which recruits and activates the catalytic subunit of DNA-dependent kinase (DNA-PKcs). Artemis, which possesses both exo- and endonuclease activities, is subsequently recruited by DNA-PKcs to clean up the DSB ends for ligation. The two DSB ends are ligated by LIG4 (present in an XRCC4/LIG4 complex), which is recruited by the Ku heterodimer. The Mre11/Rad51/Nbs1 (MRN) nuclease complex, and other proteins such as PNK, may also play a role in cleaning up the DNA ends. In situations in which there is resection at the break, polymerase X family members can ll the end prior to ligation (not shown). (b) HR. The MRN complex presumably resects the ends of the DSB, creating long 30 single-strand overhangs. Rad52 and RPA then bind to the end and single-strand portion, respectively, of the 30 overhang. Two subpathways could subsequently follow. In conservative HR (left), Rad51 replaces RPA and mediates the homology search between two sister chromatids, a process also facilitated by RAD54, followed by stand invasion. After polymerization and branch migration, the formed Holliday junctions (HJs) are resolved (it is currently unclear how), and a ligase then seals the ends to complete repair. BRCA1/2 may play a role in HR that is not completely understood. Repair by this subpathway is error-free. In nonconservative HR (right) (i.e., single-strand annealing (SSA)), the ends are brought together by homologous sequences (i.e., repeats) located on the opposite strands of the two DNA fragments (solid rectangles). After trimming of the ends (perhaps by the ERCC1/XPF endonuclease), ligation of the ends is performed by a currently unidentied ligase. Since this subpathway features deletion of a portion of DNA between the homologous segments, repair is inaccurate.

Stress Exogenous DNA Lesion DNA Repair Unrepaired Endogenous Mutations Cell death or senescence Disease (e.g., lung cancer) and aging

Figure 6 DNA repair capacity model. As detailed in the text, DNA is exposed to various exogenous and endogenous stresses (i.e., DNA-damaging agents). Resulting DNA damage is typically repaired by one of the specialized DNA repair responses. However, in instances in which either the DNA repair capacity is insufcient or the DNA damage is excessive, unrepaired lesions can promote cytotoxic or mutagenic outcomes. Such end points can drive human disease (namely cancer) and age-related disorders.

glycosylase, OGG1, may be related to lung tumorigenesis. In addition, mutations or sequence variants in genes of the NER pathway (e.g., XPC and ERCC1; Figure 4) appear to have susceptibility as well as lung cancer pharmacogenetic and survival signicance. Polymorphisms in the tumor suppressor protein p53

may also be linked to increased risk of lung cancer. Interestingly, accumulating data suggest that the chances for development of lung cancer are higher for women than men, and that this increased susceptibility may be due to higher levels of DNA adducts, decreased DNA repair capacity, increased

DNA / Structure and Function 37

frequency of mutations in tumor suppressor genes, as well as hormonal differences. Note that many inhaled occupational and environmental particles (e.g., airborne asbestos) have the capacity to be genotoxic/mutagenic. Many of these particles are harmful as a consequence of their ability to generate oxidants. Laboratory results indicate that there is increased inammation, oxidative stress, and lipid peroxidation in lung diseases such as chronic obstructive pulmonary disease, cystic brosis, and asthma. However, very little has been done to determine the effect of environmental and occupational airborne particles on DNA damage levels or the relationship of DNA repair to the prevention of such respiratory diseases. In conclusion, future work on elucidating the association of environmental exposures (including tobacco and other particles) to lung disease is paramount, as are devising strategies to suppress the harmful effects of identied compounds and clarifying the role of DNA damage and repair capacity in common respiratory ailments.
See also: Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. DNA: Structure and Function.

Taroni F and DiDonato S (2004) Pathways to motor incoordination: the inherited ataxias. Nature Reviews: Neuroscience 5: 641655. Venkitaraman AR (2004) Tracing the network connecting BRCA and Fanconi anemia proteins. Nature Reviews: Cancer 4: 266 276. Wu M (2005) DNA repair proteins as molecular therapeutics for oxidative and alkylating lung injury. Current Gene Therapy 5: 225236. Yokota J and Kohno T (2004) Molecular footprints of human lung cancer progression. Cancer Science 95: 197204.

Structure and Function

t Duisburg Essen, A Ehrenhofer-Murray, Universita Essen, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
DNA is the carrier of genetic information in all living organisms. DNA is a double helix whose strands are held together by complementary pairing between the bases adenine and thymine and between guanine and cytosine. Transcription generates an RNA template of the DNA sequence that is then translated into a protein, and the sequence of the bases determines the amino acid sequence of the protein. In eukaryotes, DNA is packaged into chromatin in the nucleus. Chromatin is a dynamic protein DNA structure that modulates access of regulatory activities (transcription, replication, and DNA repair) to the genetic material. Histone modifying complexes (e.g., for histone acetylation and methylation) and chromatin remodeling complexes cooperate to regulate chromatin access during nuclear processes on the DNA template. Among others, alterations in DNA sequence can cause respiratory diseases, such as in the hereditary disorder cystic brosis. Genetic polymorphisms also increase susceptibility to lung disease and lung cancer. Numerous changes in DNA sequence, including changes in DNA methylation, as well as structural and numerical chromosome aberrations are found in (lung) cancerous tissues. Chromatin dysfunction also affects lung disease, and transcriptional mechanisms play an important role in the pathogenesis of acute lung inammation.

Further Reading
Frosina G (2004) DNA base excision repair defects in human pathologies. Free Radical Research 38: 10371054. Husgafvel-Pursiainen K (2004) Genotoxicity of environmental tobacco smoke: a review. Mutation Research 567: 427445. Karagiannis TC and El-Osta A (2004) Double-strand breaks: signaling pathways and repair mechanisms. Cellular and Molecular Life Sciences 61: 21372147. Kastan MB and Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432: 316323. Krokan HE, Kavli B, and Slupphaug G (2004) Novel aspects of macromolecular repair and relationship to human disease. Journal of Molecular Medicine 82: 280297. Neumann AS, Sturgis EM, and Wei Q (2005) Nucleotide excision repair as a marker for susceptibility to tobacco-related cancers: a review of molecular epidemiological studies. Molecular Carcinogenesis 42: 6592. Opresko PL, Cheng WH, and Bohr VA (2004) Junction of RecQ helicase biochemistry and human disease. Journal of Biological Chemistry 279: 1809918102. Patel JD, Back PB, and Kris MG (2004) Lung cancer in US women: a contemporary epidemic. Journal of the American Medical Association 291: 17631768. Rosell R, Taron M, Ariza A, et al. (2004) Molecular predictors of response to chemotherapy in lung cancer. Seminars in Oncology 31(supplement 1): 2027. Spitz MR, Wei Q, Dong Q, Amos CI, and Wu X (2003) Genetic susceptibility to lung cancer: the role of DNA damage and repair. Cancer, Epidemiology, Biomarkers and Prevention 12: 689698. Stanton GL (2004) MGMT: its role in cancer aetiology and cancer therapeutics. Nature Reviews: Cancer 4: 296307.

Introduction
The nucleic acid DNA serves as the genetic material in all living organisms. Seminal experiments carried out in the rst half of the twentieth century led to the conclusion that DNA, and not other molecules such as proteins, carbohydrates, or lipids, is the genetic material and that it carries the hereditary factors (genes) described by Gregor Mendel in the 1860s. In 1928, Frederick Grifth established the principle of transformation the transfer of genetic material between bacteria. Subsequent experiments by Oswald Avery (1944) as well as Alfred Hershey and Martha Chase (1952) demonstrated that DNA is the

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transforming agent. Based on work from Erwin Chargaff on the base composition of DNA and X-ray diffraction pictures of DNA from Rosalind Franklin and Maurice Wilkins, James Watson and Francis Crick proposed a model for the double-helical structure for DNA in 1953.

Structure
DNA is composed of three basic units: deoxyribose (a ve-carbon sugar), phosphate, and four nitrogenous bases the purines adenine (A) and guanine (G) and the pyrimidines thymine (T) and cytosine (C) (Figure 1). These units form a long helical structure of two antiparallel strands termed the double helix.

The ribose moieties are linked by phosphodiester bonds and form the backbone of the DNA. The backbone strands are held together by complementary base pairing between the bases of the opposite strands that are attached to the ribose. The pairings depend on the shape of the bases and on their ability to form hydrogen bonds. A pairs with T, forming two hydrogen bonds, and G forms three hydrogen bonds with C (Figure 2). The base pairs are planar structures and stack on top of each other at the center of the double helix, which further stabilizes the helix by excluding water molecules. DNA is a right-handed helix with 10.4 base pairs (bp) per helix turn and has two distinct sizes of grooves running in a spiral, the major groove and the minor groove (Figure 2). The

Adenine NH2 N O O P O O CH2 N N Nitrogenous base N N N

Guanine O H N N NH2

Cytosine NH2

Thymine O CH3 H N O N O

N N

OH Phosphate Deoxyribose

Figure 1 Chemical structure of the building blocks of DNA.

... ..T A.

G ... C

...

C...G

...

... ..T A. ... ... ..T A.

P A P
C

P
G

T ... A

...

... .. C G.

C ... G

P
P G P T ... G...C ... ..T A. A C ... C ...G ...T A...

...T A...

... ..A T.

Double helix Major groove Minor groove

Backbone

Complementary base pairing

Figure 2 The structure of DNA. (Left) Simplied model of the DNA double helix. The sticks represent base pairs, and the ribbons represent the deoxyribose phosphate backbones of the antiparallel strands. (Right) Schematic of complementary base pairing in DNA. Dotted lines represent hydrogen bonds between bases. A, adenine; T, thymine; C, cytosine; G, guanine; P, phosphate.

DNA / Structure and Function 39

helix is a exible structure in that, depending on its sequence, it can be bent along the helix axis, which inuences the ability of proteins to bind DNA. The sequence of bases along the DNA strands constitutes the genetic code and determines the structure of the protein specied by a given genetic unit, a gene. Complementary base pairing is also employed to copy DNA during DNA replication and to transcribe DNA into an RNA template that is used for protein synthesis by the ribosome. The human genome consists of approximately 3 billion base pairs of DNA that are organized on 44 autosomes (22 pairs of homologous chromosomes) and two sex chromosomes, the X and the Y chromosomes. The rst draft of the human genome sequence was completed in 2001 by an international consortium, and the high-accuracy sequence was reported in 2004. The human genome carries 20 000 25 000 protein-coding genes. The coding regions comprise less than 3% of the genomic sequence and are often interrupted by noncoding regions (introns). Approximately 50% of the human genome consists of repeat sequences. They are categorized into ve classes: (1) transposon-derived repeats, also referred to as interspersed repeats, include the long interspersed repetitive elements (LINE elements), the short interspersed repetitive elements (SINE elements), long terminal repeat (LTR) retrotransposons, and DNA transposons; (2) inactive copies of cellular genes (so-called pseudogenes); (3) simple sequence repeats, which consist of direct repeats of short sequences such as (A)n, (CA)n, or (CGG)n; (4) segmental duplications consisting of blocks of 10300 kb that have been copied from one region of the genome to another; and (5) blocks of tandemly repeated sequences, for instance, at centromeres and telomeres. Centromeres are the basis for building kinetochores, the attachment sites of the microtubules during mitosis. Telomeres serve to protect the ends of the chromosomes from sequence loss and recombination. Otherwise, little is known about the function of repetitive sequences in the human genome. Simple sequence repeats (SSRs) are perfect or slightly imperfect tandem repeats of a particular short sequence. SSRs with a short repeat unit (1 13 bp) are often termed microsatellites, and those with longer repeat units (40500 bp) are termed minisatellites. The human genome consists of approximately 3% SSRs, which amounts to approximately one SSR per 2 kb of sequence. SSRs are very important in human genetic studies because they show a high degree of length polymorphism in the human population. Thus, SSRs can be used as genetic markers by measuring the number of tandem repeats of a simple sequence at a given genomic location. A

comprehensive map of SSRs is very useful in human genetic studies, for instance, to map disease mutations, and single SSRs can be used in forensics or paternity tests to identify individuals.

Regulation of DNA Function: Chromatin

DNA in the eukaryotic nucleus is packaged into a macromolecular structure termed chromatin (Figure 3). The basic repeat unit of chromatin is the nucleosome, which consists of two each of the core histones H2A, H2B, H3, and H4, around which the DNA is wrapped in two turns totaling 146 bp of DNA. Nucleosomes are positioned along the DNA strand to form a lament 10 nm in diameter. The next level of chromatin organization is the 30 nm ber, which is stabilized by the linker histone H1. The structure of this ber is alternatively proposed to be a solenoid or a two-stop helix. The 30 nm ber is further compacted into chromatin loops that are attached to a protein matrix and build individual chromosomes. In addition to the histones, there exist several types of nonhistone chromatin proteins. One major group is the high mobility group (HMG) of proteins. The HMG1- and HMG2-type proteins enhance transcription, HMG14 and HMG17 bind nucleosomes via interactions with the N-terminal tails of histones, and HMGI(Y) and HMG I-C bind to the minor groove of the DNA and are architectural components of chromosomes. Also, several transcription factors contain HMG-like domains.

Histone H1

30 nm fiber

Nucleosome Histones H3/H4 H2A/H2B

DNA

Figure 3 Model of the packing of DNA into chromatin. Blue disks represent nucleosomes.

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DNA / Structure and Function

Eukaryotic genomes are organized into functionally distinct domains that differ in their chromatin composition. So-called euchromatin mostly contains actively transcribed genes and has an open chromatin conguration. Conversely, heterochromatin is gene poor, and the genes in heterochromatic regions generally are not expressed a phenomenon termed gene silencing. Repression is achieved by the binding of specialized nonhistone proteins to the chromatin, which prevents transcription, and some heterochromatic regions also require structural RNA molecules for repression. For instance, the inactive X chromosome in female cells contains as a structural component the XIST RNA. Furthermore, pericentric heterochromatin is repressed by the action of small inhibitory RNAs. Euchromatic and heterochromatic regions can be distinguished by a cytological staining procedure that was developed by Emil Heitz in 1928. One type of change in DNA that plays a role in gene regulation involves the presence or absence of methyl groups on the bases of DNA. After replication, approximately 5% of cytosine residues within a eukaryotic genome are methylated by DNA methyltransferases, often in clusters of CpG sequence so-called CpG islands. DNA methylation alters the ability of transcription factors to bind DNA. Furthermore, methyl-cytosine binding proteins bind methylated DNA and inuence chromatin function. All DNA metabolic processes, such as DNA replication, transcription, and DNA repair in eukaryotic cells, operate on chromatin as a template, which is
Me Ac Ac

more repressive to extraneous actions than naked DNA. Two main enzymatic activities can be distinguished that regulate chromatin access in the nucleus: histone/chromatin modifying complexes and chromatin remodeling complexes.
Histone Modications

The term histone modication describes posttranslational modications on the core histones. Potential modications include lysine acetylation, arginine or lysine methylation, serine or threonine phosphorylation, and lysine ubiquitylation (Figure 4). The individual modications are attached to the side chains of the amino acids. Most known modications are found in the amino-terminal tails of the histones that protrude from the core nucleosome, but modications also exist in the core region of the nucleosome. The position of individual modications is highly conserved between different organisms (Figure 4). Posttranslational modication of histones is carried out by enzyme complexes. For instance, histone acetyltransferases (HATs) perform acetylation, and histone methyltransferases (HMTs) carry out methylation reactions. Some modications can be enzymatically reversed. For example, histone deacetylases (HDACs) remove acetyl groups. Histone modications may affect chromatin structure directly by altering DNAhistone contacts within and between nucleosomes, thus changing higher order chromatin structure. Also, protein domains are known that bind certain modications. For

Ac

Ac

Ub H2A LPKKTESH 119

H2A SGRGKQGGKARAKAKTRS 5 1 13 Me Ac P Ac Ac

Ac

Me Ac H2B

Ub TKYTSAK 120

H2B PEPAKSAPAPKKGSKKAVTKAQKKDG 5 20 24 12 15 P Me Me P P Me Me Ac Ac P Me Me Me Ac Ac

Me 79

DFKTD Me H3

Me Ac

H3 ARTKQTARKSTGGKAPRKQLATKAARKSA...GVKK 4 14 18 23 27 36 9 Me Ac Me Ac Me Ac H4

Ac

Ac

H4 SGRGKGGKGLGKGGAKRHRKVLRDN 5 12 16 20 8

Figure 4 Posttranslational modications on the four human core histones. Ac, acetylation; Me, methylation; P, phosphorylation; Ub, ubiquitylation.

DNA / Structure and Function 41

instance, a module called a bromodomain binds acetylated lysine residues in histones, and individual bromodomains in different proteins differ with regard to the precise residue they are able to bind. Similarly, some types of chromodomains bind methylated lysine side chains. Combinations of histone modications thus constitute an interaction surface for nonhistone chromatin proteins that interpret this so-called histone code and translate it into a gene expression pattern. Moreover, histone modications can also inuence DNA methylation and vice versa. HATs use acetyl-coenzyme A (acetyl-CoA) as a cofactor in the acetylation reaction in which an acetyl group is added to the e-N position of a given lysine side chain. HATs are grouped into ve families based on sequence comparison of their acetyl-CoA binding site. First is the Gcn5 family, which includes human GCN5 and PCAF. The HATs of the Gcn5 family carry a bromodomain. Second is the MYST family, named after the founding members MOZ, Ybf3/Sas3, Sas2, and Tip60. Translocations that create fusions between MOZ and other HATs are associated with certain types of leukemia. Third is the p300 family of HATs, which includes p300 and CREB binding protein (CBP), two highly related HATs. Mutations in p300 are found in epithelial cancers, and mutations in CBP are the cause of RubinsteinTaybi syndrome. Fourth, several general transcription factors, such as TAF250 or TFIIIC, contain a HAT domain as well as a bromodomain. Finally, some nuclear receptor cofactors have intrinsic HAT activity that is thought to aid in transcriptional activation. HDAC families include the HDAC I family, which resembles yeast Rpd3, and the HDAC II family, which is similar to yeast Hda1. Four class I and ve class II HDACs exist in humans. A third group of HDACs, the Sirtuin family, whose founding member is the yeast heterochromatin protein Sir2, is structurally unrelated to the other two families and, unlike the other families, requires the cofactor NAD in the deacetylation reaction. Seven human sirtuins are recognized. Mammalian sirtuins fulll diverse functions in development, heterochromatin formation, and apoptosis and are suggested to link the metabolic rate of an organism to aging. Posttranslational acetylation is not restricted to histones. Some HATs also have nonhistone targets, an activity that is termed factor acetylation. Notably, some HMG proteins are also acetylated by HATs, and the acetylation is postulated to regulate their function within chromatin. Another prominent example is acetylation of the tumor suppressor p53. p53 is a transcription factor that responds to DNA damage. Acetylation of p53 by p300/CBP and PCAF

increases its DNA binding capacity and thus its activity in transcription. p53 deacetylation is carried out by sirtuins. In contrast to acetylation, lysine methylation can result in lysine residues that are mono-, di-, or trimethylated. The HMTs use S-adenosyl-methionine as a cofactor. One class of HMTs is the SET family of proteins, named after the chromatin regulators Su(var)39, Enhancer of Zeste, and Trithorax from Drosophila. They perform methylation of lysine residues in the amino-terminal tails of the histones. Twenty-seven human homologs are found, and many of them are conrmed or potential tumor suppressors or oncogenes. In contrast to the SET HMTs, the Dot1 family of HMTs methylates the residue K79, which lies in the core region of the histones. There is no sequence similarity between the two classes of HMTs.
Chromatin Remodeling

In contrast to chromatin modication, chromatin remodeling leaves the biochemical composition of chromatin unaffected but alters histoneDNA contacts. Four basic types of remodeling are recognized: (1) nucleosome sliding, which changes the location of a nucleosome along a DNA sequence; (2) the creation of a remodeled state of the nucleosome that is characterized by altered histoneDNA contacts; (3) the removal of histones (histone eviction); and (4) histone exchange. Chromatin remodeling is catalyzed by protein complexes termed remodelers. Depending on the remodeler and the target nucleosome, the DNA sequence can be more or less amenable to accessory factors after chromatin remodeling. Chromatin remodelers use the energy of ATP hydrolysis to loosen DNAhistone contacts in order to facilitate the movement of nucleosomes. Thus, all chromatin remodeling complexes have an ATPase subunit, the motor of the complex. Two remodelers of the SWI/SNF family exist in human cells, hBRM (brahma) and BRG1 (brahma-related gene), that are highly related to each other and to yeast Snf2. Whereas BRM is dispensable for mouse development, the absence of BRG1 in mice causes death early in development. BRG1 is a central cell-cycle regulator and a tumor suppressor. A second class of remodelers contains an ATPase of the ISWI (imitation switch) protein family (termed SNF2L in mammals). In human cells, at least four complexes are observed with hSNF2h as a subunit. They are distinct in protein composition and play roles in chromatin replication and sister chromatid cohesion. A third category of remodeling ATPases is the CHD type of remodelers, which are characterized by the presence of two chromodomains. The human homolog Mi-2 is observed in several protein

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DNA / Structure and Function

complexes that also contain HDACs and methyl-cytosine binding proteins, thus providing a functional link between chromatin remodeling, histone deacetylation, and DNA methylation.

Biological Function
Transcription

DNA is the repository of genetic information in the cell. Genes are transcribed into messenger RNA (mRNA), which is translated into proteins. The genetic code consists of a linear series of triplet nucleotides present in the mRNA that reects the sequence in the DNA and species the insertion of a specic amino acid as the mRNA is translated into a growing protein chain. During transcription, eukaryotic gene promoters are rst recognized by general transcription factors (GTFs). The TATA box, a sequence approximately 30 bp upstream of the start site, is bound by TATA binding protein (TBP). TBP is part of the GTF complex TFIID that, in cooperation with other GTFs, attracts the core RNA polymerase II to start RNA synthesis. Transcription works on DNA packaged into chromatin as the native template. In this process, chromatin modiers and remodelers cooperate with sequence-specic DNA binding factors to help the transcriptional apparatus gain access to the DNA. For transcription initiation, a transcriptional activator binds to its target sequence in the promoter region of a gene. The activator then recruits HAT complexes that acetylate the histones in the chromatin at the promoter. This facilitates the recruitment of a chromatin remodeler, which then mobilizes nucleosomes in the proximity of the promoter. In the elongation phase of transcription, the RNA polymerase proceeds through chromatin along the body of the gene. It is aided in its progression by a HAT complex that is tightly associated with the elongating polymerase. Elongation is also accompanied by histone methylation, which serves to recruit chromatin remodelers that increase chromatin uidity and thus promote progression of the polymerase. Also, factors exist that remove H2A and H2B from the front of the transcribing polymerase and reassemble nucleosomes after its passage. Together, all these factors and activities contribute to successful gene expression.
Replication

divisions. The primary DNA sequence is duplicated by semiconservative DNA replication. Replication starts at origins of replication that are bound by the replication initiator, the origin recognition complex (ORC). Replication initiation is tightly coordinated with the cell cycle. The protein Cdc6 is only present in the early G1 phase of the cell cycle. Its binding to ORC allows subsequent binding of so-called licensing factors that limit initiation to once per cell cycle. Initiation is then triggered through a cyclin-dependent kinase; the DNA duplex is locally unwound, and DNA polymerases are recruited that synthesize a complementary DNA strand. Regarding transcription, DNA replication operates on the chromatin template. Accordingly, histone modications and chromatin remodeling are observed to modulate origin activity and to inuence the progression of DNA replication. Also, chromatin has to be restored on the newly replicated DNA. This process is termed chromatin assembly and is catalyzed by chromatin assembly factors that recognize replicated DNA by the presence of PCNA and deposit histones onto the DNA.
DNA Repair

The precise DNA sequence needs to be maintained during cell division and reproduction because changes in DNA sequence can have detrimental effects. Somatic mutations can cause disease of an individual, for instance, by causing genomic instability and resulting in cancer, and mutations in the germline can cause genetic diseases in the offspring. Thus, cells have developed efcient repair systems that recognize and eliminate DNA mismatches or lesions. Regarding transcription and replication, circumstantial evidence indicates a role for chromatin modiers and remodelers in regulating access of repair machineries to the DNA in chromatin.

DNA in Respiratory Diseases

Several categories with regard to how DNA is involved in respiratory diseases can be distinguished. They are briey described here and are discussed in more detail in other chapters of this encyclopedia. In one scenario, the DNA sequence of a molecule important for lung function is mutated and causes disease, as is found in hereditary disorders. A prominent example is cystic brosis (CF), an autosomal recessive disorder in which the CFTR transmembrane ion transporter is mutated, which causes the lung to produce abnormally viscous mucus. Since CF is caused by a single gene mutation, treatment by gene therapy is being pursued.

As cells divide and give rise to a whole organism, the genome must be accurately duplicated and passed on to the daughter cells in order to ensure that the genetic information remains constant over many cell

DNA / Structure and Function 43

Heritable mutations can also modulate the susceptibility of an individual to lung disease. Certain alleles of the gene encoding a1-antitrypsin (AAT deciency) increase the likelihood of developing chronic obstructive pulmonary disease, but there are probably many genetic modiers as well as environmental factors (e.g., smoking) that modulate the disease risk and that remain to be characterized. Certain genetic polymorphisms have also been associated with a predisposition to lung cancer. Subtle differences in gene expression or activity of cellular components can inuence an individuals susceptibility to tobacco smoke and other environmental factors. Candidates for susceptibility genes are alleles of enzymes that metabolize exogenous and endogenous compounds (e.g., cytochrome P450 enzymes or microsomal epoxide hydroxylase), DNA repair genes (e.g., ERCC and XRCC), and alleles of the tumor suppressor gene p53. It is likely that there are complex interactions between many susceptible genes, ethnic background, and environmental exposure that modulate lung cancer risk in the population. As in all cancer tissues, major chromosomal aberrations are also a hallmark of lung cancer cells, a phenotype that has been termed chromosomal instability. These abnormalities can be of structural or numerical nature. Cytological investigations of lung cancerous tissues have highlighted some regions in the genome as being more frequently altered (chromosomal gains or losses) in lung cancer than in other types of cancer. Research is aimed at determining which genes are affected and how they affect disease progression. Interestingly, chromosomal rearrangements involving HMGI-C and HMG-I(Y) have been identied in pulmonary chondroid hamartomas, suggesting that they cause alterations in gene expression and thus cause disease. There are many causes for genomic instability leading to cancer, including mutations in cell-cycle control and checkpoint proteins or in the repair of mutations or double-strand breaks. For instance, as in many other cancer types, lung cancer cells are frequently mutated in p53, which may cause genomic instability by abrogating p53-dependent DNA damage checkpoint control. Also, oncogenes such as the ERBB and myc gene families are occasionally amplied in lung cancer, which disrupts cell-cycle control. One route of repression of cell regulators is through DNA methylation in their promoter region, which results in gene repression mediated by methylcytosine binding proteins and subsequent recruitment of HDAC complexes. Hence, gene-promoter hypermethylation is being exploited as an early diagnostic indicator of lung cancer. Specically, aberrant promoter methylation of the CDKN2A gene,

which encodes the cyclin-dependent kinase inhibitor INK4A, is being evaluated in sputum samples for its predictive value. Similarly, methylation in the promoter region of the MGMT gene encoding the DNA repair enzyme O6-methylguanine DNA methyltransferase is being investigated. In addition to DNA sequence mutations, alterations in chromatin function also affect (lung) disease. Major genomic rearrangements as seen in cancer cells necessarily change cellular gene expression. Thus, compounds are being developed as general anticancer drugs that affect chromatin function. For instance, HDAC inhibitors are in clinical trials, although their application is not restricted to lung cancer. Transcriptional mechanisms also play a critical role in the pathogenesis of acute lung inammation. In particular, the transcription factor NF-kB, along with other proinammatory transcription factors, is activated by signaling cascades from several external stimuli, such as proinammatory cytokines, bacterial and viral products, and reactive oxygen species. NFkB interacts with HAT coactivators such as CBP and p300/PCAF and activates transcription of many cytokines and growth factors, adhesion molecules, immunoreceptors, and acute phase proteins, all of which contribute to pathogenesis. In addition, acetylation of NF-kB by CBP increases its transcriptional activity. NF-kB-mediated gene expression is further enhanced by binding of HMG-I, which thus constitutes a mediator of lung inammation. The effectiveness of corticosteroids in therapy of inammatory lung disease is attributed to their ability to suppress the action of NF-kB and other proinammatory transcription factors. They bind to nuclear hormone receptors (glucocorticoid receptor), which then directly activate anti-inammatory genes, inhibit coactivators of proinammatory transcription factors, or recruit HDACs to repress transcription.
See also: Cell Cycle and Cell-Cycle Checkpoints. Cystic Fibrosis: Overview. DNA: Repair. Gene Regulation. Genetics: Overview. Transcription Factors: Overview. Tumors, Malignant: Overview.

Further Reading
Barnes PJ, Adcock IM, and Ito K (2005) Histone acetylation and deacetylation: importance in inammatory lung disease. European Respiratory Journal 25: 552563. Becker PB and Ho rz W (2002) ATP-dependent nucleosome remodeling. Annual Review of Biochemistry 71: 247273. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333374. de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, and van Kuilenburg AB (2003) Histone deacetylases (HDACs):

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Masuda A and Takahashi T (2002) Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis. Oncogene 21: 6884 6897. Roth S, Denu JM, and Allis CD (2001) Histone acetyltransferases. Annual Review of Biochemistry 70: 81120. Sims RJ III, Nishioka K, and Reinberg D (2003) Histone lysine methylation: a signature for chromatin function. Trends in Genetics 19(11): 629639. Strahl BD and Allis CD (2000) The language of covalent histone modications. Nature 403(6765): 4145.

characterization of the classical HDAC family. Biochemical Journal 370: 737749. Ehrenhofer-Murray A (2004) Chromatin dynamics at DNA replication, transcription and repair. European Journal of Biochemistry 271(12): 23352349. International Human Genome Sequencing Consortium (2004) Finishing the euchromatic sequence of the human genome. Nature 431(7011): 931945. Luger K, Mader AW, Richmond RK, Sargent DF, and Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389(6648): 251260.

DRUG-INDUCED PULMONARY DISEASE

R A Dweik, The Cleveland Clinic Foundation, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Amiodarone
Amiodarone, an antiarrhythmic drug, can cause pulmonary toxicity in about 6% of patients receiving the drug. The diagnosis of amiodarone pneumonitis is really one of exclusion. Pulmonary embolism, congestive heart failure, and pneumonia are the main differential diagnoses in patients suspected of having amiodarone pulmonary toxicity, and should be excluded rst.
Etiology

Abstract
Drug-induced lung disease is a common iatrogenic illness. Different drugs can cause similar pulmonary syndromes/presentations. The most common presentation is an abnormality on the chest X-ray in an asymptomatic patient or a symptom-sign complex. Early diagnosis is very important and requires maintaining a high index of suspicion in the appropriate clinical settings. The nal diagnosis is usually one of exclusion. Cessation of the drug is usually enough for therapy for most drug toxicities; corticosteroid administration may be needed.

Introduction
The lung is being increasingly recognized as a target organ for drug toxicity and currently more than 150 pharmacologic agents have been reported to cause adverse pulmonary reactions. In drug-induced pulmonary disease, an abnormality on the chest X-ray lm or a symptom-sign complex is the most common form of presentation. Since the clinical presentation and radiologic and histologic ndings are mostly nonspecic, a high index of suspicion is needed to diagnose drug-induced pulmonary disease in the appropriate clinical setting. In this article, we provide a brief overview of drug-induced pulmonary disease. Table 1 summarizes the more common clinical presentations of pulmonary toxicity induced by noncytotoxic medications, including illicit drugs, while Table 2 does the same for pulmonary toxicity induced by cytotoxic agents, including radiation. Some unusual manifestations of drug-induced pulmonary toxicity are listed in Table 3. Not all the medications listed in the tables are discussed in the text. In some instances it is useful to discuss the pulmonary complications associated with a specic agent; however, we will also discuss the clinical syndromes induced by a number of different agents.

In most cases of amiodarone pneumonitis the patients have received daily doses exceeding 400 mg for two months or more. Pulmonary disease can occur with maintenance daily doses less than 400 mg but the incidence is lower (1.6%) as reported from combined placebo controlled trials involving 3439 patients. Pre-existing lung disease does not seem to increase the risk of development of toxicity (as evidenced by a lack of accelerated decrease in diffusing capacity in patients with chronic obstructive pulmonary disease (COPD) receiving amiodarone versus those receiving placebo in a randomized trial).
Clinical Features

The most common clinical presentation is a chronic interstitial pneumonitis characterized by insidious onset of dyspnea, dry cough, weakness, weight loss, and occasionally fever. Organizing pneumonia, presenting more acutely than the chronic interstitial pneumonitis, is seen in 25% of cases. A picture suggestive of adult respiratory distress syndrome (ARDS) has been described in patients undergoing general anesthesia for surgical procedures or after pulmonary angiography. Furthermore, in a retrospective study of 67 patients undergoing map-guided surgical procedures for ventricular tachycardia, the incidence of postoperative acute respiratory failure was 17% in patients previously receiving amiodarone and zero in

DRUG-INDUCED PULMONARY DISEASE 45

Table 1 Clinical presentation of pulmonary toxicity induced by noncytotoxic drugs PIE Noncardiogenic IP-F pulmonary edema Acute Chronic PVD pleural pleural effusion effusion Parenchymal hemorrhage DI-SLE BOOP Asthma

Anorexigens Dexfenuramine Fenuramine Anti-inammatory Acetylsalicylic acid Colchicine Gold Interleukin-2 Methotrexate NSAIDs Penicillamine Antimicrobial Amphotericin B Ethambutol Isoniazid Nitrofurantoin Para-aminosalicylic acid Penicillins Pentamidine Sulfasalazine Tetracycline Cardiovascular Alpha-methyldopa Amiodarone Anticoagulants Beta-blockers Dipyridamole Flecainide Hydralazine Hydrochlorothiazide Lidocaine Procainamide Propafenone Tocainide Illicit drugs Cocaine Heroin Methadone Methylphenidate Psychotropic/ antiepileptic Carbamazepine Diphenylhydantoin Phenothiazine Trazodone Tricyclics Miscellaneous Bromocriptine Contrast media Esophageal variceal sclerotherapy agents Estrogen Timolol (ophthalmic) Tocolytic agents

BOOP, bronchiolitis obliterans organizing pneumonia; IP-F, interstitial pneumonitis or brosis; PIE, pulmonary inltrate with eosinophilia; PVD, pulmonary vascular disease; DI-SLE, drug-induced systemic lupus erythematosus. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

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Table 2 Clinical presentation of pulmonary toxicity induced by antineoplastic drugs Hypersensitivity PIE Noncardiogenic IP-F Acute Chronic PVD Parenchymal BOOP Asthma inltrate pulmonary pleural pleural hemorrhage edema effusion effusion Azathioprine Bleomycin Busulfan Chlorambucil Cyclophosphamide Cytosine arabinoside Fludarabine Gemcitabine Melphalan Methotrexate Mitomycin C Nitrosoureas Paclitaxel Procarbazine Retinoic acid

BOOP, bronchiolitis obliterans organizing pneumonia; IP-F, interstitial pneumonitis or brosis; PIE, pulmonary inltrate with eosinophilia; PVD, pulmonary vascular disease. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

Table 3 Unusual manifestations of drug-induced pulmonary toxicity Presentation Alveolar proteinosis Bronchial necrosis Bronchospasm (paradoxical) Calcication (parenchymal) Cough Goodpastures syndrome Lung mass(es)7cavitation Mediastinal lipomatosis Mediastinal widening/ lymphadenopathy Mediastinitis Panlobular emphysema Pneumothorax Pseudosepsis syndrome Causative agent(s) Busulfan Radiation therapy, brachytherapy b2-Adrenergic agonists Intravenous hydrocortisone Antacids, calcium, phosphorous, vitamin D ACE inhibitors Penicillamine Amiodarone, bleomycin Corticosteroids Diphenylhydantoin, methotrexate Esophageal variceal sclerotherapy Methylphenidate Nitrosoureas, bleomycin Chronic salicylate intoxication

From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

patients not exposed to the drug p 0:03. Finally, in an unusual presentation, amiodarone pulmonary toxicity may take the form of parenchymal mass lesion(s), which may show cavitation (Table 3). Chest radiographs usually reveal a diffuse reticular pattern or bilateral airspace disease and, less commonly, nodular opacities or masses, pleural thickening, and pleural effusions. Computed tomography (CT) of the lung without the administration of

contrast (Figure 1(a)) may be helpful in making the diagnosis. Opacities caused by amiodarone toxicity have a high attenuation value (since the drug is an iodinated compound, Figure 1(b)) and appear more dense than usual opacities of other etiologies. A positive gallium scan can help differentiate amiodarone pneumonitis from pulmonary embolism or congestive heart failure (in which the gallium scan is negative) as long as infection has been excluded as a cause for the positive scan. One important limitation of gallium scan was evidenced by the lack of resolution of the abnormal uptake in three out of six patients with resolving amiodarone toxicity. Pulmonary function studies show restrictive defect and decreased carbon monoxide diffusing capacity (DLCO) and abnormal results correlate with exposure to the drug. As an example, in a trial of 519 patients, use of amiodarone for 1 year resulted in a signicant decrease in DLCO in comparison to placebo p 0:02. Although an isolated decrease in DLCO is a sensitive indicator of toxicity, it lacks specicity. In a prospective study evaluating the diagnostic utility of spirometry and DLCO in 91 patients on amiodarone, none of the 15 patients who had an asymptomatic decrease in diffusing capacity developed pulmonary toxicity in spite of continuing to take the drug in the 11 months of follow-up. Recently, measurement of serum concentrations of KL-6, a glycoprotein secreted by proliferating type II pneumocytes, has been proposed as a noninvasive marker of amiodarone toxicity. In a small study of 14 men receiving amiodarone, KL-6 levels above the

DRUG-INDUCED PULMONARY DISEASE 47

Figure 1 Radiographic appearance of amiodarone toxicity. (a) A computed tomographic scan without contrast in a patient receiving amiodarone reveals inltrates in both lung bases, due to the iodine content in amiodarone. This enhancement, however, is not pathognomonic for amiodarone toxicity. (b) The chemical structure of amiodarone showing the iodine content. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

Figure 2 Bronchoalveolar lavage ndings in amiodarone exposure/toxicity. Bronchoalveolar lavage cytologic preparation showing: (a) foamy appearance of alveolar macrophages seen by light microscopy; and (b) lamellar bodies seen by electron microscopy. Both of these ndings are characteristic of amiodarone exposure but not necessarily toxicity. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: LippincottRaven Publishers.

upper limit of 520 U ml 1 were seen in the two patients with pulmonary toxicity as opposed to normal levels in ve patients with other pulmonary abnormalities (including pneumonia, congestive heart failure, and bronchogenic carcinoma) and seven asymptomatic individuals.
Pathology

patients with or without pulmonary toxicity, but the absence of this nding makes the diagnosis of toxicity unlikely.

Management and Current Therapy

Cessation of the drug is the most important aspect of therapy. Corticosteroids have been used in uncontrolled studies and seem to be of greatest value in patients with severe disease and in those in whom the drug cannot be discontinued. Relapse of toxicity has been demonstrated after withdrawal of steroid therapy. Owing to the long elimination half-life of amiodarone, toxicity can progress for a while in spite of discontinuation of the drug. Overall, the prognosis is favorable with improvement or stabilization in threequarters of patients after discontinuation of the drug with or without steroid treatment. Unfortunately, in

Histologically, a chronic interstitial pneumonitis associated with accumulation of foamy alveolar macrophages (Figure 2(a)) in the alveolar spaces, hyperplasia of type II pneumocytes, and widening of alveolar septa are the most common ndings; diffuse alveolar damage and bronchiolitis obliterans organizing pneumonia (BOOP) are much less common. Bronchoalveolar lavage demonstrates the same foamy alveolar macrophages (Figure 2(a)) both in

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patients developing ARDS, mortality can be as high as 50%.

Drug-Induced Bronchospasm or Cough

Drug-induced bronchospasm is caused by a variety of agents (Tables 1 and 2). In most instances, the drug exacerbates bronchospasm in a patient with underlying airway hyperreactivity.
Acetylsalicylic Acid

Acetylsalicylic acid (ASA) produces bronchospasm in 0.3% of normal adults and in from 4% to 20% of asthmatic patients. The ASA triad is a syndrome characterized by asthma, nasal polyposis, and drug sensitivity. Furthermore, other nonsteroidal anti-inammatory agents (NSAIDs) can produce a similar reaction and should be avoided in aspirin-sensitive patients. The rst manifestation of the syndrome is vasomotor rhinitis with a watery discharge. Typically, this develops in the second or third decade in a person who is not atopic and who has previously taken ASA. The reaction is at rst intermittent, later perennial. It is followed by the appearance of nasal polyps, and by midlife most patients demonstrate an asthmatic response. The syndrome is not a hypersensitivity response, despite the asthma and angioedema that may be seen following absorption of the drugs. It seems to be mediated by the proinammatory and
Phospholipids Phospholipase Phospholipase inhibitors Corticosteroids Arachidonic acid Cyclooxygenase

bronchoconstrictive properties of active metabolites of arachidonic acid derived through the action of 5-lipoxygenase (Figure 3). Treatment starts with avoidance of the offending drug. In addition, leukotriene modiers have been shown to improve pulmonary function and decrease beta-agonist use in patients already receiving corticosteroids. Aspirin desensitization can improve refractory upper and lower airway symptoms but is usually reserved for severe cases or when ASA use is absolutely necessary. Nasal polypectomy is used for symptoms of nasal obstruction only; it does not alter the response to ASA, and the polyps usually recur.
Beta-Blockers

Beta-blockers can exacerbate chronic obstructive lung disease and precipitate bronchospasm. Some examples, in decreasing order of likelihood to cause bronchoconstriction, are propranolol, timolol, nadolol, atenolol, and labetolol. They should be used with extreme caution in patients with a potential for bronchospasm.
Other Agents

Dipyridamole increases the concentration of the bronchoconstrictor adenosine, which can cause signicant bronchospasm in some patients with underlying obstructive lung disease. Theophylline is the drug of choice for treatment and/or prophylaxis in these patients. Vinblastine appears to act synergistically

Lipoxygenase Lipoxygenase inhibitors (e.g., zileuton) Leukotrienes

Aspirin Nonsteroidal anti-inflammatory drugs Thromboxane Prostaglandins Prostacyclin

Receptor-level antagonists (e.g., zafirlukast, montelukast) LTC4, LTD4, LTE4

LTB4

Inflammation

Inflammation

Bronchospasm Congestion Mucus plugging

Figure 3 How aspirin and nonsteroidal anti-inammatory drugs may worsen asthma. Aspirin and nonsteroidal anti-inammatory drugs inhibit cyclooxygenase activity, diverting arachidonic acid to the alternate lipoxygenase pathway, which converts arachidonic acid to potent bronchoconstrictor mediators: leukotrienes, LTC4, LTD4, and LTE4. Lipoxygenase inhibitors and receptor-level antagonists may counteract the effect. From Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782795.

DRUG-INDUCED PULMONARY DISEASE 49

with mitomycin C to produce bronchospasm. Administration of nebulized or intravenous pentamidine or propellants can further irritate already hyperreactive airways. Paradoxical bronchospasm (Table 3) has been reported with the use of nebulized beta agonists as well as intravenous hydrocortisone (not reported with other steroid preparations). There is anecdotal evidence that patients developing paradoxical bronchospasm with beta agonists may benet from the newly available levo-albuterol preparation. Estrogen therapy may worsen bronchospasm in women with chronic obstructive lung disease or asthma, and may even be associated with the onset of asthma.
Angiotensin-Converting Enzyme Inhibitors

pathway, are not associated with this side effect and can be used in patients with ACE inhibitor-induced cough.

Pulmonary Manifestations of Anti-Inammatory Agents

Methotrexate

Angiotensin-converting enzyme (ACE) inhibitors can produce an irritating cough in about 525% of patients receiving this class of drugs. The cough occurs with all ACE inhibitors and does not respond to substitution within the same class. The pathogenesis of ACE inhibitor-induced cough probably involves accumulation of kinins and substance P (due to ACE inhibition) leading to bronchial irritation and cough (Figure 4). Another postulated mechanism is activation of the arachidonic acid pathway with consequent elevated levels of thromboxane potentiating the symptoms. In support of this hypothesis, a study using picotamide (an inhibitor of thromboxane synthase and antagonist of the thromboxane receptor) found that eight of nine patients with cough induced by enalapril had complete resolution of their symptoms. Cough usually completely resolves within 10 days of cessation of therapy. Angiotensin II receptor antagonists, which do not interfere with the synthesis

Methotrexate is currently used in low doses as an anti-inammatory drug for many conditions, especially rheumatoid arthritis and Wegeners granulomatosis. Typically, the patient is given less than 20 mg weekly. The prevalence of pulmonary toxicity in these patients is 35% and most cases occur within 2 years of initiation of therapy. In a casecontrol study, increasing age (odds ratio (OR) 5.1), presence of diabetes (OR 35.6), hypoalbuminemia (OR 19.5), use of disease modifying antirheumatic drugs (OR 5.6), and rheumatoid pleuropulmonary involvement (OR 7.1) were independent predictors for the development of pulmonary toxicity. The onset is insidious and associated with cough, dyspnea, and low-grade fever. Radiographically, the disease most commonly reveals basal or diffuse interstitial opacities that can progress to diffuse airspace disease. Pulmonary function studies classically demonstrate a restrictive pattern with a decrease in DLCO. However, there are some reports suggesting development of airow obstruction with methotrexate therapy. Unfortunately, in a prospective evaluation of 124 patients receiving low-dose methotrexate, surveillance with pulmonary function tests (spirometry, lung volumes, and DLCO) did not allow for detection of toxicity prior to the onset of symptoms.

Angiotensinogen Renin Desired effect

Kinins Adverse effect

Remikiren, enalkiren Angiotensin I Angiotensin-converting enzyme (ACE) ACE inhibitors Angiotensin II Angiotensin-receptor blockers Binding to angiotensin receptors Vasoconstriction Inactive metabolites Increased levels of kinins, substance P Bronchial irritation, cough ACE ACE inhibitors

Figure 4 A possible explanation for why angiotensin-converting enzyme (ACE) inhibitors cause cough. ACE inhibitors have the favorable effect of blocking conversion of angiotensin I to angiotensin II (left), but also lead to accumulation of kinins and substance P (right), which may explain the side effects of bronchial irritation and cough associated with this class of medication. Newer agents (angiotensin II receptor blockers, remikiren, enalkiren) in theory would not be associated with cough. From Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782795.

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Bronchoalveolar lavage uid demonstrates an increase in CD4 lymphocytes, a non-specic nding seen in a variety of inammatory lung diseases. Histologically, changes resembling hypersensitivity pneumonitis (loosely formed granulomas) overlapping with a chronic interstitial pneumonitis are seen in most patients; BOOP and diffuse alveolar damage were also demonstrated in a few cases. The majority of patients improve with discontinuation of the drug. Corticosteroids have been recommended based on a favorable clinical response in case series and the opinion of experts in the eld, but no controlled studies have examined its efcacy.
Leukotriene Inhibitors

The leukotriene inhibitors zarlukast, montelukast, and pranlukast have been associated with the development of ChurgStrauss syndrome in asthmatics. It is not clear whether the syndrome develops due to the use of the leukotriene inhibitors or it is unmasked due to the dose reduction or discontinuation of systemic steroid use achieved by the introduction of the leukotriene antagonists. The latter hypothesis is supported by the occurrence of ChurgStrauss syndrome in patients switched from systemic to inhaled corticosteroids. However, in one case report the syndrome was reported in one patient given montelukast in the absence of systemic steroid use.
Other Agents

Pulmonary edema, which is probably due to a combination of increased capillary permeability and uid overload, has been reported in association with treatment with interleukin-2. An asymptomatic, isolated decrease in diffusing capacity was demonstrated in patients receiving recombinant tumor necrosis factor for the treatment of metastatic malignancies.

Pulmonary Manifestations of Antimicrobial Agents

Nitrofurantoin

Pulmonary toxicity is the most severe adverse effect of nitrofurantoin therapy. It seems to be an infrequent complication given the extensive use of the drug. In fact, during a 16-year period, 237 cases of adverse pleuropulmonary reactions were reported in an estimated 44 million courses of the drug. Reactions to nitrofurantoin are classied into two types: acute, developing hours to days after the institution of treatment; and chronic, occurring after weeks to years of continuous therapy. The former is more common, being reported in approximately 90% of cases of toxicity in two series. In the acute form,

fever is seen in approximately 80% of cases in conjunction with cough, dyspnea, rash (20%), and arthralgias (1015%). In the patients with the chronic form, fever and rash are not seen and the respiratory symptoms develop more insidiously. Peripheral blood eosinophilia occurs in 83% of individuals with the acute form. Radiographic abnormalities are seen in the vast majority of patients and most commonly include diffuse alveolar or interstitial opacities. Pleural effusions may be present in conjunction with the parenchymal abnormalities or as an isolated nding. In the acute form, histologic ndings seem to represent a type I hypersensitivity response, including signs of pulmonary vasculitis and interstitial and alveolar monocytes or polymorphonuclear inammation with variable presence of eosinophilia. Other uncommonly reported ndings include diffuse alveolar damage, desquamative interstitial pneumonitis (DIP)-like reaction, hypersensitivity pneumonitis-like reaction, BOOP, and diffuse alveolar hemorrhage. A chronic interstitial pneumonia resembling usual interstitial pneumonitis (UIP) is the commonest pathologic nding in patients with chronic nitrofurantoin pulmonary toxicity. Discontinuation of the drug is the mainstay of therapy in both forms of nitrofurantoin pulmonary toxicity. Eighty-eight percent of patients with the acute reactions will have resolution of symptoms within 3 days of discontinuation of the drug. Clinical improvement is slower (weeks to months) in the chronic form. Benecial effects of steroid use have only been demonstrated in case reports and in a series of 447 patients its use did not accelerate recovery. In that same cohort, six cases were fatal, four of which occurred in patients with the chronic form of toxicity. Sulfasalazine has been reported to cause several pulmonary reactions including pulmonary opacities with eosinophilia, hypersensitivity pneumonitis, BOOP, DIP, pulmonary brosis, and bronchospasm. Pulmonary function studies can demonstrate both a restrictive or obstructive pattern. One patient with bronchospasm and another with bronchiolitis had resolution of their symptoms with steroid use.

Other Agents

A number of antimicrobials have been associated with an eosinophilic or hypersensitivity lung reactions including tetracycline, minocycline, sulfonamides penicillins, para-aminosalicylic acid, and ethambutol. Fatal pulmonary hemorrhage was demonstrated in 14 of 22 patients receiving amphotericin B in association with granulocyte transfusions. Although the

DRUG-INDUCED PULMONARY DISEASE 51

effect was not demonstrated in another study, a real association cannot be discounted.

Pulmonary Manifestations of Anti-Neoplastic Drugs

The delayed adverse effect of cancer treatment is an increasing health problem as therapeutic regimens for neoplastic diseases evolve and an increased number of long-term survivors occur. For instance, nearly 30% of long-term survivors of Hodgkins disease complain of dyspnea and have pulmonary function abnormalities. Furthermore, in a series of 77 patients who underwent high-dose therapy and autologous bone marrow transplantation, the incidence of drug- or radiotherapy-induced lung toxicity was 16%. The adverse pulmonary responses to various antineoplastic drugs are usually similar and most often include chronic interstitial pneumonitis/brosis, acute hypersensitivity-type reactions and noncardiogenic pulmonary edema (Table 2). Typical symptoms of the interstitial pneumonitis/brosis develop over weeks to months and include progressive exertional dyspnea, dry cough, and malaise associated with crackles on physical exam. Dyspnea, nonproductive cough, fever and occasional peripheral blood eosinophilia are the most common features of hypersensitivity-type reactions. Symptoms generally progress rapidly over the course of days. Occasionally, factors such as total cumulative dosage, patient age, or prior use of agents that may act synergistically (e.g., radiation) can be used to anticipate an untoward response. Interstitial opacities resembling pulmonary brosis are the most common radiographic ndings. Alveolar opacities can also be seen in patients presenting with hypersensitivity lung reactions. Pleural effusions are uncommon. Pulmonary function testing is usually consistent with a restrictive ventilatory defect (low vital capacity or total lung capacity) and a reduced DLCO; the ow rates are usually preserved. The list of causes of pulmonary opacities in the immunocompromised host always includes infection, which should be excluded as an etiology of opacities suspected to be due to drugs. Treatment is discontinuation of the agent. The role of steroids is not well established since no randomized trials have been conducted but they should probably be tried especially in severely symptomatic patients.
Bleomycin

Heroin and Other Illicit Drugs

The use of illicit drugs has reached epidemic proportions, resulting in an increased incidence of pulmonary toxicity. The pulmonary manifestations relate not only to the substance used, but also to the route of administration. Narcotic addiction remains a major health problem, with noncardiogenic pulmonary edema occurring as a complication of heroin, methadone, and cocaine abuse. Opiates and related drugs are well-known causes of pulmonary edema occurring in 5070% of overdoses. It has also been demonstrated with naloxone use, an opiate antagonist. Pulmonary edema develops within a few hours of use, and the patient presents with constricted pupils and depressed respiration. The chest radiograph shows alveolar opacities, usually in bat wing distribution. A high protein concentration in the edema uid suggests increase in capillary permeability as the etiologic mechanism. The therapy is supportive, with administration of oxygen, narcotic antagonists, and mechanical ventilation if needed. Cocaine is a highly addictive substance. Pulmonary complications are related to all forms of administration. Cough productive of black sputum, dyspnea, chest pain, and hemoptysis occur within minutes of crack inhalation. Pulmonary edema can occur with cocaine regardless of the route of administration and it has been reported with freebase inhalation, intravenous freebase, and even body packing (smugglers swallowing packets of cocaine, which leak into the gastrointestinal tract). Increased membrane permeability and a possible component of myocardial dysfunction are the mechanisms responsible for the development of pulmonary edema. An acute pulmonary syndrome temporally related to inhalation of crack cocaine has also been described and is characterized by fever, hemoptysis, alveolar opacities on chest radiographs, and respiratory failure. Diffuse alveolar damage is the most common pathologic manifestation and it can be associated with alveolar hemorrhage and eosinophilic inltration. The patients tend to respond to systemic corticosteroids. Other reported complications of smoking freebase cocaine include barotrauma, interstitial pneumonitis, thermal airway injury, bronchospasm, and BOOP. Pulmonary edema has been reported with amphetamine abuse. Early panlobular emphysema was described in intravenous abusers of methylphenidate and in individuals who inject talc-containing drugs intended for oral use.

Bleomycin is deposited in the skin and lungs due to decreased drug inactivation in these tissues. Thus, the lungs and the skin are the two organs displaying the most serious side effects. Approximately 10% of patients receiving bleomycin will develop pulmonary toxicity. Interstitial brosis is the most commonly mentioned adverse pulmonary response, followed by hypersensitivity pneumonitis and BOOP with

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DRUG-INDUCED PULMONARY DISEASE

parenchymal nodular densities. Symptoms usually develop within the rst 6 months of therapy and include dry cough and dyspnea. Physical examination reveals tachypnea, basilar crackles, and concomitant hyperpigmentation of the skin. Acute chest pain can also occur in 2.8% of patients during bleomycin infusion, being severe enough to require narcotics and warrant evaluations for myocardial infarction or pulmonary embolism. Factors that appear to increase the toxic potential include age, total cumulative dose, oxygen therapy, renal function, and prior radiation to the thorax. In one review, the incidence of toxicity was signicantly higher in patients older than 70 years (6% in patients 2070 years versus 15% in those above 70 years). Although toxicity can develop with doses as low as 49 units, toxicity and fatality sharply increases with doses greater than 450 units. Supplemental oxygen administration (FiO2 0.35 to 0.42) was associated with fatal pulmonary toxicity in ve patients undergoing surgical procedures, while 12 patients maintained at an FiO2 of less than 0.25 during surgery showed no toxicity. In a series of 20 patients with metastatic testicular cancer receiving bleomycin, presence of renal failure was the most signicant factor associated with the development of abnormal pulmonary function tests. Evidence for the enhancement of toxicity by concomitant use of other antineoplastic agents is inconclusive. In a series of 100 patients, radiographic abnormalities were detected by chest radiograph in 15% of cases and in 38% by chest CT. They normally include a non-specic interstitial pattern with the most severe changes appearing in the lung bases and subpleural areas. Nodular abnormalities, which can be confused with metastatic disease, can be encountered. Vital capacity, total lung capacity, and diffusing capacity have been reported to be sensitive markers for detection of early signs of toxicity but serial measurements were unable to specically predict development of toxicity. The histologic changes seen in patients with brosis are most commonly those of diffuse alveolar damage (Figure 5). Eosinophilic pneumonia is seen in patients with hypersensitivity pneumonitis. Treatment is discontinuation of the drug. Resolution of interstitial disease has been reported with steroids in case reports as well as in cases of hypersensitivity pneumonitis and BOOP. If the patients condition demonstrates reversibility, the radiographic ndings and pulmonary function test results usually improve. In a series of 180 patients treated for germ-cell tumors, the mortality related to bleomycin pulmonary toxicity was 2.8%.

Figure 5 Bleomycin toxicity. This is the typical appearance of interstitial lung disease due to drug toxicity of which bleomycin is a typical example. Characteristic features include hyperplastic alveolar epithelium, septal thickening, and lymphocytic inltration. From Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Drug-induced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: Lippincott-Raven Publishers.

Cyclophosphamide

Pulmonary toxicity is a rare complication of cyclophosphamide use either in malignant or nonmalignant disorders. It appears to occur in two distinct forms: an early-onset interstitial pneumonitis and a late-onset interstitial pneumonitis with brosis, which is seen more frequently. In the early-onset pneumonitis, patients present within 16 months of onset of therapy with dyspnea, cough, fatigue, and frequently fever. Insidious and progressive dyspnea and cough occurring after months to years of therapy are characteristic ndings of the late-onset form. There are no clearly dened risk factors for the development of toxicity. Interstitial reticular or nodular opacities are seen on the chest roentgenogram and in some cases in association with bilateral pleural thickening. Ground glass attenuation on high-resolution CT scans has also been reported. The pulmonary function tests show restrictive defects and a decreased DLCO. Histologic studies typically show organizing diffuse alveolar damage (DAD) and less frequently chronic interstitial pneumonitis and BOOP. Early-onset pneumonitis usually responds to discontinuation of the drug and, in the majority of series, steroids, whereas the course of late onset pneumonitis is most often progressive and irreversible.
Bischloroethylnitrosourea

When used as a single agent, bischloroethylnitrosourea (BCNU) has been reported to cause pulmonary toxicity in up 20% of patients. Incidence can be as high as 60% when the drug is used as part of

DRUG-INDUCED PULMONARY DISEASE 53

high-dose combination protocols for bone marrow transplantation. The incidence of toxicity increases with dose increments. There is also evidence of an increased incidence of toxicity in patients treated before the age of 5 years. Pulmonary brosis can develop as an early (within 36 months) or late (up to 17 years) complication of therapy. The course of the disease is usually insidious with progressive cough, dyspnea, and fatigue, although rapidly progressive and fatal presentations have been described. Radiographic ndings include bibasilar reticular opacities and ground glass attenuation in the lower lung zones. Less commonly, upper lobe disease, patchy consolidation, and pneumothorax can be seen. The radiograph may also be normal even in the presence of histologically proven brosis. Histologic ndings typically include interstitial pneumonitis and brosis with atypia of type II cells or diffuse alveolar damage. In patients with early lung toxicity, corticosteroids seem to be helpful. Prednisolone used at a dose of 1 mg kg 1 for 10 days followed by a slow taper resulted in complete recovery in 15 of 17 patients developing pulmonary toxicity after carmustine-based preparative regimens for bone marrow transplant. Some authors have also recommended discontinuation of therapy and institution of steroids in patients with signicant (410%) decrease in diffusing capacity. Steroids are not thought to be benecial in patients with late-onset brosis. The prognosis seems to be more favorable in cases of early-onset brosis. A mortality rate of 36% has been reported in 26 patients who developed pulmonary toxicity within 1 year of therapy. In a study of 17 survivors of childhood brain tumors who were treated with BCNU 12% died of lung brosis within 3 years of treatment and 47% after 1620 years of follow-up. Furthermore, all of the long-term survivors had evidence of lung brosis.
Busulfan

two studies, pulmonary function studies could not predict development of pulmonary toxicity. Histologic evidence of toxicity may be seen in up to 46% of all patients treated and typically include the presence of large, cytologically atypical type II pneumocytes. Alveolar proteinosis has been reported in a number of patients receiving busulfan (Table 3). Response to steroids has been reported in case series. The prognosis is generally poor with a postdiagnosis mean survival of 5%.
Mitomycin C

Busulfan is considered the prototypic drug for cytotoxic drug-induced pulmonary damage. It is estimated to occur in less than 5% of patients taking the drug. The usual case is one of long-term toxic damage to the lungs, with an insidious onset of symptoms after the patient has taken the drug for 34 years. Symptoms include cough, dyspnea, and fever and occasionally hyperpigmentation. Diffuse interstitial and alveolar opacities are typically seen on the chest X-ray, but nodular densities, pleural effusions, or a normal picture may be seen as well. The pulmonary function tests show restrictive defects, and a decrease in diffusing capacity. However, as demonstrated in

The incidence of pulmonary toxicity with mitomycin C ranges from 3% to 6%. Toxicity has been reported with mitomycin C alone or in combined treatments, particularly including vinca alkaloids. Pulmonary manifestations are somewhat unique and can include bronchospasm, acute and chronic pneumonitis, pleural abnormalities, and hemolytic uremic syndrome. Acute bronchospasm occurs in about 5% of patients and is associated with concomitant administration of a vinca alkaloid. Acute pneumonitis with concomitant vinca alkaloid administration, noncardiogenic pulmonary edema, and acute respiratory failure with diffuse alveolar damage comprise the other forms of acute presentation, occurring on average within 3 weeks of the onset of therapy. Chronic interstitial pneumonitis seems to be a less frequent complication, usually occurring in patients receiving more than 30 mg m 2 of the drug. Pleural disease presenting as exudates or pleural brosis seems to occur at a greater frequency with mitomycin C than other antineoplastic agents. Finally, a hemolytic-uremic-like syndrome associated with mitomycin C has been described. Fifty percent of patients presenting with the syndrome develop ARDS. Radiographic manifestations consist of bilateral reticular opacities predominantly in the lower lung zones and are seen in both the acute and chronic forms. Findings consistent with ARDS are also less frequently seen. The DLCO declines by 420% in approximately a quarter of patients after they have received three cycles of chemotherapy. Unfortunately, the use of serial DLCO measurements in patients receiving mitomycin C cannot predict pulmonary toxicity. Histologic ndings are typically similar to those found with other cytotoxic agents and less frequently include BOOP. Lung tissue from patients dying from the hemolytic uremic syndrome shows capillary angiomatoid changes. In cases of acute and chronic pneumonitis, prednisone therapy can result in dramatic resolution of both symptoms and radiographic abnormalities. However, mortality exceeds 70% in cases of hemolytic uremic syndrome.

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Cooper JAD, White DA, and Mathay RA (1986) Drug-induced pulmonary disease. Part 2: noncytotoxic drugs. American Review of Respiratory Diseases 133: 488505. Cottin V, Tebib J, Massonnet B, Souquet PJ, and Bernard JP (1996) Pulmonary function in patients receiving long-term low-dose methotrexate. Chest 109: 933938. Machado R, Dweik RA, Demeter S, and Ahmad M (2003) Druginduced pulmonary disease. In: Baum GL, et al. (eds.) Textbook of Pulmonary Diseases, 6th edn. Philadelphia, PA: LippincotRaven Publishers. Meyers JL (1990) Pathology of drug-induced pulmonary disease. In: Katzenstein AL and Askin F (eds.) Surgical Pathology of Non-Neoplastic Lung Disease, 2nd edn., pp. 97127. Philadelphia: Saunders. Ozkan M and Dweik RA (2001) Drug-induced lung disease: an update. Cleveland Clinic Journal of Medicine 68: 782795. Rosenow EC (1994) Drug-induced pulmonary disease. Disease a Month 40: 255310. Zitnick RJ and Cooper JA Jr (1990) Pulmonary disease due to antirheumatic agents. Clinical Chest Medicine 11: 139150.

See also: Acute Respiratory Distress Syndrome. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General; Smoking Cessation. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Pneumonia: Overview and Epidemiology. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts.

Further Reading
Carson CW, Cannon GW, Egger MJ, Ward JR, and Clegg DO (1987) Pulmonary disease during the treatment of rheumatoid arthritis with low-dose pulse methotrexate. Seminars in Arthritis and Rheumatism 16: 186195. Cooper JA Jr, White DA, and Mathay RA (1986) Drug-induced pulmonary disease. Part 1: cytotoxic drugs. American Review of Respiratory Diseases 133: 321340.

DUST MITE
A Custovic and A Simpson, University of Manchester, Manchester, UK
& 2006 Elsevier Ltd. All rights reserved.

Introduction
The term house dust mite usually refers to the mites of the family Pyroglyphidae, while domestic mites includes all mite taxa which can be found in homes (e.g., Pyroglyphidae and storage mites). In 1964 Voorhorst et al. suggested that allergen from dust mite Dermatophagoides pteronyssinus was the main house dust allergen, opposing the widely accepted hypothesis of the time that the dust antigen was produced by an abiotic chemical reaction. This marked the beginning of extensive research into mite biology and ecology, the relationship between mites and allergic diseases, and methods to reduce mite allergen exposure.

Abstract
The discovery in 1964 that allergen from dust mite Dermatophagoides pteronyssinus was the main house dust allergen marked the beginning of extensive research into mite biology, the relationship between mites and allergic diseases, and methods to reduce exposure. In order to digest food, mites produce a number of enzymes, which accumulate in their feces and in the dust reservoirs in the house, and may become inhaled and cause immune response in predisposed individuals. To date, 19 different allergens from dust mite have been characterized and found to elicit varying degrees of IgE reactivity and T-cell responses. Several mite allergens are biochemically active, including hydrolytic and nonhydrolytic enzymes. Proteolytic activity of mite allergens increases the permeability of epithelium in vitro. Opening up of tight junctions by environmental proteases and their delivery to antigen-presenting cells may be an important step in the development of sensitization and may contribute to ongoing symptoms of allergic airways disease. However, extrapolation of studies conducted in cell culture to in vivo scenarios must be made with caution. Whilst enzymatic activity may play a role in modulating allergenicity, current evidence suggests that it is not a prerequisite for allergy. There is a clear doseresponse relationship between mite allergens exposure and development of sensitization, and between mite sensitization and allergic disease. However, there is no relationship between mite exposure and the development of symptomatic disease. In contrast, in established asthma and rhinitis, mite allergen exposure in mite-allergic individuals contributes to the severity of symptoms. However, although mite avoidance should lead to an improvement of symptoms, there is little evidence to support the use of physical or chemical methods to reduce exposure.

Taxonomy, Biology, and Ecology

House dust mites belong to the family Pyroglyphidae, in the suborder Acaridida. Dust mites are not insects and belong to the same class as spiders, scorpions, and ticks. D. pteronyssinus mites are approximately 0.3 mm in length and are therefore not visible to the naked eye (Figure 1). They have eight legs and no distinct head. Pyroglyphid mites reproduce sexually; under optimal laboratory conditions, adults live for about 6 weeks, during which time a female may produce 4080 eggs. They feed on human skin scales, fungi, bacteria, and various organic detritus. Certain fungi present in house dust may partially digest human skin scales making them more palatable for dust mites. Furthermore, fungi are a food source for

DUST MITE 55

furniture in most homes in areas where mites are prevalent. In most European countries D. pteronyssinus has been found to be the most abundant species, whereas D. farinae is predominant in North America. The geographic distribution and occurrence of these two species within homes is largely dependent on the level of humidity in the mites microhabitat; D. farinae appears to survive better in drier climates than D. pteronyssinus. Blomia tropicalis from the family Glycophagidae is another mite species often found in houses in tropical and subtropical areas and is an important cause of sensitization and asthma in these areas.
Figure 1 House dust mites. Reproduced with permission of Dr B Hart, Royal Agricultural College, Cirencester, Gloucs, UK.

Mite Occurrence in Buildings

10 m 12.0 kV 1.05E3 3403/00 Zoology

Figure 2 House dust mite faecal pellets. Reproduced with permission of Dr B Hart, Royal Agricultural College, Cirencester, Gloucs, UK.

mites, and enhanced mite population growth after fungi are added to skin scales is not only due to fungal predigestion of skin scales. They have a well-developed digestive system, which produces spherical fecal pellets surrounded by a peritrophic membrane (Figure 2). It has been reported from laboratory studies that adult mites deposit on average 20 fecal pellets per day, but this may not reect accurately what is happening in wild populations. These fecal pellets are usually between 20 and 40 mm in diameter and contain a large number of allergens. The respiration is cutaneous, with skin serving as a barrier for the exchange of gas and water vapor. Dust mites live in an environment with no liquid water present. Active life stages require relatively high indoor humidity for their survival, and the level of air humidity largely governs the occurrence of mites in homes. Analysis of dust samples collected from homes in different parts of the world shows that many mite species occur in household dust. In spite of this diversity, pyroglyphid mites constitute more than 90% of the mite population in mattresses, and 7095% of the mite population in carpets and upholstered

Mites are free-living (i.e., not parasitic) and are usually found in the high-use areas of home (e.g., beds, carpets, and upholstered furniture). The conditions rendering an indoor microhabitat ideal for mite population growth are not as yet clear. The habitation of domestic dwellings results in the creation of house dust, including the accumulation of human skin scales. Suitable relative humidity and temperature in the microhabitat are essential for survival of mites. However, even in the regions where climate is conducive for mite population growth there may be huge differences in the mite density between different homes. The reasons for this variation probably include the age of the home and soft furnishings (older having higher levels), type of glazing (lower if double glazed), and presence of damp in the home. No apparent differences in the level of humidity nor the presence of adequate food are usually found between homes with high and low mite densities. The mite population of any one home is geographically isolated from that of others, but dispersal does occur on clothing. Some physical factors in the home may determine mite population density (e.g., long or loose-pile carpets contain more mites than tile or wood oors; short, tight-pile carpets harbor fewer mites than long or loose-pile carpets). Mite abundance does not appear to be affected by cleaning, and clean, well-maintained homes may still contain a large number of dust mites.
The Effect of Humidity on House Dust Mites

Whilst the abundance of dust mites in homes is related to a number of environmental factors, only the effects of temperature and humidity have been studied in detail. Mites are able to extract water vapor from unsaturated air by passive and active absorption and amount of the passive gain is directly proportional to the humidity of the environment. The optimum relative humidity for mite growth is 7595%,

56

DUST MITE

at temperatures of 15301C. The lowest relative humidity at which the passive and active uptake combined compensate for water loss under fasting conditions is known as the critical equilibrium humidity; at this point water gain is equal to water loss. Humidity below this point can dehydrate and kill mites, but will threaten the mite population only if sustained for long enough periods. At room temperature, relative humidity levels below 50% may be associated with the desiccation of mites. Studies have shown that within a room, the relative humidity of the carpet tends to be higher than the room air. Furthermore reducing the relative humidity of the room does not necessarily have an effect in mite microhabitats (carpet, mattress, and upholstery).

pellets may become inhaled and cause immune response in predisposed individuals (see Allergy: Allergic Reactions).
Proteolytic Enzyme Activity

House Dust Mite Allergens

The exact number of dust mite allergens that are clinically signicant has not yet been determined. To date, 19 different allergens from dust mite have been characterized (Table 1) and found to exhibit varying degrees of IgE reactivity and T-cell proliferative responses. Sequence polymorphisms have been identied for several mite allergens. In order to digest food, mites produce a number of enzymes, which accumulate in their feces. Mites are probably coprophagic and this accumulation of highly immunogenic enzymes is consequent to the manner in which they digest the food. These enzymes may accumulate in the dust reservoirs within the house, and as a result of any activity that causes settled dust to become airborne (e.g., vacuuming, bed making, etc.) the fecal
Table 1 House dust mite allergens Mite species Dermatophagoides spp. (D. pteronyssinus and D. farinae) Allergen Group 1 Group 2 Group 3 Der p 4 Der p 5 Der p 6 Group 7 Der p 8 Der p 9 Group 10 Group 11 Group 14 Der f 15 Der f 16 Der f 17 Der f 18w Der p 20 Eur m 2 Eur m 14

Several clinically important dust mite allergens are biochemically active, and their biological function amongst others include hydrolytic enzymes (proteases; see Cysteine Proteases, Cathepsins) and nonhydrolytic enzymes (glutathione-S-transferase and amylase). This led to the hypothesis that in addition to their intrinsic properties (e.g., protein integrity, resistance to degradation, binding capacity, and the degree of sequence similarity to host proteins), the biologic function of mite allergens may contribute to the enhanced allergenicity. Within this context, the protease activity may facilitate the allergen access to immunocompetent cells by compromising airway barrier function (e.g., by reversibly disrupting intercellular epithelial adhesion). Having facilitated the passage through epithelial barrier, the proteolytic activity of allergens may enable them to modulate immunological signaling (either by directly promoting IgE synthesis through cleavage of the low afnity IgE receptor on B cells, or indirectly through cleavage of CD25 and CD40). A typical example for such function is group 1 allergens, comprising 25 kDa acidic to neutral allergenic proteins which function as digestive enzymes. In addition, allergen groups 3, 6, and 9 have also been identied as digestive enzymes exhibiting serine protease activity. Proteolytic activity of mite allergens increases the permeability of epithelium in vitro. Der p 1 and mite

Molecular weight (kDa) 25 14 25 60 14 25 2231 26 24 36 B100 Unknown 98 53 53 60 40 14 177

Function Cysteine protease MD-2 related lipid recognition proteins Trypsin Amylase Unknown Chymotrypsin Unknown Glutathione transferase Collagenolytic serine protease Tropomyosin Paramyosin Apolipoprotein-like Chitinase Gelosolin/villin Ca-binding EF protein Chitinase Arginine kinase MD-2 related lipid recognition proteins Apolipoprotein-like

Euroglyphus maynei

DUST MITE 57

spent growth medium extract detach cultured MadinDarby canine kidney (MDCK) cells and canine tracheal epithelial cells grown on articial and natural biopolymer substrata, causing visible disruption of the epithelial architecture and lead to an increase in albumin ux across isolated sheets of bovine bronchial mucosa. Extract of D. pteronyssinus causes cell detachment of human alveolar type II epithelial-like cells and primary nasal epithelial cultures, but this is inhibited by the presence of protease inhibitors. Both cysteine and serine protease activities of mite fecal pellets lead to a concentration-dependent increase in mannitol permeability across cultured epithelial cell monolayers, but again these effects are abolished in the presence of class-specic protease inhibitors. However, it has to be emphasized that the concentrations of puried allergen and extracts used in these experiments were in the range of several hundred micrograms per milliliter. In contrast, personal inhaled exposure to Der p 1 has been estimated to be only 2 mg year 1. Proteolytically active Der p 1 may alter epithelial permeability by increasing paracellular conductance as opposed to transcellular conductance. Paracellular channels are normally sealed by tight junctions (TJ), which form the most apical element of the junctional complex between polarized epithelial cells. Both cysteine and serine proteases from D. pteronyssinus can disrupt barrier function through the degradation of TJ proteins. Immunopuried Der p 1 and serine proteases from mite fecal pellets evoke a time-dependent decrease in TJ staining which can be detected within 30 min of exposure. Increase in the mannitol permeability is related to the total number of breaks visualized by immunostaining. In addition, it was conrmed by immunoblotting that reduction in uorescence intensity was a result of proteolysis of the transmembrane proteins occludin and claudin and of the cytoskeletal protein zona occludens 1 (ZO-1). The TJ degradation and the resulting increase in transepithelial permeability are dependent upon the protease activity of the allergens and can be inhibited by the presence of protease inhibitors. Thus, it is hypothesized that the opening up of TJ by environmental proteases and their delivery to dendritic antigen-presenting cells may be an important early step in the development of sensitization and may contribute to ongoing symptoms of allergic airways disease. However, interpretation of such studies conducted in cell culture and extrapolation to in vivo scenarios with intact airway mucosa must be made with caution, as it is difcult to imply clinical relevance from such experiments given the actual human exposure levels. In in vitro studies varying concentrations of allergen with different levels of proteolytic

activity were applied to monolayers of cells. Comparable levels of activity in experiments yielded disparate levels of junctional breakdown. In order to evaluate the role of proteolytically active allergens in allergic disease, it is essential for the studies examining tight junctions in MDCK cells and bronchial epithelial cells to be replicated in primary nasal epithelial cell cultures or in ex vivo biopsy samples. Future studies also need to take into account the possible cytoprotective effect of antiproteases (e.g., a1-antitrypsin and secretory leukocyte protease inhibitor, which inhibit the protease activity of Der p 1). Thus, although enzymatic activity may play a role in modulating allergenicity, current evidence would suggest that it is not a prerequisite for allergy and this is supported by the nding that many allergens implicated in allergic rhinitis including cat, birch pollen and Der p 2 are not enzymatically active.

Dust Mites, Allergic Sensitization and Respiratory Diseases

Mite Allergen Exposure and the Development of Sensitization and Asthma

Several studies have conrmed a simple doseresponse relationship between dust mite allergen exposure and development of specic sensitization in infants and children, and early infancy has been suggested as a critical period for primary sensitization (see Allergy: Overview). The threshold concentration of 2 mg Der p 1 per gram of dust for developing mite sensitization in children already sensitized to other allergens has been suggested. In addition, sensitization to mites is a risk factor for the development of asthma in both children and adults (see Asthma: Overview). However, although a clear relationship has been demonstrated between sensitization and exposure to mite allergens, and between mite sensitization and allergic disease (especially asthma), the relationship between exposure to mite allergens and the development of manifest clinical disease is much less clear. Several longitudinal studies have examined the importance of mite allergen exposure in early life and subsequent development of asthma and mostly reported no signicant relationship between exposure and symptomatic disease. For example, data from the large prospective birth cohort (German Multicentre Allergy Study) showed: (1) a strong association between sensitization to mite and wheezing and (2) mite allergen exposure strongly related to specic sensitization. However, there was no dose response relation for mite allergen exposure and doctor-diagnosed asthma, wheezing within the last 12 months, or wheezing ever. In contrast to these

58

DUST MITE

European data, several recent studies from the developing countries in Africa (Ghana and Ethiopia) have suggested that asthmatic children are exposed to signicantly higher levels of dust mite allergen compared to controls, and that the risk for asthma in urban areas increases signicantly with increasing dust mite allergen exposure.
Mite Allergen Exposure in Established Disease

Mite allergen exposure in mite-allergic individuals is one of the numerous factors that has been related to the severity of asthma and rhinitis (see Asthma: Extrinsic/Intrinsic). If mite exposure contributes to the severity of symptoms, then reducing exposure should improve the control of disease. However, the evidence is equivocal as to whether measures to change the indoor environment are effective. Unequivocal ndings about mite exposure and allergic respiratory diseases A controlled low-dose mite allergen challenge which mimics real life increases non-specic airway reactivity in sensitized asthmatics. Peak expiratory ow rate (PEFR) variability, and pulmonary function in mite-sensitive subjects correlate with dust mite allergen levels in beds. Patients with severe asthma are signicantly more often both sensitized and exposed to high levels of mite allergen compared with patients with mild disease, and the combination of mite sensitization and high exposure, and respiratory viral infection substantially increases the risk of hospital admission. However, demonstrating the link between exposure and disease severity does not equate to demonstrating the benets of allergen avoidance. The equivocal ndings about mite exposure and allergic respiratory diseases: clinical trials of mite allergen avoidance Although complete absence of allergen exposure is usually associated with improvement in symptoms amongst sensitized patients (e.g., in patients with hay fever or seasonal asthma, the absence of exposure outside the season is associated with a dramatic improvement in symptoms), it remains unclear whether a major real-life reduction in personal exposure to mite allergens can be achieved. A Cochrane meta-analysis concluded in 1998 that current chemical and physical methods aimed at reducing exposure to mite allergens are ineffective and cannot be recommended for mite-sensitive asthmatics. The update of this meta-analysis in 2004 included 49 studies (search until June 2004), with no change in conclusions or recommendations. The largest double-blind, placebo-controlled trial on the effectiveness of mite allergen-impermeable mattress,

pillow, and duvet encasings, as a single intervention was carried out in 1122 adult patients with physician-diagnosed asthma taking regular inhaled corticosteroids and using at least 100 mg day 1 of salbutamol. There was no difference in either the primary outcomes (mean morning PEFR over a 4-week period during the run-in and at 6 months; the proportion of patients discontinuing inhaled corticosteroids in a phased reduction during months 712) or in the secondary outcomes (e.g., mean proportionate inhaled steroid reduction). Several small studies aimed at reducing mite allergen exposure have been conducted in children, and results of these have been more positive. Recently a large study adopting a wide-ranging intervention (including smoking cessation and education of carers) in children living in poor quality housing in the US reported improvements in asthma control amongst mitesensitized children. A systematic review dust mite avoidance measures for perennial allergic rhinitis has been published in 2003, suggesting no benecial effect of physical or chemical intervention. Subsequent to this metaanalysis, a study in 279 patients investigated the effectiveness of mite-allergen-impermeable encasings in mite-sensitized subjects with perennial rhinitis. Both groups reported a decrease in symptom scores during the 12-month follow-up period, with no difference in any of the outcomes between the groups. Most of the studies in adults demonstrate that in the absence of other mite control measures, allergenimpermeable covers are clinically ineffective for routine management of adult asthma or rhinitis. It remains possible that a much more extensive intervention in carefully selected subgroup of patients could have some effect, but that has not as yet been adequately addressed. Although the general consensus is that mite allergen avoidance should lead to an improvement of symptoms in mite-sensitive patients, there is little evidence to support the use of physical or chemical methods. The use of single intervention for dust mite allergy (e.g., mattress encasings) in adults with asthma or rhinitis cannot be advocated. For children, environmental control measures may be of some benet.
See also: Allergy: Overview; Allergic Reactions. Asthma: Overview; Extrinsic/Intrinsic. Cysteine Proteases, Cathepsins.

Further Reading
Aalberse RC (2000) Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228238.

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS 59

Colloff MJ (1991) Practical and theoretical aspects of the ecology of house dust mites (Acari: pyroglyphidae) in relation to the study of mite-mediated allergy. Review of Medical and Veterinary Entomology 79: 611629. Custovic A and Murray CS (2002) The effect of allergen exposure in early childhood on the development of atopy. Current Allergy and Asthma Reports 2: 417423. Custovic A, Murray CS, Gore RB, and Woodcock A (2002) Environmental allergen control. Annals of Allergy, Asthma and Immunology 88(5): 432441. Custovic A and Simpson A (2004) Environmental allergen exposure, sensitisation and asthma: from whole population to individuals at risk. Thorax 59: 825827. Custovic A and Woodcock A (2001) On allergens and asthma (again): does exposure to allergens in homes exacerbate asthma. Clinical and Experimental Allergy 31: 670673. Go /tzsche PC, Johansen HK, Schmidt LM, and Burr ML (2004). House dust mite control measures for asthma. Cochrane Database of Systematic Reviews, Issue 4, Art. No.: CD001187.pub2.DOI: 10.1002/14651858. Platts-Mills TAE and de Weck AL (1989) Dust mite allergens and asthma: a worldwide problem. Journal of Allergy and Clinical Immunology 83: 416427. Platts-Mills TAE, Vervloet D, Thomas WR, Aalberse RC, and Chapman MD (1997) Indoor allergens and asthma: report of the third international workshop. Journal of Allergy and Clinical Immunology 100(6): S1S21. Pomes A (2002) Intrinsic properties of allergens and environmental exposure as determinants of allergenicity. Allergy 57: 673679. Simpson A and Custovic A (2004) Allergen avoidance in the primary prevention of asthma. Current Opinion in Allergy and Clinical Immunology 4: 4551. Simpson A, Woodcock A, and Custovic A (2001) Housing characteristics and mite allergen levels: to humidity and beyond. Clinical and Experimental Allergy 31: 803805. Smith AM, Pomes A, and Chapman MD (2000) Molecular biology of indoor allergens. Clinical Reviews in Allergy and Immunology 18: 265283. Stewart GA and Robinson C (2003) The immunobiology of allergenic peptidases. Clinical and Experimental Allergy 33: 36. Thomas WR, Smith WA, Hales BJ, et al. (2002) Characterization and immunobiology of house dust mite allergens. International Archives of Allergy and Immunology 129: 118.

Relevant Website
http://www.allergen.org This website provides allergen nomenclature maintained by IUIS Allergen Nomenclature Sub-Committee.

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS

J B Grotberg, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Airow in the lungs results from an imposed pressure difference between the mouth and the alveoli, both for inspiration and expiration. The airways conducting the ow are a branching network of tubes which makes the relationship between pressure and ow complicated. This article covers the basic information known about these ows and how they relate to engineering principles of uid mechanics. These features include the effects of Poiseuille ow, boundary layer ow, converging/ diverging ow, ow in curved tubes and turbulent ow. Different aspects of these ows tend to dominate depending on the ow speed or Reynolds number. The concepts are integrated into a discussion of the overall ow versus pressure relationship over a wide range of Reynolds numbers. In addition, during forced expiration the outow can become limited as a result of partial collapse of the exible airways. The basis for this phenomenon is also presented.

into two daughter airways. So at any generation there are approximately 2n tubes. The trachea is the rst, and largest tube, and it is the n 0 generation. The tubes decrease in length and diameter as n increases (see Table 1). Because of this structure it is useful to review rst some basic aspects of gas ow in a single tube. The mechanics of ow pertains to any uid, which includes gases and liquids. So this subject falls within the interdisciplinary eld of biouid mechanics which joins together the physics/engineering discipline of uid mechanics with biosciences. Early contributions to biouid mechanics in many ways stemmed from this interest in dening the pressureow relationship in the lung as a means of detecting disease, monitoring gas exchange, designing respiratory devices, delivering aerosol medications, predicting toxin deposition, and advancing respiratory therapy.

Introduction
The lung airways are a branching network of tubes and each level of branching is called a generation (see Figure 1). There are n 23 generations of branches, each essentially a bifurcation of one parent airway

PressureFlow Relationships in a Single Tube

Poiseuille Flow

Consider steady uid ow in a tube of diameter d. This particular velocity prole does not change with axial position, x, so is said to be fully developed. It

60

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS

Trachea n=0

n=1

n=2 n=3

Figure 1 Lung airways as a branching network of tubes. Reproduced with permission from SOMSO Modelle (www.somso.de). & Copyright by SOMSO Modelle. Table 1 Weibel airway geometry data Generation n 0 1 2 3 4 5 6 7 8 9 10 15 20 Number of airways 1 2 4 8 16 32 64 128 256 512 1 024 32 768 1 048 576 Diameter (cm) 1.800 1.220 0.830 0.560 0.450 0.350 0.280 0.230 0.186 0.154 0.130 0.066 0.045 Length (cm) 12.000 4.760 1.900 1.760 1.270 1.070 0.900 0.760 0.640 0.540 0.460 0.200 0.083 Total XS (cm2) 2.54 2.33 2.13 2.00 2.48 3.11 3.96 5.10 6.95 9.56 13.40 113.00 1600.00 Re @ 500 cc s 1 2362.20 1745.35 1298.90 933.33 604.84 375.13 235.69 150.33 89.21 53.70 32.34 1.95 0.09 LE =LA 10.629 13.420 17.022 8.909 6.429 3.681 2.199 1.364 0.777 0.459 NA NA NA 92 13 49 091 388 221 776 809 788 399

has a parabolic shape and is known as Poiseuille ow. The relationship between the pressure drop, , is the P1 P2 DP, and the volumetric ow rate, V well-known Poiseuille law   128mL DPP V 1 pd4 where m is the uid viscosity, L is the distance in x over which the pressure drop pertains, and d is the tube diameter. Since the tube has a constant diameter for all x, there is no local acceleration (if the tube narrows) or deceleration (if the tube widens) and DP is due entirely to viscous dissipation or resistance. Resistance, R, for any type of tube ow is dened generally as the ratio of the pressure drop from vis . cous dissipation, DPV , to the ow rate R DPV =V The resistance for Poiseuille ow, RP , is then RP DPP 128mL pd4 V 2

average velocity. At any value of n, the cross-sectional =An , where An is the crossaverage velocity is Un V sectional area listed in Table 1. This dimensionless coefcient of friction, CF , for Poiseuille ow is C FP DPV L 1 64 2 d Re 1=2rU 3

where Re is the Reynolds number Re rUd=m. Some authors prefer to make their pressure drop measurements or calculations dimensionless by dividing them by DPP. Then they are assured of results whose values must be greater than or equal to unity.
Entrance Flow

Often pressureow relationships are given in dimensionless form by dividing the viscous pressure drop by (1/2)rU2, where U is the cross-sectional

For inspiratory air ow in the lung, it is more likely that Poiseuille ow is not present in the larger airways where most of the resistance occurs. Instead, from the ow division at each branch, we expect entrance ow where the velocity prole is x-dependent. The ow enters a long tube and has a at velocity prole at x 0. Further down the tube, a boundary layer dx grows along the wall as the viscous retarding forces

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS 61

slow the uid in this region. If the tube is long enough, eventually the velocity prole becomes parabolic (Poiseuille) at a distance LE from the entrance. LE depends on the Reynolds number, a reasonable approximation to this dependence is LE =d 0:03Re which holds for laminar ow rates where 50o Reo2300. The important issue for ows in the lung is that the entrance length, LE, is usually longer than the airway length, LA, in the larger airways where the major resistance to ow exists. This can be seen in Table 1 500 cm3 s1 is where the Reynolds number for V calculated at each n. Multiplying each Re value by 0.03d yields LE and the last column is the ratio LE =LA . Only at generation 8 does there start to be a ratio less than unity, and the calculation is not applicable (NA) when Reo50. The coefcient of friction for entrance ow is given by  1=2 DPE L 1 CFE b 4 d 1=2rU2 Re1=2 where the constant bB6 for a uniform tube. In dimensional terms, this relationship shows that 3=2 , which is a stronger dependence on ow DPE BV rate than the linear relationship for Poiseuille ow.
Flow in a Curved Tube

circumstances, the Re 1/2 term in eqn [5] is much larger than the Re 1 term when Rec100, which is fairly common in the large airways. So in this regime, both entrance ow and curved tube ow have an Re 1/2 dependence on CF .
Turbulent Flow

When the Reynolds numbers are large enough, the ow becomes turbulent. The critical value of Re that separates laminar from turbulent ow in a long, smooth tube is Re 2300. However, it can be a lower value in the airways where the tubes are aerodynamically short and the geometry is varied. For turbulent ow in a straight tube, the pressure drop depends on whether the tube walls are smooth or have roughness. The respective frictional coefcients are given by CFT smooth CFT rough DPTsmooth 0:32L=dRe1=4 1=2rU2 DPTrough 0:04L=d 1=2rU2

where the term 0.04 for the rough tube is an estimate when the height of the roughness is B1/100 the tube 7= 4 diameter. These relationships yield DPTsmooth BV 2 , which are stronger dependences and DPTrough BV ) or enon ow rate compared to either Poiseuille (V 3=2 ) ows. trance (V
Convergent and Divergent Flow

Airway bifurcations have axial curvature along the ow path with radius of curvature Rax. Centrifugal forces preferentially throw the faster moving uid of the midline toward the outer wall of the curve. This skews the axial velocity prole toward the outer wall compared to an equivalent, symmetric Poiseuille ow. The same mechanism creates a pair of vortices in the cross section. Individual uid particles, then, move in a helical pattern down the tube. The viscous pressure drop for fully developed ow in curved tubes depends on the Dean number, De d=2=Rax 1=2 Re=2, which involves the ratio of the tube radius to the axial radius of curvature, d/2Rax and the Reynolds number. A studied relationship for the ratio of the curved tube pressure drop, DPC , to the equivalent Poiseuille pressure drop is DPC =DPP 0:509 0:0918De1=2 . Rewritten in terms of the coefcient of friction results in CFC DPC 1=2rU2

Because the total cross-sectional area changes with generation n, the ow can be experiencing a convergence (decreasing area) or divergence (increasing area). There is a local minimum in cross-sectional area at n 3 (see Table 1). In divergent ow the prole becomes more pointed as Re increases. For Re high enough there can be backward ow near the walls. For convergent ow, the prole becomes more blunted as Re increases, and the velocity gradient near the wall is steeper. For large Re, it becomes a boundary layer whose thickness matches that of entrance ow. By similar means the coefcient of friction has an Re 1/2 dependence, as it does in eqn [4]. It also depends on the change in cross-sectional area or, equivalently, the convergence angle, a: CFconv DPconv B aRe1=2 1=2rU2 7

!   L d=2 1=4 1=2 1 0:509Re 0:0918 64 Re d Rax 5 Typical values of d=2Rax for airways can be B1/6, depending on the generation. Under these

Inspiratory Gas Flow at an Airway Bifurcation

, results from a The total ventilation gas ow rate, V combination of the pressures imposed at the alveolar and tracheal ends of the respiratory system and its

62

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS

resistance to the ow. At a typical bifurcation, the ow in the parent tube splits at the carina and a complicated ow in the daughter tubes results. The velocity prole in the daughters is a developing ow, and because of the axial curvature there are secondary swirling ows. Detailed studies have used the structure of entrance-ow viscous-pressure drop, as given in eqn [4], for inspiratory ow into a model p airway bifurcation and found b 8 2C, where C was experimentally determined to be C 1:85. So for the range of Re explored, bB21, a much stronger dependence than simple entrance ow due to high shear rates in the boundary layer. The total viscous resistance at any generation can be derived by adding up all of the individual airway resistances as parallel components. Using the equation for adding parallel resistances, we nd 1 1 1 1 2n R - Rn n ? Rn R R R R 2 8

energy change due to convective acceleration of the uid because of changes in the cross-sectional area of the conduit. It would tell us that between generation n and n 1,
2 2 Un DPK expiration 1=2rUn 1

DPK inspiration

10

Other than the region of n 3, where there is a local minimum in cross-sectional area, generally An1 4An, and from mass conservation we know that Un An Un1 An1 , so Un1 oUn for most airways. This implies that DPK o0 for inspiration, whereas DPK 40 for expiration. The magnitude of DPK can be up to B10% of DPV under various conditions.

Coefcient of Friction for an Airway Model

The pressureow relationships from experiments of inspiratory and expiratory ow in a ve-generation, rigid cast of the central airways are shown in Figure 2. Three different gases were used at various ow rates to achieve a wide range of Reynolds numbers. Now the forms of CF discussed above for single tubes become important guides in terms of their dependence on Re. Note that in Figure 2 the region of Reo200 has a slope of 1 in the loglog plot. This is the Re 1 dependence from Poiseuille ow. Then in the range 500oReo1500, the slope is 1/2, as expected when entrance ow dominates for inspiratory ow. For Re44000, the slopes of both inspiratory and expiratory curves are essentially zero, as is found in rough-walled turbulence. We note that the pressure drop at any Reynolds number is larger during expiration than during inspiration. This is due to the converging ow.

When this is done for the lung, it is readily seen that the major component of viscous pressure drop occurs within the rst 810 generations, and that Poiseuille law signicantly underestimates the losses. The smaller airways where n410 do not contribute much to the overall pressure drop. They form the silent zone of the lung, so called because diseases which affect them in early stages are not readily diagnosed by measuring overall lung resistance. The total viscous resistance of the airway tree is calculated by adding the generational resistances assumed to be in series.

Expiratory Gas Flow at an Airway Bifurcation

Expiratory ow at a bifurcation results in two vortex pairs, one pair from each daughter, due to the axial curvature of the daughters. So there are four vortices in the parent tube. The ow is also converging, so there will be a higher level of viscous dissipation in this ow compared to the Poiseuille equivalent due to the effects of convergence and swirling. Both effects have frictional coefcients that depend on Re 1/2.

Expiratory Flow Limitation

During expiratory ow, the exibility of the airway walls can become a very signicant issue in terms of restricting the ow rate. Figure 3(a) shows a exible tube arrangement for an airway, where one could consider the external pressure, Pext, to be the pleural pressure, and the ow is driven by the difference between the upstream pressure, Pu, and the downstream pressure, Pd. In forced expiration, Pext increases with Pu, so an equivalent system in a tube is to decrease Pd while keeping Pu xed. As this happens the ow increases until the internal pressure of the tube drops somewhat below the external pressure, Pext. Then the tube partially collapses, as shown in the dashed wall shape of Figure 3(a), decreasing in

Total Pressure Drop

The total pressure drop is due to kinetic energy changes, DPK, and due to frictional, viscous, effects, DPV, so that DPtotal DPK DPV 9

It is the viscous pressure drop DPV that we have discussed in all of the ows above. DPK is the kinetic

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS 63

30

HeO2 Air SF6O2

20

Sl

P/(1/2 )(V/A)2

10 9 8 7 6 5 4 3

op e 1

Steady expiratory flow

Slo

pe

1/

Steady inspiratory flow

7 8 9 102

5 6 7 8 9 103

6 7 8 9 104

Re = ( D/ ) (V/A)
Figure 2 Moody diagram of dimensionless pressure drop vs. Re for inspiration and expiration using three gases in a rigid model of the central airways. Reproduced from Isabey D and Chang HK (1981) Steady and unsteady pressureow relationships in central airways. Journal of Applied Physiology 51: 13381348, with permission from American Physiological Society.

Pext

P, A

(a) A /A0 1

cross-sectional area according to its pressurearea or tube law relationship, A(P) shown in Figure 3(b). As Pd is further decreased, the tube reduces its crosssectional area while the average velocity of the ow increases. However, their product, the volumetric , does not increase as shown in Figure ow rate, V P in Figure 3(c) has a maxi3(c). The fact that V mum, where the slope of the curve is zero, can be used to deduce a criterion for ow limitation called the wave speed theory. The outcome of the theory is that ow limitation will occur when the local ow speed, U, equals the local wave speed, C, of long uid-elastic waves that can propagate along the airway: s E UC 11 r where E is the specic elastance of the tube, E AdP=dA, from tube law shown in Figure 3(b). When U C, pressure information can no longer propagate upstream since waves carrying the new pressure information are all swept downstream. The implication for lungs is that owvolume curves at different levels of effort share a common curve toward the end of expiration when ow limitation is achieved and the ow rates are effort independent. Diseases which lower E, and hence C, will lower U at ow limitation, and expiration can be pathologically slow, as in asthma and emphysema. To counteract this, patients often breathe at a much higher functional

Pu

(b)

P Pext

Rigid tube

Flexible tubes

(c)

Pu Pd

Figure 3 Flow through a exible tube showing: (a) lateral view of collapsed segment, (b) tube law, and (c) ow limitation.

Pd

64

DYNAMICS OF GAS FLOW AND PRESSUREFLOW RELATIONSHIPS See also: Alveolar Surface Mechanics. Alveolar Wall Micromechanics. Ventilation: Control; Uneven.

residual capacity. This helps to pull outward radially on the airways to keep them from collapsing. Since airway properties and E vary along the network, the most susceptible place for the ow limiting segment seems to be the proximal airways. For example, we expect gas speeds prior to ow limitation to be largest at generation n 3 where the total cross-sectional area of the network is minimal. So ow limitation is likely near that generation. An interesting feature that can occur during ow limitation in airways is the production of utter oscillations which are heard as wheezing breath sounds. Wheezing is prevalent in asthma and emphysema patients.

Further Reading
Grotberg JB (1994) Pulmonary ow and transport phenomena. Annual Review of Fluid Mechanics 26: 529571. Grotberg JB (2001) Respiratory uid mechanics and transport processes. Annual Review of Biomedical Engineering 3: 421 457. Isabey D and Chang HK (1981) Steady and unsteady pressure-ow relationships in central airways. Journal of Applied Physiology 51: 13381348. Pedley TJ (1977) Pulmonary uid dynamics. Annual Review of Fluid Mechanics 9: 229274.

Dyspnea

see Symptoms of Respiratory Disease: Dyspnea.

E
Eicosanoids
see Lipid Mediators: Overview.

Elastin

see Extracellular Matrix: Elastin and Microbrils.

ENDOMETRIOSIS
S A Sahn and J T Huggins, Medical University of South Carolina, Charleston, SC, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Thoracic endometriosis is a clinical syndrome whereby ectopic endometrial tissue is deposited in thoracic structures. Growth of endometriotic implants is dependent on the presence of ovarian steroids. The four major presentations of thoracic endometriosis include catamenial pneumothorax, catamenial hemothorax, catamenial hemoptysis, and asymptomatic pulmonary nodules. Catamenial pneumothorax accounts for 73% of the cases of thoracic endometriosis described. Symptoms occur within 2448 h after the onset of menses. Current theories on the pathogenesis of thoracic endometriosis involve microembolization of endometrial tissue from pelvic veins and the movement of endometrial tissue into the pleural space via congenital diaphragmatic defects. The latter mechanism explains the right-side predilection of catamenial pneumothorax. The diagnosis is typically established clinically, as pathologic documentation is generally not required. The diagnosis rarely can be conrmed by pleural uid cytology, cytology from bronchoscopic aspiration, and needle aspiration of lung nodules. Elevated levels of serum CA-125 and measurements of endometrial antibody have been reported in patients with thoracic endometriosis. Treatment consists of suppression of the ectopic endometrium by interfering with ovarian estrogen secretion. Despite estrogen suppression, recurrence rates remain high, and pleurodesis should be considered in those who develop recurrent pneumothorax or hemothorax.

Introduction
Endometriosis is dened as the presence of endometrial glands and stroma outside of the uterus. This clinical entity is most often diagnosed in women between the ages of 25 and 35 years. The prevalence of endometriosis in the general population is not known. The incidence of pelvic endometriosis

is estimated at 1% for women undergoing surgery for all gynecologic indications but may be as high as 50% if the indication for surgery is to evaluate the cause for pelvic pain or infertility. Growth and maintenance of endometriotic implants are dependent upon the presence of ovarian steroids. Endometriosis most commonly involves structures within the pelvis resulting in dysmenorrhea, dyspareunia, and infertility. Endometrial tissue can occur outside the pelvis and has been described in the abdomen, central nervous system, skin, aorta, and thoracic cavity. In 1958, Maurer and colleagues were the rst to report thoracic endometriosis in a patient with recurrent spontaneous pneumothorax associated with menstruation. At thoracostomy, their patient was found to have a normal-appearing lung; however, the right hemidiaphragm had a notable defect, which was studded with endometrial tissue. They postulated that air entered the peritoneal cavity by way of the genital tract during menses. Air within the peritoneal cavity could transverse into the pleural space via the diaphragmatic defect to induce a pneumothorax. Since this initial description, thoracic endometriosis remains a rare disease with varying clinical presentations. To date, there are slightly more than 100 cases reported in the medical literature. The most common presentation of thoracic endometriosis is right-side pneumothorax. Hemothorax, hemoptysis, chest pain, and asymptomatic pulmonary nodule occur less frequently. The symptoms of thoracic endometriosis are catamenial. Catamenial is a Greek word meaning monthly or periodic. Symptoms occur with 2448 h after the onset of menses. Chest pain is the most common symptom occurring in 90% of patients with thoracic endometriosis.

66

ENDOMETRIOSIS

Successful treatment requires both eradication and suppression of existing thoracic endometrial tissue and prevention of reseeding from the pelvis. Suppressing ovulation using oral contraceptives, progestins, danazol, or gonadotropin-releasing hormone (GNRH) analogs are currently the most accepted medical strategies. Hysterectomy with bilateral salpingo-oophorectomy should be considered only in the patient who is also suffering from disabling endometriosispelvic pain. Despite estrogen suppression, pneumothorax recurrence approaches 50%. Pleurodesis should be considered in those who develop a recurrent pneumothorax or hemothorax.

(4) asymptomatic pulmonary nodules; and (5) aortic dissection. Histopathological diagnosis of thoracic endometriosis involves identifying endometrial glands and stroma within the thoracic cavity. Usually, histopathologic documentation of thoracic endometrial tissue is not required and signs are catamenial.

Clinical Features
Of the 111 cases of thoracic endometriosis reported in the English language since 1958, catamenial pneumothorax is by far the most common presentation of thoracic endometriosis occurring in 80 (73%). Catamenial hemothorax occurred in 15 (14%), catamenial hemoptysis in 8 (7%), lung nodules in 7 (5%), and aortic dissection in 1 (o1%) (Figure 1). Thoracic endometriosis most commonly presents in premenopausal women with a mean age of 35 years and a reported age range of 19 to 54 years. Postmenopausal women on hormonal replacement therapy can also develop thoracic endometriosis. The vast majority of women with thoracic endometriosis will have evidence of pelvic endometriosis. In 15% of cases, pelvic endometriosis will be lacking in those with thoracic involvement. In catamenial pneumothorax, endometriotic implants are seen in less than 15% of patients examined by thoracoscopy or thoracostomy. The symptoms of thoracic endometriosis are catamenial, presenting in 2448 h after the onset of menses. Chest pain is the most common symptom, occurring in 90% of patients with catamenial pneumothorax. In more than 90% of cases of catamenial pneumothorax, the pneumothorax is seen on the right side. Two cases of bilateral pneumothorax and one of isolated left-side occurrence has been reported. There should be a high index of suspicion for thoracic endometriosis in women of childbearing age who present with recurrent chest pain, pneumothorax, or hemoptysis. The diagnosis is established clinically; however, it is often delayed until multiple episodes have occurred as the patient and clinician fail to recognize the association between symptoms and the onset of menses.

Etiology
The causative and genetic factors for endometriosis are not well understood. Growth and maintenance of endometrial tissue is dependent upon ovarian steroids. Previous epidemiologic studies have suggested that delayed pregnancy is a risk factor for the development of endometriosis. Therefore, Caucasian women are reported to have a higher occurrence of endometriosis compared to other races. This trend, however, was a result of this group having greater access to medical care. Most believe there is no difference between the incidence of endometriosis in different ethnic groups. Genetic factors probably impact a womans susceptibility to endometriosis. A familial tendency for endometriosis has been recognized, and concordance in twins has been observed. Multiple genes interacting with environmental triggers are probably operative in conferring the disease. However, future studies are needed to identify the genes and environmental triggers involved in pathogenesis.

Pathology
Thoracic endometriosis can be classied according to its clinical presentation. These clinical presentations include: (1) catamenial pneumothorax; (2) catamenial hemothorax; (3) catamenial hemoptysis;

<1% 14% 7% 5%

n = 111 Pneumothorax Hemothorax

73%

Hemoptysis Lung nodules Aortic dissection

Figure 1 Clinical syndromes of thoracic endometriosis. Major manifestation of thoracic endometriosis is a right-sided pneumothorax.

ENDOMETRIOSIS 67

Although a histopathologic documentation of thoracic endometriosis is not required, there have been reports of the diagnosis being established by cytology of pleural uid, bronchoscopic aspirates, and needle aspiration of lung nodules. Elevated serum CA-125 (elevated CA-125 serum levels are typically seen in ovarian cancer and occasionally in benign disorders such as endometriosis) and endometrial antibody have been reported in patients with thoracic endometriosis. Pleural and pulmonary endometriotic implants have been described on computed tomography and magnetic resonance imaging scans. These scans may be negative unless obtained during menses. Bronchial arteriography has been used to establish the diagnosis in a few women. Regardless of the radiographic modality used, the appearance of thoracic endometriosis is not specic, and the diagnosis remains indirect. Thoracoscopic ndings may consist of diaphragmatic perforations and pleural implants which vary in size depending on the time of menstruation. The diaphragmatic perforations vary in size from 1 mm to 2 cm and are found predominately on the right diaphragm. Macroscopically, the endometriotic implants appear as brownyellow and sometimes red nodules surrounded by neovascularization. Although these gunshot lesions are diagnostic of endometrial foci, pleural biopsy should be performed to conrm the diagnosis.

cells may lead to the proliferation of endometriotic implants. Endometriosis in locations outside the pelvis is explained by dissemination of endometrial cells through pelvic lymphatics and veins. This microembolization phenomenon explains why endometrial tissue can be found in locations like the skin, central nervous system, vertebrae, and aorta. However, in catamenial pneumothorax, more than 90% of cases occur on the right side, suggesting another plausible mechanism for this form of thoracic endometriosis. Congenital diaphragmatic holes are more commonly found on the right. These holes allow for a direct peritonealpleural communication, thereby allowing for endometrial tissue to preferentially migrate into the right pleural space. However, fewer than 25% of patients have visible thoracic implants during thoracoscopy. Others have theorized that air originates from abdominal endometriosis which can then move preferentially through these diaphragmatic defects into the pleural space. Less-accepted theories involve high circulating levels of prostaglandin F2 during menstruation causing bronchoconstriction and leading to alveolar rupture and pneumothorax. Another speculates that swelling of intrapulmonary endometriotic implants at menses causes airway obstruction resulting in alveolar wall rupture and pneumothorax. Neither of theses theories, however, explains the right-side predilection for catamenial pneumothorax.

Pathogenesis
Several theories have been proposed to explain the pathogenesis of endometriosis. The coelomic metaplasia theory proposes that undifferentiated cells within the peritoneal cavity are capable of dedifferentiating into endometrial tissue. The implantation theory states that endometrial tissue from the uterus shed during menstruation is transported by the fallopian tubes and implants on pelvic structures. Another theory involves retrograde menstruation. This theory has been supported by the observation that females with genital tract obstruction have an increased likelihood of tubal reux; however, the incidence of retrograde menstruation is similar among those with and without endometriosis. Therefore, the development of endometriosis may be dependent upon the quantity of endometrial tissue reaching the peritoneal cavity as well as the capacity of the immune system to recognize and destroy the displaced endometrial cells. Some authors have speculated that altered immunity may play a role in the development of endometriosis. An inability of cellular immunity to recognize the presence of endometrial tissue outside the uterus may be pivotal. Various cytokines and proinammatory

Animal Models
Animal models have shown that intravenous injected endometrial tissue remain viable, invade lung parenchyma, and undergo cyclic changes during the menstrual cycle.

Management and Current Therapy

Successful treatment requires both eradication and suppression of existing thoracic endometrial tissue and prevention of reseeding from the pelvis. Medical treatment, the primary modality, consists of suppression of the ectopic endometrium by interfering with ovarian estrogen secretion. This can be accomplished by suppressing ovulation using oral contraceptives, progestins, danazol, or GNRH analogs. We do not routinely recommend hysterectomy with bilateral salpingo-oophorectomy unless the patient is also suffering from disabling endometriosispelvic pain. Progestins cause decidualization of endometrial tissue. Adverse side effects are problematic and include nausea, uid retention, abnormal uterine bleeding, and depression.

68

ENDOMETRIOSIS

Danazol, an isoxazol derivative of 17a-ethinyltesterone, induces anovulation by attenuating the mid-cycle surge of luteinizing hormone secretion, increasing free testosterone concentration, and inhibiting multiple enzymes in the steroid pathway. The recommended dose of danazol in the treatment of endometriosis is 600800 mg daily. At these doses, danazol has significant androgenic side effects, and irreversible liver toxicity has been reported. GNRH agonists decrease the secretion of follicle stimulating hormone and luteinizing hormone, resulting in hypogonadotropic hypogonadism. A persistent hypoestrogenic state ensues resulting in endometrial tissue atrophy and amenorrhea. Adverse effects associated with GNRH agonists include vaginal bleeding, hot ashes, decreased libido, depression, osteoporosis, and sleep disturbance. All medical regimens appear to be effective in the treatment of endometriosis. Several case reports have shown that GNRH agonists were effective in the treatment of thoracic endometriosis when oral contraceptives had failed. Nevertheless, recurrence

rates are as high as 50% with hormonal therapy. This suggests that endometrial implant regression is not complete and/or that recurrent embolization continues. The management of catamenial pneumothorax is similar to the acute management of other forms of spontaneous pneumothorax. Observation is appropriate for a small, asymptomatic pneumothorax. Catheter aspiration or closed tube thoracostomy should be considered for patients with symptoms or larger pneumothoraces. With these conservative approaches, pneumothorax recurrence will occur in all who do not receive appropriate estrogen suppression therapy. Moreover, despite estrogen suppression, pneumothorax recurrence approaches 50% over the next 18 months. Therefore, pneumothorax prevention strategies should be considered in those who develop a recurrent pneumothorax. Chemical pleurodesis (via tube thoracostomy, thoracoscopic talc poudrage, or surgical pleurodesis using pleural abrasion and partial parietal

Anovulatory medications Examples: Progestins, oral contraceptives, danazol, GNRH agonists Effective Continue anovulatory medications Yes Yes Continue anovulatory medications

Recurrent catamenial pneumothorax or hemothorax (50% recurrence in first 18 months) Chemical or surgical pleurodesis

Recurrent hemoptysis

Surgical or laser resection of endobronchial implants

Catamenial chest pain

Anovulatory medications Examples : Progestins, oral contraceptives, danazol, or GNRH agonists

Figure 2 Management algorithm for thoracic endometriosis.

ENDOTHELIAL CELLS AND ENDOTHELIUM 69

pleurectomy with or without talc poudrage) is highly effective in preventing catamenial pneumothorax and hemothorax. However, patients may continue to experience catamenial chest pain despite these therapeutic interventions. Catamenial chest pain is presumed to be due to cyclic proliferation of pleuropulmonary endometriotic implants in response to ovarian steroids. These symptoms can be relieved with hormonal suppression but recur if estrogen replacement therapy is initiated. It is well recognized that women suffering from thoracic endometriosis are in their reproductive years and may wish to conceive children. The use of hormonal ablation strategies would inhibit conception. On the other hand, pregnancy would be a protective state in preventing the occurrence of a pneumothorax. In women with thoracic endometriosis who are considering pregnancy, a pleurodesis procedure can be performed prior to discontinuation of estrogen suppression. Surgical resection of intraparenchymal implants and neodymiumyttrium aluminum garnet (Nd YAG) laser resection of endobronchial implants have been successful in a limited number of reports. These strategies should be entertained in those with limited disease who have recurrent hemoptysis despite appropriate estrogen suppression (Figure 2).

See also: Pleural Effusions: Hemothorax. Pneumothorax. Symptoms of Respiratory Disease: Chest Pain.

Further Reading
Elliot DL, Barker AF, and Dixon LM (1985) Catamenial hemoptysis: new methods of diagnosis and therapy. Chest 87: 687688. Fonseca P (1998) Catamenial pneumothorax: a multifactorial etiology. Journal of Thoracic and Cardiovascular Surgery 116: 872873. Hibbard LT, Schumann WR, and Goldstein GE (1981) Thoracic endometriosis: a review and report of two cases. American Journal of Obstetrics Gynecology 140: 227232. Joseph J and Sahn SA (1996) Thoracic endometriosis syndrome: new observations from an analysis of 110 cases. American Journal of Medicine 100: 164170. Lillington GA, Mitchell SP, and Wood GA (1972) Catamenial pneumothorax. Journal of the American Medical Association 219: 13281332. Olive DL and Pritts EA (2001) Treatment of endometriosis. New England Journal of Medicine 345: 266275. Olive DL and Schwartz LB (1993) Endometriosis. New England Journal of Medicine 328: 17591769. Sahn SA (2004) Thoracic endometriosis. Up to Date American Thoracic Society (electronic media). Wilkins SB, Bell-Thomson J, and Tyras DH (1985) Hemothorax associated with endometriosis. Journal of Thoracic and Cardiovascular Surgery 89: 636638. Wood DJ, Krishnan K, Stocks P, et al. (1993) Catamenial haemoptysis: a rare cause. Thorax 48: 10481049. Yamazaki S, Ogawa J, Koide S, et al. (1980) Catamenial pneumothorax associated with endometriosis of the diaphragm. Chest 77: 107109.

ENDOTHELIAL CELLS AND ENDOTHELIUM

A Hislop, Institute of Child Health, London, UK B Wojciak-Stothard, University College London, London, UK
& 2006 Elsevier Ltd. All rights reserved. Endothelial dysfunction has been implicated in several pulmonary disorders including pulmonary arterial hypertension, lung edema, acute lung injury, and acute respiratory distress syndrome (ARDS). Current research aims to identify signaling pathways leading to endothelial malfunction in search for new effective therapeutic strategies.

Abstract
The pulmonary endothelium is a single-cell layer forming the inner lining of a vast network of pulmonary arteries, veins, and capillaries. All circulating blood passes through the lungs before it enters the systemic circulation, thus the endothelium can be regarded as an endocrine organ for the whole body. In addition to allowing gas exchange the endothelium has a number of other functions. It acts as a semipermeable barrier between air and blood, regulating uid balance between the capillaries and alveolar space. The endothelial cells synthesize and transport to the smooth muscle cells relaxant and contractile mediators maintaining tone in the pulmonary circulation. It is involved in extravasation of white blood cells to areas of inammation and regulates the coagulation process important to prevent vessel injury. The agents produced by the endothelial cells have multiple tasks and there is a carefully orchestrated interaction.

Introduction
The pulmonary endothelium made up of a continuous sheet of endothelial cells is a large organ: its surface area is approximately 90 m2 (the size of a tennis court) and it lines all the blood vessels in the lung. It acts as a semipermeable barrier between the blood and the surrounding tissues regulating bidirectional exchange of uids, gases, nutrients, and other metabolic mediators. Endothelial cells produce growth factors and substances that control vascular tone and are involved in the inammatory and coagulant processes. Since the entire circulating blood passes

70

ENDOTHELIAL CELLS AND ENDOTHELIUM

through the lungs before it enters the systemic circulation, the endothelium can be regarded as an endocrine organ for the whole body. Endothelial cells are rst in line to detect and respond to changes in blood composition and ow.

Structural Considerations
The pulmonary circulation consists of three vascular segments that are arranged in series: the arteries, the microvessels or capillaries, and the veins. A second circulation in the lung, the bronchial circulation which is part of the systemic system, supplies the walls of airways and large pulmonary vessels with oxygenated blood. The pulmonary endothelial cell population is heterogeneous, and structural and functional differences exist between cells from arteries and veins, between cells from large and small vessels, and between cells within the same vascular region. Generally, the endothelial cell is attened and is only 12 mm thick and about 1020 mm in diameter. They are barely visible with the light microscope but a nucleus can be identied centrally at its thickest point. Ultrastructural studies are needed to describe the cell. It sits on a basement membrane and each cell is attached to its neighbor by tight junctions (Figure 1). These junctions are connected to the actin-based myolament system in the cytoplasm which in the resting cell is located around the cell periphery. The surface is covered by many pinocytotic vesicles rst described by Palade as plasmalemmal vesicles and now called caveolae. They are involved in transporting substances such as albumin from the circulating blood into the interstitium of the alveolar wall. The greatest density of caveolae is found in the alveolar capillaries where 20 000 caveolae per cell effectively double the surface area of the cell. WeibelPalade bodies, originally called rod shaped bodies, are found sparsely through the cytoplasm and more are found in arterial than capillary endothelium. They store P-selectin involved in leukocyte adhesion and von Willebrand factor (vWF) involved in platelet aggregation. Endothelial cells are formed by vasculogenesis from mesothelial cells surrounding the epithelial lung bud which become either hematopoietic cells or endothelial cells which join rst to form capillary tubes and then coalesce to form arteries and veins. This differentiation is the result of vascular endothelial growth factor (VEGF) produced by the epithelial cells. The airways impose organization on the randomly forming endothelial tubes and thus the preacinar pulmonary arteries and veins and airways develop together during early fetal life. Endothelial cells facilitate vessel differentiation and maturation

by secreting factors promoting smooth muscle cell proliferation and migration such as angiopoietins, endothelin, transforming growth factor beta (TGFb), and platelet-derived growth factor (PDGF). Later, in fetal life and during childhood, peripheral vessels form by angiogenesis, by sprouting from the existing blood vessels as the capillary bed forms within the alveolar walls, mediated by VEGF. During fetal life, in the peripheral vessels, the endothelial cells are not attened and obstruct the lumen increasing resistance to ow. After birth, attening their shape helps to increase blood ow to the lung. At the same time, uid is removed from the lung via the epithelial and endothelial cells.

Functions of the Endothelium in the Normal Lung

In the alveolar region, endothelial cells are primarily part of the blood gas barrier for diffusion of gases between blood and air. However, other functions may be equally as important. These include maintenance of vascular tone, barrier function, coagulation, leukocyte trafcking, production of growth factors, and cell signals with autocrine and paracrine effects (Figure 2).
Regulation of Pulmonary Vascular Resistance

The lung is a low-pressure system but there is a requirement to maintain tone in the arteries and veins. The pulmonary endothelium secretes a range of vasoconstrictors and vasorelaxants that by interacting regulate pulmonary vascular resistance (PVR) or tone. In the adult lung, the major sites of resistance appear to be the small arteries and capillaries; however, larger arteries and veins may also play a role. When oxygen tension is low, the small vessels respond by vasoconstriction a reaction different to that seen in systemic vasculature. The vasoconstriction allows blood to be diverted away from areas of low oxygen to areas of higher oxygen for the gas exchange to take place. Factors that stimulate release of vasoconstrictors also cause release of vasodilators and thus the overall response is a complex interaction. The main mediators of tone are described. Nitric oxide Nitric oxide (NO) or endotheliumderived relaxing factor (EDRF) is a powerful vasorelaxant of vascular smooth muscle cells. It is a free-radical gas produced by the vascular endothelium during conversion of L-arginine to L-citrulline by the enzyme NO synthase (NOS). Both constitutive endothelial NOS (eNOS) and inducible NOS (iNOS) are produced by the endothelial cells. eNOS is

ENDOTHELIAL CELLS AND ENDOTHELIUM 71

ctm

Nucleus

rbc bm Alveolar space (a)

j I 0.75 m

c v c bm I

0.25 m (b) 5 m wp j j j el wp

coll smc (c)

Figure 1 (a) Capillary in the alveolar wall of a normal human lung. The attened endothelial cell lines the vessel giving a thin blood gas barrier. bm, basement membrane; ctm, connective tissue matrix; j, tight cell junction; m, mitochondrion; I, epithelial type I pneumonocyte; rbc, red blood cell. (b) High-power micrograph of the endothelial cell showing numerous caveolae (c) on both surfaces and vesicles (v) within the cytoplasm. bm, basement membrane; m, mitochondrion; I, epithelial type I pneumonocyte. (c) Endothelial cell from a porcine small muscular artery. The vessel is not distended. el, elastic lamina between endothelial cells and the muscle wall; coll, collagen brils in the matrix; smc, smooth muscle cell; j, tight junction; wp, WeibelPalade body; m-mitochondrion. All electron micrographs courtesy of Ann Dewar, NHLI.

located in the caveolae. NO production in endothelial cells is inuenced by a variety of factors, including shear stress, oxygen tension, humoral factors acting via receptors, and growth factors. At a cellular level, NO production is regulated by transcription, posttranscriptional modication, substrate availability, intracellular localization, superoxide production, and cofactor availability. NO diffuses into the

smooth muscle cell where it stimulates soluble guanylate cyclase to form cGMP leading to relaxation. Hypoxia-induced pulmonary vasoconstriction and pulmonary hypertension are associated with decreasing production of NO by endothelial cells. Apart from acting as a potent vasodilator, low concentrations of NO inhibit smooth muscle cell proliferation, and prevent inammatory responses, platelet

72

ENDOTHELIAL CELLS AND ENDOTHELIUM

Physical factors Shear stress, stretch, hypoxia

Humoral factors Acetylcholine Bradykinin

Inflammatory mediators Histamine, thrombin, TNF

Paracellular permeability Antithrombotic Sepsis NO Endothelium NO PGI2 ET-1 TxA PGI2

Adhesion Neutrophils

Platelets

Caveolin Transcytosis

Selectins PAF vWF

ICAM, VCAM

PECAM

Fluid Matrix

Myofilaments

Intercellular space Dilatation SMC Constriction SMC

SMC

Barrier function

Vascular tone

Host defense

Figure 2 Diagram illustrating some of the main functions of the pulmonary endothelial cells. Under normal conditions, the endothelial cells are inuenced by humoral factors in the blood and the ow of blood. With an increase inow and thus shear stress, the presence of hypoxemia, or inammatory mediators, there is a change in balance of production of vasoactive mediators, an increase in permeability, and upregulation of adhesion molecules with platelet and neutrophil activation leading to cell migration. ET-1, endothelin-1; ICAM, intercellular adhesion molecule; NO, nitric oxide; PAF, platelet-activating factor; PECAM, platelet endothelial cell intercellular adhesion protein; PGI2, prostaglandin I2; SMC, smooth muscle cell; TNF-a, tumor necrosis factor alpha, TxA, thromboxane; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor.

and leukocyte adhesion, and increased endothelial leakage associated with pulmonary hypertension (see below). Prostacyclin Prostacyclin (Prostaglandin I2, PGI2) is a powerful vasodilator of both systemic and pulmonary vascular beds produced constitutively by the endothelium but increased in response to receptor stimulation or shear stress. It is derived from arachidonic acid, by the action of cyclooxygenases COX-1 and COX-2 and diffuses to the smooth muscle cells. The PGI2 receptor belongs to the family of G-protein-coupled receptors and its effects are mediated by cAMP. The levels of PGI2 during early fetal life are low and increase with the onset of breathing air. PGI2 also inhibits smooth muscle cell proliferation and is anti adhesive. Blood-borne bradykinin which acts through receptors on the endothelial cells causes release of NO and PGI2 and thus is a potent vasodilator in vivo; several other mediators such as potassium channels and S-nitrosothiols have also been proposed.

Thromboxane Thromboxane is another arachidonic acid metabolite produced by cyclooxygenases in endothelial cells and platelets. It is a potent vasoconstrictor, a smooth muscle mitogen, and an inducer of platelet aggregation. An increase in the production of thromboxane A2 metabolites is seen in pulmonary hypertension.

Endothelin-1 Endothelin-1 (ET-1) produced by pulmonary endothelial cells is a potent vasoconstrictor which also stimulates the proliferation of pulmonary smooth muscle cells. Release of ET-1 is stimulated by hypoxia or lung inammation. There are two types of endothelin receptors in the pulmonary vasculature. ETA and ETB receptors are found on smooth muscle cells where they mediate vasoconstriction. Endothelial cells have only ETB receptors where stimulation induces vasodilation via the release of NO, PGI2, or via ATP-gated K channels. The plasma endothelin levels are high in utero and fall rapidly during the rst week of life. They are

ENDOTHELIAL CELLS AND ENDOTHELIUM 73

increased in pulmonary arterial hypertension. ET-1 also has pro-inammatory properties in the lung. Angiotensin Angiotensin II (Ang II), the key effector of the reninangiotensin system, plays a central role in the regulation of blood pressure and electrolyte homeostasis. Angiotensin-converting enzyme, produced by endothelial cells, hydrolyzes angiotensin I to angiotensin II which acts as a powerful vasoconstrictor of pulmonary vascular smooth muscle cells. The vasoconstricting effects of this peptide are largely due to angiotensin-mediated increases in the production of ET-1. It also enhances proliferation and hypertrophy of smooth muscle cells.
Paracellular Permeability and Transcytosis

the changes in endothelial barrier function. PECAM1 is a scaffolding protein in the intercellular region that also plays a critical role in transendothelial migration of inammatory cells.
Inammatory Responses

The pulmonary microcirculation is in close proximity to the gas-exchanging alveolar epithelium. The integrity of both the alveolar endothelial and epithelial barriers is essential for lung uid balance. After birth, pulmonary endothelium takes part in the clearance of the lung uid and then endothelial cell junctions tighten to prevent leakage of plasma proteins into the interstitium and airspaces. In the normal lung, the endothelium delivers circulating substances such as albumin through transcellular trafcking of vesicles. Albumin carries a variety of peptides and amino acids from the blood through the endothelial cell via transcytosis into the interstitium. The protein binds to the caveolae on the luminal surface; these internalize, then move through the cell to the interstitium where they are released. Caveolin-1 is a scaffolding protein required for caveolae formation and caveolar transcytosis. In caveolin-1-null mice there is failure of transcytosis. In response to sepsis and intravascular inammatory mediators such as thrombin, tumor necrosis factor alpha (TNF-a), and histamine, there is increased paracellular permeability through gaps in intercellular junctions resulting in increased protein uid in the interstitial space. The intercellular gap formation is partly a result of actinomyosin-dependent contraction of endothelial cells. Pulmonary barrier function can also be affected by hypoxia and shear stress, resulting in changes in the levels of circulating VEGF, angiotensin II, and NO. VEGF, originally identied as vascular permeability factor (VPF), is a potent inducer of plasma extravasation. Angiotensin II may affect endothelial permeability via the release of prostaglandins and VEGF. By contrast, an increase in NO has been shown to prevent endothelial leakage in the lung. Changes in the levels and distribution of platelet endothelial cell intercellular adhesion protein PEC AM-1 (CD31) have also been shown to contribute to

Endothelial cells are normally nonadherent but can help recruit inammatory cells to sites of tissue injury or infection. On stimulation by damage or infection they produce cytokines, growth factors, and adhesion molecules which allow leukocytes to leave the owing blood and to roll along the vessel wall, both the leukocytes and the endothelial cells expressing adhesion molecules E-selectin and P-selectin. The leukocytes adhere to the endothelial cells depending upon upregulation of B2 integrins and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and nally migrate through the cell junctions via action of PECAM-1 which in the normal cell is concentrated at intercellular junctions. The cells move through the junction while the integrity of the endothelium is maintained.
Coagulation

The pulmonary endothelium plays a key role in maintenance of the normal coagulation. The anticoagulant/antithrombotic substances include NO, PGI2, and thrombomodulin. After damage the procoagulant/prothrombotic factors increase these include platelet-activating factor which promotes platelet adhesion and vWF which is made by the endothelial cells and stored for rapid release in the WeibelPalade bodies.

Endothelium in Respiratory Disease

Pulmonary Hypertension

Pulmonary arterial hypertension is dened as a sustained elevation of pulmonary arterial pressure to more than 25 mmHg at rest or to more than 30 mmHg with exercise. It may be idiopathic pulmonary arterial hypertension (formerly, primary pulmonary hypertension) or secondary to a variety of lung, heart, or connective tissue disorders. There may also be a lesser increase in pulmonary artery pressure associated with respiratory problems such as chronic obstructive pulmonary disease (COPD) and cystic brosis when the periphery of the lung is hypoxic leading to vasoconstriction. The main vascular changes in pulmonary arterial hypertension are an increase in arterial wall thickness due to smooth muscle cell proliferation or hypertrophy, increase in matrix deposition, and pulmonary endothelial cell dysfunction or injury. Pulmonary

74

ENDOTHELIAL CELLS AND ENDOTHELIUM See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Anticoagulants. Arteries and Veins. Bronchial Circulation. Caveolins. Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Antithrombin III. Diffusion of Gases. Endothelins. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Hypoxia and Hypoxemia. Kinins and Neuropeptides: Bradykinin. Leukocytes: Neutrophils. Lung Development: Overview. Nitric Oxide and Nitrogen Oxides. Peripheral Gas Exchange. Pulmonary Circulation. Pulmonary Vascular Remodeling. Vascular Disease. Vascular Endothelial Growth Factor.

hypertension is associated with a decrease in prostacyclin production and eNOS and an increase of thromboxane. The plasma levels of ET-1 are increased. These changes will lead to vasoconstriction and a promitogenic system. Endothelial and smooth muscle cell dysfunction has been attributed to the increase in the activity of Rho GTPase RhoA, a protein regulating actin dynamics and actomyosin contractility. Endothelial dysfunction may also contribute to the thrombotic process, a feature of most forms of pulmonary hypertension.
Acute Lung Injury/Acute Respiratory Distress Syndrome

Acute lung injury may be caused by a variety of insults including sepsis, trauma, pneumonia, and drug toxicity. There is severe oxygenation impairment, alveolar edema, and eventually pulmonary hypertension. The most extreme form of acute lung injury is acute respiratory distress syndrome (ARDS). The initial cause is an inammatory process injuring the functional processes and structure of the endothelial cells resulting in the breakdown of gas exchange and an increase in barrier permeability of the endothelium and changes in the levels of vasoactive mediators towards a balance in favor of vasoconstriction.
Therapeutic Considerations

Further Reading
Corrin B (ed.) (2000) Normal lung structure. Pathology of the Lungs, 2nd edn., pp. 134. London: Churchill Livingstone. Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 14871500. Fernandez N, Jancar S, and Sanchez CM (2004) Blood and endothelium in immune complex-mediated tissue injury. Trends in Pharmacological Science 25: 512517. Galley HF and Webster NR (2004) Physiology of the endothelium. British Journal of Anaesthesia 93: 105113. Hislop AA (2002) Airway and blood vessel interaction during lung development. Journal of Anatomy 201: 325334. Jeffery TK and Wanstall JC (2001) Pulmonary vascular remodeling: a target for therapeutic intervention in pulmonary hypertension. Pharmacological Therapeutics 92: 120. Kirchner K, Dobyns EL, and Stenmark KR (1999) Lung cell biology. In: Taussig LM and Landau LI (eds.) Pediatric Respiratory Medicine, pp. 3756. St Louis: Mosby. Lee TS, Chang CC, Zhu Y, and Shyy JY (2004) Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection. Ciculation 110: 12961302. Mehta D, Bhattacharya J, Matthay MA, and Malik AB (2004) Integrated control of lung uid balance. American Journal of Physiology 287: L1081L1090. Michiels C (2003) Endothelial cell functions. Journal of Cell Physiology 196: 430443. Moloney ED and Evans TW (2003) Pathophysiology and pharmacological treatment of pulmonary hypertension in acute respiratory distress syndrome. European Respiratory Journal 21: 720727. Orfanos SE, Mavrommati I, Korovesi I, and Roussos C (2004) Pulmonary endothelium in acute lung injury: from basic science to the critically ill. Intensive Care Medicine 30: 17021714. Piantadosi CA and Schwartz DA (2004) The acute respiratory distress syndrome. Annals of Internal Medicine 141: 460470. Predescu D, Vogel SM, and Malik AB (2004) Functional and morphological studies of protein transcytosis in continuous endothelia. American Journal of Physiology 287: L895L901. Rosenzweig EB, Widlitz AC, and Barst RJ (2004) Pulmonary arterial hypertension in children. Pediatric Pulmonology 38: 222. Stevens T, Rosenberg R, Aird W, et al. (2001) NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases. American Journal of Physiology 281: C1422C1433.

Ultimately, our aim will be to control excessive reactivity and structural remodeling and to develop therapeutic agents which exploit the interactions between the signaling pathways controlling these events. Similar treatment of both pulmonary hypertension and ARDS focuses on increasing pulmonary arterial vasodilatation by giving either inhaled or intravenous prostacyclin or inhalation of NO or its enhancement by the use of a phosphodiesterase type 5 inhibitor. More recently, ET-1 receptor antagonists have been used to treat pulmonary hypertension in adults and children. This may effect a decrease in vasoconstriction and some prevention of the muscle cell hyperplasia. Statins might be helpful in treatment of ARDS because they have antileakage and anti-inammatory effects and lead to an increase in NO production by endothelial cells, as well as increases in heme oxygenase-1 whose enzymic products, carbon monoxide and bilirubin, have potent anti-inammatory and antioxidant properties. Gene transfer of iNOS is so far only experimental.

ENDOTHELINS 75

ENDOTHELINS
N Morrell, University of Cambridge School of Clinical Medicine, Cambridge, UK J Suntharalingam, Papworth Hospital, Cambridge, UK
& 2006 Elsevier Ltd. All rights reserved.

ranging from diseases of the airways to pulmonary vascular disorders. This greater understanding has allowed for therapeutic manipulation of the endothelin system in vivo. An example of this is the use of endothelin antagonist therapy, which is now licensed for use in pulmonary arterial hypertension.

Abstract
Since their initial discovery in 1988, the importance of endothelins (ET) in both health and disease has been increasingly recognized. This family of peptides can be found throughout the body, but are found in particularly high concentrations within the pulmonary system. Here, they are synthesized on demand and act predominantly as paracrine and autocrine agents. Their actions are mediated by two endothelin receptors (ETA and ETB) that are themselves members of the G-protein-coupled receptor family. Although endothelins were initially recognized for their vasoconstrictor effects, they are now known to have mitogenic and immunoregulatory actions as well. The relative contribution of these effects in health is still unclear, but has become better understood in disease. ET-1 has been implicated in the pathogenesis of many pulmonary disorders, including pulmonary hypertension, pulmonary brosis, asthma, acute respiratory distress syndrome, lung transplant rejection, and lung cancer. This greater understanding has aided in development of drugs that can target the endothelin system; endothelin receptor antagonist therapy, for example, is now licensed for the treatment of pulmonary arterial hypertension.

Structure
The endothelin family consists of three isoforms ET1, ET-2, and ET-3, of which ET-1 is both the most prevalent and the best characterized. Although all three are 21 amino acid peptides and are structurally related, they are coded by genes from different chromosomes (chromosomes 6, 1, and 20 respectively). As Figure 1 shows, ET-1 takes the form of a hairpin loop linked by two intrachain disulde bridges with a free carboxy-terminus at one end and an amino-terminus at the other. ET-2 and ET-3 have similar forms, but differ from ET-1 by two and six amino acid residues, respectively. The functions of ET-2 and ET-3 in humans are less well characterized, so the rest of this article will concentrate on ET-1.

Regulation of Production and Activity

Although ET-1 synthesis occurs within several organs, the pulmonary system is one of the main sites of its production and clearance. Normally, ET-1 is produced predominantly by endothelial cells, with some contribution from airway epithelial and vascular smooth muscle cells. In disease, the relative contribution from these cells may vary and there may be additional production by other cells such as inammatory cells within the pulmonary system. ET-1 is initially synthesized as the inactive molecule preproET-1, a 212 amino acid peptide which undergoes posttranslational modication to its active

Introduction
In 1988, Masashi Yanagisawa and co-workers described an endothelial-derived peptide that caused potent vasospasm. The peptide was cloned, sequenced, and named endothelin (ET), and within a short time, the receptors were also characterized. Since then, work on understanding the role of endothelin has continued at a rapid rate. As a result, it is now accepted that endothelin has a wide range of actions in addition to its effects on vascular tone. It has also been implicated in many pulmonary disorders,
Leu Met Asp Lys Glu Cys Val Tyr Phe Cys His Ser Ser Cys Ser Cys

Disulfide bridges

Leu

Asp

Ile

IIe

Try

Figure 1 Structure of ET-1.

76

ENDOTHELINS
Promoters + Inhibitors

PreproET-1 Gene

PreproET-1 mRNA

PreproET-1 Peptide (212 AAs) Endopeptidases

Endothelial cell

Nucleus

Big ET-1 (38 AAs) Endothelinconverting enzyme ET-1 (21AA)

ET-1
Figure 2 Synthesis of ET-1.

Table 1 Factors regulating release of ET-1 Increased synthesis of ET-1 Growth factors (TGF-b, PDGF, EGF) Cytokines (TNF-a, IL-1b, IFN-b) Thrombin Angiotensin II Vasopressin Catecholamines Lipopolysaccharides Hypoxia Shear stress Stretch Inhibition of ET-1 synthesis Prostacyclin Adrenomedullin Heparin Nitric oxide Atrial natriuretic peptide

Biological Function
Mechanism of Action

Only a small proportion of ET-1 is released apically across the luminal surface into vessels. As such, its systemic action as a hormone is limited. Instead, the majority of ET-1 is released basolaterally and acts on neighboring cells in a paracrine manner. Under certain circumstances, it also exerts autocrine effects. ET-1 exerts its actions by binding to the endothelin receptors ETA and ETB. The expression and function of these receptors and their structure are discussed in more detail below.

form. At rst, preproET-1 is proteolytically cleaved by endopeptidases to produce big ET-1, a 38 amino acid peptide, following which it is further cleaved by soluble ET-converting enzyme (ECE) to produce ET-1 itself (Figure 2). ET-1 is not stored but is synthesized in response to demand. As such, regulation of its activity occurs at the transcriptional level. A wide variety of stimuli have been shown to either promote or inhibit the production of ET-1 (Table 1) and thus inuence its biological function. Further levels of regulatory control exist at the level of endothelin receptor expression and subsequent intracellular signal transduction. ET-1 is cleared within both the pulmonary and renal circulations. Clearance is mediated by ETB receptors which bind ET-1 and are subsequently internalized and degraded. In health, there is a balance between pulmonary production of ET-1 and its clearance in such a way that no gradient results across the pulmonary circulation.

Action on Vascular Tone

ET-1 is a potent vasoconstrictor and takes on a prominent role in the process of maintaining pulmonary vascular tone. Much of its contractile action in health is mediated through ETA receptors which are situated on vascular smooth muscle cells found largely within the proximal circulation. However, the smooth muscle cells located within the distal pulmonary circulation also host ETB receptors, which may be increased in numbers in patients with pulmonary hypertension. Binding of ET-1 to these receptors also leads to vasoconstriction directly, and is possibly more relevant during disease. Intriguingly, ETB receptors can also be found on endothelial cells where ligand binding leads to vasodilatation indirectly through the release of factors such as nitric oxide and prostacyclin. Although the net effect of all these actions still allows ET-1 to act predominantly as a vasoconstrictor, the precise interplay between these contractile and relaxant effects is dependent on

ENDOTHELINS 77

the differential expression of ET receptors under different circumstances. Within airways, ET-1 is similarly involved in bronchomotor tone. It has been shown to produce bronchoconstriction in vitro through a direct effect on airway smooth muscle cells. Both ETA and ETB receptors have been implicated, although the latter are thought to be the more dominant receptors in peripheral human airways.
Mitogenic Actions

Other Actions

ET-1 stimulates DNA synthesis and encourages proliferation of human vascular smooth muscle cells in culture. This process appears to involve both ETA and ETB receptors. When ET-1 synthesis is inhibited, by adding the ECE inhibitor phosphoramidon, this proliferative action is attenuated. This implies that not only can ET-1 be synthesized by smooth muscle cells, but also that it can then act as an autocrine mediator. Evidence from animal studies suggests that other growth factors, such as platelet-derived growth factor and epidermal growth factor, may be necessary cofactors for ET-1 to work effectively in this role. Both airway smooth muscle cells and airway epithelial cells in culture have been shown to proliferate in response to ET-1. Again, both ETA and ETB receptors are thought to be involved, although studies of human cell lines suggest that ETA may be the more relevant receptor. Furthermore, ET-1 is found to have a synergistic action when combined with other growth factors, suggesting again that it may be acting more as a co-mitogen rather than alone as a mitogen.
Monocytes Macrophages Leukocytes

The effects of ET-1 are far-reaching and not just limited to actions on smooth muscle cells and epithelial cells. ET-1, acting as a cytokine, has been shown to have a chemotactic effect on many other cell types and subsequently alter their function. Animal models of eosinophilic inammation, for example, have shown that ET-1 mRNA expression increases just prior to the inux of inammatory cells, and that the degree of inammation seen can be attenuated by the use of endothelin antagonists. Fibroblast migration and activation is also seen in response to ET-1, possibly leading to collagen deposition and altered matrix composition. These, and other actions that have been described, generally tend to show ET-1 to be a promoter of inammation within the pulmonary system. Some of these effects are due to direct interaction between ET-1 and ET receptors on the cells concerned, whilst others are mediated via the production of other cytokines such as tumor necrosis factor alpha and interleukin-1b. The extent to which these effects play a role in maintaining normal function in the lungs as opposed to playing a pathophysiological role in disease is as yet unclear. Figure 3 summarizes some of the biological functions of ET-1 within the respiratory system.

Receptors
As discussed, two endothelin receptors, ETA and ETB, appear to mediate most of the actions of endothelins in human tissue. ETA binds ET-1 and ET-2
Chemotaxis cytokine release

Airway

ET-1
Pulmonary artery

Epithelial cells

ET-1

ET-1 Mucus production Mucus gland

Endothelial cells ET-1 ET-B ET-1 Clearance No/PGI2 production Vasoconstriction proliferation ET-1

Bronchial smooth muscle Fibroblasts

Collagen deposition Bronchoconstriction proliferation

ET-A ET-B Vascular smooth muscle cells

Postcapillary venules
Vasodilatation
Figure 3 Biological functions of ET-1.

Permeability

78

ENDOTHELINS
ET1 Ca2+ H2N ET receptors Phospholipase C Receptoroperated channel G protein q GDP COOH p p q GTP DAG

GTP

GDP

Activation of protein kinase C

IP3

Activation of MAP kinase cascade

intracellular calcium Proliferation Contraction

Figure 4 Intracellular signaling mechanisms of ET-1.

with greater afnity, whereas ETB binds ET-1, ET-2, and ET-3 equally. Both receptors are members of the rhodopsin-like G-protein-coupled receptor family and preferentially interact with the Gq subfamily of guanine nucleotide binding (G) proteins (Figure 4). The binding of endothelin to ETA or ETB leads to the receptor undergoing a conformational change resulting in dissociation of the Ga subunit and activation of phospholipase C. This in turn leads to the hydrolysis of phosphoinositol 4,5-bisphosphate to the intracellular messengers 1,2 diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 increases intracellular calcium levels by inducing the release of intracellular stores, and also by opening receptoroperated calcium channels, causing an inux of extracellular calcium. This rise in intracellular calcium promotes ET-1s vasoconstrictor actions. DAG, on the other hand, activates protein kinase C which initiates several other signaling pathways further downstream including the phosphorylation of intermediates of the mitogen-activated protein-kinase cascade system. These pathways are involved in promoting cell proliferation and growth.

Endothelins in Respiratory Diseases

Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is characterized by pulmonary vascular remodeling and increased

resting pulmonary vascular tone that ultimately leads to death through decompensation of the right ventricle. Given the biological functions described above, it is clear that ET-1 could be a key factor in the development of this disease. Animal studies have demonstrated that ET-1 expression is increased in models of pulmonary hypertension. Furthermore, ET antagonists prevent development of pulmonary hypertension in these models whilst addition of such antagonists to well established pulmonary hypertension appears to partially reverse the remodeling process. As such, it appears that remodeling is a dynamic process and not a xed one, and that altering the effects of ET-1 can potentially reverse this. In humans, clinical trials of endothelin antagonists have had promising results; bosentan, a dual ETA and ETB antagonist, is now used for the treatment of idiopathic pulmonary arterial hypertension and scleroderma-associated pulmonary hypertension. Bosentan has been shown to improve functional status, reduce clinical worsening, and improve survival in patients. Studies in other conditions associated with pulmonary hypertension (e.g., congenital heart disease, HIV, and chronic thromboembolic disease) have also suggested a role for dual-receptor endothelin antagonists. However, the relative contribution of ETB receptor-mediated vasodilatation and ET-1 clearance as opposed to ETB receptor-mediated vasoconstriction in pulmonary hypertension is still under debate. In view of this, selective ETA

ENDOTHELINS 79

antagonists (e.g., sitaxsentan and ambrisentan) have also been developed and are now undergoing Phase III clinical trials.
Pulmonary Fibrosis

Lung Transplantation

Pulmonary brosis represents an abnormal woundhealing response to injury, either as a result of environmental insult or local inammation. As ET-1 is known to have proinammatory effects and is known to promote broblast migration, proliferation, and activation, it could act as either the trigger or the mediator of brotic change. Transgenic mice that overexpress ET-1 have been shown to develop pulmonary brosis, whilst increased levels of ET-1 have been found in the plasma, bronchoalveolar lavage (BAL) samples, and biopsies of patients with brosis. Some benecial effects of endothelin antagonism have been described in animal models, and so randomized placebo-controlled trials are currently underway to study the effects of bosentan on patients with idiopathic pulmonary brosis and brosing alveolitis associated with systemic sclerosis.
Asthma

Through its vasoactive properties, endothelin may play a role in the ischemia-reperfusion injury that underlies early postoperative graft failure in lung transplantation. Its proliferative actions, on the other hand, may be more relevant in the pathogenesis of bronchiolitis obliterans, a condition that often results from chronic rejection.
Lung Cancer

Several studies have demonstrated expression of ET1, big ET-1, ECE, and ETA and ETB receptors within pulmonary neoplasms. Furthermore, elevated plasma levels of big ET-1 have been correlated with a worse prognosis in non-small cell lung cancer patients. ET-1 expression by tumor cells may encourage tumor growth either through a proliferative effect on the cells themselves or through angiogenesis, allowing the tumor to extend beyond its margins.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. G-Protein-Coupled Receptors. Pulmonary Fibrosis. Pulmonary Vascular Remodeling. Surgery: Transplantation. Tumors, Malignant: Overview. Vascular Disease.

Given its vasoactive, mitogenic, and immunomodulatory actions, it is not surprising that ET-1 has been suggested as a possible mediator in the development of asthma. Certainly, bronchial epithelial cell cultures from patients with asthma do show increased expression of both preproET-1 and ET-1. In addition, both serum and BAL levels of ET-1 are increased in these patients, with levels which inversely correlate with measures of airow obstruction. Furthermore, successful treatment of asthma exacerbations is associated with a fall in these levels. Animal studies have demonstrated that endothelin antagonists modify many of the individual actions of ET-1, but whether or not endothelin antagonist therapy will be helpful in the management of asthma in the future is unknown.
Acute Respiratory Distress Syndrome

Further Reading
Barst RJ, Langleben D, Frost A, et al. (2004) Sitaxsentan therapy for pulmonary arterial hypertension. American Journal of Respiratory and Critical Care Medicine 169(4): 441447. Billington CK and Penn RB (2003) Signalling and regulation of G protein-coupled receptors in airway smooth muscle. Respiratory Research 4: 2. Boscoe MJ, Goodwin AT, Amrani M, and Yacoub MH (2000) Endothelins and the lung. The International Journal of Biochemistry & Cell Biology 32: 4162. Fagan KA, McMurtry IF, and Rodman DM (2001) Role of endothelin-1 in lung disease. Respiratory Research 2: 90101. Galie N, Manes A, and Branzei A (2004) The endothelin system in pulmonary arterial hypertension. Cardiovascular Research 61: 227237. Henry PJ (1999) Endothelin receptor distribution and function in the airways. Clinical and Experimental Pharmacology and Physiology 26(2): 162167. Jeffrey TK and Morrell NW (2002) Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Progress in Cardiovascular Diseases 45(3): 173202. Michael JR and Markewitz BA (1996) Endothelins and the lung. American Journal of Respiratory and Critical Care Medicine 154(1): 555581. Rubin LJ, Badesch DB, Barst RJ, et al. (2002) Bosentan therapy for pulmonary arterial hypertension. The New England Journal of Medicine 346(12): 896903. Teder P and Noble PW (2000) A cytokine reborn? Endothelin in pulmonary inammation and brosis. American Journal of Respiratory Cell and Molecular Biology 23: 710. Yanagisawa M, Kurihara H, Kimura S, et al. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411415.

Both elevated serum ET-1 levels and increased pulmonary immunostaining for ET-1 in patients with ARDS have implicated this peptide as a mediating factor. Endothelins may well contribute to the pathophysiology of ARDS through their effects on vascular tone, vascular permeability, and their proinammatory actions. Alternatively, they may be innocent bystanders, released as a result of endothelial dysfunction and disruption.

80

ENDOTOXINS

ENDOTOXINS
P J Bertics, M L Gavala, and L C Denlinger, University of Wisconsin Medical School, Madison, WI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Endotoxin is a major component of the outer leaflet of the outer membrane of Gram-negative bacteria and is composed of carbohydrates, fatty acids, phosphates, and associated metal ions. Endotoxin is also known as lipopolysaccharide (LPS) and varies in its carbohydrate and lipid composition between bacterial strains and species. However, several structural features are common to most LPS molecules, including a negatively charged hydrophilic heteropolysaccharide (O-antigen), a core oligosaccharide region, and a lipid A portion that usually contains two glucosamines coupled to six fatty acids. Lipid A is the toxic component of LPS and its bioactivity depends on its disaccharide, phosphate, and fatty acid content. Endotoxin is widely present in the environment, including dust, animal waste, foods, and other materials generated from, or exposed to, Gram-negative bacterial products. Occupational exposure to endotoxin (e.g., as a bioaerosol) is associated with airway disease, and endotoxin presentation to the tissues or blood initiates a profound immunological response. In the lung, the alveolar macrophage is the key in the recognition and host response to inhaled endotoxin. Endotoxin detection by the macrophage via cell surface receptors such as Toll-like receptor-4 results in the activation of multiple signaling cascades that trigger the production of both pro- and anti-inammatory mediators. Host responses following endotoxin challenge are dependent on the individual genetic background and the anatomical site and associated cell types. If the responses are too robust, adverse consequences, such as pulmonary edema and bronchospasm, can occur. Also, lobar pneumonia or endotoxemia from infections of other organ systems can cause diffuse acute lung injury, septic shock, and death. The management and treatment of individuals exposed to LPS encompass the use of antibiotics, bronchodilators, inhaled corticosteroids, and activated protein C. In cases of an overwhelming bacterial infection, supportive measures include supplemental oxygen, intravascular uid administration, vasopressors, stress-dose corticosteroids, and mechanical ventilation.

a toxin harvested from a bacterial ltrate will reproduce the disease. Gram-negative bacteria differ from other bacteria and mammalian cells by having both an inner and outer membrane that consist of lipid bilayers and transmembrane proteins. The outer leaflet of the outer membrane of Gram-negative bacteria is largely composed of an amphipathic molecule dened as LPS (Figure 1). Functionally, LPS provides a barrier to heavy metals, lipid-disrupting agents, and larger molecules (e.g., lytic enzymes and DNA). The lipid portion of LPS allows it to pack tightly to form a planar membrane, and one bacterial cell contains approximately 3.5 million LPS molecules. Using the bacterium Escherichia coli as an example, nearly 75% of the outer leaflet is composed of LPS, whereas the other 25% is largely transmembrane proteins. The orientation of the LPS in intact bacteria is believed to obscure the lipid domains beneath the polysaccharide components of the molecule. However, upon bacterial death and cell wall lysis, LPS can be released and can interact with other hydrophobic molecules, including mammalian membrane lipids and the hydrophobic domains of various serum and cell surface proteins. Released LPS is thought to play a major role in the development of the septic shock that accompanies Gram-negative bacterial infectious diseases.
Endotoxin Structure and Variation

Structure and Physicochemical Properties

Endotoxin Discovery and General Properties

The pathobiological activity of bacterial endotoxin, also known as lipopolysaccharide (LPS), was rst reported more than a century ago. Because LPS was considered an integral part of the organism, it was named endotoxin as opposed to toxins that were freely released, which were termed exotoxins. When ltrates of dead Gram-negative bacteria were infused into animals, they produced shock and death, thus fullling Kochs fth postulate, wherein

Although the exact structural composition of LPS varies between different Gram-negative bacterial species and strains, there are several structural features that are common to most LPS molecules. These structural features include a negatively charged hydrophilic heteropolysaccharide (O-antigen), a core oligosaccharide region, and a lipid A portion that generally contains two glucosamines that are coupled at the 2, 3, 20 , and 30 positions with fatty acids (Figure 2). The O-antigen is composed of repeating groups of three to ve sugars. The sugars in the O-antigen vary between bacterial strains, and they contribute to the antigenic differences observed within a species. Furthermore, bacteria with bulky O-antigen polysaccharides are often more resistant to complementmediated lysis and/or phagocytosis and exhibit an array of antigenic variants. In contrast to the Oantigen, the polysaccharides of the core region are less variable and can be divided into groups known as the outer and inner core. The outer core varies somewhat between genera and within species but is

ENDOTOXINS 81
Endotoxin (lipopolysaccharide O-antigen or LPS) n n n n Porin n proteins n Outer core Inner core Lipid A Cell wall Phospholipids Peptidoglycan

Plasma membrane

Integral proteins
Figure 1 Schematic representation of endotoxin (lipopolysaccharide (LPS)) structure and localization in Gram-negative bacteria. The diagram provides an overview of the cell wall of the bacterium E. coli, which serves as a model of comparison for the localization/ structure of endotoxin molecules from other genera. The outer membrane contains fewer proteins (e.g., the porins) as compared to the larger integral protein content of the inner membrane. Between the outer membrane and the inner membrane, there exists a peptidoglycan layer. In general, endotoxin is the major component of the outer leaflet of the outer membrane, whereas the other three leaflets of the outer and inner (plasma) membranes are composed of phospholipids resembling those of mammalian cells. The lipid A portion directly constitutes the outer leaflet of the lipid bilayer and is responsible for the toxicity associated with LPS. The inner core (pink ovals) is composed of two or three KDOs and three heptoses, which contain phosphoethanolamine and phosphate groups that bind calcium and magnesium ions and help maintain the structural integrity of the outer membrane. The outer core (gold ovals) is usually composed of hexoses (ve are shown), whereas the O-antigen (blue ovals) contains hexoses that are arranged as repeating units of trito hexasaccharides; the number of repeats (n) may be as large as 2030 units and can even vary within the same culture.

usually composed of four to six hexoses, primarily glucose, galactose, and N-acetyl-glucosamine. The inner core contains three heptoses that are coupled to phosphates and phosphoenthanolamines as well as to two or three unique sugars that form the bridge between lipid A and the polysaccharide regions of LPS; this unique sugar is known as 3-deoxy-D-mannooctulosonic acid (KDO). The inner core is highly conserved across diverse genera. The lipid A present in common enteric and some oralpharyngeal organisms usually contains six fatty acids, distributed such that four fatty acids are localized on one glucosamine and two are present on the other glucosamine (Figure 2). In contrast, the lipid A molecules synthesized by Neisseria meningitidis, Neisseria gonorrheae, and Pseudomonas aeruginosa possess three fatty acids on each glucosamine. An exception to the six fatty acid pattern is seen in Salmonella LPS, which contains seven fatty acids. Most of the fatty acids in enteric organisms contain 1216 carbons, whereas the LPS from oralpharyngeal pathogens generally have 1014 carbon fatty acids. The LPS molecules from Endobacteriaceae and P. aeruginosa possess a large O-antigen, a complete outer and inner core, two or three KDOs, and a lipid A with four hydroxymyristic acids and two acyloxyacyl fatty acids linked to the fatty acids on the 20 and 30 positions of the disaccharide. Conversely, the

LPS of nasopharyngeal pathogens, such as Neisseria species, Haemophilus inuenzae, and Bordetella pertussis, lack the O-antigen typical of the lower gastrointestinal tract organisms, and chlamydial LPS is essentially devoid of core polysaccharides except for the presence of a KDO trisaccharide. Instead of an O-antigen, other groups that confer serum resistance, such as the N-acetyl neuraminic acid found in Neisseria species, are found. The outer core hexoses, characteristic of enteric bacteria, are also missing in these nasopharyngeal organisms, but an inner core that is similar to that of E. coli and Salmonella species is present. Heptoses in the inner core from both enteric and nasopharyngeal species often contain negatively charged groups (phosphates and aminoethyl-phosphates) that tightly bind Ca2 and Mg2 .
StructureActivity Relationships

The lipid A portion is the toxic component of LPS, and every toxic lipid A is composed of a disaccharide containing either D-glucosamines or 2,3-diamino2,3-deoxy-D-glucose. Monosaccharide substructures of lipid A are B107-fold less toxic than LPS. In addition, phosphates at the 1 and 40 positions of lipid A are needed for its activity (e.g., monophosphoryl lipid A is approximately 1000-fold less toxic than LPS). Also, the number and conguration of the fatty

82

ENDOTOXINS
HO O O O O O HO O O O O NH

O O

O HO O HO O O O O O NH O O P O O

HO O O O O O O NH

O HO O HO O O O HO NH O O P O O

P
O

O O

O O

E. coli

S. minnesota

O H3N + O P
O

O O P
O

HO O O O O O O NH

O O HO O HO O O O O NH O O P O O O P O O NH3
+

O HO

N. meningitidis
Figure 2 Examples of structural variations in the lipid A portions of endotoxin molecules isolated from the Gram-negative bacteria E. coli, S. minnesota, and N. meningitidis. In each case, the remaining polysaccharides that constitute the LPS molecule, namely the inner core, outer core, and O-antigen, are attached to the free hydroxyl at the 60 position of the lipid A disaccharide (the HO group on the left-hand sugar residue in the gure).

acids attached to the disaccharide affect lipid A bioactivity. For example, lipid A substructures containing only four hydroxymyristic acids coupled to the glucosamine disaccharides are as much as 107-fold less toxic than the parent hexa-acyl synthetic lipid A. Moreover, the removal or addition of one fatty acid (i.e., penta-acyl or hepta-acyl lipid A) leads to a less toxic form of lipid A. Lastly, there is some indication that the chain length of the fatty acids is the key; that is, when the fatty acids are p12 carbons in length, the toxic activity of the LPS is reduced.

Source and Exposure

Endotoxin is found in Gram-negative bacteria and bacterial products or debris. Thus, endotoxin is widely present in the environment, including dust, animal waste, foods, and other materials generated from, or exposed to, Gram-negative bacterial products. Accordingly, individuals involved in occupations

associated with intensive livestock and/or agricultural operations are frequently exposed to elevated levels of endotoxin. Given its prevalence in the environment, endotoxin is also generally present endogenously in sites such as the airway and the gastrointestinal tract. In fact, endotoxin presentation as a bioaerosol is an important route of occupational exposure associated with the development and progression of airway disease. In addition, mechanical- or pathogen-induced (e.g., virus) injury may allow for secondary bacterial introduction and thus lead to endotoxin presentation to the tissues or blood, whereupon it initiates a robust immunological response.

Clinical Consequences
Endotoxin-Induced Cellular Responses

The innate immune system is part of an organisms rst line of defense against bacterial invasion. The

ENDOTOXINS 83

principal components of the innate immune system include antimicrobial substances produced by epithelial cells, polymorphonuclear leukocytes, and macrophages. These cells, along with B lymphocytes, are the primary targets for endotoxin-induced cellular responses. In the lung, epithelial cells are often involved in mediating the physical defenses to bacterial infection, whereas the alveolar macrophage is the key in the early recognition and host response to inhaled endotoxin. This early detection of endotoxin by the macrophage results in the activation of multiple signaling cascades that trigger the production of both pro- and anti-inammatory mediators involved in the initial immune response. When LPS is released into the blood or tissues, it is generally bound to a lipid transfer protein referred to as LPS-binding protein (LBP). LBP facilitates the binding of endotoxin to soluble or membrane-bound CD14 found on both polymorphonuclear leukocytes

and monocytes, and this LBP/LPS/CD14 complex can lead to the activation of cell surface receptors including Toll-like receptor 4 (TLR4) (Figure 3). The LBP/ LPS/CD14 complex allows monocytes/macrophages to detect LPS at concentrations as low as 10 pg ml 1. During the past several decades, LPS has been reported to activate numerous intracellular signaling events, and many of these appear to be mediated by TLR4. For example, with the aid of MD-2, which is a small secreted accessory glycoprotein, LPS recognition by TLR4 activates signaling cascades such as those associated with mitogen-activated protein (MAP) kinases, phosphatidylinositol (PI) 3-kinase, and sphingolipid metabolites, and these pathways converge to regulate LPS-induced cellular responses (Figure 3). LPS-induced activation of multiple transcription factors, including nuclear factor kappa B (NF-kB), activator protein-1 (AP-1), and the cAMP response element-binding protein (CREB), along with

LBP

LPS T L R 4 T L R 4 Plasma membrane Extracellular MD2

Other receptor systems

CD14

MD2

Intracellular

PI3K

NF- B

Sphingolipids

MAP kinases

Gene transcription mRNA stability Protein processing Cytoskeletal reorganization

Figure 3 General view of endotoxin-mediated signaling in target cells such as monocytes/macrophages. Endotoxin (LPS) interaction with LPS-binding protein (LBP) and CD14, which can be either soluble or membrane bound via a glycosylphosphatidylinositol moiety (shown as a black line), facilitates LPS recognition by receptor systems such as TLR4/MD2. Stimulation of these receptor systems by endotoxin results in the activation of multiple signaling cascades, including those involving PI3-kinase, NF-kB, sphingosine metabolites, and MAP kinase cascades (e.g., those associated with the MAP kinases p38 and extracellular signal-regulated kinases ERK1 and ERK2). These events can ultimately alter cellular transcriptional activity, mRNA stability, protein processing, and cytoarchitecture, leading to cellular responses involved in cytokine and chemokine production/release, phagocytosis, and reactive oxygen species generation. Other receptor systems, such as b-integrins, TLR2, scavenger receptors, and nucleotide receptors, have also been proposed to modulate/amplify certain LPS-induced signaling events and biological activities. Pl3K, phosphatodylinositol 3-kinase; MAP, mitogen-activation protein; TLR4, Toll-like receptor 4.

84

ENDOTOXINS

LPS-initiated effects on mRNA stability and protein processing, leads to the coordinate induction and/or release of many inammatory mediators. In addition, other membrane receptors, such as b-integrins, TLR2, scavenger receptors, and nucleotide receptors, have been proposed to act as systems that can serve to modulate or amplify the signaling induced by certain LPS molecules (Figure 3).
Physiological Responses and Respiratory Diseases

The magnitude and severity of the host response following endotoxin challenge are dependent on the individual genetic background and on the anatomical site and associated cell types. For example, Gramnegative bacteria are resident organisms of the oral mucosa and upper airway that cause minimal toxic effects if contained outside of the bloodstream, in part due to a lack of MD-2 expression by respiratory epithelial cells. In contrast, infection of lung parenchyma is quickly recognized by alveolar macrophages that produce a cascade of mediators that can be both benecial and harmful. Early host cellular responses are amplied by the release of prostaglandins, platelet-activating factor, chemokines, adenine nucleotides, and a variety of preformed cytokines (e.g., tumor necrosis factor alpha and interleukin-1b) that can promote autocrine effects on macrophage function. Several of these agents can have direct microbicidal activities, whereas others can initiate a variety of physiological responses, such as the induction of fever (which can attenuate bacterial growth) and microcoagulation. The resulting vascular congestion, cellular inammation, and secondary mediator production further limit bacterial growth. However, if these responses are too robust, adverse consequences, such as pulmonary edema and bronchospasm,

can occur manifesting as cough, dyspnea, wheeze, tachypnea, and hypoxemia in severe cases (Table 1). Clinical presentations resulting from LPS exposure are diverse, and again depend on the location involved. Inhaled LPS from various environmental or industrial sources by itself is sufcient to cause asthma exacerbations or hypersensitivity pneumonitis. With respect to bacterial infection, if the physical defense system of the upper airway or bronchial tree is impaired due to cilial damage (e.g., in response to aspiration or tobacco exposure) or defective mucus clearance (e.g., cystic brosis), then bronchitis ensues with the hallmark production of purulent sputum in the absence of radiographic ndings. Chronic inammation may result if there is ineffective bacterial removal, thereby leading to irreversible airway damage known as bronchiectasis. Conversely, overwhelming infection of the distal airways and alveoli causes pneumonia, which presents with high fever, sputum production, and characteristic dense inltrates on a plain chest lm. Finally, lobar pneumonia or endotoxemia from infections of other organ systems (e.g., peritonitis associated with a ruptured appendix) can cause diffuse acute lung injury, septic shock, and death (100 000 deaths annually in the United States) due to lung inammation incited by ltration of platelet and/or bacterial debris in the bloodstream as well as from mediator-induced vascular leak.

Management and Treatment

The therapies for LPS exposure can be classied as those associated with either airway or parenchymal disorders. Pure airway disorders are often only minimally beneted by administration of antibiotics. Wheeze and cough due to bronchospasm

Table 1 Selected physiological consequences of LPS exposure and associated strategies for therapeutic intervention Symptom or sign Fever Inammation Associated causes or mediators IL-1, IL-18 Prostaglandins, chemokines Biological effects or outcomes Bacterial stasis Phagocyte recruitment, microbial killing Therapeutic interventions Antibiotics if coupled with high suspicion of a bacterial infection Source control such as device removal or drainage if present in areas not effectively cleared (e.g., pleural empyema) Supplemental oxygen 7 mechanical ventilation Goal-directed uid resuscitation, vasopressors, stress-dose steroids Activated protein C

Tachypnea Hypotension

Hypoxia TNF-a, NO, vascular leak

Compensation for increased metabolic demands Decreased organ perfusion, lactic acidosis Disruption of microcirculation, cellular anoxia

Disseminated intravascular coagulation

PAF, platelet degranulation, thrombin

TNF-a, tumor necrosis factor alpha; NO, nitric oxide; PAF, platelet-activating factor.

ENDOTOXINS 85

can be abated with the use of bronchodilators such as albuterol. Inhaled corticosteroids may be required to attenuate inammation and other immune responses if the source of the LPS exposure cannot be eliminated. Conditions with chronic sputum production are also improved by the use of physical treatments (e.g., an expiratory utter valve or chest physiotherapy) to improve secretion management. Treatment options for parenchymal disorders, such as pneumonia and septic shock, are reviewed in Table 1. In the clinical context of an overwhelming bacterial infection, antibiotics are the mainstay of treatment. A cautionary note is that some of the most effective bactericidal antibiotics, particularly those with cell wall-directed activity such as b-lactams, also facilitate the release of LPS into the local environment or systemic circulation. If the infection breeches an anatomic space not well protected by the immune system (e.g., pleural empyema), drainage and/or a surgical procedure may be required. Supportive measures for conditions of increasing severity include supplemental oxygen, intravascular uid administration, vasopressors, stress-dose corticosteroids (improves vasopressor efcacy), and mechanical ventilation. Activated protein C, which is used for the treatment of septic shock associated with disseminated intravascular coagulation, is a unique agent in that it has both anticoagulant and antiinammatory actions. Finally, although mediator-directed neutralization therapies have been ineffective in the past, future application of genomic and proteomic data may direct patient-customized immunomodulatory treatments.
See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Adhesion, CellCell: Epithelial. Aerosols. Asthma: Occupational Asthma (Including Byssinosis); Exercise-Induced. Bronchiectasis. CD11/18. CD14. Chemokines. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Coagulation Cascade: Overview. Complement. Defense Systems. Dendritic Cells. Endothelial Cells and Endothelium. Environmental Pollutants: Overview. Histamine. Immunoglobulins. Interleukins: IL-1 and IL-18; IL-10; IL-6. Kinins and Neuropeptides: Bradykinin. Leukocytes: Mast Cells and Basophils; Neutrophils; Monocytes; Pulmonary Macrophages. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Lipoxins. Lymphatic System. Macrophage Inammatory Protein. Nitric Oxide and

Nitrogen Oxides. Occupational Diseases: Overview. Panbronchiolitis. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Viral. Signal Transduction. Toll-Like Receptors. Transcription Factors: AP-1; NF-kB and Ikb; Overview. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Chemotherapeutic Agents. Upper Respiratory Tract Infection. Vaccinations: Bacterial, for Pneumonia.

Further Reading
Arbour NC, Lorenz E, Schutte BC, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nature Genetics 25: 187191. Beutler B and Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin. Nature Reviews: Immunology 3: 169176. Denlinger LC, Fisette PL, Sommer JA, et al. (2001) The nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide. Journal of Immunology 167: 18711876. Falkow S (1988) Molecular Kochs postulates applied to microbial pathogenecity. Review of Infectious Diseases 10: S274 S276. Freudenberg MA, Keppler D, and Galanos C (1986) Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin. Infection and Immunity 51: 891895. Hotchkiss RS and Karl IE (2003) The pathophysiology and treatment of sepsis. New England Journal of Medicine 348: 138 150. Jia HP, Kline JN, Penisten A, et al. (2004) Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. American Journal of Physiology 287: L428L437. Lorenz E, Jones M, Wohlford-Lenane C, et al. (2001) Genes other than TLR4 are involved in the response to inhaled LPS. American Journal of Physiology 281: L1106L1114. Miller SI, Ernst RK, and Bader MW (2005) LPS, TLR4 and infectious disease diversity. Nature Reviews: Microbiology 3: 3646. Monick MM and Hunninghake GW (2002) Activation of second messenger pathways in alveolar macrophages by endotoxin. European Respiratory Journal 20: 210222. Proctor RA, Denlinger LC, and Bertics PJ (1995) Lipopolysaccharide and bacterial virulence. In: Roth JA, Bolin CA, Brogden KA, Minion FC, and Wannemuehler MJ (eds.) Virulence Mechanisms of Bacterial Pathogens, pp. 173194. Washington, DC: ASM Press. Raetz CRH and Whiteld C (2002) Lipopolysaccharide endotoxins. Annual Review of Biochemistry 71: 635700. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 14311433. Zhao L, Ohtaki Y, Yamaguchi K, et al. (2002) LPS-induced platelet response and rapid shock in mice: Contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system. Blood 100: 32333239.

86

ENERGY METABOLISM

ENERGY METABOLISM
E F M Wouters, University Hospital, Maastricht, The Netherlands E M Baarends, Zuyd University, Zuyd, The Netherlands
& 2006 Elsevier Ltd. All rights reserved.

Energy Balance
Energy Expenditure

Abstract
Energy metabolism is central to life and the main function of the respiratory system is to maintain aerobic metabolic processes in the body. Despite this important role, energy metabolism is poorly integrated in the diagnostic workup of chronic respiratory diseases. Increased attention during the last decade has focused on the contribution of energy imbalance in the pathogenesis of weight loss, particularly in chronic obstructive pulmonary disease (COPD) patients. Several factors contribute to the amount of energy spent by an individual: resting energy expenditure, physical activity, and to a lesser extent diet-induced thermogenesis. The article deals particularly with resting energy expenditure as well as total daily energy expenditure data, measured in COPD patients. Factors responsible for increase in resting energy expenditure as well as total energy expenditure are discussed in detail. Food intake as well as food utilization are essential components in the maintenance of energy balance. Recent progress in the eld of energy homeostasis reveals the complexity of the regulatory neuroendocrine network. The role of the anorexigenic and adipostatic hormone leptin is discussed in detail.

Total daily energy expenditure (TDEE) can be divided into three components: (1) resting energy expenditure (REE) which comprises sleeping metabolic rate and the energy expenditure for arousal, (2) dietinduced thermogenesis, and (3) physical activity-induced thermogenesis. Resting energy expenditure Indirect calorimetry is the method by which measurements of respiratory gas exchange (oxygen consumption, VO2 and carbon dioxide production, VCO2 ) are used to estimate the type and amount of substrate oxidized and the amount of energy produced by biological oxidation. Indirect calorimetry considers the whole organism in an idealized system as one big compartment containing all living cells, bathed by a homogeneous extracellular uid and renewed by a constant blood ow. Inputoutput analysis in such a system _ O and V _ CO . Since the REE allows measurement of V 2 2 accounts for the major component of TDEE in sedentary subjects and can be relatively easy measured (Figure 1), several studies have measured REE particularly in COPD, based on the growing interest in weight loss and muscle wasting in this disease condition. Data obtained by indirect calorimetry in these studies are generally related to predicted REE values. The most common approach to predict REE for an individual in clinical practice is to apply the Harris and Benedict (HB) equations, which are based on sex, age, height, and body mass. Different studies

Introduction
Although energy metabolism is central to life and the main function of the respiratory system is to exchange oxygen (O2) and carbon dioxide (CO2) in order to maintain aerobic metabolic processes in the body, energy metabolism is poorly integrated in the diagnosis and management of patients with chronic respiratory conditions. While cell physiologists approach energy expenditure as the sum of at least membrane transport, protein turnover, and metabolic cycles, the clinical physiologist considers energy expenditure in terms of basal metabolic rate, thermic effect of food, and physical activities. This article deals with the clinical physiological aspects of energy expenditure and will discuss new insights in energy intake regulation. The growing interest in metabolic aspects of chronic respiratory disease conditions such as chronic obstructive pulmonary disease (COPD) as well as the rapid growth in measurement devices for indirect calorimetry, have created new interests in calorimetry. Insights in energy metabolism significantly contribute to a better understanding of pathogenetic processes in patients suffering from respiratory pathology.

Figure 1 Picture of a resting energy expenditure measurement.

ENERGY METABOLISM 87

have investigated hypermetabolism in stable patients with COPD on base of the HB equations, most studies revealing an increased REE compared to the HB equations or to a control group. Few studies are known about the prevalence of hypermetabolism in COPD. Hypermetabolism at rest is reported in about 25% of stable COPD patients. Besides fat free mass (FFM) other factors like work of breathing, medication, and systemic inammation are reported in order to explain intersubject variability in REE in patients with COPD. The increase in oxygen cost of breathing (OCB) is assumed to be one of the main explanations for the elevated REE seen in patients suffering from COPD. Several studies have investigated the OCB in patients with COPD and all found a higher OCB in patients compared to healthy controls. This OCB is generally measured by augmenting the ventilation or ventilatory effort: it is well accepted that increasing the ventilation poses an extra elastic load on the respiratory system due to progressive dynamic hyperination. Another possible explanation for the elevated REE seen in patients with COPD may be a thermogenetic effect of pharmacological maintenance treatment as part of the symptomatic therapy in patients with chronic respiratory conditions as COPD. Bronchodilating agents such as theophyllines and b2sympathicomimetics are widely prescribed to these patients to reduce breathlessness and improve exercise tolerance. Both drugs show an elevating effect on REE in younger healthy subjects, while the increase in REE in patients with COPD is very limited (Figure 2). Finally, it is shown that COPD patients demonstrate a blunted b-adrenoreceptor-mediated thermogenic response compared to control subjects matched for age and body composition. Together, these slight effects of b-adrenergic stimulation on REE cannot fully explain the elevated REE in these patients. Another contributing factor to hypermetabolism may be related to systemic inammation. The polypeptide cytokine tumor necrosis factor alpha (TNF-a) is a proinammatory mediator produced by different cell types. TNF-a inhibits lipoprotein lipase activity and is pyrogenic. It also triggers the release of other cytokines, which themselves mediate an increase in energy expenditure, as well as mobilization of amino acids and muscle protein catabolism. Using different markers, several studies provide clear evidence for involvement of TNF-a-related systemic inammation in the pathogenesis of tissue depletion. It is demonstrated that hypermetabolism at rest is related to plasma TNF-a concentrations as well as to the level of acute phase response in COPD patients.

40 REE (%) 30 20 10 0

10

15

20 25 30 Time (min)

35

40

45

Figure 2 Change in resting energy expenditure (y-axis) during 45 min (x-axis) after nebulization of 5 mg of a b2-sympathicomimetic drug in three groups. The upper line are young healthy subjects, the lower lines are the patients with COPD and elderly healthy subjects.

Oxygen cost of breathing

Systemic inflammation

Bronchodilating agents

Increased resting energy expenditure 26% Prevalence

Figure 3 Summary of the possible factors contributing to an increased resting energy expenditure in COPD.

A summary of possible factors contributing to an increased REE in the prevalence of hypermetabolism is shown in Figure 3. Total daily energy expenditure Measurements of energy expenditure over periods of days or at least over 24 h periods were advised to better determine energy requirements in humans. Respiratory chambers allow assessment of energy metabolism in humans in sedentary conditions or during exercise testing. Using a respiratory chamber, the different components of daily sedentary physical activity can be assessed: the sleeping metabolic rate, the energy costs of arousal (basal metabolic rate minus sleeping metabolic rate), the thermic effects of the meals, and the energy cost of spontaneous physical activity. However, one of the major components of the daily energy expenditure, and certainly the most variable, is the energy expenditure associated with motion and physical activity. Day-to-day variability in energy expenditure is most likely to be related to these changes in physical activity. The doubly labeled water method allows measurement of energy expenditure in totally free-living conditions.

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ENERGY METABOLISM
2000 p < 0.01 TDEREE (kcal day1) 1500

Energy Intake

1000

500

0 Controls COPD
Figure 4 Nonresting component of total daily energy expenditure in patients with COPD and healthy subjects matched for age, sex, and body composition.

Despite the fact that measuring TDEE is methodologically difcult and expensive, recent studies have focused attention on the activity-related energy expenditure in patients with COPD. Using the doubly labeled water 2 H2 18 O technique to measure TDEE it is demonstrated that patients with COPD have a significantly higher TDEE than healthy subjects. Remarkably, the nonresting component of total daily energy expenditure is significantly higher in the patients with COPD than in the healthy subjects (Figure 4), resulting in a ratio between TDEE and REE of 1.7 in patients with COPD and 1.4 in healthy volunteers, matched for age, sex, and body weight. This increased activity related energy expenditure suggests a mechanical inefciency during activities. Otherwise, when TDEE is measured in a respiration chamber, no differences in TDEE are found between patients with COPD and healthy controls, possibly as a consequence of the limited activity in the respiration chamber. A high variability of TDEE in patients with COPD is a constant nding in different studies. This variability in TDEE has to be considered in maintaining energy balance in COPD patients, particularly when exercise is advised as part of an integrated management program. Part of the increased oxygen consumption during exercise can be related to an inefciency in muscles. Several studies indeed show a severely impaired oxidative phosphorylation during exercise in COPD. Further studies are indicated in order to investigate the possible relationship between an inefcient or relatively enhanced energy expenditure during activities and changes in substrate metabolism. In any case, there is increasing evidence suggesting that, in order to estimate energy requirements of patients with COPD, it is necessary to measure physical activity as well as metabolic efciency during exercise.

Increments in energy expenditure caused by physical and metabolic tasks are related to utilization of the chemical energy stores. Fuel intake as well as fuel utilization are essential components in the maintenance of the energy balance. Therefore, hypermetabolism can explain why some COPD patients lose weight despite an apparent normal or even high dietary intake. Nevertheless, it has been shown that dietary intake in weight-losing patients is lower than in weight-stable patients, both in absolute terms as well as in relation to measured REE. This is quite remarkable because the normal adaptation to an increase in energy requirements in healthy men is an increase in dietary intake. The reasons for a relatively low dietary intake in COPD are not completely understood. In the past, impaired dietary intake in patients with chronic respiratory conditions was related to local organ impairment such as respiratory mechanics or gas exchange abnormalities. It has been suggested that patients with COPD eat suboptimally because chewing and swallowing change breathing pattern and decrease arterial oxygen saturation. Particularly in hypoxemic patients a rapid decrease in SaO2 during a meal with slow recovery after completion of the meal is reported; the decrease in SaO2 is associated with an increased dyspnea sensation. Gastric emptying time of a meal may also affect dietary intake since gastric lling in these patients may impair diaphragmatic functioning, contributing to an increase in dyspnea. Besides these effects on the pulmonary system, recent progress in the eld of energy homeostasis has revealed a complex regulatory neuroendocrine network. The discovery of the anorexigenic and adipostatic hormone leptin and the neural circuits responsible for mediating these leptin actions has indicated the important role of the hypothalamus as the center for the integration of feeding and associated autonomic, neuroendocrine, and gastrointestinal activities. The adipocyte-derived hormone leptin represents the afferent hormonal signal to the brain in a feedback mechanism regulating fat mass. Leptin regulates adaptive feeding responses by interaction with the hypothalamic signals such as neuropeptide Y and agouti-related peptide (AGRP) resulting in a reduction in food intake as well as in an increase in energy expenditure. In addition, leptin has a regulating role in lipid metabolism and glucose homeostasis and increases thermogenesis. In Figure 5, the role of leptin in regulating energy intake and metabolism is summarized. Few data have been reported on leptin metabolism in COPD. Circulating leptin correlates well with

ENERGY METABOLISM 89

Hypothalamus Neuropeptide

Food intake Glucose metabolism

Leptin White adipose tissue

Fat Energy expenditure

Neuroendocrine function

Figure 5 The mechanism of leptin in regulating energy intake and metabolism.

body mass index (BMI) and fat percentage, as expected. In stable disease, plasma leptin corrected for fat mass was inversely correlated with dietary intake as well as with the degree of weight change after 8 weeks of nutritional supplementation in depleted patients with COPD. During an exacerbation of COPD, systemic leptin concentrations are increased and the normal feedback regulation of leptin by fat mass (FM) seems to be disrupted. In patients with emphysema as well as in patients suffering from acute exacerbations, leptin is reported to be related to systemic inammation, independently of the amount of FM. Ghrelin has recently been discovered as the missing link between enteric nutrition and central regulation of energy balance and growth. Ghrelin functions as an orexigenic or appetite-stimulating signal from the stomach. Ghrelin regulates, in an antagonistic manner to leptin, synthesis and secretion of several neuropeptides in the hypothalamus involved in the regulation of feeding and associated hypothalamic functions. Further studies are needed to unravel the pathogenesis of disturbed energy regulation in relation to systemic inammation in patients suffering from chronic respiratory diseases.

expenditure is stressed and new insights in the eld of energy homeostasis open new fascinating perspectives of research and therapeutic potential in the management of these processes in the clinical course of a variety of disorders. Future research has to integrate metabolic cellular processes in the pathogenesis of energy disorders in respiratory diseases.
See also: Primary Myelobrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Vascular Disease.

Further Reading
Baarends EM, Schols AMWJ, Pannemans DL, Westerterp KR, and Wouters EFM (1997) Total free living energy expenditure in patients with severe chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 155: 549554. Cherniack RM (1958) The oxygen consumption and efciency of the respiratory muscles in health and emphysema. Journal of Clinical Investigation 38: 494499. Coward WA, Prentice AM, Murgatroyd PR, et al. (1984) Measurement of CO2 and water production rates in man using 2H, 18 O-labeled H2O: comparisons between calorimeter and isotope values. In: van Es AJH (ed.) Human Energy Metabolism: Physical Activity and Energy Expenditure Measurements in Epidemiological Research Based upon Direct and Indirect Calorimetry, pp. 126128. Wageningen: Stichting Nederlands Instituut voor de Voeding. Creutzberg EC, Schols AMWJ, Bothmer-Quaedvlieg FCM, Wesseling G, and Wouters EFM (1998) Acute effects of nebulized salbutamol on resting energy expenditure in patients with chronic obstructive pulmonary disease and in healthy subjects. Respiration 65(5): 375380. Creutzberg EC, Schols AMWJ, Bothmer-Quaedvlieg FCM, and Wouters EFM (1998) Prevalence of an elevated resting energy

Conclusions
The growing interest in energy metabolism in respiratory medicine during the last two decades has significantly contributed to a better understanding of pathogenetic processes underlying these disease conditions. The important role of activity-related energy

90

ENVIRONMENTAL POLLUTANTS / Overview

energy expenditure in man. Human Nutrition: Clinical Nutrition 38(2): 95106. Kojima M, Hosoda H, Date Y, et al. (1999) Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 402: 656660. McGregor M and Becklake MR (1961) The relationship of oxygen cost of breathing to respiratory mechanical work and respiratory force. Journal of Clinical Investigation 40: 971980. Nguyen LT, Bedu M, Caillaud D, et al. (1999) Increased resting energy expenditure is related to plasma TNF-alpha concentration in stable COPD patients. Clinical Nutrition 18: 269274. Schoeller DA and van Santen E (1982) Measurement of energy expenditure in humans by double-labeled water method. Journal of Applied Physiology 53: 955959. Schols AMWJ, Creutzberg EC, Buurman WA, et al. (1999) Plasma leptin is related to proinammatory status and dietary intake in patients with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 160: 1220 1226. Schols AMWJ, Fredrix EW, Soeters PB, Westerterp KR, and Wouters EFM (1991) Resting energy expenditure in patients with chronic obstructive pulmonary disease. American Journal of Clinical Nutrition 54: 983987.

expenditure in patients with chronic obstructive pulmonary disease in relation to body composition and lung function. European Journal of Clinical Nutrition 52: 16. Creutzberg EC, Wouters EF, Vanderhoven-Augustin IM, Dentener MA, and Schols AM (2000) Disturbances in leptin metabolism are related to energy imbalance during acute exacerbations of chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 162: 12391245. Cunningham JJ (1991) Body composition as a determinant of energy expenditure: a synthetic review and a proposed general prediction equation. American Journal of Clinical Nutrition 54: 963969. Durnin JVGA, Edholm OG, Miller DS, and Waterlow J (1973) How much food does man require? Nature 242: 418. Friedman JM and Halaas JL (1998) Leptin and the regulation of body weight in mammals. Nature 395: 763770. Harris JA and Benedict EG (1919) A Biometric Study of Basal Metabolism. Washington: Carnegie Institute of Washington. Inui A (2001) Ghrelin: an orexigenic and somatotrophic signal from stomach. Nature Reviews: Neuroscience 2: 551560. Klein PD, James WP, Wong WW, et al. (1984) Calorimetric validation of the doubly-labelled water method for determination of

ENVIRONMENTAL POLLUTANTS
Contents

Overview Diesel Exhaust Particles Oxidant Gases Particulate Matter, Ultrane Particles Particulate and Dust Pollution, Inorganic and Organic Compounds Radon

Overview
V Stone, Napier University, Edinburgh, UK K Donaldson, University of Edinburgh, Edinburgh, UK
& 2006 Elsevier Ltd. All rights reserved.

Introduction
History

Abstract
The adverse health effects of air pollutants include respiratory and cardiovascular morbidity and mortality. The major pollutants of concern in the early twenty-rst century include particles (PM10) and gases such as ozone, oxides of nitrogen, sulfur dioxide, and carbon monoxide. Such pollutants are monitored widely and a number of authorities have generated exposure limit values or guidelines aimed to minimize the impact of these pollutants on the health of populations. Susceptibility to the health effects of pollutants varies widely according to age, health status, and socioeconomic standing. Inammation and oxidative stress are common mechanisms in the induction of adverse health effects by a number of pollutants, suggesting that these pollutants could interact to enhance the biological effect.

The term air pollution relates to any substance, gaseous, liquid or particulate, that contaminates the air. Air pollution is derived from a variety of sources, many of which are anthropogenic. In the early twentieth century most air pollutant sources were derived from industry and from the domestic burning of fossil fuels. High smoke stacks were introduced at this time to increase pollution dissipation with the belief that by the time it precipitated back down to ground level it would be sufciently dispersed to be insignificant with respect to its health effects. A number of major pollution episodes occurred and were well-documented during this time. For example, a pollution episode in the Meuse Valley of Belgium in December 1930 resulted in the deaths of both humans and cattle. Autopsy results of those who

ENVIRONMENTAL POLLUTANTS / Overview 91

Figure 1 Smog in Zebo city, China, 2004. Photograph courtesy of J Philp and C Cunningham.

died revealed the airways to be irritated and congested with mucus. Perhaps, the most famous pollution episode occurred in London in December 1952 when bad smog developed due to the entrapment of pollutants at ground level by still, cold weather and a temperature inversion. The density of particles, measured as black smoke, reached 4000 mg cm 3 (compare with current levels observed in London of 2540 mg m 3). The number of extra deaths caused by this pollution episode is debatable due to the subsequent emergence of an inuenza episode, however, estimates range from 4000 to 12 000 people. In the mid-twentieth century many governments, including that of the UK, introduced a Clean Air Act, largely in response to episodes such as those described above. This Act limited the burning of fossil fuels in towns and cities for both industrial and domestic use. This has had a dramatic effect on air pollution, leading to an overall improvement in air quality, especially for sulfur dioxide and particulates. However, the prole of airborne pollutants encountered in towns and cities in the developed world has altered over the years due to changes in the predominant sources of pollutants, namely the large and progressive increase in trafc-derived pollution. Static industrial sources remain major contributors despite the introduction of a number of systems, such as lters, to minimize emissions to the atmosphere. Trafc pollution is composed of combustion-derived products including ultrane or nanoparticles associated with metals and organic matter. In addition, trafc pollution includes gases such as carbon monoxide and

oxides of nitrogen. Pollution levels in some countries such as China however remain elevated generating visible smogs on a regular basis (Figure 1).
Pollution Monitoring

Pollution levels for a spectrum of pollutants are monitored daily in many cities in many countries. For example, the European Union (EU) has published guidelines that state air pollution limit values in an attempt to improve air quality. An EU First Daughter Directive (99/30/EC) gives the limit for each air pollutant considered, including hourly and annual means, together with a date by which these limit values must be achieved (Table 1). Each country within the EU now has an obligation to meet these objectives. In the US, air pollution is regulated by federal law of the 1990 Clean Air Act, although individual states can choose to have more stringent pollution controls. Each state has responsibility for its pollution control, since this area requires specialist knowledge of factors such as local industries, housing, and geography that effect pollution production and dissipation, and must generate a state implementation plan (SIP) to explain how the local pollution will be controlled, with each SIP being approved by the EPA.

Current Day Pollutants

Pollutant Gases

The predominant pollutant gases found in many cities include ozone (O3), oxides of nitrogen (NOx), and

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ENVIRONMENTAL POLLUTANTS / Overview

Table 1 EU limit values and EPA air quality guidelines for the major pollutants encountered in the developed world Pollutant Carbon monoxide Sulfur dioxide Symbol CO SO2 EU Air Quality Daughter Directive limit values 10 mg m 3 8 h mean Target date 2005 350 mg m 3 1 h mean not to be exceeded 424 times per year 125 mg m 3 24 h mean not to be exceeded 43 times per year Target date 2005 200 mg m 3 1 h mean 40 mg m 3 annual mean Target date 2010 120 mg m 3 8 h mean not to be exceeded 420 times per year Target date 2010 50 mg m 3 24 h mean not to be exceeded 435 times per year 40 mg m 3 annual mean Target date 2005 EPA National Ambient Air Quality Standards 40 mg m 3 1 h mean 10 mg m 3 8 h mean 316 mg m 3 24 h mean not to be exceeded 41 time per year 68 mg m 3 annual mean WHO Air Quality Guidelines 11.6 mg m 3 8 h mean 266 mg m 3 15 min mean

Nitrogen dioxide

NO2

100 mg m 3 annual mean

287 mg m 3 1 h mean

Ozone

O3

235 mg m 3 1 h mean 157 mg m 3 8 h mean

100 mg m 3 8 h mean

Particles

PM10

PM2.5

150 mg m 3 24 h mean not to be exceeded 41 time per year 50 mg m 3 annual mean 65 mg m 3 24 h mean 15 mg m 3 annual mean

50 mg m 3 24 h mean

Limit values may be exceeded during specic adverse conditions, e.g., bad weather.

sulfur dioxide (SO2). These pollutants are described in more detail elsewhere in this encyclopedia. Briey, nitrogen oxides (NOx) are a group of pollutant gases that include nitrogen dioxide (NO2) and nitric oxide (NO) formed by the high-temperature burning of fossil fuels; hence, in ambient air they are derived principally from trafc and industrial sources. Both O3 and NOx are oxidant gases, with O3 being produced by the action of ultraviolet sunlight on NOx and volatile organic compounds (VOCs) that are found in the air and are derived from combustion sources such as trafc pollution. The requirement for UV light to generate ozone means that this pollutant is most commonly associated with sunny climates; hence, busy cities in hot climates often experience high ozone levels. The development and tting of three-way catalysts to petrol cars in order to meet legislative standards is responsible for a decline in NOx pollution in many countries. In the lungs, both O3 and NOx can generate free radicals that induce oxidative damage to the lung lining uid and inammation. In experiments on human volunteers NO2 at high concentration was found to decrease lung function and increase airway responsiveness, respiratory symptoms, and the response of asthmatics to allergens. However, the concentrations required to elicit such responses are greater than those typically found in the developed

world, and are slightly higher than the values recorded during high pollution episodes. Most studies have suggested a small short-term effect of NO2 on health, although the possibility that these effects are caused by another component of trafc pollution cannot be ruled out. Elevated levels of ozone can also induce a pulmonary inammatory response and decrease lung function in humans. Symptoms observed on exposure to high levels of ozone include cough, chest pain, and breathing problems, as well as headaches and eye irritation. However, data from laboratory and epidemiological studies suggest that not all people have the same susceptibility to ozone with the effects being more pronounced in children than in adults. Those who are most susceptible are not exclusively asthmatic, although specic genotypes related to the production of antioxidants have been linked to ozone susceptibility. Sulfur dioxide differs from O3 and NOx in that it is not an oxidant gas. It is generated by the combustion of sulfur-containing fossil fuels including coal and gasoline petrol. During the London smog pollution episode, SO2 was a major pollutant, contributing to both the smog and many of the adverse health effects including respiratory irritation and toxicity. Legislation controlling the burning of fossil fuels in towns and cities, as well as the sulfur content of gasoline, has led to a significant

ENVIRONMENTAL POLLUTANTS / Overview 93

decline in SO2 levels over the last 50 years. As a consequence, far less emphasis is now placed on SO2 as an important gaseous pollutant in many towns and cities.
Particulate Matter (PM10)

Prior to the 1990s, particulate air pollution was monitored via a variety of different procedures including total suspended particulate (TSP) and black smoke. In the 1990s a worldwide initiative introduced sampling by the PM10 convention, that is, the mass of particulate matter collected with 50% efciency for particles of 10 mm aerodynamic diameter. This means that particles within PM10 in general possess a diameter of 10 mm or less; this is the point at which particles are small enough to be inhaled and deposited throughout the respiratory tract. The coarse fraction of PM10 (2.510 mm) tends to deposit in the upper airways, while the ne fraction of PM10 (less than 2.5 mm) can deposit throughout the whole respiratory system. A number of countries, including the US, now also regulate the level of PM2.5. The adverse health effects induced by inhalation of increased levels of PM10 are numerous and are described in more detail elsewhere in this encyclopedia. Epidemiological studies clearly indicate that acute effects include increased risk of hospital admission or death due to a respiratory or cardiovascular cause. Long-term adverse health effects induced by PM10 are also becoming more apparent and include lung cancer as well as the potential for increased risk of asthma. The acute effects tend to be most prominent in susceptible groups such as the elderly, and patients with pre-existing lung or cardiovascular disease. Acute effects on healthy individuals include a decrease in lung function, but occur only at much higher levels of PM10 than are usually measured in most towns and cities. As is the case with other pollutants such as ozone, the adverse effects of PM10 are believed to be the result of oxidative stress-mediated inammation. PM10 (and PM2.5) is a cocktail of components including ultrane or nanoparticles, usually derived from combustion processes, consisting of a carbon core that is often associated with a variety of metals, organic molecules, and biological molecules (e.g., bacterial endotoxin). PM10 also includes secondary particles (sulfates and nitrates) that are derived from photochemical reactions in the atmosphere, as well as biological particles (e.g., pollen and spores) and wind-blown dust. The composition of PM10 is variable over time and location, with primary sources varying between rural and urban

locations, among different towns and cities, and among countries. For example, highly industrialized cities or countries emit PM10 rich in combustion-derived nanoparticles and metals, although the type of fuel employed will tend to be low in sulfur. In contrast, developing countries may have fewer sources of pollution, but may use lower grade fuel and hence generate pollutant particles with a different chemical and size prole. Rural locations are often associated with coarse particles generated by wind-blown dust. The current conundrum for particle toxicologists is to determine which components within PM10 are responsible for inducing oxidative stress and for driving inammation. However, this is extremely difcult since the composition is highly variable and interaction between components is likely to modulate their relative toxicity. There is substantial evidence that the primary combustion-derived nanoparticles are strongly involved in driving particle-induced inammation. Other studies suggest that bacterial components such as endotoxin drive the PM10-induced inammation. These differences may be accounted for by differences in sampling location, with rural locations being more dominated than cities by endotoxin-contaminated wind-blown dust. One of the major problems in this eld is to obtain a particle sample that is representative of the particles inhaled by humans, since the sampling procedure often requires the capture of particles onto a lter. Recovery of particulate matter from the lter is rarely 100% efcient and may result in the preferential release or retainment of some components. Furthermore, the stable sampling and storage of volatile components is almost impossible, leading to their loss from the sample. The World Health Organization (WHO) has recommended that PM2.5 be routinely monitored, because epidemiological and toxicological studies suggest PM2.5 to be more closely associated with health effects than PM10. However, the WHO recognized the substantial body of evidence that some toxicological activity resides within the coarse fraction of PM10 and hence PM10 measurements should continue. Protocols to standardize PM2.5 sampling across Europe are currently under development and an EU standard for this pollutant has yet to be set. In the US, the EPA has set an air quality standard for PM2.5 of 65 mg m 3, 24 h mean.
Other Pollutants of Importance

The list of pollutants discussed above is not exhaustive, but these are the main pollutants of concern at the present time. Other important pollutants include benzene, carbon monoxide, and sulfur dioxide.

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ENVIRONMENTAL POLLUTANTS / Overview

Benzene is a volatile organic compound that is classied as a genotoxic carcinogen. In ambient air, benzene is derived from the combustion and distribution of petrol. Benzene is a genotoxic carcinogen and so a safe level of exposure cannot be recommended. Substantial effort has gone into ensuring that emissions of benzene are minimal and the EU Air Quality Directive limit value target of 5 mg m 3 must be met by 2010. Current annual mean values for benzene in countries such as the UK are 16.5 mg m 3. Carbon monoxide (CO) is a gas that is generated from the incomplete combustion of fossil fuels. Historically, CO has been mostly associated with indoor sources of pollution such as ill-maintained gas appliances. In the indoor environment such appliances can rapidly generate sufcient CO to cause death through asphyxiation. CO irreversibly binds to hemoglobin preventing oxygen binding and transport around the body, resulting in ill health or death. The main outdoor sources of CO are again trafc exhaust fumes, particularly from petrol engines. Both the EU Air Quality Directive limit values and the USA National Ambient Air Quality Standards (NAAQS) for CO are set at 10 mg m 3 (9 ppm) as an 8 h mean. Values in countries such as the UK are currently in the order of 10 mg m 3 (8 h mean).

The Adverse Health Effects of Air Pollutants

Historically, humans have always been exposed to respirable particles in the form of biological particles (e.g., bacteria), re smoke, and wind-blown dust, and have developed effective defense mechanisms to clear these substances from the lung and body. Lung lining uid and lung cells contain a number of antioxidants that provide protection against the oxidative effects of many pollutants such as PM10 and NO2. In addition, acidic pollutants can generally be neutralized by proteins and buffers within the lung lining uid. Mucus and ciliated cells protect the upper airways of the respiratory tract from particulate air pollutants. Particles and pollutants landing in the mucus are trapped and cleared from the airways by the beating motion of the cilia, allowing the particles to move via this mucociliary escalator and to be swallowed or blown from the nose. Within the respiratory regions of the lung, particles are cleared by immune cells such as macrophages or neutrophils. Biological particles are easily degraded in the phagolysosome of such cells, however, inorganic particles must be physically carried from the lung surface, either into

the lymphatic system or via the mucociliary escalator described above. Gaseous pollutants will dissolve within the mucus or lung lining uid of the lung and either react quickly with biological molecules or diffuse away. The adverse health effects of air pollutants are numerous but are frequently reported to include, especially for particles and ozone, the exacerbation of asthma symptoms in both children and adults. There are also data to suggest that some air pollutants, such as particles, actually increase the incidence of asthma. For example, children living near to a busy road, such as a motorway or freeway, are reported to have an increased risk of developing asthma. However, the body of data suggesting that pollution causes asthma is less convincing than that indicating it enhances asthma symptoms. However, the potential ability of air pollution to cause development of asthma cannot be ruled out. In relation to other causes of asthma, air pollution is not likely to be a major factor, but could contribute to other causes to increase the risk of developing this disease. Effects of pollutants on lung function in healthy people are not usually related to asthma. Such effects are relatively less important since they only truly occur at very high exposure levels, greater than those typically achieved in the ambient air environment. Chronic obstructive pulmonary disease (COPD), an inammatory airway disease most commonly observed in smokers, is made worse by exposure to pollutants such as particles. People suffering from cardiovascular disease are also more vulnerable to air pollution, resulting in an increased incidence of heart attacks and strokes. Both respiratory and cardiovascular effects can result in hospitalization or even death. A number of cohort studies suggest that particulate air pollution shortens life expectancy by 12 years, although these effects are not uniformly distributed throughout the population and depend upon a number of susceptibility factors.

Susceptibility to Air Pollutants

Not all individuals have the same susceptibility to air pollution; the very old and the very young, as well as those suffering from a pre-existing disease appear to be the most vulnerable. In 2004, the WHO published a monograph on the vulnerability and susceptibility of children to air pollution. The WHO concluded that children are more susceptible than adults due to the ongoing process of lung growth and development, incomplete metabolic systems, immature host defenses, and high rates of infection

ENVIRONMENTAL POLLUTANTS / Overview 95

by respiratory pathogens. Pre- and postnatal lung development, as well as the efciency of detoxication systems, was reported to exhibit time-dependent development, which might partly account for their enhanced susceptibility. The report also suggested that children are exposed to a higher level of air pollution than adults due to their activity patterns. The WHO report concluded that the adverse effects of pollution in children include longer term effects such as a lesser maximal functional capacity on reaching adulthood. However, not all children are the same, and the WHO recognized that the most susceptible individuals are those with underlying chronic lung disease, particularly asthma. The unborn child and infant are also at risk from air pollution, with evidence suggesting a causal link between particulate air pollution and respiratory deaths in the postneonatal period. The adverse health effects of indoor air pollution are not within the scope of this article, however, exposure to high levels of indoor air pollutants, such as environmental tobacco smoke or occupational dusts, may increase susceptibility of adults and children to outdoor pollutant exposure. Socioeconomic factors such as education and diet might also inuence susceptibility to air pollution, and might partly explain why life expectancy is shorter in people from disadvantaged population groups.

Common Mechanisms of Toxicity

Of the pollutants discussed above, particles and ozone pose the greatest health threat and so have received the greatest scientic interest with respect to adverse health effects. These pollutants demonstrate significant health effects in both epidemiological and toxicological studies. As described above ozone and particles induce oxidative stress that stimulates an inammatory response, and the combination of the oxidative stress and inammation is thought to produce the disease and ill health. Ozone is a highly reactive oxidizing gas that quickly oxidizes biological molecules that it comes into contact with, leading to oxidative stress. Many studies also provide evidence that PM10 and ultrane particles can generate free radicals and reactive oxygen species (ROSs) in vitro and in vivo. The ultrane or nanoparticles have a huge surface area per unit mass over which they can interact with biological molecules, causing oxidation. However, PM10 also contains other components capable of generating ROSs, including transition metals and quinone molecules. Transition metals, in the presence of an oxidant such as hydrogen peroxide, will redox

cycle by Fenton chemistry to generate hydroxyl radicals, one of the most potent forms of ROS found in the body. There is also evidence that metals can interact with ultrane particles to potentiate the production of ROSs in vitro and inammation in vivo. Some types of quinone molecules found within PM10 also have the capacity to redox cycle in cells, generating superoxide anion radicals and other ROSs. In addition, some quinones can arylate protein thiol groups. Since protein thiol groups play a critical role in antioxidant defense mechanisms, such as in the antioxidant glutathione, arylation of these groups by quinines may increase the susceptibility of the cells to the effects of ROSs and hence oxidative stress. Oxidative stress involves a perturbation of the antioxidant defense mechanism of the lung, allowing ROSs and free radicals to persist and cause damage to proteins, lipids, and DNA. Damage to proteins and lipids results in altered cell function that can range, according to the scale of the oxidative stress, from activation of the cell to elicit a proinammatory response to cell death. The proinammatory response entails the production of mediators, usually cytokines, which stimulate the recruitment and activation of immune cells such as macrophages and neutrophils. Inammation is a normal healthy process that is required to clear foreign objects, including bacteria and pollution particles, from the body. If the inammation is acute, then the inammation is resolved and normal tissue is regenerated. However, if the inammation is disproportionately large, prolonged, or is superimposed on top of a pre-existing inammation then the result is ill health. Oxidative stress induced by pollutants can stimulate inammation via redox-sensitive signaling pathways that culminate in activation of transcription factors such as nuclear factor kappa B (NF-kB). In an unstimulated form NF-kB resides in the cell cytoplasm associated with an inhibitor protein IkB. Via an unknown mechanism, oxidative stress stimulates the activation of NF-kB that involves the release of IkB allowing NF-kB to migrate into the nucleus where it binds to the promoter regions of proinammatory genes in order to initiate their transcription. A number of other signaling pathways that control cytokine gene expression are also sensitive to oxidative stress; such pathways include cytosolic calcium. Cytosolic calcium has been shown to be important in the upregulation of cytokine production by macrophages treated with particles. For example, in macrophages calcium is involved in stimulating the production of tumor necrosis factor alpha by ultrane particles, and interleukin 1 by PM10. The relationship between calcium and oxidative stress is

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ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

C, and Whyte G (eds.) ABC of Sports Medicine, 3rd edn., pp. 6770. London: BMJ Books. Holgate S, Samet JM, Koren HS, and Maynard RL (eds.) (1999) Air Pollution and Health: Academic Press. Kelly FJ, Dunster C, and Mudway I (2003) Air pollution and the elderly: oxidant/antioxidant issues worth consideration. European Respiratory Journal 40: 70s75s. Mehta S (2003) Air pollution and health in rapidly developing countries. Bulletin of the World Health Organization 81(10): 771. Stone V, Wilson M, Lightbody J, and Donaldson K (2003) Investigating the potential for interaction between the components of PM10. Environmental Health Preventative Medicine 7: 246253. Wilson R (1996) Introduction. In: Wilson R and Spengler J (eds.) Particles in our Air. Concentrations and Health Effects, pp. 114. Harvard University Press.

complicated. Many studies suggest that ROSs can stimulate calcium signaling; they also suggest that calcium signaling can stimulate cells to produce endogenous ROSs. Since both ozone and particles induce oxidative stress and inammation, it is possible that these pollutants may interact, either enhancing or altering the subsequent health effects. Interactions among other pollutants are also possible, with the toxic effects of one pollutant making the lung more vulnerable to the effects of the other. Investigation of such interactions is difcult. A number of epidemiological studies have struggled to attribute adverse health effects to a single pollutant, since increases in a number of pollutants often occur concurrently. For example, trafc pollution enhances both NO2 and PM10 levels, while hot sunny weather enhances ozone and PM10 levels. Toxicological studies are required to investigate the plausibility of such interactions.

Relevant Websites
http://www.defra.gov.uk This web link provides a full description of the air quality strategy for England, Scotland, Wales, and Northern Ireland as written by representatives of the UK government. It also provides access to gures relating to the actual current and historical pollution values. http://www.dh.gov.uk This website provides access to UK Policy guides, research reports and other DH-published documents about air pollution. Details regarding UK funded air pollution research projects are also listed. A link to the committee on the Medical Effects of Air Pollution (COMEAP) is also provided. http://www.epa.gov This website provides details of the US Environmental Protection Agency Air Quality Standards and includes links to the Clean Air Act. http://www.euro.who.int Air pollution information collected and reported by the World Health Organization. http://www.scielosp.org SciELO Public Health is an electronic library online covering health science articles published by scientic journals.

Conclusion
Much of the information provided here pertains to the ambient environment of the developed world and clearly illustrates a link between air pollution and disease. The developing world frequently encounters higher pollution levels with a different composition. It is possible that the lessons learned by the developed world could be used to minimize the health impact of pollutants during nancial and industrial development of other countries. However, since often nances are limited and ambitions great, other issues are likely to be considered to be of greater short-term importance (Figure 1).
See also: Carbon Monoxide. Chronic Obstructive Pulmonary Disease: Overview. Environmental Pollutants: Diesel Exhaust Particles; Oxidant Gases; Particulate Matter, Ultrane Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds; Radon.

Diesel Exhaust Particles

H-E Wichmann, GSF Institute of Epidemiology, Neuherberg, Germany
& 2006 Elsevier Ltd. All rights reserved.

Further Reading
Air Quality Expert Group (AQEG) (2004) Nitrogen Dioxide in the United Kingdom. London: Department of Environment, Food and Rural Affairs Publications. Bell ML, Davis DL, and Fletcher T (2004) A retrospective assessment of mortality from the London smog episode of 1952: the role of inuenza and pollution. Environmental Health Perspectives 112: 68. Donaldson K, Stone V, Borm PJ, et al. (2003) Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM(10)). Free Radical Biology and Medicine 34: 1369 1382. Florida-James GD, Donaldson K, and Stone V (2005) Sports performance in a polluted environment. In: Harries M, Williams

Abstract
Diesel motor emission is a complex mixture of hundreds of constituents in either gas or particle form. Diesel particulate matter (DPM) is composed of a center core of elemental carbon and adsorbed organic compounds including PAHs and nitroPAHs, and small amounts of sulfate, nitrate, metals, and other trace elements. DPM consists of ne particles including a high number of ultrane particles. These particles are highly respirable and have a large surface area where organics can adsorb easily. Exposure to DPM can cause acute irritation, neurophysiological, respiratory and asthma-like symptoms and can exacerbate allergenic responses to known allergens. Consistently lung cancer risk is elevated among workers in occupations where diesel engines have been used. However, quantication of

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles 97

the cancer risk with respect to DPM concentrations is not possible. Furthermore, ambient ne and ultrane particles, of which DPM is an important component, contribute to cardiopulmonal morbidity and mortality, and lung cancer. In conclusion, diesel exhaust poses a cancer risk greater than that of any other air pollutant, as well as causing other short- and long-term health problems. One effective way to effectively reduce emission of DPM are particle traps.

Solid carbonaceous/ ash particle with adsorbed hydrocarbon/ sulfate layer

Introduction
Diesel motor emission (DME) and especially diesel particulate matter (DPM) play an important role in the discussions regarding the risk to health posed by pollutants from ambient air or in the workplace. Several international and national bodies have dealt with the evaluation of composition, exposure, health effects, and risk assessment, as well as taking steps to prevent possible health risks by setting environmental standards for both emission and ambient concentrations.
0.2 m Sulfuric acid particles

Hydrocarbon/ sulfate particles

Composition and Physicochemical Properties

DME is a complex mixture of hundreds of constituents in either gas or particulate form. Gaseous components of DME include carbon monoxide, nitrogen compounds, sulfur compounds, and numerous lowmolecular-weight hydrocarbons such as aldehydes, benzene, polycyclic aromatic hydrocarbons (PAHs), and nitro-PAHs. The particles present in DME are composed of a central core of elemental carbon and adsorbed organic compounds, as well as small amounts of sulfate, nitrate, metals, and other trace elements. DPM consists of ne particles (particulate matter (PM2.5) including a high number of ultrane particles (o0.1 mm diameter) (Figure 1)). Collectively, these particles have a large surface area, which makes them an excellent medium for adsorbing organics. Also, their small size makes them highly respirable and they have the potential to reach the deep lung and even the blood circulation. The organics, in general, range from about 20% to 40% of the particle mass. Many of the organic compounds present on the particle and in the gases are individually known to have mutagenic and carcinogenic properties. The typical chemical composition of DPM from new heavy duty diesel vehicle exhaust is shown in Table 1: 75% (3390%) elemental carbon, 19% (749%) organic carbon, 2% (15%) metals and elements, 1% (14%) sulfate and nitrate, and 3% (110%) other.

Figure 1 Schematic diagram of diesel engine exhaust particles. Reproduced from US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002.

Table 1 Typical chemical composition of ne particulate matter in the US Eastern US Elemental carbon Organic carbon Sulfate, nitrate, ammonium Minerals Unknown 4% 21% 48% 4% 23% Western US 15% 39% 35% 15% Diesel PM2.5 75% 19% 1% 2% 3%

Reproduced from US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002.

Sources and Exposure

The most important sources of DME relevant for the general population are on-road vehicles such as

passenger cars and heavy-duty and light-duty vehicles. However, nonroad diesel engines in locomotives, marine vessels, heavy-duty equipment, etc. also play a role, especially in exposure at the workplace. DME varies substantially in chemical composition and particle sizes among different engine types, engine operating conditions (idle, acceleration, deceleration), and fuel formulations (high/low sulfur fuel). The mass of particles emitted and the organic components on the particles have been reduced over recent years. The toxicologically relevant organic compounds of DME (e.g., PAHs and nitro-PAHs) emitted from older vehicle engines are still present in emissions from newer engines, though relative amounts have decreased.

98

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

DPM mass (expressed as micrograms of DPM per cubic meter) has historically been used as a surrogate measure of exposure for whole DME. Although uncertainty exists as to whether DPM is the most appropriate parameter to correlate with human health effects, it is considered a reasonable choice until more denitive information about the mechanisms of toxicity or mode(s) of action of DME becomes available. A large percentage of the population is exposed to ambient PM2.5 and ultrane particles, of which DPM is an important constituent. Exposure estimates for the early to mid-1990s in the US suggest that national annual average DME exposure from on-road engines alone was in the range of about 0.50.8 mg DPM m 3 of inhaled air in many rural and urban areas, respectively. Exposures could be higher if there was a nonroad DME source that added to the exposure from on-road vehicles. For example, it has been estimated that in the US, on a national average basis, nonroad DME emissions are mighter than on-road emissions. For localized urban areas where people spend a large portion of their time outdoors, the exposures are higher and, for example, may range up to 4.0 mg DPM m 3 of inhaled air. In some urban situations the annual average fraction of PM2.5 attributable to DPM (on a mass basis) is about 35% at the high end, although for the US the proportion appears to be more typically in the range of 10%. The situation in Europe is different since diesel passenger cars are used more frequently. For Germany it has been estimated that on-road trafc generates 3.6 mg DPM(EC) m 3 in urban areas and 1.8 mg DPM(EC) m 3 in rural areas. On average, in Germany on-road cars are estimated to contribute about 3 mg PM2.5 m 3, which corresponds to about 20% of the mean PM2.5 concentration.

There is also evidence for an immunologic effect the exacerbation of allergenic responses to known allergens and asthma-like symptoms. A large number of epidemiological studies have been performed to investigate the health effects of ambient ne (PM2.5) and ultrane particles. They show impact on respiratory and cardiovascular endpoints such as daily mortality, hospital admissions, increase of respiratory symptoms, increased numbers of myocardial infarction, and changes of inammatory and ECG parameters. However, the contribution of DPM has not been investigated specifically in these studies. Studies in which volunteers have been exposed to concentrated ambient air have demonstrated airway effects and changes in the heart and blood vessel system that link to the adverse health effects found in the epidemiological studies. They also provide mechanistic and other toxicological information that helps explain the adverse health effects of air pollutants. The toxicological database on PM-related health effects analyzed in animal experiments is not yet able to provide definite answers regarding causal factors, whether relating to physical parameters, chemical components, or biological aspects. Nevertheless, clear differences have been identied as to induction of airway inammatory response, heart/blood vessel and allergen adjuvant effects, and between ambient PM samples from different locations. Cellular and mechanistic studies provide important information about how various particles and components related to air pollution interact with cells and cellular systems. The studies describe inammatory responses and provide support for the hypothesis that very small particles (ultrane particles) are an especially harmful component, but suggest that the more coarse and intermediate-sized PM also is important.
Chronic (Long-Term Exposure) Noncarcinogenic Effects

Health Effects
Available evidence indicates that there are human health hazards associated with exposure to DME. The hazards include acute exposure-related symptoms, chronic exposure-related noncarcinogenic effects, and lung cancer.
Acute (Short-Term Exposure) Effects

Information is limited for characterizing the potential health effects associated with acute exposure to DME. However, on the basis of available human and animal evidence, it is concluded that acute or shortterm (e.g., episodic) exposure to DME can cause acute irritation (e.g., eye, throat, bronchial), neurophysiological symptoms (e.g., lightheadedness, nausea), and respiratory symptoms (cough, phlegm).

Information from the available human studies is inadequate for a denitive evaluation of possible noncancer health effects from chronic exposure to DME. However, on the basis of extensive animal evidence, DME poses a chronic respiratory hazard to humans. Chronic-exposure, animal inhalation studies show a spectrum of dose-dependent inammation and histopathological changes in the lung in several animal species including rats, mice, hamsters, and monkeys. In epidemiological studies it has been shown that long-term exposure to ambient PM2.5 affects morbidity and mortality. In children, the lung function was impaired as well as the age-dependent lung

ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles 99

growth; the prevalence of bronchitis, sinusitis, and common cold was increased and the effects were reduced with reduced exposure. Some evidence is described for increased infant mortality in areas of high exposure. In adults, effects on chronic bronchitis and chronic obstructive pulmonary disease (COPD) were found. In addition, there is strong evidence that long-term exposure to PM2.5 significantly increases the risk for total mortality and cardiopulmonary mortality. Furthermore, in a European study significantly increased mortality was observed close to busy roads suggesting an adverse inuence of PM emitted from vehicles, 90% of which are DPM.
Chronic (Long-Term Exposure) Carcinogenic Effects

The US-EPA, like several other boards, concludes that DME is likely to be carcinogenic to humans by

inhalation. This conclusion is based on the totality of evidence from human, animal, and other supporting studies. There is considerable evidence demonstrating an association between DME exposure and increased lung cancer risk among workers in varied occupations where diesel engines historically have been used (Figure 2). Most of the individual studies and all meta-analyses showed an increased risk of lung cancer in occupations with exposure to DME. However, in most studies only data on job title and duration of stay at a workplace with exposure to DME were available. Quantication of the exposure to DPM concentration was not possible in these studies, and this is the main reason why DME has not yet been classied as carcinogenic to humans. At ambient PM2.5 concentrations also a signicantly increased risk of lung cancer mortality was observed for people living in cities with exposure to high versus low concentrations of ne particulates. In

0.5 All studies

RR estimates & 85% Cl 1 1.5

Casecontrol studies

Colhort studies Internal comparison population External comparison population Smoking adjusted

Smoking not adjusted Sub-analysis by occupation Railroad workers

Equipment operators

Truck drivers

Bus workers
Figure 2 Pooled relative risk (RR) estimates and heterogeneity-adjusted 95% condence intervals (CI) for all studies and subgroups of studies included. Reproduced from Bhatia R, Lopipero P, and Smith AH (1998) Diesel exhaust exposure and lung cancer. Epidemiology 9: 8491, with permission from Lippincott Williams & Wilkins.

100 ENVIRONMENTAL POLLUTANTS / Diesel Exhaust Particles

addition to the human evidence, there is supporting evidence of DPMs carcinogenicity and associated DPM organic compound extracts in rats and mice by noninhalation routes of exposure. Other supporting evidence includes the demonstrated mutagenic and chromosomal effects of DME and its organic constituents, and evidence that suggests bioavailability of the DPM organics in humans and animals. Although chronic high-exposure rat inhalation studies showed a significant lung cancer response, according to the US-EPA this is not thought to be predictive of a human hazard at lower environmental exposures. The response in the rat experiments mainly resulted from an overload of particles in the lung due to high exposure, and such an overload is not expected to occur in humans exposed to environmental levels.

higher than that of petrol engine exhaust. Particle traps have the potential to solve all the problems outlined above as they are very effective at retaining coarse, ne, and ultrane particulates.
See also: Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Particle Deposition in the Lung.

Further Reading
American Lung Association, Environmental Defense (2003) Closing the Diesel Divide Protecting Public Health from Diesel Air Pollution. Bhatia R, Lopipero P, and Smith AH (1998) Diesel exhaust exposure and lung cancer. Epidemiology 9: 8491. Bru hner M, Ahrens W, et al. (1999) Lung can ske-Hohlfeld I, Mo cer risk in male workers occupationally exposed to diesel motor emissions in Germany. American Journal of Industrial Medicine 36: 405414. Cohen AJ (2000) Outdoor air pollution and lung cancer. Environmental Health Perspectives 108: 743750. Garshick E, Laden F, Hart JE, et al. (2004) Lung cancer in railroad workers exposed to diesel exhaust. Environmental Health Perspectives 112(15): 15391543. Health Effects Institute (HEI) (1999) Diesel Emissions and Lung cancer: Epidemiology and Quantitative Risk Assessment. A Special Report of the Institutes Diesel Epidemiology Expert Panel. Heinrich J and Wichmann HE (2004) Trafc related pollutants in Europe and their effects on allergic disease. Current Opinion in Allergy and Clinical Immunology 4: 341348. Lipsett M and Campleman S (1999) Occupational exposure to diesel exhaust and lung cancer: a meta-analysis. American Journal of Public Health 89: 10091017. Pope CA, Burnett RT, Thun MJ, et al. (2002) Lung cancer, cardiopulmonary mortality, and long-term exposure to ne particulate air pollution. Journal of the American Medical Association 287: 11321141. Riedl M and Diaz-Sanchez D (2005) Biology of diesel exhaust effects on respiratory function. Journal of Allergy and Clinical Immunology 115(2): 221228 (quiz 229). UBA: Umweltbundesamt Federal Environmental Agency Germany (2003) Future Diesel. US-EPA (2002) Health effects assessment document for diesel engine exhaust. EPA/600/8-90/057F, May 2002. US Environmental Protection Agency (2004) Air quality criteria for particulate matter. Washington EPA/600/P-99/D02bF. WHO (2003) Health aspects of air pollution with particulate matter, ozone and nitrogen dioxide. Report from WHO Working Group meeting, 1315 January, Bonn. Copenhagen: WHO Regional Ofce for Europe. Wichmann HE (2004) Positive health effects by the use of particle traps in Diesel cars risk assessment for mortality in Germany (in German). UmweltmedForschPrax 9: 8599.

Possible Regulatory Steps

In 2003 a report by the American Lung Association and Environmental Defense concluded that Diesel exhaust contains a host of harmful contaminants that together pose a cancer risk greater than that of any other air pollutant, as well as causing other short and long term health problems. A easy and quick way to reduce DPM might be the obligatory tting of particle traps to all diesel engines. Based on an epidemiologically derived exposureresponse relationship for PM2.5 and mortality, additional data on ambient particle concentrations, and estimates on the contribution of DPM, the positive health effects of the use of particle traps have been estimated for Germany. As a result, approximately 12% of the annual deaths have been attributed to DPM from on-road vehicles. Using particle traps would prevent most of these premature deaths and would lead to an expected mean increase in life expectancy of 13 months. In Europe diesel cars are very popular. For example, in Germany the percentage of newly registered diesel passenger cars increased from 5% in 1980 to 38% in 2003, and in 2020, diesel vehicles are expected to account for 36% of total vehicle kilometers. On the one hand, total emission of DPM has decreased by 40% in the last 10 years and will decline further due to measures taken for heavy-duty vehicles; on the other hand, DPM and nitric oxide emissions from diesel passenger cars would stagnate if limit values remained unchanged. Even with the introduction of better standards for the particulate mass, the number of ultrane particles could not be controlled, and these extremely small particles pose a potential health risk that is not yet clearly understood. The carcinogenic potency of DME from current car models without particulate traps is 10 times

Relevant Websites
www.environmentaldefense.org This website is maintained by Environmental Defense Fund (EDF). It provides information on environmental rights of all people, including future generations. www.unweltdaten.de This website provides information on environmental planning and sustainability strategies, health-related environmental protection, chemical and biological safety, etc.

ENVIRONMENTAL POLLUTANTS / Oxidant Gases 101

Oxidant Gases
M Nickmilder and A Bernard, Catholic University of Louvain, Brussels, Belgium
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Ozone, nitrogen dioxide, chlorine, and trichloramine are the principal oxidant gases to which man can be exposed in the work or general environment. Ozone is the main oxidant produced in the troposphere through UV-driven reactions involving nitrogen oxides and volatile organic compounds. Nitrogen dioxide is an indoor and outdoor pollutant generated by all combustion processes when at high temperatures oxygen combines with nitrogen. Chlorine is a corrosive gas mainly used in industry but is also released by some cleaning and disinfecting products. Trichloramine is a volatile by-product of water chlorination that contaminates the air of indoor swimming pools and various other indoor environments. All these oxidant gases are potential irritants to the eyes, the upper respiratory tract, and the lungs. Depending on the inhaled dose and the individual susceptibility, they can produce a wide spectrum of short-term effects including irritation symptoms, lung function impairment, airways inammation, asthma exacerbation, and in extreme cases pulmonary edema and even death. The long-term effects of these gases are much less well documented although there is little doubt that repeated or chronic exposures to high levels of these oxidants are detrimental to the lung tissue and may lead to lung disorders, especially asthma or asthma-like syndromes (e.g., reactive airway dysfunction syndrome caused by chlorine). No specic antidote exists for these pulmonary irritants. Treatment is basically supportive according to the clinical manifestations.

considered as a transboundary problem. Current background levels are in the range of 4070 mg m 3 (0.020.035 ppm) but, as a result of its photochemical origin, it displays strong seasonal and diurnal patterns. In some areas, O3 can reach very high concentrations during the summer months (e.g., up to 41000 mg m 3 in Mexico city). NO2 is a common pollutant of ambient and indoor air and also an occupational hazard for some professions (e.g., welders). Its main indoor source is unvented combustion appliances. NO2 in ambient air largely derives from the oxidation of nitrogen monoxide, the major sources of which are combustion emissions, mainly from vehicles. Current levels of NO2 are in the order of 1050 mg m 3 but may reach several hundreds mg m 3 in areas affected by high pollution episodes. It is involved in a number of physicochemical reactions in the atmosphere, some of them leading to the formation of harmful secondary air pollutants such as O3 and nitric acid. Cl2 is an important raw material for the chemical industry. The general population can be exposed to chlorine gas in cases of accidental leakage from industrial facilities and also when using household chlorine bleach, which can release some chlorine gas. Cl2 reacts with water to form mainly hypochlorous acid (HClO) depending on the pH. NCl3 is a volatile by-product of water chlorination produced when hypochlorous acid released from

Introduction
The principal oxidant gases to which man can be exposed in the environment are ozone (O3), nitrogen dioxide (NO2), chlorine (Cl2), and nitrogen trichloride or trichloramine (NCl3). Their physicochemical properties are shown in Table 1. O3 is formed by photochemical reactions in the presence of precursor pollutants such as nitrogen oxides and volatile organic compounds (Figure 1). Because of its high reactivity with other oxidizable pollutants (e.g., NO) or materials, O3 is usually present at lower levels in indoor compared to outdoor air and in busy urban centers compared to suburban and adjacent rural areas. O3 is also subject to long-range atmospheric transport and is therefore
Table 1 Physicochemical properties of oxidant gases Formula Ozone Nitrogen dioxide Chlorine Nitrogen trichloride O3 NO2 Cl2 NCl3 MW 48 46 71 120 MP ( 1C) 193 11 101 40 BP ( 1C) 112 21 34.5 71

OH RCH3

O2 RCH2

NO RCOO RCO

OH RCHO

NO2

O2

O + NO

O3

NO2 + O2

Figure 1 Ozone formation in photochemical smog. RCOO, radical peroxy; OH, radical hydroxy; NO, nitrogen oxide; NO2, nitrogen dioxide; O3, ozone.

SW (g/100 g) 3 10 5 (Very low solubility) Low solubility 7.3 10 1 (Slightly soluble) Virtually insoluble

Color Colorless to blue Reddish to brown Greenish yellow Brownish yellow

Odor Pungent Pungent Suffocating Swimming pool

MW, molecular weight; MP, melting point; BP, boiling point; SW, solubility in water.

102 ENVIRONMENTAL POLLUTANTS / Oxidant Gases

chlorine-based disinfectants reacts with nitrogenous compounds. NCl3 is particularly concentrated in the air of poorly ventilated indoor pools where it can reach concentrations of more than 1000 mg m 3. This gas frequently contaminates many other indoor areas where chlorine bleach is used as a cleaning or disinfecting agent.

Clinical Consequences
O3 toxicity depends on its concentration, exposure duration, and respiratory rate of an individual. Because of its low solubility in water, it is carried over into the deep lung. The greatest deposition and the most severe damage are located mainly in terminal and respiratory bronchioles where O3 causes oxidative stress injury to surfactant components and epithelial cells. Short-term effects of O3 include upper respiratory tract irritation symptoms, pulmonary function changes, increased airway responsiveness, and airway inammation, characterized by both an inux of inammatory cells and an increased permeability of the airblood barrier associated with an increased ux of proteins across it (i.e., CC16 and albumin) (Figure 2). The mechanism by which O3 increases the epithelial permeability appears to involve mainly the tight junctions between cells lining the respiratory epithelium, which are disrupted primarily as a consequence of oxidative damage and secondarily by proinammatory mediators.

In children and adults, a decrease in pulmonary function can occur after only short-term exposure to O3 concentrations in the range 120240 mg m 3; for lung inammation, a threshold of around 135 mg m 3 has recently been estimated. Asthmatic subjects are generally not more sensitive to O3 than healthy subjects with regard to airway narrowing and hyperreactivity but they develop a more pronounced airway inammation. However, the pulmonary response to O3 shows a great interindividual variability, which can be explained by differences in the lung protective mechanisms but also by other factors such as age, obesity, diet, or exposure to other lung toxicants (e.g., cigarette smoke). In studies where exposure to O3 was repeated, some health effects such as lung function decline and inammatory reaction showed a progressive attenuation. However, this adaptation should not be interpreted as an intrinsic positive response, as it may impair or prevent the normal physiological responses and the ability of the lung to face new challenges such as infectious or allergic agents. In addition, there is some evidence to suggest that exposure to O3 can enhance the inammatory response to allergens, viruses, and particles. The long-term effects of O3 are much less well documented. At levels currently observed in ambient air, the evidence linking O3 exposure to asthma incidence and prevalence in children or adults is not consistent. There is some epidemiological evidence

Lymphatic drainage Airways Ep In En Blood Airways O3 Albumin Albumin Ep In En

Lymphatic drainage Blood

Albumin

CC16

CC16

CC16

O3

(a)

Lymphatic drainage

(b)

Lymphatic drainage

Figure 2 Passage of albumin and the lung-specic Clara cell protein (CC16) across the different barriers separating the airways from the blood at the level of a terminal bronchiole under normal conditions (a) and after acute exposure to O3 (b). O3 causes an increased permeability of the epithelial barriers to proteins, resulting in an increased leakage of plasma albumin into the airways and, in the opposite direction, of CC16 into the interstitium from which it is cleared by lymphatic drainage or directly into the blood across the endothelium. The thickness of the arrows is not proportional to the concentration of protein but is used to illustrate the relative permeability of the different barriers and the increased uxes casued by O3 exposure. En, endothelium; Ep, epithelium; In, interstitium.

ENVIRONMENTAL POLLUTANTS / Oxidant Gases 103

suggesting that long-term O3 exposure could reduce lung growth in children. The suggestion that prolonged O3 exposure causes chronic damage to human lung is supported by animal studies. At different levels of O3 exposure, animals show various biological responses including inammation and increased airways permeability, morphological alterations in the lung, increased susceptibility to bacterial infections, increased production of certain lung proteins active in oxidant defenses, and increase in collagen content. Furthermore, the susceptibility of lung to O3 is remarkably species and strain dependent. The effects of NO2 on health are more dependent on the level of exposure than on the duration. It is a highly reactive, poorly water-soluble gas. Biologically, its effects result primarily from a reaction with components in the cell lining uid at various sites in the respiratory tract, such as the region reaching the distal airways and air sack spaces, resulting in the formation of reactive oxygen that can damage cells or stress them to release proinammatory substances. Short-term acute effects of NO2 include lung function impairment and increase in bronchial hyperresponsiveness. Subjects with asthma are much more sensitive to NO2 exposure than healthy subjects. They show an increase of bronchial hyperresponsiveness after 180 mg m 3 of NO2, whereas healthy subjects require a 10-fold higher level to elicit the same response. In allergic asthmatics, NO2 can also enhance the response to allergens but only at concentrations that can be attained in some indoor environments (e.g., houses with unvented gas stoves). In subjects with chronic obstructive diseases, a decline of lung function has been demonstrated at a NO2 exposure of 540 mg m 3 but no threshold has really been established for this population. In nonallergic subjects, exposure to NO2 can cause a shift towards an allergic immune cell response in the cells of the airway wall, but this occurs only at very high levels ranging from a thousand to several thousand mg m 3 over several hours. In animals, long-term exposures to NO2 concentrations higher than those encountered in ambient air produce changes in lung structure and increase susceptibility to microbial lung infections. In humans, long-term exposure to NO2 may decrease lung function and increase the risk of respiratory symptoms. NO2 also exacerbates the symptoms of asthma, and this effect appears to be greater in children than in adults. However, ambient NO2 often has complex interrelations with other pollutants, such as particulate matter and O3, making it difcult to disentangle its effect per se. Furthermore, because homes with gas appliances have peak NO2 levels

causing clinical effects, the observed effects cannot always be clearly attributed to either the repeated short-term high-level exposures or long-term exposures (or possibly both). Cl2 is a potential irritant gas to the eyes, upper respiratory tract, and the lungs. Because of its solubility in water, Cl2 can damage both the airways and the alveolarcapillary structures. At physiological pH, Cl2 combines with tissue water to form mainly HClO, which diffuses into cells and forms chloramines by reaction with amino acids or proteins. These derivatives are believed to be largely responsible for the tissue damage and inammation caused by chlorine gas. Acute exposure to low concentrations (110 ppm) may cause sore throat, coughing, and eye and skin irritation. At higher levels, Cl2 causes burning of the eyes and skin, chest pain, vomiting, dypsnea, wheezing, cough, pneumonitis, and pulmonary edema. Exposure to even higher levels can produce severe injuries leading to death. Some people may develop a type of asthma due to irritating effects of Cl2, called reactive airways dysfunction syndrome (RADS). Chronic exposure of animals to Cl2 produces decreased body weight gain, eye and nose irritation, non-neoplasic lesions, and respiratory epithelial hyperplasia. Workers chronically exposed to Cl2 complain of eye irritation and respiratory effects such as throat irritation and airow obstruction. Except in cases of industrial accident, the general population is unlikely to be exposed to high levels of Cl2 but it is frequently in contact with NCl3, the main volatile by-product of water chlorination. NCl3 is a strong oxidant that is virtually insoluble in water and exerts its toxic effects more deeply in the lung than Cl2. This gas is capable of disrupting acutely or chronically the epithelial barrier in the deep lung, thereby increasing the transepithelial leakage of proteins. NCl3 is an eye and upper respiratory tract irritant, which can also produce acute lung injury in accidental exposures particularly when chlorine bleach is mixed with ammonia. In rodents, this gas has the same irritating potency as chlorine or formaldehyde and causes fatal lung edema at high doses. Information concerning the potential risks of chronic exposure to NCl3 are very scarce and are limited to studies on children attending indoor chlorinated swimming pools and on lifeguards and swimming teachers. In children, the regular attendance at chlorinated pools has recently been found to be associated with an exposure-dependent increase in lung epithelium permeability and in the risk of developing asthma. NCl3 can be a cause of occupational asthma in lifeguards.

104 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles

Table 2 Air Quality Guidelines by WHO, odor threshold, TLV, EGRP, and IDLH for oxidant gases Air Quality Guidelines O3 NO2 Cl2 NCl3 0.06 (8 h) 0.11 (1 h) 0.06 (2 h)a Odor threshold 0.05 0.12 0.31 TLV 0.1 3 0.5 ERPG 0.5 1 1 IDLH 5 20 10

a Air standard for the Brussels capital region. All values are given in ppm except air guidelines. O3: 1 ppm 1.96 mg m 3; NO2: 1 ppm 1.88 mg m 3; Cl2: 1 ppm 2.90 mg m 3; NCl3: 1 ppm 4.90 mg m 3. TLV, threshold limit values; ERPG, emergency responses planning guidelines; IDLH, immediately dangerous for life or health concentrations.

Management and Treatment

O3, NO2, Cl2, and NCl3 exert their toxicity in a dose-dependent way. No specic antidote exists for these pulmonary irritants. Treatment is basically supportive according to the clinical manifestations. It includes support of vital functions with focus on free airways, supplemental oxygen, and bronchodilators. Owing to the role of oxidative stress in pathogenesis, treatment with oxygen should only continue as long as required to keep sufcient oxygenation. Prevention of the adverse effects of these gases is mainly based on air quality guidelines from the World Health Organization (WHO) and from other national or international organizations and the establishment of odor thresholds, threshold limit values (TLV), emergency responses planning guidelines (ERPG), and immediately dangerous for life or health concentrations (IDLH) (Table 2).
See also: Asthma: Overview. Environmental Pollutants: Overview. Lung Anatomy (Including the Aging Lung).

EU Directive (1999) EU Directive 1999/30/EC of the European Council of 29 September 1999 relating to limit values for sulphur dioxide, nitrogen dioxide and oxides of nitrogen, particulate matter and lead in ambient air. Ofcial Journal of the European Communities L163: 4160. EU Directive (2002) EU Directive 2002/3/EC of the European Parliament and the Council of 12 February 2002 relating to ozone in ambient air. Ofcial Journal of the European Communities L67: 1430. Holgate ST, Samet JM, Koren HS, and Maynard RL (eds.) (1999) Air Pollution and Health. London: Academic Press. Lippmann M (ed.) (2000) Environmental Toxicants, 2nd edn. New York, NY: Wiley-Interscience. Massin N, Bohadana AB, Wild P, et al. (1998) Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools. Occupational and Environmental Medicine 55: 258263. Tickett KM, McCoach JS, Gerber JM, Sadhra S, and Burge PS (2002) Occupational asthma caused by chloramines in indoor swimming-pool air. European Respiratory Journal 19: 827832. US EPA (1997) National Ambient Air Quality Standards for Ozone. Federal Registration 62: 3885538896. WHO (2000) Classical pollutants. In: Air Quality Guidelines for Europe, European Series, no. 91, 2nd edn., pp. 173198. Copenhagen: WHO Regional Publications.

Further Reading
Bernard A, Carbonnelle S, Michel O, et al. (2003) Lung hyperpermeability and asthma prevalence in schoolchildren: unexpected associations with the attendance at indoor chlorinated swimming pools. Occupational and Environmental Medicine 60: 385394. Broeckaert F, Arsalane K, Hermans C, et al. (2000) Serum Clara cell protein: a sensitive biomarker of increased lung epithelium permeability caused by ambient ozone. Environmental Health Perspective 108: 533537. Bylin G, Cotgraeve I, Gustafsson L, et al. (1996) Health risk evaluation of ozone. Scandinavian Journal of Work and Environmental Health 22: 5104. Carbonnelle S, Francaux M, Doyle I, et al. (2002) Changes in serum pneumoproteins caused by short-term exposures to nitrogen trichloride in indoor chlorinated swimming pools. Biomarkers 7: 464478. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society (1996) Health effects of outdoor air pollution. American Journal of Respiratory and Critical Care Medicine 153: 350.

Particulate Matter, Ultrane Particles

K Donaldson and W MacNee, University of Edinburgh, Edinburgh, UK V Stone, Napier University, Edinburgh, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Particulate matter (PM), the particulate fraction, is the most harmful component of the air pollution cocktail. It is measured using the PM10 and PM2.5 conventions that approximately collect the tracheobronchial and respirable fractions, respectively. Daily increases in PM correspond to increases in deaths and hospitalizations for airway disease and cardiovascular disease 1 or 2 days later. Living in a particle-polluted area also increases the incidence of chronic lung and cardiovascular disease and

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles 105

lung cancer. The underlying causes of these adverse effects are becoming better understood. There is heterogeneity in the potency of the components of PM, with some parts that contribute to the PM mass being essentially nontoxic (e.g., sea salt). The primary combustion-derived nanoparticulate component seems to be especially important in causing oxidative stress and inammation, which may explain all the observed adverse health effects.

1.0

Inhalable

0.8 Deposition fraction

0.6 Extra-thoracic 0.4 Respirable 0.2 Tracheobronchial

Measurement
Air pollution is a cocktail of different components, both gaseous and particulate. The particulate component of environmental air pollution is measured according to a sampling convention, either as PM10 or PM2.5, which are described later. Particles deposit in different regions of the respiratory tract depending on their aerodynamic diameter (dae). Aerodynamic diameter is dened as the diameter of a sphere with the same density as water (unit density, 1 gm cm 3) and the same gravitational settling speed as the particle of interest. This settling speed is determined by the equilibrium between gravitational force (proportional to density multiplied by volume) and the aerodynamic resistance (proportional to particle diameter multiplied by velocity). The various regions of the lungs in which particles of different aerodynamic size deposit are shown in the deposition curves in Figure 1; the tracheobronchial region is the region beyond the larynx, and the respirable region is the area beyond the ciliated airways, also known as the centriacinar region. Occupational hygienists, who have long had an interest in particles and the lungs, have developed sampling conventions based on the relationship between particle aerodynamic diameter and the fraction of particles penetrating to the different regions of the respiratory tract; these International Standard Organization (ISO) conventions are illustrated in Figure 2. In air pollution measurement, the conventions of PM10 (approximately thoracic) and PM2.5 (approximately respirable) are often used. It is important to note that sampling conventions differ from deposition curves. Deposition curves show the likelihood of a particle of any size being deposited in different parts of the lung, whereas sampling conventions show the ability of samplers of a specied convention to capture particles of a certain size. There is an important relationship between the two, however, that allows sampling of particles of sizes that are relevant to health effects. These sampling conventions are the global environmental standards for measuring the mass of airborne particles, and measurements are expressed as mass per unit volume sampled conventionally mg m 3. In the UK, particulate matter (PM) and the gaseous

0.01

0.1

1 MMAD (m)

10

100

Figure 1 Deposition curves. The relationship between particle size and the efciency of deposition in various anatomical compartments of the lungs. MMAD, mass median aerodynamic diameter.

100

80 % of total particles in convention

In ha

la

Resp

60

ble

Thora

Highrisk r

( irable ) x. PM 2.5 appro

cic (ap

able espir

prox. P

40

M 10)

20

0 1 10 Aerodynamic diameter (m)

Figure 2 ISO health-based sampling conventions for particles.

100

pollutants are measured at sites in cities by Automated Urban Network (AUN) monitors run by the Department of Environment, Food and Rural Affairs (DEFRA).

Nomenclature
There are some problems of nomenclature for particles because of the continued use of older sampling conventions. For the purposes of risk assessment, the health-based conventions are preferable, but the

106 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles

following are measurement conventions of PM that are also used: Total suspended particles (TSP). A TSP monitor captures atmospheric particulate smaller than approximately 40 mm in diameter. This means that TSP is not representative of the large particles that do not enter the respiratory tract and the lungs. Black smoke. This system was used in the UK and other countries until the end of the 1980s. Air was drawn through a size-selective orice onto a white paper and the blackness of the smudge was measured by reectance. This method is obviously biased toward black (i.e., carbon-based) particles. There is a variable relationship between particles as measured by black smoke and other measures of PM. PM10. This is the commonly used size-selective sampling convention that captures particles of 10 mm with 50% efciency; it roughly corresponds to the thoracic fraction of particles as dened by ISO. PM10 is routinely measured in the UK by DEFRA in their AUN. PM2.5. This is a size-selective sampling convention that captures particles of 2.5 mm with 50% efciency; it roughly corresponds to the respirable fraction of particles as dened by ISO.

at over 1500 mg m 3. Levels of PM, along with other pollutants, have decreased markedly during the past 50 years, but the trafc-derived component, which is hypothesized to be the most harmful component, has increased, in line with increases in road trafc.

Adverse Effects of PM in Epidemiological Studies

The levels of PM in any city vary both temporally, as a level uctuating hourly around a mean, and spatially, dependent on average levels or trafc in an area or due to local industrial sources. The temporal and spatial variation in air pollution underlie the two main approaches to detecting and quantifying the adverse effects of PM (and indeed any air pollutant) in human populations. Time series studies utilize the temporal dimension and seek to relate the moving average of PM level to the moving average of a dened end point (e.g., mortality). Various lag times are used to detect the relation between the level of PM and the end point (e.g., 1 day). Environmental epidemiological studies seek to compare an end point such as the death rate in cities with high air pollution with the death rate in a city with low air pollution. Panel studies form a third category, in which small, well-characterized populations are followed for a dened end point that can be related to PM levels measured by personal or xed point samplers. When these different approaches are taken together and examined over several hundred such studies, there is good coherence between the acute effects seen in time series and panel studies and the chronic effects seen in environmental studies. More important, many of these studies show no evidence of a threshold; that is, these adverse effects are occurring at the levels of PM that pertain in our cities today. These adverse effects are summarized in Table 1; the quantitative extent of the mortality effects of PM in European studies is shown in Figure 3. Within a short lag time of 1 or 2 days following an increase in PM, there are increases in all-cause mortality, significant increases in attacks of asthma and

Levels of PM
AUN measurements show that PM10 levels can uctuate over a day, with higher levels usually occurring during rush hours on weekdays. A typical UK city in the twenty-rst century has an average PM10 level of approximately 20 mg m 3, with short-term peaks of up to 100 mg m 3. In heavily polluted cities such as Athens or Mexico City, the levels of PM10 can be much higher, commonly reaching 5075 mg m 3 on average with very high peaks. The xed monitors of the AUN, or their equivalents worldwide, form the basis for virtually all epidemiological studies of the human health effects of PM. However, personal sampling studies show higher concentrations of particles kerbside in busy streets or tunnels and also in homes where there is cigarette smoking or certain forms of cooking. In some countries, indoor burning of biomass fuels in poorly ventilated dwellings produces very high levels of exposure, but the effects of these are relatively unstudied. In the past in the UK, there was high air pollution and very severe episodes. As a comparison with the approximately 20 mg m 3 found in the air of most UK cities today, the famous great London pea-soup smogs of the 1950s had particle levels estimated

Table 1 Summary of the human adverse health end points that are increased following increases in PM, as detected in epidemiological studies Mortality from cardiovascular and respiratory causes Admission to hospital for cardiovascular causes Exacerbation of asthma in pre-existing asthmatics Symptoms and use of asthma medication in asthmatics Exacerbation of chronic obstructive pulmonary disease Lung function decrease Lung cancer

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles 107

1.030 Respiratory

The Central Role of Inammation

1.025

1.020

1.015 Relative risk All causes

1.010

1.005

1.000

0.995

Inammation plays a role in the effects of all harmful particles, and toxicology studies have emphasized the central role of inammation in the pulmonary and cardiovascular effects of PM. PM has shown a variety of proinammatory effects in CAPs exposure studies in humans and rats and in animals exposed to PM by instillation. The mechanism of the proinammatory effects of PM has been investigated extensively, and the ability of PM to cause proinammatory effects in toxicological models of the lung has been amply demonstrated. Thus, PM or surrogate particles have been shown to activate nuclear factor kappa B and other proinammatory signaling pathways in lungs and cells in culture and to increase release of proinammatory mediators such as the chemokine interleukin (IL)-8 by cells in culture. These proinammatory effects help to explain the increased exacerbations of asthma and COPD seen when pollution increases.
Inammation and Cardiovascular Effects

0.990
Figure 3 Relative risk for all cause, respiratory, and cardiovascular mortality following a 10 mg m 3 increase in PM10: results from a meta-analysis of European studies performed for a World Health Organization task group.

Cardiovascular

increased usage of asthma medication, increased deaths in chronic obstructive pulmonary disease (COPD) patients, exacerbations of COPD, and increased deaths and hospitalizations for cardiovascular disease. The adverse cardiovascular effects associated with increases in PM are well documented. Panel studies have documented associations between elevated levels of particles and onset of myocardial infarction, increased heart rate, and decreased heart rate variability. Chamber studies with concentrated airborne particles (CAPs) have also shown increased lung inammation and altered brachial artery diameter in relation to increased exposure.

PM in Toxicological Studies: Mechanism of the Inammatory Effects of PM

Toxicological studies have helped to understand the mechanism of the adverse effects on the lungs and cardiovascular system found in the epidemiology studies described previously. Toxicological studies have used both PM and surrogate particles. Surrogate particles are used because of the variability of PM and the difculty of obtaining bulk material for testing and for testing specic hypotheses related to the relative potency of the different components of PM.

Inammation can also be seen as a driver of the cardiovascular effects. There is evidence of systemic inammation following increases in PM, as shown by elevated C-reactive protein, blood leukocytes, platelets, brinogen, and increased plasma viscosity. Atherosclerosis is the underlying cause of acute coronary syndrome, the main cause of cardiovascular morbidity and mortality. Atherogenesis is an inammatory process initiated via endothelial injury and producing systemic markers of inammation that are risk factors for myocardial and cerebral infarction. Repeated exposure to PM10 may, by increasing systemic inammation, exacerbate the vascular inammation of atherosclerosis and promote plaque development or rupture. Thus, inammation in the lungs may explain both the local and the systemic cardiovascular effects of PM (Figure 4). The inammatory response to particles or the particles themselves may also affect the neural regulation of the heart, leading to death from fatal dysrhythmia. In support of this hypothesis, studies of humans and animals have shown changes in the heart rate and heart rate variability in response to particle exposures.

Relative Potency of the Components of PM in Causing Inammation and the Central Role of Oxidative Stress
PM is a complex and variable mixture of particle types (Table 2). Many of the common components of PM have low potency in causing inammation in the

108 ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles

Asthma/COPD Background inflammation in the lungs PM Worsening of lung inflammation Effects on clotting cascade Exacerbations and deaths Effects on atherogenic plaques Cardiovascular disease Susceptible lungs? Lung inflammation

Thrombosis

Deaths and hospitalizations for CV

Figure 4 Hypothetical pathways from inammation to local pulmonary and cardiovascular effects.

Table 2 Typical composition of the particles in PM PM10 component Primary combustion-derived nanoparticles Comment Large surface area per unit mass and contain transition metals and organic volatiles; derived from combustion (e.g., vehicle exhaust particles) Derived from sea spray Predominantly ammonium sulfate Predominantly ammonium nitrate Derived from the earths crust (e.g., clay) Reentrained dust from wear on road surfaces, tyre wear, etc. For example, endotoxin

Sodium and magnesium compounds Sulfate Nitrate Compounds of calcium and potassium and insoluble minerals Road dust Biologically derived materials

lungs. Among those components that contribute to the mass of PM but are unlikely to cause inammation are sea salt, ammonium nitrates and sulfates, road dust, and crustal dust. For example, in one epidemiological study, days when the origin of the particle cloud was predominantly from wind-blown dust were removed from the analysis. This greatly strengthened the relationship with adverse health effects and emphasized the importance of the combustion-derived particles. In contrast, the primary combustion-derived nanoparticles (PCDNs), derived predominantly from automobiles, especially diesel, are known to cause pulmonary inammation in humans and animals. Nanoparticles are extremely small particles with a diameter less than 100 nm, and those found in the general environment are principally derived from trafc. They have enhanced ability to generate highly reactive free radical molecules that damage and activate lung cells to produce proinammatory mediators. This is a consequence of the fact that PCDNs possess three important components: high surface

area, organic contamination, and metals. All of these components are able to generate oxidative stress, which acts on key redox-sensitive, proinammatory signaling pathways in lung cells. This leads to inammatory gene transcription, which causes the adverse health effects (Figure 5).
Oxidative Activity of PCDNs

PCDNs contain components able to generate oxidative stress when the particles deposit on the lung surface. Transition metals Formation of hydroxyl radicals (dOH), generated by ionic iron and other transition metals via the Fenton reaction, has been implicated in the oxidative stressing and inammatory effects of PM. Other transition metals, such as chromium, vanadium, and copper, are also found to occur in PM and generate dOH. Electron paramagnetic resonance spectroscopy using a spintrap has been used to directly demonstrate the generation of dOH by

ENVIRONMENTAL POLLUTANTS / Particulate Matter, Ultrane Particles 109

PM10 Primary combustion-derived nanoparticles (transition metals, particle surface, organics) Salts, crustal dust, road dust etc.

No effect Oxidative stress Lipid peroxidation

aromatic hydrocarbons (PAHs), n-alkanes, nitroPAHs, and quinones formed by combustion or derived from unburned fuel. Interaction of these organics with enzyme systems can result in oxidative stress via the production of oxidants such as superoxide anion. These also have the ability to activate oxidative stress-responsive signaling pathways in cells, leading to the release of proinammatory chemokines such as IL-8.
Translocation of Nanoparticles

Depletion of anti-oxidants

Activation of NF- B/AP-1 and other redox-sensitive signaling pathways Gene expression Inflammatory mediators Inflammation Exacerbations of airways disease/ effects on the cardiovascular system
Figure 5 Molecular pathways for the proinammatory effects of the PCDN component of PM.

PM in the presence of H2O2. In these studies, dOH formation is prevented by the transition metal chelator desferoxamine. Nanoparticle surfaces Surface area of particles is known to be a major factor in driving inammation. A large surface area lung burden of even low-toxicity particles is known to cause inammation. Nanoparticles of low-toxicity, low-solubility materials have a very high surface area per unit mass. They are also highly inammogenic by virtue of this high surface area, and oxidative stress, generated through mechanisms that are not well understood, appears to play a role. PM can contain very high numbers of PCDNs as singlet particles but also as aggregates; these aggregates can be aerodynamically larger than the classical definition of a nanoparticle (i.e., they can be larger than 100 nm). However, nanoparticles in aggregates still express toxicity consistent with their geometric surface area and not in relation to their surface area as deduced from their aerodynamic diameter as an aggregate. Organics PCDNs contain organic molecules that have the ability to generate free radicals and cause oxidative stress. These organics include polycyclic

Nanoparticles also seem to be able to traverse the epithelial barrier and gain access to the pulmonary interstitium. Particles in the interstitium are unlikely to be cleared and so the dose builds up. In the interstitium, they can activate cells, leading to more harmful interstitial inammation. In the interstitium, the particles are very close to the blood, and experimentally nanoparticles have been shown to gain access to the blood. If this is conrmed for environmental nanoparticles, then we may postulate that bloodborne nanoparticles may interact with endothelial cells and clotting proteins, thus enhancing thrombogenesis. Additionally, they may interact directly with the endothelium overlying atherogenic plaques and may even gain access to plaques. This could cause activation of cells in the plaques and hasten plaque worsening or rupture, thus making a direct contribution to acute coronary syndrome.
Endotoxins

Endotoxins are biological components, present in some particle samples, that are released from the outer membrane of Gram-negative bacteria. Chemically, they are dened as lipopolysaccharides and found in variable amounts in PM from various locations. Studies indicate that the PM endotoxin can be a major component in stimulating cells, especially macrophages, to produce inammatory mediators. Endotoxins are commonly present in the coarse fraction (PM10 and PM2.510).

Cancer and PM
Lung cancer is among the major chronic effects of living in an area with high PM10. In the famous six-cities study, more than 8000 deaths were studied in relation to the levels of air pollution in six US cities. Adjusted mortality rate ratio, corrected for smoking, for the most polluted cities compared to the least polluted showed a 25% increase in deaths from lung cancer. The risk of death from lung cancer due to living in an area with high levels of air pollution particles was further conrmed in a study using

110 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

data from the American Cancer Society. In this study, living in an area with a 10 mg m 3 elevation in ne particulate air pollution was associated with an 8% increase in the risk of lung cancer mortality. This has been supported by toxicological studies on the genotoxic effects of PM, which have demonstrated that PM has the ability to cause specic DNA adducts that form through oxidative stress pathways. The pathway from DNA adducts to accumulations of mutations, culminating in transformation to a cancer cell, is well documented. Coarse and ne PM samples have been shown to generate hydroxyl radical, the most harmful free radical formed in tissue under oxidative stress conditions, and this combines with guanine in DNA to form a specic adduct, 8-hydroxy-20 -deoxyguanosine (8-OHdG). PM samples are able to generate hydroxyl radical via transition metals, and these can chemically react with lung molecules to produce the hydroxyl radical. Organic molecules such as PAHs and quinones, both present on combustion-derived PM10, have been implicated in redox cycling and oxidative stress. When the PM is mixed with naked DNA, the 8-OHdG adduct is formed, and it is also seen in lung epithelial cells incubated with particles, a more realistic exposure than direct incubation of PM with DNA.

Interactions with Other Components of Air Pollution

Because other components of air pollution, such as ozone, also cause oxidative stress and inammation, there is potential for additive and possibly synergistic interactions between PM and the gaseous components in causing adverse effects.
See also: Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Particle Deposition in the Lung.

Donaldson K, Stone V, Borm PJ, et al. (2003) Oxidative stress and calcium signaling in the adverse effects of environmental particles (PM10). Free Radical Biology and Medicine 34: 1369 1382. Donaldson K, Stone V, Seaton A, and MacNee W (2001) Ambient particle inhalation and the cardiovascular system: potential mechanisms. Environmental Health Perspectives 109(supplement 4): 523527. Donaldson K and Tran CL (2002) Inammation caused by particles and bres. Inhalation Toxicology 14: 527. Donaldson K, Tran CL, and MacNee W (2002) Deposition and effects of ne and ultrane particles in the respiratory tract. European Respiratory Monograph 7: 7792. Dreher K (2000) Paticulate matter physicochemistry and toxicology: in search of causality a critical perspective. Inhalation Toxicology 12(supplement 3): 4557. Ghio AJ, Kim C, and Devlin RB (2000) Concentrated ambient air particles induce mild pulmonary inammation in healthy human volunteers. American Journal of Respiratory and Critical Care Medicine 162: 981988. Gilmour PS, Rahman I, Donaldson K, and MacNee W (2003) Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: L533L540. Jimenez LA, Thompson J, Brown DA, et al. (2000) Activation of NF-kappaB by PM(10) occurs via an iron-mediated mechanism in the absence of IkappaB degradation. Toxicology and Applied Pharmacology 166: 101110. Peters A, Dockery DW, Muller JE, and Mittleman MA (2001) Increased particulate air pollution and the triggering of myocardial infarction. Circulation 103: 28102815. Pope CA III, Burnett RT, Thun MJ, et al. (2002) Lung cancer, cardiopulmonary mortality, and long-term exposure to ne particulate air pollution. Journal of the American Medical Association 287: 11321141. Routledge HC, Ayres JG, and Townend JN (2003) Why cardiologists should be interested in air pollution. Heart 89: 13831388. Stone R (2002) Air pollution. Counting the cost of Londons killer smog. Science 298: 21062107. World Health Organization (2004) Health Aspects of Air Pollution. Results from the WHO Project Systematic Review of Health Aspects of Air Pollution in Europe. Copenhagen: World Health Organization.

Further Reading
Brook RD, Brook JR, Urch B, et al. (2002) Inhalation of ne particulate air pollution and ozone causes acute arterial vasoconstriction in healthy adults. Circulation 105: 15341536. Brook RD, Franklin B, Cascio W, et al. (2004) Air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association. Circulation 109: 26552671. Brunekreef B and Holgate ST (2002) Air pollution and health. Lancet 360: 12331242. Donaldson K, Jimenez LA, Rahman I, et al. (2004) Respiratory health effects of ambient air pollution particles: role of reactive species. In: Vallyathan V, Shi X, and Castranova V (eds.) Oxygen/Nitrogen Radicals: Lung Injury and Disease, Lung Biology in Health and Disease (Lenfant C, series ed.), vol. 187. New York: Dekker.

Particulate and Dust Pollution, Inorganic and Organic Compounds

W Birmili, Leibniz Institute for Tropospheric Research, Leipzig, Germany t T Hoffmann, Johannes Gutenberg Universita Mainz, Mainz, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
This article reviews the basic properties and behavior of airborne environmental particles (particulate matter, PM). The basic relevance of environmental PM to health and air quality is

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 111

outlined, and current knowledge on the sources and generation mechanisms of natural and anthropogenic PM is summarized. It is shown that the interaction between the sources, atmospheric transformation processes, and sinks of PM determines the shape of the physical particle size distribution. The general aspects of PM measurement as well as the abundance and behavior of inorganic PM compounds, notably metals, are discussed. The article concludes with an overview of carbonaceous PM and its contribution to particulate pollution, and includes the definitions and properties of the frequently discussed subfractions elemental carbon, black carbon, soot, and organic carbon.

Introduction
Airborne particles in our environment are an integral part of what we breathe in. Besides the uptake of environmental pollutants through nutrition, humans are exposed to airborne particles through the skin and, importantly, through the respiratory tract, that is, nose, thorax, the upper airways, and the lung. The biological effects of harmful pollutants strongly depend on where these pollutants are deposited in the body. In the case of airborne particulates, physical size is the main determinant of where in the airways a particle will actually be deposited. While coarse particles are deposited in the nose and the throat, ne (o1 mm grain size) and ultrane (o0.1 mm grain size) particulates may enter deep into the bronchial and even alveolar regions, where gas exchange takes place with the blood circulation. Depending on their chemical composition and morphological structure, inhaled particles may cause harmful effects such as oxidative stress and inammation, which over time may affect the bodys ability to regenerate and lead to respiratory or cardiovascular disease. Small insoluble particles have been shown to translocate to almost every organ within the human body thereby allowing for complex possibilities of disease. In this article, we introduce the natural and anthropogenic sources, the atmospheric life cycle of airborne particulates, and their properties relevant for health. Current knowledge regarding the abundance and behavior of particulate inorganic and organic species is summarized. A number of conditions found in the atmosphere, ranging from an unpolluted baseline atmosphere to polluted, near-source conditions such as those found in urban areas, are included.

twentieth century; the term was intended to be analogous to that of hydrosol, and was soon adopted for particulates suspended in the atmosphere. The physical size of a particle dictates its behavior in the atmosphere, the length of its life, its climaterelevant effects (such as light scattering or absorption, cloud formation), and its zone of deposition in the respiratory tract. Several conventions originating from public health needs have been established to quantify the entity of particles that are respirable down to a certain depth in the human airways (PM10, PM2.5). The convention PMx includes the ensemble of all particles below a certain diameter in micrometers. Often, the term PMx is used as a synonym for total particle mass concentration below this diameter. Atmosphere scientists, in contrast, tend to distinguish among particles using their mechanism of generation, that is, coarse particles (41 mm), which are mainly mechanically generated, and ne particles (o1 mm), which arise from condensation, agglomeration, and liquid phase processes. The term ultrane typically refers to particles smaller than 0.1 mm. Scientists working in the eld of atmospheric electricity would call ultrane particles intermediate ions. The relatively new term nano-particles (typically up to about 50 nm in diameter), which is used in the innovative elds of material science and molecular medicine, refers to particles and processes where atomic and molecular effects are felt.
Air Quality

Atmospheric Aerosols and Air Pollution

Definitions

The definitions relating to particulate air pollution include particulate matter (PM), suspended particulate matter, dust (typically referring to large visible particles), or atmospheric aerosol. The definition of aerosol dates back to the beginning of the

Since making use of the convenience of domestic re humankind has been exposed to particulate pollution in the form of smoke. The rst reports on urban air pollution date back to Roman times. However, frequently recurring episodes of air pollution over large areas have only been experienced since the industrialization of manufacturing processes and the agglomeration of populations in modern cities, both of which are associated with an increased generation of energy from fossil fuel. The problems caused by air pollution are manifold: odor, deterioration in visibility (dry or wet fog), damage to vegetation and buildings, adverse effects on health, and, on a larger scale, acid rain, overfertilization, and modication of the earths radiative balance. Air pollution events are usually associated with a complex and locally dependent mixture of gaseous and particulate pollutants. Two rather specic types of air pollution mixtures have become known during the twentieth century: London smog and Los Angeles smog. The term London smog (smog smoke fog) refers to a mixture of pollutants notably including sulfur dioxide (SO2) and coarse PM (e.g., y ash)

112 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

Figure 1 Good and poor visibility due to pollution aerosols in Beijing. Photographs courtesy of B Wehner, IfT Leipzig.

that is representative for a period of industrialization where the unregulated combustion of coal played the dominant role in heat and energy generation. Particle and dust sources include a plethora of industrial and domestic coal res for the generation of heat, steam, motion, and electricity. In London, the absence of systematic emission regulations until well into the 1950s meant that the city was regularly subjected to episodes of pollution, which tended to occur under cool weather conditions associated with fog and atmospheric inversion. It was only after the infamous smog event of December 1952, which caused approximately 4000 premature deaths, that legislation enforced a swift technological transition toward the cleaning of exhaust gases and the substitution of coal by natural gas as a heating fuel. While London smog is usually a phenomenon of cool weather, the Los Angeles smog, in contrast, is associated with high levels of ozone (O3) and organic PM at warm temperatures. First noted in California in the 1950s, there was initially considerable confusion about the origin of this air pollution covering the wider area of Los Angeles. By 1960, motor vehicles had been identied as the main sources of pollution, primarily by their emissions of nitrogen oxides and gaseous and particulate hydrocarbons. Nitrogen oxides react photochemically under the impact of sunlight with anthropogenic and biogenic hydrocarbons, thereby producing an excess of ground-level ozone as well as significant amounts of organic PM, giving the atmospheric boundary layer a brownish color. Owing to the complex nature of chemical reactions that lead to Los Angeles smog, abatement measures have proved difcult to establish. Indeed, reductions in nitrogen oxide emissions may under certain circumstances lead to an increase in ozone concentrations rather than to a decrease. Today, variations on air pollution abatement measures are found in many large conurbations, especially in regions where the population, energy consumption,

and volumes of motorized trafc are rapidly increasing, such as parts of China (see Figure 1), South east Asia, and India. Air pollution problems may be aggravated by local topography in cities like Mexico City, Santiago de Chile, Sa o Paulo or Kathmandu, where surrounding mountains or hills frequently block the dispersion of pollutants. Despite the spread of improved technologies for cleaning of exhaust emissions and efcient energy use, it is anticipated that air quality problems will increase on a global level along with economic growth during the decades to come.
Natural Sources of Atmospheric Aerosols

Even if the emission of all anthropogenic sources of PM could be eliminated, environmental concentrations of aerosol particles would not go down to zero because of a number of natural sources that continuously maintain a significant atmospheric aerosol population even in the remotest regions of the globe. It is a generally accepted practice to distinguish particles in terms of their class of generation mechanism. Primary particles or primary PM are those that are emitted directly from source, such as sea spray particles or desert dust. Secondary particles or secondary PM are those that are generated in the atmosphere by nucleation, condensation, or liquid phase processes from gaseous precursor species, such as particulate sulfate or secondary organic aerosol (SOA). The ocean, being the largest part of the earths surface, emits particles in several ways: Sea spray particles emerge from bursting bubbles just above the water surface, a process overwhelmingly controlled by the intensity of breaking waves and thus mainly inuenced by the wind speed. The major fraction of these particles is coarse (41 mm) and composed of the ions contained in the seawater, mainly sodium and chloride. It is noteworthy that sea spray aerosol

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 113

can be a relevant contributor to particle mass (e.g., PM10) in coastal areas. A further important oceanic source is the formation of secondary aerosol through the pathway of gaseous dimethylsulde (DMS) emissions by plankton, followed by atmospheric oxidation to methane sulfonic acid (MSA) and subsequent nucleation or condensation to atmospheric particulates. This process is part of the atmospheric sulfur cycle, and generates particles that are nano-sized at the start and grow to bigger sizes (0.11 mm) within a matter of days or weeks. Likewise, volatile iodine compounds released from marine biota, especially from macroalgae, have been observed to be efcient precursors for particle formation in coastal areas. Hypotheses about the eventual relevance of oceanic organic material for the formation of primary aerosol are currently under investigation. Continental sources of atmospheric particles are even greater. In arid regions, the wind-driven resuspension of soil material, manifested by the formation of dust storms, can be the most important source of particulate mass. Transported to the free troposphere by deep convection, Saharan desert dust, for example, regularly reaches Europe and South America where it causes overcast skies even in the absence of clouds. Enrichments of mineral dust can usually be detected by particle shape (irregular; crystal layers) and analytically by the presence of high levels of elements from the earths crust such as silicon, titanium, and the rare earth elements. Natural sources of carbonaceous and sulfate aerosols include continuous volcanic emissions or the more dramatic events of volcanic eruptions that send up large amounts of y ash into the stratospheric regions from where aerosol layers can spread across the entire globe. Finally, natural forest res, which emit mostly elemental and organic particulate carbon, are another source of natural primary aerosols. Adverse effects on human health through inhalation of smoke, however, are only expected in the immediate vicinity of the re, since atmospheric dilution reduces concentrations relatively fast downwind of a source. Important secondary sources over the continents include the formation of nitrogen-containing inorganic aerosols (especially ammonium and nitrate), and secondary aerosols from biogenic precursors, such as isoprene and terpenes, whose formation rates are especially relevant in the warm season of the year and in forested areas. In terms of their health effects, surprisingly little evidence is available for natural (especially secondary) aerosol particles. Ionic material, as well as the soluble fractions of organic carbon (OC), is readily water soluble and is therefore rapidly excreted in biological organisms. Unless associated with high

acidity, the health effects corresponding to these compounds are therefore assumed to be of minor concern.
Anthropogenic Sources of Atmospheric Aerosols

Mans impact on the environment has already heavily changed the composition of the atmosphere. The most noticeable effects are concerned with general air quality and visibility on a local to regional scale, whereas their effects upon human health, the oxidation capacity of the atmosphere, and the globes radiative balance are more complex and less evident. In contrast to the natural sources, it may be costeffective for a society to put anthropogenic PM sources under regulation mechanisms if there is evidence for a sustained disadvantage to society. Particulate pollution may originate from the wide range of industrial activities, particularly those involving the combustion of fossil fuel (steel smelters, coke generation, electric power generation), and also from waste incinerators, domestic heating, and vehicular and air trafc. Additional exposure results from domestic combustion sources (stoves). In rural valleys, wood burning may be the most relevant source for PM exposure. An important man-made source is the practice of forest clearance by burning. It is important to note that in indoor vehicular, and public transport microenvironments, emissions from vehicular sources can accumulate to the most extreme levels due to poor ventilation. Combustion aerosols are overwhelmingly carbonaceous by mass, including elemental carbon (EC) and OC, some of the latter being present in the form of toxins (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyles (PCBs)). Combustion particles are largely insoluble and thus represent a challenge for the defense mechanisms in the human lung. The anthropogenic combustion aerosols range in size from smoke and y ash particles (41 mm) through die sel soot particulates (20200 nm) to sulfuric and organic combustion nano-particles (o50 nm). Notable sources of coarse particle emissions through anthropogenic land use are wind-blown dust, agricultural aerosols, and opencast mines, which may be of considerable importance for local exposure. Anthropogenic secondary particle emissions include nitrate aerosol resulting from agricultural fertilization and the growth of livestock, sulfate aerosol stemming from the gradual oxidation of sulfur dioxide, and SOAs. Most anthropogenic effects are socially driven in that diurnal and weekly proles are observed in pollutant concentrations. In urban areas total particle mass, particle number, size distribution, and soot concentration develop a clear diurnal cycle linked to

114 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

working and trafc patterns. These idealized anthropogenic cycles are modied by meteorological effects, notably atmospheric stability, which account for varying dispersion conditions. Persistent pollution episodes are more likely to develop during the winter in cities of the Northern hemisphere. This is because in the cold season, anthropogenic emissions rise through the need for heating and atmospheric inversion situations are more frequent. Further meteorologically driven effects include the long-range transport of pollutants in tropospheric air masses, or even in the stratosphere so that particulate pollution can intrude into areas that are remote from their original source.
Lifetime of Atmospheric Aerosols and Particle Modes

The lifetime of atmospheric particles is determined by the balance between their source and removal rates. Since aerosol dynamic processes are, in general, a function of particle size, all lifetime relevant effects will depend on the particle size distribution. The basic processes that generate aerosol particles are mechanical generation (abrasion, resuspension), nucleation (formation of new individual particles exclusively from supersaturated gas phase precursors), condensation (transfer of supersaturated gases onto the surfaces of existing particles), and coagulation (build-up of larger agglomerates from small

particles). An effective pathway to build up secondary ionic particulate mass is through liquid phase processes in activated cloud droplets; this is, for example, the major pathway for particulate sulfate production. The combination of emission and transformation processes leads to most atmospheric particles being chemically mixed. Figure 2 shows two main types of particles in urban air. The main removal processes for airborne particles are dry deposition and wet deposition. Dry deposition (mainly sedimentation) to the surface (ocean, land surface, vegetation) is most relevant for coarse particles. Wet deposition includes in-cloud scavenging (i.e., activation of particles to fog or cloud droplets under supersaturation of water vapor, and subsequent growth to rain droplets) and below-cloud scavenging (i.e., removal by falling rain droplets). Wet deposition is the main removal process for particles between 0.1 and 1 mm in diameter. Depending on the humidity available in the atmosphere wet deposition limits the lifetime of accumulation mode particles to between 3 and 14 days. Coalescence is an important process to decrease the number of small particles (diameter o50 nm). The probability that a particle will coagulate with another particle increases strongly with decreasing particle size. Coalescence is also very efcient in forming agglomerations of particles in or near particle sources, such as in vehicle exhaust. At a given time, an atmospheric particle size distribution is shaped by past and present aerosol

a 100 nm b b a

a 1 m (a) (b) a b

Figure 2 Scanning electron micrographs of particles in urban air. (a) Soot agglomerates made of primary particles B70 nm in diameter. (b) Internally mixed sulfate/soot particle a indicates areas of soot, b indicates areas of ammonium sulfate). Courtesy of M Ebert, TU Darmstadt, Germany.

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 115

Homogeneous nucleation

Combustion sources

Wet deposition

Condensational growth

Cloud + fog activation

Abrasion + resuspension Defragmentation

Sedimentation

1E-3 Diameter (m) Fresh

0.01 Aged Nucleation modes

0.1 Accumulation mode

1 Droplet mode Coarse mode

10 Super coarse mode

100

Figure 3 The size spectrum of atmospheric aerosol particles featuring idealized particle modes and some of the shape-giving processes. Increased spatial and temporal variability is indicated by dot-dash and dashed line.

generation and removal processes, and liquid phase transformations such as fog and cloud processing. Figure 3 shows a multimodal paradigm of the atmospheric particle size distribution, including the various modes corresponding to distinct source and transformation processes.

Atmospheric Aerosols: General Abundance and Inorganic Compounds

Measurement and Chemical Analysis: General Techniques

A number of robust experimental techniques can be applied to determine integral as well as specic properties of PM in near real-time. These methods include: gravimetry (tempered oscillating microbalance; TEOM) to determine total particle mass concentrations; epiphaniometry to determine total particle surface area; aethalometry to determine total light attenuation; nephelometry to determine total optical scattering; optical particle sizers; ion spectrometry (to determine size distributions of charged particles); differential and scanning mobility particle sizers; aerodynamic particle sizers; and condensation nucleus counters. A relatively new but strongly emerging eld is the development and application of real-time mass spectrometric techniques for particle characterization. Two competing instrumental approaches are currently under development: individual particle analysis based on laser detection/laser ionization mass spectrometry, which is especially

suited for the characterization of inorganic aerosol constituents; and thermal desorption/electron ionization mass spectrometry, which appears to be the more promising technique for analysis of the organic aerosol fraction. Several off-line methods allow a more specic morphological analysis, but rst require the capture of particles on a lter or an impaction substrate. These methods include gravimetry (lter weighing), optical microscopy, scanning and transmission electron microscopy, and atomic force microscopy. Importantly, particle samples collected on lters and substrates can be subject to detailed elemental speciation, for example, through proton-induced Xray emission, neutron activation analysis, or atomic absorption spectroscopy. After dissolution of PM in a solvent, or evaporation, further speciation methods can be applied. Hybrid techniques usually involve a species-selective stage, such as ion chromatography, gas chromatography, liquid chromatography, capillary electrophoresis, and an actual detection stage for atoms, ions, or molecules. Sensitive multielement techniques for higher weight atoms include, for example, inductively coupled plasma atomic emission and mass spectrometry. Comprehensive details of the current aerosol measurement and chemical analytical techniques can be found in the Further reading list.
Ambient PM: General Characteristics and Inorganic Ions

Table 1 summarizes observations of integral parameters of ambient PM across Europe. Notably, mass

116 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

Table 1 Typical ambient levels of PM mass concentration and particle number concentration (D 410 nm) in Europe PM10 (mg m3) Remote Rural Urban inuenced Urban background Kerbside 8 15 25 30 40 (335) (832) (2250) (1972) (2876) PM2.5 (mg m3) 5 10 20 22 26 (222) (529) (1232) (1162) (1750) Number (103 cm 3) 1 (0.22) 2.5 (25) 6 (512) 14 (723) 50 (4060)

The values in brackets indicate the range of levels at different observation sites. From Pataud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology, Joint Research Centre.

Rural PM102.5 Urban Kerbside Elemental carbon Organic matter NO3 + NH4 2 SO4 Sea salt Mineral dust Unspecified 0 20 40 60 Mass fraction (%) 80 100

Rural PM2.5 Urban Kerbside

Figure 4 Average chemical composition of ambient PM in Europe. Data from Pataud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology, Joint Research Centre.

and number concentrations increase with increasing proximity to anthropogenic sources. Evidence is available for an overwhelming contribution of vehicular trafc to particle number concentration. Figure 4 shows the bulk chemical composition of PM102.5 (coarse PM) and PM2.5 (ne PM). Coarse PM includes large portions of mineral dust and sea salt, both the result of resuspension processes of material from the earths crust and seawater. A class of unspecied material includes strongly bound metals such as silicates, which are not accessible by standard analytical techniques. Fine PM, in contrast, is made up mainly of carbonaceous compounds and inorganic ions. In a kerbside environment (i.e., immediately next to a road), half of the ne PM may consist of carbonaceous compounds. The strong enrichments of inorganic compounds, such as sulfate, nitrate, and ammonium ions, reect the formation of secondary aerosol over continental areas. Sulfate (SO2 4 ) is known to be formed during liquid phase processes (cloud and fog processes) by oxidation of S(IV) to S(VI) by ozone (O3) and hydrogen peroxide (H2O2); an alternative pathway is direct gas-to-particle conversion of sulfuric acid (H2SO4), which is of relevance in anthropogenic plumes of smoke under pho tochemically active conditions. Nitrate (NO3 ) and ammonium (NH4 ) ions are mainly formed through oxidation of emitted nitrogen oxides (NO, NO2)

and ammonia (NH3), respectively. Particulate nitrate concentrations may be especially high downwind of agricultural areas, and may contribute to up to half of total PM10 mass concentration in rural areas. Owing to their semivolatile character, nitrates tend to partition into either the particulate or the gas phase depending on ambient temperature. Therefore, the concentration of nitrates is considerably higher in the winter than in the summer. The volatility of nitrates requires special care during experimental procedures, notably sampling and sample storage, in order to prevent sampling artifacts.
Characteristics of Metals in Ambient PM

Much of the work on the speciation of inorganic PM constituents has focused on compounds containing oxygen, carbon, nitrogen, sulfur, and halogens. This is due to their role in global warming, acid rain, declining air quality, and tropospheric ozone increase. The speciation of metals has received comparably little attention because of the relatively small concentrations in the atmosphere and the great difculties involved in sampling and analysis. However, it is now known that metallic species exert toxic effects in animals and humans, and metals have recently become the focus of health-related PM research. A recent hypothesis is that transition metals

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 117

Na Mn 101 Ca Sn Fe Ti K Hg Mg Ni Al Se Zn Cd Cu Co Pb Ag Ba Pt

102

Mass fraction

103

104

105

106

Total PM (<7.2 m)

Coarse PM (3 7.2 m)

Fine PM (<0.5 m)

Figure 5 Mass fractions of individual metals in different particle size fractions in the urban PM of Birmingham (UK). Data from Birmili W, Allen AG, Bary F, and Harrison RM. Trace metal concentrations and water solubility in size-fractionated atmospheric particles and inuence of road trafc. Environmental Science and Technology (in press).

such as iron, zinc, and copper may release free radicals in lung uid via the Fenton reaction and cause cellular inammation. The important properties of atmospheric metals are not only their ambient concentrations but also their chemical form, that is, oxidation state, solubility, and ability to form organic complexes. Since particle-bound metals need to dissolve and become free ions in the lung uid, particle solubility in lung uid is a major criterion for the bioavailability and, therefore, toxicity of environmental PM. Alternatively, metals have been exploited as tracers in order to identify individual sources of PM because it has been recognized that the search of reliable tracers may be crucial for developing cost-effective air pollution control strategies. Some elemental combinations have been identied to represent individual anthropogenic sources: oil combustion (vanadium, sulfur, arsenic), coal combustion (aluminum, scandium, sulfur), steel industry (manganese, iron, chromium), cement (calcium), waste incineration (zinc, potassium, lead, antimony), abrasive vehicle emissions (iron, barium, copper, zinc), and soil dust (aluminum, silicon, titanium, scandium, manganese). It is interesting to note that since the almost universal removal of lead from gasoline, there is no longer a significant marker available to identify vehicular exhaust PM through an inorganic multielement analysis. Figure 5 illustrates the relative abundance (mass fraction) of some metal species in urban PM. Besides the more abundant earth alkali metals, most elements occur in small traces only, usually in PM fractions below 1 per 1000. In addition, specic

differences are evident between coarse and ne PM. Metals that occur predominantly in the coarse mode are: manganese and sodium (sea water); and calcium, barium, iron, titanium, and aluminum (typical elements of the earths crust). Metals that occur predominantly in the ne mode are mercury, selenium, cadmium, tin, nickel, lead, and platinum, all of which are related to high-temperature combustion sources. It can be seen that metals reect the fundamentally different generation mechanisms of the different PM size fractions.

Carbonaceous Compounds in Atmospheric Aerosols

The concentration of inorganic carbon, essentially as carbonate, is on average negligible, at least when considering PM2.5. If inorganic carbon occurs in atmospheric PM, it is limited to the form of carbonatecontaining mineral dust, which is part of the coarse mode of PM. In contrast to their naming, EC and OC are not well-dened groups of chemical compounds. In fact, the definitions of EC and OC are strongly related to the commonly used analytical technique. The differentiation between EC and OC is based on thermal treatment of the particle-loaded lter media and measurement of the released carbon compounds as a function of temperature. The amount of carbon released below a certain temperature (typically 340 3501C) is considered to be organic (CxHyOz). The amount released above this arbitrarily chosen default temperature is considered to be EC. The original idea

118 ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution

of this classication was to obtain a measure for the pure element carbon itself, since one characteristic feature of EC is the absorption of visible light, and it is exactly this feature that is vital to understanding direct radiative forcing of atmospheric aerosol particles. Nevertheless, the terms black carbon (BC), graphitic carbon, and soot are nowadays used synonymously with EC. Other measurement techniques for the determination of the EC fraction exist and are based on the light absorption characteristics, notably the light reectance and transmittance through particles collected on a lter, and the in situ heating of particles in an air stream with photoacoustic detection. Unfortunately, none of these techniques can provide reliable information on the EC content; for example, certain higher molecular weight organic compounds can undergo charring to form pyrolytically generated EC during the thermal treatment of the aerosol samples. Not only are the names of the two classes misleading in terms of the chemical composition, but also the different carbon analysis methods do not consistently provide comparable EC or OC concentrations, as demonstrated in many comparisons between methods used and among laboratories. The degree of equivalence depends on the nature of the sample as well as on the analytical method and protocol. Unfortunately, different protocols can be based on the same method, but potentially important differences that might affect the results have not been adequately documented. Therefore, whenever EC, BC, or OC concentrations are taken from the literature, these implicit uncertainties and inaccuracies have to be taken into account.
Elemental Carbon, Black Carbon, and Soot

their formation (substance, temperature, oxygen content in ame, etc.). Primary soot particles (from diesel engines or oil burning) have diameters between 10 and 50 nm and a spherical onion-layered structure, but usually they agglomerate rapidly during the combustion process forming chain aggregates measuring from 100 nm to about 1 mm in length (Figure 2). Black carbon and soot also contain various amounts of hydrogen and other atoms as well as some extractable organic matter like PAHs or alkanes. The organic matter may be adsorbed onto the soot surface or even enclosed inside the primary soot particles. In fact, since the carbon:hydrogen ratio approaches innity within the homologous series of PAHs, the transition between organic and EC is smooth.
Organic Carbon

As mentioned above, EC (also called graphitic carbon, black carbon, or soot), is ubiquitous in combustion emissions. It is predominantly found in diesel exhaust, but it is emitted from all combustion sources such as biomass, coal, and oil burning. EC is neither pure nor highly structured carbon because real EC, such as diamond and graphite, rarely occurs as pure particles unmixed with other atmospheric constituents. The combustion-generated fraction of primary carbonaceous aerosol, commonly termed soot, can be regarded as a form of impure EC consisting of plains of hexagonal arrays of carbon atoms, similar to graphites crystalline structure. Unlike graphite these plains can be curved forming spherical bodies. If the number of graphitic layers is very small, carbon might be called amorphous; if the number is high, it might be called graphitic carbon. The size of soot particles depends on the history of

Although organic compounds typically account for 1070% of the total dry ne particle mass in the atmosphere, their concentrations, composition, and the processes that control their formation and transformation in the atmosphere are not well understood, particularly in relation to the other major ne particle constituents, such as sulfate and nitrate compounds. While the classication between EC and OC was an operational one, the further subdivision of OC is based on the different mechanisms of formation. Primary organic aerosol components include all compounds, which are released directly as PM into the atmosphere. They comprise particle phase OC from anthropogenic combustion sources (biomass burning, use of fossil fuels, and mechanically formed aerosol particles from road dusts or tire debris). In very densely populated environments even the direct input of organic particulates from domestic activities like cooking or cigarette smoking can be significant. In contrast, the natural contributions to the primary organic aerosol are less well understood. Field measurements of the content of macromolecular organic compounds in airborne particles indicate that biogenic sources play a significant role. For example, cellulose and humic-like substances have been found in atmospheric aerosol particles, pointing to a direct input of OC from vegetation, probably originating from plant surfaces. Furthermore, pollen, airborne bacteria, viruses, and spores have to be considered as contributors to the organic aerosol fraction, although some of these primary biological aerosol particles only contribute to the coarse and super-coarse particle mode. However, a detailed quantication of the different natural contributions to the atmospheric particle phase is still missing, especially due to the complex chemical composition of biological matrices.

ENVIRONMENTAL POLLUTANTS / Particulate and Dust Pollution 119

In addition, organic aerosol material is formed in the troposphere by the mass transfer of low vapor pressure substances to the aerosol phase. The condensable species are formed in the gas phase by the reaction of volatile organic compounds (VOCs) with the principal atmospheric oxidizing agents O3, OH, and NO3 radicals. To distinguish this fraction of tropospheric aerosols from the direct input of particulate organics into the atmosphere as described above, it is specied as SOA. VOCs are emitted into the troposphere from anthropogenic and biogenic sources. Anthropogenic sources comprise organics such as alkanes, alkenes, aromatics, and carbonyls, while biogenic sources include organics such as isoprene, monoterpenes, and sesquiterpenes, as well as a series of oxygen-containing compounds. Since the volatility of the oxidation products is one of the most important parameters that determines the signicance of precursor gases in terms of their aerosolforming potential, hydrocarbons with a higher number of carbon atoms in particular will contribute to SOAs under atmospheric conditions. Thus, natural SOA formation is believed to result mainly from the oxidation of C10- and C15-terpenoids, although recent investigations showed that isoprene, a biogenic C5 hydrocarbon that is released in huge amounts into the atmosphere, can also form condensable products. Among the anthropogenic precursor gases, aromatics have been shown to dominate the process of SOA formation, suggesting

that anthropogenic SOA formation in an urban airshed can be modeled on the basis of the aromatic content of the complex hydrocarbon mixture. However, based on the current knowledge about precursor emissions and the aerosol formation potential of the individual VOCs, SOA formation from natural precursors dominates the production of SOAs in the troposphere on a global scale. Interestingly, a significant anthropogenic impact on natural SOA formation is expected, resulting from the inuence of the oxidation pathway of the biogenic VOCs (reaction with O3, OH, or NO3) on the aerosol formation potential of the biogenics. Besides the direct transfer of low volatile organics into the tropospheric particle phase by gas-to-particle conversion, atmospheric processes involving heterogeneous processes are suspected to contribute to SOAs. Partially oxidized organic gases such as alcohols, aldehydes, or peroxides, which are too volatile to condense directly onto existing particles, might be absorbed into water droplets in clouds and fogs and, after undergoing chemical reactions, result in the addition of organic matter when the droplets evaporate. Another recently discovered pathway for SOA component formation involves polymerization reactions of small-chain precursor molecules, such as C2 or C3 aldehydes, in the organic particle phase; these were formerly believed not to be involved in aerosol formation. In fact, this build up of higher molecular weight compounds might also explain part

Primary OC >57 Secondary OC 11 46

Gas/particle conversion 240 210 18 35

22

12

Vegetation Biomass burning Fossil fuels

Figure 6 Estimation of global primary and secondary organic particle contributions.

120 ENVIRONMENTAL POLLUTANTS / Radon

of what is still dened as primary OC (i.e., humiclike substances). What is the significance of carbonaceous compounds from the point of view of health? The few epidemiological studies that have addressed this important question specifically suggest that combustion sources are particularly important for health. Toxicological studies have also indicated that primary combustion-derived particles have a higher toxic potential. These particles are often rich in organic compounds, which are thought to be involved in the adverse health effects of PM2.5. In addition, many organic compounds are electrophilic and reactive molecules, which can cause oxidative damage to proteins and DNA and induce cytokines and inammation. However, at present it is not possible to quantify the effect of organic compounds from the different sources on health caused by exposure to ambient PM. Based on the currently available gures, Figure 6 attempts to summarize the estimated global contributions to the organic fraction of tropospheric aerosols. It should be noted that the uncertainties regarding these gures are still very great and that some potential formation pathways, such as chemical processing in the liquid phase or polymerization of gas phase precursors, are not included. Furthermore, the specic natural sources for organic aerosol particles, such as primary organic contributions from terrestrial or marine sources, are still poorly understood.
See also: Environmental Pollutants: Overview; Diesel Exhaust Particles; Particulate Matter, Ultrane Particles.

Putaud JP, van Dingenen R, Baltensperger U, et al. (2003) A European Aerosol Phenomenology. Joint Research Centre. Seinfeld JH and Pandis SN (1997) Atmospheric Chemistry and Physics. New York: Wiley Interscience. Spokes LJ and Jickells TD (2002) Speciation of metals in the atmosphere. In: Ure AM and Davidson CM (eds.) Chemical Speciation in the Environment, 2nd edn., pp. 161187. Oxford: Blackwell. Spurny KR (ed.) (1999) Analytical Chemistry of Aerosols. Boca Raton, FL: CRC Press. Spurny KR (ed.) (2000) Aerosol Chemical Processes in the Environment. Boca Raton, FL: CRC Press. The Mayor of London (2002) 50 Years On: The Struggle for Air Quality in London since the Great Smog of December 1952. Greater London Authority.

Relevant Website
http://ies.jrc.cec.eu.int European Commission DirectorateGeneral Joint Research Center which monitors air pollution from a cruise ship.

Radon
A S Rosario and H-E Wichmann, GSF Institute of Epidemiology, Neuherberg, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Radon gas is the most important source of natural radioactivity. It is a radioactive noble gas that emanates from the ground. Radon concentrations in outdoor air are low, but in underground mines or inside houses radon concentrations can reach high levels. The biologic effects of radon are mostly due to its decay products. These are solid heavy metal isotopes that are themselves radioactive. They can be deposited on the bronchial epithelium, where they emit ionizing radiation in the form of aparticles. Alpha radiation can effectively cause permanent damage to the DNA and can eventually lead to cancer. Epidemiological studies have given conclusive evidence that radon is carcinogenic to humans and can cause lung cancer. The rst studies were performed in underground miners exposed to high levels of radon. Later studies performed in the general population showed that radon can cause lung cancer even at the lower concentrations found in houses. This indoor radon exposure is currently estimated to contribute to 510% of lung cancer deaths.

Further Reading
Birmili W, Allen AG, Bary F, and Harrison RM. Trace metal concentrations and water solubility in size-fractionated atmospheric particles and inuence of road trafc. Environmental Science and Technology (in press). Brasseur GP, Prinn RG, and Pszenny AP (eds.) (2003) Atmospheric Chemistry in a Changing World. Berlin: Springer. Brimblecombe P and Maynard RL (eds.) (2000) Urban Atmosphere and its Effect. London: Imperial College Press. Brown LM, Collings N, Harrison RM, Maynard AD, and Maynard RL (eds.) (2003) Ultrane Particles in the Atmosphere. London: Imperial College Press. Davidson CI (2000) Human exposure to toxic metals. In: Rubin ES (ed.) Introduction to Engineering and the Environment, pp. 402433. New York: McGraw Hill. Friedlander SK (2000) Smoke, Dust, and Haze. Fundamentals of Aerosol Dynamics, 2nd edn. Oxford: Oxford University Press. Gelencser A (2004) Carbonaceous Aerosol. Dordrecht: Springer. Hinds WC (1999) Aerosol Technology, 2nd edn. New York: John Wiley & Sons Inc. IPCC (2001) Intergovernmental Panel of Climate Change, Third Assessment Report. Cambridge: Cambridge University Press. Kulmala M, et al. (2004) Formation and growth rates of ultrane atmospheric particles: a review of observations. Journal of Aerosol Science 35: 143176.

Structure and Physicochemical Properties

Radon is a naturally occurring, radioactive noble gas. It is chemically inert, colorless, odorless, and soluble in water. There are three naturally occurring radon isotopes: radon-222 (radon), radon-220 (thoron), and radon-219 (actinon), of which radon-222 is the most important. It is the only gaseous member

ENVIRONMENTAL POLLUTANTS / Radon 121

Uranium-238 4500 million years Radon-222 3.82 days

Atomic mass

Radium-226 1600 years

Radon-222 3.82 days

Polonium-218 3.05 min

Lead-214 26.8 min

Figure 1 Natural decay chain of uranium-238.

of the radioactive decay chain of uranium-238 (Figure 1). Uranium is ubiquitously present in the earths crust, albeit in variable concentrations. Radon decays with a half-life of 3.8 days, emitting ionizing radiation in the form of a-particles (helium nuclei). The biologic effects of radon are mainly due to its radioactive decay products (also called radon progeny or radon daughters), in particular the shortlived decay products with a half-life of up to 30 min, such as the a-emitters polonium-218 and polonium214. These are solid heavy metal isotopes, which mostly attach to dust particles or form clusters with water molecules. Radon gas concentrations are usually measured in becquerels (Bq) per cubic meter (1 Bq m 3 corresponds to one radioactive decay per second and cubic meter of air). Another unit in current use is picoCurie (pCi) per liter (1 pCi l1 37 Bq m 3).

Source and Exposure

Radon gas is the most important source of natural radioactivity, accounting for about half of the effective radiation dose received from natural sources in the

.. .

Half-life Type of radioactive decay

Short-lived radon progeny

Bismuth-214 19.9 min

Polonium-214 164 s

Lead-210 22.3 years

Bismuth-210 5.0 days

Polonium-210 138.4 days

Lead-206 (stable)

worldwide average. It is formed in rocks and soils through the radioactive decay of radium-226, which itself is a decay product of uranium-238. As a gas, it can ascend and diffuse from the soil into the air. Alternatively, it can dissolve in ground water. The amount of radon released from the soil depends on its radium content, but also on factors affecting radon transport such as underground air and water ow and the porosity and permeability of the soil. In outdoor air, radon concentrations are usually low because of its dilution with atmospheric air. In underground mines and caves or inside buildings, however, radon concentrations can build up.
Occupational Radon Exposure: Underground Miners and Others

Underground miners, especially uranium miners, can be subject to very high radon concentrations. Other potentially exposed workplaces are radon spas or underground caves and mines (e.g., for tourist guides). Above ground, employees in water supply facilities can be exposed to high radon concentrations. Radon concentrations in typical workplaces (factories,

122 ENVIRONMENTAL POLLUTANTS / Radon

ofces, schools, etc.) tend to be lower than in the home, although the data are sparse and there can be exceptions to the rule.
Indoor Radon Exposure

For the general population, radon gas concentrations in houses pose a potential health hazard, although the concentrations are usually considerably lower than those encountered in occupationally exposed groups. Apart from the geological conditions described above, the inux of radon into houses is inuenced by meteorological factors such as wind speed and indooroutdoor differences in barometric pressure and temperature. Indoor radon concentrations are also determined by structural characteristics of the house and by ventilation habits (Table 1). Radon levels are generally highest in the basement and lower in the higher oors. Concentrations tend to be higher in single-family homes and lower in apartment blocks. Urban areas are likely to have lower radon levels than rural areas. The radon concentration inside a house is subject to diurnal and seasonal variation (usually higher at night and in the winter months). Indoor radon concentrations within a country can vary by several orders of magnitude. Variation within a country is considerably larger than variation among countries. The worldwide average indoor radon concentration is estimated to be 4050 Bq m 3, but up to several thousands or even tens of thousands of Bq m 3 have been reported in many countries.
Table 1 Factors contributing to increased indoor radon concentrationsa Geological factors High uranium/radium content of the soil, e.g., in granite rocks High porosity and/or permeability of the soil Tendency for higher concentrations in mountainous regions Presence of cracks and ssures in the basement oor or walls Insufciently sealed entrance of tubes or cables into the basement Houses without basement or without separation of the living area from the basement Living area located in a oor near the basement Single-family home Insulation windows and similar sealing efforts to enhance energy efciency Low ventilation rate

Radon concentrations typically show a skewed distribution (Figure 2). Most individuals in a population are exposed to low radon concentrations, while a small number of people are exposed to high or very high concentrations. Soil is generally the main source of indoor radon exposure. Other less important sources are building materials, radon dissolved in tap water (especially from private wells), or radon contained in coal or natural gas used for heating and cooking.

Clinical Consequences
Radon is of public health concern as it has been found to increase the risk of lung cancer. An excess of lung diseases among miners in the German Ore Mountains was observed as early as the sixteenth century by Paracelsus, and in 1913 a possible etiologic link between radon exposure and lung cancer was postulated for the rst time. Radon has been classied as carcinogenic to humans by the International Agency for Research on Cancer (IARC) in 1988 and 2001.
Mechanisms of Radon-Induced Carcinogenesis

House characteristics

Behavior
a

This list can only serve as a rough guide.

Most of the radon gas itself is exhaled again after inhalation, but its solid decay products can be deposited in the bronchi and the lung. These decay products undergo further radioactive decay on the surface of the bronchial epithelium before they can be removed from the airways by clearance mechanisms. They emit a-radiation, which is known to be more effective than other types of radiation in terms of biological damage. Whereas high doses of ionizing radiation can lead to acute effects, low doses lead to stochastic effects, that is, they increase the probability of mutations, either through direct DNA damage or through the formation of free radicals and other oxidative agents. Radiation typically causes large deletions and rearrangements, rather than simple base-pair exchanges. Alpha radiation in particular tends to cause more complex, clustered DNA damage that is more difcult for the cell to repair. If not repaired, it can be one of the critical events in the multistep process eventually leading to cancer. Radiation can affect neighboring cells (probably via cellcell communication) or descendant cells that have not themselves been irradiated. On the other hand, small doses of radiation might render the cell more resistant to subsequent carcinogenic stimuli by activating repair enzymes. There is no clear evidence that radiation causes mutations at specic sites of the genome.

ENVIRONMENTAL POLLUTANTS / Radon 123

100

200

300 m3)

400

500

Radon level (Bq

Figure 2 Distribution of indoor radon concentrations in West Germany. Ten values between 500 and 1111 Bq m 3 not shown. Data from Schmier H and Wicke A (1985) Results from a survey of indoor exposure in the Federal Republic of Germany. Science of the Total Environment 45: 307310.

In various mammalian cell systems, radon and its progeny were found to cause malignant transformation of cells. In cytogenetic studies both in vivo and in vitro, radon and its progeny have led to chromosome aberrations and increased cell proliferation in a dose-dependent fashion. An increased number of chromosome aberrations have also been found in humans exposed to high indoor radon concentrations. Experiments in rats have shown that radon inhalation can lead to the formation of tumors in the respiratory tract.
Epidemiological Studies in Underground Miners

Epidemiological Studies on Indoor Radon and Lung Cancer

The epidemiological evidence regarding the carcinogenicity of radon in humans was rst assembled in studies of large cohorts of underground miners. In a combined analysis of 11 studies performed worldwide, nearly 68 000 miners were evaluated. A statistically significant relationship between their cumulative radon progeny exposure and the subsequent lung cancer risk was found. The relationship was modeled assuming that the relative risk (the ratio of the lung cancer risk of an exposed individual to the risk of an unexposed or low-exposed individual) for lung cancer increases linearly with increasing radon exposure. This assumption was strengthened by the experimental observation that the traversal of a single a-particle through a cell has the potential to cause permanent changes to the DNA, although the existence of a threshold could not be ruled out completely.

The evidence found in the studies carried out in miners led to concern about the potential risk of elevated radon concentrations in houses. Consequently, a series of population-based (mostly case control) studies were carried out. Not all of these studies showed an association between indoor radon concentrations and lung cancer. Methodological differences among the studies partly explain the different results. The main problem, however, lies in the insufcient sample size of the single studies. Although radon is an important risk factor for lung cancer, this risk is quite small as only a small proportion of the population experiences high radon concentrations; hence, sample sizes in studies of radon exposure need to be large. For a precise quantitative risk assessment, the data from the single studies need to be combined. Such efforts have been undertaken both in North America and Europe. The results of the European analysis show a linear doseresponse relationship with no evidence of a threshold. Relative lung cancer risk increased by 8% per 100 Bq m 3 increase in indoor radon concentrations (95% condence interval: 316%). In the North American analysis, relative risk increased by 11% per 100 Bq m 3 (95% condence interval 028%). It is crucial that smoking behavior is taken into account in such studies, since smoking is a very strong risk factor for lung cancer and there is an association

124 ENVIRONMENTAL POLLUTANTS / Radon

between radon and smoking; in rural areas, smoking tends to be less frequent, while radon exposures tend to be higher. Therefore, adjustment for smoking increases the risk estimates for radon in most studies. A major problem for the casecontrol studies is the fact that cancer develops over a long time period; consequently, the level of radon exposure in houses inhabited during the previous decades has to be estimated based on measurements taken in the present. This uncertainty generally leads to risk estimates that are biased downwards. In the collaborative European analysis, accounting for these uncertainties doubled the risk estimate, that is, the linear increase in relative risk was estimated to be 16% per 100 Bq m 3 (95% condence interval: 531%). Radon seems to be more likely to cause small-cell carcinomas than other histological subtypes of lung cancer. Similarly, the risk estimate in the North American analysis increased when restrictions regarding exposure assessment were taken into account.
Lung Cancer Cases Attributable to Radon; Inuence of Smoking

Under normal conditions, frequent and thorough ventilation helps to reduce radon levels. In houses with high radon concentrations, simple and effective steps can be taken to reduce radon exposure (e.g., improved sealing of the basement or installation of ventilation systems). Radon concentrations can vary substantially even among nearby houses, and so a definite conclusion about the radon level in a given house can only be derived from direct measurement. Measurements should preferably be taken over a whole year to cancel out seasonal effects. Information on regions that are likely to suffer from higher radon concentrations, laboratories that offer radon detectors, and how to reduce radon concentrations in buildings is usually available from national radiation protection institutions. Reducing the number of smokers in the population and enabling smokers to quit will considerably diminish the number of radon-related lung cancers. As far as treatment and prognosis are concerned, radiation-induced lung cancers do not differ from other lung cancers.
See also: Environmental Pollutants: Tumors, Malignant: Overview. Overview.

Radon is considered the second most important single agent that can cause lung cancer, after smoking. Whereas smoking causes about 90% of all lung cancer cases in male populations, the proportion of cases attributable to radon is considerably lower, in the order of 510%, depending, among others, on the distribution of indoor radon concentrations in the country of interest. Only a small part of these cases occur at high radon concentrations, since the number of people exposed to very high concentrations is usually quite low. A substantial number of cases arise in houses with moderate radon concentrations. Current evidence suggests that the combined effect of smoking and radon on lung cancer risk is multiplicative or slightly less than multiplicative. This means that smoking greatly increases the radon-related lung cancer risk. Because smoking is such a strong risk factor for lung cancer and because it is still a widespread habit in the population, the absolute number of radon-induced lung cancer cases is considerably higher among smokers than among nonsmokers.

Further Reading
Burkart W (1989) Radiation biology of the lung. Science of the Total Environment 89: 1230. Darby S, Hill D, Auvinen A, et al. (2005) Radon in homes and risk of lung cancer: collaborative analysis of individual data from 13 European casecontrol studies. British Medical Journal 330: 223227. IARC International Agency for Research on Cancer (2001) Ionizing Radiation, Part 2: Some Internally Deposited Radionuclides. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 78. Lyon: IARC Press. ICRP International Commission for Radiation Protection (1987) Lung Cancer Risk from Indoor Exposure to Radon Daughters. ICRP Publication No. 60. New York: Pergamon. Jostes RF (1996) Genetic, cytogenetic, and carcinogenic effects of radon: a review. Mutation Research 340: 125139. Krewski D, Lubin JH, Zielinski JM, et al. (2005) A combined analysis of North American casecontrol studies of residential radon and lung cancer. Epidemiology 16: 137145. Little JB (2000) Radiation carcinogenesis. Carcinogenesis 21: 397404. Lubin JH, Boice JD, Edling C, et al. (1995) Lung cancer in radonexposed miners and the estimation of risk from indoor exposure. Journal of the National Cancer Institute 87: 817827. Lubin JH, Toma s ek L, Edling C, et al. (1997) Estimating lung cancer mortality from residential radon using data for low exposures of miners. Radiation Research 147: 126134. Lubin JH, Wang ZY, Boice JD, et al. (2004) Risk of lung cancer and residential radon in China: pooled results of two studies. International Journal of Cancer 109: 132137. NAS National Academy of Sciences (1999) Health Effects of Exposure to Radon (BEIR VI Report). Washington, DC: National Academy Press. Samet JM and Eradze GR (2000) Radon and lung cancer risk: taking stock at the millenium. Environmental Health Perspectives 108(supplement 4): 635641.

Management and Treatment

Increased indoor radon concentrations can and should be reduced. Ofcial action levels vary from country to country. The European Commission recommends that 400 Bq m 3 should not be exceeded in existing houses and 200 Bq m 3 in newly built houses. The US Environmental Protection Agency (EPA) sets the action level at 148 Bq m 3.

EOTAXINS 125
Schmier H and Wicke A (1985) Results from a survey of indoor exposure in the Federal Republic of Germany. Science of the Total Environment 45: 307310. UNSCEAR (United Nations Scientic Committee on the Effects of Atomic Radiation) (2000) Sources and Effects of Ionizing Radiation. UNSCEAR 2000 Report to the General Assembly. vol. I: Sources; vol. II: Effects. Wichmann HE, Rosario AS, Heid IM, et al. (2005) Increased lung cancer risk due to residential radon in a pooled and extended analysis of studies in Germany. Health Physics 88: 7179.

Relevant Websites
http://www.epa.gov US Environmental Protection Agency: y to protect human health and the environment. http://www.unscear.org This website includes an overview of average indoor radon concentrations in several countries. http://www.nap.edu The National Academy Press publishes on agriculture, behavioral science, biology, chemistry, computer science, etc.

EOTAXINS
I Sabroe, University of Shefeld, Shefeld, UK T J Williams and J E Pease, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The Eotaxins are small proteins secreted during allergic reactions that have the ability to attract eosinophils and certain other cell types from the blood. Three of these CC-chemokines have been described, Eotaxin-1, -2, and -3. They induce leukocyte recruitment by acting on a receptor, CCR3, which is highly expressed on the surface of eosinophils and also on basophils, mast cells, and a subpopulation of the Th2 lymphocytes. Neutralization of Eotaxin-1 by monoclonal antibodies has been shown to suppress eosinophil accumulation during allergic reactions in man and animal models. Small molecule antagonists are under development with the aim of blocking eosinophil recruitment and, as a consequence, inhibiting the lung dysfunction and tissue remodeling associated with eosinophil activation.

terms of amino acid sequence, they were named eotaxin-2 and eotaxin-3 on the basis of their functional homology. A recently adopted systematic nomenclature has resulted in these molecules being renamed CCL11, CCL24, and CCL26. Subsequent work has implicated the eotaxin/CCR3 axis in many aspects of leukocyte trafcking in allergic inammation, and has provided powerful arguments for targeting this pathway in the treatment of diseases such as asthma and allergic rhinitis.

Structure
The delity of the three eotaxins for their receptor CCR3 is curious, as at the primary sequence level, the mature proteins share only 3037% identity. Furthermore, the eotaxins are not all encoded on the same chromosome: the gene for CCL11 is on chromosome 17 whereas the genes for CCL24 and CCL26 are on chromosome 11. Like most chemokines described to date, the three eotaxins share a common protein fold, the Greek-key motif, consisting of three antiparallel b-pleated sheets, overlaid by a C-terminal a-helix (Figure 1(a)). Truncation studies of the eotaxins imply that the N-terminus of each chemokine is important for the activation of CCR3 and, notably, these domains contain considerable sequence divergence between the molecules. This may explain their distinct rank orders of potency/efcacy at CCR3 as measured by assays of eosinophil chemotaxis or shape change, with CCL11 being the more potent and efcacious ligand. Local pH and ionic strength have been reported to strongly inuence the ability of CCL11 to bind to CCR3, suggesting that interactions between these molecules involve charged amino acid residues, and posttranslational modications such as glycosylation may affect availability and receptor binding of the eotaxins. The interaction of CCL11 with CCR3 at the molecular level has been

Introduction
The eotaxins comprise a family of three molecules related by their biological activity. Eotaxin was rst identied in guinea pigs, where the biological activity responsible for pulmonary eosinophil recruitment in response to allergen challenge was shown to be a protein, and was then revealed by microsequencing to be a CC chemokine. This molecule induced a relatively selective recruitment of eosinophils (the rst chemokine found to do so) and was thus named according to its biological function. The human homolog of the guinea pig molecule was identied and shown also to mediate eosinophil chemotaxis. This was followed by the identication of the receptor for eotaxin, which was named CC chemokine receptor-3 (CCR3). Two further CC chemokines were subsequently identied in man that also stimulate chemotaxis of eosinophils via CCR3. Though they are quite dissimilar to eotaxin (eotaxin-1) in

126 EOTAXINS

Figure 1 (a) A cartoon representation of eotaxin-1, illustrating the Greek-key structural motif typical of chemokines. (b) A surface model of eotaxin-1, with the basic residues lining the extended groove and postulated to bind the acidic CCR3 N-terminus more darkly shaded. Both models were produced by the package PyMOL using the solution structure of Crump et al. as template (le 1EOT available from the protein data bank).

examined by the use of chimeric receptor constructs and conforms to the two-site model of receptor activation in which a high-afnity binding site within the receptor N-terminus tethers the chemokine, facilitating delivery of the ligand to a second, lower afnity site composed of the remaining extracellular loops. This latter interaction is necessary for signal transduction in response to CCL11 and correlates with recent data from studies employing a soluble protein mimic that have shown that the juxtaposition of both the N-terminus and third extracellular loop of CCR3 are required for the optimal binding of this chemokine. Residues within the N-loop of CCL11 have been shown to play a role in binding to CCR3, and binding studies utilizing a CCR3 N-terminal peptide reported that it bound within an extended groove in the CCL11 surface (Figure 1(b)).

Regulation of Production
In general, allergic inammation is associated with an induction of CCL11, and probably CCL24 and CCL26. These proteins may be directly released from early response cells (e.g., mast cells), and then synthesis upregulated in a wide range of tissue cells (e.g., epithelia, endothelial cells, airway smooth muscle) and leukocytes (including eosinophils and macrophages) as a result of an inammatory cascade. Th2 cells are able to regulate the inltration of eosinophils in allergic reactions by secreting interleukin-4 (IL-4) and IL-13, which switch on the genes for the eotaxins in others cells in the tissue. In airway smooth muscle, IL-9 can also induce CCL11 generation. Synergistic induction of CCL11 generation by

Th2 cytokines and other proinammatory molecules such as IL-1b has also been demonstrated in airway smooth muscle cells. There is evidence for separate regulation of the production of eotaxins, since intradermal allergen challenge in man results in phased induction of CCL11 and CCL24. Additionally, CCL24 is induced from monocytes by lipopolysaccharide (LPS) stimulation, whilst CCL11 is not, and since allergen exposure often occurs together with contaminants such as LPS, this provides a mechanism by which innate immunity may amplify allergic inammation. (Further complicating this picture is a change in phenotype as monocytes mature, such that IL-4, but not LPS, induces CCL24 generation from macrophages.) The opportunities for phased regulation of CCL11, CCL24, and CCL26 generation provides a potential mechanism to sequentially regulate trafcking of cells expressing CCR3.

Biological Function
Eotaxins are chemokines (chemoattractant cytokines) and, as such, their principal actions are to facilitate the recruitment of leukocytes to inammatory sites. The eotaxin family were described through their apparently selective recruitment of eosinophils. In humans and animals, intradermal injection of CCL11 and CCL24 has been shown to recruit eosinophils and, likewise, nasal administration also recruits eosinophils into nasal lavage uid. It remains unclear whether these molecules truly serve individual nonredundant functions or whether they have subtly different functions, as eotaxins can show

EOTAXINS 127

distinct patterns of cellular expression and can be differentially regulated by different cytokines. Studies of allergic inammation in human skin induced by intradermal injection of allergen have suggested that CCL11 generation correlates with early eosinophil recruitment, whilst CCL24 generation may be more associated with later phases of tissue eosinophilia. CCL11 also plays roles in constitutive trafcking of eosinophils to noninamed mucosal sites (including the gastrointestinal tract). In addition to mediating the trafcking of eosinophils to tissues, the eotaxins are also able to mobilize eosinophils from the bone marrow (facilitating local recruitment at sites of inammation by increasing systemic supply). Immature cells can also be mobilized: CCR3 has been demonstrated on CD34 stem cells and committed eosinophil progenitors. Eotaxins can also activate eosinophils, inducing cytokine generation and modulating their life span. In addition to substantial roles in the regulation of eosinophil recruitment and activation, it has become apparent that the eotaxin receptor, CCR3, is expressed on a wider range of cells (Figure 2). Regulated expression on human mast cells, basophils, and subsets of macrophages and Th2-type T cells suggests that eotaxins may play important roles in the

trafcking of these cells as well. The ability of eotaxins to mediate recruitment of so many cell types involved in allergic inammation, from Th2-type T cells to basophils, mast cells, and eosinophils, with amplication of generation of eotaxins by Th2-type cells, afrms that eotaxins are likely to play major roles in respiratory diseases.

Receptors
The eotaxins exert their common actions through their ability to bind to CCR3. In common with other chemokine receptors, CCR3 couples to G proteins (principally of the Gai subtype), to mediate activation of signaling pathways including [Ca2 ]i ux, MAP kinases, PI-3 kinases, and protein kinase C. CCR3 is very promiscuous and binds several other chemokines in addition to the three eotaxins, including members of the monocyte chemoattractant protein (MCP) family. There is some evidence that CCR3 ligands show varying abilities to activate CCR3, and there may be temporal roles for different CCR3 ligands in eosinophilic inammation. Therefore, although therapeutic antibodies against CCL11 have been developed, targeting the eotaxin/CCR3 axis in disease may be more effectively achieved using CCR3

AM

EC

MC

ASM

Eotaxins Eotaxin 3

Inhibition of CCR1, 2, and 5 may alter T cell recruitment MC Eos Th2-type T cells CCR3-mediated actions
Figure 2 Eotaxins can be generated by a range of cell types within the lung, including macrophages, epithelia, mast cells, and airway smooth muscle. Their principal action is probably to cause the recruitment of eosinophils, but they also act on other CCR3-expressing cells including mast cells and Th2-type T cells. Partial agonist and antagonist actions at other receptors may also result in alterations of patterns of leukocyte recruitment. AM, alveolar macrophage; EC, epithelial cells; MC, mast cell; ASM, airway smooth muscle; Eos, eosinophil.

Eotaxin Partial agonist of CCR2: may recruit monocytes

128 EOTAXINS

small molecule antagonists, which are in clinical development. Intriguingly, the eotaxins show varying abilities to antagonize or partially stimulate other chemokine receptors: CCL11 is a weak agonist at CCR2, whilst CCL26 can antagonize CCR1, CCR2, and CCR5 (possibly inuencing in vivo recruitment of monocytes and T cell subsets). Meanwhile, ligands of the Th1-associated chemokine receptor, CXCR3, can antagonize signaling at CCR3. The summated actions of eotaxins may therefore be a subtle blend of actions on more than one chemokine receptor, and Th1-associated chemokines may exert opposing actions on the Th2-favored CCR3 axis.

date. The failure to deplete lung tissue cells means that these studies have been inconclusive with respect to the involvement of eosinophils in bronchial hyperreactivity in man. More recent studies of the results of anti-IL-5 treatment in man indicate that the eosinophil may be more important in tissue remodeling than in hyperreactivity. Eotaxin expression is prominent in allergic diseases and the eotaxin receptor CCR3 is expressed by key cell types involved in the process. Clinical trials of small molecule CCR3 antagonists, currently under development, will provide valuable information on the importance of the eotaxins in the disease process and hold out the promise of future selective therapy for allergy and asthma.
See also: Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Asthma: Overview. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17); TECK (CCL25). G-Protein-Coupled Receptors. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Monocytes.

Eotaxins in Respiratory Diseases

Eotaxins, and their major receptor CCR3, are expressed wherever allergic inammation is present in the human respiratory tract, and levels of expression show correlations with degree of severity. Upregulation of eotaxins in allergic inammation in the nose and lung are consistent with an important role in lung disease, and these molecules may play additional roles in the regulation of sensitization. The roles of eotaxins in lung diseases have been both fairly and unfairly caught up in a current, wideranging debate on the role of eosinophils in asthma. In animal models, eotaxin has incontrovertibly important roles in eosinophil, and to some extent Th2type T cell, recruitment in allergic inammation. Depending upon the strain and exact model, removal of eotaxins by neutralizing antibodies or knockout strategies can reduce eosinophil recruitment and airway hyperreactivity (AHR), but a causal link between eosinophilia and AHR is not a feature of all models. The CCR3(/) mouse provides a further layer of complexity where routes of sensitization exert an effect on the phenotype. Intraperitoneal allergen sensitization results in increased lung mast cell numbers versus the wild-type mice and no abolition of AHR in response to allergen challenge. Where mice are sensitized epicutaneously, there is no increase in lung mast cell numbers and allergen challenge of knockouts is associated with severely impaired eosinophil recruitment and prevention of AHR. In man, the role of eosinophils in allergic airway disease remains controversial. Anti-IL-5 has been used to dramatically reduce eosinophil numbers from the circulation, but is less effective at depleting tissue eosinophils (perhaps because eosinophil progenitors, potentially originally recruited by eotaxins, are resident in lung tissues). Anti-IL-5 treatment has been associated with reductions in AHR in certain animal models, but not in small clinical studies to

Further Reading
Dent G, Hadjicharalambous C, Yoshikawa T, et al. (2004) Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum. American Journal of Respiratory and Critical Care Medicine 169: 11101117. Flood-Page PT, Menzies-Gow AN, Kay AB, and Robinson DS (2003) Eosinophils role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. American Journal of Respiratory and Critical Care Medicine 167: 199204. Forsythe P and Befus AD (2003) CCR3: A key to mast cell phenotypic and functional diversity? American Journal of Respiratory Cell and Molecular Biology 28: 405409. Gutierrez-Ramos JC, Lloyd C, Kapsenberg ML, Gonzalo JA, and Coyle AJ (2000) Non-redundant functional groups of chemokines operate in a coordinate manner during the inammatory response in the lung. Immunological Reviews 177: 3142. Humbles AA, Conroy DM, Marleau S, et al. (1997) Kinetics of eotaxin generation and its relationship to eosinophil accumulation in allergic airways disease: analysis in a guinea pig model in vivo. Journal of Experimental Medicine 186: 601612. Humbles AA, Lu B, Friend DS, et al. (2002) The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inammation and hyperresponsiveness. Proceeding of the National Academy of Sciences, USA 99: 14791484. Jose PJ, Grifths-Johnson DA, Collins PD, et al. (1994) Eotaxin: a potent chemoattractant cytokine detected in a guinea-pig model of allergic airways inammation. Journal of Experimental Medicine 179: 881887. Lamkhioued B, Abdelilah SG, Hamid Q, et al. (2003) The CCR3 receptor is involved in eosinophil differentiation and is up-regulated by Th2 cytokines in CD34 progenitor cells. Journal of Immunology 170: 537547. Ma W, Bryce PJ, Humbles AA, et al. (2002) CCR3 is essential for skin eosinophilia and airway hyperresponsiveness in a murine

EPIDERMAL GROWTH FACTORS 129

model of allergic skin inammation. Journal of Clinical Investigation 109: 621628. Menzies-Gow A, Ying S, Sabroe I, et al. (2002) Eotaxin (CCL11) and eotaxin-2 (CCL24) induce recruitment of eosinophils, basophils, neutrophils, and macrophages as well as features of early- and late-phase allergic reactions following cutaneous injection in human atopic and nonatopic volunteers. Journal of Immunology 169: 27122718. Palframan RT, Collins PD, Williams TJ, and Rankin SM (1998) Eotaxin induces a rapid release of eosinophils and their progenitors from the bone marrow. Blood 91: 22402248. Ponath PD, Qin S, Post TW, et al. (1996) Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. Journal of Experimental Medicine 183: 24372448. Rothenberg ME (1999) Eotaxin. An essential mediator of eosinophil trafcking into mucosal tissues. American Journal of Respiratory Cell and Molecular Biology 21: 291295. Rothenberg ME, Ownbey R, Mehlhop PD, et al. (1996) Eotaxin triggers eosinophil-selective chemotaxis and calcium ux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Molecular Medicine 2: 334348. Ying S, Robinson DS, Meng Q, et al. (1999) C-C chemokines in allergen-induced late-phase cutaneous responses in atopic subjects: association of eotaxin with early 6-hour eosinophils, and of eotaxin-2 and monocyte chemoattractant protein-4 with the later 24-hour tissue eosinophilia, and relationship to basophils and other C-C chemokines (monocyte chemoattractant protein3 and RANTES). Journal of Immunology 163: 39763984.

Relevant Websites
http://www.pymol.org A user-sponsored molecular visualization system on an open-source foundation providing the package PyMol. http://www.rcsb.org A protein data bank: worldwide reporitory for the processing and distribution of three-dimensional biological macromolecular structure data.

EPIDERMAL GROWTH FACTORS

J C Pache, University Hospital of Geneva, Geneva, Switzerland
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Epidermal growth factor (EGF) was the rst growth factor to be discovered. EGF precursor is a transmembranous glycoprotein from which the active EGF is cleaved by an endopepsidase. With the other members of the epidermal growth factors family, it binds to the epidermal growth factor receptor (EGFR). After ligand binding, EGFR (ErbB-1) dimerizes with itself or with its homologs ErbB-2, ErbB-3, or ErbB-4 and consequently increases its intracellular tyrosine kinase activity. This activates signaling cascades that have many effects: cell proliferation, reduced apoptosis, and angiogenesis. The alveolar septation that occurs in embryonic life relies on the presence of EGF. The bronchial wall in chronic bronchitis overexpresses EGF and EGFR. Bronchial epithelial cells, in asthma, demonstrate an increased expression of EGFR. EGFR activation occurs in non-small cell lung carcinoma by overexpression of ligand and/ or receptors, or by a partial deletion of the receptor that results in a ligand-independent activation. Many EGFR-targeted agents are in development and include antibodies directed against the receptor extracellular domain, or small molecules that interfere with the intracellular kinase domain, and various clinical trials are currently testing the relevance of these inhibitors.

newborn mice. Subsequent studies with skin explants revealed a direct effect on epidermal growth. Ligands of the EGFR include EGF, transforming growth factor alpha (TGF-a), heparin-binding EGFlike growth factor (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), and epigen. They have a high-afnity binding to the EGFR. Ligand-independent activation of EGFR may also be achieved by oxidative stress produced by cigarette smoke or activated neutrophils.

Structure
Chromosome 4 (q25q27) contains the gene for EGF, which measures approximately 120 kb. Exon 24 encodes the precursor EGF, while exons 20 and 21 encode the mature EGF. The EGF precursor molecule is a glycoprotein composed of 1207 amino acids, which consist of an intracytoplasmic (149 amino acids) and an extracellular (1032 amino acids) domain, respectively. The transmembranous part of the molecule anchores EGFR to the cytoplasmic membrane. The active EGF molecule (53 residues) is cleaved from the precursor by an endopepsidase (Figure 1). Both membrane-anchored EGF and soluble EGF bind and activate the EGFR.

Introduction
Cohen discovered epidermal growth factor (EGF) in 1962 in submaxillary glands as a factor inducing premature eyelid opening, incisor tooth eruption, and hair growth inhibition when injected in the

Regulation of Productivity and Activity

Activation of EGFR can, in turn, activate Ras signaling, but in reverse, mutant Ras upregulates EGFR

130 EPIDERMAL GROWTH FACTORS

Extracellular

Cytoplasmic

Mature form of EGF with three intramolecular disulfide bridges

EGF precursor transmembranous (prepro-EGF)

Figure 1 EGF precursor and mature molecules.

ligands. Various stimuli that include tumor necrosis factor alpha (TNF-a), cigarette smoke, and inhalation of toxins like bleomycin increase the expression of EGFR in conducting airways epithelial cells. In rodents, submaxillary glands mostly expressed EGF. It is also located in the apical membrane of the ascending limb of the kidney, in exocrine glands of the gastrointestinal tract, and in serous acini of the nasal cavity. In MadinDarby canine kidney (MDCK) cells transfected with prepro-EGF, molecule immunoreactivity appears predominantly at the apical membrane, but the metalloproteinase-dependent cleavage happens mostly at the basolateral protein. Simultaneously, EGFR locates at the basolateral cell surface. The metalloproteinase involved in prepro-EGF cleavage is unknown. TNF-a converting enzyme and metalloproteinase 17 (TACE/ADAM17) cleaves the TGF-a precursor molecule. EGFR located basolaterally binds avidly to the active ligands.

Biological Function
The EGFR gene is on chromosome 7 (p12.3p12.1), contains 26 exons, and covers approximately 110 kb. The 1186 residues molecule inserts across the cell membrane. After ligand binds the receptor, EGFR dimerizes with itself or with its three known homologs ErbB-2 (also known as neu or HER2), ErbB-3 (HER3), and ErbB-4 (HER4) to form heterodimers. This increases the quantity of dimerized receptor and the enzymatic activity of their intracellular kinase (Figure 2). The intracellular effects of EGF activation take place predominantly via tyrosine kinase activity. The

intracytoplasmic molecule acts as a docking site for many signaling proteins. After activation of the EGFR kinase and autophosphorylation, the molecule Grb2 binds to EGFR. This will result in the activation of the proto-oncogene Ras which will further activate different kinases, including extracellular signal-related kinases (ERKs) 1 and 2, leading ultimately to activation of intranuclear transcription factors. Members of the Src family of cytosolic tyrosine kinases are also involved in EGFR signaling, whereas overexpression of Src proteins strongly enhances EGF-mediated proliferation and transformation in epithelial and broblastic cells. STAT proteins are associated with the EGFR as inactive transcription factors. After activation by EGFR kinase activity, they form homo- or heterodimers, become activated, and can move to the nucleus. EGF stimulation of a cell produces strong effects on the phospholipid metabolism: EGFR directly activates phosphatidylinositol-3 kinase (PI3K), phospholipase C-g (PLC-g), and phospholipase D (PLD).

Role in Development

EGF and EGFR play predominant roles in development. The Drosophila epidermal growth factor receptor, called DER, plays multiple roles in embryonic or postembryonic stages, such as proliferation and guidance of migration. DER activates tracheal invagination and branching, as well as the specication of muscle precursor. EGFR activation can lead to cell migration by activation of many different pathways that include the activation of PLC-g.

EPIDERMAL GROWTH FACTORS 131

EGF

NH2 EGFR homodimer Cell membrane Ras P Kinase domain P P P P Grb2 P P P COOH PI3K STAT
PL C-

Raf Src MEK 1/2 Erk 1/2 P P Nucleus Cell proliferation

P P

STAT

Cell mobility
Figure 2 EGFR homodimer after EGF binding with consecutive activation of intracellular signaling molecules.

protein C (SP-C) promoter. The lungs show enlarged alveoli and subpleural and peribronchial brosis. This implicates a paracrine signal between epithelial and mesenchymal cells, which results in broblast proliferation and collagen deposition. Results of gain-of-function gene experiments focus on the role of EGF/EGFR on proliferation whose consequences will be discussed later. Loss-of-function data, in contrast, produce information on the role of EGF in development and physiology. Sialadenectomy was rst used to study the effects of reduced EGF in the body. The most affected organs were the mammary gland and the epidermis. Both organs showed a reduction in size and thickness that could be reversed by EGF administration. Genetic manipulations have produced animals null for EGF, amphiregulin, and TGF-a. The phenotypes of TGF-anull mice are mild with a waxy fur and eyes that at birth are open and opaque. EGF-null and amphiregulin-null animals have no obvious phenotype. EGFR knockout mice, dependent on their genetic background, may have a severely affected phenotype that varies from mid-gestational death to severe abnormalities of the lung. They include impaired branching, and decient alveolization and septation; type II pneumocytes have a decreased number of lamellar bodies. EGFR is primarily expressed at the cell surface, constantly internalized and recycled to the surface. Ligand activation similarly produces a rapid internalization of EGFR and eventually degradation in lysosomes, or recycling to the cell surface.

EGF stimulates DNA synthesis in cultured embryonic chick tracheal and bronchial epithelium. EGF intravenous infusion into fetal lambs induces tracheal, bronchial, and type II pneumocytes hyperplasia. Immunohistochemical studies in mouse embryo localize EGF and EGFR in epithelial and smooth muscle cells of the bronchi and bronchioles. In the human fetal and postnatal lung, EGF and EGFR colocalize within the large airways. Mature epithelial cells also express EGF which regulates proliferation of type II pneumocytes. In the fetal mouse lung, EGF stimulates branching morphogenesis, and production of surfactant proteins A and C. Abrogation of the EGFR signaling results in decreased branching morphogenesis, and a pulmonary lethal phenotype. EGF injection into neonatal mice induces precocious eyelid opening and tooth eruption, subtle effects on neurobehavioral development, and a reduction in growth rates.
Genetic Experiments

Respiratory Diseases
In children with bronchopulmonary dysplasia, bronchial cells present EGF. This suggests the implication of EGF in pulmonary remodeling processes. TGF-a and EGFR overexpress in idiopathic pulmonary brosis and in cystic brosis. In vitro, hyperoxia induces release of TGF-a by lung broblasts. In acute lung injury, TGF-a increases during hyperoxia and the consequent brosis. Fluid from bronchioloalveolar lavage of patients with acute lung injury and idiopathic brosis contains an increased level of TGF-a. In mucosal biopsies of patients with chronic bronchitis, in comparison to controls, EGF expression significantly increases in the epithelium and submucosal cells. Furthermore, in comparison to controls, smokers have an increased expression of EGFR in bronchial mucosal epithelial cells. In asthma, bronchial epithelial cells show an increased expression of EGFR which does not cause a proliferative activity. After in vitro stimulation of airway epithelial cells by various stimuli, there is an expression of

Transgenic mice have been produced in which TGF-a is expressed under the control of the surfactant

132 EPIDERMAL GROWTH FACTORS

MUC5AC and MUC2 mucin gene that depends on activation of EGFR. In chronic obstructive pulmonary disease or asthma, the activation of EGFR by increased levels of EGF therefore leads to mucin hypersecretion and airways obstruction. After epithelial cell injury in vitro or in vivo, EGFR activation promotes repair by stimulating cell proliferation and migration. After in vitro asbestos exposure, EGFR message and protein are upregulated. In addition, asbestos will cause phosphorylation of EGFR and causally activate ERK 1 and 2 as well as expression of the proto-oncogene c-fos. In asbestos or naphthalene exposed animals, the region of the bronchioles and the alveolar ducts demonstrates an increased expression of TGF-a and EGF. In mice that inhale asbestos bers, immunostain for p-ERK increases in foci of epithelial cells and is associated with proliferation and local brosis. A mouse line (9527) has been developed that expresses a mutant EGFR under the control of the lung-specic SP-C promoter. This mutant receptor (dnEGFR) lacks a portion of the intracytoplasmic phosphorylation domain, which inhibits signal transduction by the endogenous EGFR. When exposed in vivo to asbestos, the terminal bronchioles of dnEGFR mice

showed significantly less PCNA-positive cells than those of controls. After binding to a wall receptor, EGF stimulates the growth of extracellular Mycobacterium avium and M. tuberculosis. Furthermore, granulomas contain large amounts of EGF. Evidence has accumulated that overexpression of ligand and/or receptor as well as ligand-independent receptor activation occurs in many lung cancers. EGFR is overexpressed in up to 80% of squamous cell carcinomas of the lung. Deletion of the receptor extracellular portion results in a dimerization and production of an inappropriate signal, even in the absence of ligand binding. In practice, there are no universal methods to evaluate EGFR expression. Furthermore, the relation between EGFR expression and prognosis in non-small cell lung cancer is not clear; most studies do not identify EGFR as a prognostic factor. Studies on EGFR DNA may produce information on gene amplication, deletion, or mutation. Because of possible posttranslational modications that could affect the quantity of protein produced, RNA studies may not be appropriate. EGFR protein can easily be detected by immunohistochemistry (Figure 3). In addition, activation of the receptor and of the signaling

Figure 3 Squamous cell carcinoma of the lung: immunostain for EGFR (Dakocytomation) shows a membranous localization (arrows) Original magnication 400.

EPITHELIAL CELLS / Type I Cells 133

network can be studied by use of antibodies directed against phosphorylated EGFR or against p-ERK and/ or by markers of cell proliferation or maturation. Many EGFR-targeted agents are in development and/ or use in clinical trials. They include antibodies directed against the receptor extracellular domain, and small molecules that interfere with the intracellular kinase domain. Recently, studies have described mutations of the EGFR gene, located around the region encoding the ATP-binding pocket of the receptor tyrosine kinase. In a particular group of patients suffering mostly from lung adenocarcinoma, these mutations may predict the response to getinib, a tyrosine kinase inhibitor.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Bronchiolitis. Cystic Fibrosis: Overview. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Lung Development: Overview. Mucins.

Further Reading
Burgel PR and Nadel JA (2004) Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 59: 992996. Ciardiello F and Tortora G (2003) Epidermal growth factor receptor (EGFR) as a target in cancer therapy: understanding the role of receptor expression and other molecular determinants that could inuence the response to anti-EGFR drugs. European Journal of Cancer 39: 13481354. Guillermo Paez J, Ja nne PA, Lee JC, et al. (2004) EGFR mutations in lung cancer: correlation with clinical response to getinib therapy. Science 304: 14971500.

Jorissen RN, Walker F, Pouliot N, et al. (2004) Epidermal growth factor receptor: mechanisms of activation and signalling. In: Carpenter G (ed.) The EGF Receptor Family. Biologic Mechanisms and Role in Cancer, pp. 3355. Amsterdam: Elsevier. Knight DA and Holgate ST (2003) The airway epithelium: structural and functional properties in health and disease. Respirology 8: 432446. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464480. Liu JY, Morris GF, Lei WH, Corti M, and Brody AR (1996) Upregulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats. American Journal of Pathology 149: 205217. Lynch TJ, Bell DW, Sordella R, et al. (2004) Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to getinib. New England Journal of Medicine 350: 21292139. ODonnell RA, Richter A, Ward J, et al. (2004) Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax 59: 10321040. Pache JC, Janssen YM, Walsh ES, et al. (1998) Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos bers. American Journal of Pathology 152: 333340. Vignola AM, Chanez P, Chiappara G, et al. (1997) Transforming growth factor-b expression in mucosal biopsies in asthma and chronic bronchitis. American Journal of Respiratory and Critical Care Medicine 156: 591599. Whitsett JA and Zhou L (1996) Use of transgenic mice to study autocrineparacrine signaling in lung morphogenesis and differentiation. Clinics in Perinatology 23: 753769. Zanella CL, Posada J, Tritton TR, and Mossman BT (1996) Asbestos causes stimulation of the extracellular signal-regulated kinase 1 mitogen-activated protein kinase cascade after phosphorylation of the epidermal growth factor receptor. Cancer Research 56: 53345338.

EPITHELIAL CELLS
Contents

Type I Cells Type II Cells

Type I Cells
Z Borok and E D Crandall, University of Southern California, Los Angeles, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Alveolar epithelial type I (AT1) cells are large, amorphous cells with elongated thin cytoplasmic processes that cover the majority of the gas exchange surface of the lung. AT1 cells are believed to be derived from AT2 cells during development and following injury. Recent studies demonstrating differentiation of AT1 cells

from bone marrow-derived stem cells suggest other paradigms. Until recently, the only known function of AT1 cells was a presumptive passive role in gas exchange. Improvements in methods for isolation of AT1 cells and recent identication of genes and proteins in AT1 cells suggest additional functions in alveolar homeostasis for this previously elusive cell type, including water ux, active ion transport, cytoprotection, procoagulant and proinammatory properties, development, remodeling, and cell proliferation. AT1 cells have extremely high water permeability due to the presence of the water channel aquaporin-5. Identication of Na channel and Na pump subunits, and amiloridesensitive Na uptake and oubain-inhibitable Rb uptake, conrm a role in active Na transport. AT1 cells are highly susceptible to injury, and release of AT1 cell proteins into bronchoalveolar

134 EPITHELIAL CELLS / Type I Cells

lavage uid and serum may serve as an index of injury. The contributions of AT1 cell injury and repair mechanisms to pathogenesis of lung diseases are being investigated.

Introduction
Alveolar epithelial type I (AT1) cells were rst identied in rat lung by electron microsocopy in 1952. Together with type II (AT2) cells, they form a continuous surface epithelial layer lining the peripheral lung. AT1 cells cover more than 95% of the alveolar surface of the distal lung and, together with closely apposed capillary endothelial cells and thin interstitium, form the airblood barrier across which gas exchange occurs (Figure 1). Within the alveolar space, AT1 cells make contact with other AT1 cells and with AT2 cells via tight junctions located at the cell periphery. Together with AT2 cells, AT1 cells form a barrier to the passive leak of solutes and uid into the alveolar space, an important prerequisite for normal gas exchange. AT1 cells are large cells with a mean cellular volume of 4900 mm3 in the rat and 1800 mm3 in humans, and covering a surface of 4500 and 5100 mm2 in rats and humans, respectively. AT1 cells have attenuated cytoplasmic extensions that can help form the surface of more than one alveolus. Nuclei are surrounded by few mitochondria, endoplasmic reticulum with ribosomes, interspersed microlaments, and an inconspicuous Golgi apparatus. Unlike AT2 cells which contain lamellar bodies, AT1 cells do not possess any other identifying morphologic hallmarks. Throughout the cell are several ask-shaped vesicles, or caveolae, some of which

open onto the alveolar surface or interstitial space, although these are not specic to AT1 cells, being present also in endothelial cells (Figure 2). Investigation of the biological and functional properties of AT1 cells has been hampered by a paucity of specic nonmorphologic phenotypic markers for cell identication as well as lack, until recently, of reproducible methods for isolation and culture of AT1 cells.
Ontogeny

AT1 cells have been thought to arise from AT2 cells during development and following injury, although during development there is evidence that they may also arise from an undifferentiated pool of precursor cells expressing markers of both AT2 and AT1 cells. The notion of the AT2 cell as the progenitor cell in the adult is derived primarily from serial morphologic analyses of the lungs of animals exposed to sublethal concentrations of oxidant gases (e.g., NO2 and O2) and from autoradiographic analyses of regenerating lung pulse-labeled with 3H-thymidine immediately following injury, in which initial incorporation of label into AT2 cells is followed by the appearance of label in AT1 cells. Reecting this in vivo paradigm following injury, AT2 cells in culture lose their characteristic hallmarks and acquire many of the morphologic and phenotypic features of AT1 cells in situ, consistent with transdifferentiation toward an AT1 cell-like phenotype in vitro. This process is significantly inuenced by substratum, soluble factors (including homologous serum), and changes in cell shape. AT1 cells are believed to be terminally differentiated cells incapable
I A E Nu R

COL

EI I I A 1 m

Figure 1 Alveolar wall from human lung. The airblood barrier (short double arrow) consists of type I epithelial cells with long cytoplasmic extensions (I), interstitium (*) between basal laminae of epithelial and endothelial cells, and capillary endothelium (E). A, alveolus; C, plasma in the alveolar capillary; R, cytoplasm of the red cell; El, elastin; COL, collagen; Nu, nucleus of capillary endothelial cell. Reproduced from Albertine KH, Williams MC, and Hyde DM (2000) in Murray JF, Nadel JA, Mason RJ, and Boushey HA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 333. Philadelphia: Saunders, with permission.

EPITHELIAL CELLS / Type I Cells 135

Figure 2 Caveolae in rat lung. Transmission electron micrograph shows ask-shaped plasmalemmal invaginations and cytoplasmic vesicles in both capillary endothelium (right-hand side) and alveolar epithelial type I cells (left-hand side). C, capillary lumen; S, surfactant in airspace. Reproduced with permission from Newman GR, Campbell L, von Ruhland C, Jasani B, and Gumbleton M (1999) Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type 1 cell function. Cell and Tissue Research 295: 116.

of proliferation or self-renewal, although recent in vitro studies suggest that, at least in culture, AT1 cells can revert to a AT2 cell phenotype. The paradigm in which AT2 cells serve as sole progenitors of AT1 cells in the adult has been challenged by recent studies indicating that transplanted bone-marrowderived stem cells may engraft as differentiated AT1 cells in animal models following bleomycin injury. The regulation of AT1 cell differentiation at the molecular level is poorly understood. Animals in which the zinc nger transcription factor GATA-6 is repressed have an absence of AT1 cells with relatively normal AT2 cells, suggesting an important role for GATA-6 in alveolar epithelial cytodifferentiation. Several other key regulatory pathways have been implicated in AT1 cell development and differentiation (e.g., Hox genes, Wnt ligands), but their precise roles remain to be determined.
Phenotypic Markers

Ricinus communis agglutinin I (RCA1), Lycopersicon esculentum agglutinin (LEA), and Bauhinia purpurea agglutinin (BPA), although BPA is also present on macrophages. More specic phenotypic markers for AT1 cells have become available through a combination of monoclonal antibody (mAb) and molecular approaches. mAbs VIIIB2 and SF-1/RTI40 bind selectively to the apical surface of rat AT1 cells but not to AT2 cells or endothelial cells, and both increase during transdifferentiation from AT2 toward AT1 cell phenotype in vitro. VIIIB2 recognizes an as yet unidentied epitope in the apical membrane of AT1 cells in the rat and is not reactive with other lung and nonlung cell types. SF-1/RTI40 was raised using partially puried populations of AT1 cells. Screening of a rat lung library with SF-1/RTI40 led to cloning of the gene T1a that encodes a 3840 kDa glycoprotein. Within lung, T1a is selectively expressed in AT1 cells, although it is also expressed at other sites including choroid plexus, ciliary epithelium, salivary gland, and lymphatic endothelial cells. A mAb, HTI56, has been identied that binds to the apical plasma membrane of human AT1 cells. Aquaporin-5 (AQP5), a member of the aquaporin family of water channels, is a transmembrane protein of B28 kDa selectively expressed by AT1 cells in distal lung, although it is also expressed in other tissues, including salivary and lacrimal glands. Other AT1 cell markers include carboxypeptidase M, intercelluar adhesion molecule 1 (ICAM-l), receptor for advanced glycation end-products (RAGE), and caveolin-1 (an integral membrane protein associated with caveolae), none of which is unique to lung.

Function in Normal Lung

Until recently, no major functions had been attributed to AT1 cells other than a presumptive passive role in gas exchange. Early insights into AT1 cell function were gained from studies using isolated AT2 cells grown as monolayers on inexible substrata. Consistent with observations in vivo, under these conditions AT2 cells transdifferentiate toward an AT1 cell-like phenotype (AT1-like cells) and express phenotypic markers characteristic of AT1 cells in situ (Figure 3). Using this in vitro model, several putative functions were ascribed to AT1 cells, including the ability to vectorially transport Na in both an amiloride-sensitive and -insensitive manner. Consistent with a role in ion transport, subunits of the rat epithelial Na channel (ENaC) and Na pump were identied in AT1-like cells, ndings which have now been conrmed in isolated AT1 cells. Given the need for additional models for characterization of AT1 cells, both mouse (E10/C10) and rat (R3/1) cell lines

Among the earliest methods used to distinguish between AT2 and AT1 cells was differential binding of lectins to glycoproteins on their apical surfaces, although their specicity for each cell type has been questioned. Lectins that have been described predominantly on AT1 compared with AT2 cells include

136 EPITHELIAL CELLS / Type I Cells

Figure 3 Type I-like alveolar epithelial cell in culture (day 5) on polycarbonate lter. Transmission electron micrograph shows a cell with prominent nucleus and long cytoplasmic extensions resembling type I cells in vivo. Reproduced from Cheek JM, Evans MJ, and Crandall ED (1989) Type 1 cell-like morphology in tight alveolar epithelial monolayers. Experimental Cell Research 184: 380, with permission from Elsevier.

Figure 4 Expression of Na pump a1-subunit in type I (AT1) alveolar epithelial cells in situ. (a) Linear staining (red) using a mouse monoclonal antibody (Ab) localizes the Na pump a1-subunit to the basolateral surface of AT1 cells that are labeled with a polyclonal antiaquaporin-5 (anti-AQP5) Ab (green) on the apical surface. (b) Substitution of mouse monoclonal IgG for anti-a1 Ab reveals no reactivity with AT1 cells labeled with anti-AQP5 on the apical surface. (c) AT1 cells labeled on the apical surface with anti-AQP5 Ab (green) and on the basolateral surface with anti-a1 Ab (red). The AT2 cell is not reactive with anti-AQP5 Ab but expresses the a1-subunit on the basolateral surface. Reproduced with permission from Borok Z, Liebler JM, Lubman RL, et al. (2002) Sodium transport proteins are expressed by rat alveolar epithelial type I cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L606.

have been developed that have some features of AT1 cells in situ. Expression of the a-subunit of the Na pump has recently been identied in AT1 cells in situ (Figure 4). Advances in methods for isolation of AT1 cells have conrmed expression of Na channel and Na pump subunits in these cells using double-label immunouorescence in partially puried cells and reverse transcriptase polymerase chain reaction (RT-PCR) in highly puried populations. Further evidence for a

role of AT1 cells in ion transport was the observation of amiloride-sensitive Na uptake and ouabain-inhibitable Rb uptake, consistent with a role in ion transport. AT1 cells have also been shown to express b2-adrenergic receptors (b2-AR) and G-protein-coupled receptor kinase 2 which plays a role in desensitization of b2-AR. Further analyses of the specic ion channels expressed by AT1 cells will benet from the recent development of a lung slice model, in which cells that are positively identied using an AT1

EPITHELIAL CELLS / Type I Cells 137

cell-specic mAb are amenable to patch clamp analyses in situ. Recent identication of genes and proteins in AT1 cells is beginning to provide insights into additional functions of this previously elusive cell type. In studies using partially puried populations, AT1 cells were shown to have extremely high water permeability. Subsequent deletion of AQP5 in knockout mice demonstrated a dramatic decrease in airspacecapillary osmotic water permeability, conrming a role for AQP5 in osmotically driven water clearance. However, clearance of hydrostatic lung edema and active alveolar uid absorption were not affected, indicating that AQP5 is not required for active near-isosmolar uid clearance. Since AQP5 is regulated by osmotic stimuli, a role for this water channel has been suggested in regulation of cell volume or airway surface liquid. T1a is required for normal lung development. T1a knockout mice die at birth of respiratory failure and have a defect in AT1 cell differentiation, as demonstrated by fewer AT1 cells and reduced levels of AQP5. Increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells in these mice at a time when cell division has normally ceased suggest a role for T1a in regulating cell proliferation in the distal lung. Caveolae are known to function in internalization of small molecules or ions and in intracellular trafcking of macromolecules, and also play a role in integrating signaling pathways. Abundant numbers of caveolae in AT1 cells have suggested a role for AT1 cells in macromolecule transport. Studies comparing patterns of gene expression in AT1-like cells and freshly isolated AT1 cells with AT2 cells have identied several new genes associated with AT1 cells that suggest additional functions for these cells, some of which still require functional verication. Although some of these ndings await immunohistochemical conrmation and denitive functional analyses, these studies suggest roles for AT1 cells in addition to active ion transport in alveolar homeostasis, including cytoprotective functions, proinammatory and procoagulant properties, development and remodeling, tumor/growth suppression, and surfactant metabolism.

Little is known of the molecular regulation of these events. Several lung-enriched and ubiquitous transcription factors activate the T1a promoter, and binding sites for several of these are also identied in the promoter region of AQP5. However, these transcription factors also activate AT2 cell-specic genes, indicating that none of these elements alone is responsible for distinguishing between gene expression in AT2 and AT1 cells. Following lung injury, there may be loss of cell-specic proteins from the apical surface of AT1 cells, and SF-1/RTI40 has been found to be upregulated in bronchoalveolar lavage (BAL) uid in rodent models of lung injury (e.g., bleomycin, Pseudomonas pneumonia). Similarly, the human AT1 cell marker HTI56 is increased in pulmonary edema uid and serum in human patients with acute respiratory distress syndrome (ARDS), suggesting that release of a biochemical AT1 cell marker may be useful as an index of acute lung injury. There is an increasing appreciation for a central role of ongoing alveolar epithelial injury in lung diseases such as usual interstitial pneumonitis, with the concept being that progressive brosis results from failure of normal reepithelialization. It has been speculated that inhibition of the transition from AT2 to AT1 cell phenotype may play a role in the pathogenesis of this disorder due to upregulation of b-catenin and Wnt signaling in AT2 cells, which would tend to maintain a proliferative rather than terminally differentiated phenotype. The precise mediators and molecular events that are involved in failure of normal epithelial repair mechanisms are the focus of ongoing investigation.
See also: Acute Respiratory Distress Syndrome. Adrenergic Receptors. Aquaporins. Epithelial Cells: Type II Cells. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Ion Transport: ENaC (Epithelial Sodium Channel). Lung Development: Overview. Panbronchiolitis. Stem Cells.

Further Reading
Albertine KH, Williams MC, and Hyde DM (2000) Anatomy of the lungs. In: Murray JF, Nadel JA, Mason RJ, and Boushey HA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 333. Philadelphia: Saunders. Borok Z, Liebler JM, Lubman RL, et al. (2002) Sodium transport proteins are expressed by rat alveolar epithelial type I cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L599L608. Borok Z and Verkman AS (2002) Role of aquaporin water channels in uid transport in lung and airways. Journal of Applied Physiology 93: 21992206. Brody JS and Williams MC (1992) Pulmonary alveolar epithelial cell differentiation. Annual Review of Physiology 54: 351371.

Function in Respiratory Diseases

AT1 cells are highly susceptible to injury from diverse causes (e.g., bleomycin, radiation, inhaled NO2). Regardless of the cause, the response to injury is fairly stereotypic, with AT1 cells becoming detached and leaving behind denuded basement membrane. This is followed by proliferation of AT2 cells with subsequent differentiation of postproliferative AT2 cells into AT1 cells via an intermediate cell phenotype.

138 EPITHELIAL CELLS / Type II Cells

Cheek JM, Evans MJ, and Crandall ED (1989) Type 1 cell-like morphology in tight alveolar epithelial monolayers. Experimental Cell Research 184: 375387. Crandall ED and Matthay MA (2001) Alveolar epithelial transport: basic science to clinical medicine. American Journal of Respiratory and Critical Care Medicine 163: 10211029. Dobbs LG, Gonzalez R, Matthay MA, et al. (1998) Highly water-permeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung. Proceedings of the National Academy of Sciences, USA 95: 29912996. Gumbleton M (2001) Caveolae as potential macromolecule trafcking compartments within alveolar epithelium. Advanced Drug Delivery Reviews 49: 281300. Kasper M and Singh G (1995) Epithelial lung cell marker: current tools for cell typing. Histology and Histopathology 10: 155169. Kemp PJ and Kim KJ (2004) Spectrum of ion channels in alveolar epithelial cells: implications for alveolar uid balance. American Journal of Physiology 287: L460L464. Kotton DN, Summer R, and Fine A (2004) Lung stem cells: new paradigms. Experimental Hematology 32: 340343. Newman GR, Campbell L, von Ruhland C, Jasani B, and Gumbleton M (1999) Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type 1 cell function. Cell and Tissue Research 295: 111120. Schneeberger EE (1997) Alveolar type 1 cells. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientic Foundations, 2nd edn., pp. 535542. Philadelphia: LippincottRaven. Williams MC (2003) Alveolar type I cells: molecular phenotype and development. Annual Review of Physiology 65: 669695.

Introduction
The alveolar type II cell is the defender of the alveolus (Figure 1). The alveolus is the anatomic site for gas exchange, the critical function of the lung. For gas exchange to take place, the diffusion distance between inhaled gas and the red cell has to be minimized. This is accomplished by keeping the air passages open (the low surface tension at the airliquid interface provided by pulmonary surfactant), the alveolar uid volume low (transepithelial sodium transport), and inammation and edema absent (the innate immune system). Type II cells are also considered to be the progenitor cells for the alveolar epithelium. The alveolar type II cell provides many components required for the special mircoenvironment in the alveolus. Some of these components are pulmonary surfactant, the collectins SP-A and SP-D, and a variety of anti-inammatory and antimicrobial substances such as lysozyme, b-defensin 2, secretory leukocyte proteinase inhibitor (SLPI), and lipocalin 2. Finally, type II cells also provide part of the initial antioxidant defense by secreting glutathione, which is present in high concentration in alveolar uid. The domain that the type II cell defends is a special microenvironment. The alveolar unit as stated above has evolved to facilitate gas exchange, the uptake of oxygen, and the removal of carbon dioxide. The alveolar epithelium is composed of two main cell types. The type I cells are large at thin cells that cover about 95% of the surface area. They are a major part of the epithelial barrier and express genes that indicate they have significant metabolic functions. However, because of their low density of mitochondria per surface area they may lack the energetics. They represent about 11% of the cells in the alveolar wall and each type I cell in the human lung has a surface area of 7000 mm2. Type II cells are smaller cuboidal cells with the anatomic features of an active metabolic epithelial cells with a high density of mitochondria and special apical microvilli. The structural characteristics of this cell are the lamellar inclusions, which are the intracellular storage form of surfactant. Type II cells comprise 17% of the cells of the alveolar wall and cover about 5% of the alveolar surface. Each type II cell in the human lung has a surface area of 250 mm2. In the rat there is only about one alveolar macrophage per alveolus, six type II cells, and four type I cells. Hence, the initial defense to inhaled particles and gases is likely the epithelium. The alveolar uid also has important characteristics. The volume is very low, about 35 ml per adult human lung. This is about 4 ml cm 2, and the pH is acidic, about pH 6.9.

Type II Cells
R J Mason, National Jewish Medical and Research Center, Denver, CO, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The alveolar epithelium is composed of two types of epithelial cells, named alveolar type I and type II cells. Type I cells are large at cells that comprise about 95% of the alveolar surface. Type II cells are small cuboidal cells with characteristic lamellar inclusions and apical microvilli and cover about 5% of the alveolar surface. Type II cells make and secrete pulmonary surfactant. They are the progenitor cells for the alveolar epithelium in the adult lung. Type II cells also transport sodium from the apical to the basolateral surface to keep the alveolus relatively free of uid and participate in the innate immune system. Type I cells are much more susceptible to injury than type II cells. In the injured lung, type II cells spread to reform the alveolar epithelium after a loss of type I cells and then proliferate to restore the differentiated epithelium. Many interstitial lung diseases are characterized by hyperplastic type II cells which are envisioned as part of normal epithelial wound healing. However, it is also possible that type II cells contribute to the broproliferative reaction by secreting growth factors and proinammatory molecules. Finally, type II cells can give rise to alveolar cell carcinomas.

EPITHELIAL CELLS / Type II Cells 139

Type I cell

Type II cell

Figure 1 Type II cells are defenders of the alveoli by secreting surfactant, keeping the alveolar space relatively free from uid, serving as progenitor cells to repopulate the epithelium after injury, and providing important components of the innate immune system.

Type II Cell Function in the Normal Lung

Production of Surfactant

Precursor of Alveolar Epithelial Cells

The production of surfactant is the best-known function of type II cells. The surface tension in the alveoli at the end of expiration is very low (o10 mN m 1), and this provides for alveolar stability, effortless breathing, and prevents pulmonary edema and atelectasis. The low surface tension is provided by phospholipid, predominantly dipalmitoylphosphatidylcholine, and is assisted by the hydrophobic surfactant proteins SP-B and SP-C. The type II cells synthesize the phospholipids of surfactant, SP-B, and SP-C and package them together in lamellar bodies. These lamellar bodies are then secreted by exocytosis. Exocytosis is regulated by a variety of factors including stretch, intracellular calcium, extracellular ATP, b-adrenergic agonists, and agents that activate protein kinase C. Once the lamellar bodies are secreted, the contents unfold to form tubular myelin and what is termed large aggregate surfactant. This material is adsorbed into the air liquid interface at the end of inspiration. Some of the surfactant is squeezed out of the monolayer at the end of expiration, and part of this material is taken up by type II cells for reprocessing and reutilization and part is ingested by macrophages for degradation.

Another important function for type II cells is to serve as progenitor cells for all alveolar epithelial cells. Type II cells are thought to be an asynchronous transient proliferating cell population. It is thought that in vivo type II cells divide and some of the daughter cells remain as type II cells, whereas others transdifferentiate to become type I cells. In vitro there is tremendous plasticity of the epithelial phenotype, so that phenotypic type II cells and type I cells can cycle back and forth depending on the culture substratum and presence or absence of growth factors (Figure 2). However, in the fetal lung it appears that primitive lung epithelial cells can express type I cell markers without having to display type II cell markers. Hence, it is possible that not all type I cells come from differentiated type II cells. It is also not known whether or not there are bona de omnipotent stem cells in the adult lung that could give rise to alveolar epithelial cells. However, it has recently been reported that there are omnipotent epithelial stem cells in the terminal bronchioles. There is also evidence that circulating stem cells from the bone marrow can lodge in the lung and differentiate into type I and type II cells. However, this is a rare event. The relative importance of circulating stem cells in

140 EPITHELIAL CELLS / Type II Cells

In vivo In vitro In vivo

Type II cell SP-C

In vitro T1 In vivo

Type I cell

Figure 2 The phenotype of alveolar type II cells is reversible in primary culture. The type II cell phenotype is close but probably not the same as the type II cell in the normal lung, and the type I-like cells in vitro are significantly different from type I cells in vivo. The phenotypes are reversible in vitro depending on the matrix on which the cells are grown and the presence of growth factors such as KGF. The type II cells in vitro are probably like those intermediate cells observed during lung repair.

normal cell turnover and in response to injury is not known but is probably very, very limited.
Transepithelial Sodium Transport

A third function of type II cells is to keep the alveolus relatively dry by transporting sodium from the apical surface into the interstitium. Type II cells have apical sodium channels, abundant Na/K ATPase, and a rich supply of mitochondria to generate ATP, and they have been shown to transport sodium in vitro. Type I cells also have these transport components, but it does not appear that they have the energy supply as indicated by mitochondrial density to be effective sodium transporters. Beta-adrenergic agonists can increase transepithelial uid transport and alveolar uid resorption. This process requires an intact epithelium as would be present in congestive heart failure but not in adult respiratory distress syndrome or severe pneumonia where the epithelium is severely damaged.
Regulation of Innate Immunity

The nal important function of type II cells is to be an integral part of the innate immune system and to recognize and deal with inhaled microbes, toxic gases, and particulates. We inhale about 10 000 l or 16 kg of air each day. The air we exhale is cleaner in some ways than the air we inhale. The alveolus must deal with low levels of inhaled organisms without inducing much of an inammatory response but then must switch and produce a significant immune response once an overt infection is established. Type II cells participate in the innate immune function of the lung in several ways. They secrete the collectins SP-A and SP-D, which are polyvalent calcium-dependent lectins. These proteins bind and aggregate a variety of microorganisms. They also can suppress the inammatory response of alveolar macrophages under some circumstances as in the normal lung and

yet stimulate macrophages under other circumstances as in acute infection. It should be recognized that the lung should not develop an acute inammatory state or stimulate a T-cell response with every inhaled antigen or microorganism. Both the innate and the adaptive immune systems should be dampened. Inammation impairs gas exchange. For example, alveolar macrophages are relatively poor antigen-presenting cells. In addition, type II cells can secrete a variety of antimicrobial peptides such as b-defensins 2, lipocalin 2, and lysozyme. Moreover, they provide the reducing substances in the alveolar uid to neutralize oxidant gases such as the phospholipids of surfactant, reduced glutathione, ascorbate, and urate. Finally, type II cells can also signal to other more active immunoeffector cells such as macrophages and neutrophils by secreting chemokines and cytokines. Type II cells secrete a variety of cytokines and chemokines, including the interleukins IL-1b, IL-1a, IL-6, IL-8 (CXCL8), epithelial neutrophil activating peptide-78 (ENA-78; CXCL5), growth related oncogene alpha (GRO-a; CXCL1), macrophage inammatory protein-2 (MIP2; CXCL2), monocyte chemoattractant protein 1 (MCP-1; CCL2), exotaxin (CCL11), regulated upon activation normally T cell expressed and secreted (RANTES; CCL5), and undoubtedly many others. Type I cells can also secrete cytokine and chemokines.

Type II Cell Function in Respiratory Diseases

The type I cells comprise 95% of the alveolar surface and are very susceptible to injury. After damage to type I cells, the type II cells spread, proliferate, and then differentiate to reform the epithelium (Figure 3). If type II cell hyperplasia is induced by a single instillation of KGF, some of the proliferating type II cells

EPITHELIAL CELLS / Type II Cells 141

Normal

Dedifferentiation and migration

Differentiation

Proliferation

Transdifferentiation

Figure 3 Epithelial cells respond to injury or wound healing in a characteristic way, and this occurs in the lung, although it is difcult to visualize. After injury to type I cells, type II cells spread, dedifferentiate, and migrate to cover the wound. The type II cells then proliferate to form a hyperplastic epithelium and then differentiate as type II cells. Finally, some of the epithelial cells undergo apoptosis and some transdifferentiate into type I cells.

undergo apoptosis and others transdifferentiate to form type I cells with the result of normal appearing lung 1 week after instillation of KGF. However, in numerous interstitial lung diseases this process is not complete. The hyperplastic type II cells persist. The reason for this is not known but there is clearly continued proliferation as indicated by PCNA and Ki67 immunostaining as well as an apparent failure to transdifferentiate into type I cells. The role of hyperplastic type II cells in interstitial lung disease is not known. From the perspective of cutaneous wound healing they are reforming the epithelium and can limit granulation tissue and broblast proliferation. For example, in vitro type II cells release IL-1a and other substances that increase prostaglandin E2 (PGE2) production in broblasts that in turn inhibit broblast proliferation. However, type II cells can also secrete growth factors for broblasts and transforming growth factor beta (TGF-b) and may in some circumstances stimulate broblast proliferation and matrix production. It is interesting to note that the epithelial lining over broblastic foci in usual interstitial pneumonia is commonly incomplete and suggests that the incomplete epithelium may promote growth of broblastic foci and matrix production. Surfactant production is decreased in a variety of lung injuries and contributes the alveolar collapse, pulmonary edema, and increased work of breathing. Changes in phosphatidylglycerol are the earliest changes in the composition of surfactant phospholipid with lung injury, but the physiologic consequences of this change are not apparent. Similarly, transepithelial sodium transport is impaired in acute lung injury. It should be noted that the resolution of alveolar

exudation requires an intact alveolar epithelium. Until the epithelium is reformed, effective transepithelial transport cannot take place. The alveolar epithelium can also facilitate clearance of the alveolar exudate by producing tissue factor. Finally, alveolar type II cells can give rise to alveolar adenomas and adenocarcinomas. In mice nearly all of the lung adenomas express SP-C and are therefore derived from type II cells. Hence, type II cells are truly the defender of the alveolus by providing normal gas exchange and effortless breathing, dealing with low levels of toxic substances that we inadvertently inhale, and initiating an inammation response if needed to deal with a more virulent pathogen.
See also: Adhesion, CellCell: Epithelial. Epithelial Cells: Type I Cells. Extracellular Matrix: Basement Membranes. Fibroblast Growth Factors. Fluid Balance in the Lung. Keratinocyte Growth Factor. Permeability of the BloodGas Barrier. Stem Cells. Surfactant: Overview. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading
Fehrenbach H (2001) Alveolar epithelial type II cell: defender of the alveolus revisited. Respiratory Research 2: 3346. Gonzalez R, Yang YH, Grifn C, et al. (2005) Freshly isolated rat alveolar type I cells, type II cells, and cultured type II cells have distinct molecular phenotypes. American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L179L189. Mason RJ, Lewis MC, Edeen KE, et al. (2002) Maintenance of surfactant protein A and D secretion by rat alveolar type II cells

142 ERYTHROCYTES
in vitro. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L249L258. Mason RJ, Pan T, Edeen KE, et al. (2003) Keratinocyte growth factor and the transcription factors C/EBPa, C/EBPd, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. Journal of Clinical Investigation 112: 244255. Mason RJ, Williams MC, Widdicombe JH, et al. (1982) Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proceedings of the National Academy of Sciences, USA 79: 60336037. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial uid transport and the resolution of pulmonary edema. Physiological Reviews 82: 569600. Shannon JM (1994) Induction of alveolar type II cell differentiation in fetal tracheal epithelium by grafted distal lung mesenchyme. Developmental Biology 166: 600614. Shannon JM, Pan T, Nielsen LD, Edeen KE, and Mason RJ (2001) Lung broblasts improve differentiation of rat type II cells in primary culture. American Journal of Respiratory Cell and Molecular Biology 24: 235244. Veldhuizen R and Possmayer F (2004) Phospholipid metabolism in lung surfactant. Sub-Cellular Biochemistry 37: 359388. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Pediatric Respiratory Reviews 5(Supplement A): S283S287. Williams MC (2003) Alveolar type I cells: molecular phenotype and development. Annual Review of Physiology 65: 669695. Zemans RL and Matthay MA (2004) Bench-to-bedside review: the role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Critical Care 8: 469477.

ERYTHROCYTES
E Ingley and S P Klinken, Western Australian Institute for Medical Research and Centre for Medical Research, Perth, WA, Australia
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Erythrocytes are biconcave enucleate red blood cells responsible for transport of O2/CO2 between the bodys tissues and lungs; their oxygen-carrying capacity is due to their high hemoglobin content. Red blood cells are derived from hemopoietic stem cells in a process controlled by the hormone erythropoietin. Oxygenation of the erythrocyte in the lung capillaries facilitates unloading of CO2, causing shrinkage and ATP release. ATP activates endothelial cells to synthesize nitric oxide (NO), some of which becomes complexed with hemoglobin. Deoxygenation in peripheral tissues initiates NO release and CO2 loading, resulting in swelling of the red cell. Red cell defects can affect the lung. Anemias can manifest as respiratory abnormalities, although polycythemias rarely display pulmonary problems. Hemoglobinopathies, such as sickle cell disease, often show pulmonary complications causing significant morbidity and mortality. When erythrocytes contact lung broblasts, due to lung injury, they stimulate release of interleukin-8 (IL-8), enhancing inammatory responses. Cystic brosis (CF) patients develop numerous lung pathologies due to mutations in the ATP-transporting cystic brosis transmembrane conductance regulator (CFTR) gene. CF erythrocytes are unable to release ATP on mechanical deformation in the lung, preventing ATPstimulated synthesis of NO from the endothelial cells, resulting in pulmonary hypertension. Malaria-infected erythrocytes display multiple abnormalities, which result in alveolar and endothelial damage.

Introduction
Erythrocytes, or red blood cells, with their wellknown biconcave shape, are critical for exchange

and transport of O2/CO2 between the lungs and the bodys tissues and organs. This is achieved through red cells containing high levels of the oxygen carrier hemoglobin (Hb). While the eminent Egyptian physician Ibn al-Nas was the rst to describe pulmonary circulation (in the thirteenth century), Jan Swammerdam, a 21-year-old Dutch microscopist, is thought to be the rst person to observe and describe red blood cells (in 1658). These cells are found within the capillaries surrounding the alveoli of the lung (Figure 1(a)). Enucleated red cells have an average diameter of 8.4 mm and a mean volume of 90 fL; due to their highly exible membrane, erythrocytes can easily expand up to 150 fL, or be squeezed to traverse capillaries that are less than 8 mm in diameter. They are produced primarily in the bone marrow from pluripotent hemopoietic stem cells. This development of red blood cells from stem cells occurs via a process called erythropoiesis; a variety of cytokines and hormones initiate a series of proliferation and maturation steps, which result in release of reticulocytes into the bloodstream. Reticulocytes then mature into erythrocytes within 12 days (Figures 1(b) and 1(c)). The earliest committed progenitor of the erythroid lineage is the burst-forming unit-erythroid (BFU-E). This stage is not dened morphologically, rather functionally, as these cells produce large burst colonies of erythroid cells in semisolid culture media; BFU-E are highly responsive to stem cell factor (SCF) stimulation. The next recognizable stage is that of the colony forming unit-erythroid (CFU-E), which again is dened functionally via the generation of compact colonies. Erythropoietin (Epo), the hormone

ERYTHROCYTES 143

HSC

RBC

Tc

BFU-E Or Po Or R R

Pr

E E En

CFU-E Mac

Py

Py

(b) Alvll
R R Or Po Or B Po Po R

E E E E

R Or

Or

Pr Po

Tc (a) E (c)
R

Figure 1 (a) Transmission electron micrograph of airway cells showing erythrocytes (E), endothelial cells (En), type II alveolar cells with distinctive multilamellar bodies (AlvII), and the thin-walled capillaries (Tc). Scale bar 2 mm. Photograph courtesy of the School of Anatomy and Human Biology, UWA. (b) Ontogeny of erythrocytes. The development of a red blood cell (RBC) from a hemopoietic stem cell (HSC) is illustrated. Stages from the colony forming unit-erythroid (CFU-E) through to the enucleated reticulocyte (R) are found in erythroblastic islands surrounding a central macrophage (Mac). Other stages shown are burst-forming unit-erythroid (BFU-E), proerythroblast (Pr), basophilic erythroblast (B), polychromatic erythroblast (Po), orthochromatic erythroblast (Or), and the pyknotic nucleus (Py) of the enucleating erythroblast, which is engulfed by the macrophage. (c) Neutral benzidine and Wrights stain of erythroblasts at different stages of development isolated from an erythroblastic island. Hb is stained yellow/orange. Cells present are proerythroblast (Pr), basophilic erythroblast (B), polychromatic erythroblast (Po), orthochromatic erythroblast (Or), and reticulocyte (R).

principally responsible for the development of the latter stages of erythropoiesis, is produced in the kidneys in response to a low oxygen tension. Responsiveness to Epo begins at the BFU-E stage and is maximal in CFU-E through to proerythroblasts, the next stage of erythroid development. This latter phase is the rst morphologically recognizable stage and is characterized by large cells with a high nuclear to cytoplasmic ratio. The subsequent stages of development (viz. basophilic erythroblast, polychromatic erythroblast, and orthochromatic erythroblast) are typied by nuclear condensation, a decrease in cell size, and increasing Hb levels. Up to the orthochromatic erythroblast stage, cells are able to proliferate. Thereafter, nuclei become excentric, Hb synthesis increases, and eventually the nuclei are extruded, releasing reticulocytes, which in turn develop into erythrocytes. Mature red blood cells, which lack a nucleus and organelles, remain in circulation for approximately 120 days before displaying phosphatidylserine on their outer membrane, identifying them as old red blood cells that need to be removed by the reticuloendothelial system (macrophages). Nearly all of the constituents of aged erythrocytes are recycled, except for the heme component of Hb, which

is eventually excreted through the bile duct as bilirubin.

Erythrocyte Function in the Normal Lung

The specialized function of the red cell is to mediate transport of O2 from the lungs to peripheral tissues, and CO2 in the reverse direction. Erythrocytes inuence O2 transport in two main ways viz. the total concentration of Hb in the blood and its afnity for O2. Ventilation of the lungs not only mediates the O2 loading of red cells but also CO2 release, and maintenance of the bloods acidbase balance, which also inuence O2 loading (Figure 2(a)). Cardiac output and blood shunting can also affect the amount of O2 loading of erythrocytes. An essential feature of O2 transport via the red blood cell is the need to bind O2 rmly in the pulmonary capillaries under high O2 tension, and subsequently release it in the periphery under lower O2 tension. Oxygenation of the red cell is extremely rapid; almost complete oxygenation occurs within the rst third of the 780 ms transit time within the lung capillaries. To achieve maximal O2 saturation, the Hb tetramer binds O2 in a cooperative manner. The rst O2 molecule to interact with deoxygenated Hb binds

144 ERYTHROCYTES

H2CO3 HbH+ ATP HbO2 NO SNOHb CO2 HCO3 O2 CO2 O2 O2 O2

Alveoli (a) Lung capillary

H2CO3 HbH+ NO HbO2 SNOHb O2 CO2 HCO3

CO2 CO2 CO2

(b)

Tissue capillary

Tissue cells

Figure 2 Normal function of erythrocytes in the lung and peripheral tissues. (a) Erythrocytes in pulmonary capillaries release CO2 and take up O2 via their gradients. Oxygenation of Hb initiates unloading of CO2, which induces erythrocyte shrinkage. Mechanical deformation initiates release of ATP, stimulating endothelial cells to synthesize NO, which reacts with Hb to form S-nitrosohemoglobin (SNOHb). (b) Erythrocytes in the peripheral tissues release O2 and take up CO2 due to their gradients. Deoxygenation of the Hb stimulates release of NO. Loading of CO2 stimulates swelling of the erythrocyte.

relatively weakly as the Hb is in a relaxed or Rform. However, once the rst O2 molecule has bound, the other O2-binding sites of the Hb are transformed into high-afnity sites. This allosteric state of Hb is known as the tense or T-form. The extensive packing of Hb within the red cell provides numerous benets, including juxtaposition to enzymes required to regulate its O2 afnity, for example, 2,3-diphosphoglycerate, which is present at approximately equimolar concentrations with Hb in red cells. The bolus ow of red cells, as opposed to a laminar ow, means there are no stagnant ow layers along the capillary wall. The rolling action of red cells within the circulation further enhances the rate of gas exchange. Interestingly, oxygenation of Hb in the lungs promotes the binding of nitric oxide (NO) to cystineb93

of the globin moiety, forming S-nitrosohemoglobin (SNOHb) (Figure 2(a)). Deoxygenation in the periphery, accompanied by the allosteric transition of the SNOHb from the R to the T form, releases the NO group, which is known to help mediate O2 uptake by the peripheral tissues through relaxation of vessels and increasing blood ow (Figure 2(b)). Perfusion of the lung results in a relaxation of the pulmonary smooth muscle vasculature to approximately 10-fold less than that of the systemic circulation, due to induced NO synthesis in the endothelium. Red blood cells are necessary for this vasodilation as they are mechanically deformed during their traversal of the pulmonary capillary. This initiates release of ATP, which then stimulates the endothelium to synthesize NO via their P2y purinergic receptors (Figure 2(a)). Erythrocytes are the main vessel for CO2 transport to the lungs in the form of H2CO3, a reaction facilitated by the rapid hydration of CO2 by carbonic anhydrase. Red cells acquire CO2 in the periphery through a CO2 gradient; while approximately 25% of the CO2 reacts with the deoxygenated Hb to form carbamino-Hb, most is converted to H2CO3 by carbonic anhydrase, which in turn dissociates to form HCO3 and H . Deoxygenated Hb accepts the pro ton and buffers the pH of the cell. The HCO3 then diffuses out of the cell into the plasma and Cl moves in to restore the electrostatic balance; however, this upsets the osmotic equilibrium so H2O also enters, swelling the cell. Thus, gaseous exchange in red cells also results in a volume change (Figure 2(b)). The oxygenation of Hb results in the dissociation of a proton from the globin chain, known as the Bohr effect, initiating simultaneous release of CO2. The proton combines with the HCO3 to form H2CO3 and the carbonic anhydrase now converts this to CO2 and H2O, allowing release of the CO2 along its pressure gradient (Figure 2(a)). Thus, erythrocytes do not expend any energy in transporting O2 and CO2, due to the gradients of these gases and the unique properties of Hb.

Erythrocytes in Respiratory Diseases

Anemias and Polycythemias

Anemias, depending on their severity, can manifest as respiratory abnormalities, most notably dyspnea. The general decrease in O2-carrying capacity caused by anemia often results in compensatory increases in cardiac output and blood shunting, particularly in chronic anemias. Treatment of anemias caused by kidney disease with recombinant Epo results in the normalization of pulmonary functions. Anemias caused by deciencies of iron, vitamin B12, or

ERYTHROCYTES 145

folic acid, which can be corrected by appropriate supplementation, also result in the disappearance of pulmonary dysfunction. More often a primary respiratory disease will be the cause of a general hypoxic state within the erythroid compartment. Polycythemia can, on rare occasions, present with respiratory problems due to the high blood viscosity impairing blood ow. Interestingly, chronic polycythemia associated with high altitude is usually accompanied by decreased resistance in the periphery, preventing an increase in blood pressure and the appearance of respiratory problems. Hypoxemia due to pulmonary disorders that produce impaired O2 diffusion through the alveolar walls can result in a secondary polycythemia through the excessive induction of Epo. The increased hematocrit and blood viscosity results in an increase in circumference of pulmonary and tissue capillaries so the cardiac output is not significantly increased. The use of regular phlebotomy to treat polycythemia corrects the pulmonary and circulatory changes.
Hemoglobinopathies

and repair following lung injury. Resident broblasts of the lung can respond to inammatory mediators and release other molecules that regulate inammation, including interleukin-8 (IL-8), which acts as a chemoattractant for neutrophils. Erythrocytes, or factors released by them, can stimulate broblasts to release IL-8. Thus, an injury that leads to red cell contact with the extracellular matrix/broblasts has the potential to initiate an inammatory response by stimulating IL-8 secretion and neutrophil inltration.
Cystic Fibrosis

Hemoglobinopathies, including sickle cell disease and thalassemias, can result in lung pathologies/respiratory problems. Indeed, in sickle cell disease, pulmonary complications of acute chest syndrome and chronic sickle cell lung disease are significant causes of morbidity and mortality. Erythrocytes are high in antioxidants, which can prevent oxidation of iron in the heme moiety, and prevent oxidative stress damage in the lung. Interestingly, sickle cell erythrocytes have reduced antioxidant enzymes and are less effective against reactive oxygen species during oxidative injury. Sickle cells retain adhesion factors that are normally lost as reticulocytes are released into the circulation, resulting in increased adherence to the endothelial cell surface, which leads to endothelial injury in the lung. Owing to a variety of factors, sickle cells impair host defenses and, consequently, patients have an increased incidence and severity of pulmonary infections. There are no specic treatments for lung diseases associated with sickle cells; generally, treatments are aimed at reducing the proportion of sickled erythrocytes. Hydroxyurea treatment induces fetal Hb and, thus, decreases the relative concentration of the sickle (S) Hb. Epo treatment can also be employed. Transfusions and bone marrow transplantation have been successfully undertaken with patients with severe complications.
Inammatory Lung Injury

Cystic brosis (CF) patients develop numerous lung pathologies, including severe airway disease, alveolar hypoxia, and pulmonary hypertension. CF is caused by a diminution, or loss, of the cystic brosis transmembrane conductance regulator (CFTR), caused by specic genetic mutations. This potential ion channel is one of a family of molecules responsible for regulating the movement of ATP out of a cell. Red blood cells are known to release ATP upon mechanical deformation and specic inhibitors of CFTR negate this activity. Erythrocytes from CF patients are unable to display this ATP efux after physical stimulation due to their lack of functional CFTR. Furthermore, ATP released into the pulmonary circulation enhances NO synthesis in the lung. Consequently, the pulmonary hypertension seen in CF could well be due to the lack of ATP-stimulated NO synthesis in the pulmonary vasculature, normally initiated by red blood cell deformation. Additionally, in primary pulmonary hypertension, which has no known etiology, there is again a hindrance of this pathway and a lack of ATP release from red cells.
Malaria

Recent studies have indicated that erythrocytes have the potential to participate in inammation

Malaria is one of the worlds major public health concerns and significant pulmonary problems are reported; up to 5% of patients infected with Plasmodium falciparum are known to develop respiratory failure. This is caused by sequestration of parasitized red blood cells in the microvasculature of the lung; infected erythrocytes express parasite-derived proteins, which induce adhesion to endothelial cells. The development of the inammatory response to parasite antigens also leads to release of inammatory mediators, which results in alveolar and endothelial damage and capillary leakage. Many factors contribute to hypoxemia in severe malaria, including sequestration of parasitized red cells in the pulmonary vasculature, aspiration pneumonia, uid overload, and secondary Gram-negative bacteremia. Ventilation, replacement transfusion, and uid management

146 EXERCISE PHYSIOLOGY

are important for rectifying pulmonary functions during treatment with antimalarial drugs.
See also: Arterial Blood Gases. Colony Stimulating Factors. Hemoglobin. High Altitude, Physiology and Diseases. Peripheral Gas Exchange. Systemic Disease: Sickle Cell Disease.

Further Reading
Fredriksson K, Lundahl J, Palmberg L, et al. (2003) Red blood cells stimulate human lung broblasts to secrete interleukin-8. Inammation 27: 7178. Harris JW and Kellermeyer RW (eds.) (1970) The Red Cell Production, Metabolism, Destruction: Normal and Abnormal. Cambridge: Harvard University Press. Hillman RS and Finch CA (eds.) (1974) Red Cell Manual, 4th edn. Philadelphia: F A Davis Company. Ingley E, Tilbrook PA, and Klinken SP (2004) New insights into the regulation of erythroid cells. IUBMB Life 56: 177184.

James MF (1985) Pulmonary damage associated with falciparum malaria: a report of ten cases. Annals of Tropical Medicine and Parasitology 79: 123138. Klinken SP (2002) Red blood cells. International Journal of Biochemistry and Cell Biology 34: 15131518. Knight J, Murphy TM, and Browning I (1999) The lung in sickle cell disease. Pediatric Pulmonology 28: 205216. Lane DJ (ed.) (1976) Respiratory Disease. London: William Heinemann Medical Books Ltd. Pawloski JR, Hess DT, and Stamler JS (2001) Export by red blood cells of nitric oxide bioactivity. Nature 409: 622626. Pouvelle B, Buffet PA, Lepolard C, Scherf A, and Gysin J (2000) Cytoadhesion of plasmodium falciparum ring-stage-infected erythrocytes. Nature Medicine 6: 12641268. Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, and Lonigro AJ (1998) Deformation-induced ATP release from red blood cells requires cystic brosis transmembrane conductance regulator activity. American Journal of Physiology 275: H1726 H1732. Sprague RS, Stephenson AH, Ellsworth ML, Keller C, and Lonigro AJ (2001) Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension. Experimental Biology and Medicine 226: 434439.

EXERCISE PHYSIOLOGY
B J Whipp, University of Leeds, Leeds, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Exercise intolerance is a consequence of the inability to meet the energy requirements of the chosen, or imposed, task. The goal of clinical exercise testing is to stress the organ systems contributing to the intolerance to a level at which the abnormality becomes discernible from the magnitude or prole of appropriately selected response variables. Assessing the normalcy, or otherwise, of these responses to exercise requires the investigator to select and appropriately display the cluster of response variables that are themselves reective of the particular system(s) behavior. Interpretation is then based on two interrelated perspectives: discriminating a magnitude or pattern of deviation from the normal response (age, gender, and activity matched standard subject); and matching the magnitude or pattern of abnormality with that characteristic of particular impairments of physiological system function. Exercise is not a mere variant of rest: it is the essence of the machine Joseph Barcroft

systems that link and support oxygen transfer from the atmosphere to the energy transduction sites within contracting muscles. When the ability to meet the energy requirements of the chosen or imposed tasks becomes limited, exercise intolerance ensues. Physiological, as other, systems tend to fail under stress; an optimized stress prole can therefore be utilized to allow abnormalities in the magnitude or contour of change of selected variables of interest to be discerned providing clues to the source of the intolerance. This naturally requires a context of normal response.

Determinants of Normal Responses

Metabolic Considerations

Introduction
Purposeful increases in muscular activity are essential components of human cultural expression. However, the limits to plausible physical aspiration are set by the adequacy of functioning of the physiological

Exercise is fueled by the chemical energy of ingested food, which is transformed into the mechanical energy of muscular force generation. Skeletal muscle, however, is composed of different ber types with different mechanical and metabolic characteristics. For tasks requiring relatively low force generation, the more efcient and more aerobic Type I bers are predominantly recruited; with higher demands for force generation (or for rapid contractions), the less efcient and more glycolytic (anaerobic) Type II bers are also recruited. The contraction prole of

EXERCISE PHYSIOLOGY 147

Table 1 Sources of ATP resynthesis during exercise Aerobic O2 transport O2 stores  PCr stores O2 defmusc Lactate and H production O2 def (lung)

Anaerobic

the muscles must be effectively orchestrated for skillful performance. While adenosine triphosphate (ATP) is the obligatory energy resource for muscle contraction, its low concentration (B5 mM kg 1) is maintained by increased aerobic and anaerobic production (Table 1). The immediate reaction for ATP resynthesis is splitting of locally stored phosphocreatine (PCr) to creatine (Cr) and inorganic phosphate (Pi): PCr-Cr Pi DG 1

WR for moderate intensity cycle ergometry at constant pedaling frequency; not varying appreciably with tness, training, gender, or age. The typical O2 WR relationship slope (or gain) of this V (i.e., DV O2 =DWR) averages an easy-to-remember B10 ml min1 W. O ss of the standard For a 100-W WR, the V 2 70-kg slim adult cycling at 6070 rpm is composed of: O2 (B250 ml min 1); 1. resting V O2 to move 2. the unmeasured additional V legs with no load on the ywheel (0 W) (B250 ml min 1); and O ss of B1000 ml min 1, reect3. an increase DV 2 O ss ing the 100 W applied WR, to an absolute V 2 1 of B1500 ml min . O2 ss O2 ss at 0 W, the DV Although obesity increases V associated with a measured WR increment is not. And while the task performance is inefcient, in the sense that the total energy and O2 costs are high, the efciency of transducing substrate energy into effective muscular work is not (B2530% in both cases). The treadmill is less suitable for interpreting the O2 response to the apparent WR. That is, inefcient V gait patterns, the subject being partially supported by the handrails or by an investigator (e.g., during a blood-sampling procedure) modify metabolic cost of the task. The substrate mixture being oxidized inuences the O2 cost of the task. Carbohydrate is the more efcient fuel (by B6%) in terms of O2 utilization (thereby minimizing the cardiovascular demands for O2 delivery). However, fats produce appreciably less CO2 (by B40%) per unit ATP yield (thereby reducing the ventilatory demands for acid base regulation). O increases towards its steady The rate at which V 2 state (faster in trained subjects) is a major determinant of the O2def. A large O2def for a given WR requires greater utilization of stored energy resources and predisposes to early-onset lactic acidosis (low yL ). When O2 is not utilized at appropriate rates (either inadequate delivery or impediment to utilization), ATP can only be formed through the low-yield

where DG is the free energy of ATP hydrolysis. PCr consequently decreases in proportion to work rate (WR), the magnitude of decrease depending on the rate at which O2 utilization increases towards its steady-state value. Consequently, less t subjects have a greater PCr decrease for a given WR than tter subjects. The major route of ATP resynthesis, however, is oxidative phosphorylation, deriving from atmospheric O2 transported to the muscle during the exercise and O2 already stored in the body. The pul O therefore conflates the immonary O2 uptake V 2 mediate inuence of the increased cardiac output Q and the delayed inuence of the increased muscle arteriovenous O2 content difference. In the steady state, all the energy transformations derive from aerobic exchange from the transported O2 with no further contribution from O2 stores or O subsequently remains anaerobic mechanisms. V 2 steady, as long as WR remains constant. The steady CO will, in addition, state level of CO2 output V 2 depend on the substrate mixture being metabolized. Prior to the steady state, energy must be provided from other sources. The O2-equivalent of these reactions is termed the O2 deficit (O2def). While it O to attain steady state V O ss takes B3 min for V 2 2 in healthy young subjects, it takes considerably longer in older and/or sedentary subjects, whose O2def will consequently be greater. As a result of the CO2 time greater tissue capacitance for CO2, the V course will be appreciably longer; consequently, the CO2 =V O2 underrespiratory exchange ratio R V shoots during the transient. O ss increBelow the lactate threshold (yL), the V 2 ment is a relatively constant function of increasing

148 EXERCISE PHYSIOLOGY

anaerobicglycolytic pathway: C6 H12 O6 glucose-2lactate H 2ATP 2

This increase in cellular proton load generates greater stress, inuencing muscle contractile properties and ventilatory control mechanisms. The benet, however, is that the ATP supply can be maintained: 1 ATP per lactate molecule formed from glucose, or 1.5 ATP from glycogen. The bicarbonate (HCO3 ) system is the most important nonrespiratory contributor to acidbase regulation during exercise, being functionally open with respect to the atmosphere: CH3 CHOH COO H lactate proton

oxyhemoglobin dissociation curve, with little effect on O2 saturation. As there is no sustained lactic acidosis during moderate exercise, arterial pH (pHa) will be regulated at its set-point level only if PaCO2 is maintained at the appropriate level. This depends on A to the CO2 exchange rate at the lung matching V not its production rate in muscle. As a consequence of the physiologic dead space E does (VD), a proportion of the total ventilation V not contribute to V A : E 863V CO2 =PaCO2 1 VD =VT V 5

NaHCO3 sodium bicarbonate H2 CO3 carbonic acid 3

-CH3 CHOH COONa sodium lactate and

where VD/VT is the physiologic dead space fraction E and V CO2 of the breath. The relationship between V during exercise is highly linear (but with a small E axis, normally of B36 positive intercept on the V 1 l min ) up to the onset of compensatory hyperventilation. Consequently, the magnitude and prole of E =V CO is the ventilatory equivalent for CO2 V 2 closely linked to that of VD/VT: E =V CO2 863=PaCO2 1 VD =VT V 6

H2 CO3 -H2 O CO2 While each proton that combines with an HCO 3 ion produces one additional CO2 molecule to be vented VCO2 , the rate at which this CO2 is produced CO2 depends on the rate at which HCO falls. V 3
Ventilatory Considerations

The major ventilatory demands of exercise are arterial blood-gas and acidbase regulation, while optimizing the cost in terms of the respiratory muscle work. Alveolar (A), and hence arterial (a), PCO2 and PO2 can only be maintained constant during exercise A changes in precise proportion to V CO2 and V O2 , if V respectively: CO 863 V 2 A 863 V O2 V PA CO2 PI O2 PA O2 4

(The small effect of the difference in inspiratory and expiratory ventilation that occurs when R does not A is common to both reequal 1 is neglected.) As V lationships, it cannot meet the regulatory demands of both O2 and CO2 exchange when they differ during E changes in close proportion to V CO2 exercise. V with PaCO2 being the more closely regulated variable, although the control mechanisms remain poorly understood. Any consequent changes in PaO2 normally only traverse the relatively at region of the

CO2 reects a low PaCO2, high VD/VT, E =V High V E =V CO2 will be increased or both. Consequently, V under conditions which impair pulmonary gas ex E with change (high VD/VT) or induce excessive V respect to CO2 exchange (low PaCO2) caused, for example, by hypoxemia, metabolic acidosis, or anxiety. High VD/VT, however, does not necessarily reect abnormal pulmonary function, as rapid, shallow breathing yields a high VD/VT even in normal subjects. The alveolar-to-arterial partial pressure difference (A a) for the gas of interest provides an index of its exchange efciency. Thus, while alveolar hypoventilation leads to arterial hypoxemia and hypercapnia, it does not widen (A a); diffusion impairment, right-to-left shunt, and maldistribution V all do, particularly A with respect to Q A =Q of V for O2. PaO2 and PaCO2 are maintained at, or close to, resting levels during moderate exercise in normal subjects at sea level. Arterial hypocapnia, however, is a common feature of supra-yL exercise. Why, therefore, does PaO2 not systematically increase? It tends to be maintained close to resting levels at all WRs E =V O and end-tidal PO2 (PETO2) indespite both V 2 creasing progressively 4yL. That is (A a) PO2 either begins to widen at yL , or begins to widen more rapidly, normally without significant hypoxemia (although this is seen in some highly t normal subjects). In lung disease patients, pulmonary gas exchange inefciencies leads to often-marked differences

EXERCISE PHYSIOLOGY 149

between alveolar (either ideal or real) and arterial E partial pressures. And while this increases the V demand during the exercise, the ability to respond appropriately is constrained and, in the extreme, limited by the impaired pulmonary mechanics.

Normal Response Proles

Virgils dictum hoc opus hic labor est (there is the task and there is the toil) recognizes the distinction between work rate, an absolute physical construct, which we measure in units of power, and work intensity, a relative construct reecting its impact on the degree and perception of the consequent stress.
Moderate Exercise

Sub-yL WRs are considered to be of moderate intensity as they are (1) generally not stressful; (2) sustainable for prolonged periods of time; and (3) typied by steady states of cardiopulmonary and metabolic response (e.g., Figure 1(b)). The response time courses O normally increases with are different, however; V 2 CO is B50 a time constant (t) of B3040 s while tV 2 60 s, reecting the inuence of the fraction of metabolically produced CO2 taken up into muscle stores E changes marginally more during the on-transient. V E is slowly (tV E is B5565 s). Consequently, as tV appreciably longer than tV O2 , PAO2 and PaO2 fall transiently. But, owing to the relatively small kinetic E and V CO , the transient dissociation between V 2 PaCO2 increase is hardly discernible. The values of PaCO2 and PaO2 are typically averages of blood sampled over several respiratory cycles, providing a mean of the intrabreath oscillations. For example, end-inspiratory PACO2 is reduced because of the greater dilution of alveolar gas, while end-expiratory, or end-tidal, PCO2 (PETCO2) is increased because of increased CO2 ux across the alveolarcapillary membrane during exhalation (the increased mixed-venous PCO2 Pv % CO2 causing the slope of the alveolar phase to increase). The intrabreath PaO2 prole will, naturally, closely mirror that of PaCO2. In normal exercising subjects therefore, PETCO2 is typically higher than PaCO2 (by as much as 6 mmHg), depending on Pv % CO2 and the expiratory duration. PETCO2 should not therefore be used to represent PaCO2 in computing VD/VT. Algorithms for estimating PaCO2 from PETCO2 are poor in normal subjects and do not work in subjects with lung disease.
Heavy and Very Heavy Exercise

For the more stressful range of WRs above yL , at which the increase in arterial lactate (L a) and H a can stabilize (or even decrease) with time (heavy

O can also attain a delayed steady intensity), V 2 O that is a state but with a higher-than-expected V 2 result of a slow component of the V O2 kinetics which supplements the early response. This additional O2 ss=DWR values as high increment can result in DV 1 as 14 ml min W (rather than the sub-yL value of B10 ml min 1 W). At even higher WRs, beyond what is termed crit O steady states are not attainable ical power (CP), V 2 (very heavy intensity). All very heavy WRs are asso O2 , V E , and ciated with inexorable increases in V H a to the limit of tolerance (Figure 1(a)). In con CO2 prole appears more exponential-like trast, the V (Figure 1(a)). This can be easily misinterpreted, however; its prole conflates the inuence of the aerobic component with that from HCO3 -buffering (which peaks early in the transient and then declines as the rate of HCO3 decrease progressively slows) and that from hyperventilation (which develops slowly). O2 increases towards its maximal Above CP, V O2 max at a rate that is greater the higher the value V WR, progressively reducing tolerance time. O max and CP at On average yL occurs at B50% V 2 B75% (Figure 2) in groups of normal subjects. But both yL and CP occur at highly variable fractions of O max in individual subjects. Consequently, two subV 2 O2 max , as in Figure 1, jects exercising at the same %V could manifest markedly different patterns of re O2 max as an index of work sponse. The use of the %V intensity among subjects is therefore not justiable; E , pulmonary gas a common temporal prole of V exchange, and arterial acidbase response should be characterized as a common intensity. When the subjects limit of tolerance is set by E , as in chronic obstructive the ability to increase V pulmonary disease (COPD) patients, for example, O2 max than normal CP (and yL ) occurs at a higher %V (e.g., Figure 2). This provides the potential for using O max in a training regimen. a higher fraction of the V 2 Above CP, each progressive increase in WR results in O max . In contrast to normal subjects, the same V 2 however, this is also associated with effectively the E (Figure 2). same (limiting?) V Endurance training increases CP in normal subjects (Figure 3) and COPD patients. And even if the training only leads to a relatively small increase in the maximum WR (WRmax) achieved on an incremental exercise test, small changes in CP can translate into considerable improvements in the tolerable duration of some high fraction (e.g., 80%) of that maximum. Unfortunately, while CP is an important parameter characterizing the upper limit of sustainable power generation for this kind of exercise, it can at present only be determined as the power asymptote of a series of exhausting WRs.

150 EXERCISE PHYSIOLOGY

100 100

80 VE (l min 1)

80

60

60

40

40

20

20

VCO2 (l min 1) VO2max (%)

0 1.0

0.8

0.6

0.4

0.2

0.0 0 (a) 120 240 Time (s) 360 (b) 0 120 240 Time (s) 360

O , CO2 output V CO , and ventilatory V E responses to constant-load exercise on a cycle ergometer in Figure 1 Oxygen uptake V 2 2 O max ; this work rate was below the lactate threshold for subject b, but above for subject a. two subjects (a, b) exercising at the same % V 2 O slow component in subject a, and its absence in subject b. Reproduced from Whipp BJ, Ward SA, and Rossiter HB Note the marked V 2 (2005) Pulmonary O2 uptake during exercise: conflating muscular and cardiovascular responses. Medicine and Science in Sports and Exercise 37: 15741585, with permission from Lippincott Williams & Wilkins.

Response Limitations
When a subject has ostensibly exercised to the limit of tolerance, it is important to determine whether

particular systems that contribute to the energy exchange have reached their limit. For example, if the maximum voluntary ventilation (MVV) determined E, at rest is considered the maximum attainable V

EXERCISE PHYSIOLOGY 151

2250 2000 VO2 (ml min1) 1750 1500 1250 1000 750 500 250 0 200 400 600 800 10001200 MVV 2250 Peak VO2 2000 1750 1500 1250 1000 750 500 250 0 200 400 600 800 10001200 WR4 WR3 WR2 WR1 Critical power Peak VO
2

120 100 VE (l min1) 80 60 40 20 0 0

120 100 80 60 40 20 0 MVV

200 400 600 800 10001200 Time (sec)

200 400 600 800 10001200 Time (sec)

O and V E to ve progressively greater work rates performed on a cycle ergometer, to the limit of Figure 2 The responses of V 2 tolerance or for 20 min (whichever came rst) in a healthy control (left panels) and a COPD patient (right panels), matched for age. Note that, both in the control and the patient, the lowest WR (closed circles) corresponded to critical power, with consequent stability O ; at the higher four WRs, V O at end exercise attained V O max . In the control subject, V E at end exercise was progressively of V 2 2 2 higher with increasing work rate, but in no instance was the maximum voluntary ventilation (MVV) approached; in the COPD patient, E attaining MVV (i.e., with zero breathing reserve). Reproduced from Neder JA, however, all WRs above critical power resulted in V Jones PW, Nery LE, and Whipp BJ (2000) Determinants of the exercise endurance capacity in patients with chronic obstructive pulmonary disease: the powerduration relationship. American Journal of Respiratory and Critical Care Medicine 162: 497504, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

then the difference between this and the value actually attained at the end of exercise (Figure 4) can be considered to represent the subjects breathing reserve (BR). This can be zero (or even negative in a subject who bronchodilates during exercise) either as a result of MVV being low (lung disease patients) or E being high (highly t normal achievable WRs and V subjects). Also, if the maximum effort expiratory airow is considered to reect the greatest possible ow at a particular lung volume (not necessarily the case in COPD patients), then achieving these ows during exercise indicates an absence of ow reserve (Figure 5). Similarly, a tidal volume (VT) that encroaches upon the inspiratory capacity (IC) is reective of lack of volume reserve. Significant heart rate (HR) reserve at maximum exercise is usually judged relative to the subjects age-predicted maximum; unfortunately, this has high variability. These expected maxima therefore provide useful frames of reference for discriminating among potential causes of the exercise limitation.

Unlike normal (especially young) subjects, the tolerable WR range in patients with lung disease is constrained by a combination of pulmonary factors, chief of which are: 1. impaired pulmonary-mechanical and gas-ex E demands; change functions, which increase the V 2. limitations of airow generation or lung distention; E 3. increased physiological costs of meeting the V demands, in terms of respiratory muscle work, perfusion, and O2 consumption; 4. predisposition to shortness-of-breath or dyspnea, consequent to the high fraction of the achievable ow or lung distention demanded by the WR, commonly exacerbated by arterial hypoxemia and early-onset metabolic acidosis; along with 5. other factors, such as coexistent heart disease, pulmonary hypertension, nutritional deciencies, and detraining as a result of low-activity patterns of daily living the latter helping explain the high

152 EXERCISE PHYSIOLOGY

500 (WR CP) t = W Flow rate (l s1) 400 Exercise rest

10
8

10
8

6 4 2 0 2 4 200
L

6 4 2 1 2 3 4 5 0 2 4 Normal 6 8 Airways disease 1 2 3 4 5

WR (W )

300

6 8

100 0 10 Time (min)

Figure 3 Relationship between work rate (WR) and its tolerable duration (t) for exercise performed above critical power (CP), before (open circles, solid line) and after (closed circles, dashed line) a period of exercise training. Note that both relationships are well described by a hyperbolic relationship (see equation box), where W 0 is the curvature constant. Also note that, as training increases CP, a given supra-CP work rate can be sustained longer post-training. QL is the lactate threshold.

20

Figure 5 Spontaneous ow-volume curves from a normal subject (left) and a COPD patient at rest (inner black loop), at submaximal exercise (blue loop), and at maximal exercise (outer black loop) (right); inspiration downwards; expiration upwards. Note that for the COPD patient, the maximal exercise curve impacts on the volitionally generated maximal ow-volume curve, with dynamic hyperination; this is not the case for the normal subject. Modied from Leaver DJ and Pride NB (1971) Flow Volume curves and expiratory pressures during exercise in patients with chronic airway obstruction. Scandinavian Journal of Respiratory Diseases Supplement 77: 2327, with permission.

incidence of fatigue rather than dyspnea as the dominant reported cause of exercise limitation.
MVV BR BR BR VE

VO2
E response to incremental exerFigure 4 Schematic of the V O . In healthy subjects (black), note that cise, as a function of V 2 E at the lactate threshold (lled symbols) and at V O max (open V 2 symbols) becomes progressively greater as tness increases: sedentary (circles), normal tness (squares), high tness (diamonds). As maximum voluntary ventilation (MVV) is unaffected by training status, breathing reserve (BR) therefore decreases. In E at any particular V O is increased a COPD patient (blue), V 2 (reecting the characteristically increased ventilatory requirement) and MVV (dashed-dotted line) is reduced. Modied from Whipp BJ, and Pardy R (1986) Breathing during exercise. In: Macklem P and Mead J (eds.) Handbook of Physiology, Respiration (Pulmonary Mechanics), pp. 605629. Washington, DC: American Physiological Society, used with permission from the American Physiological Society.

The increased airways resistance and/or decreased pulmonary recoil pressure reduces maximum expiratory airow in COPD patients. This reduces E the effective operating range of the subjects V during exercise. The V E demands, however, are commonly greater than normal, reecting the high VD/VT (a component of which is also seen, as a result of loss of lung recoil, in the otherwise healthy elderly). This E =V O and V E =V CO which may be leads to high V 2 2 further increased from arterial hypoxemia. However, some COPD patients can have higher-than-normal PaCO2, especially those with poor peripheral chemosensitivity. E demands and The combination of increased V decreased maximum-attainable V E leads to little or no BR at maximum exercise (Figures 25). COPD patients, especially, also generate spontaneous expiratory airows during exercise which equal, or even exceed, those achieved at a given lung volume during a maximal ow-volume maneuver (Figure 5). The seeming paradox of expiratory airow during exercise exceeding that generated during a maximal expiratory effort at rest can be explained by: 1. bronchodilatation from exercise-induced increases in circulating catecholamines;

EXERCISE PHYSIOLOGY 153

2. the forced-expiratory maneuver from total lung capacity allows lung units with fast mechanical ts (the product of airways resistance and thoracic compliance) to empty at high lung volumes, leaving the longer t units to empty at lower volumes. When these less-than-maximum lung volumes are attained during spontaneous breathing, the fast t units are now recruited at these lower lung volumes, resulting in greater airow at that lung volume; and 3. maximum expiratory airow not being achieved with maximum expiratory effort (especially at low lung volumes) because of dynamic airway compression, and even closure in some cases, during the forced maneuver. The increase in end-expiratory lung volume during exercise (dynamic hyperination) in COPD patients (Figure 5) is a result of the mechanical t values being long with respect to the time available for expiration. This results in the VT encroaching upon the compliance limits, adding a restriction-like component to the obstruction. Factors that reduce breathing frequency ( fB), such as hyperoxic inspirates and/or physical training, prove effective at reducing the hyperination and improving exercise tolerance. When the upper limit for exercise tolerance is set by pulmonary determinants, other components of the bodys energy supply systems may not be stressed to their limits. Consequently, maximum HR, O2 pulse, and L a are often markedly less than predicted in COPD patients. Although the pattern of blood-gas response to exercise is highly variable in such patients, in many cases the resting levels of PaO2 can be maintained without further hypoxemia; diffusion limitation across the alveolarcapillary bed does not seem to be significantly contributory and the in dispersion does not appear to worsen. A =Q creased V Also, those patients who are capable of generating a metabolic acidosis during exercise usually evidence little or no respiratory compensation owing to the obstructive constraint. In patients with restrictive lung diseases, such as diffuse interstitial brosis, reduced airow generation (at a particular lung volume) is not of concern. Rather, the increased pulmonary elastance demands greater inspiratory muscle force and increased work of breathing. This predisposes to their typical tachypnea (fB450 min 1 being common at maximum exercise), with VT often reaching their (reduced) IC. Unlike COPD, however, the hypoxemia in these patients typically worsens as WR increases not, it matching A =Q seems, because of further impaired V but because of increasing diffusion limitation, often

leading to (A a)O2 in excess of 60 mmHg at the limit of tolerance. While exercise does not usually worsen the degree of airway obstruction in the emphysematous and/or bronchitic forms of COPD, it typically does so in patients with asthma. Although the exercise-induced bronchoconstriction which can also occur in subjects with no history (or even a recognition) of airway hyperreactivity is sometimes manifest during exercise, it is most typically a postexercise phenomenon, with high-intensity exercise the more potent trigger. A prior moderate-intensity warm-up can ameliorate the degree of bronchoconstriction during a subsequent high-intensity exercise bout, as does for a short period a prior exercise-induced bronchospastic episode itself.

Patterns of Response Indicative of Disease

While the constant-load exercise test provides a closer simulation of the demands for sustained occupational or daily living tasks, the appropriateness of the integrated system responses to exercise is best studied (at least, for the initial exercise evaluation) by means of an incremental test which spans the entire tolerance range. This allows both cause(s) and severity of exercise intolerance to be identied from considerations of the normalcy of response of variables of interest, compared with those for an appropriate reference population. It can also establish both the limits and effective operating range of system function, allowing disability and impairment to be assessed and training protocols to be optimized, and providing a frame of reference for discriminating the need for, or benets of, therapeutic interventions (including those related to major surgery). A test duration of 20 min or less is usually sufcient, comprising: 1. a resting phase, 2. a control phase of at least 3 min of unloaded exercise (or another suitably low WR) to ensure that the responses have stabilized, 3. a linearly incremental exercise phase to the limit of tolerance, and 4. a recovery phase. The results are not appreciably different when WR is increased continuously (ramp test) or by uniformly small amounts over short intervals (e.g., 1-min incremental test). An incremental phase duration of B10 min provides optimum discrimination, with 1020 W min 1 being an appropriate incremental rate for healthy nonathletic subjects but as low as

154 EXERCISE PHYSIOLOGY

5 W min 1 in a patient. A subsequent constant-load test (at a now-known intensity) may, on occasion, be needed to gain additional information about particular system response kinetics.
Useful Noninvasive Relationships

slope over the lower reaches of their WR range, which then becomes reduced in concert with ECG evidence of developing ischemia. O relationship The prole of V CO as a CO V V 2 2 2 CO function of V O2 (V slope) is one in which the V 2 response initially lags behind that of V O2 early in the transient (as some of the metabolically produced CO2 is diverted into the body stores) and then in O2 (Figure 6). creases often as a linear function of V O in this The slope of the relationship DV CO2 =DV 2 region has been termed S1, with a value typically close to unity in subjects on a typical Western diet. When the aerobically produced CO2 is supple mented by additional CO2 released from HCO3 buffering, the slope increases (Figure 6) termed S2 in this higher WR region. As the amount of CO2 released in the proton-buffering process is a function not of the magnitude of HCO3 decrease but its rate of decrease, S2 is highly dependent on the WR incrementation rate. At slow incrementation rates, additional CO2 from compensatory hyperventilation CO2 in the S2 range. The presence of an supplements V isocapnic buffering region with more rapid WR incrementation rates obviates the concern for hyperventilation being the cause of the slope increase. Having ruled out both aerobic metabolism and hyperventilation as the source of the increased S2, one is left with the remaining source HCO3 de E retains the same relacrease reecting yL . Why V CO2 in this supra-yL region as below yL, tionship to V E increases as a function of V O2 but not that is, V CO2 (Figure 6) is not fully understood. But when the V V-slope plot may not be partitioned into two defensibly linear segments, the unit tangent to the curve may be used as a second-order estimate of yL . At a CO , however, the beginning of an increase in higher V 2 E =V CO and decrease in PETCO2 provides evidence V 2 of compensatory hyperventilation. This is termed the respiratory compensation point (RCP). CO2 relationship and ventilatory equi E V V E V CO2 relationship is linear up to valents The V RCP (Figure 6), with a slope of B25 in healthy E =V CO2 young subjects but increasing with age. V CO however declines hyperbolically with respect to V 2 E V CO slope itself), re(toward the value of the V 2 E interecting the inuence of the small positive V E =V CO is closely cept. As discussed previously, V 2 linked to VD/VT in the regulation of PaCO2 and pHa E =V CO2 reect either (eqn [6]). Thus, high values of V a low PaCO2, high VD/VT, or both. But, without arterial blood sampling, how can a low PaCO2 be E =V CO ? It cant ruled out as the cause of the high V 2 at least not conclusively! This is especially so when

The response proles and maximum values of certain noninvasive variables, in addition to the standardly assessed electrocardiogram (ECG) and arterial blood pressure, have proven especially informative with respect to inferring system pathophysiology, in the context of the available normal predicted values. O WR relationship In response to a constant V 2 O attains a constant steady-state value (over WR, V 2 the WR range for which steady states are attainable). For a ramp test (e.g., Figure 6), a constant rate of change of WR yields a constant rate of change O2 , after a small lag-phase (reecting the sysof V tem response kinetics). The gain of this response O =DWR has been shown not to differ from DV 2 that of the steady-state response (normally 9 11 ml min 1 W). The incremental gain is therefore often used as an index of the work (in)efciency. But while the rate of change, in the linear region of the ramp, is normally the same as that for the steady O2 at any WR is lower. state test, the actual value of V However, in many patients with cardiopulmonary diseases, this incremental gain can be very low (e.g., 8 ml min 1 W 1 or less), suggestive of impaired aerobic exchange. The highest value achieved with good subject ef O 2 V O peak or V O2 max fort is termed the peak V 2 O2 does not continue to increase with further when V O peak is not increase in WR. While the good effort V 2 different with different ramp slopes, the WRmax is progressively greater the faster the ramp; this must be accounted for when choosing a %WRmax for a subsequent constant-WR strategy. Abnormalities of the O response predominantly reect inadequacies of V 2 cardiovascular function. Consequently, poorly t (but otherwise normal) subjects typically evidence O peak and yL but with a normal DV O2 =DWR. low V 2 Patients with lung disease, other than those with significant pulmonary vascular dysfunction, present a O response pattern similar to the sedentary normal V 2 O peak that is even more signisubject but with a V 2 ficantly reduced, that is, when impaired lung mechanics limit the tolerable WR range. Patients with cardiac, peripheral, and/or pulmonary vascular disorders, on the other hand, often also manifest ab O2 WR slope throughout the increnormally low V mental test. Those with coronary heart disease commonly present with a relatively normal response

EXERCISE PHYSIOLOGY 155

B 1000 VCO2 (ml min1) A VO2 (ml min1) 1000 B

500

500 S1 = f( ) 0

VO

1000 B

WR (W)

80

60 50 40 30 20

70 60 50 C 40 A VE (l min1)

50 40 30 20 10 1000 0 0 S2 =C

A=

VE /VO , VE/VCO 2 2

B = VO peak (max ?)
2

C = VE (in)efficiency
1000

VO2 140 A 130 120 110 B

VCO 10

D = HR max
D

HR, VO2/HR (min1, ml bt 1)

60 PET CO2, PET O2 50 40 30 20

180 150

E = O2-P max F = R pattern G = Breathing pattern VE limitation ?

5 E 100

VO2

1000

60

WR (W)

80

1.6 1.4 VT (ml)

1500

1000 G 500

RER

1.1 F 0.9 0.6

VO

1000

VE

60

Figure 6 Left and middle panels show a multipanel display of the system responses of a series of physiological variables to an incremental cycleergometer exercise test performed to the limit of tolerance. The selected clusters of variables are those that have been shown to be particularly useful both for establishing parameters of system function (e.g., the estimated lactate threshold (yL), the gain O =DWR) and peak of the V O response, and the ventilatory efciency (DV E =DV CO ) and for assessing the normalcy, or otherwise, (DV 2 2 2 of the magnitude and prole of system responses. The values commonly derived from such a display are designated A, B, C, D, E, F and G in the data display; their physiological equivalents are given in the right panel. Left panel, top to bottom: the steady-state level of CO2 CO , ventilatory equivalents for O2 and CO2 (V E =V O ,V E =V CO ), end-tidal PCO2 and PO2 (PETCO2, PETO2), and the respiratory output V 2 2 2 O vs. WR; V E vs. V CO ; heart rate (HR) and O2-pulse (V O / O . Middle panel, top to bottom: V exchange ratio (RER) as a function of V 2 2 2 2 E. HR) vs. WR; and tidal volume (VT) vs. V

PETCO2 is low simultaneously, less so if it is normal, but it is an unlikely cause if PETCO2 is high. Patients with lung disease consequently manifest E 2V CO slope and high values for both the V 2

CO2 measured at its minimum or at yL . Patients E =V V with heart disease also evidence high values of these variables during exercise, predominantly (it appears) to an increased VD/VT resulting from uneven

156 EXERCISE PHYSIOLOGY

distribution of pulmonary perfusion. The magnitude of the increase is correlative of poor prognosis, espe O peak are also low. cially when yL and V 2 E =V O2 following its initial The increase in V VD/VT -linked decline is used as an index of the ad E drive above yL . ditional V End-tidal gas tensions These are easy to measure and extremely difcult to interpret. As described above, PETCO2 normally increases progressively with increasing WR up to yL (Figure 6); it then stabilizes with increasing WR in the region of isocapnic buffering (as the effect of the increasing Pv % CO2 is offset by the decreasing TE), and subsequently decreases as frank compensatory hyperventilation is manifest. PETO2, in contrast, decreases progressively up to yL, at which point it begins to increase sys E increase exceeds that of tematically as the rate of V V O2 (Figure 6), that is, there is no iso-oxic equivalent of the isocapnic period. Mean PaCO2, however, differs from mean PACO2 inhomogeneities and/or right-to A =Q as a result of V left shunt, leading to PETCO2 being commonly less than PaCO2 in patients with COPD or pulmonary vascular disease, for example. Consequently, a PETCO2 which is less than mean PaCO2 during exercise (or even equal to it) is reective of abnormal gas exchange. Breathing pattern While some individuals entrain fB to some unit multiple of their cycling or stride fre E during exercise normally increases prequency, V dominantly through changes in VT over the relatively linear thoracic compliance range, thereby minimizing airow-related work of breathing. At higher WRs, E are usually mediated largely further increases in V through increases in fB, obviating large increases in elastic work of breathing over the shallow thoracic E is commonly used to compliance region. The VTV discern both the breathing pattern response and the E and/or VT reach limiting values. extent to which V Patients with COPD have, in addition to an aug E response, a disproportionately large fB mented V increase during the exercise and little or no BR at the limit of tolerance. This is also true of the patients with restrictive disorders, but in this case VT increases to the level of the IC early in exercise with fB often in excess of 50 min 1. O HR relationship and O2 pulse HR during exV 2 O2 to an ageercise is normally a linear function of V dependent maximum (averaging B220age, but with a large standard deviation of B10 min 1) and a slope that is an inverse function of a physical tness. As this linear relationship has a positive intercept on the O =HR increases HR axis, the O2 pulse O2 -P V 2

hyperbolically as WR increases. But the O2-P is of further interest, as it is numerically equivalent to the product of stroke volume and the arteriovenous O2 content difference. And so, if O2-P fails to increase with increasing WR, then either both variables are constant or one is increasing while the other decreases either way a reection of poor cardiovascular functioning. The rate at which O2-P increases is not only abnormally low but actually becomes at in patients with impaired cardiovascular function, including heart failure, hypertrophic cardiomyopathy, and coronary artery disease, and also patients with significant pulmonary circulatory pathology.

Conclusion
A clinical exercise tests is a device for educing aws or abnormalities of physiological system function that contribute to exercise intolerance. The results of an appropriately designed test can also provide a frame of reference for the need for, and any benets of, interventions designed to improve performance. Appropriately interpreting deviations from an expected response prole (i.e., characteristic of a reference population) needs a clear justication of their underlying physiological determinants. Commercially available carts now readily provide a wealth of relevant evidence. But this is information; appropriate interpretation remains the investigators challenge.
See also: Asthma: Exercise-Induced. Carbon Dioxide. Energy Metabolism. Hypoxia and Hypoxemia. Neurophysiology: Neuroanatomy. Space, Respiratory System. Ventilation: Overview; Control.

Further Reading
American College of Sports Medicine (1995) Guidelines for Exercise Testing and Prescription. Baltimore: Williams & Wilkins. American Thoracic Society/American College of Chest Physicians (2003) Statement on cardiopulmonary exercise testing. American Journal of Respiratory and Critical Care Medicine 167: 211277. Casaburi R and Petty TL (eds.) (1993) Principles and Practice of Pulmonary Rehabilitation. Philadelphia: Saunders. Gallagher C (1990) Exercise and chronic obstructive pulmonary disease. Medical Clinics of North America 74: 619641. Johnson BD, Badr MS, and Dempsey JA (1994) Impact of the aging pulmonary system on the response to exercise. Clinics in Chest Medicine 15: 229246. La nnergren J and Westerblad H (2003) Limits to human performance caused by muscle fatigue. Physiology News 53: 710. Leaver DJ and Pride NB (1971) Flowvolume curves and expiratory pressures during exercise in patients with chronic airway obstruction. Scandinavian Journal of Respiratory Diseases Supplement 77: 2327. Neder JA, Jones PW, Nery LE, and Whipp BJ (2000) Determinants of the exercise endurance capacity in patients with chronic obstructive pulmonary disease: the powerduration relationship.

EXTRACELLULAR MATRIX / Basement Membranes 157

American Journal of Respiratory and Critical and Care Medicine 162: 497504. Roca J and Whipp BJ (eds.) (1997) Clinical Exercise Testing, European Respiratory Monograph, vol. 2, no. 6. Shefeld: European Respiratory Journals. Rowell LB (1993) Human Cardiovascular Control. New York: Oxford University Press. Wagner PD (1996) Determinants of maximal oxygen transport and utilization. Annual Review of Physiology 58: 2150. Wasserman K, Hansen JE, Sue DY, Stringer WW, and Whipp BJ (2004) Principles of Exercise Testing and Interpretation. Philadelphia: Lippincott Williams & Wilkins. Weisman IM and Zeballos RJ (eds.) (1994) Clinical exercise testing. Clinics in Chest Medicine 15. Whipp BJ and Pardy R (1986) Breathing during exercise. In: Macklem P and Mead J (eds.) Handbook of Physiology, Respiration (Pulmonary Mechanics), pp. 605629. Washington, DC: American Physiological Society. Whipp BJ and Sargeant AJ (eds.) (1999) Physiological Determinants of Exercise Tolerance in Humans. London: Portland Press. Whipp BJ, Ward SA, and Rossiter HB (2005) Pulmonary O2 uptake during exercise: conflating muscular and cardiovascular responses. Medicine and Science in Sports and Exercise 37: 15741585.

EXTRACELLULAR MATRIX
Contents

Basement Membranes Elastin and Microbrils Collagens Matricellular Proteins Matrix Proteoglycans Surface Proteoglycans Degradation by Proteases

Basement Membranes
J H Miner and N M Nguyen, Washington University School of Medicine, St Louis, MO, USA
& 2006 Elsevier Ltd. All rights reserved.

Introduction
Basement membranes (BMs) are thin sheets of specialized extracellular matrix that were rst identied by transmission electron microscopy as continuous ribbon-like structures adjacent to a subset of cells. They are evolutionarily ancient structures, being present even in primitive organisms such as sponges and Hydra. In mammals, BMs underlie endothelial and epithelial cells and surround all muscle cells, fat cells, and peripheral nerves. They play important roles in ltration, in compartmentalization within tissues, and in maintenance of epithelial integrity, and they inuence cell proliferation, differentiation, migration, and survival. In the lung, BMs are associated with bronchial and vascular smooth muscle cells, bronchial epithelium, nerve, and pleura, and they are part of the airblood barrier between microvascular endothelial cells and alveolar epithelial cells (Figure 1). The biochemical characterization of BM proteins was facilitated by the EngelbrethHolmSwarm (EHS) tumor, a transplantable tumor of mouse origin that produces large amounts of BM components that can be easily isolated. Studies of EHS tumor matrix proteins led to the identication of the four

Abstract
Basement membranes (BMs) are thin sheets of specialized extracellular matrix that underlie endothelial and epithelial cells and surround all muscle cells, fat cells, and peripheral nerves. They play important roles in ltration, in compartmentalization within tissues, and in maintenance of epithelial integrity, and they inuence cell proliferation, differentiation, migration, and survival. In the lung, BMs are associated with bronchial and vascular smooth muscle cells, bronchial epithelium, nerve, and pleura, and they are part of the airblood barrier between microvascular endothelial cells and alveolar epithelial cells. Collagen IV and laminin are the major components of all BMs which also contain entactin/nidogen and various heparan sulfate proteoglycans, including perlecan. Two collagen IV heterotrimeric protomers, (a1)2a2 and a3a4a5, are found in alveolar basement membranes, but the latter does not appear to have an important function in the lung. However, the a3 chain contains the epitope that is attacked in Goodpastures syndrome, an autoimmune disease characterized by pulmonary hemorrhage and glomerulonephritis. Laminins containing the a5 chain are widespread in both developing and adult lung. Preventing expression of laminin a5 via targeted mutagenesis interferes with lung lobe septation and maturation of the lung parenchyma.

158 EXTRACELLULAR MATRIX / Basement Membranes

BM ALV BM RBC IM

FIB

AT 1

AT 1

EC ALV BM IM

Figure 1 Schematic diagram of the airblood interface. A basement membrane (BM) surrounds the capillary and separates the endothelial cell (EC) from the thin-walled alveolar epithelial cell. ALV, alveolus; AT 1, alveolar type I cell; RBC, red blood cell; FIB, broblast; IM, interstitial matrix.

major classes of proteins present in all BMs: type IV collagen, laminin, entactin/nidogen, and sulfated proteoglycans. Though all BMs contain these four components, in vertebrates there are different isoforms of each that are differentially distributed in BMs, leading to significant molecular heterogeneity among BMs. This presumably translates into functional heterogeneity. In this article, we focus on the isoforms of type IV collagen and laminin that are expressed in the lung and how they relate to lung function and disease.

Type IV collagen -chain

7S

Collagenous Trimerization

NC1

(a)

Protomers 2 1 1 4 3 5 6 5 5

Structure
Type IV Collagen

Type IV collagen describes a family of six genetically distinct a-chains, a1a6, which heterotrimerize with each other to form protomers (Figure 2). Protomers represent the building blocks of the collagen IV network. Mature collagen IV a-chains are B180 kDa and contain three major domains: a short N-terminal 7S domain, a long central collagenous domain, and a C-terminal noncollagenous 1 (NC1) domain of B230 amino acids. The collagenous domain is dened by the presence of stretches of Gly XY triplet repeats, which allows formation of stable, stiff triple helices. X and Y can be any amino acid but are most commonly proline. Collagen IV is unique compared to other collagens (there are 26 other types in humans) because its chains all contain multiple interruptions of the Gly XY repeats within the

(b)

Figure 2 Structure of type IV collagen. (a) Each of the six individual a-chains contains an N-terminal 7S domain, a central collagenous domain with multiple interruptions, and a C-terminal noncollagenous domain (NC1). (b) Three a-chains assemble in a dened fashion to form the three types of protomers. All three are found in the lung, though the (a5)2a6 protomer is restricted to bronchial smooth muscle BMs.

collagenous domain. These interruptions are presumed to impart exibility to the trimer and thus to the BM of which it is a part.
Laminin

Laminins constitute the major noncollagenous component of BMs. Laminins are heterotrimeric

EXTRACELLULAR MATRIX / Basement Membranes 159

1,2,3B,5 LN LE L4 L4 LN 1,2 L4 L4 LN 1,3 1,2 3A,4 1,3 3 3A 2

Coiled-coil

LG1 LG2 LG3 LG4

LG5

LG1 LG2 LG3 LG4

LG5

LG1 LG2 LG3

LG Lm-1,2,3,4,6B,7B,10,11,12,15 Lm-6A,7A,8,9,14 Lm-5A

Figure 3 General structure of laminins. Laminin (Lm) heterotrimers (with Arabic numbers) exist in three shapes: cruciform, Y- or Tshaped, and rod-shaped. The globular domains of the short arms, laminin N-terminal (LN) and laminin four (L4), are separated by rodlike laminin EGF-like repeat (LE) domains. The abg chains trimerize via the coiled-coil domains to form the long arm. The C-terminal LG domain is unique to a chains.

Table 1 Laminin heterotrimers and chain composition Laminin-1 Laminin-2 Laminin-3 Laminin-4 Laminin-5A Laminin-5B Laminin-6A/B Laminin-7A/B Laminin-8 Laminin-9 Laminin-10 Laminin-11 Laminin-12 Laminin-14 Laminin-15 a1b1g1 a2b2g2 a1b2g1 a2b2g1 a3Ab3g2 a3Bb3g a3A/Bb1g1 a3A/Bb2g1 a4b1g1 a4b2g1 a5b1g1 a5b2g1 a2b1g3 a1b2g3 a1b5g3

laminin a3b3g2, or laminin-5), the N-terminal short arms are truncated and/or cleaved by proteases.

Regulation of Production and Assembly

Type IV Collagen

glycoproteins comprised of one a, one b, and one gchain (Figure 3). Fifteen laminin isoforms have been identied from combinations of ve a-, four b-, and three g-chains (Table 1). The a-, b-, and g-chains share structural homologies, including the laminin N-terminal domains (LN), laminin epidermal growth factor (EGF)-like repeats domains (LE), laminin 4 domains (L4), and the a-helical coiled-coil long arms. Unique to a-chains are the large C-terminal laminin globular (LG) domains that consist of ve smaller units (LG15). These a-chain LG domains are sometimes cleaved by proteases to make them smaller. For a subset of laminin chains (especially

The six collagen IV a-chain genes are arranged in head-to-head pairs on three different chromosomes, and each pair is thought to be regulated by a bidirectional regulatory element. Col4a1 and Col4a2 are the most widely expressed pair. The a1- and a2(IV)-chains are produced by the EHS tumor and are found in virtually all BMs throughout the body. Col4a3 and Col4a4 have a more restricted expression pattern, but the a3- and a4(IV)-chains are especially prominent in kidney glomerular and lung alveolar BMs. Col4a5 and Col4a6 have different but overlapping expression patterns. The a6(IV)-chain is found in smooth muscle and in some kidney BMs, always localizing with the a5(IV)-chain, but a5(IV) is present at additional sites wherever a3 and a4(IV) are present, including kidney glomerular and lung alveolar BMs. Biochemical studies have revealed the molecular basis for these ndings. The six a-chains can trimerize in only three specic combinations to make three different protomers: (a1)2a2, a3a4a5, and (a5)2a6 (Figure 2). Specicity for trimerization is encoded by residues in the NC1 domains. The protomers

160 EXTRACELLULAR MATRIX / Basement Membranes

assemble in the endoplasmic reticulum and are then secreted into the extracellular matrix. A collagen IV network forms from these protomers via both NC1 domain interactions in which two protomers participate, and N-terminal 7S domain interactions in which four protomers participate. Covalent cross-linking of 7S domains and of lateral protomer associations provides increased stability to the network.
Laminin

alv aw alv bv

Laminins form by intracellular self-trimerization of individual a-, b-, and g-chains (100400 kDa) into a cruciform structure (400800 kDa) with a common triple helical coiled-coil in the long arm that terminates in the large LG domain contributed by the a chain (Figure 3). Secreted laminins self-assemble into a ne network through interactions at the N-terminal globular domains, and this network then connects with the collagen IV network via entactin/nidogen and proteoglycans to stabilize the BM. Numerous cell types including epithelial, endothelial, and mesenchymal cells are capable of synthesizing laminins. In the developing lung, laminin a1 is expressed by both epithelial and mesenchymal cells and is deposited in the BM of developing airways. By the second trimester of gestation, laminin a1 can be weakly detected by antibody staining but not by in situ hybridization. Laminin a1 is absent from late embryonic and postnatal lung. Laminins a2a5 are present in both embryonic and adult lung but differ in source and distribution. Laminin a2 is expressed by mesenchymal cells and deposited in the airway BM. Laminins a3A and a3B are expressed by epithelial cells and deposited in the epithelial BM of the airways and alveoli. Laminin a4 is expressed by mesenchymal cells and endothelium is detected in vascular BMs and in BMs surrounding smooth muscle cells of the large airways. Laminin a5 is expressed by epithelial and endothelial cells and is present in epithelial BM of the airways, alveoli, blood vessels, and pleura (Figure 4). It is of note that while epithelial BMs often contain multiple laminin a chains, and thus multiple laminin heterotrimers, the only laminin a-chain present in the visceral pleura is laminin a5. Laminins b3 and g2 are exclusively found in laminin-5 with laminin a3 and parallel laminin a3 in expression and distribution. Laminins b1, b2, and g1 exhibit a wide pattern of expression and distribution. Laminin chain expression and production are regulated by multiple factors, such as growth factors, transcription factors, cytokines, and hormones. In injury states, transforming growth factor beta

alv

Figure 4 Laminin a5 is widely deposited in lung BMs. A frozen section of adult mouse lung was stained with a rabbit antimouse laminin a5 antibody. Alveolar, bronchial epithelial, smooth muscle, and endothelial BMs contain laminin a5. alv, alveolus; bv, blood vessel; aw, airway. Original magnication 200.

(TGF-b) is a potent stimulator of extracellular matrix production, but its effect on laminin synthesis varies according to cell type and laminin chain examined. In general, epithelial and endothelial cells increase laminin production in response to TGF-b, while no response or a decrease in laminin production is seen in broblasts. In cell culture or in differentiating or diseased animal tissues, interleukin-1b, glucose, interferon a and g, and glucocorticoids have all been shown to increase laminin transcription.

Biological Function
Collagen IV

For many years the collagen IV network was thought to serve as a required scaffold for BM assembly. However, targeted mutation of the linked mouse Col4a1 and Col4a2 genes shows that BMs can form in the total absence of collagen IV, though they exhibit impaired integrity, and homozygous mutant embryos die just past mid-gestation. This suggests that the main function of a1 and a2(IV) is to maintain the stability of BMs. This may also be true for the a3-, a4-, and a5(IV)-chains. In the absence of any one of them due to mutation of the encoding gene, the a3a4a5 protomer cannot form, so all three chains are missing from sites where they normally colocalize. The physiological result of this is a disease called Alport syndrome, dened as hereditary glomerulonephritis associated with sensorineural hearing loss and lens abnormalities. The major histopathological nding is thickening and splitting of the glomerular BM, suggesting that its integrity is

EXTRACELLULAR MATRIX / Basement Membranes 161

impaired. However, despite the abundance of the a3a4a5 protomer in the normal lung, there is no obvious lung phenotype associated with Alport syndrome in humans or in animal models for the disease.
Laminin

cells. Thus, laminin a5 has a crucial role in maturation of the lung parenchyma.

Receptors
The primary receptors that bind collagen IV and laminin are the integrins. Integrins are a family of ab heterodimeric transmembrane glycoproteins that serve as adhesion receptors, signaling receptors, components of mechanotransducers, and modulators of cell proliferation and apoptosis. Pulmonary epithelial cells express many integrins, but those that interact with laminin are integrins a3b1 and a6b4. a1b1, a2b1, and a3b1 have been reported to interact with type IV collagen. The importance of integrins to lung development is demonstrated in mice lacking integrin a3, which exhibit reduced airway branching and proximalization of distal epithelium. Additional, nonintegrin receptors for laminin include dystroglycan, cell surface heparan sulfate proteoglycans (syndecans), and the Lutheran blood group glycoprotein.

Numerous studies have shown that laminins are important for maintenance of alveolar epithelial cell (AEC) phenotype, promotion of adult AEC proliferation, and protection of AECs from apoptosis. Laminins affect lung development at multiple stages and in multiple compartments. With regard to the lung, laminin-1 (a1b1g1) is the most studied laminin, but it is present only during early stages of lung development. Nonetheless, in vitro studies with lung bud cultures show that addition of laminin-1 antibodies or proteolytic fragments disturbs lung bud branching morphogenesis. On the other hand, laminin a2 is important for bronchial smooth muscle cell differentiation in vitro, and ablation of the entactin/nidogen binding site in laminin g1 causes impaired sacculation. Targeted mutagenesis in mice has been used extensively to study the biological functions of laminins. Lama1 / , Lamb1 / , and Lamc1 / embryos die very early in development long before lung formation. Mutation of the Lama2, Lama4, or Lamb2 genes in mice does not cause embryonic or perinatal death and does not result in any described lung phenotype. Lama3 / , Lamb3 / , and Lamg2 / mice die 13 days after birth from nonrespiratory causes, but their lungs have not been closely examined. Most Lama5 / mice die between embryonic day 14 and 17, after completion of the pseudoglandular stage of lung development but before saccular and alveolar stages. Examination of Lama5 / mice through E16.5 revealed normal branching morphogenesis but incomplete to absent lobar septation, lack of visceral pleura BM formation, and ectopic deposition of laminin a4 in airway BMs. Production of a lung-epithelial cell-specic laminin a5 knockout mouse shows a role for laminin a5 in the latter stages of lung development. These mice exhibit abnormal sacculation/alveolization with enlarged, emphysematous-appearing distal airspaces, and they died within hours after birth from respiratory compromise. Distal epithelial cell differentiation and maturation were severely impaired, with markedly reduced alveolar type II cells and a near absence of alveolar type I cells. In addition, cell proliferation was decreased, and apoptosis was increased. The density of the capillary network was diminished, and this was associated with decreased production of vascular endothelial growth factor by lung epithelial

Collagen IV in Respiratory Disease

There are no apparent lung abnormalities typically associated with Alport syndrome, despite the absence of the a3a4a5 protomer. This is probably due to compensation by the a1/a2(IV) network, which is normally present throughout the lung. However, there is a rare X-linked form of Alport syndrome that is associated with diffuse leiomyomatosis (DL). This usually affects esophageal smooth muscle, but benign tumors of bronchial smooth muscle have also been reported. Alport syndrome with DL is always accompanied by deletions that encompass the 50 end of COL4A5 and extend into the COL4A6 gene no farther than the second intron. Interestingly, deletions that extend farther into COL4A6 do not result in DL, suggesting it is not merely the absence of a6 in smooth muscle that causes the tumors. The autoimmune disorder Goodpasture syndrome severely affects both the kidney glomerulus and the lung parenchyma. In Goodpasture syndrome, antibodies to a cryptic epitope in the NC1 domain of collagen a3(IV) bind both to the kidney glomerular BM and to the lung alveolar BM and cause glomerulonephritis and pulmonary hemorrhage, respectively. The mechanism of exposure of the epitope is unknown, but there has been speculation that tobacco smoke, hydrocarbons, or endogenous oxidants may be involved.

Laminin in Respiratory Disease

While laminins are both numerous and prominent in the lung, as of yet, no clinical human pulmonary

162 EXTRACELLULAR MATRIX / Elastin and Microbrils

disease has been denitively associated with laminins. Lung injuries, whether from infection, acute respiratory distress syndrome, toxins, or non-specic causes, can manifest abnormalities and discontinuities of the BM, as well as elevated laminin fragments and laminin concentration in bronchoalveolar lavage uid. However, no causative relationship has yet been discovered.
See also: Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Angiogenesis, Angiogenic Growth Factors and Development Factors. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Lung Development: Overview. Matrix Metalloproteinases. Mesothelial Cells. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumors, Benign. Vascular Endothelial Growth Factor.

Elastin and Microbrils

E C Davis and B Quiney, McGill University, Montreal, QC, Canada
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Elastin is an extracellular matrix protein that is critical for lung development and function. Elastin comprises B2.5% of the dry weight of the lung and is distributed as elastic bers throughout virtually all pulmonary structures. Closer investigation of elastic bers reveals that they actually contain two components, a core of insoluble elastin and a peripheral mantle of 10-nm microbrils. The microbrils are composed of a number of proteins; however, most of these proteins are only associated proteins and not true constituents. The main constituent protein of microbrils is brillin. As the name implies, elastin provides the lung with the property of elastic stretch and recoil. Since this property is clearly essential for lung function, abnormal assembly or destruction of lung elastic bers can lead to devastating pulmonary disease.

Further Reading
Aberdam D, Virolle T, and Simon-Assmann P (2000) Transcriptional regulation of laminin gene expression. Microscopy Research and Technique 51: 228237. Colognato H and Yurchenco PD (2000) Form and function: the laminin family of heterotrimers. Developmental Dynamics 218: 213234. Crouch EC, Martin GR, Brody JH, and Laurie GW (1997) Basement membranes. In: Crystal RG and West JB (eds.) The Lung: Scientic Foundations, pp. 769791. Philadelphia: LippincottRaven. Hudson BG (2004) The molecular basis of Goodpasture and Alport syndromes: beacons for the discovery of the collagen IV family. Journal of the American Society of Nephrology 15: 25142527. Hudson BG, Tryggvason K, Sundaramoorthy M, and Neilson EG (2003) Alports syndrome, Goodpastures syndrome, and type IV collagen. New England Journal of Medicine 348: 2543 2556. Miner JH and Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annual Review of Cell and Developmental Biology 20: 255284. Nguyen NM, Kelley DG, Schlueter JA, et al. (2005) Epithelial laminin a5 is necessary for distal epithelial cell maturation VEGF production and alveolization in the developing murine lung. Developmental Biology 282: 111125. Nguyen NM, Miner JH, Pierce RA, and Senior RM (2002) Laminin a5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung. Developmental Biology 246: 231244. Sheppard D (2002) Functions of pulmonary epithelial integrins: from development to disease. Physiological Reviews 83: 673686. Yurchenco PD, Amenta PS, and Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biology 22: 521538.

As early as 1891, the distribution and organization of elastic bers in various tissues was demonstrated by light microscopy using characteristic staining reactions. Several of these histological stains, such as Weigerts resorcin-fuchsin (from 1898) and Verhoeffs iron hematoxylin (from 1908), are still routinely used today. By the 1960s, the morphological appearance of elastin in different tissues was being documented in detail: interwoven rope-like bers in elastic ligaments, three-dimensional networks in the lung, concentric sheets or lamellae in the great vessels, and an anastomosing, honeycomb pattern in elastic cartilage. At approximately the same time, the presence of small laments at the surface of developing elastic bers was also noted by electron microscopists. These ne extracellular laments became so routinely observed in a variety of tissues that they were assigned a specic name, collectively termed microbrils (Figure 1). The microbrils were established as distinct components of elastic bers based on characteristic staining properties that were different from those of elastin and on their separation from elastic bers and partial characterization. Even at this early date, it was evident that the microbrils appeared rst in the extracellular matrix followed by the deposition of elastin, suggesting that they played some role in elastogenesis. In 1986, the major core constituent of microbrils was identied and named brillin. Since then, considerable research has been conducted on the molecular assembly of brillin into microbrils, the role of microbrils in elastic ber assembly, additional functions of elastin and microbrils apart from their structural role in elastogenic

EXTRACELLULAR MATRIX / Elastin and Microbrils 163

Ep

Ep

EI

Coll

F
(b) SMC mf

Ep

mf

SMC
F

EI

(a)

(c)

Coll

Figure 1 Electron micrographs of elastic bers in the lung. (a) Elastic bers (black spots) form an extensive network interposed between the airway epithelium (Ep) and the subjacent smooth muscle cell layer (SMC). Fibroblasts (F) in this region are responsible for much of the elastic synthesis. Higher magnication of the elastic bers in longitudinal (b) and cross-section (c) shows the close association of elastin (El) with the lamentous microbrils (mf). An abundance of collagen (Coll) is also seen in this area.

tissues, and genetic diseases linked to the elastin and brillin genes.

Structure
Tropoelastin is the monomeric form of elastin. The human tropoelastin gene (ELN) is a single copy on chromosome 7 (band q11.2). The gene contains 34 exons interspersed between large introns, giving a primary transcript of approximately 40 kb (Figure 2). The primary transcript undergoes considerable alternative splicing, generating multiple heterogeneous tropoelastin isoforms. The production of these different isoforms is spatially and temporally regulated, suggesting that the ratio of tropoelastin isoforms may be important for the structural function of the mature elastin polymer. In humans, the most frequently observed isoform lacks exon 26A, which has been reported to be expressed only in certain disease states. The tropoelastin protein ranges from 62 to 75 kDa, depending on the isoform, and is characterized by a distinct, periodic organization of hydrophobic and cross-linking domains. The protein is particularly rich in glycine, valine, alanine, and proline, with more than 75% of the amino acids being nonpolar or

uncharged. The only two cysteine residues are located in the C terminal, where they form an intramolecular disulde bond. Once outside the cell, individual tropoelastin monomers are cross-linked to form an extremely durable, insoluble polymer. Three brillin genes exist in humans with distinct but overlapping expression patterns. Of these, brillin-1 is the major structural component of microbrils. The human brillin-1 gene (FBN1), consisting of 65 exons that extend over 200 kb, is located on chromosome 15 and encodes a protein of B350 kDa. The protein is composed of multiple domains, the most abundant being tandem repeats of calcium-binding epidermal growth factor (EGF)-like modules (Figure 2). Within each of these modules are six cysteine residues that form three intradomain disulde bonds, giving the protein a rigid, rod-shaped structure. At the cell surface, brillin monomers are thought to align in a head-to-tail fashion, with a lateral packing of eight monomers to form the microbril.

Regulation of Production
Elastin is synthesized and secreted by several different cell types, depending on anatomic location within

164 EXTRACELLULAR MATRIX / Elastin and Microbrils

Tropoelastin 2 4 6 8 10 12 15 18 20 22 24 26 exon 26A exon 36 28 30 32 36 Exon 36
G L K + S 0H
S-S

Signal peptide Alternatively spliced exons Hydrophobic exons Cross-linking exons

C A G G F P

C G + R + K

+ R

+ K

(a)

(b)

Fibrillin-1

RGD

N-terminus

8-cysteine/TGFbinding domain Hybrid domain Transglutaminase cross-link

EGF-like domain Calcium-binding EGF-like domain N-linked sugar

C-terminus Proline rich region (c)

Figure 2 Schematic representation of the exon organization of tropoelastin, the amino acid sequence of exon 36 of tropoelastin, and the domain structure of brillin-1. (a) Tropoelastin contains alternating hydrophobic and cross-linking domains. Multiple exons are subjected to alternative splicing, with exon 26A being most commonly spliced out. (b) The C-terminal exon of tropoelastin contains the only two cysteine residues. These form an intramolecular disulde bond creating a hairpin loop and a positively charged pocket. This region is thought to be important for elastic ber assembly. (c) Fibrillin-1 has 47 calcium-binding EGF-like domains, interspersed with TGF-b binding modules. The protein has many N-glycosylation sites and an integrin-binding RGD motif in the center of the molecule.

the lung. These cells include chondroblasts, myobroblasts, interstitial broblasts, and mesothelial, endothelial, and smooth muscle cells. Apart from cleavage of the signal sequence as the completed polypeptide enters the endoplasmic reticulum, the tropoelastin monomer remains relatively unchanged as it traverses the secretory pathway en route to the cell surface, with no glycosylation or proteolytic processing. Once secreted, individual monomers are cross-linked between lysine residues on adjacent monomers, forming desmosine cross-links that are unique to elastin. The enzyme responsible for crosslink formation is lysyl oxidase. In contrast to tropoelastin, the brillin monomer is N-glycosylated and undergoes C-terminal proteolytic processing by a furin/PACE-type protease. Mutations in FBN1 that alter these posttranslational modications can result in intracellular retention of the brillin monomer or defective microbril assembly. Once secreted, pericellular proteoglycans such as heparan sulfate proteoglycan and chondroitin sulfate proteoglycan may

aid in the assembly of the brillin monomers into microbrils. Based on biomechanical approaches, it has been suggested that transglutaminase cross-links play a major role in strengthening the microbrillar network and, in terms of microbril assembly, may facilitate correct alignment of the brillin monomers at xed positions during initial formation. As in other elastogenic tissues, elastin in the lung is primarily produced during late fetal, neonatal, and postnatal development. Elastin is rst expressed in smooth muscle cells of the developing intralobar pulmonary arteries near airway branch points during the pseudoglandular stage. Expression increases during the canalicular and saccular stages of development as elastic bers are assembled between the smooth muscle cells and epithelium of developing airways. Elastin expression reaches a peak during alveolarization in the neonate, with high levels seen in the alveolar walls, particularly at bends, and in the alveolar septal tips. At the end of development, prominent elastic bers can be seen at these sites as

EXTRACELLULAR MATRIX / Elastin and Microbrils 165

Airway Alveolar tips

(a)

Vessels

Pleura

Alveolar walls

(b)

(c)

Figure 3 Light microscope images showing the appearance of elastic bers in various regions of the lung using Harts stain for elastin. (a) Elastic bers form a linear, lamentous network underlying the airway epithelium. Concentric layers of elastin are also seen surrounding the pulmonary vessels. (b) A continuous sheet of elastin is found subjacent to the mesothelial cells of the visceral pleura. (c) Elastic bers are present in the alveolar walls and septal tips. At the septal tips, the elastin forms a ring around the mouth of each alveolus.

well as in longitudinal bands subjacent to the airway epithelium, as concentric lamellae in the arteries and arterioles, and as a continuous sheet under the mesothelium of the visceral pleura (Figure 3). At different stages of lung development, therefore, different cells are responsible for the production and assembly of elastin into the distinct architectural forms necessitated by function. During development, elastin expression is controlled by pretranslational regulation of the amount of the elastin mRNA transcript available. One mechanism for upregulating elastin expression as the lung develops is mechanical stress. Fetal and neonatal breathing movements are important for lung development and coincide with the onset of elastin synthesis. Furthermore, in vitro studies have shown that intermittent stretching of organotypic cultures of mixed fetal rat lung cells can increase elastin gene expression. Specic soluble factors can also inuence the level of elastin expression. Stimulating factors include insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-b1, glucocorticoids, and retinoids, and repressing factors include EGF, basic broblast growth factor, phorbol esters, tumor necrosis factor alpha, interleukin-1b, and vitamin D. Following development, elastin expression ceases.

Since the protein is incredibly long-lived, with a halflife that exceeds the life span of the organism, little or no elastin synthesis is needed in the healthy adult. Elastin expression can be reactivated following lung injury, such as in brosis or pulmonary hypertension. The elastin synthesized during repair, however, is often disorganized with tortuous, clumped, and frayed bers. The cessation of lung elastin production after development is controlled strictly by a posttranscriptional mechanism. Although tropoelastin premessenger RNA is transcribed at the same rate in neonatal and adult cells, the fully processed transcript does not accumulate in adult lung. In the cytosol of adult cells, a trans-acting protein has been reported to bind to exon 30 and destabilize the elastin transcript. TGF-b1, which is known to stabilize tropoelastin mRNA, acts by interfering with the exon 30binding protein interaction. Although cross-linking experiments have suggested that the binding protein is approximately 52 kDa, the identity of this protein remains to be determined.

Biological Function
Elastin is the key structural protein of the lung that allows for repeated deformability: stretch during the

166 EXTRACELLULAR MATRIX / Elastin and Microbrils

active process of inspiration and elastic recoil during the passive process of quiet expiration. The property of distensibility is important for the function of not only the terminal airspaces but also the conducting airways, pleura, and pulmonary vasculature. The function of the microbrils appears to be as a scaffold for the deposition, orientation, and assembly of the tropoelastin monomers. This notion is supported by observations that elastin is always seen in the presence of microbrils, microbrils appear prior to elastin in the extracellular matrix during development, and tropoelastin can bind to brillin-1 with high afnity. The appearance of elastin in the airspaces coincident with the formation of the septal walls led early investigators to hypothesize not only that elastin may play a mechanical role in the lung but also that elastin could be the driving force behind alveolar development. Support for this hypothesis came from the development of transgenic mice in which elastic ber assembly was disrupted. In mice decient in platelet-derived growth factor-A, myobroblasts fail to form in the alveolar tips and elastin is not produced. Until postnatal day 4, lung development appears normal. As development continues, however, the distal airspaces become distended and thin walled, and alveolar sacs fail to form. Progressive disruption of normal development continues and the mice die by 6 weeks of age. Not only is the physical presence of elastin needed for alveolar development but it must be properly assembled too. Evidence to support this concept has come from studies of bulin-5. Fibulin-5 is an elastic

ber-associated protein essential for normal elastic ber assembly. In bulin-5 null mice, expanded distal airspaces are observed by 2 weeks of age. Elastic bers are present in the alveolar walls and tips; however, they are abnormal in distribution and appearance. These results indicate that elastin must be both produced and correctly assembled by the myobroblasts in the alveolae for the normal development of these structures. Ultimately, the earliest role of elastin in lung development was revealed by investigating lungs of mice decient in elastin. In the elastin-null mice, distal airways failed to branch, possibly through a signaling mechanism from the elastin to the mesenchymal cells surrounding the developing airways (Figure 4). Elastin-null mice die by postnatal day 3 due to overproliferation of smooth muscle cells in the aortic wall, precluding any further studies of postnatal lung development in these animals. Similar to elastin, studies of transgenic mice have revealed an additional function for brillin apart from merely aiding in elastic ber assembly. Mice homozygous for a centrally deleted FBN1 allele die by postnatal day 10 due to aortic rupture. Immediately after birth, however, these mice showed a significant increase in distal airspace caliber, with only the rare formation of a secondary alveolar septum. This phenotype was independent of elastic ber assembly since smooth muscle cell differentiation and distribution appeared normal in the mutant lung and elastin was localized to the alveolar walls and septal tips. Additional research showed that brillin can bind to and regulate the activity of TGF-b1. In

(a)

(b)

Figure 4 Lung development is severely disrupted in the absence of elastin. Comparison of lungs from postnatal day 1.5 mouse pups from wild-type (a) and elastin-null (b) mice shows a dramatic difference in lung structure. In the absence of elastin, terminal airway branching is greatly impaired. The defect is even evident in the intact lung (insets show whole heart/lung blocks). Note the expanded airspaces in the peripheral lung tissue of the elastin-null mouse compared to the wild-type animal (arrows).

EXTRACELLULAR MATRIX / Elastin and Microbrils 167

mice harboring the mutant brillin-1 gene, therefore, dysregulation of TGF-b1 activation and signaling leading to an increase in cell death was found to be the cause of the lung phenotype. A similar lung defect was found in the tight-skin mouse, a model for emphysema, in which a genomic duplication in FBN1 generates a larger than normal brillin protein, leading to abnormal microbrils with altered molecular organization. It is thought that in the neonatal tight-skin mouse lung, the inability of normal growth factor signaling, together with defective elastic ber assembly, contributes to the cessation of alveolarization seen in these mice.

Receptors
Both tropoelastin and brillin have been reported to interact with cell surface proteins. Until recently, the binding of tropoelastin to cells has been solely attributed to the elastin/laminin receptor complex. This complex includes the 67 kDa elastin-binding protein (EBP), an alternatively spliced enzymatically inactive form of b-galactosidase. EBP recognizes a hexapeptide repeat (VGVAPG) in the central region of tropoelastin and has been implicated in protecting tropoelastin from degradation and aiding in the secretion and assembly of the monomer into the elastic ber. Puried EBP has yet to be examined in vitro for its ability to bind tropoelastin. Interestingly, mice lacking the b-galactosidase gene have no reported vascular defects. A direct interaction of tropoelastin via the cell surface avb3 integrin was reported despite the fact that tropoelastin does not contain an RGDintegrin binding sequence. A single specic binding site was localized to the C-terminal 16 residues of tropoelastin, a highly charged region encompassed by exon 36 (Figure 2). In contrast to tropoelastin, brillin-1 contains an RGD sequence located in the fourth eight-cysteine motif. This RGD sequence is responsible for the binding of brillin-1 to both the avb3 and a5b1 integrins and has been suggested to provide an important link between elastic bers and the cell surface. Such a function is supported by the appearance of abnormal cellelastic ber connections in mice with mutations in the brillin-1 gene. Incomplete inhibition of binding by competing RGD peptides or antibodies against the integrin proteins suggests that a second non-RGD-mediated receptor for the brillin molecule exists in certain cell types.

Elastin and Microbrils in Respiratory Disease

Elastic ber abnormalities leading to respiratory disease can result from either destruction or

overproduction of elastin in the adult or by aberrant initial assembly. Destruction of elastin in the lung is a key factor in the pathogenesis of emphysema. For the most part, this occurs due to an imbalance between elastases and anti-elastases. Enhanced elastase activity can occur by the recruitment of elastase-secreting inammatory cells into the lung, often as a result of air pollution or cigarette smoking, or by deciency in the elastase inhibitor, a1-antitrypsin. a1-Antitrypsin deciency is a rare hereditary disorder, but it is likely widely under- and misdiagnosed. An overproduction of elastin can also adversely affect lung function. Inhalation of mineral dust, which often occurs as an occupational hazard, can reinitiate tropoelastin gene expression leading to an aberrant accumulation of mature elastin and, ultimately, granulomatous lung diseases or pulmonary brosis. Abnormal assembly of elastic ber during development can also occur in premature infants subject to mechanical ventilation and oxygen supplementation due to respiratory complications at birth. In this respiratory disorder, termed bronchopulmonary dysplasia, patients have decreased alveolar number, impaired lung development, and irregular elastic ber structure. Altered lung structure and function can also occur as a consequence of mutations in the elastin or brillin genes. In humans, heterozygous null mutations of ELN cause supravalvular aortic stenosis (SVAS), a generalized segmental stenotic arterial disease characterized by overproliferation of vascular smooth muscle cells. No lung phenotype has been reported in these patients. In contrast, a distinct class of ELN mutations cause autosomal dominant cutis laxa, with the main manifestation being lax, redundant, and inelastic skin. In these patients, a mutant tropoelastin protein is synthesized that appears to act through a dominant negative mechanism to disrupt normal elastic ber assembly and function. Unlike SVAS, some cutis laxa patients present with pulmonary emphysema, suggesting that the quality of the elastic ber is more important for lung development than the quantity. Mutations in the brillin-1 gene cause Marfan syndrome, which is an autosomal dominant disorder characterized by tall stature and other skeletal defects, aortic dilatation, mitral valve prolapse, and ectopic lentis. Some individuals with Marfan syndrome present with pneumothorax secondary to rupture of emphysematous bullae in the lung. Approximately 1015% of patients have emphysema, although this is likely underdiagnosed. Similar to that described previously for mouse models with defective brillin-1 production, the lung phenotype in Marfan patients likely results from reduced targeting and sequestration of latent TGF-b1

168 EXTRACELLULAR MATRIX / Collagens

in the lung tissue, leading to increased TGF-b1 activation, inhibition of alveolar development, and eventual emphysema.
See also: Acute Respiratory Distress Syndrome. Alveolar Wall Micromechanics. Breathing: Breathing in the Newborn. Bronchopulmonary Dysplasia. Chest Wall Abnormalities. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General. Environmental Pollutants: Particulate Matter, Ultrane Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds. Extracellular Matrix: Degradation by Proteases. Fibroblasts. Genetics: Overview. Lung Anatomy (Including the Aging Lung). Lung Development: Overview. Myobroblasts. Occupational Diseases: Overview; Asbestos-Related Lung Disease; Silicosis. Pectus Excavatum. Platelet-Derived Growth Factor. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Airway; Vascular. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models.

Collagens
S E Dunsmore, Brigham and Womens Hospital at Havard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Collagens, the most abundant proteins in the animal kingdom, are best known for providing mechanical strength to skin, bone, and other tissues. In the lung, collagens limit expansion of the parenchyma and prevent airway collapse. Structurally, collagens are characterized by a triple helical region containing a Gly-x-y motif. Collagens are found in the extracellular matrix where they are assembled into brils and other supramolecular structures. Normal growth and development are dependent on the proper assembly of collagens. Collagens interact with three types of receptors: integrins, discoidins, and glycoprotein VI. Respiratory disease can result from inappropriate synthesis, assembly, or deposition of collagen. Current research indicates that collagen fragments may be important endogenous inhibitors of tumorigenesis and mediators of lung transplant rejection.

Introduction Further Reading

American Thoracic Society Ad Hoc Statement Committee (2004) Mechanisms and limits of induced postnatal lung growth. American Journal of Respiratory and Critical Care Medicine 170: 319343. Dietz HC and Mecham RP (2000) Mouse models of genetic diseases resulting from mutations in elastic ber proteins. Matrix Biology 19: 481488. Foster JA and Curtiss SW (1990) The regulation of lung elastin synthesis. American Journal of Physiology 259: L13L23. Gardi C, Martorana PA, de Santi MM, van Even P, and Lungarella G (1989) A biochemical and morphological investigation of the early development of genetic emphysema in tight-skin mice. Experimental and Molecular Pathology 50: 398410. Kielty CM, Sherratt MJ, Marson A, and Baldock C (2005) Fibrillin microbrils. Advances in Protein Chemistry 70: 405436. Kielty CM, Sherratt MJ, and Shuttleworth CA (2002) Elastic bres. Journal of Cell Science 115: 28172828. Lindahl P, Karlsson L, Hellstrom M, et al. (1997) Alveogenesis failure in PDGF-A-decient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development. Development 124: 39433953. Mariani TJ, Sandefur S, and Pierce RA (1997) Elastin in lung development. Experimental Lung Research 23: 131145. Milewicz DM, Urban Z, and Boyd C (2000) Genetic disorders of the elastic ber system. Matrix Biology 19: 471480. Mithieux SM and Weiss AS (2005) Elastin. Advances in Protein Chemistry 70: 437461. Neptune ER, Frischmeyer PA, Arking DE, et al. (2003) Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome. Nature Genetics 33: 407411. Starcher BC (2000) Lung elastin and matrix. Chest 117: 229S234S. Wendel DP, Taylor DG, Albertine KH, Keating MT, and Li DY (2000) Impaired distal airway development in mice lacking elastin. American Journal of Respiratory Cell and Molecular Biology 23: 320326.

Collagen came into use as a word in the English language around 1865. It is derived from the French term collage ` ne, which stems from the Greek word for glue, Kolla. Early definitions of collagen centered on its role as a constituent of connective tissue which yielded glue or gelatin upon boiling and indeed, chemical means of solubilizing collagen were examined in some of the rst reports of experimentation with the molecule. By the early 1940s, the techniques of electron microscopy and X-ray diffraction were being used to dene the structure of collagen brils. The triple-chain helical nature of the collagen molecule became evident in 1955, but it was not until 1969 that the existence of more than one type of collagen was recognized. Knowledge of the existence and structure of different collagen types was advanced by the application of recombinant DNA technology which was rst used in 1978 to isolate the gene for the proa2(I) chain of chicken type I procollagen. Analysis of the human genome indicates that there may be more than 40 collagen genes encoding the subunits of at least 27 different collagen types. Collagens are traditionally divided into two classes, brillar (Table 1) and nonbrillar. Fibrillar collagens (types I, II, III, V, XI, XXIV, and XXVII) are the major structural components of connective tissues, cartilage, and bone. Types I and III collagens are the most abundant proteins in the lung accounting for about 1520% of the dry weight of the tissue. Nonbrillar collagens may be further subdivided into basement membrane (types IV and VII) (Table 2), short-chain (types VI, VIII, and X) (Table 3), FACIT

EXTRACELLULAR MATRIX / Collagens 169

Table 1 Fibrillar collagens Collagen type I Gene symbol COL1A1 COL1A2 COL2A1 COL3A1 Chain composition [a1(I)]2a2(I) [a1(I)]3 [a1(II)]3 [a1(III)]3 Supramolecular structure 67 nm banded brils Tissue localization Bone, tendon, dermis, cornea Cartilage, vitreous body of eye Dermis, aorta, uterus, intestine Placenta, bone, dermis, cornea Cartilage, intervertebral disk Cornea, bone Cartilage, eye, ear, lung, colon Lung distribution Interstitium, vasculature, pleura, trachea, bronchi Bronchial and tracheal cartilage Interstitium, vasculature, pleura, trachea, bronchi Interstitium, vasculature, basement membranes Bronchial and tracheal cartilage May not be present Expressed by chondrocytes and epithelial cells

II III

67 nm banded brils 67 nm banded brils

COL5A1 COL5A2 COL5A3 COL11A1 COL11A2 COL2A1 COL24A1 COL27A1

[a1(V)]2a2(V) [a1(V)a2(V)a3(V)] [a1(V)]3 [a1(XI)a2(XI)a3(XI)]

9 nm banded brils

XI

Fine brils (similar to type V collagen brils) Presumed to form homotrimeric brils Presumed to form homotrimeric brils

XXIV XXVII

[a1(XXIV)]3 [a1(XXVII)]3

Table 2 Basement membrane collagens Collagen type IV Gene symbol COL4A1 COL4A2 COL4A3 COL4A4 COL4A5 COL4A6 COL7A1 Chain composition [a1(IV)]2a2(IV) [a1(IV)]2a3(IV) [a1(IV)]2a4(IV) [a1(IV)]2a5(IV) [a1(IV)]2a6(IV) [a1(VII)]3 Supramolecular structure Nonbrillar meshwork Tissue localization Basement membranes Lung distribution Underneath airway and alveolar epithelium

VII

Anchoring brils

Skin, amniotic membrane, mucosal epithelium

Underneath airway epithelial cells

Table 3 Short-chain collagens Collagen type VI Gene symbol COL6A1 COL6A2 COL6A3 COL8A1 COL8A2 COL10A1 Chain composition [a1(VI)a2(VI)a3(VI)] Supramolecular structure 510 nm beaded microbrils Nonbrillar hexagonal lattice Nonbrillar hexagonal lattice Tissue localization Uterus, dermis, cornea, cartilage Endothelial cells, Descemets membrane Calcifying cartilage Lung distribution Interstitium, vasculature Vasculature

VIIIa

[a1(VIII)]2a2(VIII)

Xa
a

[a1(X)]3

Not present in adult lung

Many molecules such as collectins, complement related factors, metabolic regulators (ACRP30, Hib27, acetylcholinesterase), and elastic ber-associated molecules (emilins) are structurally homologous to collagens VIII and X possessing similar triple helical and C-terminal noncollagenous (NC1) domains.

170 EXTRACELLULAR MATRIX / Collagens

Table 4 FACIT (Fibril-associated Collagens with Interrupted Triple helices) collagens Collagen type IX Gene symbol COL9A1 COL9A2 COL9A3 COL12A1 COL14A1 Chain composition [a1(IX)a2(IX)a3(IX)] Supramolecular structure Associates with type II collagen brils Associates with type I collagen brils Associates with type I and II collagen brils Associates with brillin-1 rich microbrils and Dbanded type II collagen brils N-terminal-interacting oligomers Tissue localization Cartilage, vitreous body of eye Dermis, tendon, cartilage Dermis, tendon, cartilage Heart, kidney, smooth muscle Lung distribution Tracheal and bronchial cartilage Interstitium of brotic lungs Interstitium of brotic lungs Minimal expression

XII XIV

[a1(XII)]3 [a1(XIV)]3

XVI

COL16A1

a1(XVI)

XIX

COL19A1

a1(XIX) [a1(XIX)]2 [a1(XIX)]3 [a1(XIX)]4 [a1(XIX)]5 [a1(XIX)]6 a1(XX) a1(XXI)

Basement membrane of differentiating muscle cells

Minimal expression in adult lung

XX XXI

COL20A1 COL21A1

Presumed to bind to collagen brils Unknown

Corneal epithelium Placenta, vasculature of heart, stomach, kidney, skeletal muscle Myotendinous junction, articular cartilagesynovial uid junction, hair follicles

May not be present Minimal expression

XXII

COL22A1

a1(XXII)

Presumed to bind to basement membrane components

Not expressed

(types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII) (Table 4), transmembrane (types XIII, XVII, XXIII, and XXV) (Table 5), and multiplexin (types XV and XVIII) collagens (Table 6). Type IV collagen is the most abundant nonbrillar collagen in the lung constituting approximately 5% of parenchymal collagen. Type IV collagen is responsible for the tensile strength of the bloodgas barrier and plays a large part in preventing stress failure of the pulmonary capillaries under normal conditions.

proteins so that this amino acid is used as a specic index of collagen synthesis and concentration. The three polypeptide chains associate via the Gly-x-y sequences to form triple helices. All collagens contain at least one triple helical region. In this conformation, peptide bonds linking adjacent amino acids are buried within the interior of the molecule which renders the triple helical region highly resistant to proteolysis (Figure 1).

Regulation of Production and Activity Structure

Collagens consist of three polypeptide chains containing the amino acid sequence (Gly-x-y). Although x and y may be any amino acid, proline and hydroxyproline predominate in these positions such that approximately every third x is proline and every third y is hydroxyproline. Hydroxyproline is not unique to collagen; however, concentrations present in collagen are much higher than for other The pattern of collagen synthesis is tissue specic and is regulated at both the transcriptional and translational levels. Collagens are also subject to numerous posttranslational modications. In most cases, biological activity is dependent upon the assembly of supramolecular structures (Figure 2). Fibril formation by types I, II, and III collagen is well characterized. Likewise, the macromolecular assembly of the open network structure of type IV collagen has been

EXTRACELLULAR MATRIX / Collagens 171

Table 5 Transmembrane collagensa Collagen type XIII Gene symbol COL13A1 Chain composition [a1(XIII)]3 Supramolecular structure Homotrimeric transmembrane Homotrimeric transmembrane Homotrimeric transmembrane Homotrimeric transmembrane Tissue localization Adhesive junctions in cartilage, bone, skeletal muscle, lung, intestine, skin Hemidesmosomes Placenta, kidney, skin, neural cells Alzheimer amyloid plaques, exclusively produced by neuronal cells Lung distribution Sites of branching morphogenesis Not expressed Not expressed Not expressed

XVII XXIII XXV

COL17A1 COL23A1 COL25A1

[a1(XVII)]3 [a1(XXIII)]3 [a1(XXV)]3

Several molecules with small triple helical domains: ectodysplasin A, the class A macrophage scavenger receptors, the MARCO receptor, and the colmedins (gliomedin, UNC-122, COF-2, CG6867) are classied as collagenous transmembrane proteins.

Table 6 Multiplexin collagens Collagen type XV Gene symbol COL15A1 Chain composition a1(XV) Supramolecular structure Forms bridge between basement membrane and collagen brils Unknown Tissue localization Vascular, neuronal, mesenchymal basement membranes, most epithelial basement membranes Endothelial and epithelial basement membranes Lung distribution Vasculature with the exception of alveolar capillaries Alveolar capillaries

XVIII

COL18A1

a1(XVIII)

extensively studied. Details of the molecular assembly of type VI collagen into beaded microbrils, type VII collagen into anchoring brils, and types VIII and X collagen into nonbrillar hexagonal lattices have been reported. Transmembrane collagens form homotrimers with the collagenous domain extracellularly oriented. FACIT collagens associate with interstitial collagen bers. In the case of type IX collagen, this association occurs through covalent cross-linking of lysine/hydroxylysine residues in the N-terminal of the second collagenous domain of type IX collagen to sequences in the C-telopeptide region of the type II collagen bril. Collagenases (matrix metalloproteinase (MMP)-1, MMP-8, and MMP-13) are the only proteases capable of degrading brillar collagen. These MMPs cleave at a site within the triple helical region of brillar collagen producing fragments that are threefourth and one-fourth the size of the original molecule. This single cleavage event destroys the triple helical structure of the collagen bril and renders the molecule biologically inactive. In contrast, biologically active fragments may be produced from proteolytic degradation of the noncollagenous domains

(NC1) of types IV, XV, and XVIII collagen. Although the proteolytic events that generate these fragments are not entirely clear, the potent effects of endostatin on tumor growth and angiogenesis have been of great interest.

Biological Function
Collagens are essential for normal growth and development. A variety of clinical disorders primarily manifested in the skeleton, skin, and joints are attributed to mutations in the various collagen genes. Collagens also play important roles in wound repair. Fibrous collagen activates platelets to initiate formation of a hemostatic plug. Cell migration following injury is directed by collagens which are important constituents of granulation tissue. Collagens can function as survival factors and generally prevent apoptosis of adherent cells. Recently, it has become evident that collagen fragments may have biological functions. Endostatin, a proteolytic fragment of collagen type XVIII, is a potent inhibitor of angiogenesis.

172 EXTRACELLULAR MATRIX / Collagens

Glycine O H2N OH NH2 (a) OH OH NH2 OH Proline O Hydroxyproline O

(GlyProHyp)2 O H2N
NH
OH

O HN O HN HN O OH N H

OH

(b)
Figure 1 Constituents of the collagen triple helix. (a) The three most common amino acids found in the collagen triple helix: glycine (Gly), proline, and hydroxyproline. (b) Collagen chains associate via Gly-x-y sequences to form triple helices. In the triple helices, side chains of proline and hydroxyproline (shown in pink) are on the outside of the molecule, and peptide bonds between the amino acids (shown in blue) are on the inside of the helix. Hydrogen bonds (denoted by green dashed line) stabilize the triple helix and occur between the HN-group of glycine and the OQC group of the amino acid in position x.

Angiogenesis is also inhibited by restin, a collagen XV fragment, and by arrestin, canstatin, and tumstatin, type IV collagen fragments. These fragments have been identied in serum and in the extracellular matrix and may be important endogenous inhibitors of tumorigenesis. A type XXV collagen fragment, CLAC-P, has been linked to neuronal degeneration in Alzheimer disease. CLAC-P binds to and stabilizes aggregates of amyloid-b peptides.

Receptors
Four integrin receptors, a1b1, a2b1, a10b1, and a11b1, interact with collagens. These integrins contain an I domain which recognizes the hexapeptide motif GlyPheHypGlyGluArg found in the triple helical region of collagens. Integrins a1b1 and a2b1 are widely expressed and differ in binding afnities for brillar and basement membrane collagens. Fibrillar collagens preferentially associate with integrin a2b1 while integrin a1b1 has a higher afnity for type IV collagen. Tissue distribution of integrins a10b1 and a11b1 is limited. Chondrocytes express a10b1, and its primary ligand is type II collagen. Integrin

a11b1 is predominantly found in muscle tissue where it preferentially interacts with type I collagen. Two kinases, focal adhesion kinase and integrin-linked kinase, interact with the cytoplasmic tail of the b1 subunit to modulate signaling pathways that regulate cell proliferation, migration, and survival. Collagens also bind to two discoidin domain receptors (DDRl and DDR2) which are expressed in a variety of tissues including the lung. Binding of collagen types I, II, III, IV, V, VI, and VIII to DDR1 has been documented. DDR2 appears to have a higher afnity for brillar collagens and in particular for collagen types I and III. DDRs are receptor tyrosine kinases, but their intracellular signaling partners are incompletely characterized. Both DDR1 and DDR2 null mice are smaller than wild-type littermates, however, suggesting that the interaction of collagen with DDRs is functionally important. Fibrous collagen binds to glycoprotein VI to induce platelet aggregation. Glycoprotein VI is found exclusively on platelets and signals through a well-known cascade involving Src kinases, phosphatidylinositol 3-kinase, phospholipase Cg2, and intracellular calcium to effect platelet activation.

EXTRACELLULAR MATRIX / Collagens 173

Fibril Types I, II, III, V, and XI collagen

FACIT Types IX, XII, and XIV collagen

Beaded microfibrils Type VI collagen

Open network Type IV collagen

Anchoring fibrils Segment long spacing-like structures Type VII collagen

Transmembrane Types XIII, XVII, XXIII, and XXV collagen

Hexagonal lattice Types VIII and X collagen

Figure 2 Collagen supramolecular structures. Lysyl oxidase-induced cross-links are shown in orange. FACIT cross-links are depicted in green.

The biologically active collagen fragments (endostatin, restin, arrestin, canstatin, tumstatin) have been shown to bind to a variety of receptors including integrins (a1b1, a2b1, a3b1, a5b1, avb5, avb1, and a6b1), glypican-3, and vascular endothelial growth factor receptor 2. It is not clear, however, which of these receptors mediates the in vivo effects of these collagen fragments.

Collagens in Respiratory Disease

Pulmonary brosis, the end result of a variety of clinical entities, is the respiratory disease most often associated with collagens. The primary pathological

defect in this disease is the inappropriate synthesis and deposition of interstitial collagens in the lung parenchyma. Blood oxygenation is adversely affected by the loss of capillary beds and disruption of the bloodgas diffusion barrier. In the airways, increased subepithelial deposition of collagen is observed in patients with asthma, chronic obstructive pulmonary disease (COPD), or obliterative bronchiolitis. Although the functional significance of increased collagen deposition in these diseases remains unclear, airway brosis often correlates with disease severity. Collagens are associated with a group of immune disorders termed collagen vascular diseases. Systemic lupus erythematosus, rheumatoid arthritis, Sjo grens

174 EXTRACELLULAR MATRIX / Collagens

syndrome, systemic sclerosis, polymyositis, and dermatomyositis are examples of collagen vascular diseases with pulmonary complications. Pleuritis and pulmonary infections are common in systemic lupus erythematosus patients. Rheumatoid arthritis patients may also suffer from pleuritis, but interstitial lung disease or brosis is the predominant pulmonary manifestation. Interstitial lung disease is common in patients with Sjo grens syndrome or systemic sclerosis and may occur in patients with polymyositis and/or dermatomyositis. Although grouped together because of the common connective tissue or collagen manifestations, many of these diseases are also characterized by the presence of autoantibodies to nuclear proteins. Pulmonary hypertension can result from a variety of causes including genetic defects, hypoxia, and chronic thrombotic disease. In all cases, type I collagen is increased in the vasculature. In primary pulmonary hypertension, type I collagen is increased in the neointima of the vessels. When vascular remodeling is induced by hypoxia, however, increased collagen is observed in the medial and adventitial layers of the vessels. Osteogenesis imperfecta and EhlersDanlos syndrome are the most common manifestations of collagen gene mutations. Approximately 60% of osteogenesis imperfecta patients who have altered or decient type I collagen present with severe chest wall deformities. Pulmonary compromise is the leading cause of death in these patients. Pulmonary complications are rare in EhlerDanlos syndrome although cavitary lesions, pneumothorax, and pulmonary hemorrhage are occasionally observed in patients with the type IV subtype in which the gene encoding type III collagen is mutated. Pulmonary hemorrhage also occurs in patients with Goodpasture syndrome which is characterized by the production of autoantibodies to type IV collagen. Recent evidence suggests that type V collagen may be a major antigen involved in lung allograft rejection. In animal models, type V collagen-reactive T cells perpetuate the graft rejection response. Oral administration of type V collagen can induce tolerance to lung allografts and prevent the development of obliterative bronchiolitis. Although preliminary, these results may be very important in the future therapy of chronic respiratory disease as the efcacy of lung transplant as a treatment modality is presently limited by the high rate of graft rejection.
See also: Acute Respiratory Distress Syndrome. ADAMs and ADAMTSs. Adhesion, CellCell; Vascular; Epithelial. Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Alveolar Hemorrhage.

Alveolar Wall Micromechanics. Apoptosis. Arteries and Veins. Autoantibodies. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Overview. Complement. Connective Tissue Growth Factor. Cytoskeletal Proteins. Drug-Induced Pulmonary Disease. Extracellular Matrix: Basement Membranes; Matrix Proteoglycans; Degradation by Proteases. Fibroblasts. Granulomatosis: Lymphomatoid Granulomatosis; Wegeners Disease. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Matrix Metalloproteinases. Myobroblasts. Neurobromatosis. Occupational Diseases: Overview; Coal Workers Pneumoconiosis; Asbestos-Related Lung Disease; Silicosis. Permeability of the BloodGas Barrier. Platelets. Polymyositis and Dermatomyositis. Progressive Systemic Sclerosis. Pulmonary Alveolar Microlithiasis. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Pulmonary Vascular Remodeling. Radiation-Induced Pulmonary Disease. Signal Transduction. Systemic Disease: Sarcoidosis; Diffuse Alveolar Hemorrhage and Goodpastures Syndrome. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Aouacheria A, Cluzel C, Lethias C, et al. (2004) Invertebrate data predict an early emergence of vertebrate brillar collagen clades and an anti-incest model. Journal of Biological Chemistry 279: 4771147719. Boot-Handford RP and Tuckwell DS (2003) Fibrillar collagen: the key to vertebrate evolution? A tale of molecular incest. BioEssays 25: 142151. Chu M-L and Prockop DJ (2002) Gene structure. In: Royce PM and Steinmann B (eds.) Connective Tissue and Its Heritable Disorders, pp. 223248. New York: Wiley-Liss. Eble JA (2005) Collagen-binding integrins as pharmaceutical targets. Current Pharmaceutical Design 11: 867880. Exposito JY, Cluzel C, Garrone R, and Lethias C (2002) Evolution of collagens. Anatomical Record 268: 302316. Franzke CW, Bruckner P, and Bruckner-Tuderman L (2005) Collagenous transmembrane proteins: recent insights into biology and pathology. Journal of Biological Chemistry 280: 40054008. Gelse K, Poschl E, and Aigner T (2003) Collagens: structure, function, and biosynthesis. Advanced Drug Delivery Reviews 55: 15311546. Kielty CM and Grant ME (2002) The collagen family: structure, assembly, and organization in the extracellular matrix. In: Royce PM and Steinmann B (eds.) Connective Tissue and Its Heritable Disorders, pp. 159221. New York: Wiley-Liss. Knupp C and Squire JM (2005) Molecular packing in networkforming collagens. Advances in Protein Chemistry 70: 375403. Myllyharju J and Kivirikko KI (2004) Collagens, modifying enzymes and their mutations in humans, ies and worms. Trends in Genetics 20: 3343. Ortega N and Werb Z (2002) New functional roles for non-collagenous domains of basement membrane collagens. Journal of Cell Science 115: 42014214. Suki B, Ito S, Stamenovic D, Lutchen KR, and Ingenito EP (2005) Biomechanics of the lung parenchyma: critical roles of collagen and mechanical forces. Journal of Applied Physiology 98: 18921899.

EXTRACELLULAR MATRIX / Matricellular Proteins 175

Verrecchia F and Mauviel A (2004) TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression. Cellular Signalling 16: 873880. Vogel WF (2001) Collagen-receptor signaling in health and disease. European Journal of Dermatology 11: 506514. White DJ, Puranen S, Johnson MS, and Heino J (2004) The collagen receptor subfamily of the integrins. International Journal of Biochemistry and Cell Biology 36: 14051410.

Matricellular Proteins
P Bornstein, University of Washington, Seattle, WA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The term matricellular was coined to identify a group of proteins that are secreted and function in the extracellular matrix, but do not play a structural role as an integral component of a physical entity such as a ber or basement membrane. Instead, matricellular proteins primarily serve to modulate cell function. These functions are achieved in part by interaction with a variety of cell-surface receptors, with the resulting engagement of signal transduction pathways. Matricellular proteins are also capable of interacting with structural matrix proteins, such as collagens and bronectin, and with a number of bioactive proteins, that is, cytokines, growth factors, and proteases. As a consequence, these bioactive molecules can either be sequestered in the matrix, presented to their appropriate receptors, or in the case of proteases, inhibited by direct binding or cleared from the pericellular environment by formation of protein enzyme complexes that are recognized by a scavenger receptor. It should be noted, however, that the distinction between matricellular and structural matrix proteins is not sharp, since structural proteins such as collagens, laminins, and bronectin also inuence cell function by interaction with cell-surface receptors, and they also bind bioactive proteins. The classication of proteins as matricellular is based on similarity of mode of action rather than on homology in amino acid sequence and three-dimensional structure. Thus, the thrombospondins, SPARC family members, tenascins, CCN proteins, and osteopontin, the core group of matricellular proteins considered in this chapter, are structurally unrelated. The partial dependence of the function of matricellular proteins on the proteins with which they interact gives rise to the characterization of their function as contextual. The relevance of these proteins to the biochemistry and physiology of the lung will therefore depend to a large extent on the co-expression of interacting proteins. Matricellular proteins are frequently expressed in pulmonary brosis and in neoplasms of the lung, and early evidence supports the ability of these proteins to inuence the progress of these disorders.

cellular behavior, had its origin in studies of thrombospondin (TSP)-1, SPARC (secreted protein, acidic, and rich in cysteine), and tenascin-C. In contrast to typical adhesive matrix proteins such as bronectin and type I collagen, these proteins were found to be anti-adhesive in that a variety of cells attached poorly to the proteins in vitro, and attachment of cells to collagen or bronectin was reduced when these matricellular proteins were added. It is also known that these proteins are expressed predominantly, but transiently, in tissues that are undergoing rapid change, for example, during embryonic development, and in wound healing, angiogenesis, and bone remodeling. Therefore, it was surmised that matricellular proteins serve to modulate cell attachment and detachment, and thus regulate cell migration during morphogenesis and tissue repair. Subsequent studies indicated that the properties of osteopontin and the six members of the CCN family (an acronym derived from the initial names of the rst three members in this family: Cyr61, connective tissue growth factor, and Nov) justied their inclusion in the group of matricellular proteins. It is likely that additional proteins and/or proteoglycans will be found to qualify for inclusion in this group. An additional reason to consider inclusion of matricellular proteins in a separate category of extracellular proteins was provided by studies of the functions of TSP1 and TSP2, rst in vitro and subsequently deduced from the phenotypes of knockout mice. The diversity of the functions that were attributed to TSP1, based on studies in vitro, was puzzling and could not be readily explained by the existence of a close homolog, TSP2, or by modication of the protein, either by alternative splicing or by limited proteolysis. This problem was compounded when the full panoply of effects from a lack of TSP2 was revealed in the phenotype of the TSP2 knockout mouse. Similarly, the phenotype of the SPARC-null mouse, which is still under active investigation, has turned out to be far more complex than initially anticipated. This wide range of functions can be more readily understood when one postulates that the complexity of the pericellular environment can provide an explanation for the diversity of function displayed by these and other matricellular proteins.

The Structure of Matricellular Proteins Introduction

The realization that proteins exist in the extracellular milieu, distinct from cytokines, growth factors, and proteases, whose primary function is to modulate
Thrombospondins 1 and 2

Matricellular proteins typically display a multidomain structure, resulting from the process of exon shufing during evolution, and differences among

176 EXTRACELLULAR MATRIX / Matricellular Proteins

Thrombospondin 1 and 2 S NH2 S PC I I I II II II III III III III III III III COOH

S NH2 S Thrombospondin 3, 4, and 5

Figure 1 A schematic representation of the structures of individual chains in the thrombospondins. TSP1 and TSP2 are trimers and TSPs 35 are pentamers. The structure of the NH2-terminal domains is not known and the amino acid sequences in this domain vary considerably among the TSPs. The oligomerization domain, containing interchain disulde bonds, is followed by a procollagen homology domain (PC), also known as a von Willebrand type C repeat, and in the case of TSP1 and TSP2, by three TSR (thrombospondin structural repeats) or properdin-like repeats. All TSPs have type II or EGF-like repeats, type III or calcium-binding repeats, and a COOHterminal domain.

II

II

II

II

III III III III III III III

COOH

homologous members of a subfamily among different species (orthologs) can be attributed to selection for the properties unique to the individual paralogs (related proteins within a single species). The TSP family (Figure 1) consists of ve members. TSP1 and TSP2 are disulde-bonded trimers with a chain molecular weight (MW) of 145 000, while TSP3, TSP4, and TSP5 (also known as COMP, cartilage oligomeric protein) are pentamers of about 105110 000 molecular weight. The differences in the MWs of the pentameric TSPs result from a variation in their NH2terminal domains. Currently, only TSP1 and TSP2 can be considered to be matricellular proteins. While functional information for the pentameric TSPs is limited, at least TSP3 and TSP5 appear more likely to play a structural role in the matrix. The conservation in amino acid sequence between mouse TSP1 and TSP2 increases monotonically from 32% identity in the NH2-terminal domain to 82% identity in the COOH-terminal domain. As shown in Figure 1, a TSP1 or TSP2 chain is composed of an NH2-terminal domain, a disulde knot or oligomerization region that links the three chains in the protein, a PC (procollagen/von Willebrand type C) domain, the types I, II, and III repeats, and a COOHterminal domain. The type I repeats, also known as the properdin or thrombospondin structural repeats (TSR) repeats, are broadly distributed in nature and are found in other vertebrate proteins (properdin, ADAMTS proteases, F-spondin), Drosophila (F-spondin, papilin), Caenorhabditis elegans (Fspondin), and the malarial parasite, Plasmodium falciparum (circumsporozoite protein). The type II

or EGF-like repeats are also found in many other proteins including brillins, tenascins, and in the extracellular domain of many transmembrane proteins. On the other hand, sequences in the type III repeats and the COOH domain are unique to members of the TSP family. Rotary shadowing electron microscopy of TSP1 molecules revealed a bola-like structure in which the three NH2-termini are coalesced into a single globe that is connected by protein strands to three separate COOH-terminal globes. The latter were thought to represent only the COOH-terminal domains, but recent biophysical and crystallographic studies indicate that the COOH-terminal half of each chain is folded into a compact structure that is stabilized by the binding of calcium ions. Thus, the strand visible by rotary shadowing may be limited to the procollagen domain and the TSR repeats in the calcium-replete structure. The crystal structure of the second and third TSR repeats of human TSP1 has revealed a novel, antiparallel, three-stranded fold with alternating stacked layers of tryptophan and arginine residues.
SPARC and Family Members

SPARC is a 32 kDa glycoprotein that is widely expressed during development and growth of vertebrates, but expression in the adult is more restricted to tissues such as bone and the lens of the eye. Reexpression occurs following injury and in tumors. SPARC orthologs also exist in Drosophila and C. elegans. The structure of SPARC and its paralogs are shown schematically in Figure 2. The crystal

EXTRACELLULAR MATRIX / Matricellular Proteins 177

SPARC (285) Hevin (634) QR1 (660) Testican1 (421) tsc 36 (288) SMOC-1 (434)
Figure 2 A schematic representation of the structures of six members of the SPARC family. There are two additional testicans and two additional tsc proteins. The individual domains in the proteins are represented by different colors and are not drawn to scale. The number of amino acids in each protein is indicated in parentheses. Yellow: the acidic domain, which is highly variable in length; blue: follistatin domain; orange: the highly conserved E-C domain, containing two EF hand calcium-binding structures (black bars); white: thyroglobulinlike domain. The green, hatched, and cross-hatched domains are specic for the indicated proteins. Figure kindly provided by Dr E Helene Sage.

structure of two of the three domains of human SPARC, the best-studied member of this ten-protein family (Figure 2) has been solved. While the acidic NH2-terminal domain was poorly structured and could not be resolved by X-ray analysis, the structure of the middle, follistatin domain was shown to resemble that of serine protease inhibitors of the Kazal family. The COOH-terminal domain consists of two EF-hand calcium-binding sites and a number of ahelices. These two domains interact through a small interface. The highly variable, acidic NH2-terminal domains of different SPARC family members are thought to determine the properties that are unique to each member of the family.
Tenascins

molecule can vary considerably in molecular weight (from 190 to 300 kDa in tenascin-C) as a result of extensive alternative splicing.
Osteopontin

The tenascins (TNs) are a family of four or possibly ve structurally related extracellular proteins that exist as trimers due to the presence of an NH2-terminal TN assembly (TA) domain (Figure 3). Chain association initially occurs by interaction of heptad repeats and further stabilization is achieved by formation of interchain disulde bonds. Despite the presence of additional domains in human TN-X, this TN and avian TN-Y may be orthologs, based on similarities in sequence and expression patterns. In the case of tenascins-C and -W, two trimers associate to form hexamers (see rotary shadowing image in Figure 3(a)), which is the basis for the term hexabrachion, applied initially to tenascin-C; this protein has also been termed cytotactin. The structures of the tenascins are described in detail in the legend to Figure 3. Whereas the formation of heterotypic multimers is theoretically possible, the tenascins are thought to be homo-trimeric or -hexameric in the sense that all subunit chains are the products of the same gene. However, the chains in any given

Osteopontin (OPN) is sometimes classied as a member of an integrin-binding family of ve N-linked glycoproteins that includes bone sialoprotein, dentin matrix protein I, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein. The genes encoding these proteins are closely linked on human chromosome 4. However, since the other members of the family appear to perform structural roles in the matrix and do not share the wide range of nonstructural functions displayed by OPN, only the latter will be considered here. The name OPN was coined to suggest a bridging function for the protein between hydroxyapatite and cells in bone. In addition to calcium-containing minerals, OPN binds to collagen, bronectin, and osteocalcin. OPN is a highly acidic protein that is rich in aspartic and glutamic acids and serine (Figure 4). Additional negative charges are introduced by phosphorylation of serine and threonine residues. Judging by its highly acidic character and amino acid sequence, OPN is likely to be highly exible, with very little a-helical or b-sheet structure. The molecular weight of the protein varies as a result of alternative splicing, as well as variable N- and O-glycosylation and phosphorylation. In addition, OPN is subject to cleavage by thrombin between Arg168 and Ser169 (in the human protein). Thrombin cleavage modulates its interactions with cell-surface receptors, and consequently inuences cell adhesion and migration. Recently, OPN has also been shown to be a substrate for matrix metalloproteinases (MMPs), although the function of MMP-derived fragments is uncertain. In

178 EXTRACELLULAR MATRIX / Matricellular Proteins

Region of oligomer assembly

Spliced FN-III domains

Conserved FN-III domains

EGF L repeats

Fibrinogen globe

(a) TA domain TN-C: Heptad TN-R: EGFL repeats

(b) FN-III domains

1 2 3 4 5 A1 A2 A3 A4 B AD2 AD1 C D 6 7 8

FG globe

TN-W:

WA WB WC WC WD

TN-X:

XA XA

SPX

1 X1 X2 X3 2

X4 X5

X6 X7 X8 X9 X10 X11 X12

XR1 XR2 XR3 XR4 XR5 XR6 XR7 XR8 XR9

XR X13 X14 X15 X16 X17 X18 X19 X20 X21 10

TN-Y: (c)

YA

SPX

1 YB1 YC1 YD1 YE1 YE2 YE3 YD2 YE4 YE2 YD1 YE5 8 2

Figure 3 The structures of the members of the tenascin family. (a) Rotary shadowing EM image of two mouse TN-C hexamers. (b) A model of a TN-C hexamer. The domains are color-coded as in (c). FN III, bronectin type III repeats; EGF-like, epidermal growth factorlike. (c) Models of the structures of the four or ve members of the tenascin family. Despite their apparently different structures, mammalian TN-X and avian TN-Y may be orthologs (see text). The TN assembly (TA) domain (green) is comprised of four heptad repeats and a number of cysteines that form interchain disulde bonds. In the cases of TNs X and Y, the white ovals indicate that the cysteines required to link trimers in higher order multimers are missing. The EGF-like repeats that are common to all vertebrates (salmon ovals) vary from 18.5 in TN-C to one and two halves in TN-Y. In the case of TN-C, an additional repeat specic to mammals (navy blue oval) is present. The FN-III repeats (blue, red, or purple rectangles) fall into several different categories that reect their composition or origin during evolution. A serine- and proline-rich domain (purple diamond) is found in TNs X and Y. A brinogen globe (yellow circle) is common to all TNs. Reproduced from Developmental Dynamics; Jones FS and Jones PL; copy right & 2000, Wiley-Liss. Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

comparisons of the OPN sequences among vertebrate species, a polyaspartic acid sequence, the thrombinand MMP-cleavage sites, and a GRGDS integrinbinding sequence are highly conserved.

CCN Proteins

The CCN proteins are a family of six homologous, secreted proteins that share a very similar structure.

EXTRACELLULAR MATRIX / Matricellular Proteins 179

Osteopontin

Thrombin cleavage site ( v 3, v 5, v 1, 8 1) PPP P P (CD44) P P P P P P PP COOH 300 Ca2+-binding domain

RGD

NH2

1 Ca2+-binding domain

169

SVVYGLR ( 9 1, 4 1, 4 7)

MMP cleavage site

Figure 4 A schematic structure of human OPN. The boxes from N- to C-terminus indicate domains that have some conrmed function as follows: (1) the signal sequence; (2) conserved caseinkinase-2 phosphorylation sites; (3) calcium-binding domain containing a polyaspartic acid sequence; (4) RGD site; (5) MMP cleavage site; (6) calcium-binding domain; and (7) heparin-binding domain. The positions of phosphorylated serines (P) are indicated. The site of thrombin cleavage occurs between amino acids 168 and 169 in the human protein. The approximate sites of interaction with a number of integrins and CD44 are shown. Kindly provided by Dr Cecilia Giachelli.

Cyr61, CTGF, and Nov are now termed CCN1, CCN2, and CCN3; CCN 46 were previously known as WISP (Wnt-1-induced secreted protein) 46. With the exception of CCN5, which lacks a C-terminal module, each of the proteins contains a signal peptide, an insulin-like growth factor-binding protein domain, a von Willebrand factor C repeat, a TSR or TSP1 repeat, and a C-terminal domain (Figure 5). Each of these modules is, in turn, encoded by a separate exon. This conservation of structure extends to the presence of 38 homologously placed cysteines in all CCN proteins except CCN6, which contains only six cysteines in its von Willebrand factor C repeat. However, the complexity of the family is increased considerably by the additional presence of variants that are either truncated N- or C-terminally by limited proteolysis, or that originate by alternative splicing. CCN variants or isoforms can perform functions that differ considerably from those of the parent protein.

Biological Functions of Matricellular Proteins

Thrombospondins 1 and 2

TSP1 was rst discovered as a major component of a-granules in platelets and was thought to be important in the formation of a platelet plug in response to injury. However, TSP1-null mice show no defects in clot formation and do not have a bleeding diathesis.

Instead, it seems likely that a major function of platelet TSP1 is to serve as a chemotactic factor that attracts inammatory cells (monocytes/macrophages), which in turn initiate wound repair. The phenotypes of TSP1- and TSP2-null mice have played an important role in our understanding of the function these proteins. This is true because it is difcult to determine the biologically relevant functions of these proteins by experiments in vitro. Thus, such experiments cannot replicate the interactions with multiple cell-surface receptors (Table 1) and other extracellular proteins displayed by TSP1 and TSP2, nor do they take into account the temporal and spatial specicity of their expression. A characteristic of matricellular proteins such as TSP1 and TSP2 is that the consequences of their absence is often not evident in uninjured adult animals. TSP1-null mice appear normal, but display a mild spinal lordosis, and have a higher than normal fetal mortality. A major manifestation is increased susceptibility to pneumonias, apparently caused by the normal ora of the upper respiratory tract. While the problem has not been studied carefully, a lack of the chemotactic activity of TSP1 for inammatory cells may be a factor. In addition, TSP1 functions to bind and activate latent transforming growth factor (TGF)-b1, and there is evidence for a deciency of active TGF-b1 in some tissues, particularly the lung, of TSP1-null mice, which could contribute to an increased predisposition to infection. The best-studied function of TSP1 is its ability

180 EXTRACELLULAR MATRIX / Matricellular Proteins

CCN2 CCN3 CCN5 CCN1 CCN4 (a) CCN6

Exon 1 Promoter Signal peptide NH2 No. of cysteine residues (b) 12 IGFBP

E2

E3

E4

E5 3-UTR

VWC

TSP1

Cysteine knot COOH 6 10

10(6)

Variable

Figure 5 The CCN proteins. (a) Dendogram showing the evolutionary relationships of the six CCN proteins. (b) Schematic representations of a typical CCN gene and protein. Each CCN gene consists of ve exons, each of which encodes a module in the protein. IGFBP, insulin-like growth factor binding protein; VWC, von Willebrand factor type C repeat; TSP1, thrombospondin-1 or TSR repeat. All CCN proteins contain the same number of cysteines in homologous modules, except CCN6, which contains 6 instead of 10 cysteines in the VWC module. Reproduced from Perbal B and Takigawa M (eds.) (2004) CCN Proteins: A New Family of Cell Growth and Differentiation Regulators. London: Imperial College Press, with permission from Imperial College Press.

Table 1 Interactions of thrombospondins with cell-surface receptorsa NH2-terminal domain Syndecans Sulfatides Calreticulin LRP1 a3b1c a4b1 a6b1
a

Type I repeats CD36 HIVgp120b ab1d ab1d

Type II repeats ab1 ab1d

d

Type III repeats avb3 a5b1

C-terminal domain CD47

Unless otherwise specied, both TSP1 and TSP2 interact with these receptors. No receptors have yet been found to interact with the procollagen domain. b 120 kDa glycoprotein on the surface of human immunodeciency virus. c Applies only to TSP1. d The sequences in the type I and II repeats are pan-b1 specic, i.e., they serve as ligands for several ab1 integrins.

to inhibit angiogenesis. As a consequence, tumors grow more rapidly and metastases are increased in the absence of TSP1. A number of studies have implicated the TSR repeats of TSP1 in its antiangiogenic properties. These repeats interact with the scavenger receptor CD36 on endothelial cells, and this interaction culminates in activation of caspases and apoptosis. TSP2-null mice also appear overtly normal and are normally fertile. However, their skin is unusually fragile, and tendons and ligaments are lax to an extent that the end of the tail can be tied into a knot. These ndings suggest a defect in collagen

brillogenesis; indeed, collagen brils in skin and tendon are generally wider in diameter and are irregular in contour by electron microscopy. The basis for these changes in collagen structure is not known, but an increase in levels of matrix metalloproteinase (MMP)-2 levels may be partially responsible. Both TSP2-null broblasts in culture and injured tissues in TSP2-null mice show increased levels of MMP2. The cause of this increase can be attributed to the fact that TSP2 serves as a clearance factor for MMP2. The TSP2MMP2 complex is recognized by the LRP1 scavenger receptor, endocytosed, and degraded in lysosomes. MMP2 and MMP9 levels are strikingly

EXTRACELLULAR MATRIX / Matricellular Proteins 181

increased in the myocardium of angiotensin II-induced hypertensive TSP2-null mice, and a high proportion of these mice die of cardiac rupture. TSP2, like TSP1, is an inhibitor of angiogenesis. While there is a modest increase in vascularity in uninjured skin, vessel density is markedly increased in healing excisional skin wounds and in the foreign body capsule surrounding implanted biomaterials in TSP2-null mice. As a consequence, these mice heal their wounds more quickly, and studies are under way to determine whether inhibition of TSP2 levels by local gene therapy can prolong the functional lifetime of implanted biosensors and delivery devices.
SPARC and Family Members

and grow normally. However, the major, striking manifestations of the SPARC-null mouse, such as cataracts, increased adiposity, and premature osteopenia, are lacking in the hevin-null mouse. Very little is known about the functions of other SPARC family members.
Tenascins

SPARC was rst identied, more or less simultaneously, as a secreted protein in cultures of endothelial cells, as a major noncollagenous protein in bone (termed osteonectin), and as a putative constituent of basement membranes (BM-40). However, its presence as an integral protein in basement membranes has not been conrmed. Puried SPARC protein inhibits cell attachment by interfering with focal adhesions and causes cells to arrest in the G1 phase of the cell cycle. SPARC also interacts with at least two growth factors (vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF)) and this binding probably accounts for its anti-angiogenic activity in vitro. As with targeted disruption of several other matricellular protein genes (gene knockouts), the study of SPARC-null mice has been highly informative and has revealed functions of SPARC that were unsuspected. The complex phenotype of the SPARC-null mouse is characterized by formation of cataracts at an early age, laxity, reduced tensile strength and collagen content of skin, accelerated closure of excisional dermal wounds, a marked increase in adipose tissue, severe premature osteopenia, and a propensity for increased growth and metastatic spread of a variety of ectopically transplanted tumors. Studies are under way to identify cell-surface receptors for SPARC and to elucidate the biochemical mechanisms responsible for the unusual ndings in the SPARC-null mouse. Hevin, also known as SC1, a close structural paralog of SPARC, is widely expressed in brain and in high endothelial venules, from which it receives its name. Hevin shares some functions with SPARC, including its ability to inhibit focal adhesions and cell attachment and proliferation. Hevin-null mice also heal excisional skin wounds more rapidly than normal. Hevin is thought to be involved in regulation of cell migration during embryonic development, although hevin-null mice appear normal at birth

The phylogenetic distribution of TNs, which contain a sequential arrangement of EGF-like and bronectin-III repeats and a brinogen domain, appears to be limited to vertebrates. In keeping with their classication as matricellular proteins, TNs are expressed primarily during development, in response to injury or mechanical stress, and in tumors. Indeed, TN-C was rst discovered as a glioma-associated extracellular protein. Despite a number of studies to determine whether the level of expression of TN-C can serve as a prognostic indicator, no consistent association has been found between the levels of TN-C and the invasiveness of tumors. Like TSPs and SPARC, TN-C shows counter-adhesive properties. There is evidence that TN-C interferes with the synergy between the major bronectin receptor, a5b1, and its co-receptor, syndecan-4, leading to reduced activity of Rho and disassembly of actin stress bers. However, like the TSPs, the effects of TN-C, as well as that of other TNs, can be cell specic. The TN-C-null mouse was initially described as normal, but subsequent analyses have revealed subtle defects. Thus, TN-C-null mice are more susceptible to snake venom-induced glomerulonephritis and to chemically induced dermatitis, ndings that are consistent with an anti-inammatory function of the protein. The lungs in TN-C-decient mice also show a defect in vascular branching. TN-C, -R, and -Y are all expressed in the central nervous system in patterns that are distinct, both with respect to anatomical location and appearance during development. It is thought that the alternatively spliced forms of these TNs may have subtly different properties. TN-C-null mice display abnormalities in motor coordination and exploratory behavior, and TN-R-null mice have reduced velocities in axonal conduction. TN-X is widely expressed in connective tissues, primarily in skin, tendon, heart, and smooth and skeletal muscle. The TN-X gene is part of a multigene cluster that includes the steroid 21-hydroxylase gene (CYP21A2), and a defect in TN-X was rst identied in a patient who also had adrenal hyperplasia due to CYP21A2 deciency. In this patient a single deletion removed the entire CYP21A2 gene and truncated the contiguous TN-X gene. Patients with TN-X deciency have a form of the

182 EXTRACELLULAR MATRIX / Matricellular Proteins

EhlersDanlos syndrome (benign joint hypermobility syndrome) that is characterized by hypermobile joints, hyperelastic skin, and easy bruisability. There is evidence that heterozygosity for the TN-X-null allele results in haploinsufciency. TN-X mice also show a constellation of ndings that mimic the hypermobility-type EhlersDanlos syndrome. These mice have hyperextensible skin and a reduced collagen content in dermis and tendon. Although the synthesis of collagen by cultured TN-X broblasts was normal, these broblasts failed to incorporate the collagen into the matrix. Tissues and broblasts from TN-X-null mice have increased levels of both proand active MMP2 and MMP9, and these proteases may be partly responsible for the adhesive defect seen in TN-X-null broblasts and the increased tumor spread and metastases in these animals.
Osteopontin

in macrophages. OPN also plays an important role in the immune system by stimulating T cell-mediated immunity, as well as B cell activation and antibody production.
CCN Proteins

In keeping with the expression patterns of other matricellular proteins, the distribution of OPN is limited in healthy adult animals and humans, but expression is markedly increased in disease and in response to injury. In normal bone, OPN is synthesized by both osteoblasts and osteoclasts and is thought to regulate the mineralization of bone matrix and bone remodeling, both by direct interaction with bioapatite and by binding to integrin cell-surface receptors. OPN similarly inhibits and promotes regression of ectopic calcication in vascular tissues by physical interaction with hydroxyapatite crystals and by induction of carbonic anhydrase II, thus promoting acidication of the extracellular milieu. In the kidney, OPN regulates renal inducible nitric oxide synthase (iNOS) and the deposition of urinary calcium oxalate. OPN is found normally in urine, saliva, milk, and bile, where the protein is also thought to inhibit the formation of concretions. Studies of OPN-null mice have provided support for these functions. Thus, mice that are null for matrix Gla protein, which develop precocious vascular calcication that is associated with increased OPN deposition, are even more prone to ectopic calcication when crossed with OPN-null mice. In acute and chronic injury both pro- and antiinammatory modes of action of OPN have been documented. As a pro-inammatory protein, OPN functions as a chemoattractant for macrophages and T cells and enhances the expression of Th1 cytokine by interaction with a number of cell-surface receptors, including CD44 and several integrins. As an anti-inammatory agent, OPN inhibits the induction of iNOS mRNA and thus blocks formation of NO and the production of reactive oxygen species

The constellation of domains present in CCN proteins is limited to vertebrates, although Drosophila appears to have a truncated homolog and individual domains are more widely distributed among metazoan species. CCN proteins are normally expressed during embryonic development, and in adult animals in the nervous, vascular, and musculoskeletal systems, and in the adrenal gland. However, in keeping with their matricellular properties, the expression of these proteins is enhanced during tissue repair, and in brosis, inammation, and tumor growth. CCN proteins are involved in regulation of cell-cycle progression, cell adhesion and migration, and control of both production and degradation of ECM proteins. As a result, these proteins participate in diverse processes such as angiogenesis, chondro- and osteogenesis, and tissue remodeling. CCN1 inuences the function of broblasts, endothelial and smooth muscle cells, platelets, and monocytes by interaction with at least ve different integrins. In some cases, heparan sulfate proteoglycans participate as co-receptors. In the case of actively motile cells, such as broblasts and smooth muscle cells, CCN1 stimulates formation of lopodia and lamellipodia that enable migration. Signal transduction leads to increased expression of MMP1 and 3. Adherence to CCN1 also protects endothelial cells from apoptosis upon serum withdrawal. CCN1 is rapidly activated transcriptionally by basic broblast growth factor, PDGF, and TGF-b1. In keeping with its important role as a proangiogenic factor, CCN1null mice die in early to midgestation as a result of defective vascular bifurcation and placental development, as well as compromised vascular and cardiac integrity. The histopathological ndings resemble those in av-null mice and thus identify CCN1 as a major ligand for av integrins. CCN2 was initially termed connective tissue growth factor, but it is generally recognized that its function in stimulating cell proliferation is indirect. CCN2 is also proangiogenic and stimulates endothelial adhesion, migration, and proliferation. Stimulation of migration is associated with dissolution of focal adhesions and disassembly of the actin cytoskeleton. The protein is expressed in the skeletal system and promotes synthesis of type II collagen and proteoglycans. CCN2-null mice die soon after birth from respiratory failure caused by extensive

EXTRACELLULAR MATRIX / Matricellular Proteins 183

skeletal defects in the thorax and elsewhere. Matrix remodeling in the hypertrophic zones of growth plates in long bones is defective, and this can be attributed to decreased expression of VEGF and reduced numbers of osteoblasts and osteoclasts that produce MMPs. Less is known about the functions of CCN3 (previously termed Nov) and CCN4-6 (WISP 1-3). CCN3 interacts with several integrins as well as with extracellular proteins, and appears to be involved in regulation of cell growth and differentiation. The protein is proangiogenic and induces neovascularization in corneal implants. Recently, CCN3 was shown to interact with the gap junction protein, connexin 43. This interaction may play a role in the growth control exerted by gap junction proteins. The expression of CCN4 is restricted to osteoblasts and to osteoblast progenitor cells of the perichondrial mesenchyme. CCN4 binds decorin and biglycan, two small, leucinerich proteoglycans, and promotes proliferation of mesenchymal cells and differentiation of osteoblasts, while repressing differentiation of chondrocytes. The protein functions similarly during fracture repair. The expression and function of CCN5 are also restricted largely to the regulation of osteoblast function. Mutations in the gene encoding CCN6 in humans cause recessive progressive pseudorheumatoid dysplasia, a disorder characterized by postnatal degeneration of articular cartilage.

Acknowledgments
I thank Drs Helene Sage, Cecilia Giachelli, Fred Jones, and Lester Lau for their review of parts of the article.
See also: Adhesion, Cell-Matrix: Integrins. Angiogenesis, Angiogenic Growth Factors and Development Factors. Extracellular Matrix: Collagens; Matrix Proteoglycans; Surface Proteoglycans.

Further Reading
Adams JC (2001) Thrombospondins: multifunctional regulators of cell interactions. Annual Review of Cell and Developmental Biology 17: 2551. Bornstein P (2001) Thrombospondins as matricellular modulators of cell function. Journal of Clinical Investigation 107: 929934. Bornstein P, Armstrong LC, Hankenson K, Kyriakides TR, and Yang Z (2000) Thrombospondin 2, a matricellular protein with diverse functions. Matrix Biology 19: 557568. Bornstein P and Sage EH (2002) Matricellular proteins: extracellular modulators of cell function. Current Opinion in Cell Biology 14: 608616. Brekken RA and Sage EH (2001) SPARC, a matricellular protein: at the crossroads of cellmatrix communication. Matrix Biology 19: 815827. Chiquet-Ehrismann R and Chiquet M (2003) Tenascins: regulation and putative functions during pathological stress. Journal of Pathology 200: 488499. Denhardt DT, Noda M, ORegan AW, Pavlin D, and Berman JS (2001) Osteopontin as a means to cope with environmental insults: regulation of inammation, tissue remodeling, and cell survival. Journal of Clinical Investigation 107: 10551061. Giachelli CM and Steitz S (2000) Osteopontin: a versatile regulator of inammation and biomineralization. Matrix Biology 19: 615622. Ivkovic S, Yoon BS, Popoff SN, et al. (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130: 27792791. Jones FS and Jones PL (2000) The tenascin family of ECM glycoproteins: structure, function, and regulation during embryonic development and tissue remodeling. Developmental Dynamics 218: 235259. Lau LF and Lam SCT (1999) The CCN family of angiogenic regulators: the integrin connection. Experimental Cell Research 248: 4457. Mo F-E, Muntean AG, Chen CC, et al. (2002) CYR61 (CCN1) is essential for placental development and vascular integrity. Molecular and Cellular Biology 22: 87098720. ORegan A (2003) The role of osteopontin in lung disease. Cytokine and Growth Factor Reviews 14: 479488. Perbal B and Takigawa M (eds.) (2004) CCN Proteins: A New Family of Cell Growth and Differentiation Regulators. London: Imperial College Press. Sullivan MM and Sage EH (2004) Hevin/Sc1, a matricellular glycoprotein and potential tumor-suppressor of the SPARC/BM40/osteonectin family. International Journal of Biochemistry and Cell Biology 36: 991996. Zweers MC, Hakim AJ, Grahame R, and Schalkwijk J (2004) Joint hypermobility syndromes: the pathophysiologic role of tenascin-X gene defects. Arthritis and Rheumatism 50: 2742 2749.

The Role of Matricellular Proteins in Respiratory Diseases

There is a small but growing literature that documents the association of increased expression of tenascin C, SPARC, TSP1, osteopontin, and CCN 1 and 4 with a variety of disorders, including neonatal respiratory distress syndrome, pulmonary tumors, and brosing diseases of the lung. In the majority of published studies the relation of these proteins to the disease states has been limited to the documentation of a spatial association. Since matricellular proteins are typically expressed in response to a variety of injuries, this association is to be expected. Nevertheless, in some cases the expression of the matricellular protein appears to have predictive value for patient outcome. Furthermore, preliminary evidence suggests that these proteins can exert regulatory functions that can either promote or inhibit the progression of the disease. Such opposing effects very likely reect the complex roles that matricellular proteins play in inammation, and in the control of cell adhesion, migration, division, and viability.

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Matrix Proteoglycans
C W Frevert, VA Puget Sound Medical Center, Seattle, WA, USA T N Wight, The Hope Heart Institute, Seattle, WA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Matrix proteoglycans, complex molecules composed of a core protein and glycosaminoglycan side chains, impart biomechanical properties to lung tissue and are important biological modiers which regulate processes such as lung development, homeostasis, inammation, and wound healing. The diverse roles of matrix proteoglycans suggest that these molecules play a critical role in normal and diseased lungs.

are four classes of glycosaminoglycans: (1) hyaluronan, (2) chondroitin sulfate (CS)/dermatan sulfate (DS), (3) heparan sulfate (HS)/heparin, and (4) keratan sulfate (KS). All four classes of glycosaminoglycan are found in normal lungs and all except hyaluronan are bound to core proteins. The predominant glycosaminoglycan in normal lungs is HS (4060%) followed by CS/DS (31%), hyaluronan (14%), and heparin (5%). Sulfation of glycosaminoglycans, a structural modication that occurs in the Golgi during chain elongation, has important biological consequences as specic sulfation patterns on glycosaminoglycans form the binding sites for a number of proteins including morphogens, growth factors, cytokines, and chemokines.

Introduction
Proteoglycans are a family of charged molecules containing a core protein and one or more covalently attached glycosaminoglycan side chains. The complexity and diversity of proteoglycans is derived from approximately 30 different core proteins of varying size and structure (10500 kDa) and the considerable variability in the number (1 to 4100) and types of glycosaminoglycan side chains attached. Proteoglycans are found in the extracellular matrix, plasma membrane of cells, and intracellular structures. Matrix proteoglycans such as perlecan, collagen XVIII, and agrin are found in the basal laminal of cells, and decorin, biglycan, and versican are found in the interstitial spaces of the lungs. The glycosaminoglycan side chains and core proteins of matrix proteoglycans regulate a number of biomechanical and cellular processes in normal and diseased lungs.

Glycosaminoglycan Structure
Hyaluronan is a repeating disaccharide structure composed of glucuronic acid and N-acetyl glucosamine that is nonsulfated and is not attached to a core protein (Table 1). CS has a disaccharide repeat pattern similar to hyaluronan, but it contains galactosamine instead of glucosamine. The galactosamine can have a sulfate ester attached at the 4- or 6-position (e.g., chondroitin 4-sulfate) and the glucuronic acid can have a sulfate ester placed at the 2-position. The degree of sulfation of CS is variable and can differ within a single preparation and from one tissue to another. Dermatan sulfate is a copolymer composed of two types of disaccharide repeats, D-glucuronate-N-acetyl galactosamine and L-iduronate-N-acetyl galactosamine. The formation of iduronic acid residues occurs by the conversion of glucuronic acid already incorporated into the growing polymer by an epimerization reaction that is tightly coupled to the sulfation process. KS has a repeating disaccharide residue containing galactose and N-acetyl glucosamine. The linkage of KS to the core protein differs from other glycosaminoglycans because it is not formed on the typical xyloseserine linkage. KS expression in lungs is

Glycosaminoglycan Side Chains

Glycosaminoglycans are linear polymers of repeating disaccharides with a high negative charge imparted by sulfate and/or carboxyl groups in their structure. There

Table 1 Structure of glycosaminoglycans Glycosaminoglycan Hyaluronan Chondroitin sulfate Dermatan sulfate Keratan sulfate Heparan sulfate Heparin Monosaccharide (1) D-glucuronic acid D-glucuronic acid D-glucuronic acid or L-iduronic acid D-galactose D-glucuronic acid or L-iduronic acid D-glucuronic acid or L-iduronic acid Monosaccharide (2) N-acetyl glucosamine N-acetyl galactosamine N-acetyl galactosamine N-acetyl glucosamine N-acetyl glucosamine N-acetyl glucosamine Sulfation patterns No sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 2-O-sulfation, 4-O-sulfation, 6-O-sulfation 6-O-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation 2-O-sulfation, 3-O-sulfation, 6-O-sulfation, N-sulfation

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limited but this glycosaminoglycan is present in the cartilage of the trachea. Heparin and HS are glycosaminoglycans of closely related structure and contain repeating disaccharide units composed of glucosamine and either D-glucuronic acid or L-iduronic acid. Glucuronic acid is the predominant uronic acid component in HS, whereas iduronic acid is the largest component in heparin. Heparin and HS contain a number of glucosamine residues that contain N-sulfate groups instead of N-acetyl groups. HS contains a lower degree of O-sulfation than heparin and therefore has fewer sulfates per disaccharide than heparin. In mature HS about 4050% of the amino sugars are converted from the N-acetylated form to the N-sulfated form. The N-sulfated residues occur predominantly in contiguous sequences or S-domains, which are separated by 15 disaccharides that are made in large part by N-acetyl-rich domains. This results in HS being a more complex molecule than heparin.

structural similarity to the protease inhibition domain in tissue inhibitor of metalloproteinase-1. Decorin and biglycan are small leucine-rich CSPGs with core proteins of similar size and structure (Table 2). The core protein of decorin has one CS/DS chain, whereas biglycan has two CS/DS chains. Versican is a large CSPG with a core protein consisting of an N-terminal globular domain (G1) which binds hyaluronan and a C-terminal binding domain (G3) which contains lectin-like, EGF-like, and CRP-like subdomains. The two globular domains, G1 and G3, are separated by two domains that contain CS chains. Alternative splicing of versican mRNA leads to four isoforms, V0, V1, V2, and V3, which carry varying amounts of CS side chains, and mRNA for all isoforms except V2 have been found in normal lungs.

Distribution of Matrix Proteoglycans

Basement membrane proteoglycans. The basement membrane or basal lamina of the cell contains proteoglycans that interact directly with cells (Figure 1). Proteoglycans found in the basement membrane include the HSPGs, perlecan, agrin, and collagen XVIII. Collagen IV is the only collagen which is more abundant in the basement membrane than collagen XVIII. Interstitial proteoglycans. The large CS proteoglycan, versican, is located in the interstitial spaces in the lungs where it interacts with hyaluronan (HA), which xes this proteoglycan in tissue as a very high-molecular-weight aggregate (Figure 1). Decorin and biglycan are also located in the interstitial spaces of the lungs where decorin binds collagen bers with a regular periodicity. Basal surface of cells. Syndecans are a small family of transmembrane HSPGs which interact with a number of matrix proteins (Figure 1). Whereas syndecans are not considered matrix proteoglycans (and are therefore not discussed in detail in this article), their interaction with matrix transmits signals plays a role in focal adhesion and cell death pathways. Syndecans are described in greater detail in Extracellular Matrix: Surface Proteoglycans.

Core Proteins of Matrix Proteoglycans

A number of HS proteoglycans (HSPGs), CS proteoglycans (CSPGs), and DS proteoglycans (DSPGs) are found in the matrix of lungs (Table 2). Perlecan is a large proteoglycan consisting of ve domains, which contain low-density lipid (LDL) receptor class A modules, Ig-like repeats, laminin-like repeats, and EGF-like repeats. Electron micrographs of perlecan showed a protein with the appearance of beads on a string, which resulted in the name perlecan. Whereas HS is the predominant glycosaminoglycan on perlecan, CS chains are found on domain V of recombinant perlecan. Collagen XVIII is an HSPG with features typical of collagen including sensitivity of the core protein to collagenase. This HSPG is of particular interest because of the presence of endostatin, a 22 kDa antiangiogenic peptide located in the C-terminal domain of collagen XVIII. Agrin is an HSPG that contains nine follistatin-like domains which share similarity to Kazal-type protease inhibitors, including pancreatic trypsin inhibitor, follistatin, thrombin inhibitor, and elastase inhibitor. The N-terminal laminin-binding domain of chick agrin has a high

Table 2 Matrix proteoglycans Proteoglycan Perlecan Collagen XVIII Agrin Decorin Biglycan Versican Core protein (kDa) 450 180 220 40 40 350 Location Basal lamina Basal lamina Basal lamina Interstitium Interstitium Interstitium GAG type HS/CS HS HS/CS CS/DS CS/DS CS

Biomechanical Properties of Proteoglycans

Extracellular matrix proteoglycans confer important biomechanical properties to lung tissue. Glycosaminoglycans are strongly hydrophilic and their high negative charge attracts cations such as Na , which pulls water into the matrix through osmotic activity. The large amount of water pulled into the

186 EXTRACELLULAR MATRIX / Matrix Proteoglycans

Alveolar space

Type II cell Epithelium

Alveolar macrophage

Interstitial space Syndecan-1

Perlecan Collagen

Versican-HA Interstitial cell Decorin Syndecans

Endothelium

Vascular space
Figure 1 Matrix proteoglycans in normal lungs. Perlecan, a HS proteoglycan, is found in the basal lamina of epithelial and endothelial cells. The CS proteoglycans, versican and decorin, are found in the interstitial space of the lungs. Versican binds to the glycosaminoglycan, hyaluronan, to form high-molecular-weight complexes. Decorin binds to collagen and helps stabilize the collagenelastin network. Syndecans are membrane proteoglycans that interact with matrix proteins. This gure is not meant to represent the concentrations of the different proteoglycans in normal lungs and is meant to only show their location in lung tissue.

matrix by glycosaminoglycans results in swelling of the matrix, which allows the matrix to withstand compressive forces. Proteoglycans interact with other matrix molecules such as collagen, laminin, and elastin to form a structured matrix. Work performed in decorin knockout mice suggests that the stabilization of the collagenelastin network by this proteoglycan contributes to lung elasticity and alveolar stability. Proteoglycans also provide a selective charge ltration barrier for the exchange of oxygen and plasma constituents and studies suggest that disruption of this charge barrier could lead to marked alterations in vascular permeability.

Proteoglycans as Determinants of Cellular Phenotype

Proteoglycans are important determinants of cellular phenotype, playing a critical role in development, cellular proliferation, cell death, inammation, and wound healing. A critical function of proteoglycans

in matrix is mediated by glycosaminoglycan side chains which bind to growth factors, cytokines, and chemokines. The binding of these proteins to glycosaminoglycans was initially believed to be a non-specic ionic interaction between positively charged amino acids on proteins and the negatively charged sulfates on glycosaminoglycans. It is now known that considerable specicity exists in the interaction between proteins and glycosaminoglycans with unique binding sites being identied on glycosaminoglycans for proteins such as thrombin and growth factors. The degree of specicity between proteins and glycosaminoglycans is still not known but there is speculation that there may be a specic sulfation pattern (e.g., binding site) for each protein that binds to a glycosaminoglycan. Current evidence suggests that the specicity observed in proteinglycosaminoglycan interactions plays an important role in the function of proteins in lung tissue. Listed below are ve potential functions of protein glycosaminoglycan interactions.

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1. Determination of protein binding sites. Because of the specicity observed in proteinglycosaminoglycan interactions, matrix proteoglycans position proteins such as chemokines and broblast growth factors to distinct anatomical locations (e.g., basement membrane or intersitial spaces). 2. Storage/sequestration. Glycosaminoglycans provide a site for proteins to bind, which sequesters a protein and prolongs its retention in lung tissue. The binding of chemokines to glycosaminoglycans provides a site for dimerization of chemokines which is a mechanism shown to increase the amount of chemokine binding in the lungs. Undersulfation of the basement membrane matrix of alveolar type II cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these alveolar epithelial cells. Increased sulfation of glycosaminoglycans under type I cells sequesters broblast growth factors, limiting their effect on this cell. In contrast, decreased sulfation of the matrix under type II cells allows broblast growth factors to activate signaling pathways in this cell. 3. Formation and stabilization of morphogen and chemokine gradients. The binding of morphogens and chemokines to glycosaminoglycans has been proposed as a mechanism whereby gradients develop in tissue. The binding afnity of chemokines to glycosaminoglycans, which is in the low micromolar range, provides a mechanism whereby the chemokine is able to diffuse and form chemotactic gradients in tissue. 4. Cell signaling. The binding of growth factors and cytokines to glycosaminoglycans has been shown to play a role in cellular activation. For proper presentation to broblast growth factor receptors, members of the broblast growth factor family interact with HSPG. 5. Protection from proteolysis. The binding of a protein to a glycosaminoglycan may sequester proteolytic cleavage sites, which protects proteins from proteolysis. Glycosaminoglycans have been shown to protect chemokines and growth factors from proteolytic cleavage.

a shift from the normal lung with HS predominating to the diseased lung where the predominant GAG is CS and DS. An increased deposition of the CSPG, versican, is a feature common to a number of interstitial lung diseases, including acute respiratory distress syndrome (ARDS), idiopathic pulmonary brosis, bronchiolitis obliterans organizing pneumonia, and lymphangioleiomyomatosis. Often associated with the increased expression of versican in the lungs is an increased deposition of hyaluronan and the CSPGs, decorin and biglycan. Patients with severe asthma have increased expression of versican and biglycan in their airways, which is correlated with airway responsiveness. Because of the potential for matrix PG and hyaluronan to affect mechanical properties and cellular function in the lungs, changes in their expression will most likely affect the clinical course of lung diseases. However, further work is required to determine the role of proteoglycans in the pathogenesis of lung disease.
See also: Alveolar Wall Micromechanics. Extracellular Matrix: Matricellular Proteins; Surface Proteoglycans. Fibroblast Growth Factors. Smooth Muscle Cells: Airway.

Further Reading
Allen BL, Filla MS, and Rapraeger AC (2001) Role of heparan sulfate as a tissue-specic regulator of FGF-4 and FGF receptor recognition. Journal of Cell Biology 155(5): 845858. Bensadoun ES, Burke AK, Hogg JC, and Roberts CR (1996) Proteoglycan deposition in pulmonary brosis. American Journal of Respiratory and Critical Care Medicine 154(6 Pt 1): 18191828. Cavalcante FS, Ito S, Brewer K, et al. (2005) Mechanical interactions between collagen and proteoglycans: implications for the stability of lung tissue. Journal of Applied Physiology 98(2): 672679. Frevert CW, Kinsella MG, Vathanaprida C, et al. (2003) Binding of interleukin-8 to heparan sulfate and chondroitin sulfate in lung tissue. American Journal Respiratory Cell and Molecular Biology 28(4): 464472. Fust A, LeBellego F, Iozzo RV, Roughley PJ, and Ludwig MS (2005) Alterations in lung mechanics in decorin-decient mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 288(1): L159L166. Giri SN, Hyde DM, Braun RK, et al. (1997) Antibrotic effect of decorin in a bleomycin hamster model of lung brosis. Biochemical Pharmacology 54(11): 12051216. Halfter W, Dong S, Schurer B, and Cole GJ (1998) Collagen XVIII is a basement membrane heparan sulfate proteoglycan. Journal of Biological Chemistry 273(39): 2540425412. Karlinsky JB, Bucay PJ, Ciccolella DE, and Crowley MP (1991) Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans. American Journal of Physiology 261(2 Pt 1): L148L155. Noonan DM, Fulle A, Valente P, et al. (1991) The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. Journal of Biological Chemistry 266(34): 2293922947.

Proteoglycans and Disease

Changes in the composition of glycosaminoglycans and proteoglycans in the lungs have been reported in animal models and human lung disease. A consistent nding in animal models such as exposure to lipopolysaccharide, bleomycin, and silica is an increase in the synthesis of CS and DS. This change occurs within the rst 72 h following exposure and results in

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Sannes PL, Khosla J, Li CM, and Pagan I (1998) Sulfation of extracellar matrices modies growth factor effects on type II cells on laminin substrata. American Journal of Physiology 275(4 Pt 1): L701L708. Sasaki T, Fukai N, Mann K, et al. (1998) Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO Journal 17(15): 42494256. Smits NC, Robbesom AA, Versteeg EM, et al. (2004) Heterogeneity of heparan sulfates in human lung. American Journal of respiratory Cell and Molecular Biology 30(2): 166173. Spillmann D, Witt D, and Lindahl U (1998) Dening the interleukin-8-binding domain of heparan sulfate. Journal of Biological Chemistry 273(25): 1548715493. Tapanadechopone P, Hassell JR, Rigatti B, and Couchman JR (1999) Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan. Biochemical and Biophysical Research Communications 265(3): 680690. Westergren-Thorsson G, Chakir J, Lafreniere-Allard MJ, Boulet LP, and Tremblay GM (2002) Correlation between airway responsiveness and proteoglycan production by bronchial broblasts from normal and asthmatic subjects. International Journal of Biochemistry & Cell Biology 34(10): 12561267.

Surface Proteoglycans
P W Park, Y Chen, and K Hayashida, Baylor College of Medicine, Houston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

denes proteoglycans as heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), or keratan sulfate proteoglycans (KSPGs). Some proteoglycan core proteins carry both heparan sulfate (HS) and chondroitin sulfate (CS) chains. Surface proteoglycans include the syndecan and glypican families with four and six members in mammals respectively, NG2 (AN2 in mice), betaglycan, thrombomodulin, a5b1 integrin, and CD44. The rst studies on GAGs and proteoglycans date back to 1916 when heparin, a highly sulfated version of HS, was unexpectedly identied as a potent anticoagulant in liver extracts (hence the name heparin) by a medical student who was trying to isolate a procoagulant molecule. HS was rst thought to be a contaminant in the heparin preparation, but was later distinguished from heparin in 1948 by the difference in the extent of sulfation and greater structural variability. For a long time, biological functions of proteoglycans were largely speculative and, in fact, most proteoglycans were thought to be specic to cartilage, functioning as cushions in joints for variable, compressive loads. Recent studies have revealed that surface proteoglycans also function as key modulators of many molecular interactions, including those relevant to lung biology.

Structure Abstract
Cell surface proteoglycans, such as syndecans, glypicans, and CD44, regulate a wide variety of molecular interactions that mediate cell adhesion, migration, proliferation, and differentiation. Through these activities, surface proteoglycans modulate many pathophysiological processes, including development, inammation, tissue repair, cancer metastasis, and infection. Proteoglycans are composites of glycosaminoglycans (GAGs) attached covalently to core proteins. The majority of the ligandbinding activities of proteoglycans are mediated through GAGs. Recent studies have revealed that surface proteoglycans play key roles in lung biology. In humans, expression of several surface proteoglycans is reduced in lung cancers, suggesting that these proteoglycans are potential prognostic markers. Mice lacking certain GAG biosynthetic enzymes die soon after birth from conditions resembling acute respiratory distress syndrome (ARDS), indicating that proteoglycans are essential for lung development and function. Furthermore, inammatory responses in mice made null for syndecan-1 and CD44 are drastically altered in various models of lung inammation. These data highlight the physiological significance of surface proteoglycans in lung development and in the pathogenesis of respiratory diseases.

Introduction
Nearly all mammalian cells express proteoglycans on their cell surface. The chemical nature of the glucosaminoglycans (GAGs) attached to core proteins

A proteoglycan consists of a core protein and one or several covalently attached GAG chains. GAGs are attached to and polymerized on certain Ser residues of a Ser-Gly dipeptide sequence, often repeated two or more times. GAGs are linear polysaccharides consisting of repeating disaccharide units that are dened by the composition and chemical linkage of the amino sugar and uronic acid monosaccharides in the disaccharide unit. The signature disaccharide repeat of an HS/heparin polysaccharide is GlcUAb1-4GlcNAca14, CS (including dermatan sulfate) is GlcUAb13GalNAcb14, keratan sulfate (KS) is Galb1-4GalNAcb13, and hyaluronan (HA) is GlcUAb13GlcNAcb14. Except for HA, these primary structures are modied in the Golgi apparatus by several sulfation and epimerization reactions that are catalyzed by distinct enzymes. Because the polymerization and modication reactions do not go to completion, the biosynthetic process generates an exceptionally diverse array of GAG structures, both in length and extent of modication. For example, HS, the most structurally heterogeneous GAG, varies in length from 50 to 150 disaccharides with cell type and core protein. However, a mere HS decasaccharide can potentially assume over 106 distinct sequences, which is already in vast excess of the estimated gene products

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that the whole human genome can generate. This enormous structural diversity largely explains why and how HS binds to so many proteins. Except for HA, HS, CS, and KS are all found covalently linked to specic core proteins in vivo as proteoglycans. Surface proteoglycans harbor HS, CS, or both. Core proteins of syndecans, NG2, CD44, betaglycan, and thrombomodulin are type I transmembrane proteins, whereas core proteins of glypicans are attached to the surface through a glycosylphosphatidylinositol (GPI) anchor. As shown in Figure 1, these core proteins have distinct structural designs and GAG-attachment sites. For example, syndecan core proteins contain an extracellular domain extended in conformation due to a high Pro content, whereas glypican extracellular domains are globular because they contain 14 highly conserved Cys residues that form intramolecular disulde

bonds. HS chains are attached distal to the plasma membrane on all syndecans and CS chains are also attached proximal to the cell surface on some syndecans (e.g., syndecan-1). GAG-attachment sites in NG2 (CS) and CD44 (HS/CS) are located in the middle portion of the core protein, whereas those of glypicans (HS), thrombomodulin (CS), and betaglycan (HS/CS) are proximal to the cell surface. The short cytoplasmic domain of syndecans contains several signaling and scaffolding motifs, such as three highly conserved Tyr and one highly conserved Ser for potential phosphorylation, and a C-terminal PDZ-binding domain. The NG2 cytoplasmic domain also contains a PDZ-binding domain and several Thr residues that may be phosphorylated. The CD44 cytoplasmic domain contains an ezrin, radixin, moesin (ERM) motif that may link surface CD44 to the actin cytoskeleton. No obvious signaling or

Syndecans (core proteins: 22 46 kDa) HS HS

Glypicans (core proteins: 62 66 kDa)

CD44 (core protein: varies with alt. splicing)

S-S HS or CS CS CS CS HS HS GPI

Betaglycan (core protein: 94 kDa)

Thrombomodulin (core protein: 62 kDa)

NG2 (core protein: 200 kDa)

CS HS or CS HS or CS

CS

Figure 1 Core proteins of syndecan and glypicans: structural designs and GAG-attachment sites. HS, heparan sulfate; CS, chondroitin sulfate.

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scaffolding motifs have so far been found in the cytoplasmic domains of thrombomodulin and betaglycan.

Regulation of Production and Activity

The temporal and spatial expression patterns of surface proteoglycans are highly regulated. For example, syndecan-1 is rst detected at the four-cell stage in mouse embryos, suggesting that its expression is zygotically activated. In adult lung, syndecan-1 is the predominant HSPG expressed on the basolateral surface of airway and alveolar epithelia. Lung epithelia also express syndecan-4, although its expression is much lower than that of syndecan-1. Several inammatory mediators (e.g., transforming growth factor beta (TGF-b), antimicrobial peptides) and pathological conditions (e.g., cancer, myocardial infarction) regulate the expression of surface proteoglycans at the transcriptional and posttranscriptional level. Furthermore, surface expression of syndecans and CD44 can be regulated by a proteolytic cleavage mechanism called ectodomain shedding. Ectodomain shedding is of paramount importance to proteoglycan biology because it both rapidly changes the surface phenotype of affected cells by reducing the amount of surface GAGs and generates soluble proteoglycan ectodomains replete with all their GAGs that can function as autocrine or paracrine effectors. Glypicans are also released from the cell surface by cleavage of their GPI anchor by phopholipases in vitro, suggesting that glypicans also function as soluble molecules in vivo. Other surface proteoglycans are also found in soluble form, but how they are released from the cell surface is not known. Because GAG structures dictate ligand-binding activities of proteoglycans, regulation of GAG biosynthetic enzymes is also thought to modulate proteoglycan function.

affecting the stability, conformation, and oligomerization state of either ligand or signaling receptor. Furthermore, binding of some chemokines to surface proteoglycans generates a chemotactic gradient that directs migration of inammatory cells to specic sites of tissue injury. Surface proteoglycans also function as soluble molecules because they can be released from the surface by ectodomain shedding and other mechanisms. Once solubilized, surface proteoglycans show functions similar to or distinct from their immobilized counterparts. The biological significance of surface proteoglycan functions is best exemplied by phenotypes of mice made null for the various biosynthetic enzyme and core protein genes. For example, mice lacking N-deacetylase/N-sulfotransferase-1 or C5 epimerase, enzymes required for the synthesis of sulfated HS, die shortly after birth due to respiratory failure. CD44 null mice show defects in the tissue distribution of myeloid progenitors. Glypican-3 null mice show phenotypes resembling SimpsonGolabiBehmel syndrome, a human overgrowth disorder, and they also show abnormal lung development. No apparent congenic abnormalities have so far been found in syndecan-1, -3, and -4, and glypican-2 and -4 null mice. However, syndecan-1 and -4 null mice show abnormal phenotypes when subjected to experimental models of inammatory diseases, and syndecan-1 null mice, in particular, exhibit various lung phenotypes as described below.

Surface Proteoglycans in Respiratory Diseases

Several lines of evidence indicate that surface proteoglycans are central players in the pathogenesis of major respiratory diseases (Figure 2). In lung cancer, surface expression of syndecan-1 and glypican-3 is significantly reduced in lung carcinomas. In general, low expression of surface proteoglycans is associated with a poor prognosis in lung and other cancers. High levels of soluble syndecan-1 ectodomains are also associated with a poor outcome in lung carcinomas, suggesting that aberrant activation of syndecan-1 shedding may promote lung cancer progression. The biological basis for the correlation between surface proteoglycan expression and lung cancer has yet to be clearly dened, but it is speculated that normal expression of surface proteoglycans is required to regulate cellular activities central to cancer progression, such as adhesion, migration, and proliferation. Lung inammation is exacerbated in CD44 null mice instilled intratracheally with bleomycin and in syndecan-1 null mice instilled intranasally with

Biological Functions
Surface proteoglycans, especially those harboring the highly structurally diverse HS chains, bind and regulate a plethora of biological molecules through their GAG chains. The list includes growth factors, cytokines, chemokines, proteinases and inhibitors, antimicrobial peptides, coagulation factors, surfactant proteins, extracellular matrix components, and many more. Surface proteoglycans can serve as primary receptors for some ligands (e.g., lipoprotein lipase). However, in most cases, surface proteoglycans function as co-receptors that capture ligands and catalyze the encounter between ligands and their respective signaling receptors. Surface proteoglycans can also regulate receptorligand interactions by

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Asthma: inhibit CC chemokine-mediated Th2 cell homing, attenuate allergic lung inflammation Ectodomain Lung infection: inhibit host defense, promote infection Lung cancer: promote cancer growth, metastasis

Shedding

Acute lung injury: generate CXC chemokine gradient that guides neutrophil transepithelial migration

Lung infection: promote microbial adhesion and invasion Lung cancer: regulate cell proliferation and migration Lumen

Airway epithelia

Figure 2 Functions of surface proteoglycans in lung diseases.

allergens, suggesting that these surface proteoglycans function as anti-inammatory molecules in idiopathic interstitial pneumonia and asthma. In the mouse model of interstitial pneumonia, CD44 is required to clear apoptotic neutrophils and accumulated HA fragments at sites of tissue injury, and to assure the correct activation of TGF-b1. In the mouse model of asthma, syndecan-1 attenuates allergic lung inammation by inhibiting the recruitment of Th2 cells to the lung, a central process in asthma pathogenesis. Here, syndecan-1 shedding by airway epithelial cells is activated by allergen challenge and shed ectodomains attenuate lung inammation by binding and inhibiting the CC chemokines CCL7, CCL11, and CCL17 through their HS chains. Syndecan-1 shedding has also been found to modulate lung inammation in bleomycin-induced acute lung injury. Upon injury, newly synthesized KC (a CXC chemokine, mouse functional homolog of human interleukin-8 (IL-8)) binds to syndecan-1 HS on alveolar epithelial cells and the syndecan-1/KC complex is shed by MMP-7. This process generates a KC gradient that guides the transepithelial migration of neutrophils into the alveolar compartment. Interestingly, syndecan-1 null mice are more susceptible to bleomycin-induced tissue damage and lethality, indicating that syndecan-1 shedding protects the host in acute lung injury by regulating and conning inammation to specic sites of tissue injury. In fact, these and other available data suggest that activation of epithelial syndecan-1 shedding is an innate host

response that assures the adequate and correct functioning of host inammatory responses. In lung infection, accumulating evidence indicates that many bacterial and viral pathogens exploit surface proteoglycans to promote their pathogenesis. First, respiratory pathogens, such as Mycobacterium spp., Bordetella pertussis, Haemophilus inuenzae, parainuenza virus, and respiratory syncytial virus, bind to the HS moiety of surface HSPGs to promote their adhesion and also their invasion of host cells if they are intracellular pathogens. In fact, microbial exploitation of surface HSPGs for host cell adhesion and invasion is a mechanism common to many intracellular pathogens, including nonrespiratory pathogens such as Neisseria gonorrhoeae, Leishmania spp., and herpes simplex virus. Second, several respiratory pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae, also take advantage of airway syndecan-1 by inducing ectodomain shedding to promote their lung pathogenesis. These pathogens activate syndecan-1 shedding through secreted virulence factors, and the shed ectodomains inhibit various host defense factors (e.g., antimicrobial peptides, collectins) through their HS chains. These processes modify the normally sterile pulmonary environment to favor infection over eradication. Consistent with this mechanism, syndecan-1 null mice resist intranasal P. aeruginosa, S. aureus, and S. pneumoniae lung infections, and wild-type mice are protected from infection by intranasal administration of ectodomain

192 EXTRACELLULAR MATRIX / Degradation by Proteases

shedding and HS antagonists. Together, these studies underscore the importance of surface proteoglycan functions in the pathogenesis of major respiratory diseases. Current efforts aimed at further dissecting the molecular mechanisms of how surface proteoglycans function in lung biology should yield relevant information needed to design new therapeutic approaches to attenuate, halt, or reverse various lung disorders.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chemokines. Chemokines, CXC: IL-8. Extracellular Matrix: Matricellular Proteins. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Degradation by Proteases
P J Stone and S M Morris, Boston University School of Medicine, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Matrix degrading enzymes are proteinases that are capable of processing substrates that are bound to surfaces or lie beneath the surface of tissue. The in vitro method of assessing matrix proteinases using puried substrates is relatively straightforward. However, extrapolation of the result to the case of the complex substrate or in vivo situation is often difcult and may lead to inaccurate predictions. An example of this is two proteinases that have similar elastolytic potency with puried elastin substrates but substantially different emphysema-inducing potencies in animal models.

Further Reading
Berneld M, Go tte M, Park PW, et al. (1999) Functions of cell surface heparan sulfate proteoglycans. Annual Review of Biochemistry 68: 729777. Berneld M, Kokenyesi R, Kato M, et al. (1992) Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annual Review of Cell Biology 8: 365393. Conrad HE (1998) Heparin-Binding Proteins, pp. 1527. San Diego, CA: Academic Press. Esko JD and Selleck SB (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annual Review of Biochemistry 71: 435471. Filmus J and Selleck SB (2001) Glypicans: proteoglycans with a surprise. Journal of Clinical Investigation 108: 497501. Forsberg E and Kjelle n L (2001) Heparan sulfate: lessons from knockout mice. Journal of Clinical Investigation 108: 175180. Gallagher JT (2001) Heparan sulfate: growth control with a restricted sequence menu. Journal of Clinical Investigation 108: 357361. Li Q, Park PW, Wilson CL, and Parks WC (2002) Matrilysinmediated shedding of syndecan-1 regulates chemokine mobilization and transepithelial efux of neutrophils in acute lung injury. Cell 111: 635646. Park PW, Pier GB, Hinkes MT, and Berneld M (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411: 98102. Park PW, Reizes O, and Berneld M (2000) Cell surface heparan sulfate proteoglycans: selective regulators of ligand receptor encounters. Journal of Biological Chemistry 275: 2992329926. Reizes O, Park PW, and Berneld M (2000) Cell surface heparan sulfate proteoglycans. In: Beckerle M (ed.) Cell Adhesion: Frontiers in Molecular Biology, pp. 155188. Oxford: Oxford University Press. Rostand KS and Esko JD (1997) Microbial adherence to and invasion through proteoglycans. Infection and Immunity 65: 18. Sanderson RD (2004) Heparan sulfate proteoglycans and heparanase: partners in osteolytic tumor growth and metastasis. Matrix Biology 23: 341352. Teder P, Vandivier RW, Jiang D, et al. (2002) Resolution of lung inammation by CD44. Science 296: 155158. Tyrrell DJ, Horne AP, Holme KR, Preuss JM, and Page CP (1999) Heparin in inammation: potential therapeutic applications beyond anticoagulation. Advances in Pharmacology 46: 151208.

Definition of Matrix Degrading Enzymes

Proteolytic enzymes (proteinases) catalyze the thermodynamically favored (but, normally, extremely slow) hydrolysis of peptide bonds in their selected substrates. The catalytic hydrolysis results in the addition of one water molecule across a peptide bond, usually in a number of steps, resulting in a new N-terminal amino group and a new C-terminal carboxyl group. Proteolytic processing often regulates processes involving those substrates and their products. Antiproteases regulate proteinase activity by slowing or inactivating the catalytic site or interfering with proteinasesubstrate interactions. Matrix degrading enzymes are proteinases that are capable of processing substrates that are bound to surfaces or lie beneath the surface of tissue. Thus, matrix processing enzymes are able to catalytically hydrolyze peptide bonds within a polymeric extracellular matrix protein, such as elastin. The core protein chain of proteoglycans can be degraded by a number of proteinases, including human neutrophil elastase. In addition, the glycosaminoglycan chains attached to the core protein can be degraded by a number of glycosaminoglycan degrading enzymes. Chronic excess elastin degradation is thought to play a central role in the pathogenesis of pulmonary emphysema.

Classication of Matrix Degrading Enzymes

Proteinases are usually categorized by their active site components that catalyze peptide bond hydrolysis: serine proteinases, (matrix) metalloproteinases, cysteine proteinases, and aspartyl proteinases. Serine

EXTRACELLULAR MATRIX / Degradation by Proteases 193

proteinases, metalloproteinases, and cysteine proteinases are discussed in detail elsewhere. Excess proteolysis by these enzymes has been linked to a number of pathologic processes. More than 500 genes in the human genome encode proteinases or proteinase-like sequences.

Identication of Matrix Degrading Enzymes and Their Sources

Sources of matrix degrading enzymes in the lung include blood-derived inammatory cells, microorganisms, and endogenous lung cells. Fibroblasts and smooth muscle cells can be induced to remodel surrounding extracellular matrix by expressing proteinases, including serine and metalloproteinases. Studies using transgenic animal models in which genes for individual proteinases are knocked out have been used to help identify suspect host proteinases in lung diseases such as pulmonary emphysema. Studies in which the substrate preferences of proteinases are altered by site-directed mutagenesis of individual amino acids in the proteinase may further pinpoint the role of these proteinases.

materials in the sampled biologic uid that are similar to substrate degradation products. For example, the measurement in the urine of desmosine, a crosslink amino acid characteristic of elastin, is rendered difcult by the presence of chemically similar nitrogen metabolites. In principle, the amount of desmosine in the urine should reect the degradation of elastin in the organism, since desmosine does not appear to be metabolized by the liver. However, the kidneys may slow by sequestration the excretion of increased levels of desmosine and release the excess desmosine over several days following in vivo treatment with elastase. On the other hand, measurement of desmosine in lung uids such as sputum or bronchoalveolar lavage uid should be relatively simple, but the correlation with lung elastin degradation is challenging, due to sampling and dilution effects. Two elastase enzymes exhibit different emphysemainducing potency An example of the discrepant results between in vivo and in vitro assays is provided by studies of two elastolytic enzymes, porcine pancreatic elastase and human neutrophil elastase, that have been used to produce experimental emphysema in rodent models by introduction into the lungs. It was observed that hamsters receiving nearly equal molar amounts of porcine pancreatic elastase or human neutrophil elastase exhibited a 59% increase in airspace size with the former but only a 19% increase with the latter. There was also a positive correlation between the increase in airspace size and the urine desmosine levels for the rst 3 days after treatment with elastase. Elevated urine desmosine levels were evidence for increased elastin destruction. Urine desmosine levels for animals receiving saline only, human neutrophil elastase, or porcine pancreatic elastase were 74, 212, and 816 ng per animal per day for the rst 3 days. However, both porcine pancreatic elastase and human neutrophil elastase exhibited similar enzymatic half-lives in the lung as measured in lavage uid, 51 and 45 min, respectively.
Assays Using Puried Substrates: The Two Elastases Exhibit Similar Activity

Assessment of Matrix Degradation

There is a wide range of assays to assess matrix degradation. These include in vitro methods that use a puried substrate, or a complex substrate derived from a living system that contains the substrate of interest. There are also in vivo methods in which degraded substrate is measured in biological uids or tissues. While the in vitro method using puried substrate is relatively straightforward, extrapolation of the result to the case of the complex substrate or in vivo situation is often difcult and may lead to inaccurate predictions.
In Vivo Assays

Elevated expression of a matrix degrading proteinase in vivo may not necessarily result in a proportionate increase in degradation product. Factors such as compensating increases in proteinase inhibitor levels or lack of access of proteinase to the substrate may intervene. Degradation may also be decreased by the distance between the source of the proteinase and its substrate. On the other hand, the presence of a protected microenvironment could favor degradation, such as one created by an adherent neutrophil releasing proteinase in close juxtaposition to the substrate. Measurement of substrate degradation in vivo is often complicated by the presence of many interfering

The elastin solubilizing activities of these two elastases were compared using puried elastin substrates. With elastin puried from calf neck ligament using a relatively gentle autoclaving method, human neutrophil elastase and porcine pancreatic elastase exhibited comparable elastolytic activity at 371C, that is, 367743 and 381746 mg elastin solubilized per nmole of elastase h 1 (mean7SD), respectively.

194 EXTRACELLULAR MATRIX / Degradation by Proteases Assays Using Reconstituted Systems Elucidate Differences in Potency of the Two Elastases

Cell culture models When the same amount of porcine pancreatic elastase or human neutrophil elastase was placed in a tissue culture ask containing a 5-week-old cell culture of primary smooth muscle cells with the serum washed away, the porcine pancreatic elastase solubilized 117728 mg elastin per nmol elastase and the human neutrophil elastase solubilized only 1173 mg elastin per nmol elastase or 971% of the 117 mg elastin solubilized by porcine pancreatic elastase. Yet, with elastin puried from the cultures porcine pancreatic elastase and human neutrophil elastase exhibited comparable levels of elastin solubilizing activity, 297738 and 2707133 mg elastin solublized per nmol of elastase h 1, respectively. Thus, human neutrophil elastase exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity with intact cell cultures as compared with the puried elastin substrate, whereas porcine pancreatic elastase exhibited only a 1.5- to 2.5-fold reduction. Desmosine cross-links were used as the marker for elastin in the protein mixture. This effect could not satisfactorily be explained as preferential inhibition of human neutrophil elastase activity in the culture system, because the amount of protein solubilized by human neutrophil elastase was 59% that of porcine pancreatic elastase. The mean elastin content of porcine pancreatic elastase-solubilized protein was 110% that of the elastin content of the corresponding cell culture; the value for human neutrophil elastase-solubilized protein was only 16%. The amount of elastin per microgram of solubilized protein for human neutrophil elastase was 15% that for porcine pancreatic elastase. Additional insights into the mechanism behind this greatly decreased activity of human neutrophil elastase in solubilizing elastin in complex substrates were obtained by immunocytochemical study of these cultures employing antibodies to human neutrophil elastase and porcine pancreatic elastase. This study revealed that porcine pancreatic elastase penetrates the smooth muscle cell cultures more readily than does human neutrophil elastase (Figure 1). Because the amount of elastin in these cultures increases with increasing distance from the free surface, the lesser amounts of elastin solubilized by human neutrophil elastase may be partly due to poor penetration of human neutrophil elastase into the living extracellular matrix, resulting in limited access to elastin substrate. Ultrastructural and immunocytochemical studies indicated, however, that even when human neutrophil elastase does have access to elastin substrate, it is less efcient than porcine pancreatic elastase at penetrating and degrading individual elastic bers (not shown).

(a)

(b)
Figure 1 Smooth muscle cell cultures treated with (a) porcine pancreatic elastase and (b) human neutrophil elastase. Elastase was localized with a primary antibody specic for each enzyme followed by a secondary antibody conjugated to colloidal gold followed by silver enhancement to produce a black precipitate at the site of the elastase. Equivalent amounts of enzyme and equal exposure times resulted in complete penetration of the culture by porcine pancreatic elastase, while human neutrophil elastase remained near the top of the culture. Scale bar 10 mm.

Human lung tissue A similar approach was used in comparing the activity of human neutrophil elastase and porcine pancreatic elastase in solubilizing elastin in human lung tissue. Lung tissue was exposed in vitro to human neutrophil elastase or porcine pancreatic elastase. Although both enzymes solubilized protein at similar rates, porcine pancreatic elastase solubilized elastin ve times faster than did human neutrophil elastase, based on measurement of soluble desmosine cross-links characteristic of elastin. Only 1% of the protein solubilized by human neutrophil elastase was elastin, whereas elastin accounted for 8% of the protein solubilized by porcine pancreatic elastase. Ultrastructurally, human neutrophil elastase-exposed tissue exhibited fewer damaged elastic bers as well as some bers that were damaged at the edges, whereas the interior of the ber appeared intact. Elastic bers in tissue exposed to porcine pancreatic elastase always showed extensive damage in both the exterior and interior of the ber. Immunocytochemical studies in which antibodies to human neutrophil elastase and porcine pancreatic elastase were applied to thin sections of Lowicryl-embedded tissue indicated that both of these elastases could be detected in association with elastic bers, but only in areas of the ber that showed morphological evidence of elastase injury (Figure 2). Factors that might explain the relatively low elastolytic activity of human neutrophil elastase in vivo or in reconstituted systems include the lower ability

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 195

(a)

(b)

Figure 2 Elastic bers in human lung tissue exposed to (a) porcine pancreatic elastase or (b) human neutrophil elastase. Primary antibody specic for each elastase was followed by a secondary antibody conjugated to colloidal gold. Elastolytic digestion is evident throughout the elastic ber in tissue treated with pancreatic elastase and colloidal gold particles are seen over the entire ber. The elastic ber exposed to neutrophil elastase shows damage and colloidal gold particles only at the periphery. (a) Scale bar 0.5 mm; (b) scale bar 0.25 mm.

of human neutrophil elastase to move through tissue as well as penetrating individual elastic bers. Purication of elastin involves the homogenization of tissue and the formation of nely divided elastin that is more susceptible to degradation by human neutrophil elastase. Other possibilities include the presence of sulfated proteoglycans in the extracellular matrix, such as proteoglycans containing chondroitin sulfate and heparan sulfate that are inhibitors of human neutrophil elastase and might exert a protective effect.
See also: Chronic Obstructive Pulmonary Disease: Emphysema, General. Extracellular Matrix: Elastin and Microbrils; Matrix Proteoglycans; Surface Proteoglycans. Matrix Metalloproteinases. Serine Proteinases.

Further Reading
Lopez-Otin C and Overall CM (2002) Protease degradomics: a new challenge for proteomics. Nature Reviews 3: 509519. Morris SM and Stone PJ (1995) Immunocytochemical study of the degradation of elastic bers in a living extracellular matrix. Journal of Histochemistry and Cytochemistry 43: 11451153.

Morris SM, Stone PJ, and Snider GL (1993) Electron microscopic study of human lung tissue after in vitro exposure to elastase. Journal of Histochemistry and Cytochemistry 41: 851866. Owen CA, Campbell MA, Sannes PL, Boukedes SS, and Campbell EJ (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. Journal of Cell Biology 131: 775789. Shapiro SD, Goldstein NM, Houghton AM, et al. (2003) Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. American Journal of Pathology 163: 23292335. Starcher B and Peterson B (1999) The kinetics of elastolysis: elastin catabolism during experimentally induced brosis. Experimental Lung Research 25: 407424. Stone PJ, Bryan-Rhad J, Lucey EC, et al. (1991) Measurement of urinary desmosine by isotope dilution and high performance liquid chromatography: correlation between elastase-induced air-space enlargement in the hamster and elevation of urinary desmosine. American Review of Respiratory Disease 144: 284290. Stone PJ, McMahon MP, Morris SM, Calore JD, and Franzblau C (1987) Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase: an in vitro model of elastolytic injury. In Vitro Cellular & Developmental Biology 23: 663676.

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

K Lewandowski and M Lewandowski, Elisabeth Krankenhaus, Essen, Germany
& 2006 Elsevier Ltd. All rights reserved. advanced intensive care, extracorporeal membrane oxygenation may be established as an additional therapeutic option during the acute phase. Extracorporeal membrane oxygenation is able to partly take over oxygenation and carbon dioxide removal and thereby allow respirator settings to be adjusted to the aims of lung protective mechanical ventilation. The key features of an extracorporeal circuit are catheters for vascular access, tubing for blood drainage and return, pump, membrane lung, heat exchanger, and monitoring unit. Venovenous circuits are established

Abstract
For patients with severe acute respiratory failure whose pulmonary gas exchange cannot be improved sufciently by means of

196 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

if the major goal is respiratory support while venoarterial techniques are considered if combined cardiac and respiratory failure is an issue. Bleeding remains the most prevailing complication in adult patients with acute respiratory distress syndrome (ARDS) undergoing extracorporeal gas exchange, and seems partly related to anticoagulation. Surface-heparinized extracorporeal circuits and membranes markedly reduce the daily blood loss and survival rates are higher than in patients treated with nonheparinized systems. Survival rates of adult ARDS patients treated with extracorporeal gas exchange are 3350%. In neonates with severe respiratory failure, survival rates of 68% and higher are reported.

Introduction
Extracorporeal gas exchange has an established role as salvage therapy for severe respiratory or cardiac failure in adults, children, and neonates unresponsive to optimal ventilator and intensive care management. In patients with acute respiratory distress syndrome (ARDS), the technique may be helpful in establishing lung protective mechanical ventilation and thereby avoiding ventilator-associated lung injury. Further indications of extracorporeal gas exchange include treatment of acute exacerbations of chronic lung diseases and bridging therapy for lung transplant recipients until an organ becomes available. It is the purpose of this article to review the history, rationale, technique, and outcome of this demanding procedure.

History
Gibbon began developing the heart-lung machine in 1937 to permit open-heart surgery. He designed a system in which anticoagulated blood was directly exposed to oxygen (lm or bubble oxygenators). However, due to the direct contact between blood and the gaseous phase severe hemolysis, thrombocytopenia, hemorrhage, and organ failure complicated the treatment and limited the use of this device to a few hours. In 1956, Clowes and his associates developed an articial lung that separated the gaseous from the liquid phase by a membrane. This membrane oxygenator, with subsequent improvements in materials, provided faster and more efcient blood oxygenation with fewer complications than the lm or bubble oxygenators and became practical for cardiopulmonary bypass taking more than a few hours. In 1972, Hill and colleagues reported the survival of a 24-year-old polytraumatized patient with ARDS who had been treated with extracorporeal membrane oxygenation (ECMO) during the acute phase of the disease. Four years later, Bartlett and colleagues described the rst newborn to be treated with ECMO, who survived. These enthusiastic publications nourished the hope that an effective new symptomatic therapy for

severe hypoxia in ARDS was now available. A large randomized multicenter trial was launched in 1974 to test venoarterial ECMO versus conventional therapy in adult ARDS patients. The study revealed that mortality rates in the ECMO therapy group were as high as 90% and not significantly different from those in the conventionally treated group. In the following years, Kolobow and Gattinoni presented an advanced technique where oxygen uptake and carbon dioxide (CO2) removal were dissociated. Oxygenation was primarily accomplished through the nearly motionless natural lung via apneic oxygenation or low-frequency positive-pressure ventilation (LFPPV), while CO2 was cleared through the articial lung (extracorporeal CO2-removal, ECCO2R). ECCO2-R was performed at low extracorporeal blood ows (2030% of cardiac output) so that a venovenous bypass technique instead of a venoarterial one sufced; this turned out to be less harmful to blood cells, coagulation, and internal organs. With this technique, survival rates of up to 49% were reported. In 1994, a second randomized controlled trial tested the LFPPV-ECCO2-R technique against advanced standard treatment. Again, survival rates in the ECCO2-R group were not significantly different from those in the conventionally treated group (42% conventional vs. 33% ECCO2-R). In 1983, Swedish researchers further improved the ECMO technique by binding the heparin molecule covalently to all synthetic surfaces of the circuit. It was thereby possible to avoid full systemic anticoagulation.

Uses in Respiratory Medicine

Terminology

Several acronyms have been used to describe the variety of techniques that have been invented to extracorporeally oxygenate blood and remove CO2. When the acronym ECMO was coined in the 1970s, it stood for a high-ow venoarterial bypass system that was aimed primarily at blood oxygenation. Subsequently, this method was rened and other forms of extracorporeal bypass techniques were introduced (see Table 1). The acronym ECMO survived the changing technologies and today is a general byword for the wide range of methods that are currently in use for extracorporeal blood oxygenation and CO2 removal. However, the question of which acronym to use is of less importance than characterizing the applied extracorporeal circulation and pulmonary support system. At least three criteria need to be reported: the vascular access used (venovenous, venoarterial, or arteriovenous), the proportion of

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 197

Table 1 Common acronyms for various types of extracorporeal gas exchange Acronym ECMO ECCO2-R PECOR ECLA ECLS AVCO2, av-ECLA, pECLA Meaning Extracorporeal membrane oxygenation Extracorporeal CO2 removal Partial extracorporeal CO2 removal Extracorporeal lung assist Extracorporeal life support Pumpless extracorporeal lung assist System Traditional high-ow venoarterial bypass system aiming primarily at blood oxygenation Low-ow venovenous bypass technique for extracorporeal gas exchange and CO2 removal Low-ow venovenous bypass to eliminate a part of the bodys CO2 load in patients with chronic lung diseases Venovenous low-ow bypass system in patients who were not endotracheally intubated and mechanically ventilated Prolonged but temporary (130 days) support of heart or lung function using mechanical devices Arteriovenous pumpless low-ow bypass system for patients with respiratory failure

cardiac output pumped, and the ventilatory regimen of the natural lung.
Rationale of ECMO

Within the last decades it has become evident from laboratory and clinical research that in patients with severe acute respiratory failure, mechanical ventilation contributes to the progression of the disease. The term ventilator-induced lung injury (VILI) was coined to describe the non-specic radiographic, physiologic, and pathologic manifestations of acute lung injury and its complications. VILI is viewed as a systemic disease that closely resembles the symptoms and the macroand microscopical features of experimental acute lung injury and is not markedly different from the diffuse alveolar damage present in human ARDS. It may be associated with pulmonary and systemic infections, multisystem organ dysfunction, volutrauma, barotrauma, and increased mortality. Establishing a lung-protective-ventilation strategy in severe acute respiratory failure may be incompatible with the goal of maintaining sufcient gas exchange. ECMO is able to take over part of oxygenation and CO2 removal and thereby may allow respirator settings to be adjusted to the mechanical and gas exchange properties of the diseased lung so that the aims of lung-protective mechanical ventilation, even in severe acute respiratory failure, can be achieved.
ECMO Technique

Important components of an extracorporeal circuit for membrane oxygenation include catheters for vascular access, tubing for blood drainage and return, pump, membrane lung, heat exchanger, and monitoring unit. The circuit design is essentially the same in neonatal, pediatric, and adult ECMO. Nowadays a wide array of vascular access cannulas is available

for surgical introduction or percutaneous cannulation of the femoral, iliac, and jugular veins or the femoral, carotid, axillar, or iliac arteries; they are usually made of polyvinylchloride. Extracorporeal blood ow may be routed in three possible ways: venoarterial, venovenous, and arteriovenous. Venoarterial access is the traditional method and is required when there is combined cardiac and respiratory failure. Venoarterial ECMO is the most common mode of extracorporeal support in children and has so far accounted for two-thirds of treatments in this group. Venovenous access is the method of choice for respiratory support in all age groups as long as adequate cardiac function permits. In the majority of patients, especially in adults with primary respiratory disease, venovenous ECMO has proved to be satisfactory. Arteriovenous access is applied when a pumpless ECMO technique is used. This system works with the pressure gradient between artery and vein. To move the blood through the circuit, either nonocclusive or occlusive roller pumps or centrifugal pumps are employed. However, most centers prefer to use the roller pump because it generates only mild pressure uctuations during blood inow and outow, and produces little hemolysis. The membrane oxygenator with a surface area of up to 4.5 m2 is the heart of the extracorporeal circuit. Most available devices work in a similar fashion. Blood ows through the tubing into the membrane lung, which is either assembled like an envelope or consists of thousands of microporous bers of a hollow ber device. Oxygen ows through the membrane lung in the opposite direction to ow of the bloodstream, diffuses into the blood, and CO2 is eliminated blood then enters the tubing and is perfused via the heat exchanger and via the return cannula into the patient. The initial contact of blood with foreign surfaces induces profound activation of the coagulation

198 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

system. To avoid clotting, either systemic heparin should be administered or the entire circuit needs to be coated with heparin. Finally, to make the extracorporeal system safe, it is crucial that a sophisticated level of monitoring is employed. This should include online circuit pressure monitors and ow monitors, bubble detectors, and premembrane and postmembrane oxygen saturation assessment. Figure 1 shows an example of a typical venovenous bypass.
Complications of ECMO

anticoagulation. It has been shown that the use of surface-heparinized ECMO circuits and membranes reduces the daily blood loss, the amount of substituted red cells, and the necessary i.v. heparin dose. Survival rates are higher than in patients treated with nonheparinized systems.
Outcome from ECMO

Known hazards of the ECMO technique can be classied into mechanical and patient-related medical complications. Mechanical complications include oxygenator failure, tubing/circuit disruption, pump or heat exchanger malfunction, and problems associated with cannula placement or removal. Patient-related medical problems are bleeding, neurologic complications, additional organ failure (e.g., renal, cardiovascular, liver), barotrauma, infection, and metabolic disorders. Available literature suggests that bleeding remains the most important complication in adult patients with ARDS undergoing extracorporeal gas exchange and seems partly related to constant

Over the last decade, survival rates of adult ARDS patients treated with extracorporeal gas exchange in prospective observational studies have stabilized at a level of 5070%. This is in sharp contrast to the extremely low survival rates of 10% reported in the rst US ECMO study of 1974 and also markedly better than those of the second randomized controlled trial of 1994 (33%). In 1996, the results from a UK ECMO randomized controlled trial in 185 neonates with severe respiratory failure showed a survival rate of 68% in the ECMO group. Survival was significantly worse in the conventionally treated control group (41%). Since long, extensive information regarding survival in ECMO-treated adults, neonates, and children with severe acute respiratory failure has been provided by the Extracorporeal Life Support Organization

Respirator

Membrane oxygenator

Roller pump

Figure 1 Schematic drawing of a low-ow venovenous ECMO circuit. In the typical low-ow venovenous bypass often used today, venous blood is drained through two heparin-coated catheters percutaneously inserted from both groins into the inferior vena cava and then connected by a Y-piece. The oxygenated blood is returned into the superior vena cava through a catheter, which is advanced percutaneously via the right internal jugular vein. A femoral-jugular venovenous bypass is established using a near-occlusive roller pump and a parallel conguration of two hollow ber oxygenators. The gas ow rate is set to the required CO2 levels. Oxygenation is accomplished through the mechanically ventilated natural lung as well as by arterializing the circulating blood via the membrane oxygenator. Reproduced from Lewandowski K (2000) Extracorporeal membrane oxygenation for severe acute respiratory failure. Critical Care 4: 156168, with permission from BioMed Central Ltd, Current Science Group.

EXTRACORPOREAL MEMBRANE-GAS EXCHANGE 199

(ELSO) Registry. Yearly survival rates have remained constant at 80% for neonatal cases regardless of diagnosis. In children, a survival rate of slightly more than 50% has been documented since 1989, which is a great improvement on the 30% survival rate recorded previously.
Other Gas Exchange Devices

Superior vena cava

Strictly speaking, the intravenous oxygenator (IVOX) and the Hattler catheter, an intravenous membrane oxygenator, are not extracorporeal membrane-gas exchangers. Nevertheless, they are interesting in that they represent options to reduce the technical complexity, the pump-associated complications, and costs and manpower usually associated with ECMO. The IVOX device is a membrane oxygenator system (surface area 0.210.52 m2) congured as a complex of thin hollow bers mounted on a catheter. It is positioned in the vena cava and venous blood circulates through the pores of the oxygenator while pure oxygen is supplied to the hollow bers by an external pump. Compared with ECMO the gas exchange capacity appears to be significantly inferior. The average O2 transfer in preliminary human studies was 50100 ml min 1 and the average CO2 elimination was 4874 ml min 1. Clinicians rarely used this device; however, it has served as a prototype for future models, such as the Hattler Catheter. The features of the Hattler Catheter (see Figure 2) are somewhat similar to those of the IVOX. However, the key component of the Hattler catheter is a central pulsating balloon, helically surrounded by 1000 hollow ber membranes. Through three exteriorized gas pathways, O2 is introduced into the body and CO2 is removed from the blood. The Hattler catheter is inserted simply through a vein in the leg, which can be done using percutaneous insertion, and placed in the vena cava with its tip located in the vena cava superior. The balloon pulsates with a frequency of 150 per min to move the bers and mix the blood in that region. Oxygen, which is suctioned through the bers, diffuses from the membranes into the blood. CO2 diffuses into the membranes and is removed by a vacuum tube. The device is not able to replace fully the function of the lungs, but is only able to supplement oxygenation of the body. Human lungs have a gas exchange area of 100 m2 while the Hattler catheter works with just 0.210.23 m2 of exchange area. The gas exchange capacity was determined to be 115305 ml min 1 m 2 membrane surface for O2 and 128340 ml min 1 m 2 for CO2. Finally, the pumpless arteriovenous extracorporeal lung assist (av-ECLA), also termed pumpless

Hollow fiber membranes

Pulsating central balloon

Right atrium Inferior vena cava

Insertion through peripheral vein site Exteriorized gas pathways

Figure 2 The Hattler Catheter in situ. Modied from http:// www.alung.com/cath.html, with permission from Professor B G Hattler.

extracorporeal lung assist (pECLA), is another true extracorporeal device for gas exchange (see Figure 3). The system is driven by the pressure gradient between the femoral artery and the respective vein. This is technically feasible because the drop in pressure induced by the oxygenator is approximately only 15 mmHg. Extracorporeal blood ow ranges between 1 and 2.6 l and allows for sufcient CO2 elimination (minimally 38 ml l 1 membrane oxygenator blood ow) and a remarkable increase in oxygenation (minimally 45 ml l 1 membrane oxygenator blood ow). However, these levels will only be reached if a relatively high blood ow through the oxygenator and a sufcient mean arterial blood pressure of around 70 mmHg can be established. Preliminary results in a limited

200 EXTRACORPOREAL MEMBRANE-GAS EXCHANGE

Femoral artery

Flow sensor

Oxygen inflow

Femoral vein

Figure 3 A pumpless extracorporeal lung assist placed between the patients legs. The device is an arteriovenous shunt with an interposed membrane oxygenator. Reproduced from Reng CM, Philipp A, Kaiser M, et al. (2000) Pumpless extracorporeal lung assist and adult respiratory distress syndrome. Lancet 356: 219220, with permission from Elsevier.

number of patients with ARDS have revealed encouraging results.

See also: Acute Respiratory Distress Syndrome. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading
Bartlett RH, Roloff DW, Custer JR, et al. (2000) Extracorporeal life support. The University of Michigan experience. Journal of American Medical Association 283: 904908. Clowes GH, Hopkins AL, and Neville WE (1956) An articial lung dependent upon diffusion of oxygen and carbon dioxide through plastic membranes. Journal of Thoracic and Cardiovascular Surgery 32: 630637. Dreyfuss D and Saumon G (1998) Ventilator-induced lung injury: lessons from experimental studies. American Journal of Respiratory and Critical Care Medicine 157: 294323. Gattinoni L, Pesenti A, Mascheroni D, et al. (1986) Low-frequency positive pressure ventilation with extracorporeal CO2-removal in severe acute respiratory failure. Journal of American Medical Association 256: 881886. Gibbon JH (1937) Articial maintenance of circulation during experimental occlusion of pulmonary artery. Archives of Surgery 34: 11051131. Hemmila MR, Rowe SA, Boules TN, et al. (2004) Extracorporeal life support for severe acute respiratory distress syndrome in adults. Annals of Surgery 240: 595607. Hill JD, OBrien TG, Murray JJ, et al. (1972) Prolonged extracorporeal oxygenation for acute post-traumatic respiratory failure (shock-lung syndrome). New England Journal of Medicine 286: 629634. Kolobow T and Bowman RL (1963) Construction and evaluation of an alveolar membrane articial heart-lung. Transactions of the American Society of Articial Internal Organs 9: 238243.

Kolobow T, Gattinoni L, Tomlinson T, and Pierce JE (1977) Control of articial breathing using an extracorporeal membrane lung. Anesthesiology 46: 138141. Lewandowski K (2000) Extracorporeal membrane oxygenation for severe acute respiratory failure. Critical Care 4: 156168. Lewandowski K, Rossaint R, Pappert D, et al. (1997) High survival rate in 122 ARDS patients managed according to a clinical algorithm including extracorporeal membrane oxygenation. Intensive Care Medicine 23: 819835. Liebold A, Reng CM, Philipp A, et al. (2000) Pumpless extracorporeal lung assist experience with the rst 20 cases. European Journal of Cardio-Thoracic Surgery 17: 608613. Morris AH, Wallace CJ, Menlove RL, et al. (1994) Randomized clinical trial of pressure-controlled inverse ratio ventilation and extracorporeal CO2 removal for adult respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 149: 295305. Pesenti A, Bombino M, and Gattinoni L (1998) Extracorporeal support of gas exchange. In: Marini JJ and Slutsky AS (eds.) Physiological Basis of Ventilatory Support, pp. 9971020. New York: Dekker. Reng CM, Philipp A, Kaiser M, et al. (2000) Pumpless extracorporeal lung assist and adult respiratory distress syndrome. Lancet 356: 219220. UK Neonatal ECMO Trial Group (1996) UK collaborative randomized trial of neonatal extracorporeal membrane oxygenation. Lancet 348: 7582. Zapol WM, Snider MT, Hill JD, et al. (1979) Extracorporeal membrane oxygenation in severe acute respiratory failure. A randomized prospective study. Journal of American Medical Association 242: 21932196. Zwischenberger JB and Alpard SK (2002) Articial lungs: a new inspiration. Perfusion 17: 253268.

Relevant Website
http://www.med.umich.edu Extracorporeal Life Support Organization (ELSO) Registry website with information regarding survival in ECMO-treated adults, neonates, and children with severe acute respiratory failure.

F
FIBRINOLYSIS
Contents

Overview Plasminogen Activator and Plasmin

Overview
A A-R Higazi, HadassahHebrew University Medical Center, Jerusalem, Israel
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The brinolytic system consists of serine proteases, their substrates, receptors, and inhibitors. Activation of the brinolytic cascade leads to the generation of the protease, plasmin, which cleaves the insoluble polymeric network composed primarily of brin. Imbalances in the system that result in the inhibition of plasmin formation lead to the deposition of brin in tissue as well as in vessels, common features of diverse pulmonary diseases. In addition to brin cleavage, each component in the plasminogen activator (PA) system is involved in a wide range of (patho)physiological processes. This involvement may result from: (1) the catalytic activity of the proteases, for example, the activity of plasmin in tissue remodeling by degrading the extracellular matrix; (2) interaction with cellular integrins and integrin ligands on cells or in matrix to regulate cell adhesion and migration and hereby immunity, tumor invasiveness, and metastasis; (3) direct or indirect induction of signal transduction by the proteases and/or their inhibitors, which can be mitogenic or regulate vascular reactivity. The multifunctionality of the components of the PA cascade has led to extensive research by groups from diverse elds. Notwithstanding the dramatic increase in recent knowledge, our understanding is far from complete and additional research is needed to clarify several aspects of the system, especially novel ways to regulate its activity in vivo.

coagulation and the brinolytic systems under pathologic conditions. This includes both increases in the expression of tissue factor (TF), the primary initiator of clotting, and suppression of brinolytic activity due, in large part, to increased synthesis and release of plasminogen activator inhibitor type 1 (PAI-1). Increased concentrations of the urokinase plasminogen activator (uPA) have also been reported in lung diseases characterized by brin deposition, such as pneumonia, but the presence of PAI-1 and TF seems to overcome its brinolytic effect. Similarly, pleural injury is characterized by early and persistent brin deposition and disordered brin turnover plays a significant role in the pathogenesis of pleural inammation, repair, and scarring.

Components of the Fibrinolytic System

The brinolytic system consists of serine proteases and their inhibitors. The enzymes of the brinolytic
PA PAI-1 Plasminogen () Fibrin clot Plasmin () SFDPs

Introduction
The brinolytic system is designed to cleave the insoluble polymeric network of brin into soluble fragments (Figure 1). In addition to providing the structure of blood clots, brin deposition is a common feature of many lung diseases, including acute lung injury (ALI), acute respiratory distress syndrome (ARDS), pneumonia, and pleural injury. Fibrin deposition in the lung results from both systemic and local (alveolar, interstitial) imbalance between the

2-AP

Figure 1 The brinolytic cascade. The plasminogen activators (PAs), tPA, uPA, scuPA (or pro-uPA), or scuPA/uPAR complex (among others), activate the proenzyme plasminogen to the powerful proteolytic enzyme plasmin. Plasmin cleaves the insoluble network of brin, converting it into soluble brin degradation products (SFDPs). Plasmin can cleave other proteins and activate other proteases, such as metalloproteinases. Plasmin can compromise the vascular integrity at sites of injury and thereby accelerate dissolution of the vascular basal lamina. Plasminogen activator inhibitor type 1 (PAI-1) inhibits tPA and uPA. a2-antiplasmin (a2-AP) inhibits the activity of plasmin.

202 FIBRINOLYSIS / Overview

cascade are expressed, in part, as proenzymes that are activated under certain physiological and pathological conditions. Activation of the system ends with the conversion of the proenzyme plasminogen into the active serine protease plasmin. The classic function of the system is summarized in Figure 1. It is designed to generate the proteolytic enzyme plasmin, which cleaves brin into soluble fragments. However, each component in the system has other activities and is involved in a wide range of pathophysiological processes.
Plasminogen and Plasmin

Plasminogen is a zymogen or proenzyme. Plasminogen is activated to plasmin by cleavage of the Arg561 Val562 peptide bond by plasminogen activators (PAs). The resultant plasmin is a serine protease that is considered to be the major extracellular protease in plasma. In addition to brinolysis, plasmin can directly degrade the extracellular matrix (ECM) through its activity on bronectin, laminin, and type IV collagen, as well as indirectly by activating prometalloproteases (MMPs), such as stromelysin and procollagenase, thereby facilitating cell migration and tissue remodeling. Plasmin also activates latent cell-associated transforming growth factor beta 1, which regulates the inammatory response, among other effects. Plasmin can also activate the classic and alternate complement pathways and generate bradykinin from kininogen. Plasmin(ogen) in thrombotic diseases The wide specicity of plasmin poses advantages and disadvantages to the host. Clearance of intravascular brin averts tissues ischemia and lysis of extravascular brin limits ingress of inammatory cells that use it as a matrix and thereby promotes tissue healing. On the other hand, the pleiotropic effects of plasmin are problematic when PAs are used in therapeutic concentrations to treat myocardial infarction or ischemic stroke. For example, tissue-type plasminogen activator (tPA) increases the risk of bleeding by cleaving brinogen (among other plasma proteins), activating MMP-9 and extravasating into extravascular tissue where it encounters additional substrates, a situation of relevance to the lung in settings in which the endothelial and epithelial barrier is impaired. Plasmin(ogen) in infectious diseases There is considerable evidence that surface-bound plasmin may facilitate bacterial invasion of pericellular matrix. Pathogenic bacteria, such as group A, C, and G streptococci and Staphylococcus aureus, or mycoplasma express plasminogen-binding sites and some

organisms, such as Mycobacterium tuberculosis, have been shown to both bind and activate plasminogen. Plasminogen and plasmin bind to cells and to bacteria through lysine binding sites in their kringle domains. Several bacterial plasminogen receptors have been reported, including a-enolase. In addition, a pseudo-lysine arrangement has been implicated in the high-afnity binding of plasminogen to the M-like protein of group A streptococci. Whether plasmin(ogen) is directly involved in the immune response during infectious diseases is under study. Binding of plasmin(ogen) to macrophages and other leukocytes increases cell surface proteolytic activity, thereby enhancing migration and ECM degradation. Furthermore, peritoneal recruitment of monocytes and lymphocytes in response to thioglycollate inammation is impaired in plasminogen knockout (plasminogen / ) mice. On the other hand, no changes were found in the number and distribution of lung macrophages between WT and plasminogen / mice after infection by Mycobacterium avium. Differences related to tissue (lung vs. peritoneum) and the inammatory agent (thioglycollate vs. M. avium) may be responsible for the discrepancy. Certain fragments of plasmin(ogen) devoid of any catalytic activity have been shown to have a signal transduction effect that inhibits angiogenesis. These fragments (including kringles 13) known as angiostatin have also been found to inhibit the PA activity of the scuPA/uPAR complex and to regulate contraction of isolated arterial rings (Higazi, unpublished data).
Urokinase Plasminogen Activator (uPA) and Its Receptor (uPAR)

uPA uPA is synthesized and released as a 411 amino acid proenzyme composed of an N-terminal fragment (ATF, amino acids 1135) that contains the growth factor domain (GFD, amino acids 143) required for binding to uPAR, a kringle (amino acids 44135), and a C-terminal proteolytic domain (lowmolecular-weight uPA; amino acids 136411). uPA is synthesized as a single-chain proenzyme (scuPA) with little or no intrinsic enzymatic activity. scuPA is activated when it is cleaved by plasmin at Lys158Ile159 to generate a two-chain molecule (tcuPA) or by binding, as a single-chain molecule, to uPAR. The scuPA/uPAR complex and tcuPA have similar catalytic rate constants, but differ markedly in kinetic parameters and reversibility of activation and inactivation by the PAI-1 where the scuPA/suPAR complex is more resistant to PAI-1 inhibition than tcuPA. Furthermore, the scuPA/uPAR complex is more

FIBRINOLYSIS / Overview 203

active than tcuPA in models of pulmonary microembolism and pleural effusion. uPAR uPAR is a glycolipid-anchored (GPI) 281 amino acid protein composed of three domains (DI III) that are partial repeats, each containing two to three paired disulde bonds linked by exible linker sequences. Binding of uPAuPAR to various transmembrane adapters, such as integrin ligands/integrins and the low-density lipoprotein receptor-related protein (LRP), has been implicated in cell adhesion and intracellular signaling, although a direct productive association of GPI-uPAR with intracellular signaling proteins in lipid rafts has not been excluded. uPA/uPAR in lung disease uPA and uPAR have been implicated in diverse physiological and pathological processes involving cell migration through brin and subcellular matrices, that are of direct relevance to lung injury and repair, for example, angiogenesis, wound repair, inammation, immunity, and tumor metastasis. uPA / mice have defects in brinolysis, wound healing, neointima formation, immunity, and dissemination of certain tumors. In uPAR / mice leukocyte mobilization to sites of infection is impaired. uPA / mice have increased susceptibility to lethal pulmonary infection by certain organisms. Furthermore, deciency of uPA has been associated with more severe pulmonary hypertension and impaired vascular remodeling during chronic hypoxia in mice. Nonproteolytic effects of uPA and uPAR As mentioned above, uPA is active as a proteolytically cleaved two-chain molecule or as a single-chain molecule bound to uPAR. Proteolytic conversion to

tcuPA also dramatically increases its susceptibility to irreversible inhibition by PAI-1. Inactive PAI-1uPA uPAR complexes are rapidly internalized by LRP. Thus, PAI-1 initiates an LRP-dependent decrease in cell surface tcuPA and uPAR. Interestingly, uPAR is elevated on the surface of LRP-decient cells and this increase correlates with increased cell mobility. The binding of uPA to uPAR stimulates intracellular signaling and also induces conformational changes in uPAR, which increase its afnity for vitronectin (VN) and promotes its interaction with a variety of integrins. uPAR occupancy can also activate certain integrins and growth factor receptors. Another pathway to initiate intracellular signaling by uPA is by direct interaction with LRP (without the intervention of uPAR). The interaction of uPA with LRP regulates vascular contractility, including that of pulmonary arteries (Higazi, unpublished data). This pathway is inhibited by PAI-1 and by a PAI-1-derived peptide that does not affect the catalytic activity of uPA (Figure 2).
Tissue-Type Plasminogen Activator

Tissue-type plasminogen activator (tPA) is the second principal plasminogen activator in mammals. tPA / mice accumulate brin in several organs and their capacity to lyze pulmonary microemboli is impaired. tPA also modulates vascular (including pulmonary arterial) contractility through LRP (Higazi, unpublished data) and exerts proteolytic and nonproteolytic toxic effects on extravascular tissues, best characterized in the central nervous system. PAI-1 and a PAI-1-derived peptide (Figure 2) inhibit tPA-mediated vasoactivity without affecting its brinolytic activity. As in the case of uPA / mice, bleomycin-induced pulmonary brosis in tPA / mice is enhanced.

Docking site

EEIIMD

Catalytic site tPA or uPA

PAI-1 PAI-1 tPA or uPA (b) PA-EEIIMD (c)

(a)

Figure 2 PAI-1 interactions with PAs. (a) PAI-1 binds to tPA or uPA through two independent sites: the catalytic site and a docking site. The docking site, comprising amino acids 296299 in tPA and 179184 in uPA, lies outside the catalytic site. PAI-1 binds to the docking site through amino acids 350355. (b) The PAI-1-derived peptide EEIIMD, which corresponds to amino acids 350355, interacts with the docking site in uPA and tPA and competes for the binding of PAI-1. (c) Binding of EEIIMD to uPA or tPA may induce conformational changes that inhibit their effect on vasoactivity and other signal transduction pathways.

204 FIBRINOLYSIS / Overview Plasminogen Activator Inhibitor Type 1

PAI-1 is a member of the serine protease inhibitor (SERPIN) family and is the primary physiological inhibitor of both tPA and uPA. It is a trace protein in plasma where it circulates in a complex with the adhesive glycoprotein vitronectin. Binding of PAI-1 to VN stabilizes the inhibitor in its active conformation, and this binding is reversed when the inhibitor forms complexes with tPA or uPA. tPA and uPA bind PAI-1 through two independent sites (Figure 2), the catalytic site and a docking site. The docking site is located outside the catalytic site and is composed of amino acids 296299 in tPA and 179184 in uPA. PAI-1 binds to the docking site of tPA or uPA through the amino acids 350355 (Figure 2). The peptide EEIIMD, which corresponds to amino acids 350355 in PAI-1, interacts with the docking site in uPA and tPA and competes for the binding of PAI-1. EEIIMD inhibits tPA- and uPA-mediated vasocontractility without affecting their catalytic activities. We hypothesize that binding of EEIIMD to the PAI-1 docking site on tPA or uPA produces conformational changes in the plasminogen activators that neutralize their signal transduction capacity (Figure 2).
Fibrinogen

pulmonary brosis through other mechanisms, including the regulation of collagen turnover by activation of collagenases and metalloproteinases. An alternative explanation for the increased pulmonary brosis reported in PA knockout mice may be their involvement in signal transduction, specifically in regulation of vasoactivity; since PAI-1 abolishes tPA- and uPA-induced signal transduction and vasoactivity, this hypothesis could also explain the phenotype seen in PAI-1 decient and overexpressing mice.
See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Overview; Fibrinogen and Fibrin. Fibrinolysis: Plasminogen Activator and Plasmin. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Proteinase Inhibitors: Alpha-2 Antiplasmin. Serine Proteinases. Thrombolytic Therapy.

Further Reading
Bdeir K, Murciano J-C, Tomaszewski J, et al. (2000) Urokinase contributes to brinolysis in the pulmonary microcirculation. Blood 96: 18201826. Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890897. Gunther A, Mosavi P, Heinemann S, et al. (2000) Alveolar brin formation caused by enhanced procoagulant and depressed brinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 161: 454462. Gyetko MR, Chen GH, McDonald RA, et al. (1996) Urokinase is required for the pulmonary inammatory response to Cryptococcus neoformans. A murine transgenic model. Journal of Clinical Investigation 97: 18181826. Higazi AA-R, Cohen RL, Henkin J, et al. (1995) Enhancement of the enzymatic activity of single-chain urokinase plasminogen activator by soluble urokinase receptor. Journal of Biological Chemistry 270: 1737517380. Idell S (1994) Extravascular coagulation and brin deposition in acute lung injury. New Horizons 2: 566574. Khalil N, Corne S, Whitman C, and Yacyshyn H (1996) Plasmin regulates the activation of cell-associated latent TGF-beta(1) secreted by rat alveolar macrophages after in vivo bleomycin injury. American Journal of Respiratory Cell and Molecular Biology 15: 252259. Lahteenmaki K, Kuusela P, and Korhonen TK (2001) Bacterial plasminogen activators and receptors. FEMS Microbiology Reviews 25: 531552. Lijnen HR (2001) Plasmin and matrix metalloproteinases in vascular remodeling. Thrombosis and Haemostasis 86: 324333. Lijnen HR and Collen D (2005) Molecular and cellular basis of brinolysis. In: Hoffman Hematology: Basic Principles and Practice, 4th edn., pp. 19551959. MacKay AR, Corbitt RH, Hartzler JL, and Thorgeirsson UP (1990) Basement membrane type IV collagen degradation: evidence for the involvement of a proteolytic cascade independent of metalloproteinases. Cancer Research 50: 59976001. Miles LA, Dahlberg CM, Plescia J, et al. (1991) Role of cell-surface lysines in plasminogen binding to cells: identication of a-enolase as a candidate plasminogen receptor. Biochemistry 30: 16821691.

Fibrinogen is a 340 000 kDa protein consisting of three pairs of disulde-bonded nonidentical polypeptide chains. Fibrinogen is proteolyzed by thrombin during coagulation, releasing brin monomer. Fibrin monomers polymerize into an insoluble network that gives tensile strength to the blood clot and is the substrate of the brinolytic system. Fibrin(ogen) binds to integrin and nonintegrin receptors on many cell types. Fibrin(ogen) enhances broblast migration and proliferation and is involved in the early stages of the immune inammatory responses by stimulating leukocyteendothelial cell adhesion and migration. Furthermore, brin colocalizes with collagen-rich brotic lung lesions, suggesting the involvement of brin(ogen) in the progression of inammatory diseases in general and in the broproliferative response after lung injury specifically. Involvement of brin(ogen) in lung brosis was initially supported by studies showing brin within brotic lesions and other studies showing that bleomycin-induced pulmonary brosis is exacerbated in tPA / and uPA / mice. Fibrosis is also greater in PAI-1 overexpressing mice and attenuated in PAI-1decient animals. However, genetic ablation of brinogen does not alter either the early inammatory response to bleomycin injury or the development of collagen-rich pulmonary brosis. Therefore, plasminogen and plasminogen activators may regulate

FIBRINOLYSIS / Plasminogen Activator and Plasmin 205

Nassar T, Akkawi S, Shina A, et al. (2004) The in vitro and in vivo effect of tPA and PAI-1 on blood vessel tone. Blood 103: 897902. Ploplis VA, French EL, Carmeliet P, Collen D, and Plow EF (1998) Plasminogen deciency differentially affects recruitment of inammatory cell populations in mice. Blood 91: 20052009. Plow EF, Herren T, Redlitz A, Miles LA, and Hoover-Plow JL (1995) The cell biology of the plasminogen system. EMBO Journal 9: 939945. Wilberding J, Ploplis V, McLennan L, et al. (2001) Development of pulmonary brosis in brinogen-decient mice. Annals of the New York Academy of Sciences 936: 542548. Wistedt AC, Kotarsky H, Marti D, et al. (1998) Kringle 2 mediates high afnity binding of plasminogen to an internal sequence in streptococcal surface protein PAM. Journal of Biological Chemistry 273: 2442024424.

Plasminogen Activator and Plasmin

S Shetty and S Idell, University of Texas Health Center, Tyler, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The plasminogen activation system involves several components, including two plasminogen activators, urokinase-type (uPA) and tissue-type (tPA), a plasma membrane-associated receptor (uPAR), and two specic inhibitors, plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2). The plasminogen activator/plasmin system plays an important role in both physiological and pathophysiological processes involving degradation of intravascular thrombi and extracellular matrices that may contribute to wound healing in the setting of tissue inammation and repair and desmoplasia in the setting of neoplasia. Recent studies have revealed that the plasminogen activation system, through interaction with integrins and other components of the extracellular matrix, is also involved in the regulation of cellular signaling, proliferation, adhesion, and migration in a manner that is independent of plasminogen activator-mediated proteolytic activity. Overexpression of uPA, uPAR, and PAI-1 is commonly associated with acute or chronic lung injury and lung carcinomas. The expression of components of the plasminogen activation system is regulated both at the transcriptional and posttranscriptional level by a variety of proinammatory cytokines or other proinammatory mediators through interactions of specic transcription factors or mRNA binding proteins with plasminogen activation system component DNAs or mRNAs, respectively. Most recently, it has been found that uPA itself can stimulate its own expression as well as that of uPAR and PAI-1. The induction of components of the plasminogen activation system by uPA could likewise contribute to the alteration in brin turnover that may occur in the injured lung and in lung neoplasia.

effected by either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA). Plasmin is, in turn, capable of degrading protein constituents of the extracellular matrix and basement membranes. Extracellular proteolytic enzymes such as serine proteinases including uPA and tPA and matrix metalloproteinases play an important role in tissue remodeling. uPA and tPA are produced by a wide range of normal and transformed cells. tPA appears to play a major role in intravascular thrombolysis whereas uPA is mainly involved in tissue remodeling during evolving inammation and repair or in neoplasia. The proteolytic activities of these proteases are tightly regulated by serine protease inhibitors (serpins), including plasminogen activator inhibitors (PAI)-1 and PAI-2. A shift towards proteolysis is observed during tissue remodeling and angiogenesis. A shift towards proteolysis is likewise observed in malignant tissues and neoplastic cells enabling tumors to invade adjacent normal tissues, to induce tumor-associated angiogenesis, and to promote metastatic spread. The balance of proteolysis and inhibition of proteolysis is therefore contingent on variable expression of PAs and PAIs and may therefore inuence the virulence of tumors in terms of both local extension and distant spread.

Biochemical Properties of uPA

The uPA is secreted as a 53 kDa prouPA otherwise known as single-chain uPA, which binds to its cell surface receptor uPAR and is later converted by plasmin to the relatively more active two-chain form of uPA, which in turn converts membrane-bound plasminogen to plasmin. The two polypeptide chains of uPA are linked by several disulde bonds (Figure 1). The uPA molecule contains three functional domains. It contains an N-terminal receptor binding growth factor (EGF)-like domain and a C-terminal serine protease domain, which are interconnected by the kringle domain. In addition to effecting the activation of plasminogen, uPA also activates prohepatocyte growth factor/scatter factor (HGF/SF) and transforming growth factor beta (TGF-b).

Biochemical Properties of tPA

tPA is a 70 kDa protein secreted in a proenzyme form that is also converted by plasmin to the active twochain form. The tPA molecule has four functionally distinct domains. These include a bronectin-like or nger domain, an EGF-like domain, two kringle domains, and a serine protease domain. The presence of receptors for tPA has been reported in several cell

Introduction
The zymogen plasminogen is converted to the active serine protease plasmin by proteolytic cleavage

Kringle region
M G R P C L P W N S V Y C Q W T V P S G R A L R K 120 K R P 60 G N L R D V P Y Q N F E R H C C Y G M 100 N N V H L G H K G L G E D Y C C A 140 D G T K K P S S P K S K D D T

80 Q Q T Y H A H R F G 160 G I E T T

E N Q P

W F A A I Y R

S D Q L A G C Q

T T

L Q

R P I

K R F

Growth factor domain

Q 40 H C E K I P C C D C N S N V P T H S L C Q R 400 K N W H C I E 20 V L T G G H E S I W E N N N P N G K Y F S L S L F A H L NH2 S V COOH R T F K

G G

Y V G

C F I D Y P H T F A K P P S L C S A E W V I 200 M E C E N S P Y S N K R T L N 300 N G G D D T E F Y P K Q L G Q S G Y T F R E M P K I I G E 260 E K T S C Q T M R L G L L L Q L T S C V K A 360 L I K V V T M G P D I L G Y Y V N I 320 H G S W G P H S S G S R H D Q H E G R G Q E C Q A V 380 C C T L A T T S L D P K D K T K W Q P D A A C L M K 340 I C L 280 L I S

T R S P Q

G S G

T V Y

180 R V S G G R H

K K E

D Y

I V Y L G 220 R

F E V E N L I 240 L H K D Y D A S

Serine protease part

Figure 1 Primary structure of uPA. The diagrammatic model shows the primary structure of uPA and illustrates the three major domains of the molecule. Reproduced from Collen D and Lijnen HR (1994) American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L319L328, used with permission from The American Physiological Society.

FIBRINOLYSIS / Plasminogen Activator and Plasmin 207

types. tPA has high afnity for brin, which promotes its activation. tPA is mainly associated with intravascular brinolysis, but is also represented at sites of extravascular inammation, including pleural and parenchymal lung injuries. tPA is induced by thrombosis such as may occur in tissue ischemia and inammation.

Plasminogen Activator Inhibitors

Plasminogen activator inhibitors belong to the serpin protease inhibitor (SERPIN) superfamily. The inhibitors include PAI-1, PAI-2, and protease nexin I. These inhibitors exhibit a peptide bond for targeting proteases and stably coupling them with a 1:1 stoichiometry. These are very fast-acting inactivators of the plasminogen activators and are present in most body uids and tissues.

The N-terminal domain has uPA binding activity, while the other two domains bind to vitronectin, a key constituent of the extracellular matrix. uPAR has an approximate molecular weight of 50 kDa and is highly glycosylated. uPAR is associated with the plasma membrane through a glycosylphosphotidyl inositol (GPI) anchor. Several cytokines and growth factors including TNF-a, TGF-b, HGF, vascular endothelial growth factor (VEGF), and broblast growth factor (FGF) induce uPAR expression in a variety of cell types.

Pathophysiological Role of the Plasminogen Activator System

The plasminogen activator system has been shown to inuence a broad array of cellular responses that extend well beyond traditional brinolysis. Alteration of the plasminogen activator system assumes particular importance in promoting brin deposition and remodeling in diverse forms of tissue injury. Similarly, it has been shown that expression of brinolytic components is altered in the setting of lung cancer as well as other solid neoplasms.

Plasminogen Activator Inhibitor 1

PAI-1 is a 50 kDa glycoprotein serpin secreted by several cell types. PAI-1 can bind to free and receptorbound uPA, thereby inhibiting uPA-mediated extracellular matrix degradation. The receptor-bound PAI-1 also mediates internalization of trimeric cell surface uPAPAI-1uPAR complexes and the subsequent recycling of uPAR to the cell surface. PAI-1 is also involved in the regulation of cell adhesion and migration. PAI-1 is rapidly induced by TGF-b, representing a potentially critical mechanism that promotes an antibrinolytic balance of the plasminogen activator system and favors maintenance of transitional brin and brotic repair.

Specic Derangements of the Plasminogen Activator System Associated with Acute and Chronic Lung Injury
It is now well known that uPA is the major plasminogen activator expressed in alveolar lining uids in the normal lung. In the absence of disease, brinolytic activity is readily detectable in bronchoalveolar lavage specimens obtained from normal healthy subjects. Conversely, brinolytic activity is depressed in the alveolar compartment in ARDS. This defect in local brinolysis in part accounts for the orid extravascular brin that is consistently observed in the alveolar and interstitial neomatrix of the injured lung. The brinolytic defect in the alveolar space in ARDS is largely attributable to increased local expression of PAI-1. PAI-2 and antiplasmins also contribute to the decrease in brinolytic activity in ARDS, so that the plasminogen activator system is downregulated in series at the levels of plasminogen activators and plasmin (Figure 2). Similar abnormalities were found in the bronchoalveolar lavage specimens of patients with idiopathic pulmonary brosis, sarcoidosis, and other forms of interstitial lung diseases. In ARDS and interstitial lung diseases, the local defect in uPA-dependent brinolytic activity is largely attributable to local overexpression of PAI-1.

Plasminogen Activator Inhibitor 2

PAI-2 is a 47 kDa protein and is less potent in inhibiting receptor-bound uPA than PAI-1. PAI-2 is rapidly induced by proinammatory mediators including tumor necrosis factor alpha (TNF-a) and LPS. PAI-2 protects cells from TNF-a-mediated apoptosis. PAI-2 exists both in intracellular and secreted form and the latter participates in the control of tissue remodeling and brinolysis. PAI-2 is generally undetectable in the circulation, except in pregnancy and in association with selected neoplasms. PAI-2 also occurs in extravascular uids, including bronchoalveolar lavage from patients with acute respiratory distress syndrome (ARDS) or chronic lung injury or in pleural effusions from patients with exudative pleuritis.

The uPA Receptor

The receptor for uPA, uPAR, is a plasma membrane protein that consists of three cysteine-rich domains.

208 FIBRINOLYSIS / Plasminogen Activator and Plasmin

Fibrinolysis

PAI Antiplasmins Coagulation

ARDS Pneumonias Interstitial lung disease

Figure 2 Alterations of local brinolysis and coagulation in acute and chronic lung injuries.

Fibrin turnover is similarly altered in the setting of pleural injury, which is likewise characterized by intrapleural brin deposition. In pleural effusions of patients with exudative pleuritis due to pneumonia, metastatic cancer, malignant mesothelioma, or emphysema, brinolytic activity is generally undetectable. The decrease in brinolytic activity under these circumstances is also due to overexpression of PAI-1, which is increased several-fold versus the usual plasma level. Both uPA- and tPA-related brinolytic activities are also downregulated by PAI-2 and antiplasmins within the pleural compartment. These abnormalities favor the maintenance of intrapleural brin in evolving pleural loculation. Reversal of these abnormalities by the intrapleural administration of brinolysins has been part of the therapeutic armamentarium for over 50 years, although recent multicenter clinical trials are now being conducted to evaluate their efcacy for patients with organizing pleural loculation and brosis. In both parenchymal and pleural injuries, a variety of resident cell types express components of the plasminogen activator system. Alveolar macrophages, lung epithelial cells, lung broblasts, and pleural mesothelial cells are sources of uPA, tPA, and brinolytic inhibitors. These cells also express the urokinase receptor. In these cells, inammatory mediators released during lung injury inuence the regulation of uPA, uPAR, and PAI-1 and PAI-2. For example, TNF-a and TGF-b increase uPA, uPAR, and PAI-1 expression by lung epithelial cells and lung broblasts.

growth and spread of solid tumors. Abnormalities of the plasminogen activator system have likewise been implicated in the pathogenesis of tumor invasiveness and metastatic spread. Mechanisms by which the uPAuPAR system contributes to the pathogenesis of tumor invasiveness include proteolytic degradation of basement membrane and extracellular matrix constituents and intracellular signaling that initiates mitogenic or altered adhesion responses. Several reports link the expression of uPA, uPAR, PAI-1, and PAI-2 to both tumor cell invasiveness and to differential prognosis in cancer patients. For instance, high levels of uPA have been correlated with poor outcome or shorter survival in a variety of tumors, including those of the breast and lung as well as a variety of other solid neoplasms. Expression of PAI-1 has likewise been linked to aggressive tumor behavior and uPAR expression also appears to be a prognostic marker in some forms of malignancy, including breast or colorectal cancer. Increased expression of uPA, uPAR, and PAI-1 has been demonstrated in different histologic forms of lung carcinoma and linked to poor prognosis, suggesting that clinical outcome extends beyond predictable alterations in brinolytic capacity and may, rather, be broadly related to pathophysiologic responses initiated by these proteins through cellular signaling.

Autocrine Regulation of the Plasminogen Activator System

Recent reports conrm that uPA itself can stimulate its own expression or that of uPAR or PAI-1 in lung epithelial cells in culture (Figure 3). While clear evidence of the role of these pathways in lung or pleural injury has yet to be determined, they may likewise contribute to the derangements of brin turnover that have been reported to occur in the injured lung and in lung neoplasia. Tumor cell invasion is facilitated by saturation of uPAR with either exogenous or overexpressed endogenous uPA. Tumor cells generally exhibit several characteristics such as anchorage-independent growth, high migratory capacity, the ability to produce relatively large quantities of tumor proteases, and enhanced proliferation. In several human tumors, the balance between proteases encompasses alterations of the uPAuPARPAI-1 system. The efciency of plasminogen activation by uPA is enhanced several-fold when uPA is bound to uPAR. The uPA/uPAR complex also functions as a vitronectin receptor, which could thereby inuence tumor cell motility and invasiveness. It now appears that uPA, uPAR, and PAI-1 are integrally involved in the pathogenesis of lung carcinomas, raising the

Disordered Fibrinolysis in Lung and Pleural Neoplasia

There are consistent abnormalities in brin turnover in inammation, evolving brosis, and cancer. Extravascular brin deposition in solid neoplasms promotes the desmoplastic response as well as the

FIBRINOLYSIS / Plasminogen Activator and Plasmin 209

Fibrinolysis

The uPAR promoter contains AP-1, SP-1, AP-2, and SP-1/-3 binding sites.

Remodeling of transitional (alveolar & interstitial) fibrin

Autoregulation of uPA Regulation of uPAR and PAI-1

Posttranscriptional Regulation of the Plasminogen Activator System

uPA, uPAR, PAI-1, and PAI-2 mRNAs are all regulated by posttranscriptional mechanisms that operate at the level of mRNA stability. The uPA mRNA 30 untranslated region (30 UTR) contains multiple instability determinants and specic mRNA-binding protein recognition sequences. Interaction of two specic mRNA-binding proteins has been reported for uPA mRNA. Levels of tPA protein are determined by activation of tPA mRNA, which is controlled by poly(A) tail length. PAI-1 mRNA stability is altered via TGFb, cAMP, and insulin-like growth factor. At least two stability determinants have been identied within the PAI-1 30 UTR. Multiple PAI-1 mRNA-binding proteins interacting with the PAI mRNA 30 UTR have been reported. PAI-2 mRNA is also induced by PMA and TNF-a at the posttranscriptional level. Posttranscriptional regulation of PAI-2 mRNA is regulated by determinants present in both the coding region and 30 UTR. uPAR mRNA is also induced by several agents including PMA, LPS, or TGF-b at the posttranscriptional level. Stability of uPAR mRNA is regulated by determinants present both in the coding and 30 UTR. A 50 kDa protein interacts with the uPAR mRNAcoding region and a 40 kDa protein with 30 UTR determinants to regulate uPAR mRNA stability.
See also: Acute Respiratory Distress Syndrome. Epidermal Growth Factors. Fibrinolysis: Overview. Fibroblast Growth Factors. Gene Regulation. Proteinase Inhibitors: Antichymotrypsin. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Vascular Endothelial Growth Factor.

Signaling of proinflammatory and profibrogenic cytokines

Figure 3 Role of brinolytic pathways in lung injury and repair. Urokinase-type plasminogen activator (uPA), its receptor (uPAR), and its major inhibitor plasminogen activator inhibitor-1 (PAI-1).

important possibility that overexpression of uPA, uPAR, and PAI-1 by these cells and clinical prognosis may rely upon autocrine or paracrine induction by uPA.

Transcriptional Regulation of the Plasminogen Activator System

Expression of uPA, uPAR, PAI-1, and PAI-2 genes is regulated at both transcriptional and posttranscriptional levels. uPA gene expression is induced by growth factor, hormones, UV light, changes in cell morphology and contact, and, as noted above, by uPA itself. The best-characterized regulatory regions of the uPA promoter are the Ets/AP-1a composite site, the AP-1b site, and the connecting 74 bp cooperation mediator region. Ets1 and Ets2 have been shown to activate the uPA promoter. Similarly, tPA expression is enhanced by a wide range of stimuli, including retinoids and steroids. The transcription factors SP1, CREB1, NF1, and ATF2 are responsible for induction of tPA mRNA expression. PAI-1 gene expression is likewise regulated by many growth factors and cytokines, including TGF-b, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), FGF, interleukin-1 (IL-1), and TNF-a and hormones. SP-1, AP-2 proteins as well as a 72 kDa unknown component have been found to interact with the PAI-1 promoter. Transcription factor nuclear factor I and the ubiquitous factor (USF) are responsive to TGF-b induction in the natural promoter. PAI-2 gene expression is regulated by multiple growth factors, hormones, cytokines, and vasoactive peptides. Site-directed mutagenesis studies revealed the involvement of AP-1a and CRE-like sites for both basal and PMA-induced PAI-2 transcription. Finally, the uPAR gene is induced by various signals including PMA, TGF-b, TNF-a, cAMP, and HIV infection.

Further Reading
Andreasen PA, Kjoller L, Christensen L, and Duffy M (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. International Journal of Cancer 72: 122. Besser D, Verde P, Nagamine Y, and Blasi F (1996) Signal transduction and the uPA/uPAR system. Fibrinolysis 10: 215237. Collen D and Lijnen HR (1994) American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L319L328. Collen D and Lijnen HR (1994) Fibrinolysis and the control of hemostasis. In: Stamatoyannopoulos GS, Nienhuis AW, Majerus PW, and Vermus HV (eds.) The Molecular Basis of Blood Diseases, pp. 725752. Philadelphia: Saunders. Dickenson JL, Bates EJ, Ferrante A, and Antalis TM (1995) Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis: evidence for an alternate biological function. Journal of Biological Chemistry 270: 2789427904. Garcia R, Schweigerer L, and Medina MA (1998) Modulation of the proteolytic balance plasminogen activator/plasminogen activator

210 FIBROBLAST GROWTH FACTORS

inhibitor by enhanced N-myc oncogene expression or application of genistein. European Journal of Cancer 34: 17361740. Idell S (1995) Coagulation, brinolysis and brin deposition in lung injury and repair. In: Phan SH and Thrall RS (eds.) Pulmonary Fibrosis: Lung Biology in Health and Disease. vol. 80, pp. 743776. New York: Dekker. Irigoyen JP, Munoz-Canoves P, Montero L, Koiczak M, and Nagamine Y (1999) The plasminogen activator system: biology and regulation. Cell and Molecular Life Sciences 56: 104132. Mazar AP, Henkin J, and Goldfarb RH (1999) The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis. Angiogenesis 3: 1532. Nykjaer A, Conese M, Christensen EI, et al. (1997) Recycling of the urokinase receptor upon internalization of the uPA: serpin complexes. EMBO Journal 16: 26102620. Pedersen H, Brunner N, Francis D, et al. (1994) Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Research 54: 46714675. Planus E, Barlovatz-Meimon G, Rogers RA, et al. (1997) Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading. Journal of Cell Science 110: 1091 1098. Scheneiderman J, Adar R, and Savion N (1991) Changes in plasmatic tissue-type plasminogen activator and plasminogen activator inhibitor activity during acute arterial occlusion associated with severe ischemia. Thrombosis Research 62: 401408. Shetty S and Idell S (2000) Posttranscriptional regulation of the brinolytic system in lung and pleural disease. Research in Advanced Pathology 1: 116. Shetty S and Idell S (2002) New perspectives on the regulation of brinolysis in the lung. Recent Research Development in Biological Chemistry 1: 223230. Xing RH, Mazar A, Henkin J, and Rabbani SA (1997) Prevention of breast cancer growth, invasion, and metastasis by anti-estrogen tamoxifen alone or in combination with urokinase inhibitor B-428. Cancer Research 57: 35853593.

FIBROBLAST GROWTH FACTORS

D Ornitz, Washington University School of Medicine, St Louis, MO, USA P Sannes, North Carolina State University, Raleigh, NC, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Fibroblast growth factors (FGFs) are a large group of small polypeptide growth factors, some of which play key roles in pulmonary biology. Their molecular sizes vary from 17 to 34 kDa and they share a wide range of amino acid homology with highly conserved gene structures and amino acid sequences. They also share the common property of avidity for heparin, which enables them to bind to components of extracellular matrices and cell surfaces. FGFs form a trimolecular complex with heparin-like molecules (e.g., heparan sulfate) and one of four polypeptide tyrosine kinase receptors. The chemical heterogeneity of heparan sulfate and alternative splicing of FGF receptors both determine FGF binding specicity and receptor dimerization and activation. Functions for FGFs are, in many cases, narrowly dened to specic stages of development or repair, while others are broadly applied to physiological maintenance and responses to injury. FGF signaling has been shown to affect cell proliferation, cell survival, chemotaxis, migration, and cell adhesion. FGFs are known to play specic roles in tumorigenesis and pulmonary brosis, and have unique capacities to protect against DNA damage induced by oxidants and some environmental toxicants.

Fgf-like sequences have been identied in the unicellular organisms Drosophila and Caenorhabditis elegans. It is believed that the Fgf gene expansion coincided with a phase of global gene duplication that occurred during the period leading to the emergence of vertebrates. Early work on FGFs was focused, not surprisingly, on broblasts, although it soon became apparent that the FGFs impacted a wide variety of cell types and functional modalities. In the lung, FGFs were rst noted in bleomycin-induced brosis where they were found to stimulate DNA synthesis in type II alveolar epithelial cells. FGFs are essential for lung development and in the adult are expressed in a variety of respiratory diseases, with some having distinctly protective qualities.

Structure
FGFs are encoded by 22 distinct genes. The FGFs have a central core region of increased similarity, containing 28 highly conserved amino acid residues and six identical residues. Ten of the conserved residues interact with one or more of the various FGF receptors and are separate from regions that bind heparin/heparan sulfate. FGF1 and FGF2 have been studied in the greatest detail. X-ray crystallography has identied a b-trefoil 12-core structure containing 12 antiparallel and fourstranded b sheets in a triangular array. Two of the 12 strands in FGF2 contain basic amino acids that are the primary binding sites for heparin. Fgf genes typically contain three coding exons, with exon 1 containing the initiation methionine. However, several have an additional 50 transcribed

Introduction
The broblast growth factor (FGF) family members have wide-ranging and sometimes overlapping functions. FGFs have been identied in vertebrates and invertebrates, and even in some viral genomes.

FIBROBLAST GROWTH FACTORS 211

1 5 0.5 16 kb 0.5 24 kb 2 3 3

1 4 sub exons Encodes the N-terminal domain divergent between Fgf s (a)

Encodes the FGF conserved core

Encodes the C-terminal domain divergent between Fgf s

N-terminus SP (b)

Core

C-terminus

Figure 1 The prototypical Fgf gene (a) contains 3 coding exons, including a conserved core (2) and 50 and 30 exons encoding N-terminal (1) and C-terminal (3) domains, respectively, that are divergent between Fgfs. Open boxes represent untranslated regions; solid boxes represent coding sequence. The 50 transcribed sequence encodes an N-terminal signal peptide (SP) in most of the FGFs (b).

sequence that initiates from upstream codons. The genes vary in size from under 5 to over 100 kb (Figure 1). Fgf genes are scattered throughout the genome, suggesting that the Fgf gene family was generated by gene and chromosomal duplications and translocations during evolution.

Pleura Mesenchyme FGF9 FGFR2b

Regulation of Production and Activity

Most of the FGFs (FGFs 38, 10, 15, 1719, and 21 23) have N-terminal signal peptides and are readily secreted from cells. Several FGFs lack these peptides and are released from cells by nonclassical mechanisms (FGFs 1 and 2), several FGFs have internal signal sequences (FGFs 9, 16, and 20), and several FGFs are retained intracellularly (FGFs 1114). Some (FGFs 2 and 3) even possess nuclear-localization signals and can be found in the nucleus, although the functional consequences of this remain unclear. FGF2 is found prominently in the basal laminae of epithelium and is bound to the structural heparan sulfate proteoglycan (Perlecan).

Airway epithelium

FGF10

FGF9
Figure 2 FGF regulatory pathways in lung development. The expression of Fgf10 in lung mesenchyme is regulated by FGF9 produced by both pleura and airway epithelium. FGF10 in turn inuences epithelial proliferation and bud formation through the FGFR2b receptor expressed only by epithelium.

Biological Function
FGFs play critical roles in the morphogenesis and maturation of the developing lung, the specicity and impact of which are determined by spatiotemporal patterns of expression. Initial bud formation and epithelial proliferation are stimulated by FGF10, while FGF7 promotes epithelial differentiation and, along with FGF2, expression of surfactant proteins-A (SPA), SP-B, and SP-C. FGF7 and FGF10 share an unusual ability to signal through epithelial splice forms

of FGF receptors (FGFR2b). The expression of FGF7 and FGF10 is regulated in part by FGF9, which signals from lung epithelium and mesothelium to mesenchyme (Figure 2). This stimulatory regulation is opposed by sonic hedgehog (Shh), bone morphogenetic protein-4 (BMP-4), and transforming growth factor beta 1 (TGF-b1). FGF10 is essential for lung bud initiation and its expression is selectively induced by retinoic acid. Furthermore, FGF10 acts as a strong chemoattractant to distal but not proximal epithelium. These effects are greatly inuenced by the presence of extracellular matrices, such as heparan sulfate proteoglycans (HSPGs) and Netrin-1 and -4. Genetic manipulation of FGF expression during development has served to emphasize further the

212 FIBROBLAST GROWTH FACTORS

importance of spatiotemporal factors. Inducible transgenic expression of FGF7 results in increased lung size and cystadenomatoid malformations prenatally, while postnatally, FGF7 induces marked epithelial cell proliferation, adenomatous hyperplasia, and mononuclear inltration. Fgf9-null mice exhibit hypoplastic lungs and early postnatal death. Interestingly, their lungs have reduced mesenchyme and decreased airway-branching but show significant distal airspace formation and pneumocyte differentiation. Prenatal disruption of Fgf10 causes adenomatous malformations, perturbed branching morphogenesis, and respiratory failure at birth. Postnatal transgene interference results in differentiated papillary and lepidic pulmonary adenomas. Notably, the epithelial cells lining the tumors have high expression of thyroid transcription factor (TTF)-1 and SP-C, but not Clara cell secretory protein, suggesting that FGF10 promotes differentiation of peripheral alveolar type II cells. Fgf18-null mice have lungs that are smaller than normal without impairment of proximal or distal airways. They appear to have reduced alveolar space, thicker interstitial compartments, and many embedded capillaries, suggesting an important role for FGF18 during later stage embryonic lung development. In the adult lung, FGF1, FGF2, and FGF7 may be important in maintaining normal physiological homeostasis. These FGFs, when overexpressed, stimulate proliferation of type II alveolar cells, and FGF7 increases mRNA levels and secretion of SP-A and SP-D too. FGF7 can also increase the expression of Na -K -ATPase and aquaporin 5 and regulate ion and uid balance. FGF2 has been shown to decrease elastin production in broblasts; induced expression of FGF3 at low levels results in increased free alveolar macrophage inltration and at high levels in diffuse alveolar type II cell hyperplasia.

I SP S-S

II S-S

III S-S TM KI

Figure 3 FGFRs contain two or three extracellular immunoglobulin-like domains (I, II, or III) dened by disulde bonds (S-S), a transmembrane domain (TM), and kinase (KI) domain. Alternative mRNA splicing species the sequence of the C-terminal half of the immunoglobulin III resulting in isoforms IIIb or IIIc.

not react with the same mesenchymal ligand. Instead, the mesenchymally expressed variant has specicity for a different FGF that is sourced from epitheliallike tissue. FGFRs typically contain two or three immunoglobulin-like domains and a heparin-binding sequence. The heparin-binding character of both FGFs and FGFRs is a critical determinant of binding specicity and receptor activation. Heparin and related heparan sulfate proteoglycans on cell surfaces and/or in extracellular matrices also serve as binding or storage sites for FGFs and protect them from thermal denaturation and proteolysis.

Relevance to Respiratory Diseases

Expression of FGF2 has been linked to progression of lung tumors and ultimately outcomes in patient. Increased serum levels of FGF2 found in small cell lung carcinoma correlated inversely with responsiveness to chemotherapy. On the other hand, expression of FGF2 in tumor-associated stromal cells and vessels correlated inversely with progression of non-small cell lung cancer (NSCLC). Expression of FGFR1 strongly correlated with NSCLC responsiveness to the anticancer drug, doxorubicin. The angiogenic properties of FGF2 have been shown to be of particular importance in pulmonary adenocarcinomas, where they have a strong correlation with metastasis and patient survival. Similarly, in mesothelioma pleural effusions, elevated FGF2 levels correlated with poor patient survival. Interestingly, elevated levels of extracellular FGF1 and FGF2 appear to correlate with resistance to anticancer therapy. Pulmonary brosis seems to be connected with FGFs as well. FGF2 has been shown to be increased in macrophages in alveolar brosis and in mast cells in idiopathic pulmonary brosis, chronic beryllium disease, and silicosis. Experimentally, brogenic exposures to hyperoxia or paraquat are associated with rises in expression of FGF2 and its FGFR1 receptor or FGF1, respectively. FGF7 has been shown to prevent bleomycin-induced lung injury and brosis,

Receptors
FGFs bind to tyrosine kinase receptors (FGFRs). Binding specicity of FGFRs 13 is determined by alternative splicing of immunoglobulin-like domain III (Figure 3). FGFR4 has only one known spliced form in the alternatively spliced region of immunoglobulin-like domain III. The ligands specic for each splice form tend to be expressed in adjacent tissues with resultant directional signaling. The exon encoding immunoglobulin-like domain IIIb is expressed by epithelium while the exon encoding immunoglobulin-like domain IIIc is expressed in mesenchymally derived cells. Receptor isoforms expressed by epithelium bind mesenchymal sourced ligands. Interestingly, the alternative splice form of the same receptor expressed in neighboring mesenchyme will

FIBROBLASTS 213

in part by reducing pulmonary edema, transforming growth factor beta and platelet-derived growth factor-BB expression, and alveolar type II cell loss. FGF7 has been shown to have rather unique properties of protection against certain cellular injuries. It facilitates repair of radiation- or oxidant-induced DNA damage in pulmonary epithelial cells by mechanisms involving tyrosine kinases, protein kinase C, and DNA polymerases. This repair also involves antiapoptic effects of FGF7 by virtue of its receptor interaction with a p21-activated protein kinase 4 and inhibition of caspase-3. Alpha-naphthylthiourea-induced lung leakage is attenuated by FGF7 by inducing alveolar type II cell hyperplasia and attendant Na K -ATPase activity. FGF7 also protects against Pseudomonas aeruginosa-induced lung injury and reduces lung damage due to acid instillation. FGF7 enhances postpneumonectomy lung growth by alveolar proliferation and prevents ventilator-induced lung injury. Perhaps similarly to the latter, FGF10 prevents stretchinduced alveolar epithelial cell DNA damage through mitogen-activated protein kinase (MAPK) activation. FGF10 has also been shown to substantially rescue pulmonary hypoplasia induced by nitrofen.

Acknowledgments
This work was supported in part by a grant from the March of Dimes (DMO) and PHS grant HL-44497 (PLS).
See also: Epithelial Cells: Type II Cells. Extracellular Matrix: Basement Membranes; Matrix Proteoglycans; Surface Proteoglycans. Keratinocyte Growth Factor. Lung Development: Overview; Congenital Vascular Disorders.

Further Reading
Bellusci S, Grindley J, Emoto H, Itoh N, and Hogan BL (1997) Fibroblast growth factor 10 (FGF10) and branching

morphogenesis in the embryonic mouse lung. Development 124: 48674878. Cardoso WV (2001) Molecular regulation of lung development. Annual Review of Physiology 63: 471494. Colvin JS, White A, Pratt SJ, and Ornitz DM (2001) Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development 128: 20952106. Deterding RR, Havill AM, Yano T, et al. (1997) Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor. Proceeding of the Association of American Physicians 109: 254268. Itoh N and Ornitz DM (2004) Evolution of the Fgf and Fgfr gene families. Trends in Genetics 20: 563569. Liu Y, Stein E, Oliver T, et al. (2004) Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis. Current Biology 14: 897905. Lu Y, Pan ZZ, Devaux Y, and Ray P (2003) p21-activated protein kinase 4 (PAK4) interacts with the keratinocyte growth factor receptor and participates in keratinocyte growth factor-mediated inhibition of oxidant-induced cell death. Journal of Biological Chemistry 278: 1037410380. Metzger RJ and Krasnow MA (1999) Genetic control of branching morphogenesis. Science 284(5420): 16351639. Ornitz DM and Itoh N (2001) Fibroblast growth factors. Genome Biology 2: 112. Ornitz DM, Xu J, Colvin JS, et al. (1996) Receptor specicity of the broblast growth factor family. Journal of Biological Chemistry 271: 1529215297. Powers CJ, McLeskey SW, and Wellstein A (2000) Fibroblast growth factors, their receptors and signaling. Endocrine Related Cancer 7: 165197. Song S, Wientges MG, Gan Y, and Au JL (2000) Fibroblast growth factors: an epigenetic mechanism of broad spectrum resistance to anticancer drugs. Proceedings of the National Academy of Sciences, USA 97: 86588663. Takeoka M, Ward WF, Pollack N, Kamp DW, and Panos RJ (1997) KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells. American Journal of Physiology 272: L1174L1180. Weaver M, Batts L, and Hogan BL (2003) Tissue interactions pattern the mesenchyme of the embryonic mouse lung. Developmental Biology 258: 169184. Wu KI, Pollack N, Panos RJ, Sporn PH, and Kamp DW (1998) Keratinocyte growth factor promotes alveolar epithelial cell DNA repair after H202 exposure. American Journal of Physiology 275: L780L787.

FIBROBLASTS
F Chua and G J Laurent, The Royal Free Hospital, London, UK
& 2006 Elsevier Ltd. All rights reserved. provides crucial structural support for various cells and exerts profound inuences on their function. These interactions regulate normal airway and alveolar morphogenesis during fetal development, and regain prominence during repair responses following injury to these tissues. In addition to matrix formation, immune functions have also been ascribed to broblasts, including antigen presentation and the secretion of bioactive molecules. These roles highlight the versatility and indispensability of broblasts to the maintenance of normal lung physiology. Consequently, broblast dysfunction has been implicated in the pathogenesis of pulmonary disorders in which there is

Abstract
Fibroblasts are found in abundance in the airway and distal lung. They orchestrate the continual production and turnover of the pulmonary extracellular matrix, the correct organization of which is vital for efcient gas exchange. The lung matrix also

214 FIBROBLASTS
disproportionate extracellular matrix deposition (e.g., brotic lung diseases) or destruction (e.g., emphysematous and cystic lung disorders). In these settings, uncontrolled broblast activation and proliferation or, conversely, the loss of broblast number or function, contribute to the initiation and progression of abnormal lung repair. Substantial research efforts are being directed at identifying molecular targets and novel strategies to treat disorders characterized by pathological matrix remodeling.

LR Ep Eo

Introduction
Fibroblasts are derived from mesenchymal progenitors and are relatively easy to isolate from the lung and other tissues. In fact, the rst electron micrograph of an intact cell was that of a broblast cultured from a chick embryo, reported by Porter and colleagues in 1945. They become most prominent in late fetal life, coinciding temporally with periods of heightened matrix production. In the adult lung, broblasts represent approximately one-third of all lung cells. With an elongated, spindle-shaped appearance and an estimated volume of approximately 1600 mm3, the broblast is also one of the largest cells in the lung. Parenchymal lung broblasts are located mainly within the interstitial space, while airway broblasts have a predominantly subepithelial distribution (Figures 1 and 2). Wherever their location, these cells are inherently associated with connective tissue bers and possess specialized cytoplasmic protrusions and invaginations that facilitate interactions with other cells, vascular and neutral structures, as well as the surrounding extracellular matrix (ECM).

Fib 10 m (a)

Ep

Ery Endo

Col Fib El BM 2 m (b)

Figure 1 Transmission electron micrograph of (a) human airway and (b) alveolar wall. (a) A biopsy specimen of an asthmatic airway with thickened lamina reticularis (LR), epithelial cells (Ep), a broblast/myobroblast (Fib), and an eosinophil (Eo). Courtesy of Professor P K Jeffrey. (b) An interstitial broblast (Fib) closely associated with elastin bers (El), collagen brils (Col), an endothelial cell (Endo), and the basement membrane (BM). The attenuated cytoplasm of an epithelial cell (Ep) is also seen, as well as an intracapillary erythrocyte (Ery). Courtesy of Ann Dewar and Professor P K Jeffrey.

Fibroblast Functions in the Normal Lung

Lung broblasts undergo activation prior to discharging many of their most important cellular functions. Relatively passive tissue broblasts transdifferentiate into contractile myobroblasts, with the acquisition of enhanced synthetic and migratory potential, as well as the expression of alpha-smooth muscle actin (a-SMA). Activation of broblasts can occur through several different mechanisms: stimulation by growth factors in an autocrine or paracrine manner (e.g., transforming growth factor beta or TGF-b), signaling interactions with various ECM proteins (e.g., bronectin), dynamic cellcell communications (e.g., alveolar epithelial cells), or changes in physicochemical gradients in their immediate environment (e.g., mechanical stress or hypoxia).

Synthesis of Extracellular Matrix Proteins

The mammalian pulmonary ECM constitutes roughly 25% of the dry weight of the lung and is the result of a ne balance between the synthesis and degradation of a variety of proteins. Activated

broblasts are the archetypal producers of this ECM and synthesize, amongst others, a family of collagens (predominantly brillar types I and III), elastin, proteoglycans, laminin, bronectin, and tenascin. Collagens constitute the commonest ECM proteins, and line conducting airways, interstitium, and vascular walls. They contain a characteristic right-handed triple helix that is formed by three tightly binding polypeptide a-chains. The helical regions of these chains are composed of repeating Gly-X-Y triplets, with approximately every third X being proline and every third Y, hydroxyproline. Although ve types of brillar collagens are found in the human lung (types I, II, III, V, and XI), their relative abundance may vary quite significantly. For example, types I and III form almost 90% of all collagens within the pulmonary interstitial compartment and present in a fairly consistent ratio of 2:1 under homeostatic conditions.

FIBROBLASTS 215

Injury

Soluble signals (e.g., TGF- , PDGF, GM-CSF, IL-4)

Physicochemical stimuli (cellECM interactions, stretch, hypoxia)

Fibroblast

Proliferation and migration

Apoptosis

Transdifferentiation and synthesis

Immune functions

Extracellular matrix remodeling

Excess matrix deposition (interstitial lung diseases, airway remodeling)

Inadequate matrix repair (pulmonary emphysema, cystic fibrosis)

Figure 2 Fibroblasts play a key role in the pathogenesis of a number of pulmonary diseases. In response to diverse stimuli, they undergo functional alterations crucial for pathological matrix remodeling characteristic of these disorders.

The most intense period of collagen ber synthesis occurs during the perinatal period. However, the synthetic capacity of broblasts persists well into adulthood. Consequently, daily rates of collagen turnover reaching as high as 10% have been reported in the lungs of adult rodents and rabbits. Even though the rate of collagen synthesis may decrease with increasing age, the degradation rate of newly synthesized procollagen may remain high. It has been estimated that normal adult lung broblasts can degrade up to 1 106 molecules of procollagen per hour. In brotic states, enhanced collagen synthesis and its decreased degradation are thought to underlie the excessive accumulation of ECM that characterizes such disorders. While collagen bers are the main determinants of the expansile properties of the lung, elastic bers provide the resiliency for restoring shape and volume known as pulmonary elastic recoil. Elastin accounts for 2030% of the dry weight of the lung ECM and contains regions of extreme hydrophobicity essential for its designated biological roles. It is synthesized as a 70 kDa soluble precursor (tropoelastin) that is rapidly cross-linked and polymerized into a random network of elastin molecules. Proteoglycans are a distinct group of complex macromolecules containing a core protein linked to variable numbers of

glycosaminoglycan polysaccharide side-chains. Aggregating proteoglycans regulate ECM hydration and contribute to its compressive properties. In the alveolar wall, the major proteoglycans are heparin sulfate, chondroitin sulfate, and dermatan sulfate. On the other hand, heparin sulfate and laminin are the central proteoglycans in the alveolar basement membrane.

Secretion of Inammatory Mediators and Growth Factors

Fibroblasts also produce a variety of inammatory mediators and growth factors. In turn, they are subject to regulation by these and other signals produced by neighboring cells or released from the ECM. TGFb is one prime example. Many broblast-dependent processes are governed by the activation of TGF-bmediated autocrine and paracrine pathways. Indeed, TGF-b appears to be the most effective inducer of extracellular matrix production yet identied. During broblast differentiation, TGF-b upregulates the expression of myobroblast contractile proteins essential for phenotypic switching and also inhibits broblast/ myobroblast apoptosis. Insulin-like growth factor II (IGF-II) and interleukin-4 (IL-4) are other important

216 FIBROBLASTS

mediators of broblast activation. On the other hand, platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-a), and connective tissue growth factor (CTGF), a member of the PDGF family, appear primarily to promote broblast proliferation. Fibroblast-derived soluble factors such as chemokines and lymphokines can also set up chemical gradients to attract and stimulate inammatory cells migrating into the matrix. Secretory products of these cells may then act directly on broblasts, or indirectly to mobilize stored growth factors with matrix-modifying properties. Fibroblastmatrix interactions are mediated by specic cell surface receptors such as a1b1 and a2b1 integrins that facilitate broblast interactions with brillar collagens (types I and III, respectively), and a5b1 and aVb3 integrins that promote broblastbronectin interactions. These processes are temporally and spatially relevant to the overall ability of the lung ECM to respond to constitutive and adaptive stimuli. Tissue inhibitors of metalloproteinases (TIMPs) produced by broblasts oppose the degradation of ECM by matrix metalloproteinases (MMPs), a family of secreted and membrane-localized Zn2 -dependent enzymes. A correct balance in relative MMPTIMP activity is critical for the physiological turnover of pulmonary ECM. In addition to these proteins, matrix synthesis and breakdown are also controlled by the activity of morphogens and other mediators such as retinoids, patched (PTCH), epidermal growth factor (EGF), and hepatocyte growth factor. Overall, a high degree of molecular regulation ensures that airway branching and alveologenesis occur normally and that repair responses following tissue injury are not subverted by unwanted broproliferation.

Fibroblasts in Respiratory Diseases

Fibroblasts in Interstitial Lung Diseases

Fibroblasts are central to the pathogenesis of chronic interstitial lung diseases (ILDs) such as idiopathic pulmonary brosis (IPF), pulmonary sarcoidosis, and asbestosis. One current hypothesis suggests that repeated or sequential episodes of lung injury provide the pathological signals to drive uncontrolled broproliferation, a process that ultimately results in ECM overproduction. Recent studies have indicated that aberrant alveolarbroblast (epithelial mesenchymal) interactions in the parenchymal environment may also contribute to such brogenic responses. Indeed, myobroblasts in the interstitial space have been shown to produce soluble factors that, amongst other things, induce alveolar epithelial cell apoptosis, a pathologic determinant of pulmonary brosis.

Numerous cytokines and growth factors are known to regulate matrix accumulation at the molecular level. The most widely described group of probrotic mediators belongs to the TGF-b family of proteins. These polypeptides promote broblast activation, growth, and collagen synthesis and counterregulate the function of MMPs and TIMPs. Increased TGF-b expression has been identied in lungs from patients with a variety of brotic disorders. In rats, the overexpression of constitutively active TGF-b following direct gene delivery to the distal lung leads to the development of severe and protracted pulmonary brosis. Conversely, neutralization of TGF-b activity or signaling inhibits lung collagen accumulation as well as histologic parameters of pulmonary brosis. New insights have also been gained into signal transduction pathways downstream of the TGF-b receptor complex. Early ndings in the bleomycin model of pulmonary brosis have shown that excessive ECM accumulation is associated with decreased expression of Smad3 (a TGF-b receptor-associated signaling molecule) whereas inhibition of pulmonary brosis correlates with increased expression of Smad7, a protein that inhibits signaling of the same pathway. At least 20 other cytokines, including IGF-1, endothelin-1 and TNF-a, have been shown to exert probrotic effects in vitro and have been implicated in disease pathogenesis in vivo. In addition, abnormalities of the coagulation and brinolytic systems also appear to play a role in promoting brotic lung repair. More specifically, thrombin has been shown to stimulate broblast mitogenesis via the production of autocrine mediators (e.g., PDGF-AA) and promote connective tissue synthesis by the activation of cell surface protease-activated receptors (PARs). Decreased protein C activation leading to impaired brinolysis and enhanced procoagulability has also been described in the lungs of patients with ILDs. In the quest to further delineate broblast activation pathways in pulmonary brosis, emerging evidence from animal studies suggests that bone marrowderived broblast progenitors termed brocytes may populate the repairing lung during the brotic response. These cells express, amongst others, CD45 and collagen I as well as the chemokine receptors CCR7 and CXCR4. In mice treated with bleomycin, increased sequestration of such cells occurs in areas of enhanced brotic matrix deposition. Although the mechanisms by which these cells trafc to the lung are still unclear, a role for the CXCL12 chemokine (a ligand for CXCR4) has been implicated. Antibrotic molecules also appear to act by suppressing broblast function. In a preliminary study, the administration of interferon gamma (IFN-g), a cytokine that inhibits broblast differentiation,

FIBROBLASTS 217

proliferation, and collagen production in vitro, was associated with improved lung function in a small group of patients with corticosteroid-resistant IPF. The prostaglandin PGE2, a potent inhibitor of broblast proliferation and matrix production in vitro, may also exert similar actions in the lung. Fibroblasts obtained from patients with IPF have a diminished capacity to synthesize PGE2 under basal conditions and when stimulated with TGF-b. Antibrotic properties have also been ascribed to IL-7, a molecule that is able to inhibit TGF-b signaling in lung broblasts and attenuate bleomycin-induced pulmonary brosis in vivo. The role of the broblast in acute broproliferative lung injury, in particular the acute respiratory distress syndrome (ARDS), is also increasingly appreciated. Abnormal lung matrix turnover in this devastating disorder is now believed to occur much earlier than previously thought. In ARDS, the formation of hyaline membranes, a type of provisional matrix, within damaged alveoli is a key feature of the accelerated broproliferative response. However, the molecular controls that distinguish this form of acute brosis from the more chronic pattern in ILDs have not been elucidated.
Fibroblasts in Asthma

proinammatory mediators, including chemoattractants such as eotaxin and monocyte chemotactic protein-1. In the chronically inamed airway, the constant exchange of information between broblasts and inltrating leukocytes sets up a fertile ground upon which airway remodeling is augmented and propagated. Although these observations provide strong circumstantial evidence linking pathological airway remodeling to bronchial hyperresponsiveness and airow obstruction, the mechanisms underlying these processes are far from established. Studies in individuals with chronic asthma have shown that circulating brocyte-like cells expressing CD34, collagen type I, and a-SMA accumulate in areas of collagen deposition within the bronchial subepithelial environment following allergen challenge. When stimulated in vitro by brogenic mediators such as endothelin-1 and TGF-b, such cells acquire a myobroblast phenotype and secrete bronectin and collagen type III. In a corresponding murine allergen model, the use of in vivo cell-labeling techniques has enabled the trafcking of similar brocytes to be monitored as they move from the circulation to the bronchial submucosa where they subsequently differentiate into tissue-embedded myobroblasts.
Fibroblasts in Chronic Obstructive Pulmonary Disease (Chronic Bronchitis and Emphysema)

Dramatic changes in the anatomy of the asthmatic airway are well described, but interest has historically focused on smooth muscle cell and glandular hyperplasia. However, there has been a recent surge in the appreciation of bronchial myobroblast accumulation and subepithelial brosis in the airways of chronically asthmatic patients. In these individuals, the lamina reticularis, the layer between the epithelial basement membrane and the underlying smooth muscle layer, may be increased by two- to three fold in thickness. This anomaly is the direct result of quantitative increases in a variety of extracellular matrix proteins, including collagen types I, III, and V, elastin, and bronectin. Most airway broblasts are also found within this layer. In fact, the concept of the epithelialmesenchymal trophic unit in the airway preceded an analogous description in the pulmonary interstitium. These sub-basal lamina broblasts lie in close proximity to bronchial epithelial cells, and are thought to be the primary elements of the broproliferative machinery responsible for matrix overproduction. In addition, thickening of the subepithelial basement membrane itself is well documented. T-helper type 2 cytokines, particularly IL-4 and IL-13, can promote broblast activation, extracellular matrix accumulation, and the secretion of

In chronic obstructive pulmonary disease (COPD), most of the changes in the extracellular matrix occur in the peripheral airways and surrounding parenchyma. Although airway remodeling is less well understood than in asthma, increased thickness of the inner (lamina reticularis) and outer (adventitial) walls of the airways has been documented. However, less is known about the composition of individual matrix proteins or the specic changes in broblast number or phenotype associated with this process. In addition to changes in the airway wall, emphysematous lesions of the lung parenchyma are also associated with destruction of alveolar connections to the outer bronchial wall, a phenomenon implicated as a contributor to overall airway obstruction. However, a causative link between airway ECM alterations and clinical airow limitation in COPD remains unproven. The predominant hypothesis of emphysema suggests that a persistent proteaseantiprotease imbalance in the distal lung favors net lung matrix degradation, particularly of elastin and its associated proteins. This view emphasizes the notion that both uncontrolled matrix breakdown and inadequate tissue repair are required to produce the abnormal enlargement of alveoli and loss of alveolar septal tissue that characterize this condition. Cigarette smoke constituents and

218 FIBROBLASTS

cadmium are among the agents known to be deleterious to the lung matrix. Their ability to inhibit various broblast functions including proliferation, matrix production, and chemotaxis in vitro has broader implications for the capacity of the lung matrix to regenerate following degradative injury. Paradoxically, discrete areas of increased interstitial ECM may be evident in lungs affected by the emphysematous process. It is possible that while the overall extent of matrix loss may overwhelm the tissue restorative potential in such lungs, focal ECM renewal may occur where reparative efforts are particularly successful. The increased expression of TGF-b and epidermal growth factor (EGF) in epithelial and submucosal cells of patients with COPD has been reported. These mediators may contribute to the peribronchiolar brosis present on histological analysis although the nature of the localized brogenic response is inadequately understood.

See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Extracellular Matrix: Basement Membranes; Elastin and Microbrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast Growth Factors. Interferons. Matrix Metalloproteinases. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
Chambers RC and Laurent GJ (1997) Collagens. In: Crystal RG, West J, Weibel E, and Barnes P (eds.) The Lung: Scientic Foundations, 2nd edn., vol. 49, pp. 709727. Philadelphia: Lippincott-Raven. Dunsmore SE, Chambers RC, and Laurent GJ (2003) Matrix proteins. In: Gibson GJ, Geddes DM, Costabel U, Ster PJ, and Corrin B (eds.) Respiratory Medicine, 3rd edn., pp. 8292. London: Elsevier. Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2003) Bone-marrow-derived progenitor cells in pulmonary brosis. Journal of Clinical Investigation 113: 243252. Hogaboam CM, Smith RE, and Kunkel SL (1998) Dynamic interactions between lung broblasts and leukocytes: implications for brotic lung disease. Proceedings of the Association of American Physicians 110: 313320. Huang M, Sharma S, Zhu LX, et al. (2002) IL-7 inhibits broblast TGF-beta production and signaling in pulmonary brosis. Journal of Clinical Investigation 109: 931937. Keerthisingam CB, Jenkins RG, Harrison NK, et al. (2001) Cyclooxygenase-2 deciency results in a loss of the anti-proliferative response to transforming growth factor-b in human brotic lung broblasts and promotes bleomycin-induced pulmonary brosis in mice. American Journal of Pathology 158: 14111422. McAnulty RJ and Laurent GJ (2002) Fibroblasts. In: Barnes PJ, Drazen J, Rennard S, and Thomson N (eds.) Asthma and COPD: Basic Mechanisms and Clinical Management, pp. 139 144. London: Academic Press. McGowan SE and Torday JS (1997) The pulmonary lipobroblast (lipid interstitial cell) and its contributions to alveolar development. Annual Review of Physiology 59: 4362. Nakamura Y, Romberger DJ, Tate L, et al. (1995) Cigarette smoke inhibits lung broblast proliferation and chemotaxis. American Journal of Respiratory and Critical Care Medicine 151: 1497 1503. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating brocytes trafc to the lungs in response to CXCL12 and mediate brosis. Journal of Clinical Investigation 114: 438446. Powell DW, Mifin RC, Valentich JD, et al. (1999) Myobroblasts. I. Paracrine cells important in health and disease. American Journal of Physiology 277(1 pt. 1): C1C19. Schmidt M, Sun G, Stacey MA, Mori L, and Matoli S (2003) Identication of circulating brocytes as precursors of bronchial myobroblasts in asthma. Journal of Immunology 171: 380389. Sempowski GD, Beckmann MP, Derdak S, and Phipps RP (1994) Subsets of murine lung broblasts express membrane-bound soluble IL-4 receptors. Role of IL-4 in enhancing broblast proliferation and collagen synthesis. Journal of Immunology 152: 36063614. Sime PJ, Xing Z, Graham FL, Csaky KG, and Gauldie J (1997) Adenovector-mediated gene transfer of active transforming growth factor-b1 induces prolonged severe brosis in rat lung. Journal of Clinical Investigation 100: 768776.

Therapeutic Considerations

There is as yet no specic therapy to prevent or reduce the pathological accumulation of airway or parenchymal lung matrix. Corticosteroids remain the cornerstone of treatment of symptomatic asthma and COPD, but their ability to alter the pathobiology of brotic lung diseases is unproven. The propensity of these and other immunosuppressive agents to cause severe adverse effects significantly limits their clinical use. Attention has recently shifted to agents that may interfere directly with the brogenic process in the lung. Although distinct subpopulations of broblasts are suspected to initiate or intensify airway or parenchymal lung brosis, the mechanisms by which they operate are still unclear. In vitro and in vivo studies have highlighted the potential value of angiotensin-converting enzyme inhibitors/angiotensin II receptor antagonists, PGE2, colchicine, N-acetylcysteine, and lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in this context. The few agents to have produced any measurable benet in very small numbers of patients with pulmonary brosis include pirfenidone, relaxin, and IFN-g. However, their true clinical value remains unknown. The use of increasingly sensitive molecular techniques and rened disease models is yielding important information regarding the role of the broblast in major pulmonary disorders. These insights may help to dene aspects of broblast biology that are amenable to therapeutic manipulation. Such efforts are necessary in order to improve the efcacy and specicity of future strategies aimed at interfering with the brotic process in the lung.

FLUID BALANCE IN THE LUNG 219

Stenmark KR and Mecham RP (1997) Cellular and molecular mechanisms of pulmonary vascular remodeling. Annual Review of Physiology 59: 89144. Swiderski RE, Bencoff JE, Floerchinger CS, Shapiro SD, and Hunninghake GW (1998) Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary brosis. American Journal of Pathology 152: 821828. Uhal BD, Joshi I, Hughes WF, et al. (1998) Alveolar epithelial cell death adjacent to underlying myobroblasts in advanced brotic human lung. American Journal of Physiology 275: L1192 L1199. Vlahovic G, Russell ML, Mercer RR, and Crapo JD (1999) Cellular and connective tissue changes in alveolar septal walls in emphysema. American Journal of Respiratory and Critical Care Medicine 160: 20862092.

FLUID BALANCE IN THE LUNG

A E Taylor, University of South Alabama, Mobile, AL, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Airway edema is a very common occurrence in patients with heart failure and also in different forms of lung inammation (sepsis). The lungs circulation, airways, and alveoli have tremendous surface areas and can fully oxygenate the venous blood returning from the body into the right atrium and also rid the body of the high amounts of CO2 contained in the venous blood. However, the large gas-exchanging areas in the lungs can also create a problem when left-sided heart failure is present. During pathology, the pulmonary capillary hydrostatic pressure (PPC ) increases in the lungs circulation and uid leaves the circulation (JV ) to enter the lungs interstitium. This is opposed by the protein osmotic pressure in the plasma (pP ). The lymph ow increases (JL ) and removes a large amount of this uid. The interstitial protein osmotic pressure (pT ) will also decrease as proteins are removed by lymph ow and diluted by protein-free uid entering the lungs interstitium. Interstitial uid pressure (PT ) also increases, which opposes the increased microvascular pressure. Increases in PT and JL coupled with the decreased interstitial protein osmotic pressure (pT ) have been dened as edema safety factors because they provide a substantial protective effect against alveolar edema formation. When PPC increases, the sum of these forces can provide a safety factor of approximately 23 mmHg before the lung tissues ll with edema uid.

Physiologists derived this equation for muscle uid exchange, and sd is a recent addition to the uid ux equation. Since KFC and PPC are the primary components in eqn [1] that produces JV , early studies developed ingenious ways to measure them. First, an organ was isolated and perfused, and the arterial and venous pressures and ow were measured. Then, arterial and venous pressures were adjusted until the organ did not gain or lose weight. The blood ow and arterial pressure (PA ) were decreased and venous outow pressure (PV ) increased to maintain a noweight-change condition in the organ an isogravimetric state. This procedure was done several times, and the intercept of PV versus ow curve on the pressure axis was PPC since the resistance in the arterial and venous systems were known. Normal ow

Tissue

(7) PPC PT (5) (28)

P T

(5)

(5) (Surface tension) Alveolus

(17) (6) PLYM

(+1) Net pressure

The following equation describes the microvascular ltration rate (JV ) occurring across the lungs microcirculation and is shown schematically in Figure 1: JV KFC PPC PT sd pP pT 1

Capillary

Lymphatic filling pressure (PT PLYM)

Figure 1 Schematic representation of the Starling forces operating between pulmonary capillaries and the interstitium. Note that the alveolar surface tension is exactly opposed by the subatmospheric interstitial uid pressure ( 5), and the lymphatic lling pressure (PT PLYM ) [ 5( 6)] is positive, which promotes the lling of lymphatic vessels. Reproduced from Taylor AE, Rehder K, Hyatt RE, and Parker JC (1989) Clinical Respiratory Physiology, Ch. 10, p. 178. Philadelphia, PA: Saunders, with permission from Elsevier.

where KFC is the ltration coefcient of the capillary wall; PPC is the hydrostatic pressure in the lungs microvessels; PT is the interstitial pressure of the lung; pP and pT are protein osmotic pressures of the plasma and tissues, respectively; and sd is the reection coefcient for the plasma protein. If sd is 1 for the proteins, the membranes are impermeable to the protein, and if sd is 0, the protein is freely permeable.

220 FLUID BALANCE IN THE LUNG

was again instituted, and knowing PA , both PV and PPC could be calculated. These techniques were used to evaluate the forces (PPC and PT ), ows ( JV and JL ), and plasma (pP ) and lymph (pL ) protein osmotic pressure. In addition, we developed a protein osmometer to measure pT and pP . Thus, for the rst time, techniques were in place to apply similar techniques in lung preparations to measure all components of eqn [1]. The rst quantitative study of edema formation conducted in dog lungs was done by Guyton and Lindsey in 1959. Left atrial pressures were increased to different levels in in situ dog lungs, and then the lungs were removed and weighed, both before and after they were dried. Surprisingly, the amount of uid in the lungs interstitium as determined by the lungs wet/dry weight ratios did not increase until left atrial pressures (LAPs) exceeded 25 mmHg. However, once LAPs exceeded this value, a large increase in pulmonary edema formation occurred. This study produced two major conclusions. First, no substantial amount of edema accumulated in lung tissues until LAPs exceeded 25 mmHg. Second, edema did not occur at lower LAPs, but the reason for this was not clear. We now know that interstitial uid pressure (PT ) increases as uid enters the lungs interstitium, lung lymph ow ( JL ) removes some of the microvascular ltration, and tissue plasma proteins are also removed by the lymph ow, which decreases the tissue osmotic pressure (pT ). The average value of these edema safety factors, which act to oppose edema formation when microvascular pressures increase, is 50% of the edema safety factor in lungs as a result of the decreased tissue protein osmotic pressure (pT ). The increased lymph ow ( JL ) and the increased tissue hydrostatic pressures (PT ) each provide approximately 22% of the safety factors. The sum of these forces provides an edema safety factor of approximately 23 mmHg. This means that lungs are normally protected against pulmonary edema formation by these safety factors; however, when the microvascular pressure increase exceeds approximately 25 mmHg, intra-alveolar edema forms very rapidly even in undamaged lungs. When the endothelial barrier is damaged by sepsis and other inammatory responses, severe lung edema occurs even at very low PPC because the microvascular walls are extremely permeable to all plasma proteins. KFC will also increase when the endothelial barrier is damaged, which increases the amount of uid entering the tissues as PPC increases. Also, the coefcient that determines the activity of osmotic pressure gradient acting across the microvascular wall (sd ) is decreased, causing pP pT to become very small. In this condition, even though the lymph

ow ( JL ) increases to very high levels, the ow is insufcient to remove the excessive amount of uid that enters the lungs tissue. Severe intra-alveolar edema that contains high protein concentrations will result and cause death if the process causing the edema cannot be corrected. The major conclusion of this rst quantitative study of pulmonary edema formation was that future pulmonary edema studies had to be performed to determine the values of KFC , lymph ow, PT , PPC , pP , pL , and sd . The techniques needed to measure these requirements were rst studied in isolated and perfused animal lungs that were continuously weighed in order to accurately measure the rate of edema formation. This was accomplished by simply placing the pump-perfused lungs on a sensitive weight device. Second, ways to measure lymph ow ( JL ) and its plasma protein osmotic pressure (pT ) were also developed. The existing microvascular pressure (PPC ) was measured using gravimetric procedures and the interstitial pressure (PT ) was measured using previously implanted devices; lung weight changes were also continuously measured. The parameters shown in eqn [1] also provide some small amount of edema protection across the pulmonary circulation following elevation of capillary pressures when lungs are severely damaged by pathology but it is more difcult to reverse than hydrostatic edema. Finally, a biophysical factor, the osmotic reection coefcient (sd ), which is multiplied by (pP pL ), is close to 1 for normal capillaries but has been shown to be reduced to less than 0.3 when the pulmonary vascular walls are severely damaged, that is, if sd 0:9, then the proteins in plasma and tissues would exert 90% of the measured protein osmotic pressure. This important factor can be measured using the following equation, which requires that the concentration of a protein in both lymph (CL ) and plasma (CP ) be measured when lymph ow is high and CL =CP approaches a constant but before edema results: CL =CP 1 s=1 sex 2

where x 1 sJL =PS, and PS is the permeability surface area product of the lungs microcirculation. Importantly, when JL is very high, eqn [2] reduces to CL =CP 1 s and sd can be easily determined by using the following equation and analyzing the lymph and plasma concentrations of the proteins under study as they attain a constant value: s 1 C L =C P 3 Obviously, all components responsible for producing volume ow occurring across the capillary wall

FLUID BALANCE IN THE LUNG 221

were nally dened, and now each protein in plasma can be measured and studied in both normal and damaged lungs. Importantly, safety factors allow PPC to vary from moment to moment in normal lungs; that is, PT increases, JL increases, and the reduction in tissue proteins (pT ) all change in a direction to oppose further ltration into the lungs tissue. As long as PPC is not greatly elevated, the lungs will not become edematous but will continue to properly exchange gases with the circulation and atmosphere unless the edema becomes severe. It is important to understand that when the endothelial barrier in the lungs circulation is damaged, edema is likely to develop, even if the PPC remains low. The following calculations can be used to illustrate this point. Using eqn [1], we can calculate JV , assuming the following forces are normal transcapillary ltration values: PT 5 mmHg, pP 28 mmHg, pT 17 mmHg, sd 1, KFC 0:2 ml min 1, and PPC 7 mmHg. Thus, JV 0:2 ml min 1. This is the normal amount of uid entering the lungs tissues each minute, and it can easily be removed by lymph ow. However, when capillary walls are damaged, a very different effect occurs. Assume the following forces (in mmHg): PPC 7, PT 5, sd 0:5, pP 28, pT 19, and KFC is doubled. Then, JV 3 ml min 1, as compared to 0.2 ml min 1 in normal lungs. Note that when the endothelial barrier is damaged and KFC is doubled, tissue protein osmotic pressure increases, and sd is reduced to 0.5 with damaged capillaries. This change in capillary permeability causes a 15-fold difference in transcapillary ltration. Edema will certainly develop in the lung because lymph ow is unable to remove this amount of excess uid and protein entering the tissues, and the edema uid continues to enter the tissues and alveoli. The magnitude of safety factors increases when left atrial pressures rise during chronic heart failure and in intact lungs of in vivo animals such as sheep, but acute studies are usually conducted in experiments using isolated perfused lungs. However, it is well-known that patients with left atrial pressure of 40 mmHg should have extensive pulmonary edema, but edema does not occur! Previous animal studies have shown that chronic elevations of left atrial pressure cause 25 times as much lymph ow as does acute elevation of left atrial pressure. A larger lymph ow safety factor is present during chronic elevation of left atrial pressure which produces a 25-fold increase in conductivity of pulmonary lymphatics. Therefore, the safety factors that oppose edema formation must become far greater than the 20 25 mmHg occurring in acute studies. However,

chronically elevated left atrial pressure is due to the extremely large lymph ows. The safety factor might well be 4045 mmHg, that is, double the 23 mmHg total safety factor seen in acute pulmonary edema formation because of the huge lymph ow. However, there are vertical gradients in PPC , PT , pT , and JL in upright intact lungs that are very different from the average values used in the examples given previously, as shown in Figure 2. For example, the capillary pressure increases from 2 to 12 mmHg from the apex to the base of a lung, which is 10 cm high. These different capillary pressures result in a 10 mmHg difference in force acting at the bottom of the lung compared to the apex, and they will produce a greater transcapillary ltration in the more dependent regions of the lungs. Since a greater ltration occurs in the bottom of the lung relative to the top, pT would be less in the base tissues (17 mmHg) than at the apex (19 mmHg) of the lung. Lymph ow would also be higher at the base of the lung compared to the apex, and the lymphatic system can remove a larger portion of the ltration. PT decreases from 8 to 0 mmHg from the top to the bottom of the lung, which opposes ltration at the bottom of the lung. The combination of greater transcapillary ltration and lymph ow in the basal regions of the lung results in decreased pT and increased PT . The differences between these capillary, tissue forces, and lymph ows at the apex and base of the

Top

PT 8

PC 2

P 1

C=

28)

19

10 cm

18

Heart level

0 Bottom

17

12

Figure 2 Schematic representation of tissue uid pressure (PT ), plasma protein osmotic pressure of the tissue uids (pT ), and capillary pressure (PC ) at the bottom, midpoint, and top of an upright lung. The imbalance in tissue forces (DP) is the same at the top and bottom of the lung. pC is the plasma protein osmotic pressure. However, the ltration coefcient will be higher at the bottom of the lung than the top and more transcapillary ltration will occur. Lymph ow will increase at the bottom of the lung and remove some of the capillary ltration. Reproduced from Taylor AE, Khimenko PL, Moore TM and Adkins WK (1997) Fluid balance. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung Scientic Foundations, 2nd edn., p. 1557. New York: Raven Press, with permission from LippincottRaven Publishers.

222 FLUID BALANCE IN THE LUNG

lungs (i.e., PPC , PT , pP , pT , and even KFC , JV , and JL ) are usually studied in animal lungs laid at on a scale. However, it is necessary to know how these forces change in the upright lung in order to accurately evaluate the uid exchange in the upright human lung. The differences given here are sufcient for a general analysis of transcapillary ltration, but for more precise measures, lungs must be studied in an upright position since the KPC will be much smaller at the top of the lung as compared to the lower portion. Although isolated and perfused lungs have provided investigators with new insight into the factors responsible for both producing and opposing pulmonary edema, a more clinical-type animal model has also been developed to study edema formation in awake and standing sheep. The LAPs were measured and the sheeps lung lymph was also collected. LAPs were usually elevated while the sheep were being studied, lymph collected, and its plasma proteins measured. Then, sheep were subjected to sepsis or some form of inammation, such as a period of ischemia in the lungs, followed by reperfusion, and the JL and its protein concentrations were measured before and after treatment with different interventions to determine whether specic interventions could prevent and/or reverse the damage to the sheeps microvascular membrane and lessen pulmonary edema. Obviously, when the pulmonary capillaries are normal, CL =CP will decrease when LAPs are increased; however, when the capillaries are damaged, lymph ow and the plasma protein concentrations will also increase even when LAP increases. This animal model was, and still is, a most useful experimental procedure because the correction of sepsis and other forms of lung pathology can be sufciently dened to perhaps provide clinically useful data; certain interventions have been found that prevent and even reverse capillary wall damage in the awake-sheep model. Finally, what is known about the reabsorption of edema uid once it lls large portions of the lungs? Obviously, reducing capillary pressure will decrease ltration, and although it is controversial, it is now believed that the pulmonary epithelial cells can actively move ions from the intra-alveolar uid into the interstitial tissues of the lung which could be removed from the lung by the lymphatics, or the transported uid can also enter the blood that ows through the lung, if the forces are consistent with capillary reabsorption. It appears that amiloride-sensitive Na channels are located in the epithelial cells of the alveoli and when airways are activated by Na / K ATPase, alveolar uid removal is accelerated and can be removed by capillary absorption or lymph

ow. Ouabain, which blocks these channels, and amiloride, an Na channel blocker, increase lung wet/dry weight ratios exposed to ischemia/reperfusion injury from 10 (damage with only ischemia/ reperfusion) to 18, when both Na channels and the Na /K ATPase are blocked. However, if the capillary endothelial barrier is damaged, it cannot absorb the transported uid, which complicates data interpretation. Future studies must be performed to gain a much better understanding of the transport systems in the lungs epithelial barrier to clarify its role in edema clearance. It is not clear how much uid can be absorbed by active transport from the airways and whether the transport is upregulated in edema.
See also: Acute Respiratory Distress Syndrome. Carbon Dioxide. Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deciency. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Infant Respiratory Distress Syndrome. Ion Transport: Calcium Channels; ENaC (Epithelial Sodium Channel). Lymphatic System. Nitric Oxide and Nitrogen Oxides. Oxidants and Antioxidants: Antioxidants, Enzymatic. Permeability of the BloodGas Barrier. Pleural Effusions: Pleural Fibrosis. Pulmonary Circulation. Vascular Disease.

Further Reading
Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in uid transport in lung and airways. Journal of Applied Physiology 93: 21992206. Guyton AC and Lindsey AE (1959) Effect of elevated left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circulation Research 7: 649657. Khimenko PL and Taylor AE (1999) Physiology of body uids. In: Webb AR, Shapiro MJ, Singer M, and Suter PM (eds.) Oxford Textbook of Critical Care, pp. 2427. Oxford: Oxford University Press. Moore TM, Shirah WB, Khimenko PL, et al. (2002) Involvement of CD40CD40L signaling in postischemic lung injury. American Journal of Physiology 283: L1255L1262. Pappenheimer JR, Renkin EM, and Borrero LM (1951) Filtration, diffusion and molecular sieving through peripheral capillary membranes. A contribution to the pore theory of capillary permeability. American Journal of Physiology 167: 1346. Parker JC and Townsley MI (2004) Evaluation of lung injury in rats and mice. American Journal of Physiology 286: L231 L246. Patlak CS, Goldstein DA, and Hoffman JF (1963) The ow of solute and solvents across a two-membrane system. Journal of Theoretical Biology 5: 426442. Rippe B and Haraldsson B (1992) Transport of macromolecules across microvascular walls: the two-pore theory. Physiology Review 74(1): 163219. Rippe B and Taylor A (2001) NEM and lipin increase albumin transport in lung microvessels. American Journal of Physiology 280: H34H41.

FLUID BALANCE IN THE LUNG 223

Staub NC (1978) Lung water and solute exchange. In: Lefant C (ed.) Lung Biology in Health and Disease, vol. 7. New York: Dekker. Taylor AE (1997) Microvascular uid and solute exchange. In: Encyclopedia of Human Biology, 2nd edn., pp. 25812592. San Diego: Academic Press. Taylor AE and Ballard ST (1992) Microvascular function. American Review of Respiratory Disease 146: S24S27. Taylor AE, Khimenko PL, Moore TM, and Adkins WK (1997) Fluid balance. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung Scientic Foundations, 2nd edn., p. 1557. New York: Raven Press. Taylor AE and Parker JC (1985) Pulmonary interstitial spaces and lymphatics. In: Fishman AP and Fisher AB (eds.) Handbook of Physiology, Respiratory System I, pp. 167230. Baltimore: Williams & Wilkins. Taylor AE, Rehder K, Hyatt RE, Parker JC (1989) Clinical Respiratory Physiology, Ch. 10, p. 178. Philadelphia, PA: Saunders. Uhley HN, Leeds SE, Sampson JJ, and Friedman M (1961) Some observations on the role of the lymphatics in experimental acute pulmonary edema. Circulation Research 9: 688693.

G
GASTROESOPHAGEAL REFLUX
P O Katz and N Tajong, Albert Einstein Medical Center, Philadelphia, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
This article reviews the presentation, epidemiology, diagnosis, and therapy of gastroesophageal reux and its relationship to lung disease. Focus is on cough, asthma, and idiopathic pulmonary brosis. The approach to treatment can be applied to any patient with a pulmonary disease or symptom believed to be associated with gastroesophageal reux disease.

Gastroesophageal Reux Disease

Gastroesophageal reux disease (GERD) is a very common yet complex disease with widely variable presentations and complications that extend beyond the esophagus. The most common esophageal presentation is heartburn, which is caused by reux of acid and gastric contents into the esophagus. Approximately 50% of Americans experience heartburn once a month with 25% using over-the-counter antacids three or more times a month with 47% of the patient population experiencing daily symptoms. Recent reviews have suggested the annual cost of treating GERD with both over the counter (OTC) and prescription drugs to be over 8 billion US dollars. Normal physiology involves transient relaxation of the lower esophageal sphincter (LES), especially after meals. This allows gastric contents (acid) to enter the esophageal lumen, which can lead to mucosal damage in susceptible patients. The main determinants of injury to esophagus are the duration of contact as well as epithelial resistance to injury, and factors other than acid. The duration of contact depends on the number of reux episodes and the efciency of esophageal peristalsis, which normally clears the refluxed gastric contents. Patients with GERD can have a wide range of clinical presentations ranging from esophageal symptoms with or without mucosal damage to evidence of mucosal damage without symptoms, or few esophageal symptoms with extensive extraesophageal manifestations. Typical symptoms of GERD include heartburn, which is described as a retrosternal

burning sensation with radiation to throat. Often there is regurgitation of acidic gastric contents into the mouth with complaints of sour taste. Symptoms are worse after high-fat meals, in the supine position, after heavy meals, spicy foods, alcohol consumption, and citrus products. Of all GERD symptoms a history of heartburn and regurgitation has the highest specicity for diagnosing GERD. Since there is no accepted gold standard the diagnosis of GERD can be made using endoscopy, pH monitoring, radiologic testing, provocative testing, and therapeutic trial of acid suppression. The rst line approach to the patient with typical GERD symptoms is a therapeutic trial of acid suppression usually with a proton pump inhibitor (PPI) once or twice daily. Some advocate twice daily dosing for 12 weeks, while others recommend once a day for 48 weeks. Unfortunately, there are no denitive studies suggesting the optimal drug, dose, or duration of trial; however, multiple studies have supported this approach to patients with typical symptoms of GERD. Patients who fail to experience symptom relief despite a therapeutic trial or those with unusual or alarm symptoms (weight loss, dysphagia) are referred for upper endoscopy to rule out other etiologies and to grade the severity of esophageal mucosal damage. Upper endoscopy is also indicated for patients with longstanding disease to screen for Barretts esophagus. The main drawbacks of endoscopy are that it lacks sensitivity for GERD as many studies reveal that only 3040% of patients with heartburn will have erosive esophagitis. The measurement of esophageal pH by an intraluminal monitor is another method for diagnosing GERD. A pH probe is placed 5 cm above the manometrically located lower esophageal sphincter with continuous recordings of pH in the distal esophagus over a dened period. Recent advances have allowed this to be accomplished with a tubeless recording device that is taped to the esophagus. Monitoring is for 48 h. Patients symptoms are recorded so that correlation of symptoms with acid reux can be made. Limitations of pH monitoring include patient acceptance and reproducibility, which may be as low

226 GASTROESOPHAGEAL REFLUX

Table 1 Atypical symptoms or signs of gastroesophageal reux disease EENT a Pharyngitis Hoarseness/voice changes Otitis Sinusitus Vocal cord granulomas Subglottic stenosis Laryngitis Laryngeal cancer Cough
a

Pulmonary Asthma Cough Idiopathic pulmonary brosis Chronic bronchitis Pneumonia Unclassied Unexplained chest pain Sleep apnea Dental erosions

EENT, eyes, ears, nose, and throat.

Stimulation of cough sensory nerve ends in the esophagus may cause transmission to and from the cough center via the vagus nerve. This nervous impulse may also be reexively transmitted via vagal efferents from the brain, which stimulate secretion of neurotransmitters in the lower respiratory tract causing cough. Lastly, stimulation of nerve endings in the esophagus may send impulses directly to the trachea, causing cough, independent of the central nervous system (Figure 1). Esophageal dysmotility is also seen in patients with chronic cough and may be an underestimated mechanism for GERD-induced cough and present new possibilities for treatment of these difcult patients.
Clinical Presentation

as 85% in patients with respiratory symptoms, and that the test only measures acid reux into the esophagus. Combined impedance and pH monitoring will clarify the role of nonacid reux. Barium studies can diagnose anatomical abnormalities that may contribute to GERD, but are of limited use in the evaluation of GERD without alarm symptoms as they lack sensitivity for mucosal disease. Provocative testing involves the infusion of 0.1 N hydrochloric acid into the esophagus (Bernstein test) with a positive test dened as reproduction of patients typical symptoms with the infusion. This test is highly specic for acid reux, but not sensitive and so reserved for situations when a pH monitor is not available.
Respiratory Complications

The likelihood of GERD as etiology of cough is high when typical symptoms such as heartburn, sour taste, and regurgitation are present. It is not unusual to have cough as the initial presentation of GERD, even in the absence of heartburn. Cough related to reux is traditionally thought to be nocturnal, but this association is hard to prove. Patients with microaspiration-triggered cough may present with more reux symptoms, usually occurring prior to cough. It is important to note that cough may induce reux, further complicating the presentation.
Diagnosis

The respiratory manifestations of acid reux disease include bronchial asthma, cough, pulmonary brosis, and pneumonia. A more extensive list of extraesophageal manifestations of GERD is seen in Table 1.

Chronic Cough
Chronic cough is a problem that plagues 340% of the population. The three most common etiologies include asthma, esophageal reux, and rhinitis. Approximately 20% of nonsmoking patients with chronic cough and normal chest X-ray have esophageal reux, either as sole cause or in combination with the other causes of chronic cough. The pathophysiology of GERD-induced cough is not well understood, but involves microaspiration with exposure of esophageal contents into the bronchial tree as well as vagally mediated tracheoesophageal reex. Exposure of esophageal contents to the airway directly irritates cough receptors in small and larger airways through vagal mediation. The tracheobronchial reex has been theorized to cause GERD-induced cough in several ways.

There is no accepted gold standard for diagnosing cough induced by GERD. Excluding causes of chronic cough such as asthma and postnasal drip is crucial. The most reliable test to determine if cough is due to GERD is resolution of cough with treatment of reux. Many authors including us support a therapeutic trial of antisecretory therapy after other nonreux causes have been excluded, in light of the fact that GERD is so prevalent in this population. One study found that of 17 patients with a positive pH test, six (35%) had striking resolution in their symptoms within 2 weeks of omeprazole therapy (40 mg b.i.d.), which was sustained over 1 year. This approach if successful avoids the cost of diagnostic procedures and limits them to those who do not respond to an empiric trial. Twenty-four hour esophageal pH measurement is the most sensitive and specic test for diagnosing GERD-induced cough with positive and negative predictive values of 89% and 100%, respectively. Abnormal ndings on pH study include increased frequency of reux episodes, decreased acid clearance times, and increased acid exposure times. However, an abnormal pH study does not reliably predict which patients with cough and abnormal pH results

GASTROESOPHAGEAL REFLUX
Esophagus CNS Tracheobronchial tree Microaspiration Airway

227

Reflux

Mediator release Inflammation

Airway vagal afferents Esophageal vagal afferents CNS Airway vagal efferents

Edema Mucus Smooth muscle

Heightened bronchial reactivity

Figure 1 Gastroesophageal reux disease may lead to microaspiration with direct effects on the epithelium. Reuxate can stimulate vagal afferents in the esophageal or tracheobronchial tree. Axon reexes may cause direct pulmonary neuroinammatory changes or can lead to central nervous system (CNS) stimulation. The addition of nerve growth factor can lead to switching of airway A bers to tachykinin-expressing bers, which result in greater CNS stimulation and peripheral neuroinammatory effects. Reproduced from Stein M (2003) Symposium: possible mechanisms of inuence of esophageal acid on airway hyperresponsiveness. American Journal of Medicine 115(3A): 55S59S, with permission from Elsevier.

will respond to treatment. In a recent study 11 of 17 patients with abnormal pH did not respond to treatment with omeprazole. Some with a normal pH study (thus physiologic reux) may still have GERD as cause of cough. In this group of patients there is a temporal relationship between cough during or within 5 min of periods when esophageal pH is less than 4 despite normal reux time. Endoscopy may reveal the mucosal damage caused by GERD but has little role in the diagnostic evaluation of GERD-induced cough. Esophageal manometry has a limited role, but may be important as an adjunct to pH monitoring in selected groups of patients.

Asthma
Airway hyperresponsiveness is now the accepted underlying pathophysiology in asthma. The association between asthma and GERD is derived from multiple studies showing a high prevalence of acid reux in asthmatics, as well as the observed improvement in some asthma patients with treatment of acid reux. As many as 80% of adult asthmatics have abnormal acid reux. Compared to normal controls asthma patients have decreased lower esophageal sphincter pressures, more frequent reux episodes, and take longer to clear the esophagus of acid. A large study of veterans found a higher likelihood of asthma in patients with esophagitis and stricture, and a more recent study found asthmatics to have significantly more frequent and more severe GERD symptoms

than nonasthmatics. More evidence for the relationship between asthma and GERD was noted in a large prospective study of asthmatics, which not only found heartburn to be common in asthmatics, but also found that heartburn was independently associated with future hospitalizations in asthmatics. A population-based study in three European countries found that subjects with nocturnal GERD were more likely to be diagnosed with asthma and have evidence of airway reactivity as measured by peak expiratory ow rate. The mechanism by which GERD causes respiratory symptoms is unknown, but esophageal acidinduced airway hyperresponsiveness is the most accepted theory. This is thought to occur via microaspiration of reuxate, esophageal triggered vagal reexes, and esophageal triggered necroinammation. The interplay between these mechanisms is thought to result in airway hyperreactivity (Figure 2). Microaspiration may lead to airway inammation through damage of epithelial cells, which release inammatory cytokines, and this leads to increased vagal tone. Vagally mediated airway reexes due to acid reux have been demonstrated in many studies including one in which 20 patients with asthma and GERD were compared to nonasthmatics with GERD using acid infusion. The peak expiratory ow decreased in the asthma group with acid challenge compared to the nonasthmatic group and it was found that administration of vagolytic doses of atropine ablated the response to acid infusion. Increased airway hyperresponsiveness, as measured

228 GASTROESOPHAGEAL REFLUX

Esophagus Axon reflex CNS Tracheobronchial tree Neurokinins Airways

Axon reflex

Inflammation

Reflux

Microaspiration NGF

Edema

Mucus Vagal afferents CNS Vagal afferents Vagal efferents NGF Bronchial hyperreactivity Smooth muscle Mediators

Figure 2 Both esophageal afferents and airway afferents lead to central nervous system (CNS) stimulation with resultant increases in vagal efferent impulses. This leads to increased hyperresponsiveness. NGF, nerve growth factor. Reproduced from Harding S (1997) Ambulatory Esophageal pH Monitoring: Practical Approach and Clinical Applications, pp. 149164. Baltimore: Williams & Wilkins, with permission.

by maximal expiratory ow at 50% vital capacity, is seen with infusion of 0.1 N HCl in the esophagus. This response can be blocked by pretreatment with atropine, further implicating the vagal link. Medications used to treat asthma may also exacerbate reux and produce further symptoms. Inhaled beta2 agonists were shown to cause a dose-dependent decrease in LES pressures as well as a dose-dependent decrease in esophageal body amplitudes. A study on the effects of oral steroids on reux in asthma subjects showed significant increase in esophageal acid contact time at the proximal and distal pH probes. There was no significant increase in asthma or GERD symptoms, or basal stimulated gastric acid secretion. These results were limited by the small sample size.
Diagnosis of GERD-Related Asthma

is similar to chronic cough in that most experts advocate a trial of PPI and pursue further diagnostic testing only if this fails.

Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary brosis (IPF) is a severe form of interstitial lung disease of unknown etiology that has been associated with GERD. The evidence for this association comes from observational and epidemiologic studies as well as from animal models. The histology of this disease is characterized by interstitial brosis, inammation, and honeycomb changes interspersed by normal lung epithelium. Various animal studies dating back to the 1950s demonstrated similar changes in the lung epithelium when exposed to 0.1 N hydrochloric acid as well as gastric acid. A case-control study using patients with esophagitis and strictures were compared to controls for the occurrence of laryngeal, pharyngeal, sinus, and pulmonary diseases. Patients with erosive esophagitis were more likely to have various pulmonary diseases, including pulmonary brosis with odds ratio 1.36 for IPF. Further association of pulmonary brosis with GERD was demonstrated in a study that showed that the incidence of GERD was significantly higher in patients with conrmed

The diagnosis of GERD-related asthma relies mainly on the clinical presentation. As in chronic cough asthmatics can have GERD in the absence of typical symptoms. This so-called clinically silent GERD was demonstrated in a study in which 62% of asthmatics had acid reux on pH study without clinical symptoms. Many patients come to attention after usual therapies for asthma have failed. The workup

GASTROESOPHAGEAL REFLUX

229

pulmonary brosis than in controls. Sixteen of 17 IPF subjects had abnormal acid exposure compared to 4 of 8 controls. Twenty-ve per cent of the patients with abnormal pH results had no symptoms of acid reux. Thus far, the data linking GERD to IPF is compelling, but more studies are needed. Large-scale randomized trials looking specifically at GERD and IPF as well as prospective therapeutic trials would shed more light on this fatal disease.

for successful treatment, as simple normalization of acid control may not be sufcient. Complete elimination of esophageal acid exposure may be needed in patients with extraesophageal disease for adequate symptom control.
Suggested Approach to Treatment of Pulmonary Symptoms due to GERD

Treatment
There have been few clinical trials of treatment involving patients with extraesophageal manifestations of GERD, specifically asthma, cough, and voice changes. The majority are uncontrolled and do not address long-term maintenance. Treatment is based on the principles advocated for treating patients with heartburn and erosive esophagitis, observations from available clinical trials, and clinical experience. It appears in general that patients with extraesophageal manifestations of GERD must be treated with higher doses of pharmacologic therapy, principally with PPIs (twice daily), and with longer periods of treatment (up to 34 months) to achieve complete relief of symptoms as compared to patients with heartburn and erosive esophagitis (usually 8 weeks). Important insights into treatment of patients with extraesophageal GERD can be gleaned from one well-designed study in which 30 patients with documented asthma and GERD proven by prolonged pH monitoring were treated with increasing doses of omeprazole. Starting at 20 mg daily, the medication was increased by 20 mg after each 4-week treatment period for 3 months or until esophageal acid exposure was reduced to normal. Normalization of esophageal acid exposure resulted in improvement in pulmonary symptoms in 70% of patients. Several observations emerge from this trial: 8/30 (28%) of patients needed greater than 20 mg of omeprazole a day to normalize esophageal acid exposure; and many patients required the entire 3-month period of treatment to achieve optimal symptom relief with improvement progressing continuously over the 3-month period, conrming the observations of others. A favorable response to omeprazole was seen in patients who presented with frequent regurgitation (greater than once a week) and those with abnormal proximal acid exposure demonstrated by ambulatory pH monitoring. This study emphasizes the importance of adequate esophageal acid control to achieve improvement in patients with extraesophageal symptoms. Further studies are needed to determine the optimal reduction in esophageal exposure required

It is our current practice to begin therapy with PPIs twice daily, before breakfast and dinner for 23 months (Figure 3). Clinical experience suggests patients with GERD will respond to this length of therapy, though many will require longer treatment periods to achieve optimal results. Patients who have a good initial but incomplete response to this therapeutic trial are to be continued at the same dose for an additional 48-week period to assess continued improvement. If satisfactory symptom relief is not achieved at this point, we evaluate the patient with prolonged reux pH monitoring studies while on continued therapy with PPI to assess the adequacy of intragastric acid suppression and elimination of distal and proximal esophageal acid exposure. If acid suppression is incomplete, additional medical therapy is indicated prior to assuming that the patient is a medical failure. If the patient has a poor response to the initial therapeutic trial, prolonged ambulatory pH monitoring should be performed while on therapy to evaluate drug efcacy.

Possible GERD symptoms

Trial of PPI Rx

Success (confirm dx)

Persistent symptoms

Ambulatory MII-pH monitoring on Rx

Acid GERD with symptoms

No GERD

Nonacid GERD with symptoms

Figure 3 GERD diagnostic algorithm. PPI Rx, proton pump inhibitor reux.

230 GASTROESOPHAGEAL REFLUX

Recent studies from our laboratory have shown that 70% of patients with GERD treated with twicedaily PPI therapy will continue to have gastric acid breakthrough (pH o 4) for at least 1 h in the overnight period. Reux will occur in 3050% of these patients during this breakthrough period. Another study from our laboratory found that 30% of patients with extraesophageal reux (asthma, cough, LPR) continue to have abnormal acid exposure (GER) despite therapy twice daily with a PPI. These patients require more aggressive medical therapy, as even small amounts of acid exposure may be injurious to the mucosa of the larynx and airways. Doubling the dose of PPI (e.g., omeprazole 40 mg b.i.d.) may be sufcient to control esophageal acid exposure effectively. However, some patients will have continued nocturnal gastric acid breakthrough and esophageal reux despite high-dose PPI and may require the addition of an H2-receptor antagonist to achieve optimal acid suppression. Referral for ambulatory pH monitoring to document the effectiveness of therapy and assessment of the presence of continued acid exposure or nonacid reux is indicated if patients are refractory to PPIs. We do not routinely recommend adding a prokinetic agent to PPIs. We reserve prokinetic agents for patients in who adequate acid exposure cannot be accomplished with acid suppressive agents, and for the patient with documented delayed gastric emptying. Clinical experience suggests that most patients with extraesophageal GERD will have chronic GERD and will require long-term medical therapy and/or consideration of antireux surgery for longterm control. The dose of PPI and other medications, and the decision to perform antireux surgery should be individualized to maintain symptom relief and mucosal healing. Current evidence suggests that long-term medical therapy is safe and tolerance or tachyphylaxis extremely rare. Patients who choose long-term medical therapy can be condent of excellent long-term control of acid-induced mucosal injury without the worry of serious complications.
See also: Asthma: Overview; Extrinsic/Intrinsic. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Laryngitis and Pharyngitis. Pulmonary Fibrosis. Upper Respiratory Tract Infection.

Further Reading
Avidan B, Sonnenberg A, Schnell TG, and Sontag SJ (2001) Temporal associations between coughing or wheezing and reux in asthmatics. Gut 49(6): 767772.

Canning BJ and Mazzone SB (2003) Reex mechanisms in gastroesophageal reux disease and asthma. American Journal of Medicine 115(3A): 45S48S. Diette GB, Krishnan JA, et al. (2002) Asthma in older adults, factors associated with hospitalization. Archives of Internal Medicine 162: 11231132. El-Serag HB and Sonnenberg A (1997) Comorbid occurrence of laryngeal or pulmonary disease with esophagitis in the United States Military Veterans. Gastroenterology 113: 755760. Field and Stephen K (1999) A critical review of the studies of the effects of stimulated or real gastroesophageal reux on pulmonary function in asthmatic adults. Chest 115: 848856. Fontana GA and Pistolesi (2003) Cough 3: chronic cough and gastroesophageal reux. Thorax 58(12): 10921095. Gislason T, Jansen C, Vermeire P, et al. (2002) Respiratory symptoms and nocturnal gastroesophageal reux: a population-based study of young adults in three European countries. Chest 121: 158163. Harding S (1997) Ambulatory Esophageal pH monitoring: Practical Approach and Clinical Applications, pp. 149164. Baltimore: Williams & Wilkins. Harding SM, et al. (1995) GERD induced bronchospasm. American Journal of Respiratory and Critical Care Medicine 151: A589. Harding SM, Richter JE, et al. (1996) Asthma and gastroesophageal reux: acid suppression therapy improves asthma outcome. American Journal of Medicine 100: 395405. Harding SM and Richter JE (1997) The role of gastroesophageal reux in chronic cough and asthma. Chest 111: 13891402. Harding and Susan M (2003) Recent clinical investigations examining the association of asthma and gastroesophageal reux. American Journal of Medicine 115(3A): 39S44S. Irwin RS, Azwacki JK, Curley FJ, French CL, and Hoffman PJ (1989) Chronic cough as the sole presenting manifestation of gastroesophageal reux. American Review of Respiratory Diseases 140: 12941300. Irwin RS, Curley FJ, and French CL (1990) Chronic cough: the spectrum and frequency of causes, key components of the diagnostic evaluation, and outcome of specic therapy. American Review of Respiratory Diseases 141: 640647. Irwin RS and Richter JE (2000) Gastroesophageal reux and chronic cough. American Journal of Gastroenterology 95(8): S9S14. Kastelik JA, Redington AE, Aziz I, et al. (2003) Abnormal esophageal motility in patients with chronic cough. Thorax 58(8): 699702. Katz PO, Anderson C, Khoury R, and Castell DO (1998) Gastrooesophageal reux associated with nocturnal gastric acid breakthrough on proton pump inhibitors. Alimentary Pharmacology and Therapeutics 12: 12311234. Khoury R, Mohiuddin M, Katz P, et al. (1998) Inuence of body position on nighttime recumbent reux in patients with GERD. Gastroenterology 114(4): A174. Kiljander TO (2003) The role of proton pump inhibitors in the management of gastroesophageal reux disease-related chronic cough. American Journal of Medicine 115(3A): 65S71S. Morice AH and Kastelik JA (2003) Cough: chronic cough in adults. Thorax 58(10): 901907. Peghini PL, Katz PO, Bracy NA, and Castell DO (1998) Nocturnal recovery of gastric acid secretion with twice-daily dosing of proton pump inhibitors. American Journal of Gastroenterology 93: 763767. Schnatz PF, Castell JA, and Castell DO (1996) Pulmonary symptoms associated with gastroesophageal reux: use of ambulatory pH monitoring to diagnose and to direct therapy. American Journal of Gastroenterology 91(9): 17151718.

GENE REGULATION 231

Stein M (2003) Symposium: possible mechanisms of inuence of esophageal acid on airway hyperresponsiveness. American Journal of Medicine 115(3A): 55S59S. Zaid Y and Johnson DA (1999) Diagnostic evaluation in gastroesophageal reux disease. Gastroenterology Clinics of North America 28(4): 809827.

GENE REGULATION
M A Perrella, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

expression of genes can be regulated, with emphasis at the level of gene transcription and mRNA processing. Figure 1 provides an overview of the gene regulation process.

Abstract
The genome of an organism contains the complete set of genetic information passed on from generation to generation. The gene is a subunit of this genetic information, consisting of a sequence of nucleic acids. These nucleic acids provide a code, which in many circumstances lead to the production of functional proteins. The inheritable information contained in our genes determines the development of a person or organism, and their eventual appearance, functional status, and even their susceptibility to contract diseases. Variations in gene content or alterations in gene expression may increase a persons risk for disease development. Through the study of gene sequences as well as their regulation, medical science has the potential to make major breakthroughs in the management (detection, prevention, and therapy) of various disease processes. The present article provides an overview of how gene expression can be regulated: from gene transcription in the nucleus, to various mRNA processing events, to export and decay of mRNA in the cytoplasm.

Transcriptional Regulation
A gene is a subunit of genetic information consisting of a sequence of nucleic acids. These nucleic acids provide a code, which in many circumstances leads to the production of functional proteins. If the protein binds in a sequence-specic manner to DNA, it is referred to as a trans-acting factor. The segment of genomic DNA, typically 612 bp in length, that acts as a binding site for trans-acting factors is called a cis-acting element. The interaction of trans-acting factors and their cognate cis-acting elements contribute to gene regulation, and is discussed in detail below. Sequence-specic trans-acting factors are often composed of functional modules. For example, a DNA-binding domain may be linked to one or more activation or repression modules, as well as modules for dimerization or regulation. These factors are frequently classied according to the type of DNAbinding domain, usually a short motif in this domain responsible for DNA binding. Examples of these DNA-binding domains include zinc nger, steroid receptors, helixturnhelix, helixloophelix, and leucine zippers. For further details regarding transcription factors and their cognate DNA-binding sites, please refer to Transcription Factors: Overview or the articles on specic transcription factors of interest. Gene regulation by transcription involves the synthesis of RNA, which is catalyzed by RNA polymerase (type II in higher eukaryotes). Genes that contain RNA polymerase II promoters have a short conserved sequence, termed the initiator (Inr) at the start point of transcription. Most of these RNA polymerase II promoters contain a sequence approximately 25 bp upstream of the transcription start point called the TATA box. The TATA box sequence consists entirely of A . T nucleotides, and it tends to be surrounded by G . C rich sequences. The TATA box (one of the rst cis-acting elements to be identied) is

Introduction
Genes are the genetic material that helps to determine how a person or organism develops, their appearance and functional status, and what diseases they may eventually contract. The complete set of genes within the body of a person is known as the genome. A project was initiated in 1990 to sequence the entire human genome and provide a roadmap of the genetic material that makes up our bodies. The information provided by the genome project is helping to give scientists and physicians a better understanding of how diseases occur, and is being used to develop medications that specifically target the cause of a disease, not just the symptoms. Diseases may be caused by variations in gene content or by alterations in gene expression, which increases a persons risk for disease development. Through the study of gene sequences and their regulation, medical science has the potential to make major breakthroughs in the pathophysiology, early detection, treatment, and prevention of distinct disease processes. The present article focuses on the mechanisms by which the

232 GENE REGULATION

Enhancer Transcription factors (trans -acting factors) Promoter Exons/introns

General transcription apparatus Pol ll TBP TATAA Exon 1 Intron 1 Exon 2

Regulatory sequences (cis -acting elements)

Transcription Pre-mRNA Exon 1 Intron 1 Exon 2

RNA processing mRNA

MeGppp

Exon 1

Exon 2

AAAA 3 poly(A) tail

5 cap Transport into cytoplasm Translation

Figure 1 Overview of gene regulation. In conjunction with the general transcription apparatus, trans-acting factors bind to their cognate regulatory sequences (cis-acting elements) in the 50 -anking sequence of a gene to initiate and drive transcription. The general transcription apparatus contains RNA polymerase II (Pol II) and TATA-binding protein (TBP) and associated factors that form a transcriptional complex at the TATA box (TATAA), and subsequently initiates transcription (bent arrow). Trans-acting factors bind to cisacting elements contained in promoter and enhancer regions of the gene. These two regions of the 50 -anking sequence of a gene can be separated by several kilobases. The mRNA that is transcribed undergoes a variety of processing events, including the splicing out of introns and joining of exons to generate a smaller mRNA that contains the intact coding sequence, the addition of a 50 methylated cap (MeGppp) and a poly(A) tail (AAAA), and the export of the mature mRNA into the cytoplasm for translation of the functional protein.

considered to be involved in the positioning of eukaryotic RNA polymerase II (a sequence-specic DNA binding transcription factor or trans-acting factor) for correct initiation. RNA polymerase II cannot initiate transcription by itself, but is dependent on auxiliary transcription factors. For RNA polymerase II, binding of TFIID to a region that extends upstream of the TATA box is the rst step in initiation. TFIID consists of the TATA-binding protein (TBP) and TAFs (TBP-associated factors). Binding of TBP to the TATA box is the rst step in initiation, followed by TAFs binding to the initiation complex in a dened order. When RNA polymerase II binds to this complex, transcription is initiated. The polymerase plus the auxiliary factors constitutes the general or basal transcription apparatus. Promoters that do not contain a TATA box are called TATA-less promoters. In TATA-less promoters, the same general apparatus, including TFIID, are needed. However, in these promoters TFIID binds to the Inr via one or more of the TAFs (and not TBP) directly recognizing the Inr element. In promoters that contain a TATA box, transcriptional initiation typically occurs at a dened start point. The TATA box aligns the RNA polymerase by its interaction

with TFIID allowing initiation to occur at the proper site. However, in the case of TATA-less promoters, they often lack a unique start point, with transcriptional initiation occurring at one of a number of potential start points. It is also of interest that in TATA-less promoters, binding sites for a subset of sequence-specic activators, for instance Sp1, are commonly located in the proximal promoter region (within the rst 250 bp upstream from the transcription start site). These activators, in conjunction with their cognate DNA binding sites, interact with the basal transcriptional machinery to efciently regulate gene transcription in TATA-less promoters. The largest subunit of RNA polymerase II contains a C-terminal domain (CTD) that consists of multiple repeats of a 7 amino acid sequence. The CTD is unique to RNA polymerase II, and CTD can be highly phosphorylated on serine and threonine residues. This phosphorylation process is involved with the transcriptional initiation process. Once initiation has taken place, RNA polymerase II moves along the DNA template, synthesizing RNA until it reaches a termination sequence. The production of eukaryotic mRNA involves additional processing after transcription is complete, allowing production of mature

GENE REGULATION 233

mRNA. Further details regarding these procedures will be described in a later section on mRNA processing. In conjunction with RNA polymerase II, expression of eukaryotic genes is tightly regulated by additional trans-acting factors interacting with their cognate cis-acting elements. These elements are contained in promoter or enhancer regions of the gene. The promoters are typically located immediately upstream of the transcription initiation site, while enhancers may act at long distances, either upstream or downstream, of the RNA start site. The enhancer region(s) may be separated by several kilobases from the promoter. While enhancers are made up of the same type of cis-acting elements, the density of these elements is greater in the enhancer than in the promoter. This gives an enhancer the ability to form a regulatory complex. When a group of trans-acting factors assembles on an enhancer region to regulate gene transcription, this complex is referred to as an enhanceosome. Together, sequence-specic transcription factors and their associated DNA binding sites play an important role in determining the regulation of gene expression.

Chromatin
Beyond the interaction of trans-acting factors with cis-acting elements, the chromosomal environment also plays an essential role in the transcriptional regulation of gene expression. In the cell, DNA is packaged into chromatin by histone and nonhistone proteins. Moreover, whether a gene is expressed depends on the structure of chromatin. Thus, the control of chromatin structure has a great inuence on the regulation of gene expression. Changes in chromatin structure can occur in a very localized way (affecting a promoter of a specic gene), or these changes may affect large regions of a chromosome. Changes that affect large regions of a gene control the potential of that gene to be expressed. For instance, by affecting a large region of a gene the expression of the gene may be silenced, by preventing transcription factors and RNA polymerase from binding to DNA and activating gene transcription. On the other hand, changes in chromatin structure can also occur at an individual promoter, controlling whether the expression of a particular gene is activated or repressed.

histones have an effect on chromatin structure and the initiation of gene transcription. One such modication, acetylation, has been shown to be associated with gene activation. On the other hand, some repressors of transcription function by deacetylating histones. Thus, the acetylation process is reversible, with each modication being catalyzed by a specic enzyme. Enzymes that support the acetylation of histones are referred to as histone acetyltransferases (HATs), while enzymes that subsequently remove the acetyl groups are histone deacetylases (HDACs). Another modication of DNA that is associated with altering gene expression is methylation. Methylation of both histones and DNA, by methyltransferase enzymes, is a feature of inactive chromatin. If methylation of DNA occurs in the vicinity of a promoter, then transcription cannot proceed. Most of the methylation sites in DNA are CpG islands. The methylation of these CpG islands can regulate transcription by either preventing a transcription factor from binding to DNA, or by causing specic repressors to bind DNA. Thus, by regulating methyltransferase enzyme activity, the methylation status of DNA can be altered and gene expression regulated. Phosphorylation of histone H1 occurs during mitosis of the cell cycle, and it has been shown that histone H1 is a good substrate for Cdc2 kinase, which is a critical regulator of the cell cycle. Another process that has been shown to be associated with histone phosphorylation (H3) is chromatin remodeling. Loss of a kinase that phosphorylates histone H3 has devastating effects on chromatin structure. Taken together, modications of histones and DNA including acetylation, methylation, and phosphorylation have a dramatic effect on gene regulation.

Nonhistone Proteins or Architectural Transcription Factors

As mentioned previously, a group of trans-acting factors that assembles on an enhancer region of a gene to regulate gene transcription is referred to as an enhanceosome. The combinatorial assembly, or disassembly, of an enhanceosome is critical to allowing tight control over the regulation of gene expression. The effective formation of an enhanceosome depends on appropriate DNA conformation to allow the binding of transcription factors, and thus the assembly of a functional complex. Architectural transcription factors are a group of proteins that lack the intrinsic ability to directly transactivate a gene, yet they have been shown to be important in the regulation of specic genes, in part, by controlling DNA conformation. These architectural transcription factors, which

Histone Proteins
Events leading to alterations in chromatin structure depend on interactions with histones. For example, recently it has been determined that modications of

234 GENE REGULATION

are nonhistone proteins, contribute to the regulation of chromatin structure and thus the regulation of gene expression. An example of a group of architectural transcription factors is the high-mobility group (HMG) proteins. HMG proteins can be separated into three different superfamily members: HMG-I/Y (HMGA), HMG1/2 (HMGB), and HMG-14/-17 (HMGN), each of which is characterized by a functional sequence motif. For example, HMG-I/Y proteins bind to AT-rich regions in the minor groove of DNA. Each HMG-I/Y protein has three separate DNA-binding motifs, which are referred to as AT-hooks, separated by exible linker peptides. A single AT-hook motif preferentially binds to a stretch of DNA containing 46 bp of AT-rich sequence. Binding of HMG-I/Y proteins to DNA can promote DNA structural changes including bending, straightening, unwinding, and loop formation. Depending on the target gene, these DNA conformational changes introduced by HMG-I/Y can either facilitate or prevent the assembly of enhanceosomes, and thus affect gene transcription. Beyond modifying the structure of DNA, architectural transcription factors such as HMG-I/Y are also known to recruit transcription factors into an enhanceosome complex. By binding in the minor groove of DNA, HMG-I/Y is able to recruit transcription factors to the major groove. Taken together, studies have demonstrated that HMG-I/Y is able to orchestrate the regulation of gene expression by forming an enhanceosome complex to efciently drive transcription by altering DNA conformation and recruiting transcription factors to DNA. Figure 1 depicts a simplied two-dimensional view of gene transcription, with one or a few transcription factors binding to regulatory elements of a gene, subsequently leading to the up- or downregulation of that gene. However, a much more complex process occurs when taking into account chromatin and the role of architectural transcription factors. In this model, a group of transcription factors assembles into a complex that regulates gene expression through proteinDNA and proteinprotein interactions. This complex of factors assembles on an enhancer region of a gene (enhanceosome), and then through DNA binding interacts with the promoter region to regulate gene transcription. Taking into account our studies and the work of others, we have devised a model of nitric oxide synthase (NOS)2 enhanceosome formation (Figure 2) that may form during lipopolysaccharide (LPS) and inammatory cytokine stimulation. NOS2 is a highly inducible gene that is regulated at the level of gene transcription. NOS2 plays an important role in vivo in disease processes such as endotoxemia and sepsis, where an

HMG-I/Y IRF RNA Pol II HMG-I/Y

p50/p65

HMG-I/Y TBP
TA TA A

DNA
Figure 2 Assembly of an enhanceosome complex by HMG-I/Y. A model of NOS2 enhanceosome formation by an inammatory stimulus is depicted. In this model, HMG-I/Y proteins bind DNA, recruit transcription factors to DNA (such as the p50 and p65 subunits of nuclear factor kappa B (NF-kB) and IRF family proteins), and facilitate interactions between these transcription factors and the transcriptional initiation complex that includes the TATA-binding protein (TBP) and associated factors and RNA polymerase II (RNA Pol II). Reproduced from Carvajal IM, Baron RM, and Perrella MA (2002) High mobility group-I/Y proteins: potential role in the pathophysiology of critical illness. Critical Care Medicine 30(supplement 1): S36S42, with permission from Lippincott Williams & Wilkins.

overproduction of nitric oxide contributes to disease pathophysiology. This model is meant to provide a theoretical image of how architectural factors (such as HMG-I/Y) can interact with transcription factors and DNA to orchestrate the assembly of an enhanceosome complex (through DNAprotein and proteinprotein interactions) that allows the initiation of transcription along with the general transcriptional machinery and RNA polymerase II. The above example demonstrates how HMG-I/Y is able to enhance the transcription of NOS2. However, HMG-I/Y is also able to suppress transcription in other genes. For example, HMG-I/Y is able to prevent induction of the interleukin-4 promoter by competing with the nuclear factor of activated T cells (NF-AT) proteins for DNA binding. Thus, nonhistone architectural transcription factors, such as HMG-I/Y, are able to play a significant role (either positive or negative) in the transcriptional regulation of gene expression.

mRNA Processing
For most genes, transcription is the major control point and the most common level of gene regulation. However, control points exist beyond transcriptional initiation and synthesis, including mRNA processing,

GENE REGULATION 235

export of mRNA from the nucleus to the cytoplasm, and mRNA degradation in the cytoplasm. This section focuses on mRNA processing and degradation. As mentioned above, the largest subunit of RNA polymerase II contains a CTD that is unique to RNA polymerase II. This CTD can be highly phosphorylated on serine and threonine residues. CTD is critical for transcriptional initiation; however, it may also be involved in processing RNA after its synthesis by RNA polymerase II is complete. For instance, guanylyl transferase binds to phosphorylated CTD and adds a G residue (in the reverse orientation to all other nucleotides) to the 50 end of the newly synthesized RNA. This structure is termed a cap. The 50 cap is a substrate for methylation events (13 methyl groups are added to the new terminal G). An additional set of proteins binds to CTD, and then binds with splicing factors, allowing a coordination of transcription and splicing. RNAs that are derived from interrupted genes require splicing to remove the introns and thus generating a smaller mRNA that contains the intact coding sequence. Thus, CTD appears to help coordinate transcription with additional RNA processing. Finally, following the synthesis of RNA, a stretch of approximately 200 bases of adenylic acid, termed poly(A), is added to the 30 end of mRNA. The 30 poly(A) tail, along with the 50 cap, stabilizes the mRNA against degradation (Figure 3). Once processing is complete, the mature RNA must then be exported from the nucleus to the cytoplasm, where translation of the mRNA into a protein takes place. Besides stability, the 30 poly(A) tail may also play a role in nuclear mRNA processing,

export of the mRNA into the cytoplasm, and translation events. While the regulation of gene expression may involve any or all of these stages of RNA processing, changes do not uncommonly occur in splicing (i.e., removal of introns and joining of exons in RNA). Most genes are transcribed into RNA that gives rise to a single type of mRNA, with no variation in exons and introns. However, in some genes, alternative splicing of RNA occurs giving rise to more than one isoform of mRNA. These alternative spliced messages may lead to the expression of different protein products. In some cases, multiple products of gene transcription are made in a cell; however, in other circumstances the splicing process is regulated so that certain transcripts are generated in specic cell types or under specic cellular conditions.

mRNA Stability
In many genes, the regulation of gene expression and subsequent protein production is achieved by controlling the mRNA levels. Thus, the level of cytoplasmic mRNA is a balance between nuclear events (rate of nuclear mRNA synthesis, modication, and export) and the rate of cytoplasmic mRNA degradation. Thus, mRNAs of genes with a short half-life respond more acutely to changes in transcription compared with genes having stable mRNAs with a long half-life. Different decay pathways have been identied that regulate mRNA degradation. Nevertheless, the process of regulated mRNA decay involves various structural components of the message including the 50 cap, the 50 -untranslated region (UTR), the coding region, the 30 -UTR, and the 30 poly(A) tail. Most mammalian mRNAs are polyadenylated, and the poly(A) tails of these genes are typically bound to a poly(A)binding protein (PABP). This complex of the poly(A) tail and PABP provides stability to the mRNA and prevents rapid degradation by exoribonucleases in a 30 -50 direction (Figure 3). Deadenylation of the poly(A) tail is the rst step in the degradation of many mammalian mRNAs. In deadenylation-dependent pathways, poly(A) shortening is often followed by removal of the 50 cap, thus also allowing 50 -30 degradation and a bidirectional decay of the message (Figure 3). mRNA decay may also occur in a deadenylation-independent manner. In this pathway, endonucleolytic cleavage of the message occurs, followed by exoribonuclease decay. The deadenylationdependent pathway appears to be the more prevalent pathway of mRNA degradation. The interaction of specic cis-acting structural elements and their associated trans-acting factors

MeGppp

5-UTR

Coding region

3-UTR PABP AUUUA AAAA Poly(A) + PABP protects against 3 5 exonuclease

5 cap protects against 5 3 exonuclease

Stable

3 5 5 3 Degrade Exonuclease Exonuclease AUF1 AUUUA 5-UTR Coding region 3-UTR

BP PA

pp

AA

Figure 3 mRNA degradation. The 30 poly(A) tail (AAAA) bound by poly(A)-binding protein (PABP), along with the 50 cap (MeGppp), stabilizes the mRNA and protects against exonuclease activity. However, binding of a trans-acting factor (e.g., AUF-1) to the A U-rich elements triggers destabilization by deadenylation and loss of PABP, and removal of the 50 cap. This allows both 30 -50 and 50 -30 exonuclease activity to degrade the mRNA.

M e

AA

236 GENE REGULATION

allow stabilizing or destabilizing inuences on mRNA. The biological activity of these interactions at the mRNA level relies on a combination of primary and secondary structural elements, assembled in a complex, and recognized by RNA-binding proteins. Protein binding to the 30 -UTR may thus be sequence specic (as in DNA-mediated regulatory signals) or promoted by structural stem-loops formed in the mRNA. Thus, 30 -UTR plays a major role in the control of mRNA decay. Well recognized are the A U-rich elements (ARE), containing AUUUA motifs (Figure 3). The ARE are potent destabilizing elements that play a critical role in many genes with short half-lives. As stated, these elements are located in the 30 -UTR, and several ARE-binding proteins have been described that either support mRNA decay (e.g., AUF-1 in several interleukins [IL-1,2,3,6], c-myc, and b1-adrenergic receptor; and tristetrapolin (TTP) in granulocyte-macrophage colony-simulating factor (GM-CSF), growth-related oncogene alpha (GRO-a), and tumor necrosis factor alpha (TNF-a) mRNAs) or mRNA stability (e.g., HuR in VEGF and ELAV proteins in interferon-a). Regulatory elements may also exist in the coding region (CRD-1 and CRD-2 in c-fos mRNA) and the 50 -UTR (JNK-response elements, which bind stability determinants nucleolin and YB-1 in IL-2 mRNA). While it is clear that the binding of trans-acting factors to cis-acting elements in mRNAs plays an important role in mRNA decay or stability, the details regarding the signals that this interaction provides to the degradation machinery is still being elucidated. Moreover, questions remain regarding the mechanisms of mRNA degradation in a gene-specic (individual gene) or function-specic (class of genes) manner.

gene silencing mechanism that is induced by processing of double-stranded RNA (dsRNA) by Dicer into short dsRNAs of approximately 2125 bp in length. These small dsRNAs are referred to as small interfering RNAs (siRNAs). Following unwinding of the siRNAs, one strand of the siRNA duplex is incorporated into an RNA-induced silencing complex (RISC). The RISC targets the cognate mRNA for degradation. Taken together, these small RNAs are capable of altering gene expression from the level of gene transcription, to mRNA stability, to translational events.

Future Directions
At all levels of investigation within an organism, scientists and physician scientists hope to have an impact on understanding the development and function of an organism, and to help determine how and why a disease process may occur. Through the study of gene sequences as well as their regulation, medical science has the potential to make major breakthroughs in the pathophysiology, early detection, treatment, and prevention of distinct disease processes. This overview aims to provide a basic understanding of the potential importance of studying gene regulation, and its potential impact on the biomedical sciences and discovery opportunities in the future.
See also: Transcription Factors: Overview; AP-1; ATF; Fox; NF-kB and Ikb; POU; PU.1.

Further Reading
Bevilacqua A, Ceriani MC, Capaccioli S, and Nicolin A (2003) Post-transcriptional regulation of gene expression by degradation of messenger RNAs. Journal of Cellular Physiology 195: 356372. Carvajal IM, Baron RM, and Perrella MA (2002) High mobility group-I/Y proteins: potential role in the pathophysiology of critical illnesses. Critical Care Medicine 30(supplement 1): S36S42. Guhaniyogi J and Brewer G (2001) Regulation of mRNA stability in mammalian cells. Gene 265: 1123. He L and Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nature Reviews: Genetics 5: 522531. Kadonaga JT (2004) Regulation of RNA polymerase II transcription by sequence-specic DNA binding factors. Cell 116: 247257. Lemon B and Tjian R (2000) Orchestrated response: a symphony of transcription factors for gene control. Genes and Development 14: 25512569. Lewin B (2004) Transcription. In: Challice J (ed.) Genes VIII, pp. 241278. Upper Saddle River, NJ: Pearson Prentice Hall. Matzke M, Aufsatz W, Kanno T, et al. (2004) Genetic analysis of RNA-mediated transcriptional gene silencing. Biochimica et Biophysica Acta 1677: 129141.

RNA-Mediated Gene Silencing

Recently, a new avenue of gene regulation has developed, using regulatory RNAs to induce gene silencing. Gene silencing by nucleotide sequence-specic interactions mediated by RNA can function in at least three ways. First, RNAs can recognize DNA and produce alterations in the genome, such as DNA methylation and histone modications, to target specic regions of the gene and modify chromatin structure altering gene expression at the transcriptional level. Second, micro (mi)RNAs bind to the complementary 30 -UTRs and act as translational repressors; miRNAs are generally 2125 bp noncoding RNAs that are cleaved from precursors (by RNase III activity termed Dicer) and form stemloop structures. Third, the most recognized form of RNA gene silencing is RNA interference (RNAi). RNAi is a sequence-specic

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Reeves R and Beckerbauer L (2001) HMGI/Y proteins: exible regulators of transcription and chromatin structure. Biochimica et Biophysica Acta 1519: 1329. Smale ST (1997) Transcription initiation from TATA-less promoters within eukaryotic protein-coding genes. Biochimica et Biophysica Acta 1351: 7388.

GENETICS
Contents

Overview Gene Association Studies

Overview
E K Silverman, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The major techniques used in respiratory genetic epidemiology include familial aggregation studies, linkage analysis, and association studies. These approaches are being used to localize genetic susceptibility determinants for many respiratory diseases.

Introduction
Genetic determinants likely have some degree of inuence on every major respiratory disease. In some cases, such as cystic brosis, the importance of genetic factors is obvious; however, even diseases with a major environmental basis, such as respiratory infections, likely are inuenced by genetic susceptibility as well. Traditionally, genetic disorders have been classied as monogenic or complex. Monogenic disorders are strongly inuenced by a single major gene. These typically rare conditions include cystic brosis and a1-antitrypsin deciency. Complex disorders are likely inuenced by multiple genetic and environmental inuences, as well as gene-by-gene and geneby-environment interactions. However, diseases which are classied as complex may be inuenced by major genes in monogenic subtypes, such as Netherton disease and atopy, or cutis laxa and emphysema. In addition, monogenic disorders may have variable expression that is determined by environmental factors and genetic modiers. Thus, even classical monogenic disorders may have features of a complex disorder, and complex disorders may have monogenic subtypes. The identication of genetic determinants typically involves demonstration of a phenotype/genotype correlation. A phenotype is an observable trait in an

individual, which may be the presence/absence of disease or a quantitative disease-related characteristic. Quantitative disease-related phenotypes are often known as intermediate phenotypes, because they can be viewed as capturing intermediate pathways between a genetic determinant and a disease. Intermediate phenotypes are likely often more powerful for susceptibility gene identication than the assessment of the presence or absence of a disease, which is subject to diagnostic bias. For example, serum IgE levels and airway responsiveness are intermediate phenotypes that have been widely studied in asthma genetics. Although intermediate phenotypes can be quite useful, the genetic determinants of an intermediate phenotype may not overlap completely with the genetic determinants of the disease of interest. Thus, demonstrating a relationship of purported susceptibility genes identied from studies of intermediate phenotypes to disease status is still important. The two general approaches to susceptibility-gene identication are candidate-gene approaches and positional cloning. Candidate-gene approaches assess the relationship between variants in a gene hypothesized to be important in the disease of interest, based on the current concepts of disease pathophysiology (or based on other evidence, such as geneexpression differences). Positional cloning involves a more systematic search through the genome without a preconceived opinion about the location of a novel susceptibility gene; such genome-wide approaches using linkage analysis and association studies will be discussed below.

Familial Aggregation
Several genetic epidemiological methods, which are generally referred to as familial aggregation studies, linkage analysis, and association studies, are used to determine whether genetic factors inuence a phenotype. Familial aggregation studies determine

238 GENETICS / Overview

whether a phenotype tends to be more similar in relatives compared to unrelated individuals. Familial aggregation can be caused by genetic or environmental factors. For instance, nuclear family members typically share a household environment, and diseases that would be inuenced by that household environment (e.g., lung cancer and cigarette smoke exposure) will aggregate in families even without genetic inuences. On the other hand, genetic factors are unlikely to be involved if familial aggregation is not present. Familial aggregation can be demonstrated in several ways. Comparison of disease prevalence in relatives of affected individuals to control subjects can be used to assess the statistical significance and magnitude of the risk to relatives. Family studies can be used to assess heritability, the estimated fraction of phenotypic variation that is due to genetic causes. Twin studies, which compare concordance between monozygotic (identical) and dizygotic (fraternal) twins, can also be used to assess the heritability of a phenotype. Higher concordance in monozygotic than dizygotic twins suggests genetic inuences, but the potentially more similar environmental exposures in monozygotic than dizygotic twins should also be considered.

Assessment of Genetic Variation

The human genome includes approximately 3 billion nucleotide bases on 22 pairs of autosomal chromosomes and two sex chromosomes. There are approximately 30 000 genes in the human genome; much of the human genome sequence is intergenic and of uncertain function and significance. There are approximately 10 million locations in the 3 billion-letter genetic alphabet at which the genetic sequence commonly varies between people. When there are at least two alternate genetic variants (referred to as alleles) at a location in the genome that each occur with allele frequencies above 1%, that locus is said to be a polymorphic locus, or polymorphism. If there is a genetic variant with allele frequency below 1%, it is often referred to as a mutation, although this 1% threshold is somewhat arbitrary. Genetic variation determines important inherited differences between people, but also includes nonfunctional, evolutionarily neutral changes. Most of the approximately 10 million single nucleotide polymorphisms (SNPs) in the genome probably have no functional effects. Functional genetic variants include nonsynonymous SNPs that change the amino acid sequence of the corresponding protein, but regulatory variation is possibly even more critical for complex diseases. Although more difcult to prove a functional effect,

regulatory variation can affect the magnitude, timing, and location of gene expression. The mechanisms of regulatory variation are still being determined, but important regulatory variants could be present in multiple locations, including the promoter region, the introns, the 30 untranslated region, or in enhancers that are located far upstream or downstream from a gene. Identication of evolutionarily conserved sequences across multiple species appears to be a promising approach to identify sites of potential regulatory genetic variation. There are several common types of genetic variation, including SNPs, short tandem repeat polymorphisms (STRs), and insertion-deletion variants (indels). SNPs occur approximately once every 300 nucleotide bases, but the occurrence of SNPs varies widely throughout the genome; some genes have many hundreds of SNPs while others have very few. Most genetic determinants of respiratory diseases (coding or regulatory) are likely to be SNPs, which represent the most common type of genetic variation. The optimal genotyping approach for SNPs has not been determined; many different methods are available, and most of them rely on the polymerase chain reaction (PCR). Historically, differences in recognition patterns for cleavage of DNA by microbial restriction enzymes were used to assess for the presence of an SNP; this approach is laborious and requires identication of a restriction enzyme recognition sequence at a specic locus of interest. Commonly used approaches for SNP genotyping today include hybridization (e.g., TaqMan), minisequencing (e.g., Sequenom), and oligonucleotide ligation reactions (combined with minisequencing in genotyping platforms such as Illumina). STR polymorphisms are less frequent than SNPs in the genome; they include 2, 3, or 4 bp repeat sequences that have a high mutation rate. The number of repeats at an STR polymorphism often varies between individuals, so they are often informative in linkage studies that attempt to track specic transmitted chromosomal regions through families. Genotyping of STR markers is reasonably standardized; uorescent labels are attached to PCR primers that anneal to nonrepetitive unique DNA sequences surrounding the repeat sequence, and the length of the repeat is assessed after PCR. Although previously genotyped using polyacrylamide gel-based approaches, capillary electrophoresis has increased the throughput and reliability of STR genotyping. Indels are also reasonably common; they typically include short insertions or deletions of 1 to 4 nucleotide bases in length. Genotyping of indels can be performed by assessing the length of the sequence, analogous to STR genotyping.

GENETICS / Overview 239

Genetic variants can be analyzed individually, or as a set of adjacent variants on a particular chromosome known as a haplotype. Haplotypes can be determined from extended pedigree information or by intensive molecular characterization; quite often, statistical methods are used to impute the likely haplotypes for genetic association analysis.

Linkage Analysis
Linkage analysis is a commonly used genetic epidemiological approach to identify the general location of a genetic determinant of a phenotype of interest. Genetic variants (traditionally short tandem repeat markers, but more recently SNPs as well) are genotyped within families, and linkage to a hypothetical disease gene locus is assessed. If two genetic loci are located on different chromosomes (or far apart on one chromosome), the transmission of the variants at those loci will be independent and will follow Mendelian segregation. On the other hand, if those two loci are located close enough together such that recombination is reduced below the Mendelian expectation of 0.5 (50%), then they are linked. The null hypothesis that is being tested in linkage analysis is that the recombination fraction between two genetic loci is equal to 0.5; in effect, there is random genetic assortment of these loci. The statistical certainty of rejecting this null hypothesis of no linkage can be assessed by a p value or by a Lod score. A Lod score is the logarithm of the odds for linkage. Lod scores above 3 generally correspond to significant linkage, although more stringent thresholds have been suggested, varying with the type of families that are included. Genetic loci that are separate by relatively large physical distances can still show evidence for linkage; this is both a strength and a weakness of this approach (Table 1). The strength lies in the fact that

only a modest number of genetic variants need to be genotyped to perform a genome scan for linkage, typically on the order of 400 STR markers. It is a weakness in that the physical location of the susceptibility locus could reside within a large number of nucleotide base pairs. With a classical monogenic disorder, specic recombination events can be identied and additional families can be included to narrow the physical region of interest. In complex traits, where recombination events are not determined with certainty due to potential complicating factors such as environmental phenocopies (subjects with the disorder of interest due to purely environmental rather than genetic causes) and genetic heterogeneity (multiple genetic mechanisms for a disorder), localization only to broad genomic regions (e.g., 20 million base pairs) is typical. Linkage analysis can be performed with modelbased approaches, which require assumptions regarding a variety of disease allele parameters, such as allele frequency and penetrance. Although these model-based or parametric approaches have been extremely successful for monogenic disorders, they have been less successful in complex disorders, at least in part, because the model-based parameters for complex disorders are often completely unknown. Model-free or nonparametric approaches have been more widely used in complex disorders; these methods typically assess allele-sharing between affected relatives. In addition to presence/absence of phenotypes, linkage to quantitative phenotypes can also be performed, for example, using variance component approaches.

Genetic Association Analysis

Genetic association studies assess whether a specic genetic variant (an allele) tracks with a disease, that is, whether there is significant evidence for linkage

Table 1 Comparison of linkage and association analysis Issue Subjects included Genetic markers for a genome scan Driving property of statistical test Resolution for localization of a susceptibility gene for a complex disorder Utility in monogenic disorders Utility in complex disorders Linkage analysis Families (nuclear families or extended pedigrees) 400 STRs or 150010 000 SNPs Meiotic recombination in families Approx. 20 000 000 bases Association analysis Cases/controls or families (often parent/child trios) 100 000 to 1 000 000 SNPs Linkage disequilibrium in populations Approx. 200 000 bases

Very high Useful, but replication has been difcult

Potentially useful; typically performed after linkage studies Useful, but replication has been difcult

240 GENETICS / Gene Association Studies

disequilibrium. As discussed in the article Genetics: Gene Association Studies, both case-control and family-based approaches can be used to assess for the presence of linkage disequilibrium between a measured genetic variant and a hypothetical disease locus. Linkage disequilibrium measures the tendency of alleles at nearby genetic loci to track together. As opposed to linkage analysis, which determines whether a particular location in the genome is shared more often than expected by chance among relatives with a particular phenotype, association analysis determines whether a specic allele tracks with disease more often than expected by chance in a population of individuals. Linkage disequilibrium is variable across the genome, but it is uncommon for signicant linkage disequilibrium to extend beyond 200 kilobases. Thus, the resolution of genetic association studies is typically ner than genetic linkage studies. Although it is difcult to select the right genetic variant to test for association, if a valid association is uncovered, the functional variant is likely nearby if such an association can be found. Although still quite expensive, genome-wide association studies are becoming feasible. These studies will require genotyping hundreds of thousands of SNPs to provide adequate genomic coverage (fewer in Caucasian and Asian populations, and more in African populations due to the differing extents of average linkage disequilibrium in those populations). Such largescale association studies have the potential to identify many genetic determinants of complex respiratory diseases.
See also: Cystic Fibrosis: Overview. DNA: Structure and Function. Gene Regulation. Genetics: Gene Association Studies. Neurobromatosis. Proteinase Inhibitors: Alpha-2 Antiplasmin. Pulmonary Function Testing in Infants. Systemic Disease: Sarcoidosis; Langerhans Cell Histiocytosis (Histiocytosis X); Sickle Cell Disease. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Gene Association Studies

n, Brigham and A A Litonjua and J C Celedo Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
With the completion of the Human Genome Project and the rapid progress in genotyping technology, the number of genetic association studies in respiratory diseases has increased exponentially in recent years. Most of these association studies have employed a population-based case-control design, and very few have provided results that have been replicated by others. While disagreement in results between studies may be due to true differences in genetic variation and environmental exposures among populations, attention must be paid to potential problems that lead to lack of replication. These potential problems include differences in phenotype definitions, lack of attention to HardyWeinberg equilibrium in controls, small sample sizes, multiple testing, and potential population stratication. While methods have now been developed to test and control for population stratication, family-based methods have also been developed in response to this problem. This article provides an overview of the design and conduct of genetic association studies for complex respiratory diseases.

Introduction
Genetic association studies can be dened as investigations that assess the relationships between genetic variants and either aspects of a disease or the disease itself. In its simplest form, association studies determine whether a particular form of a genetic marker occurs more frequently in subjects with a particular trait or disease than in those without the trait or disease of interest. Association studies play an important role in identifying genetic determinants of complex human diseases. The rapid progress of the Human Genome Project has propelled the use of genetic association studies as a tool to better understand complex respiratory disorders. The most popular design that has been employed is the population-based case-control study. Because this design is susceptible to potential bias by population stratication (see below), family-based designs have been developed. Each of these designs have their advantages and disadvantages, and the investigators choice of the most efcient design will depend on factors such as the age of onset of the disease and the availability of subjects and specimens. In general, when a significant association is found between a genetic variant (e.g., a polymorphism) and a disease, there are three possible explanations: (1) the nding is a false positive due to a spurious association; (2) the association is due to close proximity to a functional variant in the same locus or in an

Further Reading
Hartl DL and Clark AG (1997) Principles of Population Genetics, 3rd edn. Sunderland, MA: Sinauer Associates. Hirschhorn JN and Daly MJ (2005) Genome-wide association studies for common diseases and complex traits. Nature Reviews: Genetics 6: 95108. Khoury MJ, Little J, and Burke W (2004) Human Genome Epidemiology. Oxford: Oxford University Press. Rao DC and Province MA (eds.) (2001) Genetic Dissection of Complex Traits. San Diego, CA: Academic Press. Silverman EK, Shapiro SD, Lomas DA, and Weiss ST (2005) Respiratory Genetics. London: Hodder-Arnold. Thomas DC (2004) Statistical Methods in Genetic Epidemiology. Oxford: Oxford University Press.

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adjacent locus (linkage disequilibrium (LD); see below); or (3) the locus is functional and directly affects phenotype expression. Spurious associations may arise due to chance or bias. In genetic association studies, the main type of bias that must be considered is that which results from population stratication, which may be thought of as a form of confounding by ethnicity. Population stratication results when cases and controls are not drawn from the same underlying population, and two necessary conditions exist: (1) the prevalence of the disease of interest is different in the two populations, and (2) the frequency of alleles is different in the two populations. Population-based case-control designs are susceptible to this form of bias, whereas family-based designs are generally immune to the effects of population stratication. Chance alone may explain false positive results in genetic association studies, and the importance of replication of initial ndings in independent populations cannot be overemphasized. When potential sources of bias have been adequately controlled, the most likely explanation for a positive association between a genetic marker(s) and a disease of interest is LD between the marker(s) studied and a nearby functional locus. LD is the association between particular alleles at two or more loci because of their proximity (tight linkage) on the same chromosome. When alleles at two distinct loci occur more frequently together than expected based on their frequencies, these alleles are said to be in LD. Finally, a positive genetic association may represent the nding of a true disease susceptibility locus. However, this needs to be evaluated by functional studies in humans and/or animal models of the disease of interest.

then the trait may be heritable. For dichotomous traits, studies typically examine the proportion of relatives of the probands who also have the trait and compare this with the proportion of relatives of control subjects who have the trait. Studies of monozyotic and dizygotic twins are helpful in providing clues to the heritability (the proportional contribution of genetic factors) of a trait. A higher concordance of disease in monozyotic twins than in dizygotic twins suggests that the disease has a significant genetic component. Finally, segregation analysis can be used to evaluate the most likely mode of inheritance of a disease of interest; this can be particularly helpful if a monogenic variant of a complex disease is suspected. In this method, a variety of models (both genetic and nongenetic) are t to family data and the results are assessed to determine which model best ts the data. Because segregation analysis entails the use of family data, it is generally performed as part of gene mapping studies, before genetic marker information is available.

Phenotypes for Gene Association Studies

The phenotype of an individual refers to the observable trait or characteristic of that individual, for example hair color or height. In the case of association studies, phenotypes that are studied are usually the presence or absence of a disease. More recently, studies have also begun to investigate genetic modiers of a disease (e.g., modiers of disease presentation of a-1-antitrypsin deciency or genes associated with the severity of asthma). The choice of phenotype is critical to the success of gene association studies. It may appear to be straightforward and appealing to use the disease diagnosis as the phenotype, since most studies are investigating the risk factors for the condition of interest. However, diagnostic misclassication is problematic and can seriously affect the power of the study. Disease diagnosis is a qualitative (all or none) rather than a quantitative (continuous) trait, and generally quantitative phenotypes are more powerful in identifying disease susceptibility genes. Furthermore, complex diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are heterogeneous in their presentation. For example, one must decide whether they are studying atopic vs. nonatopic asthma or COPD that primarily presents as emphysema or as chronic bronchitis. In narrowing the phenotype definition, it is necessary to collect information on disease-associated traits, or intermediate phenotypes. Intermediate phenotypes are appealing because they are objective, are measured

Genetic Contribution to Disease

Before embarking on a gene association study, it is rst necessary to determine whether the disease of interest has a significant heritable component. Several methods exist to determine the genetic contribution to a disease, and in many instances, these studies have already been performed and results have been published. One method to determine whether a disease has a significant genetic component is to perform familial aggregation studies. For continuous traits, these studies determine the correlations of the trait between relatives of different types (e.g., between siblings, between parents and children) and compare these correlations. For example, if the correlation of the trait among siblings is stronger than that found between spouses (who are not biologically related),

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Table 1 Intermediate phenotypes for respiratory gene association studies Phenotype Symptoms Disease severity or control Lung function Bronchoconstrictor response Bronchodilator response Atopy Biomarkers of inammation Lung specic Generalized Imaging Tool or measurement Self-administered questionnaires; interviewer-administered questionnaires; video questionnaires Questionnaires on medication use, healthcare utilization including urgent care and emergency room visits Spirometry: FEV1, FVC, FEV1/FVC, FEV2575/FVC, PEF Challenge testing using non-specic (e.g., methacholine, histamine) or specic (e.g., platelet activating factor) agents FEV1 change after administration of bronchodilator (albuterol) Total and specic IgE; skin test reactivity; eosinophil count Markers measured in sputum, exhaled gases and exhaled breath condensates, BAL uid Markers measured in blood and urine CT analysis of parenchyma and airways

with more precision and reliability than physicians diagnoses and most are continuous measures (quantitative traits), thus potentially increasing the power for the analyses. Table 1 summarizes some intermediate phenotypes that may be used in respiratory disease genetics. Questionnaires for symptoms and disease control, spirometry, bronchoconstrictor and bronchodilator responses, and atopy measures have been extensively used in association analyses, while imaging modalities and biomarker measurement hold promise for better phenotyping in the future. Information on symptoms is collected with the use of questionnaires. Several standardized, and in some instances validated, questionnaires are available. These include the questionnaires from the American Thoracic Society and Division of Lung Diseases (ATS-DLD), the International Study of Asthma and Allergy in Childhood (ISAAC), the Medical Research Council (MRC) of Great Britain, and the International Union against Tuberculosis and Lung Disease (IUATLD) to name a few. These have been in use for many years in the eld of respiratory epidemiology. For asthma in particular, the ISAAC questionnaire has incorporated a video questionnaire, designed to overcome language and cultural differences in the interpretation of the symptom of wheeze, making standard definitions for multinational studies a possibility. In addition to collecting information on baseline symptoms, information on disease severity or control, and medication use may also be collected. These will be important for studies investigating genetic modiers of disease or determinants of frequency of exacerbation of a whole range of respiratory diseases. Spirometry is widely used in gene association studies in respiratory diseases. Field studies using spirometry have been conducted for many decades, making it applicable for measurement of lung function in families and populations. Measures of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), forced

expiratory ow 2575% (FEF2575), which is the ow between 25% and 75% of the vital capacity, and the peak expiratory ow (PEF) can all be determined from the spirometric maneuver. Similary, bronchoconstrictor response testing and bronchodilator response testing are invaluable in dening phenotypes for respiratory disease. While some studies have incorporated one or the other measure in the phenotype definition, it is likely that these traits are dened by different sets of genes. Because asthma and atopy are closely associated, measurements of total and specic immunoglobulin E (IgE) in serum and skin test reactivity to a range of allergens have been used to dene the phenotype in many asthma genetic association studies. Additionally, studying the genetic determinants of these traits may also uncover mechanisms involved in the development of asthma. Inammation is a major process in many respiratory disorders. While the exact cell type and the location of the inammatory inltrate vary in each disorder, biomarkers have been used in studies of the presence of disease, disease severity, or response to treatment, and have been useful in elucidating the pathophysiologic processes occurring in each one. A wide range of cytokines, products of oxidation, and other mediators have been studied. These can be roughly categorized as biomarkers of generalized or lung-specic inammation. Early studies used biomarkers measured in blood components and urine, because obtaining these samples was reasonably simple. However, these biomarkers may not faithfully reect the inammatory process occurring in the lung, and their levels are confounded by comorbid conditions. While specimens obtained using the beroptic bronchoscope, including bronchoalveolar lavage (BAL), are specic, the procedure is too invasive to be of routine use in human genetic association studies, where large numbers of cases and controls, or family

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members need to be phenotyped. Sputum induction is a reasonably noninvasive method of obtaining samples, and many biomarkers that are measured in blood have been shown to be measurable in these sputum samples. There is more research interest, however, in the newer noninvasive methods of sample collection. Collection and examination of both exhaled gases and exhaled breath condensates are promising methods of obtaining respiratory samples. However, standardization of equipment and techniques is needed before these methods can be advocated as means of phenotyping subjects. The advent of high-resolution computed tomographic (HRCT) scanning has allowed better anatomical understanding of the changes occurring in various lung diseases. Assessment of small airways dimensions has aided in the diagnoses of various lung disorders, such as constrictive bronchiolitis, hypersensitivity pneumonitis, sarcoidosis, and several occupational lung disorders. HRCT has also been used to assess the extent and distribution of parenchymal abnormalities in pulmonary emphysema. Other imaging modalities (e.g., hyperpolarized helium-3 gas magnetic resonance imaging) of the lung are rapidly being developed, and these hold great promise in noninvasively assessing the structural characteristics of the lung to aid in phenotyping subjects for respiratory genetics studies. The absence of so-called gold standards for the diagnosis of respiratory disorders has led to the use of variable definitions in association studies. This variability in dening phenotypes is surely one reason for the lack of replication in association studies, and collecting information on objective intermediate phenotypes helps to standardize phenotype definition across studies. A strategy for dening phenotypes has been to combine a physicians diagnosis with symptoms and an objective intermediate phenotype (e.g., spirometry in the case of COPD, bronchoconstrictor response or bronchodilator response in the case of asthma).

Selection of Candidate Genes and Polymorphisms

Genetic association studies can examine alleles in candidate genes or in a genomic region(s) of interest (ne-mapping association studies). Because ne-mapping association studies are more expensive and complex, candidate gene association studies are frequently conducted. A candidate gene is dened as a gene that is identied either by its protein product that suggests that it could be the determinant of the disease in question (a biological candidate), or by its position in a chromosomal region that has been

linked with the disease (positional candidate). Examples of biological candidates are the b2 -adrenergic receptor gene (ADRB2) for asthma because of the central role of b agonists in asthma therapy; oxidant antioxidant genes such as glutathione-S-transferase gene for emphysema based on the pathobiology of the disease; and the tumor necrosis factor alpha (TNF-a) gene in pulmonary brosis because of the role of cytokines and inammation in the development of brosis. Positional information derived from linkage studies is another way to identify candidate genes. There are fewer examples of this, since most of the linkage analyses have been performed in the two most common complex respiratory disorders: asthma and COPD. In asthma, several genome scans have been conducted, and some chromosomal regions that have shown consistent linkage to asthma-related phenotypes in several populations (e.g., 5q, 6p, 12q, and 13q). Thus, genes located in these regions are all potential candidate genes for association studies of asthma. To date, there have only been three genome scans for COPD-related phenotypes, with regions in chromosome 2q in one study and chromosome 4 in another having log10 of the odds of linkage (LOD) scores above 3.3. Once candidate genes have been selected for study, the next step is to nd variants in the gene. Variation in DNA sequence in a gene that has a frequency of greater than 1% is called a polymorphism. There are different types of polymorphisms in the genome: single nucleotide polymorphisms (SNPs), repeat polymorphisms, and insertions or deletions. By far, the most common type of variation is the SNP, which is a single base variation in DNA sequence. SNPs occur commonly, at about 1 out of every 300 bases in the human genome, and it is estimated that there are about 10 million SNPs across the human genome. Sources of information on SNPs include the National Center for Biotechnology Information (NCBI) SNP database and the International Haplotype Map (HapMap) Project. SNPs are ubiquitous and are found in both coding and non-coding regions of a gene. When an SNP occurs in a coding region of a gene, it may have one of three consequences. The rst is a nonsense SNP, which is a single nucleotide variation that results in a STOP codon, thus giving rise to a nonfunctional protein. The second is a missense (also called nonsynonymous) SNP, in which the single nucleotide variation results in the coding of a different amino acid, thus giving rise to a protein with altered structure or function. Thirdly, SNPs in coding regions may also be synonymous SNPs, in which the single nucleotide variation does not change the amino acid sequence of the protein. Most SNPs

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are located in the noncoding regions of the genome, but may still be important because they may lie in gene promoter or regulatory regions that may affect gene expression. For practical reasons, genotyping all known SNPs in a gene for an association study is often not feasible. Thus, choosing a subset of SNPs for genotyping is an important step for a successful association study, and investigators should have an idea of how they want to prioritize available SNPs. Priority should be given to SNPs that have known function, nonsynonymous SNPs in coding regions, and SNPs known to alter gene splicing or expression. SNPs that have previously been associated with the disease of interest should also be chosen, as this would allow comparison of results across studies. Preference should also be given to SNPs that are common (i.e., SNPs with minor allele frequency of at least 5%), so that statistical power of the study will not suffer. Additionally, consideration should be given to SNPs that can be reliably genotyped (e.g., HapMap SNPs). Finally, since SNPs in a gene may be closely correlated with other SNPs in the same gene because of LD, it is possible to choose a subset of SNPs that captures most of the information that one would get by typing the whole set. In the extreme case (complete LD), an allele found at one locus can predict completely which allele will be found at another locus, making one of the loci redundant for the purpose of genotyping. Empirical studies have reported blocklike segments in the genome with high LD. These studies have also shown that areas of the genome have a much smaller number of haplotypes (the linear, ordered arrangement of alleles in a segment of a chromosome) than would be predicted by the number of markers alone. Thus, these studies have motivated a marker selection strategy for association studies, whereby a subset of SNPs is chosen that can stand as a proxy for many other SNPs. These tag SNPs would then capture most of the common variation within the region of interest. There are various algorithms that choose tag SNPs based on LD or based on haplotypes. However, these assume that there is prior knowledge of the LD patterns of the region of interest. This data may arise from resequencing a subset of subjects in a study or from large genotyping studies in reference samples such as the HapMap project.

designs. Population-based designs entail collection of unrelated individuals, some who have the disease of interest (cases) and others who do not (controls), while family-based designs entail collection of information from families of individuals who have the disease (probands). Each of these designs has its advantages and disadvantages. Ultimately, the choice of which study design to use will depend on a host of factors such as the expense of collecting phenotype information and samples, genotyping costs, and characteristics of the disease of interest (e.g., for late-onset diseases, obtaining parental genotype data may not be feasible).

Population-Based Designs
Probably the simplest design for gene association studies is the case-control design, where a comparison is made in the frequency of SNP alleles in two well-dened groups of individuals: cases who have the disease or the trait of interest, and controls who are known to be unaffected. This is no different from the standard case-control design used in epidemiologic studies, and the nding of an increased frequency of an SNP allele or genotype in cases compared with controls indicates that the presence of the SNP may increase the risk of disease (Figure 1). Because of the ease of the concept and the familiarity with this design, it is no surprise that this is the most popular design that researchers have adopted. However, many of the limitations of this design in the epidemiologic setting also apply in association studies, and basic principles of performing good casecontrol association studies are often overlooked. In selecting cases and controls, the source population should be well dened. Cases should be a representative sample of all the cases in this precisely dened and identied population. Hence, the source population is determined by the eligibility criteria for cases to enter the study. For example, if in a casecontrol association study for asthma, cases were made up of admissions to a city hospital, the source population would be dened as all the patients admitted to that hospital who would have been given a diagnosis of asthma, if indeed they had asthma. Controls should be sampled from this source population. Controls should also be representative of the source population with respect to exposure. In the particular case of gene association studies, this means that allele frequencies found in controls should reect those in the source population. Obviously, it is impossible to determine whether this is indeed the case, but random sampling of the source population when selecting controls assures a greater likelihood of the representativeness of the control group.

Study Designs for Gene Association Studies

There are two basic study designs for gene association studies: population-based designs and family-based

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A Case Control Allele counts

cells AA Case Control Genotype counts AB BB

(Observed Expected) 2 Expected

Figure 1 Population-based case-control association studies. Associations may be analyzed as either allele counts or genotype counts. In either case, the analysis consists of comparing the observed counts to the expected counts in a w2 analysis with either one (allele counts) or two (genotype counts) degrees of freedom. When nongenetic covariates, such as age, are collected in a study, these may be accounted for in the analysis by tting multiple logistic regression models with the case status as the dependent variable and the genetic and nongenetic variables as the independent variables.

A second type of population-based design is the cohort study. A cohort study consists of assembling a group of subjects, who are then followed in time to determine the frequency with which disease develops (e.g., identifying infants at birth and following them for the development of asthma; or assembling a group of smokers and seeing who will develop clinically significant emphysema). Cohort studies are more expensive and labor intensive, but are less prone to ascertainment bias than case-control studies. It is also possible to select cases and controls from existing cohorts (nested case-control studies). Numerous population-based association studies have been published, particularly for asthma and COPD, yet very few of these studies have been replicated, and in some instances, contradictory results have been found. There are many reasons for the lack of replication in association studies, and some of these include: (1) no assessment and control for population stratication, (2) small sample sizes, (3) multiple testing, (4) differences in phenotype definitions between studies, and (5) failure to consider linkage disequilibrium with adjacent loci. As explained earlier, population stratication is a potential problem in population-based case-control association studies, and is one reason for false positive ndings. In the design phase of a study, this potential problem may be minimized by careful matching of cases and controls on ethnicity. However, subtle differences in the ethnic make-up of the population may be easily missed and formal assessment and control (if it is present) of population stratication is needed for a well-designed casecontrol association study. Determining whether HardyWeinberg equilibrium (HWE) is present

among controls is important (Figure 2). True genetic associations can cause deviations from HWE among cases, so it should be tested in control subjects. If genotypes are not in HWE, this is a clue of potential genotyping error (most commonly) or population stratication. Finally, a set of markers that are not known to be linked to or associated with the disease of interest can be genotyped, and an analysis of association between these markers and the trait of interest can be used as a formal test of population stratication. It is currently unclear how many unlinked markers need to be genotyped to exclude significant population stratication. If indeed stratication is found, then methods such as genomic control, exist to control this in the analysis. While there are numerous association studies in respiratory disease in the literature, many of these have been composed of small numbers of cases and controls. Ideally, case-control association studies should have adequate numbers of subjects. The numbers should be guided by several factors such as the frequency of the polymorphisms and the effect estimates related to these polymorphisms, but investigators should aim to collect as many cases and controls as is feasible. Having large numbers of cases and controls improves the chances for replication of a positive association. Multiple testing is another problem in case-control association studies, and arises from testing multiple phenotypes, multiple SNPs in genes, and/or multiple genes. While it is clear that the classical Bonferroni correction is too conservative, the best method for addressing the multiple testing issue for association studies remains unresolved. Probably the best strategy so far is replication of results in an independent population.

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Fundamental principle in population genetics Simple explanation for how genetic diversity for Mendelian traits can be maintained in a population
Named after the English mathematician G. H. Hardy (18771947) and the German physiologist W. Weinberg (18821937) who independently formulated the model and deduced its theoretical predictions of genotype frequency A locus with two alleles, A and a, where: p = frequency of A q = frequency of a Equilibrium distribution of genotypes is defined as: Genotype Frequency AA p2 Aa 2pq aa q2

Absence of HardyWeinberg equilibrium among control subjects in a population-based case-control study is likely due to either genotyping error or population stratification, while its absence in parents of offspring in a family-based study of association may be due to genotyping error.
Figure 2 The HardyWeinberg principle.

As indicated in the earlier section on phenotypes, there is a lack of standard definitions for respiratory phenotypes used in association studies. Thus, differences in phenotype definition can lead to the contradictory ndings from study to study. Obtaining information on intermediate phenotypes that are collected in a standardized manner may help in replication studies. Despite attention to these details of the case-control association study, ndings may still not be replicated in other populations. It is possible that one locus may be associated with a trait in one population, but another locus is associated with the same trait in another population (locus heterogeneity). It is also likely that different environmental stimuli in separate populations interact with the same locus to produce different associations. In this case, the associations seen in each population may be real, but differing environmental exposures may produce dissimilar, sometimes even conicting, results. Finally, different patterns of LD may exist in different populations, and if the causative SNP (or SNPs) is not the one being tested, these different patterns of LD will also produce dissimilar results. To summarize, various issues may affect the validity of gene association studies. Careful attention to these study design particulars is needed to have a well designed study. Finally, even if a study is well designed, the need for replication of ndings in independent populations cannot be overemphasized.
Family-Based Association

The problem of potential population stratication in case-control designs was the main impetus to the

development of methods that incorporated family designs in association studies. One of the earliest approaches to the problem of population stratication is the transmission disequilibrium test (TDT), reported by Spielman and colleagues in 1993. The rationale behind the TDT is that in the absence of both linkage and association between the marker locus being studied and the true disease locus, marker alleles will be transmitted randomly from parents to offspring. In the original TDT design, a pedigree with both parents and a single affected offspring was required, and it compared the transmission of marker alleles to affected offspring against the nontransmission of alleles by means of a simple w2 statistic (Figure 3). Many extensions and variations to the TDT have been developed since the original report, which now allow the incorporation of multiallelic markers, multiple markers, missing parental information, extended pedigrees, quantitative traits, and non-genetic covariates. Compared with case-control association studies, family-based association studies have three main advantages: (1) control for potential population stratication, (2) increased ability to detect genotyping errors because of availability of parental data, and (3) reduced costs of phenotyping, particularly for the TDT design (only affected offspring is phenotyped). It should be remembered, however, that parental data may not be available for family-based association studies because of multiple factors, including late-onset of the disease of interest and divorce. In addition, more individuals have to be genotyped for a familybased study than in a case-control study, although this is offset somewhat by the need to genotype a set of unlinked markers in the population-based design.

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(1) (2)
Hartl DL, Clark AG (1997) The HardyWeinberg principle. In: Hartl DL and Clark AG (eds.) Principles of Population Genetics, 3rd edn. Sunderland: Sinauer Associates. Hoffjan S, Nicolae D, and Ober C (2003) Association studies for asthma and atopic diseases: a comprehensive review of the literature. Respiratory Research 4(1): 14. Horvath S and Laird NM (1998) A discordant-sibship test for disequilibrium and linkage: no need for parental data. American Journal of Human Genetics 63: 18861897. Horvath S, Xu X, and Laird NM (2001) The family based association test method: strategies for studying general genotype phenotype associations. European Journal of Human Genetics 9: 301306. Khoury MJ, Beaty TH, and Cohen BH (1993) Fundamentals of Genetic Epidemiology. New York: Oxford University Press. Laird NM, Horvath S, and Xu X (2000) Implementing a unied approach to family based tests of association. Genetic Epidemiology (Supplement) 19: 3642. Lake SL, Blacker D, and Laird NM (2000) Family based tests of association in the presence of linkage. American Journal of Human Genetics 67: 15151525. Lange C, DeMeo D, Silverman EK, Weiss ST, and Laird NM (2004) PBAT: tools for family based association studies. American Journal of Human Genetics 74(2): 367369. Meyers DA, Larj MJ, and Lange L (2004) Genetics of asthma and COPD: similar results for different phenotypes. Chest 126(2 supplement): 105S110S. Molno NA (2004) Genetics of COPD. Chest 125(5): 19291940. Monks SA and Kaplan NL (2000) Removing the sampling restrictions from family-based tests of association for a quantitativetrait locus. Amercian Journal of Human Genetics 66: 576592. Newton-Cheh C and Hirschhorn JN (2005) Genetic association studies of complex traits: design and analysis issues. Mutation Research 573: 5469. Risch NJ (2000) Searching for genetic determinants in the new millennium. Nature 405(6788): 847856. Rothman KJ and Greenland S (1998) Case-control studies. In: Rothman KJ and Greenland S (eds.) Modern Epidemiology, 2nd edn., pp. 93114. Philadelphia: Lippincott-Raven. Rothman KJ and Greenland S (1998) Cohort studies. In: Rothman KJ and Greenland S (eds.) Modern Epidemiology, 2nd edn., pp. 7991. Philadelphia: Lippincott-Raven. Schulze TG and McMahon FJ (2002) Genetic association mapping at the crossroads: which test and why? Overview and practical guidelines. American Journal of Medical Genetics 114: 111. Silverman EK (2002) Genetic epidemiology of COPD. Chest 121(3 supplement): 1S6S. Steen KV and Lange C (2005) PBAT: a comprehensive software package for genome-wide association analysis of complex family-based studies. Human Genomics 2(1): 6769. Tabor HK, Risch NJ, and Myers RM (2002) Opinion: candidategene approaches for studying complex genetic traits: practical considerations. Nature Reviews: Genetics 3(5): 391397. Villar J, Flores C, and Mendez-Alvarez S (2003) Genetic susceptibility to acute lung injury. Critical Care Medicine 31(4 supplement): S272S275. Weiss KM and Terwilliger JD (2000) How many diseases does it take to map a gene with SNPs? Nature Genetics 26(2): 151157.

A B

A B

A Untransmitted allele A B b d

Test statistic T= (b c )2 2 1 b+c

Transmitted allele

A B

a c

Figure 3 Transmission disequilibrium test. Family-based association studies may be analyzed by the transmission disequilibrium test (TDT) or the various extensions of the TDT. In the simplest case of a trio (i.e., genotypes from both parents and from the affected offspring), the TDT compares the transmission of an allele to the affected offspring against the nontransmission of the allele. In the illustration above for family (1), the A allele is transmitted to the offspring by each parent; thus, in the table, b 2, and a, c, and d are all equal to 0. For family (2), the A allele is transmitted by the father to the offspring and the B allele is untransmitted, while one A allele is transmitted by the mother and the other A allele is untransmitted. Thus, for this trio, the table should be lled out with a 1, b 1, and c and d are equal to 0. These counts are performed for all the trios in the study and these are summed over all the tables and analyzed as a w2 test with one degree of freedom. It is clear from this illustration that homozygous parents do not contribute to the test statistic.

Ultimately, investigators have to carefully weigh the advantages and disadvantages of a particular study design for a disease or trait of interest.
See also: Asthma: Overview. CD1. Gene Regulation. Genetics: Overview.

Further Reading
Abecasis GR, Cardon LR, and Cookson WOC (2000) A general test of association for quantitative traits in nuclear families. American Journal of Human Genetics 66: 279292. Cardon LR and Bell JI (2001) Association study designs for complex diseases. Nature Reviews: Genetics 2(2): 9199. Cardon LR and Palmer LJ (2003) Population stratication and spurious allelic association. Lancet 361(9357): 598604. Clark AG (2004) The role of haplotypes in candidate gene studies. Genetic Epidemiology 27: 321333. Davies JC (2004) Modier genes in cystic brosis. Pediatric Pulmonology (Supplement) 26: 8687. Garcia CK and Raghu G (2004) Inherited interstitial lung disease. Clinics in Chest Medicine 25(3): 421433. (Erratum in: Clinics in Chest Medicine 25(4): xi.) Haines JL and Pericak-Vance MA (eds.) (1988) Approaches to Gene Mapping in Complex Human Diseases. New York: Wiley-Liss.

Relevant Websites
http://www.hapmap.org International Haplotype Map Project. http://www.ncbi.nlm.nih.gov National Center for Biotechnology Information.

248 G-PROTEIN-COUPLED RECEPTORS

G-PROTEIN-COUPLED RECEPTORS
S B Liggett, University of Maryland, Baltimore, MD, USA D W McGraw, University of Cincinnati, Cincinnati, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
G-protein-coupled receptors (GPCRs) are a large and diverse superfamily of cell surface receptors that transduce their signals via membrane-associated heterotrimeric guanine nucleotidebinding proteins (G-proteins). It is estimated that there are over 1000 GPCRs that may make up to 510% of the human genome. Members of the G-protein superfamily share a common structural motif of seven transmembrane spanning domains that includes an extracellular N-terminus and an intracellular C-terminus, interspersed by seven domains that span the cell membrane in tandem, connected by three extracellular loops and three intracellular loops. Although the general scheme of signal transduction is similar for all members of the GPCR superfamily, there is considerable structural diversity in the receptor proteins that provides for plasticity at nearly every step of regulation including receptor synthesis, ligand binding, G-protein coupling, and intracellular trafcking (desensitization, resensitization, and downregulation). Through activation or inhibition of their downstream messengers, GPCRs regulate a wide array of physiological and compensatory functions in the lung. GPCRs are currently important targets for drugs used to treat lung diseases such as asthma, chronic obstructive pulmonary disease, and pulmonary hypertension, and are a major focus of pharmaceutical development. Because so many aspects of GPCR function are governed by receptor structure, it is not surprising that an increasing amount of data indicate that naturally occurring genetic variation in GPCRs may encode receptors that differ in signal transduction, and thereby contribute to differences in physiology or therapeutic responses.

nucleotides, odorants, light, and undoubtedly many others. That so many different signaling events are accomplished via the same basic mechanism suggests that the process is highly efcient. While the general scheme of signal transduction is similar for all GPCRs, significant structural diversity has evolved among members of the GPCR family which provides for diversity at virtually every step of the process.

Structure
All GPCRs share a common structural topology that has been derived from their homology to bovine rhodopsin, the only mammalian GPCR for which high-resolution three-dimensional crystal structure has been resolved, and by studies utilizing proteolytic digestion, peptide mappings, and site-directed mutagenesis. This unique structure consists of seven hydrophobic regions that adopt a-helical conformations which serve as transmembrane spanning domains. This structure orients the receptor in the cell membrane such that the N-terminal end is extracellular, the C-terminal end is intracellular, and the seven transmembrane (7-TM) spanning domains are linked by three extracellular loops and either three or four intracellular loops depending upon the presence of a palmitoylated cysteine residue in the proximal C-terminal tail (Figure 1). The presence of specic residues within a GPCR may additionally permit the receptor to undergo a variety of regulatory posttranslational modications including N-linked glycosylation, palmitoylation, formation of disulde bonds, and phosphorylation. GPCR families have been categorized into ve or six different groups based upon similarities in their structural features or sequence, as well as their function. Over 90% of known GPCRs are in family A, which includes rhodopsin, olfactory, biogenic amine, nucleic acid, and bioactive lipid and peptide receptors. The biogenic amine group includes the well-characterized adrenergic, muscarinic, prostanoid, histamine, and adenosine receptors. Family A receptors have a short N-terminal extracellular domain and several conserved amino acids within the transmembrane spanning domains. In most cases, the transmembrane spanning domains of family A members serve as the ligand-binding site, while the intracellular connecting loops serve as coupling and regulatory domains. Family B receptors include the glycoprotein hormone receptors (e.g., luteinizing hormone, thyroid stimulating hormone, and follicle stimulating

Introduction
G-protein-coupled receptors (GPCRs) represent a large and diverse superfamily of integral membrane receptor proteins that transduce extracellular stimuli to an intracellular signal via a comparatively smaller group of membrane-associated heterotrimeric guanine nucleotide-binding proteins (G-proteins). Since the cloning of bovine rhodopsin and the b2-adrenergic receptor (b2-AR), several hundred GPCRs have been isolated by molecular cloning techniques, with estimates that the human genome may have over 1500 members of the superfamily based on bioinformatics techniques of the genome sequence. GPCRs are thus the largest superfamily of plasma membrane receptors, comprising up to 510% of the human genome. GPCRs respond to a wide array of external stimuli including neurotransmitters, hormones, chemokines,

G-PROTEIN-COUPLED RECEPTORS 249

NH2

Extracellular

Intracellular

HOOC
Figure 1 Schematic representation of the putative membrane topology GPCRs. Seven hydrophobic transmembrane spanning are connected in tandem by three extracellular and three intracellular loops. The N-terminal tail is extracellular and the C-terminal tail is intracellular.

hormone receptors), secretin, calcitonin, and parathyroid hormone receptors. Family B receptors are characterized by a long extracellular N-terminal domain (4400 amino acids) that interacts with portions of the extracellular connecting loops to form the ligand-binding domain. Family C receptors contain an even longer extracellular N-terminus (4600 amino acids) where the ligand-binding domain is exclusively located. This group includes the metabotropic glutamate (mGlu) and g-aminobutyric acid (GABA) neurotransmitter receptors, the extracellular calcium sensing receptor, and members of the taste receptor group. Additional families of GPCRs include those related to fungal pheromone P- and a-factor receptors (family D), fungal pheromone A- and M-factor receptors (family D), and those related to slime mold cAMP receptors (family E). A growing number of new GPCR families continue to be reported, including those of the frizzled and smoothened families. It is thus likely that any classication of GPCRs will continue to expand and change, especially since many GPCRs identied by cloning or bioinformatics are presently orphan receptors with no function yet ascribed to them.

Signal Transduction
The classic feature of GPCRs is their utilization of G-proteins for intracellular signal transduction. Signal transduction is initiated when the receptor adopts an activated conrmation that can then bind to one or more members of the heterotrimeric G-protein family. In an early model of receptor signaling, the

activated conformation was thought to be achieved upon receptor binding to agonist. However, numerous GPCRs have been observed in vitro to couple to G-proteins and transduce signals in the absence of agonists. Such behavior is better explained by a multistate model wherein there is equilibrium between active and inactive receptor conformation states that is inuenced by the presence or absence of agonists. In this scenario, ligands that promote stabilization of the active conformation are agonists, those that do not affect the active/inactive equilibrium are neutral antagonists, and those that favor the inactive conformation are inverse agonists. Once a GPCR adopts an active conformation, it can associate with one or more of the B20 members of the G-protein family to form the ternary complex (Figure 2). Each G-protein heterotrimer is composed of a Ga subunit that can bind guanine nucleotides (GTP and GDP) in combination with nondissociable Gb and Gg subunits. Binding of the activated GPCR is accompanied by Ga subunit-mediated exchange of GDP for GTP and subsequent separation of the G-protein into Ga and nondissociable Gbg subunits. Depending upon the G-protein family members involved, these Ga and Gbg subunits act individually or synergistically to activate or inactivate various downstream effectors such as adenylyl cyclase, phospholipase C, and ion channels. Subsequent hydrolysis of GTP to GDP, which enables the Ga subunit to reassociate with Gbg subunits, completes the receptoractivated portion of the cycle. A family of proteins termed regulators of G-protein signaling (RGS protein) serve a major role in this GTP hydrolysis.

250 G-PROTEIN-COUPLED RECEPTORS

GTP G (GDP) GDP G (GTP)

Signal RGS G (GDP)

Signal

interaction is via the agonist-promoted binding of the receptor and scaffolding proteins such as b-arrestin. Taken together, the term GPCR may not be most appropriate, but will undoubtedly be retained for historical reasons. Rather, 7-TM receptors best describe the superfamily.

Regulation of Production and Activity

Following activation of signal transduction, many, but not all, GPCRs display a characteristic reduction in signaling intensity. This phenomenon, termed desensitization, is a negative feedback mechanism to prevent or limit the consequences receptor overstimulation. Overall GPCR activity thus reects the balance between activating events, such as coupling of the receptor to its G-protein, and the events responsible for its desensitization. Although a combination of events may be responsible for the latter, three predominant mechanisms appear to be common among GPCRs: (1) uncoupling of the receptor from its G-protein by receptor phosphorylation, (2) internalization of the receptor from the cell surface to internal compartments, and (3) downregulation of the total number of receptors due to decreased production or increased degradation. These events are temporally distributed with phosphorylation occurring most rapidly, followed by internalization (within minutes) and downregulation (hours). These events are dictated not only by factors such as the nature of the agonist or length of exposure, but also by the presence or absence of requisite structural features (e.g., appropriate serine/threonine residues to act as substrates for phosphorylation) within the receptor itself. For susceptible GPCRs, phosphorylation is the most rapid desensitization event. Phosphorylation may be mediated by second-messenger-dependent kinases such as PKA or PKC, or by a group of secondmessenger-independent kinases termed G-proteincoupled receptors kinases (GRKs) of which seven family members have been identied. The critical substrates for phosphorylation appear to be serine and threonine residues within the intracellular loops and C-terminal tail regions, although the specic residues differ for the two groups of kinases. GRK-dependent phosphorylation promotes binding of cytosolic proteins from the arrestin family, which serves to uncouple the protein from its G-protein. Unlike second messenger-dependent phosphorylation, activation of downstream effectors is not requisite for GRK-mediated phosphorylation but an activated, or agonist-occupied, receptor is. Because of signal amplication, second-messenger-dependent phosphorylation is thought to be the primary desensitization event at low levels of agonist, whereas higher

Figure 2 Signal transduction by GPCRs. Receptors may exist in an activated or inactivated state. Agonist binding stabilizes or induces the activated state and enables the receptor to associate with its G-protein. Receptor activation causes the Ga subunit to exchange GDP for bound GTP, and to dissociate from the Gbg subunits. Either of subunit may then act to stimulate or inhibit downstream effectors such as adenylyl cyclase or phospholipase C.

The downstream second messengers and effectors activated by a particular GPCR are determined in large extent by the G-protein to which the receptor preferentially couples. Well-recognized examples of G-protein signal pathways include GS stimulation of adenylyl cyclase, Gi inhibition of adenylyl cyclase, and Gq/11 stimulation of phospholipase C. Other G-protein families include GT, GO, GH, and GZ. Studies using mutagenesis and chimeric receptors have demonstrated that specicity of GPCR coupling to G-proteins appears to be dictated in large part by the structure and conformation of the second and third intracellular loops and the portion of the cytoplasmic tail near the membrane. While it was thought early on that a given GPCR activated a single predominant G-protein and its associated signaling pathway, it has since been recognized that many GPCRs can couple to multiple G-protein families to simultaneously activate or inhibit additional signaling cascades. It is interesting to note that for some receptors that can couple to multiple Gproteins, synthetic agonists can bind in such a way to favor conformational states that provide preferential coupling to one G-protein over the other. Moreover, within these major G-protein families, it is now known that multiple isoforms of the a, b, and g subunits exist. For example, at least three different isoforms of Gai exist. Evidence suggests these subunit isoforms may differ in their signal transduction capacity and specicity, thereby providing for additional levels of complexity and plasticity in GPCR signal transduction. Finally, it is now recognized that signaling, in its most liberal interpretation, does not necessarily require receptorG-protein interaction. For example, the b2-AR couples to the sodium hydrogen exchanger regulatory factor via direct interaction between the factor and a small region in the distal portion of the C-terminal cytoplasmic tail of the receptor. Another method of signaling without the need for efcient G-protein

G-PROTEIN-COUPLED RECEPTORS 251

levels of agonist additionally activate the more receptor-specic GRK-dependent kinases. Evidence is mounting that other kinases (e.g., tyrosine kinases and casein kinase 1a) may phosphorylate GPCRs as well, although the effect of these events on GPCR signaling is less well known. Internalization is a mechanism of desensitization whereby a GPCR is translocated from the plasma membrane to an internal cellular compartment where it is recycled or degraded. It is also where dephosphorylation occurs, and is often considered a mechanism of resensitization, if the receptor is rapidly recycled to the cell surface rather than degraded. The process is governed by the interaction of multiple determinants that include receptor structure, the nature of the activating ligand, the cell type expressing the receptor, and the surrounding cellular environment. No consensus structural motif for internalization has been identied, and the relevant structural determinants appear to vary among GPCR family members. For some GPCRs, internalization requires arrestin interaction; thus receptor phosphorylation and internalization can be intimately linked. Downregulation occurs following prolonged receptor activation and differs from internalization in that the total receptor pool (not just the cell surface receptors) is reduced. Downregulation may also be evoked by second messengers (i.e., cAMP/PKA activation by other GS-coupled receptors downregulates b2-AR). Molecular mechanisms responsible for mediating downregulation include decreased receptor synthesis rates due to transcriptional, translational, or posttranslational changes, and increased rates of receptor degradation. The genetic or structural determinants that affect downregulation may therefore reside outside the coding region of the gene, such as in the promoter or 30 untranslated region. However, as with other aspects of GPCR regulation, structural features of the receptor protein are also important. This is exemplied in the human b2-adrenergic receptor where a single N-linked glycosylation site is important for downregulation, and naturally occurring polymorphisms in the coding region differ in their resistance or sensitivity to downregulation.

important targets for therapeutic agents, with estimates that up to 50% of modern drugs target GPCRs. Important examples for pulmonary disease include the use of b2-AR agonists, muscarinic antagonists, and leukotriene antagonists for the treatment of asthma and chronic obstructive pulmonary disease, and the use of endothelin receptor antagonists and prostacyclin receptor agonists for treatment of pulmonary hypertension. The intimate relationship between GPCR structure and its function further suggests that differences within a GPCR due to genetic variation (mutations or polymorphisms) could potentially impact physiology, pathophysiology, and therapeutic responses. For example, polymorphisms in the b2-AR appear to have affects on risk, disease phenotypes, and response to therapy in the treatment of asthma.
See also: Acetylcholine. Adrenergic Receptors. Asthma: Overview. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Cyclic Nucleotide Phosphodiesterases. Fluid Balance in the Lung. Histamine. Ion Transport: Overview. Lipid Mediators: Lipoxins. Neurophysiology: Neural Control of Airway Smooth Muscle. Signal Transduction. Smooth Muscle Cells: Airway; Vascular.

Further Reading
Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Phamacology Reviews 53: 124. Filipek S, Stenkamp RE, Teller DC, and Palczewski K (2003) G protein-coupled receptor rhodopsin: a prospectus. Annual Review of Physiology 65: 851879. Filipek S, Teller DC, Palczewski K, and Stenkamp R (2003) The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors. Annual Review of Biophysics and Biomolecular Structure 32: 375397. Foord SM, Bonner TI, Neubig RR, et al. (2005) International Union of Pharmacology. XLVI. G protein-coupled receptor list. Phamacology Reviews 57: 279288. Kenakin T (2002) Drug efcacy at G protein-coupled receptors. Annual Review of Pharmacology and Toxicology 42: 349379. Lefkowitz RJ (2004) Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends in Pharmacological Sciences 25: 413422. Liggett SB (2000) Pharmacogenetics of beta-1- and beta-2adrenergic receptors. Pharmacology 61: 167173. Marchese A, Chen C, Kim YM, and Benovic JL (2003) The ins and outs of G protein-coupled receptor trafcking. Trends in Biochemical Sciences 28: 369376. Palczewski K, Kumasaka T, Hori T, et al. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289: 739745. Pitcher JA, Freedman NJ, and Lefkowitz RJ (1998) G proteincoupled receptor kinases. Annual Review of Biochemistry 67: 653692. Small KM, McGraw DW, and Liggett SB (2003) Pharmacology and physiology of human adrenergic receptor polymorphisms. Annual Review of Pharmacology and Toxicology 43: 381411.

Biological Function
GPCRs regulate a wide variety of physiological and compensatory functions in the lung including maintenance of bronchomotor and vasomotor tone, secretory and absorptive function of the bronchial and alveolar epithelium, cell growth and regeneration, ciliary function, and mediator release by both resident and circulating inammatory cells. It is thus not surprising that GPCRs have been and continue to be

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GRANULOMATOSIS
Contents

Lymphomatoid Granulomatosis Wegeners Disease

Lymphomatoid Granulomatosis
M I Schwarz and G P Cosgrove, University of Colorado Health Sciences Center, Denver, CO, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Lymphatoid granulomatosis (LG) is a rare lymphoproliferative disorder of unknown etiology affecting multiple sites, most notably within the lungs, skin, and central nervous system. The clinical presentation is similar to a systemic vasculitis, thereby necessitating a denitive evaluation to differentiate the two entities, as LG is now believed to be a malignant lymphoma. Characteristic of LG is a triad of histology including: (1) polymorphic lymphocytic inltrates with small lymphocytes, plasma cells, and atypical mononuclear cells termed immunoblasts, (2) angioinvasion with transmural inltration of arteries and veins by T lymphocytes, previously termed angiitis, and (3) focal necrosis within the vessel walls, termed granulomatosis. In association with the majority of cases is the concurrent or recent infection with EpsteinBarr virus (EBV). Optimal therapy has yet to be determined but chemotherapy, usually corticosteroids with cyclophosphamide, has been successful in some patients, especially in the more aggressive variants. In light of the association of EBV and clonal proliferation of B lymphocytes, novel antiviral therapy and B-cell-specic therapies have been used with success.

malignant lymphoma, with B- and T-cell proliferation. More specifically, it is thought to be a T-cellrich B-cell lymphoreticular disorder. The majority of cases are caused by neoplastic transformation of B lymphocytes by EpsteinBarr virus (EBV) infection, with reactive T-lymphocyte proliferation. Isolated cases of T-lymphocyte predominant proliferation have been reported in the absence of evidence to suggest recent EBV infection. In these cases, the disorder may represent an isolated peripheral T-cell lymphoma.

Pathology
A triad of histology characterizes LG: (1) polymorphic lymphocytic inltrates with small lymphocytes, plasma cells, and atypical mononuclear cells termed immunoblasts, (2) angioinvasion with transmural inltration of arteries and veins by T lymphocytes, previously termed angiitis, and (3) focal necrosis within the vessel walls, termed granulomatosis (see Figure 1). The T lymphocytes appear to be reactive and typically are polyclonal without significant atypia. The large, atypical mononuclear cells, or immunoblasts, frequently have significant cellular atypia and represent transformed B lymphocytes

Introduction
Lymphomatoid granulomatosis (LG) is an uncommon, multisystem, immunoproliferative disorder of uncertain etiology. It was rst described as a distinct clinicopathologic entity by Liebow and colleagues in 1972. LG is characterized by angiocentric lymphoproliferation of T and B lymphocytes, with the lung being the most common organ of involvement. Divergent pathogenic mechanisms such as autoimmunity, postviral lymphoproliferation, and malignant conversion of B and T lymphocytes have been suggested. As a result, the terms angiocentric immunoproliferative lesion and angiocentric lymphoma have also been used to describe this disorder. The preferred term remains LG.

Etiology
The pathogenesis of LG remains unclear but the current body of evidence suggests that it is a form of a

Figure 1 High-resolution computed tomogram in a 58-year-old female with LG. Multiple subcentimeter nodules adjacent to the bronchovascular bundle (black arrows) as well as within the interlobular septa were identied (white arrows). A condent diagnosis was obtained following a surgical lung biopsy.

GRANULOMATOSIS / Lymphomatoid Granulomatosis

253

secondary to EBV infection, based on immunophenotyping and EBER1/2 RNA in situ hybridization. Histopathologic grading systems based on the presence of EBV-positive cells, cellular atypia, and the degree of necrosis have been described, but their clinical usefulness is limited due to inconsistency and poor correlations with outcome.

Table 1 Common presenting symptoms and signs in patients with lymphomatoid granulomatosis Symptom/sign Cough Fever Rash/nodules Malaise Weight loss Neurologic abnormalities Central nervous system symptoms Cranial nerve abnormalities Peripheral nerve abnormalities Dyspnea Splenomegaly Chest pain Hepatomegaly Lymphadenopathy 152 cases (%) 56 58 39 35 35 30 19 11 7 30 18 13 12 8

Clinical Features
LG is a rare disease affecting multiple organs during its course. Based on the original report by Liebow and colleagues, and then a continuation by Katzenstein and colleagues, patients of all ages may be affected. The usual age of onset is between 40 and 60 years with a mean of 48 years. Rarely, it may also affect children. Males are affected twice as often as females. LG occurs in individuals who are otherwise healthy, with the exception of sporadic cases in which it is associated with primary or secondary immunodeciency disorders such as WiskottAldrich syndrome or postchemotherapy immunodeciency or organ transplantation, HIV, and human T-cell leukemia virus infections. This association has led some to suggest that patients who develop LG have a selective immunodeciency for controlling EBV. Pulmonary manifestations occur in all patients at some point during the course of their disease. The most common extrapulmonary sites of involvement are within the skin and nervous system. Manifestations within the spleen, liver, lymph nodes, kidney, adrenal gland, and heart were also noted at the time of autopsy by Katzenstein and colleagues but did not result in clinically significant symptoms or signs. It is in this manner that it contrasts to Wegeners granulomatosis. Pulmonary involvement manifests as cough, dyspnea, and chest pain (see Table 1). Systemic symptoms include weight loss, fever, and night sweats. Skin lesions are occasionally the only presenting manifestation, but they are often found in conjunction with the pulmonary disease in 2040% of cases. These are both dermal and subcutaneous nodules, which may be generalized but are more commonly localized to the lower extremities. Although typically nontender, ulcerative, tender, and erythematosus lesions resembling erythema nodosum may occur. Neurologic involvement occurs in up to 30% of cases and can result in peripheral neuropathy, cranial nerve palsies, or direct involvement of the brain with seizures. Destructive nasopharyngeal and paranasal sinus involvement may also occur and, in the setting of nodular pulmonary disease, cannot be distinguished from Wegeners granulomatosis in the absence of a surgical lung biopsy. The presence of clinical manifestations in organ systems other than

Reproduced from Cancer; Katzenstein AL, Carrington CB, and Liebow AA; Copyright & 1979, Wiley. Reprinted with permission of John Wiley & Sons, Inc.

the lungs is associated with more malignant lymphomatous disease and a poorer prognosis. There are no characteristic laboratory abnormalities associated with LG. Non-specic immunodeciencies, such as anergy and impairment in in vitro antigen stimulation assays, were reported by Sordillo and colleagues. Hypergammaglobulinemia (IgG and IgM) was noted in 47% of patients reported by Katzenstein and colleagues. Serologies for systemic autoimmune diseases and systemic vasculitides are typically unremarkable. EBV is detected in the majority of patients. Chest radiography shows primarily middle- and lower-zone involvement with multiple nodules, which have a tendency to coalesce and cavitate (see Figure 2). Alveolar inltrates and diffuse reticular opacities may also occur. Hilar and mediastinal adenopathy does not occur in LG, in contrast to other pulmonary lymphomas, primary or secondary. High-resolution computed tomography demonstrates diffuse, poorly dened nodular inltrates in a peribronchovascular distribution or within the interlobular septa. Thin-walled cysts in association with nodular inltrates may also be seen. An accurate diagnosis of LG is dependent upon histologic inspection of specimen from an involved site, most commonly being the lung. In light of similar clinical and radiographic abnormalities, it must be differentiated from Wegeners granulomatosis, lymphocytic interstitial pneumonia, malignant primary or secondary pulmonary lymphoma, or extranodal NK/T-cell lymphoma, nasal type, which is also associated with EBV infection but rarely involves the

254 GRANULOMATOSIS / Lymphomatoid Granulomatosis

Figure 2 Histologic appearance of lymphomatoid granulomatosis. Photomicrograph demonstrating prominent perivascular lymphocytic inammation (a). The cellular composition was determined using immunohistochemistry. CD3 ( ) T lymphocytes (b) were more prominent than CD20 ( ) B lymphocytes in this case (c). Few atypical cells, the absence of necrosis, and a negative EBER1/2 RNA in situ hybridization resulted in a classication of low-grade LG. Original magnications: (a) 200 ; (b and c) 100 .

lung. Immunohistochemistry studies serve to dene the repertoire of B and T lymphocytes and in situ hybridization for EBV provide corroborative evidence to support a diagnosis of LG.

Acknowledgment
This work is supported by the NIH (NHLBI SCOR HL67671).
See also: Granulomatosis: Wegeners Disease. Leukocytes: T cells. Tumors, Malignant: Lymphoma. Vasculitis: Overview.

Animal Models
Animal or other biologic systems modeling LG have yet to be developed. The disease has been reported in felines, canines, and horses, but these were spontaneous cases similar to the human disease.

Further Reading
Cosgrove GP, Fessler MB, and Schwarz MI (2003) Lymphoplasmocytic inltrations of the lung. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, 4th edn., pp. 825847. Malden, MA: Blackwell Science. Fauci AS, Haynes BF, Costa J, Katz P, and Wolff SM (1982) Lymphomatoid granulomatosis. Prospective clinical and therapeutic experience over 10 years. The New England Journal of Medicine 306(2): 6874. Isaacson PG (1990) Lymphomatoid granulomatosis. British Medical Journal 300(6724): 612. Jaffe ES and Wilson WH (1997) Lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications. Cancer Surveys 30: 233248. Jaffe ES and Wilson WH (2001) Lymphomatoid granulomatosis. In: Jaffe ES, Harris NL, Stein H, et al. (eds.) World Health Organization Classication of Tumors. Pathology and Genetics of Tumors of Haematopeotic and Lymphoid Tissues, 1st edn., p. 185. Lyon, France: IARC Press. Katzenstein AL, Carrington CB, and Liebow AA (1979) Lymphomatoid granulomatosis: a clinicopathologic study of 152 cases. Cancer 43(1): 360373. Lee JS, Tuder R, and Lynch DA (2000) Lymphomatoid granulomatosis: radiologic features and pathologic correlations. American Journal of Roentgenology 175(5): 13351339. Liebow AA, Carrington CR, and Friedman PJ (1972) Lymphomatoid granulomatosis. Human Pathology 3(4): 457558. Myers JL, Kurtin PJ, Katzenstein AL, et al. (1995) Lymphomatoid granulomatosis. Evidence of immunophenotypic diversity and relationship to EpsteinBarr virus infection. The American Journal of Surgical Pathology 19(11): 13001312. Sordillo PP, Epremian B, Koziner B, Lacher M, and Lieberman P (1982) Lymphomatoid granulomatosis: an analysis of clinical and immunologic characteristics. Cancer 49(10): 20702076. Wilson WH, Kingma DW, Raffeld M, Wittes RE, and Jaffe ES (1996) Association of lymphomatoid granulomatosis with

Prognosis and Current Therapy

The clinical course of LG varies from benign, selflimited disease to malignant lymphoma. The majority of cases are progressive and requires therapy. Survival ranges from 14 to 72 months from the time of diagnosis. In those with malignant variants, mortality approaches 60% at 1 year. Poor prognostic features include central nervous system involvement, highgrade atypia, younger age, and hepatosplenomegaly. In those without extensive systemic involvement, prolonged survival may occur without treatment. Optimal therapy has yet to be determined for those with progressive disease. Aggressive therapy consisting of a combination of corticosteroids and cyclophosphamide resulted in durable remission in approximately 50% of patients. Antiviral therapy, specifically interferon-a2b, has been used in light of the association of LG and EBV. In a small series by Wilson and colleagues, 3/4 patients achieved durable remission at 40 months following therapy. More recently, therapy directed against transformed B lymphocytes, using the anti-CD20 antibody, rituximab, has shown promise with minimal toxicity. Future studies will need to address the efcacy of such novel therapies as interferon-a2b and rituximab in a larger cohort of patients.

GRANULOMATOSIS / Wegeners Disease 255

EpsteinBarr viral infection of B lymphocytes and response to interferon-alpha 2b. Blood 87(11): 45314537. Zaidi A, Kampalath B, Peltier WL, and Vesole DH (2004) Successful treatment of systemic and central nervous system lymphomatoid granulomatosis with rituximab. Leukemia & Lymphoma 45(4): 777780.

Wegeners Disease
W L Gross, P M Aries, and P Lamprecht, University Hospital Schleswig Holstein, Luebeck, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Wegeners granulomatosis (WG) is a potentially life-threatening chronic inammatory disease of as yet unknown etiology, characterized by necrotizing granulomatous lesions and an autoimmune vasculitis mediated by antineutrophil cytoplasmatic autoantibody (ANCA). The histopathologic hallmark is the triad of granulomatous inammation, necrosis and vasculitis. WG usually takes a biphasic course starting in the upper and/or lower respiratory tract (so-called localized WG) with subsequent generalization with prominent clinical features of systemic vasculitis. Approximately 95% of patients with active generalized WG are ANCA positive, which is usually directed against Wegeners autoantigen proteinase 3 (PR3). In vitro and animal models suggest that the interaction of ANCA with cytokine-primed neutrophils results in premature activation, respiratory burst, and degranulation of neutrophil granulocytes with subsequent endothelial cell damage and necrotizing vasculitis. Altered T-cell responses with predominant Th1-type cytokine release might facilitate autoantigen recognition in WG. WG is a multifocal inammatory disease that affects most often the upper and lower tract and the kidneys, but involvement of any other organ such as eye, skin, heart, gastrointestinal tract, central and peripheral nervous system may occur and give rise to serious or life-threatening complications. In few cases involvement of the upper and/or lower respiratory tract may be the only manifestation for years (localized WG). Cyclophosphamide plus steroids (NIH-scheme) is the standard therapy for the induction of remission in severe organ- or life threatening disease followed by azathioprine or other drugs for the maintenance of remission. In localized and early systemic WG trimetoprim-sulfamethoxazole and/or less aggressive immunosuppressions (e.g., MTX) can induce remission.

disease courses and even long-term variants restricted to the upper respiratory tract in the absence of kidney involvement. Fifty years ago, Carrington and Liebow introduced the term limited WG in order to dene disease stages based on clinical and pathological ndings. Today, the European Vasculitis Study Group (EUVAS) distinguishes localized (i.e., WG restricted to the respiratory tract) and early systemic WG (i.e., nonimminent WG without renal organ involvement) from generalized WG. In most patients the disease progresses to generalized WG, but in a smaller fraction of patients the disease remains localized or early systemic for years. In generalized WG the upper and/or lower respiratory tract and the kidneys are involved in 80% of the patients. Rapidly progressive glomerulonephritis and pulmonary hemorrhage (pulmonary-renal syndrome) are typical symptoms of life-threatening full-blown WG (Table 1). Highly specic antineutrophil cytoplasmatic autoantibodies targeting Wegeners autoantigen PR3 (PR3-ANCA) are detected in about 95% of patients with generalized WG. A large number of in vitro studies and two recently published in vivo studies provide evidence that systemic vasculitis is induced by antineutrophil cytoplasmatic autoantibody (ANCA).

Etiology
The etiology of WG is still subject to intensive research. The detection of ANCA directed to proteinase 3 (PR3-ANCA) is highly specic for WG, but incomplete knowledge is achieved about how granulomatous lesions, autoimmune vasculitis, and PR3ANCA evolve. Infections and other environmental inuences may play a role in triggering or maintaining disease activity on the basis of a genetic predisposition. Recently, a strong association of HLADPB1*0401 has been reported. Epidemiological studies have revealed an association of noninfectious agents such as silica and exposure to farming with WG. Chronic nasal carriage of Staphylococcus aureus is an independent risk factor for a relapse. Animal models and anecdotal reports suggest that further infectious agents might have a role in triggering disease activity but denitive proof has been lacking so far. The risk of relapses in the respiratory tract can significantly be reduced by the use of trimethoprimsulfamethoxazole.

Introduction
Wegeners disease (WG) is a potentially organ- and life-threatening autoimmune disease of as yet unknown etiology characterized by granulomatous lesions and a necrotizing vasculitis predominantly affecting small vessels, that is, arterioles, capillaries, and venules. A prevalence of 23.7 per million adults has been reported in Europe. The peak incidence is in the fourth and fth decade of life. The overall incidence ranges between 2.9 and 12/106/year depending on the geographic region. In recent years, early diagnosis has led to the recognition of less aggressive

Pathology
WG is characterized by the classic pathological triad of granulomatous inammation predominantly of the

256 GRANULOMATOSIS / Wegeners Disease

Table 1 Differential diagnosis of the pulmonary syndrome: distinction is dependent on pathomechanism Diagnostic feature Antibasalmembrane-antibodies Immunocomplexes Immunohistochemistry Linear immunodeposits Granular immunodeposits Disease Goodpasture syndrome Idiopathic alveolar hemorrhagic syndrome Systemic lupus erythematodes Essential mixed cryoglobulinemic vasculitis nlein purpura HenochScho IgA-nephritis Idiopathic alveolar hemorrhagic syndrome Wegeners granulomatosis Microscopic polyangiitis ChurgStrauss syndrome

ANCA (PR3-ANCA/ MPO-ANCA)

Negative or minimal immunodeposits (pauci-immune)

respiratory tract, necrotizing glomerulonephritis, and systemic vasculitis. The classical triad is most often found in specimens from open lung biopsies. However, not all elements of the triad may be seen and only one or two features may be detected in the biopsies. Renal granulomas are rare and found in o10% of kidney biopsies. Granulomatous lesions are built up by CD4 and CD8 T cells, CD28 T cells, histiocytes, CD20 B lymphocytes, neutrophil granulocytes, macrophages, and multinucleated giant cells surrounding an area of central necrosis. The term geographic necrosis is used for a conuent or serpiginous pattern of the central necrosis. Geographic necrosis and histiocytes lined up in a pallisading fashion are characteristic morphological features most often found in open lung biopsies. Scattered eosinophils are seen and sometimes may cause difculties in distinguishing WG from ChurgStrauss syndrome. The necrotizing vasculitis affects predominantly small vessels and mediumsize arteries, that is, capillaries, venules, arterioles, and arteries. Endothelial cells are the target of the initial injury. The earliest changes affect the vascular endothelium with swelling, necrosis, and platelet deposition. In the lung capillaries, venules and arterioles are inltrated by polymorphonuclear leukocytes. Pulmonary microvascular necrotizing vasculitis (capillaritis) is the cause of pulmonary hemorrhage. In the kidney, rupture of the basement membrane subsequent to neutrophil degranulation gives rise to glomerular capillary thrombosis followed by segmental necrosis of the tuft. Necrotic material, blood, and brin spill into the Bowmans space. As a consequence, accumulation and proliferation of monocytes and parietal (Bowmans) epithelial cells with the formation of crescents is seen. The growing crescent compresses the capillary tuft, leading to segmental and global loss of circulation through the glomerulus. In WG, the histological picture of vasculitis is polymorphic. Apart from necrotizing vasculitic lesions, leukocytoclastic vasculitis of the skin may be seen as cutaneous vasculitis

manifestation. Necrotizing granulomatous arteritis involving medium-sized vessels is commonly found close to large necrotizing granulomatous foci in the lung. Nongranulomatous brinoid necrosis may affect bronchial arteries. Finally, vasculitis morphologically identical to that seen in polyarteritis, both in size of the vessels involved and the presence of brinoid necrosis, may also be seen in WG in some cases. No or only a few immunodeposits are detected in glomerular lesions, hence the name pauci-immune vasculitis.

Clinical Features
Localized Wegeners Granulomatosis

Localized WG has been dened by the EUVAS as WG restricted to the upper and/or lower respiratory tract (Table 2). Retrospective studies revealed that 50% of WG patients initially present with solitary ear, nose, and throat (ENT) involvement. The duration of the localized disease stage may last from months to several years. Typically symptoms are nasal obstruction by mucosal swelling, serosanguinous discharge, and epistaxis. Nasal crusting, mucosal ulcerations, hoarseness, sinusitis, or mastoiditis are further typical symptoms of upper respiratory tract involvement. Persistent disease activity causes saddle-nose deformity, septum perforation, and subglottic tracheal stenosis (Figure 1). Proptosis may be a sign of retrobulbary granuloma, often due to per continuum inltration from the sinus. Granulomatous inammation of the lower respiratory tract may lead to bronchus stenosis, atelectasis, and obstructive pneumonia. Localized WG usually progresses to early systemic or generalized WG in over 95% of cases within weeks or months.
Generalized Wegeners Granulomatosis

Early systemic WG has been dened by the EUVAS as WG with any organ involvement except renal or

GRANULOMATOSIS / Wegeners Disease 257

Table 2 Clinical subgroups of Wegeners granulomatosis according to the definitions of the European Vasculitis Study Group (EUVAS) Subgroup Localized WG Early systemic WG Generalized WG Severe renal WG Refractory WG Organ involvement Upper and/or lower respiratory tract Any except renal involvement or imminent organ failure Renal with serum creatinine p500 mmol l 1 and/or other imminent organ failure Renal with serum creatinine 4500 mmol l 1 Progressive disease despite therapy with GC ad Cyc Constitutional symptoms No Yes Yes Yes Yes Presence of ANCA Yes/No Usually yes Yes Yes Yes/No

WG, Wegeners granulomatosis; GC, glycocorticoids; Cyc, cyclophosphamide.

Figure 1 Wegeners granulomatosis with saddle-nose deformity.

Figure 2 Wegeners granulomatosis with scleritis.

imminent vital organ failure. Generalized WG has been dened as WG including renal involvement and/ or imminent organ failure. Classical WG usually takes a biphasic course, starting in the respiratory tract (localized WG), followed by subsequent generalization of the disease. Generalization is often heralded by constitutional symptoms (weight loss, fever, and night-sweat) and rheumatic complaints such as migratory-arthralgia and myalgia. Otological manifestations include sensorineural hearing loss, vestibular impairment, and facial nerve palsy due to otitis media, mastoiditis, or neuropathy. Relapsing polychondritis may occasionally be encountered. Retroorbital masses, mostly developing by granulomatous inammation per continuum of the sinus, may cause proptosis, compression of the optic nerve, and progressive visual loss. Dacryocystoadenitis and nasolacrimal duct inammation are peculiar organ manifestations of WG and are usually not found in other vasculitides. Episcleritis presenting as red eye, uveitis, retinal exudates, optic nerve vasculitis, and retinal artery occlusion reect occular vasculitic lesions (Figure 2). Pulmonary involvement is one of the cardinal features in WG and occurs in over 80% of cases

during the course of the disease. Even if pulmonary symptoms are clinically absent, 30% may have radiographically demonstrable pulmonary lesions. Abnormalities of pulmonary function such as reduced CO diffusion may be early signs of pulmonary involvement. In case of subglottic stenosis an inspiratory stridor or severe dyspnea may be present. Frequently, early complaints related to the lower respiratory tract are cough, dyspnea, pleuritic pain, or hemoptysis. Bland hemoptysis or alveolar hemorrhage with respiratory insufciency and anemia reects pulmonary capillaritis and has a high mortality rate (Figure 3). Multiple pulmonary nodules may be asymptomatic; however, solitary granulomatous masses can cause severe bronchial stenosis. Spontaneous pneumothorax may be induced by rupture of a subpleural cavity nodule. Pleural effusions and mediastinal or hilar adenopathy are less common. Cardiac manifestations are pericarditis with pericardial effusion, myocarditis, and coronaritis with or without myocardial infarction and rarely subsequent cardiomyopathy. Renal involvement ranges from asymptomatic hematuria to rapidly progressive glomerulonephritis.

258 GRANULOMATOSIS / Wegeners Disease

Cytokine-primed neutrophils release PR3 from azurophilic granula, and express PR3 on the cell surface. Binding of PR3-ANCA to the membrane-located PR3 induces further premature activation and degranulation of the cells with subsequent endothelial damage and induction of necrotizing vasculitis (ANCA-cytokine-sequence theory) (Figure 4). Recently, murine transfer models showing the in vivo induction of a necrotizing systemic vasculitis and glomerulonephritis by myeloperoxidase-specic (MPO)-ANCA and exacerbation of inammatory panniculitis by PR3-ANCA have further underscored the pathophysiological role of ANCA in inducing vasculitis.
Figure 3 Wegeners granulomatosis: clinical presentation with the recent development of hemoptysis. CT scan shows classical signs of diffuse alveolar hemorrhage.

Diagnosis
Diagnosis of WG is based on clinical manifestations, serological parameters, and histomorphological ndings. The classication criteria of the American College of Rheumatology (ACR), dening clinical criteria for distinguishing patients with WG from those with other vasculitides, were proposed in 1990 before the implementation of ANCA as a routine diagnostic procedure and are not intended to be used as diagnostic criteria (Table 3). Non-specic serological parameters indicative of inammation such as elevated erythrocyte sedimentation rate and C-reactive protein are usually found. With regard to ANCA highest specicity (99%) and sensitivity (73%) can be reached when the results of the immunouorescence (IFT) and enzyme-linked immunosorbent assay (ELISA) technique are combined. In IFT a cytoplasmic pattern C-ANCA is found in 50% of patients with localized WG and in 95% with generalized WG. The target autoantigen detected by ELISA is PR3 in X95% of WG patients. Few patients (p5%) present with an MPO-ANCA. Tissue biopsies should be obtained from easily accessible symptomatic organs such as the upper or lower respiratory tract. Transbronchial biopsies are seldom representative, whereas nasal biopsies produce a much higher yield of representative biopsies. However, most often specimens from open lung biopsies exhibit the classic pathological triad of granulomatous inammation, necrosis, and vasculitis. Kidney biopsies typically provide evidence of segmental necrotizing glomerulonephritis with little or no immunoglobulin deposition (pauciimmune) which is characteristic of ANCA-associated vasculitis. Disease activity, extent, and damage can be scored with validated instruments such as the Birmingham vasculitis activity score (BVAS), the disease extent index (DEI), and the vasculitis damage index (VDI) (Table 4). ANCA titers can be used for the follow-up of patients: a fourfold or greater rise in the titer may indicate an imminent relapse and should be

A nephritic urine sediment (microscopic hematuria with red cell casts and glomerular selective or unselective proteinuria) and reduction of creatinine clearance demonstrate renal involvement. In over 50% of cases neurological involvement is seen as a consequence of vasculitis or compression by mass-like lesions or frank vasculitis. In most patients, sensory peripheral neuropathy or motor mononeuritis multiplex occurs within the rst 2 years. An effect on the CNS is less frequent (about 10%). Further manifestations, including involvement of the gall bladder, ovaries, parotis, and breast among others, add to the broad spectrum of symptoms.

Pathogenesis
American pathologist Fienberg suggested that WG starts as granulomatous disease in the upper and/or lower respiratory tract with subsequent development of vasculitis. A predominance of T-helper-1 (Th1)type cytokines is seen in early granulomatous lesions in localized WG whereas this is less evident in generalized WG. Disease progression is associated with a switch or further complexity of the collective T-cell response with the appearance of a subset of Th2-type cells. This switch or increasing complexity of the cytokine prole might be a consequence of further Bcell expansion and T-cell-dependent PR3-ANCA production during disease progression. Infections such as S. aureus, other environmental inuences, or Wegeners autoantigen PR3 itself are thought to play a role in triggering and/or maintaining disease activity in Wegeners granulomatosis on the basis of a genetic predisposition to an exaggerated Th1-type response early in the disease process. Neutrophils become activated by tumor necrosis factor alpha (TNF-a).

1 Resting stage

2 PR3 expression on the cell surface sPR3 PR3 TNFTNF-

3 Expression of adhesion molecules

PR3 ANCA

IL-8, IL-1

PR3 PR3

IL-8, IL-1 LFA-1 LFA-1

siCAM-1

PR3

CAM-1

E-selectin

VCAM-1

Endothelial cells: resting 4 Ligation and activation

Activated 5 Destruction and granuloma formation

PR3 PR3 ANCA PR3 , AT F RII LTB4 O2

HLE PR3

PR3-ANCA-IC

.1AT sPR3

Figure 4 ANCA-cytokine sequence theory. 1, Resting polymorphonuclear granulocytes (PMNs) with proteinase-3 (PR3) stored in vesicles. 2/3, Activation of PMNs and endothelial cells by proinammatory cytokines resulting in expression of adhesion molecules and translocation of the intracytoplasmic PR3 on the cell surface. 3/4, Adhesion of PMN on endothelial cells. 4/5, Binding of ANCA to membrane-located PR3 leads to further activation and is followed by degranulation of PMNs. Thus, toxic radicals and lytic proteins are released near the endothelial cells, out of reach for proteinase inhibitors like alpha-1 antitrypsin. Subsequently, endothelial damage and necrotizing vasculitis takes place.

260 GRANULOMATOSIS / Wegeners Disease

followed by close monitoring of the patient for clinical signs of WG activity.

Therapy
In severe organ- and life-threatening WG induction of remission with the NIH-standard or so-called

Table 3 Definitions and criteria for Wegeners granulomatosis The American College of Rheumatology proposed clinical classication criteria (vasculitis must be present) for Wegeners granulomatosis (1990): * Nasal or oral inammation (painful or painless oral ulcers or purulent or bloody nasal discharge) * Abnormal chest radiograph showing nodules, xed inltrates, or cavities * Abnormal urinary sediment (microscopic hematuria with or without red cell casts) * Granulomatous inammation on biopsy of an artery or perivascular area Definition and classication of Wegeners granulomatosis according to the Chapel Hill Consensus (1994): * Granulomatous inammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels (i.e., capillaries, venules, arterioles, and arteries) * Necrotizing glomerulonephritis is common * Cytoplasmic pattern ANCA (C-ANCA) with antigen specicity for proteinase 3 (PR3) is a very sensitive marker for Wegeners granulomatosis

Faucis scheme remains the gold standard, with oral cyclophosphamide (2 mg kg 1 day 1 po for 3 days) and steroids (initially 12 mg kg 1 day 1 prednisolone iv or orally, with subsequent tapering of dose) given for 36 months. Data from a recent EUVAS trial (CYCAZAREM) provided evidence that azathioprine is equally effective for the maintenance of remission. Therefore, azathioprine should be regarded as the treatment of choice for maintenance therapy. The use of mesna is recommended for prophylaxis of the cyclophosphamide urotoxicity. Several trials analyzed the potential benet of plasmapheresis in patients with severe renal involvement. Preliminary data from the latest EUVAS trial (MEPEX) analyzing initial plasmapheresis versus intravenous high-dose methyl-prednisolone (1 g) in addition to standard treatment with cyclophosphamide and steroids showed higher efcacy of plasmapheresis in patients with a creatinine of 4500 mmol l 1. Disease and treatment-related mortality was shown to be high in the subgroup of patients in this trial. In non-life-threatening, nonrenal early systemic WG, MTX can be used. The efcacy of other agents (leunomide, mycophenolate mofetil, and cyclosporin A) has been reported in case reports or smaller case series. The optimal duration of maintenance therapy is the subject of another presently ongoing EUVAS study (REMAIN) comparing 2 years versus 5 years of maintenance remission with azathioprine.

Table 4 Organ manifestations of Wegeners granulomatosis and common disease activity, extent and damage scores Organ manifestation ELK classication Disease extent index (DEI) 2 2 2 na 2 2 2 2 2 2 Birmingham vasculitis activity score (BVAS) 6 6 12 na 6 6 2 9 9 6 Vasculitis damage index (VDI) 6 7 3 1 2 7 5 6 1 15

ENT Lung Kidney Mucous membrane Skin Eye Joint/muscles Central nervous system Peripheral nervous system Cardiovascular system/ peripheral vascular disease Intestine Constitutional symptoms Side effect of therapy Maximal score

E L L na S Ey A C P H

Gi B na na

2 1 na 21

9 3 na 63

4 na 6 64

na, not applicable. ELK: reecting the disease extent without weighting scoring. DEI: every organ involved that is attributable to active WG is added up. BVAS: disease features are scored when they are due to active vasculitis. Scores have been weighted according to the severity. Each section has a different maximum score. VDI: this is for recording organ damage that has occurred in patients since the onset of vasculitis.

GRANULOMATOSIS / Wegeners Disease 261

For prophylaxis, treatment with trimetoprim-sulfamethoxazole reduces S. areus colonization in the respiratory tract and thereby reduces the risk of respiratory relapses. Trimetoprim-sulfamethoxazole also induces remission in localized WG. The primary goal of all therapies is to achieve remission. Using the BVAS, remission is dened as the absence of signs of new or worse disease activity and persistent disease activity for no more then one item on the BVAS evaluation form. Some disease manifestations regress completely, but others do not such as nasal septal perforation or persistent defect proteinuria after glomerulonephritis. Despite the efcacy of the above-mentioned regimen in the majority of patients, up to 10% achieve no complete remission and/or early relapses occur in up to 50% of patients. Thus, the aim of new treatment options is to combine improved efcacy with less toxicity. TNF-a inhibitors inximab and etanercept, anti-CD20 antibody rituximab, azathioprine pulse therapy, highdose cyclophosphamide, anti-thymocyte globin, intravenous immunoglobulins, 15-deoxyspergualin, and humanized monoclonal anti-CD52 or anti-CD4 antibodies have been reported to be benecial in refractory WG in smaller case series. One trial found no significant differences between the addition of etanercept to standard treatment and standard treatment alone in the rates of sustained remission in nonrefractory WG (WGET study by eight American centers). However, it has been shown that TNF-a production is elevated in active and refractory WG. TNF-a levels are normalized with successful standard treatment. Therefore, as suggested by these in vitro data, patients responding to standard treatment will have no further benet from the addition of a TNF-a blocking agent, whereas patients refractory to standard treatment will benet from the addition of a TNF-a inhibitor. In line with this consideration are data from an open-label, multicenter, prospective clinical trial by Booth, et al. who successfully induced remission with iniximab in addition to standard or existing treatment regimens both in active and refractory patients (n 32). Further, higher rates of nonsustained remission in the WGET trial might have been caused by early tapering of steroids in that study.
See also: Alveolar Hemorrhage. Corticosteroids: Therapy. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpastures Syndrome. Vasculitis: Microscopic Polyangiitis.

Further Reading
Jagiello P, Gencik M, Arning L, et al. (2004) New genomic region for Wegeners granulomatosis as revealed by an extended association screen with 202 apoptosis-related genes. Human Genetics 114: 468477. Jayne D for the European Vasculitis Study Group (EUVAS) (2001) Update on the European Vasculitis Study Group trials. Current Opinion in Rheumatology 13: 4855. Jayne D, Rasmussen N, Andrassy K, et al., for the European Vasculitis Study Group (2003) A randomized trial of maintenance therapy for vasculitis associated with antineutrophil autoantibodies. New England Journal of Medicine 349: 3644. Jennette JC and Falk RJ (1997) Small-vessel vasculitis. New England Journal of Medicine 337: 15121523. Jennette JC, Falk RJ, Andrassy K, et al. (1994) Nomenclature of systemic vasculitides. Proposal of an International Consensus Conference. Arthritis and Rheumatism 37: 187192. Komocsi A, Lamprecht P, Csernok E, et al. (2002) Peripheral blood and granuloma CD4 CD28 T-cells are a major source of IFN-g and TNF-a in Wegeners granulomatosis. American Journal of Pathology 160: 17171724. Lamprecht P, Erdmann A, Muller A, et al. (2003) Heterogeneity of CD4 and CD8 memory T cells in localized and generalized Wegeners granulomatosis. Arthritis Research and Therapy 5: R25R31. Leavitt RY, Fauci AS, Bloch DA, et al. (1990) The American College of Rheumatology 1990 criteria for the classication of Wegeners granulomatosis. Arthritis and Rheumatism 33: 11011107. Pster H, Ollert M, Frohlich LF, et al. (2004) Antineutrophil cytoplasmic autoantibodies against the murine homolog of proteinase 3 (Wegener autoantigen) are pathogenic in vivo. Blood 104: 14111418. Popa ER, Stegeman CA, Kallenberg CGM, and Cohen Tervaert JW (2002) Staphylococcus aureus and Wegeners granulomatosis. Arthritis Research 4: 7779. Reinhold-Keller E, Beuge N, Latza U, et al. (2000) An interdisciplinary approach to the care of patients with Wegeners granulomatosis. Long-term outcome in 155 patients. Arthritis and Rheumatism 43: 10211032. Stone JH, Hoffman GS, Merkel PA, et al. for the International Network for the Study of the Systemic Vasculitides (INSSYS) (2001) A disease-specic activity index for Wegeners granulomatosis: modication of the Birmingham Vasculitis Activity Score. Arthritis and Rheumatism 44: 912920. Xiao H, Heeringa P, Hu P, et al. (2002) Jennette: antineutrophil cytoplasmic autoantibodies specic for myeloperoxidase cause glomerulonephritis and vasculitis in mice. Journal of Clinical Investigation 110: 955963.

Relevant Website
http://www.vasculitis.org Homepage of the The European Vasculitis Study Group (EUVAS) with information about trial protocols, trial progress, meetings, nomenclature, disease scoring and ANCA-testing.

H
HEMOGLOBIN
R Winslow, University of California, San Diego, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Hemoglobin is the red oxygen-carrying pigment of the blood, contained within red blood cells (erythrocytes). It is composed of four subunits, two a and two b polypeptide chains, each of which contains a heme group with iron (Fe) which is the site of oxygen binding. The detailed structure of each of the chains has been dened and the reversible binding of oxygen is explained by two distinct structures of hemoglobin. The synthesis of the a and b chains of hemoglobin is controlled by specic genes that are activated at different times of fetal development. A large number of mutations of the chains have been detected. Most of them are of no clinical consequence, but some, like the sickle cell anemia mutation, can cause severe disease. Mutations that affect the relative quantities of the chains can also cause disease (the thalassemias).

a and two b) of about 140 amino acids each. Each chain carries a tetrapyrrole iron-containing prosthetic group, heme, which can bind one molecule of oxygen. All vertebrate and most nonvertebrate animals use hemoglobin to transport oxygen from the atmosphere to tissues. In humans several hemoglobins occur normally, but all contain four subunits: the differences are limited to the primary structure (amino acid sequence) of globin. Each polypeptide chain is the product of a specic gene. For the composition of hemoglobin A, the major component of A adult human red blood cells, is aA 2 b2 . During human fetal development at least three different non-a globin chains are produced, each a product of a separate gene (Figure 1). The z and e chains are found in very early fetuses and g chains later and in newborns. By about 6 months after birth, the newborn exhibits only the adult pattern of hemoglobin (Table 1).
Primary Structure

Structure
Hemoglobin is a globular protein whose molecular mass is about 64 kDa. The protein portion (globin) is composed of four subunit polypeptide chains (two
100

Hemoglobin was one of the rst proteins whose primary structure (amino acid sequence) was determined

Polypeptide chains present (%)

80

60

40

20

3 Gestation in months

6 Birth

3 Age in months

Figure 1 Ontogeny of the human globin chains. The earliest detectable globins are the e chains of hemoglobins Gower 1 and 2. The z chain of hemoglobins Portland 1 and 2 appear only transiently. By 3 months of gestation, hemoglobin F predominates; by 6 months after birth, hemoglobin A constitutes about 97% of the total. The d chains are synthesized at approximately the same time as the b chain. See Table 1 for the chain compositions of hemoglobin A.

264 HEMOGLOBIN

(Figure 2). The high-resolution X-ray crystallographic analysis by Perutz and co-workers allowed an extensive integration of sequence and functional data. This integration leads to an appreciation of structure function relationships so delicate that substitution of
Table 1 The hemoglobins of human red blood cells A0 F A2 Portland 1 Portland 2 Gower 1 Gower 2 a2b2 a2g2 a2d2 g2z2 z4 e4 a2z2 Main component of human red blood cells Fetal hemoglobin Minor component of human red blood cells Embryonic hemoglobin Embryonic hemoglobin Embryonic hemoglobin Embryonic hemoglobin

one amino acid in 280 can lead to serious clinical consequences, as in sickle cell anemia.
Secondary Structure

About two-thirds of the residues of each chain are arranged in seven and eight helices of the a and b chains, respectively (see Figure 3). The helices are designated from A to H, and vary from 7 to 20 residues in length. There are 3.6 residues per turn of helix, which are stabilized by hydrogen bonds formed longitudinally along their axes. The remaining onethird of the residues are distributed over corners or nonhelical regions of the chain.

Val Val Ala Ala Tyr Val C Phe Tyr Leu Leu Val Val Thr Ser Ala Thr Leu Leu His Asn Ala Lys Lys Lys Lys Lys

His Ala Leu Gly Gly Pro Pro Ser Ser Lys Lys Asn Asp F Leu Phe Arg His Cys Val Glu Glu Phe Val Tyr Tyr

Leu Leu Try Try Ala Gly Thr Try D His Thr Gly Ala Ala Gly Ser Ala Val Val Leu Leu Phe Phe Leu Val Arg His

Ser Thr Gly Gly Glu Glu Thr Thr Gly Pro His His Val Leu Ala Thr G Asp Asp Leu Val H Thr Thr Ala Ala

A Pro Pro Lys Lys Ala Ala Lys Gln Ser Asp Gly Gly Ala Ala Leu Leu Pro Pro Val Cys Pro Pro Ser Gly

Ala Glu Val Val Leu Leu Thr Arg Ala Ala Lys Lys His His Ser Ser Val Glu Thr Val Ala Pro Val Val

Asp Glu A Gly Asn Glu Gly Phe Phe Val Lys Lys Val Leu Asp Glu Asn Asn Leu Leu Val Val Ser Ala

Lys Lys Ala Val Ar g Ar g Pro Glu Met Val Val Asp Asp Leu Leu Phe Phe Ala Ala His Gin Thr Asn

Thr Ser B His Met Leu His Ser E Gly Ala Leu Asp Asn His His Lys Arg Ala His Ala Ala Val Ala

Asn Ala Ala Phe Leu Phe Phe Asn Asp Gly Met Leu Ala Cys Leu Leu His His Ser Ala Leu Leu

Val Val Gly Asp Leu Val Gly Pro Ala Ala Pro Lys His Asp Leu Leu Leu Phe Leu Tyr Thr Ala

Lys Thr Glu Glu Ser Val Asp Asp Gln Lys Leu Phe Asn Gly Lys Lys Ser Gly Pro Gly Asp Gln Ser His

Figure 2 The amino acid sequences of the normal adult globin chains. Helical regions are designated by letters A to H. The numbers above each row refer to residues of the a chain, those below the rows to the non-a chains. Residues that are common to more than one chain are enclosed in boxes.

HEMOGLOBIN 265
C

C D

A N

Figure 3 Schematic diagram of a globin chain. Any of the globin chains (or myoglobin) could t the diagram with only minor alteration. The a-helical regions are lettered, and heme is shown as a disk. The only covalent link between heme and globin is between the iron atom and histidine F8, the proximal histidine.

Myoglobin and all the various hemoglobin chains resemble each other to a remarkable degree. To emphasize this resemblance and to facilitate comparison among chains, residues are given designations according to their position in or between helical regions. For example, residue 10 of the a chain is valine A7(10)a because it occupies position 7 from the beginning of the A helix. Although the corresponding residue in the b chain is valine A7(11)b, it occupies the eleventh position from the N-terminus of that chain. Similarly, the residues in the interhelical regions are given designations using letters of adjacent helices: the third residue in the CD region of the a chain, for example, is histidine CD3(45)a, and in the b chain, serine CD3(44)b.
Tertiary Structure

The three-dimensional folding of a globin chain is also shown in Figure 3. The similarity among the various chains is sufcient so that the gure could represent any of them with only minor alterations. Heme is represented as a at ring between the E and F helices. Proline does not t into a helix because of its pyrrolidine ring; it cannot form hydrogen bonds with the next coil in the helix. Therefore, it is likely

to occur at corners and interhelical regions. Other corners are stabilized by a variety of stereochemical mechanisms, some of which involve portions of chains that lie nearby. Interactions can also occur between helical segments that lie in proximity to one another. For example, the B and E helices are stabilized in each helix by glycine residues, which afford a compact t at their crossing. A very important force stabilizing the globin chain structure is the presence of heme itself. Free globin is much less soluble than the molecule containing heme. It is embedded rmly into a pocket in the chain as shown in Figure 3. It has polar and nonpolar ends, one formed by propionic acid groups and the other by vinyl and methyl groups, respectively. The amino acid side groups lining the heme pocket are largely nonpolar and allow the heme to enter, vinyl end rst. It is then anchored there by a large number of interactions between the pyrrole ring and amino acids of the heme pocket. In addition, it is linked covalently to histidine F8, which is called the proximal histidine. The distal histidine E7 is not linked covalently to the heme group, but its presence in the heme pocket is important in maintaining spatial relationships. The hydrophobic nature of the heme pocket is extremely important in the maintenance of

266 HEMOGLOBIN

the heme iron in its reduced (Fe2 ) state, which is required for oxygenation. A number of mutations in the environment of the heme pocket can lead to the entry of water, oxidation of the iron, and as a result, methemoglobin formation. The distal histidine (E7) acts as a gate to allow ligand to enter the heme pocket only when it swings out. In the a chain (or myoglobin), a hydrogen bond forms between Ne of histidine E7 and the oxygen molecule.
Quaternary Structure

The association of globin chains into a tetrameric structure leads to cooperativity, the Bohr effect, the 2,3-diphosphoglycerate (2,3-DPG) effect, and suitable oxygen afnity so that tissue oxygen requirements can be met (see OxygenHemoglobin Dissociation Curve). The molecule has a twofold axis of symmetry, each half containing one a and one b chain, which are held tightly together by a large number of atomic interactions. There are four areas of contact between the subunits: a1b1, a2b2, a2b1, and a1b2. The a1b2 and a2b1 regions are somewhat weaker than the a1b1 and a2b2 contacts and allow movement during oxygenation. Isolated hemoglobin chains are less stable than the fully associated tetramers. Thus, the regions of contact between them are important in maintaining normal solubility. Several unstable mutants involve the a1b1 and, to a lesser degree, the a1b2 contacts. The central cavity of hemoglobin is lled with water, allowing entrance of charged molecules that affect molecular function, such as 2,3-DPG and salts. Most of the charged amino acid side groups are directed toward the outside of the molecule and enter into electrostatic relations with solvents, allowing very high solubility within the red blood cells. Buried in the midst of the globin chains are the four heme groups containing ferrous iron, protected against oxidation by a hydrophobic environment. Two quaternary conformations The function of hemoglobin in oxygen binding can be most easily understood on the basis of at least two separate quaternary conformations, corresponding to the oxy and deoxy states. Perutz and his associates described these two structures using X-ray crystallography and related them to the R (for relaxed) and T (for tense) conformations. They found that the combination of deoxyhemoglobin with oxygen produced shifts of the , and a change in the tilt of helical regions of 2 to 3 A the heme group relative to the globin chain. This is accompanied by shifts of the iron atom within the porphyrin and of the heme-linked histidine. In the deoxy subunit the e-methyl group of valine E11 (67) blocks the heme pocket. In the oxy form (R)

this pocket widens, making room for the binding of an oxygen molecule. There is little difference at the a1b1 (and a2b2) contact, but a significant change at the a1b2 (and a2b1) contact occurs. The region is dovetailed so that the CDa region of one chain ts into the FG region of the other. During transition from R to T, the dovetailing between CDa and FG changes so that the hydrogen bond linking aspartate G1(94)a to asparagine G4(102) is replaced by another between tyrosine C7(42)a and aspartate G1(99). Finally, large differences occur in the areas of the C-terminal residues. In deoxyhemoglobin the tyrosines (HC2) occupy pockets between helices F and H, and their phenolic groups are hydrogen-bonded to valines FG5. The Cterminal residues are constrained by participation in salt bridges. In oxyhemoglobin, the phenolic groups of tyrosine HC2a are ejected from their pockets, and the salt bridges are broken. Perutz and co-workers proposed a model that relates these molecular changes to oxygen binding. The rst, or trigger, step results from the combination of iron with oxygen. When this occurs, the atomic radius of iron decreases (high-to-low spin transition), allowing it to move closer to the plane of the por , but it phyrin ring. This movement is less than 1 A is sufcient to change the distance between the heme-linked histidine and the plane of the porphyrin ring, initiating the sequence of changes leading to the T-to-R transition. This description applies to the a chain; but in the b chain, the hydrophobic side group of valine E11 is in the heme pocket, providing an additional block to oxygen binding. Movement of the porphyrin ring relative to the heme-linked histidine results in a narrowing of the pocket between helices F and H into which the phenolic group of tyrosine HC2 ts, which leads to its ejection. Rotation of this group distorts the C-terminal amino acids so that the salt bridges are no longer possible, and they rupture. Each subunit undergoes change in the tertiary structure with successive oxygenation, as described above; after two or three of them have done so, there is a change in the quaternary structure. This change involves a rotation of the two halves of the molecules (a subunits) relative to one another, and a sliding occurs at the a1b2 and a2b1 interfaces. The equilibrium between the R and T conformations can be inuenced by a number of small molecules that bind at specic sites. These heterotropic or allosteric effectors are mainly hydrogen ion (H ), chloride (Cl ), 2,3-DPG (within the red cell), and carbon dioxide (CO2), but many other compounds bind to hemoglobin as well (see OxygenHemoglobin Dissociation Curve).

HEMOGLOBIN 267

Hemoglobin Variants: The Hemoglobinopathies

More than 500 structural variants of human hemoglobin have been identied. Most of them have been discovered because of altered electrophoretic mobility. The great majority of these mutations have no clinical consequences, but some affect critical properties such as solubility, oxygen afnity, or oxidation rate. Fewer a chain than b chain variants have been found, most likely because a chain mutations would affect fetal hemoglobin as well as adult hemoglobin (see Figure 1). While most of these variants are the result of point mutation, other genetic mechanisms, such as deletions, insertions, additions, and unequal crossing-over have occurred as well. In addition to these structural mutations, the regulation of the quantities of the various gene products is subject to genetic control as well. Altered regulation of a or b chain expression occurs in the thalassemias. The hemoglobin variant with the greatest clinical impact S is hemoglobin S (aA 2 b2). In this mutation, the normal glutamic acid at b6 is replaced by valine, giving that region of the molecule a slightly more hydrophobic environment such that polymers with adjacent molecules can form when the molecules are in the deoxygenated conformation. The polymers become extensive, eventually deforming the red blood cell, causing them to occlude small vessels. This leads to lack of oxygenation of the tissue and, if not reversed, death of the tissue. The clinical entity caused by this process is called sickle cell anemia, and is characterized by marked anemia and intermittent episodes of severe pain. The discovery of the hemoglobin S mutation was the rst instance of a genetic disease caused by a point mutation.
See also: OxygenHemoglobin Dissociation Curve. Systemic Disease: Sickle Cell Disease.

Globin the protein part of hemoglobin (without the heme prosthetic group) Heme an iron-containing tetrapyrrole prosthetic group that binds one molecule of oxygen Hemoglobinopathy a clinical disease caused by any mutation either of a structural hemoglobin peptide subunit or of an imbalance in their synthesis Point mutation substitution of one amino acid for another Polypeptide chain a protein subunit consisting of a series of amino acids Primary structure amino acid sequence Prosthetic group a nonprotein group, contained within a protein; heme is a prosthetic group Quaternary structure arrangement of protein subunits into a fully functional molecule R state the high oxygen afnity hemoglobin conformation Secondary structure arrangement of amino acids into helical segments Sickle cell anemia a genetic disease caused by mutation of glutamic acid to valine at position 6 of the b chain Tertiary structure arrangement of helical segments into a three-dimensional protein structure Thalassemia a genetic disease in which the synthesis of polypeptide chains of hemoglobin is unbalanced, leading to hemolysis and anemia T state the low oxygen afnity hemoglobin conformation a chain one of the peptide subunits of adult human hemoglobin (A) a helix a helical sequence of amino acids in a peptide chain b chain one of the peptide subunits of adult human hemoglobin (A) g chain one of the peptide subunits of the human fetal hemoglobin (F) d chain one of the peptide subunits of the human adult minor hemoglobin (A2) e chain one of the peptide subunits of the human fetal hemoglobin (Gower 1 and 2) z chain one of the peptide subunits of the human fetal hemoglobin (Portland 1 and 2)

Further Reading
Bolton W and Perutz M (1970) Three dimensional Fourier synthesis of horse deoxyhemoglobin at 2 Angstrom units resolution. Nature 228: 551552. Bunn HF and Forget BG 2005 In: Dyson J (ed.) Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia: Saunders. Ingram VM (1957) Gene mutations in human haemoglobin: the chemical difference between normal and sickle cell haemoglobin. Nature 180: 326328. Perutz M (1989) Myoglobin and haemoglobin: role of distal residues in reactions with haem ligands. Trends in Biochemical Sciences 14: 4244.

Glossary
Cooperativity the increase of O2 afnity with successive ligation of O2 molecules

268 HEPATOCYTE GROWTH (SCATTER) FACTOR

Hemorrhage

see Alveolar Hemorrhage.

Hemothorax

see Pleural Effusions: Hemothorax.

HEPATOCYTE GROWTH (SCATTER) FACTOR

S E Mutsaers, The University of Western Australia, Nedlands, WA, Australia S E Herrick, University of Manchester, Manchester, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Hepatocyte growth factor (HGF), also known as scatter factor and tumor cytotoxic factor, is a multifunctional polypeptide secreted as an inactive single-chain propeptide which is cleaved extracellularly by serine proteases to give an active heterodimer consisting of an a- and b-chain. The a-chain contains an N-terminal hairpin loop and four kringle domains, whereas the b-chain resembles the catalytic domain of serine proteases but does not have enzymatic activity. Cells of mesenchymal origin are the primary producers of HGF, whereas epithelial and endothelial cells are the main responding cell types. HGF acts through the cell-surface receptor, c-met, to cause proliferation and migration, induce tubulogenesis, inhibit apoptosis, and promote invasiveness. During development, HGF plays a major role in epithelial mesenchymal interactions essential to organogenesis especially of the lung, heart, liver, and kidney. In the adult, HGF maintains normal tissue function and injury leads to increased systemic HGF levels and tissue mRNA expression with associated tissue regeneration. Studies in experimental models suggest that administration of HGF may provide therapeutic benet for conditions including pulmonary brosis, emphysema, asthma, and pulmonary hypertension. However, excessive HGF activity or cmet expression is associated with tumor growth and metastasis and c-met mutations have been detected in a variety of human cancers including mesothelioma and non-small cell lung carcinomas, and may contribute to the invasive growth of lung cancer.

SF. Subsequently, molecular cloning and physiological activity demonstrated that HGF and SF were identical. Another factor found to be identical to human placental HGF is tumor cytotoxic factor, initially puried from the conditioned medium of human embryonic lung broblasts, IMR-90 cells. HGF/ SF forms part of a family of plasminogen-related growth factors along with HGF-like/macrophage stimulating protein (an effector of macrophage chemotaxis and phagocytosis), plasminogen (a proenzyme of the serine protease family), and apolipoprotein (a family of glycoproteins associated with lipid transport), as they all are thought to have evolved from the same ancestral gene. HGF is produced mainly by mesenchymal cells with activity mediated through the c-met receptor found principally on epithelial and endothelial cells.

Structure
The gene-encoding HGF maps on chromosome 7q21.1 in humans and is composed of 18 exons interrupted by 17 introns spanning approximately 70 kb. In the mouse, the HGF gene maps on chromosome 5; however, the primary structure of HGF is 490% homologous in humans and rodents. The signal peptide involved with intracellular processing and secretion and the 50 -untranslated region are contained in the rst exon, whereas the next ve pairs of exons correspond to the amino-terminal and four successive kringle domains, respectively. The 12th exon encodes the region that links the heavy and light chain, and the remaining six exons specify the serineprotease domain. The promoter sequence of the HGF gene contains a number of putative regulatory elements which include a transforming growth factor beta (TGF-b) inhibitory element, interleukin (IL)-6, cAMP, oestrogen and vitamin D responsive elements, and potential binding sites for tumor necrosis factor alpha, IL-6, IL-1, and liver-specic, B-cell-specic and macrophage-specic transcription factors. HGF is derived from a biologically inactive singlechain precursor (pro-HGF) of 728 amino acids (Figure 1). Amino acid sequence is highly homogeneous with plasminogen. The mature active form is

Introduction
Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional polypeptide that has been implicated in embryo development, tissue repair, and cancer growth. HGF and SF were independently isolated based on two different biological activities. In the mid-1980s, HGF was initially isolated from rat platelets as a factor that stimulated DNA synthesis in primary cultures of rat hepatocytes, in human plasma from patients with fulminant hepatic failure and in rabbit serum. Around the same time, humanembryo-broblast conditioned medium was found to contain a factor which induced scattering of tightly packed colonies of epithelial cells and so was named

HEPATOCYTE GROWTH (SCATTER) FACTOR 269

Kringle 2 Pro-HGF Processing Arg-Val Hairpin loop -Chain Processing site Mature HGF Kringle 1 Kringle 3 -Chain Kringle 4

Figure 1 Structure of the mature active HGF molecule following processing from its biologically inactive single-chain precursor, proHGF. The mature HGF consists of a heavy a-chain and a light b-chain held together by a disulde bond. The a-chain contains an N-terminal hairpin loop and four kringle domains, whereas the b-chain resembles the catalystic domain of serine proteases but does not have enzymatic activity. Adapted from Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(12): 123, with permission from Elsevier.

a heparin-binding, heterodimeric glycoprotein consisting of a heavy 69 kDa a-chain and a light 34 kDa b-chain held together by a disulde bond. The heavy a-chain contains an N-terminal hairpin loop, homologous to the plasminogen activation peptide and four kringle domains involved in proteinprotein interactions, whereas the b-chain resembles the catalytic domain of serine proteases but does not have enzymatic activity. The rst and second kringle domains are particularly necessary for the correct biological function of the molecule. Naturally occurring variants of HGF have been identied resulting from alternative RNA splicing. Natural variants, NK1 and NK2, which contain the N-terminal hairpin loop and either the rst or the rst two kringle domains, respectively, retain the ability to interact with the c-met receptor. However, although NK1 demonstrates the full spectrum of HGF responses when expressed as a transgene in vivo, NK2 antagonizes the pathological consequences of HGF overexpression. A third natural variant, an isoform of 723 amino acids (dHGF) with a deletion of ve residues located in the rst kringle, retains all the biological activity of fulllength HGF.

495, respectively. HGF activation is induced by a number of factors including urokinase plasminogen activator (uPA), tissue plasminogen activator, HGFactivator (HGF-A), matriptase, kallikrein, and blood coagulation factor XIIa. HGF induces the expression of uPA and so participates in a positive feedback loop. Activation of HGF is a nely regulated process, given that some of these molecules require activation from inactive zymogens and are also under inhibitory control. For example, HGF-A, the most powerful activator, is also secreted as a proenzyme, which requires activation by serine proteases such as thrombin. Furthermore, the identication of endogenous HGF-A inhibitors (HAIs) has provided further information about the regulation of HGF-A activity. Currently, two types of HAIs, namely HAI-1 and HAI-2, have been found. Active HGF mainly exerts its actions on epithelial cells through the c-met receptor; however, non-epithelial cells such as hematopoietic, endothelial, lymphoid, neural, muscle, and mesothelial cells may also respond.

Biological Function
HGF regulates a wide range of cellular processes such as cell survival, proliferation, migration, and differentiation (Figure 2). It acts as an endocrine, paracrine, and autocrine factor and can be detected in sera, bronchoalveolar lavage, cerebrospinal and synovial uids, although levels change with age and during pregnancy. Mice genetically decient in HGF are embryonic lethal with severe placental insufciency, reduction of sensory nerves, and developmental defects in the liver and muscle. Transgenic mice that overexpress HGF develop diverse tumors, polycystic kidney disease, and inammatory bowel disease. In development, HGF is involved in the formation of multiple tissue/organ systems such as the lung, placenta, tooth, kidney, liver, heart, pancreas, prostate, mammary gland, ovary, testes, muscle, and

Regulation of Production and Activity

A variety of cells including platelets, hepatocytes, monocytes, broblasts, and certain tumor cells produce pro-HGF following stimulation by inammatory mediators such as IL-1b and prostaglandin E2 but also branched amino acids, heparin, basic broblast growth factor (bFGF), epidermal growth factor, and platelet-derived growth factor. Following secretion of pro-HGF from the cell, it becomes bound to the cell surface or to extracellular matrix (ECM) components, in particular heparan sulfate and dermatan sulfate containing proteoglycans. The active disulde-linked heterodimer is formed by proteolytic cleavage of inactive single-chain pro-HGF between arginine and valine residues, at position 494 and

270 HEPATOCYTE GROWTH (SCATTER) FACTOR

Kringle 2 Kringle 1 Hairpin loop HGF

Kringle 3 -chain Kringle 4

HGF HGF

-chain

c-Met

Plasma membrane
P P S Y Y Y Y S Y Y Y Y P P P P P

PH
P

PI3-K Ras SOS

p85 P
P

Ga

PLCb1
P P

PI3-K p85 Rap1

Grb2 Bag-1 STAT

Grb2
P

Crk

Shp2 MAPK

Antiapoptosis Angiogenesis Morphogenesis Motogenesis Neurite extension

Mitogenesis

Figure 2 Biological activities elicited by HGF mediated through intracellular signaling of the c-met receptor. Adapted from Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(12): 123, with permission from Elsevier.

neuronal tissue. During organogenesis, HGF plays a key role in epithelialmesenchymal transition. In situ hybridization analysis has demonstrated HGF and c-met gene expression in mesenchyme and epithelium, respectively, of the developing lung. Furthermore, HGF has been shown to be a mesenchyme-derived morphogen-regulating lung morphogenesis by mediating epithelial branching in collaboration with FGF family members. HGF is also involved in regulating bone remodeling where HGF is secreted by osteoclasts and activates its receptor on both osteoclasts and osteoblasts. In the brain, HGF is a new member of the family of neurotrophic factors. In lung and breast cancer, HGF concentration correlates with disease relapse and reduced overall survival suggesting that HGF may promote tumor progression. In the lung, squamous cell carcinoma cells over expressing c-met show increased tumorigenesis and metastases in response to HGF in vivo. HGFs ability to upregulate uPA and its receptor uPAR may increase the invasive potential of tumor cells. However, HGF has also been shown to have cytotoxic activity on certain tumor cells implying HGF inhibits some cancer growth. It is likely that the level of c-met signaling is key to the control of carcinogenesis.

HGF plays a key role in the proper maintainance and repair of adult tissue including the lung. It is a potent mitogen for epithelial and hematopoietic cells and chrondrocytes and is involved in tubule formation of many epithelial cell lines including those of the lung, mammary gland, kidney, pancreas, and liver. Following cutaneous wounding, overexpression of HGF induces keratinocyte proliferation and enhanced expression of matrix metalloproteinases leading to increased re-epithelialization, and through upregulating vascular endothelial growth factor expression, increased blood vessel formation, and granulation tissue expansion. In contrast, neutralization of HGF leads to retarded wound healing associated with reduced vascularization, granulation tissue formation, and re-epithelialization. Administration of antibodies to HGF in normal mice leads to the onset and progression of tissue brosis, and renal dysfunction again conrming one of the key activities of HGF is the preservation of normal tissue structure and function. Prevention of brosis and normal regeneration of lung, liver, heart, and kidney has been associated with the antiapoptotic activity of HGF and the protection of epithelial cells against DNA damaging agents. In addition, HGF prevents the initiation and progression of chronic brosis

HEPATOCYTE GROWTH (SCATTER) FACTOR 271

by directly antagonizing the probrotic actions of TGF-b1 possibly through inhibiting myobroblast activation.

HGF Receptor
The HGF receptor is the Met protein product of the c-met proto-oncogene (p190 met). c-Met consists of a transmembrane 145 kDa b-chain and an extracellular 50 kDa a-chain to form a dimeric 190 kDa protein with structural features of a tyrosine kinase receptor (Figure 2). The a-chain is heavily glycosylated and is disulde-linked to the b-chain. The latter contains the kinase domain, the tyrosine autophosphorylation sites, and the multifunctional docking sites that comprise a specic stretch of amino acids located in the carboxy-terminal part of the protein. HGF also binds to cell-surface heparan sulfate proteoglycans that serve as low-afnity HGF receptors and modulate the interaction between HGF and c-met receptor. The various biological functions of HGF on target cells must be attributable to different intracellular signal transduction pathways as only one receptor has so far been identied. HGF binding, in the presence of ATP and Mg2 , triggers receptor dimerization and autophosphorylation at a conserved two-tyrosine motif within the receptor docking site resulting in the recruitment of intracellular signaling molecules containing the src homology (SH) domain. c-Met signals in the large part via the Ras signaling pathway after binding the docking protein Grb2. Data have shown that activation of the Ras pathway leads to cell proliferation, whereas the PI3 kinase pathway is needed to induce motogenesis. Both pathways are essential for invasive growth. Dysregulation of c-met signaling is observed in carcinogenesis. Point mutations in c-met detected in the catalytic domain of the receptor have been associated with carcinomas, and overexpression of c-met gene in transgenic mice leads to carcinomas of the thyroid gland, breast, liver, pancreas, and ovary. Increased expression of c-met is associated with a poor prognosis.

Hepatocyte Growth (Scatter) Factor in Respiratory Diseases

HGF acts as a lung-regenerating factor after injury as it stimulates proliferation of alveolar type II epithelial cells, thus contributing to regeneration of alveolar structures. Hence, HGF levels are often elevated in disease conditions such as in bronchoalveolar lavage uid of patients with idiopathic pulmonary brosis, sarcoidosis, and the interstitial lung disease associated with rheumatoid arthritis. However,

excessive levels of activated HGF are reported in mesotheliomas and non-small cell lung carcinomas and may contribute to the invasive growth of lung cancer. It was reported that HGF mRNA and HGF activity increased in whole lung 36 h after intratracheal administration of hydrochloric acid, and an increase in whole lung HGF expression has been reported in a rat model of ischemia-reperfusion. HGF is also upregulated following bleomycin administration in mice. However, if levels are further increased through administering HGF systemically or intratracheally, the brosis is attenuated. In contrast, administration of neutralizing antibodies to HGF markedly increased collagen accumulation in the lungs of bleomycin-injured mice. Administration of HGF to the airways of mice also reduced airway inammation, hyperresponsiveness, and remodeling following sensitization and challenge with ovalbumin. Recent studies have demonstrated a potential therapeutic application for HGF in emphysema. Human broblasts from patients with emphysema produce less HGF than from normal patients. When overexpressed in an animal model of emphysema, HGF improved pulmonary function by inhibiting alveolar cell apoptosis, enhancing alveolar regeneration, and promoting angiogenesis. HGF also promoted compensatory growth in remnant lung tissue of emphysamateous rats following lung volume reduction surgery. The major sources of HGF are thought to be macrophages and interstitial broblasts and the cellular target, alveolar epithelial cells. Evidence suggests that the increase in HGF following lung injury is controlled by the plasminogen activation system, primarily through plasmin-mediated breakdown of the ECM releasing proteoglycanbound HGF into the alveolar space. Interestingly, the lung may also be a source of HGF after injury to other organs. For instance, after partial hepatectomy, unilateral nephrectomy, or induction of hepatitis in rats, HGF mRNA in the intact lung increased at 6 h. These ndings suggest that the lung may contribute to organ repair and regeneration in an endocrine fashion through production of circulating HGF. The factors that stimulate upregulation of HGF expression following lung or other organ injury have not been fully elucidated. There is a vast amount of evidence that shows raised HGF levels in lung disease but no studies exploring the therapeutic value of HGF in human clinical conditions. In vivo gene transfection with HGF gene attenuated the medial hypertrophy of pulmonary arteries and enhanced the ameliorating effect of prostacyclin for pulmonary hypertension in monocrotoline-treated rats. Thus, gene therapy with HGF and prostaglandin I2 (prostacyclin) synthase may be a

272 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

promising strategy for severe pulmonary hypertension. In addition, administration of HGF might exhibit a potent function in vivo for protection and improvement of acute and chronic lung injuries induced by inammation and/or oxidative stress. Overall, there is tremendous potential for HGF to be used as a novel therapeutic agent in combating various inammatory and brotic disorders including those of the lung.

Conclusion
HGF has trophic, mitogenic, regenerative, and therapeutic effects on lung, kidney, pancreas, spleen, liver, heart, and spinal cord. While HGF was originally identied as a potent mitogen for mature hepatocytes, the biological functions of this factor reach far beyond its original identication. Due to its wealth of regeneration potential, use of HGF for purposes of therapeutics is being given increasing attention.
See also: Adhesion, CellCell: Vascular. Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Chemokines. Chronic Obstructive Pulmonary Disease: Emphysema, General. Coagulation Cascade: iuPA, tPA, uPAR. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibrinolysis: Plasminogen Activator and Plasmin. Fibroblasts. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Pulmonary Macrophages. Lung Development: Overview. Mesothelial Cells. Mesothelioma, Malignant. Oncogenes and Proto-Oncogenes: Overview. Platelet-Derived Growth Factor. Platelets. Pleural Effusions: Overview. Pulmonary Fibrosis. Serine Proteinases. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumors, Malignant: Overview; Metastases from Lung Cancer. Vascular Disease.

Further Reading
Dohi M, Hasegawa T, Yamamoto K, and Marshall BC (2000) Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary brosis. American Journal of Respiratory and Critical Care Medicine 162(6): 23022307.

Funakoshi H and Nakamura T (2003) Hepatocyte growth factor: from diagnosis to clinical applications. Clinica Chimica Acta 327(12): 123. Hattori N, Mizuno S, Yoshida Y, et al. (2004) The plasminogen activation system reduces brosis in the lung by a hepatocyte growth factor-dependent mechanism. American Journal of Pathology 164(3): 10911098. Higashio K, Shima N, Goto M, et al. (1990) Identity of a tumor cytotoxic factor from human broblasts and hepatocyte growth factor. Biochemical and Biophysical Research Communications 170(1): 397404. Jiang W, Hiscox S, Matsumoto K, and Nakamura T (1999) Hepatocyte growth factor/scatter factor, its molecular, cellular and clinical implications in cancer. Critical Reviews in Oncology/ Hematology 29(3): 209248. Ma PC, Maulik G, Christensen J, and Salgia R (2003) c-Met: structure, functions and potential for therapeutic inhibition. Cancer and Metastasis Reviews 22(4): 309325. Matsumoto K and Nakamura T (1997) Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration. Biochemical and Biophysical Research Communications 239(3): 639644. Ono M, Sawa Y, Mizuno S, et al. (2004) Hepatocyte growth factor suppresses vascular medial hyperplasia and matrix accumulation in advanced pulmonary hypertension of rats. Circulation 110(18): 28962902. Rubin JS, Bottaro DP, and Aaronson SA (1993) Hepatocyte growth factor/scatter factor and its receptor, the c-met protooncogene product. Biochimica et Biophysica Acta 1155(3): 357371. Stella MC and Comoglio PM (1999) HGF: a multifunctional growth factor controlling cell scattering. The International Journal of Biochemistry & Cell Biology 31(12): 13571362. Stoker M and Perryman M (1985) An epithelial scatter factor released by embryo broblasts. Journal of Cell Science 77: 209223. Stuart KA, Riordan SM, Lidder S, et al. (2000) Hepatocyte growth factor/scatter factor-induced intracellular signalling. International Journal of Experimental Pathology 81(1): 1730. Ware LB and Matthay MA (2002) Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inammation, and repair. American Journal of Physiology. Lung Cell Molecular Physiology 282(5): L924L940. Yanagita K, Matsumoto K, Sekiguchi K, et al. (1993) Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury. Journal of Biological Chemistry 268(28): 2121221217. Yanagita K, Nagaike M, Ishibashi H, et al. (1992) Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochemical and Biophysical Research Communications 182(2): 802809.

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

J B West, University of California San Diego, La Jolla, CA, USA
& 2006 Elsevier Ltd. All rights reserved. reduces the inspired partial pressure of oxygen. Physiological consequences include impairment of physical performance, mental performance, and sleep. The deleterious effects of high altitude are reduced by the process of acclimatization, the most important feature of which is an increase in ventilation. Other features include polycythemia, changes in oxidative enzymes in cells, and reduced intercapillary distances in some tissues. However acclimatization does not return the body to its sea level status. Three principal high-altitude diseases affect people who

Abstract
Exposure to high altitude causes generalized hypoxia throughout the body because the reduction in barometric pressure

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 273

normally live near sea level. Acute mountain sickness causes headache, fatigue, insomnia, and anorexia but is generally a selflimiting disease that resolves after 23 days. High-altitude pulmonary edema is a much more serious condition characterized by severe dyspnea, which may progress to coughing up frothy uid. The pulmonary hypertension that occurs at high altitude because of the alveolar hypoxia damages some pulmonary capillaries, a condition known as stress failure. High-altitude cerebral edema is uncommon but potentially fatal and causes confusion, ataxia, and eventually coma. Permanent residents of high altitude sometimes develop chronic mountain sickness characterized by severe polycythemia and a constellation of neurological symptoms.

0 800 Barometric pressure (mmHg)

Altitude (ft) 20 000 10 000 Sea level Denver Commercial aircraft cabin Pikes peak 150 Inspired PO2 (mmHg)

600 100

400 Highest human habitation 200 0 Mt Everest 0 0 2000 4000 6000 Altitude (m) 8000 50

Physiology of High Altitude

What is Meant by High Altitude?

In this context, there is no precise definition of high altitude but, in general, it refers to altitudes that affect the human body. At altitudes of 15002500 m, physiological changes such as increased ventilation are seen. From 2500 to 3500 m, high-altitude illness is common if ascent is rapid. At higher altitudes of 35005500 m, altitude illnesses are frequently seen but long periods of altitude exposure are possible. Above that, progressive deterioration is inevitable. A surprising number of people live, work, or play at high altitude. For example, 140 million people reside at altitudes over 2500 m mainly in the Americas, Asia, and eastern Africa. Some people work at very high altitudes, e.g., in mines at over 4500 m and in observatories at over 5000 m. Mountaineers ascend to altitudes of over 8000 m. All of these groups are prone to high-altitude diseases with occasional fatal outcomes.
Barometric Pressures at High Altitude

Figure 1 Relationship between altitude and barometric pressure. Note that at 1520 m (5000 ft) in Denver, CO, the PO2 of moist inspired gas is about 130 mmHg, while on the summit of Mt Everest it is only 43 mmHg. Reproduced from West JB, Respiratory Physiology The Essentials, 7th edn., Lippincott Williams & Wilkins, 2005, with permission from Lippincott Williams & Wilkins.

and Himalayas are at low latitudes where the barometric pressure for a given altitude tends to be relatively high.
Physiological Effects of High Altitude

As the altitude increases, the barometric pressure falls because of the smaller column of air above. Figure 1 shows the relationship between altitude and barometric pressure in those regions of the world where people normally go to high altitude. At an altitude of 3000 m, which commonly occurs in ski resorts, the barometric pressure and inspired PO2 are only about 70% of the sea level value. The highest altitude at which humans permanently reside is about 5000 m, where the inspired PO2 is only onehalf of the sea level value. The highest point in the world, Mt Everest (altitude 8848 m), has a barometric pressure only one-third of the sea level value. The barometric pressures shown in Figure 1 are higher than those of the standard atmosphere, which is used by the aeronautical industry and included in some medical textbooks. The reason is that regions of the world with high mountains such as the Andes

The hypoxia of high altitude affects almost all the organ systems in the body. However, in the present context the most important changes are in physical performance, mental performance, and sleep. Physical performance as measured by maximal oxygen consumption falls as the inspired PO2 is lowered. At an altitude of 3000 m, the maximal oxygen consumption is reduced to about 85% of the sea level value and at an altitude of 5000 m, it is down to about 60%. On the summit of Mt Everest, the maximal oxygen consumption is only about 20% of the sea level value. These substantial reductions in physical performance are accompanied by a greatly increased sense of fatigue. The impaired physical performance is presumably mainly caused by the reduced availability of oxygen to mitochondria, which interferes with the normal function of the electron transport chain. However, some investigators believe that central inhibition from the brain may play a part in limiting exercise capacity. Mental performance is impaired at high altitude. For example, visual perception is altered and night vision is reduced at altitudes as low as 2000 m. People working at an altitude of 4000 m have greater mental fatigue, reduced attention span, and make an unusually large number of arithmetical errors. Mountaineers at altitudes over 6000 m show impairment of short-term memory and manipulative skills, and it is

274 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

interesting that some residual impairment of these functions can be demonstrated after return to sea level when compared with the pre-exposure values. Sleep is poor at high altitude and a common complaint is that people do not feel refreshed in the morning. Periodic breathing is almost universal above an altitude of 4000 m and the periods of apnea may result in arousals.
Acclimatization to High Altitude

When lowlanders (that is, people who normally live near sea level) go to high altitude, a series of adaptive changes occur, collectively known as acclimatization. The most important of these is an increase in pulmonary ventilation, but other changes also occur including polycythemia, increase in the density of capillaries in peripheral tissues, and changes in oxidative enzymes inside cells. Hyperventilation is the most important feature of acclimatization to high altitude. The mechanism is stimulation of the peripheral chemoreceptors by the low PO2 in the arterial blood. There is a prompt increase in ventilation on ascent to high altitude but the ventilation continues to rise over several days apparently because of increased sensitivity of the peripheral chemoreceptors to the low PO2. The extent of the hyperventilation at extreme altitude is remarkable. For example, on the summit of Mt Everest the alveolar ventilation is increased about vefold, which drives the alveolar PCO2 down from its normal value of about 40 mmHg to below 10 mmHg. Polycythemia is often thought to be an important feature of acclimatization but for visits of a week or so to high altitude it plays almost no role because of the delay in the production of new red cells. However, the hematocrit often increases within a day or so of going to high altitude because of a reduced plasma volume. This is partly caused by dehydration and partly by hormonal changes regulating plasma volume. Longer-term stays at high altitude are accompanied by polycythemia which increases the oxygen carrying capacity of the blood. However, residence at very high altitudes may cause such high hematocrits (over 70%) that the accompanying viscosity of the blood actually reduces exercise performance. Some misconceptions about acclimatization have developed, particularly among nonmedical people. Although acclimatization greatly reduces the incidence of high-altitude diseases, it does not return the body to its sea level status. In other words, even in the well-acclimatized person at high altitude, the hypoxia impairs physical and mental performance, and sleep. As an example, astronomers working on

the summit of Mauna Kea, Hawaii (altitude 4200 m) have such a low alveolar and arterial PO2 even when fully acclimatized that if this were caused by chronic obstructive pulmonary disease at sea level, they would be entitled to continuous oxygen therapy under Medicare. This emphasizes the great hypoxic stress of these altitudes. A recent technical advance has improved the quality of life and efciency of people who are required to work at high altitude. If the oxygen concentration of a room is raised from its normal value of 21% to 25% or higher, the deleterious effects of high-altitude hypoxia can be mitigated. A radiotelescope operated by the California Institute of Technology at an altitude of 5050 m in north Chile has rooms where the oxygen concentration is maintained at 27%. In effect, this reduces the equivalent altitude (based on the level of the inspired PO2 ) to about 3200 m, which is easily tolerated. The oxygen is produced from air by electrically powered concentrators that inject the oxygen into the ventilation of the room. The result has been a tremendous increase in productivity and quality of life for people who must work at these great altitudes. The same technique has been used in a mine in north Chile where the dormitories are at an altitude of 3800 m. Miners who have difculties with sleeping have much improved nights if they sleep in an oxygen-enriched room.
Physiological Changes at Extreme Altitudes

Mountaineers who ascend to extremely high altitudes such as those near the summit of Mt Everest undergo extraordinary physiological changes. There is an enormous increase in alveolar ventilation, which, on the Everest summit, drives the alveolar PCO2 down to about 78 mmHg. This very high ventilation maintains the alveolar PO2 at about 35 mmHg, which is just sufcient to maintain life. The very low PCO2 is accompanied by an extreme respiratory alkalosis with an arterial pH (based on the measured alveolar PCO2 and base excess of the blood) of over 7.7. Even so, the maximal oxygen consumption is only about 1 l min 1, a miserably low value but just sufcient to allow a climber to reach the summit. It is a remarkable coincidence that a climber at the highest point on Earth appears to be so close to the limit of tolerance to oxygen deprivation.

Diseases of High Altitude

There are three major diseases affecting lowlanders who go to high altitude: acute mountain sickness, high-altitude pulmonary edema, and high-altitude cerebral edema. In addition, there are several other

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 275

pathological conditions such as high-altitude retinal hemorrhage. In special situations, lowlanders may develop a condition known as subacute mountain sickness. Permanent residents of high altitude, especially in the Andes, sometimes develop chronic mountain sickness.
Acute Mountain Sickness

This condition is common in people who ascend from near sea level to altitudes of 3000 m and above. However, it occasionally occurs at altitudes as low as 2000 m. The most important features are headache, lightheadedness, breathlessness, fatigue, insomnia, anorexia, and occasionally nausea. Commonly, the symptoms begin 23 h after ascent and often the rst night at high altitude is particularly unpleasant. However, the condition is usually self-limiting and most of the symptoms disappear after 23 days, although the insomnia may persist. Acute mountain sickness responds well to descent. The best way to reduce the incidence of acute mountain sickness is by gradual ascent, which allows time for acclimatization. Handbooks for trekkers often advise that above an altitude of 3000 m the ascent rate should be no more than 300 m per day with a rest day every 23 days. However, this is rather conservative and many people are able to ascend more rapidly. An interesting feature of acute mountain sickness is that even a brief recent exposure to high altitude reduces the incidence of the disease. The cause of acute mountain sickness is poorly understood. Of course hypoxia is a major factor because this is the only unavoidable physiological stress at high altitude. Other factors such as cold, high winds, and intense solar radiation can be removed by appropriate protection. Some people think that the respiratory alkalosis that occurs as a result of the hyperventilation may be partly responsible. This would t with the time course because the alkalosis also becomes less evident after 23 days as a result of renal compensation. There is some evidence that mild cerebral edema plays a role. In fact, there is a continuum between severe acute mountain sickness and high-altitude cerebral edema, which is discussed below. The cause of the cerebral edema is not fully understood but contributing factors may be an increased cerebral blood ow or increased permeability of the cerebral capillaries. There is some evidence of slight brain swelling and since the cranium is essentially rigid, even a small increase in brain tissue volume may cause a large rise in pressure. Note that at high altitude there are two opposing inuences on cerebral blood ow. The arterial hypoxemia causes

cerebral vasodilatation whereas the low PCO2 and alkalosis result in cerebral vasoconstriction. The incidence of acute mountain sickness can be reduced by taking the carbonic anhydrase inhibitor acetazolamide. This is not necessary if one ascends slowly but, for example, a rapid ascent to high altitude as in ying from Lima to Cuzco in Peru, or into La Paz, Bolivia inevitably means an abrupt exposure to high altitude. The dose of acetazolamide is 250 mg once or twice a day. Some people recommend 125 mg at night to reduce the incidence of periodic breathing and thus improve sleep. The mechanism of action of acetazolamide is probably the metabolic acidosis, which stimulates ventilation. Side effects to the drug are common including paresthesia of ngers and toes, diuresis, and an unpleasant taste to carbonated drinks. In addition, acetazolamide is a sulfonamide drug and therefore some people have a hypersensitivity to it. Dexamethasone has also been used to reduce the incidence of acute mountain sickness, the recommended dosage being 2 mg every 68 h. Some studies have shown that gingko biloba reduces the incidence of acute mountain sickness but additional work is necessary to establish its effectiveness. At the present time, it does not seem to be as effective as acetazolamide. Acute mountain sickness usually does not need treatment although the headache may respond to aspirin, acetaminophen, or ibuprofen. If the condition is severe, oxygen can be administered. Acetazolamide 250 mg three times a day or dexamethasone 4 mg four times a day are also useful for severe mountain sickness. The best treatment is descent, which generally causes a rapid improvement.
High-Altitude Pulmonary Edema

Whereas acute mountain sickness is usually self-limiting and a minor nuisance, high-altitude pulmonary edema is a serious condition that is occasionally fatal. The incidence depends very much on the ascent rate. Commonly, the incidence is about 12% but in reports of people ascending rapidly to 4500 m, the incidence was as high as 10%. The condition typically occurs about 12 days after reaching high altitude and an interesting feature is that if it does not occur within a week it is unlikely to occur at all unless the subject ascends even higher. One form of high-altitude pulmonary edema is seen in high-altitude residents who travel to low altitude for a few days and then return to high altitude. This is called reascent high-altitude pulmonary edema. It is not easy to predict who is at risk from highaltitude pulmonary edema but people who have developed it on one occasion are more likely to do so

276 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

on a subsequent ascent. There is some evidence that an upper respiratory tract infection may increase the likelihood, particularly in children. High-altitude pulmonary edema may be preceded by acute mountain sickness but this is certainly not always the case and failure to realize this has resulted in some missed diagnoses. A major symptom of high-altitude pulmonary edema is dyspnea with orthopnea being common. Exercise tolerance is reduced. Frequently, there is a dry cough initially but this may progress to one that produces frothy pink sputum because of blood staining. On examination tachypnea and tachycardia frequently occur, there may be a mild pyrexia, and crackles can be heard over the lung elds by auscultation. Occasionally, the patient is aware of uid in his or her lungs because of gurgling sounds. The pathogenesis of high-altitude pulmonary edema has been puzzling in the past and has stimulated much recent research. Left ventricular failure can be ruled out because cardiac catheterization has shown normal wedge pressures. However, there is strong evidence of an association with pulmonary hypertension. This is brought about through vasoconstriction of small pulmonary arteries as a result of the alveolar hypoxia. The evidence for the importance of pulmonary hypertension in the pathogenesis can be summarized as follows. Susceptible individuals tend to have an unusually vigorous hypoxic pulmonary vasoconstrictor response, and also unusually high pulmonary artery pressures have been measured prior to the onset of edema. Cardiac catherization studies in patients with high-altitude pulmonary edema have shown pulmonary artery systolic pressures as high as 144 mmHg with a usual range of 6080 mmHg. The normal value is in the 20s. Exercise at high altitude is recognized as a provocative factor because this increases pulmonary artery pressure. Patients with a restricted pulmonary vascular bed, for example unilateral absence of a pulmonary artery, are especially at risk. Finally, pulmonary vasodilator drugs such as nifedipine are useful both in the prevention and treatment of the disease. An important nding from bronchoalveolar lavage studies was that the alveolar uid has a large concentration of high molecular weight proteins and cells, and therefore is of the high-permeability type. After a day or so the edema uid may contain markers of an inammatory response but, importantly, this is not seen in the early stages. There are also changes in blood coagulation and platelet activation later in the disease. The absence of an inammatory response early on implicates a mechanical origin. There is now convincing evidence that the pathogenic mechanism is uneven hypoxic pulmonary vasoconstriction, which results in the high pressure in the

pulmonary arteries being transmitted to some of the capillaries. Direct evidence of uneven hypoxic pulmonary vasoconstriction has recently been reported. It is not particularly surprising that this occurs because it is known that in the adult lung, the distribution of vascular smooth muscle is uneven with some small pulmonary arteries having very little. As a result vasoconstriction cannot occur, or is weak, in these regions. The resulting high pressure in some of the capillaries means that the stresses in the capillary wall become extremely high, and the vessels therefore develop ultrastructural damage. It is not surprising that the stresses are high because the bloodgas barrier in some places is extraordinarily thin, being only 0.20.4 mm in thickness. Since the wall stress is inversely proportional to the wall thickness, other factors being equal, it is easy to see how the circumferential or hoop stresses in the capillaries become enormously high. Ultrastructural changes in pulmonary capillaries in animal preparations have been demonstrated when the capillary pressure is raised to high levels, though not as high as apparently can occur in high-altitude pulmonary edema. The ultrastructural changes include disruption of the capillary endothelial layer, alveolar epithelial layer, and, in some cases, all layers of the wall. The result of this ultrastructural damage is that uid containing high molecular weight proteins and cells leaks out of the capillaries. In addition, exposure of the endothelial basement membranes causes platelet adhesion, which may be responsible for the blood coagulation and inammatory response that develops later in the disease. The damage to the capillaries is termed stress failure because it results from the very high stresses in their walls. The sequence of events in the pathogenesis of high-altitude pulmonary edema is outlined in Figure 2. As stated above, high-altitude pulmonary edema normally comes on about 24 days after ascent, and if it does not occur within the rst week it is very unlikely to occur at all. The explanation for this may be remodeling in the arteries in response to the high pulmonary artery pressure. This is well known to occur in animals in experimental conditions where there is alveolar hypoxia. The remodeling causes an increase in vascular smooth muscle in the small pulmonary arteries and this presumably protects the capillaries that would otherwise be at risk. A related explanation may be responsible for the reascent highaltitude pulmonary edema mentioned above. In this case, the abundant smooth muscle in the small pulmonary arteries of high-altitude residents presumably undergoes involution when they go to a lower altitude and this makes them susceptible again to high-altitude pulmonary edema.

HIGH ALTITUDE, PHYSIOLOGY AND DISEASES 277

Alveolar hypoxia Hyperresponsive pulmonary vasculature Descent, oxygen, nifedipine

Hypoxic pulmonary vasoconstriction (uneven)

Exercise

Increased capillary pressure (some capillaries)

Restricted vascular bed (unilateral PA)

Damage to capillary wall (stress failure)

High permeability edema (patchy distribution)

Exposed basement membranes

Neutrophil activation Release of inflammatory markers

Platelet activation Fibrin thrombi

Figure 2 The sequence of events in the pathogenesis of high-altitude pulmonary edema. Reproduced from West JB and MathieuCostello O (1992) High altitude pulmonary edema is caused by stress failure of pulmonary capillaries. International Journal of Sports Medicine 13(supplement 1): S54S58, with permission.

The pathogenic mechanism outlined above is consistent with the prevention and treatment of the disease. Subjects should ascend slowly to allow some remodeling of the pulmonary circulation to occur. Strenuous exercise, particularly immediately after ascent, is a risk factor because it raises the pulmonary artery pressure and therefore it should be avoided. Pulmonary vasodilator drugs such as the calcium channel blocker nifedipine given prophylactically reduce the incidence of high-altitude pulmonary edema. The best treatment is to remove the patient to a lower altitude as quickly as possible. Recovery is usually rapid if this is done. Of course oxygen should be administered if it is available to relieve the alveolar hypoxia. Nifedipine has been shown to relieve symptoms, a suggested dose being 20 mg of the slowrelease preparation orally every 68 h. Other vasodilators such as nitric oxide may be effective but are generally difcult to use in the eld. Some recent research suggests that salmeterol and sildenal may also be helpful.
High-Altitude Cerebral Edema

This is an uncommon but potentially fatal condition. It is usually associated with acute mountain sickness and indeed many people think that it is the far end of the spectrum of this condition. The incidence is uncertain but is perhaps as high as 12% in people who ascend above 4500 m. Headache is common as part of the acute mountain sickness. However, the onset of high-altitude cerebral edema is indicated by ataxia, mental confusion, and

mood changes. Ultimately coma may supervene, followed by death. Hallucination has been described in some patients. On examination ataxia can be demonstrated, there is often papilledema and occasionally there are focal neurological signs affecting the cranial nerves. Hemiparesis has been described. The pathogenesis of high-altitude cerebral edema is the subject of intense study but is still not clear. Certainly, there is evidence of cerebral edema. Autopsies have shown edema of the brain with swollen attened gyri, and in a few patients magnetic resonance imaging has revealed intense T2 signals in white matter, particularly in the splenium and corpus callosum. This is consistent with cerebral edema. Why the edema occurs is unclear. Possibly an increased cerebral blood ow plays a role. As indicated earlier, arterial hypoxemia causes cerebral vasodilatation and increased retinal blood ows have been demonstrated at high altitude. It is also possible that there is an increased permeability of the bloodbrain barrier. By far the most important treatment is to remove the patient to a lower altitude as quickly as possible. This often results in rapid improvement. Oxygen should be administered if this is available. Dexamethasone is also valuable, the suggested dose being 8 mg initially followed by 4 mg every 6 h. Sometimes, as on an expedition, rapid removal to a lower altitude is not practicable. In this case, portable hyperbaric bags such as the Gamow bag have a place. The patient is placed inside the bag and the pressure is raised with a foot pump. This simulates removal to a lower altitude. The Gamow bag can

278 HIGH ALTITUDE, PHYSIOLOGY AND DISEASES

also be used for treatment of high-altitude pulmonary edema.

Chronic Mountain Sickness

This is seen in permanent residents of high altitude, often after many years of residence. The condition is characterized by severe polycythemia with very high hematocrits that may exceed 70%. Values above 80% have even been recorded. The polycythemia is accompanied by cyanosis and a constellation of neurological symptoms including headache, fatigue, somnolence, and mood changes. Treatment is difcult. Patients usually improve if they go down to a lower altitude but often this is not practicable for economic reasons. Therapeutic phlebotomy provides temporary relief but the polycythemia returns. Some success has been obtained with respiratory stimulants such as medroxyprogesterone acetate, the rationale for using these being that some patients develop hypoventilation. An interesting feature of this condition is that while it is relatively common in the Andes at high altitude, it is much rarer in Tibet. This is of great interest to anthropologists some of whom believe that the residents of the Tibetan plateau have progressed further in genetic adaptation to high altitude than the highlanders of the Andes. It is argued that the Andean residents have only been at high altitude for some 10 000 years or so whereas Tibetans have been high-altitude residents for a very much longer period.
Subacute Mountain Sickness

cyanosis, and congestive heart failure. This is particularly evident in Han Chinese infants in Tibet. Interestingly, Tibetan infants rarely develop the condition. Again the pathogenesis is apparently right heart failure as a result of the severe pulmonary hypertension.
Retinal Hemorrhage

This condition is common in members of climbing expeditions who go above 5000 m. The ame-shaped hemorrhages can be seen by ophthalmoscopy but usually cause no visual impairment. The ophthalmic appearances become normal when the climbers return to sea level.
See also: AcidBase Balance. Arterial Blood Gases. Diffusion of Gases. Exercise Physiology. Fluid Balance in the Lung. OxygenHemoglobin Dissociation Curve. Peripheral Gas Exchange. Permeability of the BloodGas Barrier. Pulmonary Circulation. Vascular Disease. Ventilation: Control.

Further Reading
Anand IS, Malhotra RM, Chandrashekhar Y, et al. (1990) Adult subacute mountain sickness a syndrome of congestive heart failure in man at very high altitude. Lancet 335: 561565. Hackett PH and Roach RC (2001) Current concepts: high-altitude illness. New England Journal of Medicine 345: 107114. Hackett PH and Roach RC (2004) High altitude cerebral edema. High Altitude Medicine and Biology 5: 136146. Hornbein TF and Schoene RB (2001) High Altitude: An Exploration of Human Adaptation. New York: Dekker. Moore LG, Niermeyer S, and Zamudio S (1998) Human adaptation to high altitude: regional and life cycle perspectives. American Journal of Physical Anthropology Yearbook 41: 2564. Pollard AJ and Murdoch DR (2003) The High Altitude Medicine Handbook, 3rd edn. Oxford, New York: Radcliffe Medical Press. Reeves JT and Leo n-Velarde F (2004) Chronic mountain sickness: recent studies of the relationship between hemoglobin concentration and oxygen transport. High Altitude Medicine and Biology 5: 147155. Schoene RB (2004) Unraveling the mechanism of high altitude pulmonary edema. High Altitude Medicine and Biology 5: 125135. Ward MP, Milledge JS, and West JB (2000) High Altitude Medicine and Physiology, 3rd edn. London: Arnold. West JB (1984) Human physiology at extreme altitudes on Mount Everest. Science 223: 784788. West JB (2005) Respiratory Physiology The Essentials, 7th edn. Baltimore: Lippincott Williams & Wilkins. West JB and Mathieu-Costello O (1992) High altitude pulmonary edema is caused by stress failure of pulmonary capillaries. International Journal of Sports Medicine 13(supplement 1): S54S58.

This term is confusing and there are currently efforts to replace it with better descriptors. It refers to two separate conditions. One affects young adults who spend long periods of time at extremely high altitudes. The best example has been soldiers in the Indian army who have been posted to altitudes of approximately 6000 m for several months. They develop dyspnea and dependent edema, and this may be associated with a cough and even effort angina. On investigation, there are signs of right heart hypertrophy and the condition is thought to be caused by right heart failure as a result of the severe pulmonary hypertension over a long period. Interestingly, cattle that are grazed at high altitude develop a similar condition known as Brisket disease, so-called because the edema is prominent in the soft tissue of the neck or brisket. The other group of patients to whom the term subacute mountain sickness is given are infants at high altitude who develop severe respiratory distress,

HISTAMINE 279

HISTAMINE
W W Busse and J E Knuffman, University of Wisconsin Medical School, Madison, WI, USA
& 2006 Elsevier Ltd. All rights reserved.

The Molecule
Histamine (b-imidazolethylamine) comes from the Greek histos which means tissue, derived undoubtedly from the molecules presence in a variety of tissue types. Negatively charged side chains of proteoglycans within the cytoplasmic matrix associate with histamine, including heparin and chondroitin 4-sulfate. Histamine is derived from decarboxylation of the amino acid histidine by the enzyme L-histidine decarboxylase (HDC). This decarboxylation occurs primarily in the Golgi apparatus of mast cells and basophils. Histamine is stored in its active form within granules in the cytoplasm of these cells. In humans, the basophil contains most of the histamine content of the body. In addition to mast cells and basophils, dendritic cells and T cells also express HDC, although histamine is not stored in the latter. Histamine is also synthesized in platelets, endothelial cells, enterochromafn-like cells, and neuronal cells (Figure 1). Histamine reaches peak serum concentrations in about 5 min and returns to baseline values in less than 30 min, giving it a half-life of about 20 s. Mast cells contribute local histamine release in asthma pathophysiology, but account for only about 2% of the histamine found in the circulation, the basophil producing most of the remainder. The metabolism of the histamine molecule is divided into two separate pathways. One path accounts for 70% of the molecules metabolism and involves a methylation step utilizing histamine N-methyltransferase. This methylation can be followed by an oxidation step before the nal product, N-methylimidazole acetic acid, is excreted via the renal system. The other route of histamine metabolism involves direct oxidation of histamine followed by successive phosphate transfers to arrive at the nal metabolite, riboside-N-3imidazole. Less than 5% of histamine is excreted

Abstract
Histamine is a molecule that has long been recognized as crucial to the allergic response. In this article, the authors discuss the metabolism of histamine, its interaction with cells of the immune system and, briey, how development of antagonists of the histamine receptor can modulate the molecules effector functions. Specic focus is given to the various types of histamine receptors and histamines release from the mast cell. Histamines role in T-cell function is also addressed. In the upper airway, histamine is a primary mediator of symptoms comprising allergic rhinitis such as nasal and ocular pruritus, sneeze, and nasal congestion. Regarding allergen-induced lower airway symptoms, histamine contributes to bronchoconstriction leading to wheeze and cough.

Introduction
Histamine is one of the principle molecules associated with the allergic-response mechanism, and was one of the rst products identied as originating from mast cells. First described in the early 1900s by Drs Dale, Laidlaw, and Barger, histamine has been extensively studied, not only for its own effects on the body, but also for the effects of its antagonists on preventing certain responses. As early as the late 1940s, there were 20 formulations available to modulate histamines actions. In this chapter, the highlights of histamine that will be covered include a discussion of the molecule and its receptors, release from and interaction with cells of the immune system, as well as a brief discussion of clinically useful antagonists of the histamine receptor.

Histamine

Bronchoconstriction

Increased mucus production

Vasodilatation

Itch

Wheeze

Chest tightness

Cough

Mucus plugging

Nasal congestion

Nasal/ocular

Urticaria

Figure 1 Putative effects of histamine.

280 HISTAMINE

unchanged in the urine, but elevations are more prolonged than that of serum, making measurement of urinary histamine a more useful clinical tool.

Table 1 Histamine receptors Receptor HR1 Chromosome 3 G protein Gq/11 Location Nasal mucosa, endothelium, lung, lymphocytes, monocytes, neurons, hepatocytes, gastric mucosa, heart, CNS Gastric mucosa, lung, uterus, heart, CNS Central and peripheral nervous system, perivascular autonomic nerve terminals, GI tract Hematopoietic cells, colon, heart, lung, thymus, spleen, small bowel

The Histamine Receptors

Histamine receptors are 7-transmembrane receptors, which mediate a variety of physiologic responses and are encoded for by up to 1000 genes. At this point in time, there are four histamine receptors (HR14) that have been identied, with a fth less sufciently characterized, molecularly. All histamine receptors are associated with a G-protein-coupled receptor, a very common target for modern pharmacotherapy. Histamine interacts with peptide stretches located on the third and the fth transmembrane regions of the receptor. The H1 receptor gene is encoded on chromosome 3 and is involved in several physiologic and pathophysiologic roles, the most notable being that of allergic disease. The basis of this receptors function is to mobilize intracellular calcium levels. This calcium release is coupled to phospholipase C (PLC) catalyzing breakdown of membrane phospholipids. Phosphatidyl 4,5-biphosphate (PIP2) is broken down into 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 mediates calcium release which leads to activation of numerous potential second messengers (cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), just to name two) which further promote downstream cellular processes. In the lung, the H1 receptor mediates the contraction of airway smooth muscle and has been implicated in heightened airway hyperresponsiveness. Other effects of the H1 receptor include urticaria and increased vascular permeability which have clinical applications in the treatment of chronic urticaria and allergic rhinitis. The H2 receptor is encoded for on chromosome 5 and relies not only the phosphoinositide metabolic pathway but also the adenylate cyclase system that uses cAMP as a mediator. H2-receptor stimulation leads to gastric acid secretion, inotropic and chronotropic cardiac stimulation, and downregulation of the immune system. Other effects mediated by H1 and H2 receptors include vasodilatation and increased mucus production. The third histamine receptor to be identied, H3, is encoded for on chromosome 20. It has been implicated in the modulation of histamine storage and release specifically in the central and peripheral nervous systems. It is believed that the H3 receptor functions as a negative feedback mechanism on histamine synthesis. The H4 receptor gene is found on chromosome 18 and is significantly homologous to the H3 receptor, creating

HR2 HR3

5 20

Gas Gi/o

HR4

18

Gi/o

overlap in the experimental use of H3 and H4 agonists and antagonists, presently. There is a fth histamine receptor labeled H(ic), which is not well dened currently (Table 1).

Cellular Histamine Release

To understand the effects of the histamine molecule, it is necessary to review the mechanisms which mediate its release from pertinent cells, namely mast cells and basophils. As mentioned above, the mast cell accounts for much of the local histamine release pertinent to the asthmatic and rhinitis processes. The initial step leading to histamine release begins with a cross-linking, by antigen, of adjacent IgE molecules on the mast cell surface. The Fc portions of these IgE molecules bind to a high-afnity Fc receptor which is specic for these heavy chain regions. The relative strength of this bond is high, meaning that although serum IgE levels are among the lowest of all immunoglobulin subtypes, the effects of IgE are quite powerful. This Fc receptor, FceRI, is constitutively expressed on the surfaces of mast cells and basophils and is central in mediating the effector functions of these cells (Figure 2). The FceRI is composed of three transmembrane components: a, b, and g. The a subunit mediates ligand binding through two Ig-like domains. The b subunit is a 4-pass transmembrane protein containing an immunoreceptor tyrosine-based activation motif (ITAM) near its cytoplasmic carboxyterminus and is involved in the subsequent signaling cascade. There are two identical, linked g chains involved in

HISTAMINE 281
IgE molecule binding site S N S S SS N

N S

N C Lyn

C C ITAMs C Syk

Figure 2 High-afnity IgE receptor, FceRI. IgE, produced from B cells, binds in the region of the Ig-like domains, created by disulde linkages in the a subunit. The tyrosine kinase, Lyn, phosphorylates ITAMs on the cytoplasmic portions of the b and g chains. Another tyrosine kinase, Syk, is activated when it associates with the phosphorylated ITAMs on the g chains. Syk, then, is able to continue the intracellular signaling process.

intracellular signaling which contain one ITAM apiece on their cytoplasmic aspects. There are at least three main pathways leading to the effector function of the mast cell. The rst two involve the formation and release of cytokines such as tumor necrosis factor (TNF) and lipid-derived products, such as prostaglandin D2, leukotriene C4 (LTC4), LTD4, and LTE4, among others. The third pathway involves the release of granule contents, namely histamine and others. Going back to the FceRI complex described above, there exists a tyrosine kinase on the cytoplasmic aspect of the b chain called Lyn. Soon after antigen cross-linking of IgE has taken place, Lyn phosphorylates the ITAMs located on the other cytoplasmic b chain and the g chain. At that juncture, another tyrosine kinase called Syk phosphorylates an isoform of PLC, introduced previously during the discussion of the H1 receptor. PLC allows for an overall increase in intracellular calcium levels which contributes to the activation of protein kinase C (PKC). PKC is important because it is this molecule that dissolves the actinmyosin complexes near the cellular membrane, allowing the granules containing histamine, and other preformed mediators, to fuse with the plasma membrane. After the fusion is complete, these mediators are released into the surrounding tissues to exert their effects. Besides histamine, other preformed mediators from mast cells and basophils include heparin, chondroitin sulfate, tryptase, chymase, acid hydrolases, cathepsin G, carboxypeptidase, and lysophospholipase, among others (Figure 3).

The Interaction of Histamine with Cells of the Immune System

In the asthmatic process, histamine release from mast cells is associated with airway obstruction, mucosal edema, and mucus hypersecretion, undeniably related to its inherent properties of promoting smooth muscle contraction in the airway as well as increasing vascular permeability. These effects are largely mediated through HR1, although HR2 plays a significant role in mucus secretion. The cellular components involved in promoting asthma and rhinitis symptoms is a topic of intense research. A number of immune cell types express H1 and H2 receptors, such as regulatory T cells, B cells, helper T cells, and monocytes. An important concept to introduce is the Th1/Th2 dichotomy. CD4 T cells can differentiate into either the Th1 or Th2 subsets, based on the prole of cytokines secreted. Th1-like cytokines include IFN-g and TNF-b which are linked to cell-mediated immune responses, while Th2-like cytokines include IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, linked to a humoral response pattern including the allergic phenotype. Dendritic cells play a sizable role in inuencing which subset a certain CD4 T-cell clone becomes, potentially based on early antigen presentation in the periphery. When immature dendritic cells are exposed to IFN-g, the IL-12 production is increased, which is important for cultivating a Th1 response. Similarly, the effects of increased levels of IL-10 on mature dendritic cells can result in suppressed levels of IL-12 and a resultant Th2 response.

282 HISTAMINE
PIP2

Mast cell plasma membrane

Fc RI complex

PLC Activated Syk

IP3

DAG

MAP kinase cascade Ca2+ Lipid mediator synthesis and secretion Cytokine expression and secretion Histaminecontaining granule PKC

Actinmyosin complexes dissolved

Histamine released after granule fuses with mast cell membrane

Figure 3 Intracellular signaling in the mast cell. After the binding of antigen cross-linked IgE to the FceRI and subsequent activation of the Syk tyrosine kinase, several distinct intracellular signaling pathways result. Syk may activate a mitogen-activated protein (MAP) kinase cascade leading to the production of lipid mediators and the production of cytokine gene expression. Syk also activates an isoform of phospholipase C (PLC) which breaks down phosphatidylinositol biphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). DAG, along with increased intracellular calcium stimulated by IP3, both activate protein kinase C (PKC), which breaks down actinmyosin complexes near the mast cell membrane allowing fusion of granules with it. These granules contain histamine and a number of other preformed mediators. After fusion with the cell membrane and subsequent exocytosis of the granule contents occur, histamine is released to the extracellular tissue.

With the Th1/Th2 paradigm in mind, studies evaluating histamines effects can be placed into perspective. Some antigen-presenting cells (APCs) such as immature dendritic cells can express all four histamine receptors. It has been shown that in these cells, the H1 and H3 receptors can promote antigen-presenting properties as well as Th1 phenotype while H2-receptor stimulation contributes to an APC more capable of secreting IL-10 consistent with a Th2 cell type. In addition, the administration of histamine has been shown to inhibit IL-12 and other pro-inammatory cytokines in mature dendritic cells by way of the H2 receptor. With regard to T cells, it has been shown that the Th2 subtype has a significantly higher expression of HR2 and that these cells express more Th2 cytokines, such as IL-4 and IL-13. The Th1 cells show higher expression of HR1 and are capable of producing more IFN-g. The differences between HR1 and HR2 potentially explain part of the concept of tolerance invoked by HR2 stimulation and autoimmunity fueled by HR1 stimulation.

Histamine Antagonists Use in the Respiratory, Gastrointestinal, and Integumentary Systems

A number of pharmacologic agents that antagonize the histamine receptors, namely HR1 and HR2, are available for clinical use. Many of these uses apply to rhinitis, allergic conjunctivitis, urticaria, and gastroesophageal reux disease. Some in vitro data with antihistamines in asthma have been intriguing, but studies looking at antihistamines for treating asthma in humans to date have been less encouraging. With the advent of the low or nonsedating, second-generation antihistamines aimed at HR1, treatment of rhinitis has become far more tolerable for the average patient. Two of these second-generation HR1 antagonists, astemizole and terfenadine, were removed from the market due to concerns over prolongation of the QT interval, especially when combined with azole antifungal agents or macrolide antimicrobials (Table 2). In general, the HR1 antagonists have proven extremely useful in treating the symptoms of allergic

HISTAMINE 283
Table 2 Histamine receptor antagonists HR1 antagonists First generation Pheniramine Brompheniramine Chlorpheniramine Triprolidine Dimetindene Hydroxyzine Meclizine Buclizine Diphenylpyraline Azatadine Cyproheptadine Second generation Acrivastine Oxatomide Cetirizine Fexofenadine Ebastine Astemizole Loratadine Diphenhydramine Clemastine Dimenhydrinate Doxylamine Phenyltoloxamine Carbinoxamine Pyrilamine Tripelennamine Antazoline Promethazine Methdilazine HR2 antagonists Cimetidine Ranitidine Famotidine Nizatidine

is focused on evaluating histamines modulation of the immune system especially during its formative period. Knowledge of the mechanisms of pharmacotherapy currently available for the treatment of upper airway symptoms will undeniably lead to a further understanding of histamines role in the lower airway as well.
See also: Allergy: Overview; Allergic Rhinitis. Asthma: Overview; Acute Exacerbations; Exercise-Induced. Chymase and Tryptase. Dendritic Cells. Gastroesophageal Reux. G-Protein-Coupled Receptors. Interleukins: IL-4; IL-6; IL-9; IL-10; IL-12; IL-13. Leukocytes: Mast Cells and Basophils; T cells. Lipid Mediators: Leukotrienes. Neurophysiology: Neural Control of Airway Smooth Muscle. Reactive Airways Dysfunction Syndrome. Signal Transduction.

Desloratadine Mizolastine Terfenadine Ketotifen Azelastine Levocarbastine

Further Reading
Abbas AK and Lichtman AH (eds.) (2003) Immediate hypersensitivity. In: Cellular and Molecular Immunology, 5th edn., pp. 432452. Philadelphia, PA: Saunders. Akdis CA and Blaser K (2003) Histamine in the immune regulation of allergic inammation. Journal of Allergy and Clinical Immunology 112: 1522. Hart PH (2001) Regulation of the inammatory response in asthma by mast cell products. Immunology and Cell Biology 79: 149153. Idzko M, La Sala A, Ferrari D, et al. (2002) Expression and function of histamine receptors in human monocyte-derived dendritic cells. Journal of Allergy and Clinical Immunology 109: 839846. Leurs R, Smit MJ, Timmerman H, et al. (1996) Histamine receptors and histamine. In: Simons FER (ed.) Histamine and H1Receptor Antagonists in Allergic Disease, pp. 190. New York: Dekker. Lieberman P (2001) Prevention and therapy of Immunologic diseases: antiinammatory medications. In: Rich RR (ed.) Clinical Immunology, 2nd edn., pp. 109.1109.11. New York: Mosby. MacGlashan D (2003) Histamine: a mediator of inammation. Journal of Allergy and Clinical Immunology 112: S53S59. Mazzoni A, Young HA, Spitzer JH, Visintin A, and Segal DM (2001) Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization. Journal of Clinical Investigation 108: 18651873. Simons FER (2003) Antihistamines. In: Adkinson NF, Yunginger JW, Busse WW, et al. (eds.) Middletons Allergy: Principles and Practice, 6th edn., pp. 834863. New York: Mosby. Simons FER (2004) Drug therapy: advances in H1-antihistamines. New England Journal of Medicine 351: 22032217. Togias A (2003) H1 receptors: localization and role in airway physiology and in immune functions. Journal of Allergy and Clinical Immunology 112: S60S68.

rhinitis, specifically addressing sneezing, pruritus, and rhinorrhea. They have been shown to improve quality of life. They are not used solely for the purpose of relieving nasal congestion like nasal corticosteroids. Several prescription antihistamines are currently marketed in combination with pseudoephedrine, as the latter medication can address nasal congestion. In addition to their clinical efcacy in treating allergic rhinitis, HR1 antagonists are widely used in the treatment of chronic urticaria and also given in addition to intramuscular epinephrine for cases of anaphylaxis. Side effects from the rstgeneration HR1 antagonists may include somnolence, impaired mental efcacy, irritability, and blurred vision among a host of other central nervous system (CNS) adverse effects. Although the HR2 antagonists have an adjunctive role in the treatment of chronic urticaria, these medications are widely used in treating conditions of excessive acid production from the gastrointestinal tract.

Conclusion
The properties of histamine, its receptors, and a brief introduction to its role in the immune response have been addressed in this article. Current research

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HUMAN IMMUNODEFICIENCY VIRUS

M Lipman, The Royal Free Hospital, London, UK
& 2006 Elsevier Ltd. All rights reserved. Table 1 Adult AIDS indicator diseases (1993) Candidiasis of the esophagus, trachea, bronchi, or lungs Cervical cancer, invasive Coccidioidomycosis, disseminated or extrapulmonary Cryptococcosis, extrapulmonary Cryptosporidiosis with diarrhea for over 1 month Cytomegalovirus disease outside the liver, spleen, or lymph nodes Herpes simplex skin ulcers for over 1 month or involving the lungs or esophagus HIV-related encephalopathy Isosporiasis with diarrhea for over 1 month Kaposis sarcoma Lymphoma: Burkitts or immunoblastic or primary (i.e., not involving other parts of the body) brain Disseminated mycobacteriosis (including tuberculosis) Pneumocystis jirovecii pneumonia (PCP) Recurrent bacterial pneumonia within a 12-month period Progressive multifocal leukoencephalopathy (PML) Recurrent Salmonella septicemia Toxoplasmosis of the brain HIV wasting syndrome Adapted from Centers for Disease Control, Atlanta, USA.

Abstract
Human immunodeciency virus (HIV) is a human retrovirus belonging to the lentivirus family. It is responsible for the progressive immune dysfunction that leads to acquired immunodeciency syndrome; and has resulted in over 20 million deaths. Many of these are due to respiratory disease with pathogens such as bacteria (pneumococcus and Gram-negative organisms), the fungus Pneumocystis jirovecii, and Mycobacterium tuberculosis. The immune dysregulation induced by HIV also leads to a variety of noninfectious pulmonary complications. Highly active antiretroviral therapy has transformed HIV care: the rates of severe opportunist disease have plummeted in countries where the drugs are widely available.

Introduction
Human immunodeciency virus (HIV) is a human retrovirus belonging to the lentivirus family. It is responsible for the progressive immune dysfunction that leads to acquired immunodeciency syndrome (AIDS); and has resulted in over 20 million deaths. Two subtypes (HIV-1 and HIV-2) have been identied. HIV-1 is responsible for the vast majority of infections. HIV-2 is mainly prevalent in West Africa, and has a similar, though slower, clinical course. This article focuses on HIV-1 infection in adults. Life-threatening opportunist illnesses associated with HIV infection were rst reported in 1981. These included the fungal pneumonia due to Pneumocystis jirovecii (formerly known as Pneumocystis carinii) and the herpes virus-driven tumor Kaposis sarcoma (KS). The term AIDS was created early within the epidemic as an epidemiologic surveillance definition to capture the diseases which resulted from profound immune dysregulation. This has been modied over time to incorporate the expanding spectrum of recognized opportunist illness (Table 1). By the end of 2004 it was estimated that approximately 39 million children and adults were HIV infected. This number has increased year on year, as for every 3 million deaths there are 5 million new infections worldwide. Of these, 800 000 are children or neonates. Most HIV-positive individuals live in either middle or lower income countries, in particular in sub-Saharan Africa, with limited healthcare resources. HIV/AIDS is the fourth biggest global killer, and the leading cause of death in Africa. HIV infection is mainly spread through sexual contact. Worldwide over 70% of infections are

acquired heterosexually. In higher income countries, homosexual/bisexual males account for the largest number of infected individuals, although in many Western nations the principal route of new transmission is now heterosexual sex. Injecting drug use is responsible for about 10% of all HIV infections; while fewer than 5% of cases result from infected blood and blood products. Vertical mother-to-child HIV transmission is an important route of spread, especially in lower income countries.

Etiology and Pathology

Infectious HIV can be recovered from most body uids including blood, semen, and cervical or vaginal secretions. At initial infection, HIV spreads to the local lymph nodes, circulating immune cells and thymus. The acute viremia may be partly cleared by a host immune response. However, persistent trapping of HIV by dendritic cells exposes vulnerable host cells to infection, leading to ongoing viral replication and dissemination. The virus preferentially infects activated HIV-specic CD4 T lymphocytes, though it can also directly attack other T and B cells as well as monocytes, tissue macrophages, and cells expressing surface CD4. Viral entry requires binding of the HIV external envelope glycoprotein gp120 to the CD4 receptor as well as protein co-receptors such as chemokine receptor 5 (CCR5), or other co-receptors including

HUMAN IMMUNODEFICIENCY VIRUS 285

CXCR4, dependent on host cell type. Once HIV is inside the cell it can, using viral reverse transcriptase, integrate its genetic material into the host genome. The virus (in the form of proviral DNA) remains latent in many cells until the cell itself becomes activated. This constitutes a long-term reservoir of potentially infectious virus. Cytokine or antigen stimulation (e.g., with HIV or other viruses) will switch on viral transcription into new RNA which ultimately produces more infectious virus particles. These can leave the cell and infect other CD4-bearing cells. Following an acute seroconversion illness (often clinically evident as a u-like syndrome in up to 50% of cases), an HIV-infected individual will remain well despite there being detectable circulating HIV. As the virus targets the very cells responsible for immune regulation, persistent infection of CD4 cells leads to increasing degeneration of the immune system. This is mirrored by CD4 cell depletion and the onset of clinical symptoms. The immune dysregulation ultimately results in opportunistic infections and tumors. There is much debate over the precise mechanisms involved. Possibilities include enhanced cell apoptosis, syncytia formation with cell death, chronic immune activation, and HIV glycoprotein binding to uninfected CD4 cells causing widespread activation of cytotoxic T lymphocytes (CTL). The end result is that in the majority of infected people the blood CD4 count falls over time. The situation in the lung is similar in that profound changes occur in the resident alveolar and interstitial cell populations. Several different pulmonary cell populations can be infected by HIV, and studies show that there are higher viral loads in lung cells compared to blood during episodes of respiratory disease. In many patients, the presence of HIV in the lung leads to an asymptomatic macrophage alveolitis and CD8 lymphocytosis. The CTL are directed against broblasts, macrophages, and lymphocytes. With progressive failure of CD4 regulation, and consequent changes in the cytokine milieu, the residual cells are less able to maintain the normal homeostasis which usually can mount a strong local immune response against pathogens yet also exhibit tolerance toward selected antigen. The consequence of progressive HIV disease is therefore an increased incidence of disseminated pulmonary infection, the development of specic respiratory illness through defects in the normal immune surveillance mechanisms, and failure of the local, controlled immune response. Although blood contains approximately only 2% of total body lymphocytes, the decline in blood absolute CD4 and CD4 percentage of total lymphocytes are useful indicators of the risk of developing a severe illness. Individuals can be stratied as having

relatively intact (blood CD4 4 500 cells ml 1), moderate (200500 cells ml 1), or severely impaired immunity (o200 cells ml 1). This is useful as a pragmatic guide when planning treatments and assessing disease episodes. For example the risk of individuals developing Pneumocystis pneumonia rises precipitously as the blood CD4 count falls below 200 cells ml 1. Hence specic primary prophylaxis has been recommended at this level in all adults. High plasma HIV loads are also associated with more rapid disease progression and risk of death. HIV-related clinical symptoms can provide important prognostic information independent of CD4 count, oral thrush and constitutional illness being the strongest clinical predictors of progression to AIDS. The US Centers for Disease Control and Prevention (CDC) clinical classication of disease describes the various levels of HIV infection, though does not predict individual outcome. It is based on evidence of HIV infection with clinical indicators of impairment in cell-mediated immunity (Table 2). The 1993 CDC classication includes an immunologic criteria for AIDS (CD4 counto200 cells ml 1 or CD4 percentageo14%) irrespective of clinical symptoms (Table 3). Antiretroviral therapy directly targets HIV at different stages within its life cycle. Currently drugs
Table 2 Centers for Disease Control (CDC) classication of HIV infections (1985)a Group I Group II Group III Group IV Subgroup A Acute primary infection Asymptomatic infection Persistent generalized lymphadenopathy (PGL) Other disease Constitutional disease, e.g., weight loss 410% body weight or 44.5 kg fevers 438.5, diarrhea lasting 41 month Neurological disease: myelopathy, peripheral neuropathy, HIV encephalopathy Secondary infectious diseases: C1: AIDS dening secondary infectious disease, e.g., Pneumocystis jirovecii pneumonia, cerebral toxoplasmosis, cytomegalovirus retinitis C2: other specied secondary infectious diseases, e.g., oral candidiasis, multidermatomal varicella zoster Secondary cancers, e.g., Kaposis sarcoma, non-Hodgkins lymphoma Other conditions, e.g., lymphoid interstitial pneumonitis

Subgroup B

Subgroup C

Subgroup D Subgroup E
a

The CDC definition of the stages of HIV disease was updated in 1993 (see Table 3). However, the older classication remains in use for some research studies and is therefore reproduced here. Adapted from Centers for Disease Control, Atlanta, USA.

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Table 3 1993 Revised CDC classication system for HIV infection 1. Classication categoriesa Category A: one or more of the conditions listed below in an adolescent or adult (13 years or older) with documented HIV infection. Conditions listed in categories B and C must not have occurred. Asymptomatic HIV infection Persistent generalized lymphadenopathy (PGL) Acute (primary) HIV infection with accompanying illness (sometimes known as seroconversion illness) or history of acute HIV infection Category B: symptomatic conditions in an HIV-infected adolescent or adult that are not included among conditions listed in category C and that meet one of the following criteria: (1) the conditions are attributed to HIV infection or are indicative of a defect in cell-mediated immunity, or (2) the conditions are considered by physicians to have a clinical course or to require management that is complicated by HIV infection. This category includes all such symptomatic conditions, with the exception of those placed in category C. Examples of conditions in this category include, but are not limited to: Bacillary angiomatosis Candidiasis (thrush) in the mouth and/or upper throat Candidiasis of the vagina and/or vulva which is persistent, frequent, or responds poorly to treatment Cervical abnormalities of moderate or severe extent or cervical cancer Constitutional symptoms such as fever (38.5 1C) or diarrhea lasting longer than 1 month Herpes zoster (shingles) involving at least two distinct episodes or more than one dermatone (skin area) Idiopathic thrombocytopenia purpura Listeriosis Oral hairy leukoplakia Pelvic inammatory disease, particularly if complicated by tuboovarian abscess Peripheral neuropathy Category C: includes the following conditions listed in the AIDS (see Table 1) surveillance case definition. For classication purposes, once a category C condition has occurred, the person will remain in category C. 2. Clinical categoriesb CD4 T cell categories X500 cells ml 1 200499 cells ml 1 o200 cells ml 1
a

(A) Acute (primary) HIV, asymptomatic or PGL A1 A2 A3

(B) Symptomatic (not A or C see explanation) B1 B2 B3

(C) AIDS indicator conditions C1 C2 C3

For classication purposes, category B clinical conditions take precedence over those in category A. For example, someone previously treated for oral or persistent vaginal candidiasis (and who has not developed a category C disease) but who is now asymptomatic should be classied in clinical category B. b This classication straties patients clinically (A to C) and immunologically (1 to 3). Groups A3 and B3 satisfy the immunologic but not the clinical criteria for AIDS. Category B consists of symptomatic conditions that are not included in category C but can be either attributed to, or are complicated by, HIV infection. Examples include thrush (oral or persistent vulvovaginal); moderate or severe cervical dysplasia; thrombocytopenia; and peripheral neuropathy. Adapted from Centers for Disease Control, Atlanta, USA.

block entry to host cells, reverse transcription, and viral production and virion release. In 1996 effective drug combinations were introduced. This highly active antiretroviral therapy (HAART) has had a profound impact on those who can access medication. Major opportunistic infections as well as mortality have been reduced by over 80%. Diseases associated with severe immunosuppression such as cytomegalovirus (CMV) retinitis and Mycobacterium avium intracellulare complex (MAC) are much less common. However there has been an increase in AIDS-dening malignant disease, for example, non-Hodgkins lymphoma (NHL), as well as non-AIDS deaths due to liver and cardiovascular disease. The drugs are also not without significant side effects. Despite the efforts of the World Health Organization (WHO), HAART remains largely conned to the wealthier nations and for the majority of infected individuals, the natural history of HIV is the same as

that seen prior to HAART. It is likely that almost all will progress, with a median time from infection to AIDS of approximately 10 years. However the estimated range is very wide, at 18 months to 25 years. Without HAART, 94% of AIDS patients will die within 5 years. Respiratory disease occurs at all stages of HIV infection. A wide variety of infectious and noninfectious diseases can occur (Table 4). Prior to HAART, Pneumocystis jirovecii pneumonia was the commonest life-threatening disease, accounting for up to 40% of all AIDS-dening illnesses. Tuberculosis (TB) is now the leading cause of HIV-related death in Africa. Even with HAART, it is the most frequent global presentation of severe opportunistic infection. TB can present at any stage of HIV disease, and acts synergistically to suppress CD4 counts and elevate viral loads. Although TB can affect immunocompetent individuals, the impairment of cell-mediated

HUMAN IMMUNODEFICIENCY VIRUS 287

Table 4 Common etiologies of HIV-related pulmonary disease Bacteria Streptococcus pneumoniae Hemophilus infuenzae Staphylococcus aureus Pseudomonas aruginosa Escherichia coli Nocardia asteroides Mycobacterium tuberculosis Mycobacterium avium intracellulare complex Mycobacterium kansasii Fungi Pneumocystis jirovecii Cryptococcus neoformans Histoplasma capsulatum Candida albicans Aspergilus spp. Penicillium marneffei Parasites Toxoplasma gondii Cryptosporidium spp. Strongyloides stercoralis Viruses Cytomegalovirus Adenovirus Noninfectious causes Lymphoid interstitial pneumonitis Non-specic interstitial pneumonitis Pulmonary hypertension Emphysema Lung cancer Kaposis sarcoma Lymphoma, Hodgkins and non-Hodgkins Immune reconstitution inammatory syndrome

Adapted from Centers for Disease Control, Atlanta, USA.

immunity in HIV greatly increases individual susceptibility to the development of active TB. It is estimated that HIV has increased worldwide rates of TB by 10%. In Africa up to one-third of cases of TB occur in HIV-positive individuals. Within 1 year of HIV infection, subjects living in TB-endemic areas have a doubling of risk of active disease; whilst subsequent risk per year is similar to that found in HIVnegatives over a lifetime (510%). Pneumocystis pneumonia (PCP) is historically important, though the use of HAART and targeted specic primary prophylaxis has greatly reduced its incidence. Patients who present with PCP in higher income countries are usually those who are unable or unwilling to access medical care. Pulmonary CD4 cell depletion as well as probable CD8 dysfunction contributes to its pathogenesis. Acute bronchitis is seen in up to 30% of individuals with early HIV infection. Bacterial pneumonia has an attack rate approximately six times that found in the HIV uninfected population. The risk of specic conditions is determined to some extent by individuals as well as geographic factors. Injecting drug users have a high incidence of wasting syndrome, recurrent bacterial pneumonia, and TB, whilst subjects from populations with high background rates of TB are at greatly increased risk of this disease. Thus it is the combination of locally impaired pulmonary immunity together with the presence of specic environmental organisms that appears to determine the spectrum of opportunist respiratory disease present within a community. Respiratory disease in children is also common. PCP is often the presenting illness in undiagnosed HIV-infected babies and neonates. Recurrent, severe bacterial infections occur frequently; and, together with TB and cytomegalovirus (CMV) infection, are typical post-mortem ndings. Lymphoid interstitial pneumonitis is almost exclusively seen in children.

Clinical Features of Respiratory Disease

The clinical presentation of both infectious and noninfectious respiratory illness can be very similar. Typically an individual may complain of cough and breathlessness. A short history (a few days), or one associated with fever and purulent sputum, suggests a bacterial cause. A longer duration with minimal respiratory signs and evidence of significant immunosuppression elsewhere, for example, oral thrush, implicates PCP. Marked weight loss and constitutional symptoms may point to an underlying mycobacterial etiology. Gradual breathlessness over several months is present in conditions such as lymphoid interstitial pneumonitis (LIP) and HIVrelated pulmonary hypertension. However, clinical features are rarely cause-specic: for example, several pathogens including viruses, bacteria, protozoa, and fungi can mimic PCP. Early investigation, with the aim of targeting treatment, is thus a critical part of respiratory disease management. In practical terms, useful information can be derived from knowledge of an individuals previous medical history. For example, PCP is much more likely if a patient has a low CD4 count (o200 cells ml 1) and either no history of adequate specic Pneumocystis prophylaxis or poor adherence to a prescribed regimen. TB is especially likely (at any CD4 count) in subjects who have lived in endemic areas or have a personal history of mycobacterial exposure. It should be remembered that two or more pathogens are recovered from up to 20% of patients during a respiratory illness.
Bacterial Pneumonia

This may be the rst clinical manifestation of HIV infection. Its presentation is no different to that seen in an immunocompetent population. Typical organisms are listed in Table 4. At higher blood

288 HUMAN IMMUNODEFICIENCY VIRUS

CD4 counts Streptococcus pneumoniae is the commonest cause. More unusual bacteria occur with increasing frequency at later stages of HIV infection. Such individuals are also at greater risk of recurrent pneumonia, septicemia, and the development of rapid-onset bronchiectasis. Up to a quarter of HIV infected children will have episodes of bacterial septicemia, usually secondary to pulmonary disease. Gram-negative septicemia is a well-recognized cause of HIV-related death. The commonest radiological feature is focal consolidation, found in 50% of cases. However, bacterial pneumonia may present as solitary or multiple pulmonary nodules, interstitial opacities, or with a pleural effusion. Diagnosis rests on clinical suspicion, and culture of sputum, lung uid, and blood. Rapid antigen testing for pneumococcus can be helpful. However a large number of bacterial pneumonias are treated empirically with no specic organism isolated.
Mycobacterial Disease

the reduced T-cell number and function seen in patients with advancing HIV. Such tests, however, may have a role in predicting the future risk of developing mycobacterial disease, which is especially important in this population. Other mycobacterial opportunist pathogens such as MAC and Mycobacterium kansasii usually occur when the blood CD4 count is less than 100 cells ml 1. Their clinical presentation may be similar to TB (with cough, sputum, weight loss, and fever) though can be very non-specic. MAC in particular is seen in a disseminated form and can be diagnosed in such cases on the basis of culture from any normally sterile site such as blood or bone marrow.
Pneumocystis Pneumonia

HIV infection has transformed the clinical presentation and management of mycobacterial disease. The changes produced in local pulmonary immunity lead to a failure of granuloma formation and thus a high incidence of extrapulmonary and disseminated disease. In such cases, the radiological features of pulmonary TB are often atypical: instead of post primary features of upper zone inltrates with cavitation, there is non-specic mid and lower zone shadowing, pleural effusions, and thoracic lymphadenopathy. This pattern is much more common as the blood CD4 count falls. The cornerstone of TB diagnosis is sputum smear microscopy and culture. Three early morning (or overnight) samples should be obtained if the patient is productive. If not, then either induced sputum or bronchoalveolar lavage (BAL) will be helpful. These should be carried out in negative pressure isolation to reduce the risk of TB transmission. However, it is common for TB to be recovered from more than one body site; and blood, urine, and, if clinically indicated, spinal uid cultures also should be collected. New methods of rapid diagnosis include nucleic acid amplication (NAA) tests as well as serological and immunological techniques. The NAA tests are increasingly useful, as they have a greater sensitivity than microscopy, can distinguish TB from other mycobacteria, and allow early detection of drug resistant strains. Immune-based tests (often using T cell responses to mycobacteria-specic antigens) appear to be less sensitive in HIV-infected individuals than the immunocompetent. This is not surprising given

PCP typically presents with a history lasting several days to weeks of dry cough, breathlessness, fever, and malaise. There are often surprisingly few pulmonary ndings, though the chest radiograph typically reveals bilateral mid and lower zone interstitial and alveolar shadowing. In up to 10% of cases this may be very subtle and the radiograph will appear normal. Computed tomography (CT) scanning can be helpful in such cases, demonstrating the ground glass appearance consistent with alveolar pathology. It should be noted that this is not specic for PCP and that provided the patient is t enough, lung sampling is necessary to make a denitive diagnosis. Pneumocystis is difcult to culture, and PCP diagnosis relies on direct visualization of the cysts. Immunouorescence techniques increase the sensitivity of microscopy; and NAA tests are generally not required for diagnosis. Sampling can be performed using induced sputum, BAL, or tissue biopsy. The procedure of choice may be determined by local expertise. Induced sputum has a yield of 5080% but a low negative predictive value, and is easiest to perform. BAL is more sensitive (over 90%) though requires greater technical expertise, facilities, and expense. Transbronchial and open lung biopsies are bigger procedures, with greater patient risk. They tend to be reserved for cases that remain diagnostically challenging despite imaging and BAL. The distribution of Pneumocystis and its inammatory response within the interstitium and alveolar space mean that exercise testing can be helpful in those patients with a compatible history and a normal chest radiograph. Here, after exercise (such as step-ups or cycling on a xed frame for 510 min to increase heart rate to 80% of maximum predicted), exercise desaturation (o90%) is more likely to be due to PCP than bacterial or other etiologies.

HUMAN IMMUNODEFICIENCY VIRUS 289 Other Infections

The following pulmonary infections occur in individuals with markedly impaired pulmonary immunity. This is usually reected in blood CD4 counts o100 cells ml 1. CMV is often recovered from the lung of patients with respiratory disease. The significance of this is unclear. When associated with PCP, the overall outcome appears to be worse, although there is little evidence to show that specic CMV treatment is benecial. The presence of CMV may therefore reect the extent of profound local immunosuppression rather than active disease per se. Toxoplasma gondii pulmonary disease presents with cough and fever. The lung may be the only site of disease or, just as with the fungus Cryptococcus neoformans, can be associated with severe, disseminated infection. Radiographic appearances are nonspecic and the diagnosis can be made using cytology or NAA techniques. Pulmonary cryptococcosis is typically conrmed using antigen tests (on lung uid as well as blood) and culture.
Noninfectious Conditions

LIP is reported in up to 40% of children aged over 1 year with perinatally acquired HIV infection. It presents with slowly progressive breathlessness associated with cough, wheeze, and digital clubbing. It is much less common in adults, though can be mistaken for PCP and other infections. The diagnosis may require conrmation by tissue biopsy, where massive inammatory cell inltration will be evident throughout the alveolar and interstitial spaces. A less aggressive form of disease is seen in adults presenting with widespread CD8 T cell inltrates involving the parotids, muscle, and bone as well as lung. This diffuse inltrative syndrome can occur in subjects with either relatively well-preserved immunity or who have had a good response to antiretroviral treatment. A further and less well-characterized condition is that of non-specic interstitial pneumonitis (NSIP). This has been used to describe individuals who have typically mild symptoms of dry cough, breathlessness, and occasionally fever but where no infectious cause has been determined. The ndings are often associated with pulmonary function abnormalities such as a reduced transfer factor. Chest radiology may show inltrates or nodularity. It is thought that NSIP is due to the increased non-specic proinammatory state present within the HIV-infected lung. Pulmonary hypertension of unknown etiology appears to be more common in HIV-infected individuals. It presents as increasing breathlessness, with ultimately right-sided cardiac failure. The plexiform arteriopathy found on histology is thought to arise

from HIV protein- and cytokine-mediated vasoconstriction and endothelial proliferation. This needs to be distinguished from other pulmonary vascular abnormalities such as those associated with injected foreign material (e.g., talc in injecting drug users), chronic thromboembolic disease, and hepatopulmonary syndromes in cirrhotics. HIV-infected cigarette smokers appear, in some studies, to have an accelerated loss of lung volume and early onset bullous emphysema. This may be due to increased local production of matrix metalloproteases, which in turn results from dysregulation of HIV-infected macrophages. KS is the tumor most strongly associated with HIV infection. The relative risk compared to HIV-negative populations is measured in the hundreds. Its incidence has declined in countries where HAART has been introduced, though it remains the commonest HIVrelated cancer. Pulmonary KS will often present as progressive breathlessness, cough, and hemoptysis in patients with KS elsewhere. Typical other sites include the skin, mouth (especially hard palate), and abdominal viscera. Chest radiology often shows reticulonodular shadowing in a bronchocentric distribution, intrathoracic adenopathy, or large (blood-stained) pleural effusions. Diagnosis can usually be made on the basis of compatible symptoms with radiology or direct visualization of lesions at bronchoscopy. Infection with human herpes virus 8 (HHV-8), a sexually transmitted virus, is necessary for an individual to develop KS. This to some extent explains the tumors high prevalence in HIV-infected populations; though it is likely that HIV proteins are also responsible for accelerating tumorigenesis. Other HHV-8 associated diseases include primary effusion lymphoma and angiofollicular lymphoid hyperplasia (Castlemans syndrome), both of which are more common in HIV-positive populations. NHL is the second most common HIV-associated tumor. It is typically of B cell origin; and in many cases is associated with EpsteinBarr virus infection. Approximately 10% of NHL involves the lung. Its presentation is often non-specic and the radiological features are equally varied. These include inltrates, mass lesions, and effusions. Diagnosis is often based on tissue biopsy. Lung cancer appears to be about four times more common in HIV-infected smokers. Unlike viral-associated tumors, there does not seem to be an obvious relationship with the degree of immunosuppression. The tumors are predominantly peripheral non-small cell lung cancers; and patients tend to be younger than HIV-negative subjects. The diagnosis is usually established by sputum or pleural uid cytology, histologic and cytologic evaluation of samples obtained

290 HUMAN IMMUNODEFICIENCY VIRUS

via bronchoscopy, or by percutaneous ne-needle aspiration. The immune restoration inammatory syndrome (IRIS) is a clinical phenomenon associated with the use of HAART. This may take a number of different forms; and similar presentations have been seen in posttransplant populations as well as HIV-negative individuals on treatment for TB. In general terms, patients experience either a recurrence of previously controlled symptoms of an opportunistic infection shortly after starting HAART, or the antiretrovirals unmask subclinical disease. IRIS has been reported with a great number of infectious agents, typically those due to intracellular pathogens. These include MAC, TB, CMV, herpes, and hepatitis viruses. In fact, one problem with IRIS is that it is a diagnosis of exclusion (of other intercurrent events, and relapse of the original disease process). As such it may be overdiagnosed clinically. Patients with mycobacterial disease seem especially prone to IRIS. Up to 15% of HIV-negative individuals experience a transient deterioration in symptoms after starting antimycobacterial treatment (the paradoxical reaction). This more than doubles when HAART is coadministered, and the reaction is often much more severe. There are no good predictors of who is at risk of IRIS; though given its association with changes in local immunity it would seem reasonable that a rapid reduction in HIV load and possibly increases in blood CD4 count (as a reection of cell trafcking to the site of activity) may be relevant. Symptoms can last anything from a few days to more than a year. They are most often at the original site of disease. In cases where HAART activates latent infection, the location may be very atypical, for example, focal bone disease due to MAC.

3.

4.

5.

6.

7.

Management and Current Therapy

The detailed discussion of specic treatments is covered in other sections of this encyclopedia. Some general principles which apply to HIV management are as follows: 1. Clinical suspicion can guide initial therapy and some treatments should not be deferred for outstanding investigations if the patient is sick (e.g., treatment of PCP in an individual with compatible symptoms and a low CD4 count). 2. Rapid investigation is vital there may be a short period of time during which pulmonary samples or tissue biopsies can be obtained. At a later stage, the procedure may be deemed too risky (e.g., bronchoscopy in a hypoxic patient) or 8.

the prescribed treatments may reduce the sensitivity of the laboratory tests. Get to know what resources are available within the local pathology services. Diagnostic tests for relatively rare conditions are dependent on the expertise of those performing and interpreting them. Thus, there may be a high rate of false negative results in a center with little experience of a particular condition. Several different pathologies may coexist at the same time; hence nonresponse should prompt early reassessment and investigation. Drugdrug interactions and adverse effects are common in HIV-infected individuals. This is especially the case for HAART and anti-TB agents; whilst anti-PCP treatments can cause adverse effects in up to 50% of individuals. Specialist input from a team experienced in HIV disease is crucial. In practice this may require contributions from multiple disciplines which then need to be coordinated as one seamless, management plan. Prevention is better than cure. Prophylactic regimens should be considered in all individuals at risk of significant opportunist infection. HAART has transformed the spectrum of respiratory illness and its management. It undoubtedly prevents disease by improving local and specic immune responses. Individuals who have a sustained increase in blood CD4 count, an undetectable viral load, and good drug concordance can stop specic infection prophylaxis. For example, PCP prophylaxis can be discontinued at CD4 counts above 200 cells ml 1 (i.e., similar levels to that at which it is usually instituted). Immunization schedules with noninfectious vaccines may be useful. Pneumococcal immunization, despite its rather limited efcacy, has been shown to reduce rates of bacterial infection and decrease mortality. At present its uptake in HIV-infected populations is low. Scrupulous infection control is critical. Although many infections are not clearly transmissible between subjects within a healthcare setting, there is an increased incidence of bacterial nosocomial pneumonia, and TB is a constant concern in HIVinfected subjects. Respiratory precautions should be adopted when managing patients with pulmonary symptoms. These include the use of isolation facilities, with negative pressure ventilation if they are admitted to a ward with other immunocompromised individuals. The use of particulate dust lter masks by staff is important if the patient is undergoing investigation, for example, bronchoscopy. Procedures that involve the production of an aerosol (such as sputum induction or bronchoscopy)

HUMAN IMMUNODEFICIENCY VIRUS 291

should be performed in negative pressure facilities with air vented directly to the outside. Avoidance of unnecessary procedures is also relevant; and the importance of careful, repeat sputum examinations for acid-fast bacilli cannot be overstated.
Tuberculosis

HIV-related TB usually responds to standard short course (6 month) four drug rifamycin-based therapy. There are some concerns regarding risk of relapse with this duration of therapy. Indication for treatment prolongation are persistence of culture positive samples (usually sputum) after 2 months of treatment with what should be effective therapy; low CD4 counts (o100 cells ml 1) with no option for HAART, in areas where TB is endemic; extrapulmonary disease with cerebral involvement; and mycobacterial drug resistance. In general, there is a good response to standard therapy, though short-term mortality is increased in patients with advanced immunosuppression. A key issue concerns the optimum time at which to start HAART. Ensuring adequate TB treatment is most important, though if HIV-infected individuals with CD4 counts o100 cells ml 1 defer antiretrovirals for too long, there is a high risk of further opportunist infections. The early use of HAART needs to be balanced against a possible increase in adverse effects, drugdrug interactions, reduced adherence through large pill burden, and a potential for significant IRIS during this period. There is no standard approach and most clinicians tend to assess such cases on an individual basis, using the small amount of evidence that currently exists. Drug-resistant TB is increasingly common. It is almost always due to inferior treatment either from inadequate prescribed therapy or poor patient concordance. It is associated with HIV infection, though this may result from the social clustering of infectious cases in both conditions. Multidrug-resistant TB, where the organism is resistant to at least isoniazid and rifampicin, is difcult to treat, has a high mortality, and has been implicated in several outbreaks of nosocomial TB. HIV infection is a risk factor for acquired rifampicin monoresistance. This may be due to underdosing or impaired absorption of medication. Treatment adherence is important for all TB patients. Directly observed therapy as part of a package of care may be helpful when there is possible noncompliance. TB control requires organized contact tracing, chemoprophylaxis for those at risk (i.e., HIV-infected individuals with a history of significant TB exposure and positive skin/immune-based tests),

and effective local measures to reduce spread. The issue of chemoprophylaxis in high-income countries is thorny. Some authorities recommend that it should be offered to HIV-positive patients with declining CD4 counts who have a positive puried protein derivative skin test. Historically, in resource-poor settings, treatment and prevention of established TB was an effective strategy. However, HIV infection had made this approach much less benecial, and notwithstanding the problems associated with widescale implementation of HAART, HIV itself must also be treated.
Pneumocystis Pneumonia

In practical terms, if PCP seems the most likely diagnosis, treatment should be started early and a diagnostic procedure planned for the next few days, as Pneumocystis can be recovered during the rst week of treatment. Respiratory support has altered the management of severe PCP. With all currently available drug therapies (including cotrimoxazole, pentamidine, and clindamycin plus primaquine) there is a period of several days before clinical improvement occurs. The use of adjuvant steroids (to reduce interstitial leak) and support techniques such as continuous positive airways pressure (CPAP) circuits can help to maintain respiratory health during this time, often avoiding the need for full mechanical ventilation in those with severe PCP. Patients with rst-episode PCP and/or a good quality of life are usually those considered for formal ventilation. Pneumocystis prophylaxis can be given in either a primary form (when an individual is deemed to be at high risk of future disease) or as secondary prophylaxis, following an episode of PCP. This is a sensible policy as the risk of relapse/reinfection is estimated at almost 50% over a 12-month period if HAART and prophylaxis are not available. The increase in use of agents such as co-trimoxazole (a combination of trimethoprim and sulfamethoxazole) in this context and also in low-income countries to reduce risk of infection and long-term mortality, has led to concerns regarding the development of drug-resistant Pneumocystis. Mutations have been observed in the organisms dihydropteroate synthase gene; and these do appear to be associated with use of such sulfacontaining drugs. There is no good evidence that this leads to worse outcomes or that these effective drugs should not be used to treat active disease; though this seems a future possibility.
Noninfectious Conditions

In higher-income countries up to 50% of HIV-infected individuals are cigarette smokers. This has

292 HYPERBARIC OXYGEN THERAPY

been associated with an increased incidence of upper and lower respiratory tract infection, recurrent PCP, and overall accelerated mortality. Given the compelling evidence for high rates of lung cancer and chronic obstructive pulmonary disease, it would seem that smoking cessation strategies should be prioritized as part of a healthy living program. This is especially important in the context of HAART prolonging survival at the expense of an increase in blood lipids and possibly cardiovascular events. HAART has undoubtedly improved the outcome for HIV-related tumors. This is reected in the reported reduction in KS and NHL. Lung cancer, however, is different. Despite HAART and cancer chemotherapy, the outlook tends to be poor. The good scientic rationale for treating HIV-associated pulmonary hypertension with HAART and endothelin-1 inhibitors has been proven in practice. Survival is no longer measured in months; and the quality of life for individuals tolerating therapy is greatly improved.
See also: Bronchoalveolar Lavage. Interstitial Lung Disease: Hypersensitivity Pneumonitis. Laryngitis and Pharyngitis. Pneumonia: Overview and Epidemiology; Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Mycobacterial; The Immunocompromised Host. Vaccinations: Viral. Viruses of the Lung.

Further Reading
Agostini C, Zambello R, Trentin L, and Semenzato G (1996) HIV and pulmonary immune responses. Immunology Today 17: 359364. Badri M, Wilson D, and Wood R (2002) Effect of highly active antiretroviral therapy on incidence of tuberculosis in South Africa: a cohort study. Lancet 359: 20592064. Bonnet F, Lewden C, May T, et al. (2004) Malignancy-related causes of death in human immunodeciency virus-infected patients in the era of highly active antiretroviral therapy. Cancer 101: 317324.

Castro M (1998) Treatment and prophylaxis of pneumocystis carinii pneumonia. Seminars in Respiratory Infections 13: 296303. Huang L, Crothers K, Atzori C, et al. (2004) Dihydropteroate synthase gene mutations in pneumocystis and sulfa resistance. Emerging Infectious Diseases 10: 17211728. Interdepartmental Working Group on Tuberculosis (1998) UK Guidance on the Prevention and Control of Transmission of (1) HIV-Related Tuberculosis and (2) Drug-Resistant, including Multiple Drug-Resistant Tuberculosis: The Prevention and Control of Tuberculosis in the United Kingdom. London: Department of Health. Lawn SD, Bekker LG, and Miller RF (2005) Immune reconstitution disease associated with mycobacterial infections in HIVinfected individuals receiving antiretrovirals. Lancet Infectious Diseases 5: 361373. Masur H, Kaplan JE, and Holmes K (2002) Guidelines for preventing opportunistic infections among HIV-infected persons 2002: recommendations of the US Public Health Service and the Infectious Diseases Society of America. Annals of Internal Medicine 137: 435478. Mayaud C, Parrot A, and Cadranel J (2002) Pyogenic bacterial lower respiratory tract infections in human immunodeciency virus-infected patients. European Respiratory Journal 20(supplement 36): 28s39s. Pozniak AL, Miller RF, Lipman MC, et al. on behalf of the BHIVA Guidelines Writing Committee (2005) BHIVA treatment guidelines for tuberculosis (TB)/HIV infection 2005. HIV Medicine 6 (supplement 2): 6283. Smith CJ, Levy I, Sabin CA, et al. (2004) Cardiovascular disease risk factors and antiretroviral therapy in an HIV-positive UK population. HIV Medicine 5: 8892. Stebbing J, Gazzard B, and Douek DC (2004) Where does HIV live? New England Journal of Medicine 350: 18721880. Subcommittee of the Joint Tuberculosis Committee of the British Thoracic Society (2000) Management of opportunist mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax 55: 210218. UNAIDS (2004) Report on Global AIDS Epidemic. Geneva: UNAIDS. White NC, Agostini C, lsrael-Biet D, Semenzato G, and Clarke JR (1999) The growth and the control of human immunodeciency virus in the lung: implications for highly active antiretroviral therapy. European Journal of Clinical Investigation 29: 964 972. Zumla A, Johnson MA, and Miller R (eds.) (1997) AIDS and Respiratory Medicine. London: Chapman & Hall.

HYPERBARIC OXYGEN THERAPY

I Grover and T Neuman, University of California, San Diego, CA, USA
& 2006 Elsevier Ltd. All rights reserved. were treated with pressurized air. In 1955, the modern era of hyperbaric oxygen therapy was born. Today, patients are treated in either monoplace chambers or multiplace chambers. The pressurized oxygen exerts its effects by several different mechanisms, including creating a diffusion gradient for inert gases, oxygenating ischemic tissues, limiting reperfusion injuries, inactivating certain toxins, and supporting angiogenesis and leukocyte function. There are 13 indications for which scientic evidence supports the use of hyperbaric oxygen therapy: arterial gas embolism; decompression sickness; carbon monoxide poisoning; clostridial myonecrosis; crush injuries, compartment

Abstract
Hyperbaric oxygen therapy is the use of oxygen at increased atmospheric pressure. The history of hyperbaric oxygen therapy can be traced back to the late seventeenth century, when Henshaw treated patients in a pressurized chamber. Initially, patients

HYPERBARIC OXYGEN THERAPY 293

syndrome, and other acute ischemias; enhancement of healing in selected problem wounds; exceptional blood loss anemia; intracranial abscess; necrotizing soft tissue infections; refractory osteomyelitis; delayed radiation injury; preservation of skin grafts and aps; and thermal burns.

Using hyperbaric therapy for treatment of multiple disorders dates back to 1662, when Reverend Henshaw used a pressurized chamber for therapeutic purposes. In 1889, a British engineer, Ernest Moir, became the rst person to treat decompression sickness (DCS) with recompression. When he took over as the superintendent of the Hudson River tunnel project, workers had a 25% mortality rate from DCS. After he installed a recompression chamber at the work site, the mortality rate from DCS declined to 1.7%. In 1955, Ite Boerema, from the University of Amsterdam, performed an experiment using hyperbaric oxygen to keep exsanguinated pigs alive by the oxygen dissolved in plasma alone. At the same time, W H Brummelkamp at the University of Amsterdam discovered that hyperbaric oxygen could be used to inhibit anaerobic infections. In Scotland, studies suggested that hyperbaric oxygen was benecial in the treatment of carbon monoxide poisoning in humans. There are 13 indications for the use of hyperbaric oxygen therapy (HBOT) which are backed by scientic evidence. HBOT consists of treating a patient with 100% oxygen at pressures above 1.4 atm absolute (4 m of seawater) in a hyperbaric chamber. The oxygen is delivered to the patient by inhalation. There are two types of chambers in which patients are treated: monoplace and multiplace. They (Figure 1) are used to treat a single patient. They are pressurized with oxygen or air, and attendants do not have to go into the chamber with the patient. Monoplace chambers are less expensive than multiplace chambers, and they require less operating space. Multiplace chambers (Figure 2) are larger and used to treat multiple patients simultaneously. These chambers are pressurized with air, and oxygen is delivered to the patients via plastic hoods or face masks. Multiplace chambers have the advantage of treating multiple patients at a single time. Also, critically ill patients can be treated with a nurse and other support personnel in the chamber. Hyperbaric oxygenation has two primary mechanisms of action that are the basis of its use: the mechanical effect and the effect of an increased partial pressure of oxygen. The mechanical effects of pressure shrink gas bubbles and gas-lled spaces in the body following Boyles law (volume is inversely proportional to absolute pressure). There are multiple other physiologic effects that supernormal partial pressures of oxygen exert on the body. These benets

Figure 1 Monoplace chamber. Photo courtesy of OxyHeal Health Group, Inc.

Figure 2 Multiplace hyperbaric chamber. Photo courtesy of OxyHeal Health Group, Inc.

include angiogenesis, broblast growth and collagen production, improved osteoclast function, enhanced removal of carbon monoxide (CO) from hemoglobin, inhibition of a-toxin production in clostridial myonecrosis, improved leukocyte killing, decreased neutrophil adherence to capillary walls, increased production of superoxide dismutase, and vasoconstriction in normal vessels. These effects are the physiologic rationale for the use of HBOT in the conditions discussed next.

Indications for Hyperbaric Oxygen Therapy

Gas Embolism

A gas embolism occurs from any number of mechanisms, including mechanical ventilation, penetrating

294 HYPERBARIC OXYGEN THERAPY

chest trauma, chest tube placement, bronchoscopy, and pulmonary barotrauma from scuba diving. Gas bubbles enter the arteries or veins and cause a number of different symptoms, including loss of consciousness, altered mental status, focal neurological deficits, hypotension, pulmonary edema, or cardiac arrhythmias including cardiac arrest. A patient who exhibits neurologic symptoms or cardiac symptoms from a gas embolism should be treated with HBOT. First, HBOT exerts a mechanical effect on bubble size with recompression. As the pressure in the chamber increases, the bubble size will decrease. Second, the increased amount of oxygen in the blood creates a diffusion gradient to help further reduce the size of the bubbles. Third, HBOT inhibits neutrophil adherence to capillary walls, reducing the inammatory response caused by damage to the endothelium from the presence of a gas bubble. Finally, the increased oxygen carried by the plasma may help oxygenate partially ischemic tissues.
Decompression Sickness

carboxyhemoglobin level greater than 40% should be treated with HBOT. HBOT benets the CO-poisoned patient by decreasing the half-life of carboxyhemoglobin from more than 300 minutes breathing room air to 23 minutes breathing 100% oxygen at 3 atm absolute (ATA). The half-life of carboxyhemoglobin remains 90 minutes if breathing 100% oxygen at sea level. Also, CO has been shown to inhibit cellular oxidative metabolism by binding to cytochrome c oxidase. HBOT at 3 ATA has been shown to expedite the dissociation of CO from cytochrome c oxidase, thereby allowing the process of oxidative phosphorylation to return to normal. Other benets of HBOT in CO poisoning are the prevention of lipid peroxidation of the cell membrane and prevention of the adherence of neutrophils to the vascular endothelium by inhibiting the b2 integrin system.
Clostridial Myonecrosis

DCS encompasses a myriad of syndromes caused by bubbles of inert gas that are generated from rapid decompression during ascent from diving, ying, or in a hyperbaric or hypobaric chamber. When these bubbles occur in large enough numbers to cause pain or impede normal organ function, symptoms of DCS arise. As with gas embolism, the treatment for DCS is HBOT. The mechanism of action of HBOT is the same as for gas embolism.
Carbon Monoxide Poisoning

Carbon monoxide is a colorless, odorless, and tasteless gas that is a leading cause of injury and death from poisoning worldwide. Patients who suffer from CO poisoning can present with tachycardia, tachypnea, chest pain, headache, altered mental status, seizures, amnesia, or peripheral neuropathy. Laboratory abnormalities include elevated carboxyhemoglobin levels and a metabolic acidosis. There are ve randomized clinical trials in the literature using HBOT in patients with CO poisoning. Although there have been contradictory results from these studies, the best was done by Weaver and coworkers and was published in 2002. This study showed a statistically significant reduction in neuropsychological sequelae in patients treated with HBOT. Patients with evidence of metabolic acidosis, ischemic chest pain and/or electrocardiographic evidence of ischemia, abnormal psychometric testing, history of unconsciousness, carboxyhemoglobin level greater than 15% in a pregnant patient (because of increased binding of CO to fetal hemoglobin), or a

Clostridial myonecrosis is a severe, life-threatening infection caused by anaerobic, spore-forming, grampositive, encapsulated bacilli of the genus Clostridium. These infections are rapidly progressive and treatment involves the combined use of surgery, antibiotics, and HBOT. HBOT has been shown to be bacteriostatic to clostridia in vivo and in vitro by the formation of oxygen free radicals. The clostridia organisms have no free radical-degrading enzymes, such as superoxide dismutases, catalases, and peroxidases, thereby making them susceptible to the oxygen free radicals produced during HBOT. Also, it has been shown that oxygen tensions of 250 mmHg, which can be achieved on 100% oxygen at 3 ATA, inhibit the production of a-toxin, which is one of the main hemolytic and tissue-necrotizing toxins produced by the clostridial organisms.
Necrotizing Soft Tissue Infections

Necrotizing soft tissue infections are an increasing problem in current medical practice. These infections are caused by aerobic or anaerobic bacteria, but most commonly they are caused by mixed bacterial ora. Often, hosts are immunocompromised, contributing to the rapid spread of the infection. Necrotizing infections are hypoxic and an occlusive endarteritis caused by the infection contributes to the wound hypoxia. There are a limited number of neutrophils at the wound site due to intravascular sequestration of the polymorphonuclear leukocytes (PMNs). The PMNs that are present function poorly due to hypoxia and a decreased oxidationreduction potential (Eh) from the accumulation of metabolic products from the aerobic organisms. HBOT is recommended

HYPERBARIC OXYGEN THERAPY 295

as an adjunct to surgical debridement and antibiotics. With HBOT, there is increased tissue oxygenation, which inhibits anaerobic bacteria growth by direct toxic mechanisms and by improving the Eh as well. Also, with improved tissue oxygenation, PMN function improves. Patients are treated twice a day until the infection is controlled and then daily until no further debridement is required.
Crush Injury and Other Acute Traumatic Peripheral Ischemias

angiogenesis, intracellular leukocyte bacterial destruction, and infection resistance, are all reliant upon oxygen. This is why hypoxic wounds may not heal. HBOT increases tissue oxygenation, thereby reversing the effects of the hypoxic wound environment.
Exceptional Anemia

Traumatic injury causes a wound with damaged blood vessels. Tissue ischemia and a hypoxic wound environment can then ensue. This hypoxic wound environment causes increasing edema, which contributes to a vicious cycle of wound ischemia and edema. Several surgical and orthopedic conditions have this pathophysiology: crush injuries, compartment syndromes, threatened grafts and aps, threatened replantations, burns, and frostbitten extremities. As such, HBOT is used as an adjunct for the treatment of these conditions. Initially, HBOT will provide oxygen to hypoxic tissues. PMN function is improved, broblast migration and proliferation improve, collagen is laid down, and neovascularization proceeds because tissue oxygen tensions are increased due to HBOT. Another benet of HBOT is edema reduction. Hyperoxygenation during HBOT causes vasoconstriction, which has been shown to reduce blood ow by 20%. Venous outow continues unabated, so there is an overall edema reduction. Oxygen delivery to tissues is unaffected by vasoconstriction because of the hyperoxygenation from HBOT. Finally, HBOT limits the reperfusion injury by preventing lipid peroxidation of the cell membrane; antagonizing the b2 integrin system, thereby inhibiting the sequestration of neutrophils to the vascular endothelium; and providing additional oxygen for reperfused tissues to generate scavengers such as superoxide dismutase, catalase, peroxidase, and glutathione.
Enhancement of Healing in Selected Problem Wounds

Occasionally, a profoundly anemic patient will be unable to be transfused with packed red blood cells either because of religious objections or because the patient cannot be cross-matched. If the patients hemoglobin is so low that oxygen delivery is insufcient to offset the basic metabolic demands of the body, then oxygen debt will accumulate. As oxygen debt increases, signs of end-organ failure develop. HBOT is a method that repays the accumulated oxygen debt. The patient can be treated with frequent HBO treatments until blood becomes available or the patient produces sufcient hemoglobin.
Intracranial Abscesses

Intracranial abscesses encompass several disorders: cerebral abscess, subdural empyema, and epidural empyema. HBOT is recommended as an adjunct to antibiotics and surgical debridement. Benets of HBOT in these disorders include high concentrations of oxygen in brain tissue that inhibit the mainly anaerobic organisms that cause intracranial abscesses, decreased edema around the abscess site by mechanisms mentioned previously, and improved leukocyte function.
Refractory Osteomyelitis

Problem wounds can result in significant morbidity and mortality, and they represent an increasing burden on the healthcare system as the population ages. Diabetic ulcers are an example of problem wounds that are difcult to heal by conventional methods. These wounds are hypoxic due to the small vessel disease caused by diabetes. Since wound healing has been shown to be oxygen-dependent, these hypoxic wounds heal, if at all, at a much slower rate. The processes that contribute to wound healing, such as broblast replication, collagen placement,

Patients suffering from refractory osteomyelitis (i.e., osteomyelitis that does not respond or reoccurs after appropriate antibiotics and surgical debridement) usually have systemic problems or local wound factors that inhibit their ability to heal these infections. HBOT is an adjunct to systemic antibiotics and surgical debridement. The benets of HBOT include enhancement of osteogenesis by improving the oxygen-dependent osteoclast function of removing necrotic bone. Additionally, PMNs destroy bacteria by improved oxidative killing when oxygen tensions in the infected bone are raised to normal or supernormal levels. Studies have also shown that aminoglycoside transport across the bacteria cell wall is oxygendependent. HBOT improves aminoglycoside efcacy in hypoxic wound environments by improving delivery of the antibiotic across the bacterial cell wall. Finally, HBOT reduces tissue edema, limits the inammatory response, and promotes neovascularization and wound healing, as described previously.

296 HYPERBARIC OXYGEN THERAPY Delayed Radiation Injuries

Delayed radiation injury leads to progressive endarteritis that results in tissue hypoxia and secondary brosis. Once the oxygen demand of the tissues outstrips oxygen supply, tissue breakdown occurs. The most common delayed radiation injuries that benet from HBOT are prevention of osteoradionecrosis of the mandible in patients requiring dental work following radiation to the oropharynx, treatment of radiation cystitis, and treatment of radiation proctitis. HBOT promotes angiogenesis, which leads to increased cellularity in irradiated tissues. Studies have shown that HBOT can return to radiated tissue 80% of the capillary density of nonradiated tissue.

the lens. Claustrophobia may result from the patient being treated in a relatively small enclosed space. These symptoms can be treated with anxiolytics. Oxygen toxicity causes effects in the central nervous system (CNS) and pulmonary systems. The manifestation of CNS oxygen toxicity is seizures. Oxygen toxicity seizures have an incidence of 1 in 10 000 treatments at 2.4 ATA. Pulmonary oxygen toxicity is another possible side effect. The symptoms are substernal chest pain, dry cough, and a decrease in vital capacity. Finally, there is a theoretical risk of pneumothorax and arterial gas embolism if the patient were to have trapping of gas in his or her lungs or if the patient held his or her breath during ascent.
See also: Carbon Monoxide. Diving. Lung Abscess.

Contraindications and Side Effects

HBOT is very safe and there are few contraindications to therapy. The major absolute contraindication to HBOT is an untreated pneumothorax because it could progress to a tension pneumothorax during the ascent phase of a treatment when gas volume will expand due to the decrease in barometric pressure. Relative contraindications include pregnancy because of unknown risks to the fetus, chronic sinusitis because of the risk of sinus barotrauma, emphysema with CO2 retention because these patients may lose their stimulus to breath, high fever because this can predispose the patient to seizures from oxygen toxicity, a history of spontaneous pneumothorax because of the risk of recurrent pneumothorax, a seizure disorder because of the risk of oxygen-induced seizures, or a history of middle ear surgery because otic barotrauma could damage the repaired structures. Several medications are contraindicated in HBOT, including doxorubicin due to cardiac toxicity, bleomycin due to pulmonary toxicity, cis-platinum due to problems with wound healing, disulram, and sulfamylon. The most common side effect of HBOT is barotrauma of the middle ear. This occurs in 2% of patients and is a result of eustachian tube dysfunction or an inability to properly perform the Valsalva maneuver. Disruption of the round window is a rare consequence of HBOT and results from a forceful Valsalva maneuver. Sinus squeeze is another form of barotrauma that results from the expansion of trapped gases in the sinuses on ascent. Another side effect of treatment is a temporary refractive change. These changes typically occur after numerous treatments and consist of progressive myopia. It is theorized that the changes are due to oxygen uptake by

Further Reading
Boerema I, Meijne NG, Brummelkamp WH, et al. (1960) Life without blood. A study of the inuence of high atmospheric pressure and hypothermia on dilution of the blood. Journal of Cardiovascular Surgery 1: 133146. Brummelkamp WH (1965) Considerations on hyperbaric oxygen therapy at three atmospheres absolute for clostridial infections type welchii. Annals of the New York Academy of Sciences 117: 688699. Feldmeier JJ (ed.) (2003) Hyperbaric Oxygen 2003: Indications and Results. The Hyperbaric Oxygen Therapy Committee Report. Kensington MD: Undersea and Hyperbaric Medical Society. Kindwall EP and Whelan HT (eds.) (2002) Hyperbaric Medicine Practice, 2nd rev. edn. Flagstaff AZ: Best. Marx RE, Ehler WJ, Tayapongsak P, and Pierce LW (1990) Relationship of oxygen dose to angiogenesis induction in irradiated tissue. American Journal of Surgery 160: 519524. Marx RE, Johnson RP, and Kline SN (1985) Prevention of osteoradionecrosis: a randomized prospective clinical trial of hyperbaric oxygen versus penicillin. Journal of the American Dental Association 111: 4954. Moon RE (1997) Treatment of decompression sickness and arterial gas embolism. In: Bove AA (ed.) Diving Medicine, 3rd edn., pp. 184204. Philadelphia: Saunders. Thom SR (1993) Functional inhibition of leukocyte beta-2 integrins by hyperbaric oxygen in carbon monoxide mediated brain injury in rats. Toxicology and Applied Pharmacology 123: 248256. Weaver LK, Hopkins RO, Chan KJ, et al. (2002) Hyperbaric oxygen for acute carbon monoxide poisoning. New England Journal of Medicine 347: 10571067. Zamboni WA, Roth AC, Russell RC, et al. (1989) The effect of acute hyperbaric oxygen therapy on axial pattern skin ap survival when administered during and after total ischemia. Journal of Reconstructive Microsurgery 5: 343347. Zamboni WA, Wong H, Stephenson L, et al. (1997) Evaluation of hyperbaric oxygen for diabetic wounds: prospective study. Undersea Hyperbaric Medicine 24: 175179.

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 297

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME

J D Brewer and M D P Davis, Mayo Clinic, Rochester, MN, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Hypocomplementemic urticarial vasculitis syndrome (HUVS) is a form of urticarial vasculitis (UV) characterized as a clinicopathologic entity comprised of chronic urticaria clinically and vasculitis pathologically. When patients with UV have an associated hypocomplementemia, they are more likely to manifest systemic complications of the disease and are characterized as having HUVS. HUVS is affected by both genetic and environmental factors; however, the exact cause remains unknown. HUVS is a rare disease with approximately 100 cases described, is more common in women, usually affects people in their 40s, and is rare in children but associated with a more complicated course when found in the pediatric population. Patients with HUVS have chronic urticaria for more than 6 months, hypocomplementemia, and a various array of systemic manifestations including musculoskeletal, respiratory, renal, gastrointestinal, cardiac, ophthalmologic, central nervous system, and rheumatologic features. Biopsies of active urticarial lesions show leukocytoclastic vasculitis with extravasation of white blood cells and endothelial cell damage. Patients almost invariably have low levels of the C1q component of complement as well as the presence of an IgG antibody directed against a collagen-like region of C1q, also refered to as C1q precipitans. Biopsies of certain organs, especially the kidney, may show deposits of these immune complexes within the basement membrane of inamed tissue. The pathogenesis of HUVS is largely unknown, although hypotheses exist about exacerbating factors of the disease. HUVS is believed to be an autoimmune disease, much like systemic lupus erythematosus, with the formation of autoantibodies that cause immune complexes, deposition in certain parts of the body, and activation of the immune system causing inammation and destruction. For example, immune complexes are thought to deposit in the lung, causing aggregation and activation of neutrophils, which actively secrete elastase, causing destruction of the lung architecture and the development of chronic obstructive pulmonary disease (COPD). There is no specic therapy for HUVS; however, many therapeutic agents have been found to be benecial. Pruritus is treated with antihistamines and cutaneous manifestations have responded to dapsone, colchicine, antimalarials, nonsteroidal anti-inammatory drugs (NSAIDs), interferon alpha, and corticosteroids. Severe systemic aspects of HUVS have been treated with chemotherapeutic/immunosuppressive agents such as azathioprine, cyclophosphamide, mycophenolate mofetil, cyclosporine, and methotrexate, as well as combinations of previously mentioned agents. The prognosis of HUVS is relatively good; however, morbidity and mortality do occur, and are more likely to be seen in pediatric patients. Death from disease is rare, and is most commonly due to laryngeal edema or COPD.

Introduction
Urticarial vasculitis (UV) is a clinicopathologic entity requiring both chronic urticarial wheals clinically

and the evidence of cutaneous vasculitis or leukocytoclastic vasculitis (LCV) histopathologically. Biopsy of the urticarial lesions reveals this vasculitis as having leukocytoclasis and vessel wall destruction, which may or may not be accompanied by brinoid deposits. UV is sometimes associated with hypocomplementemia, and hence many authors divide UV into normocomplementemic urticarial vasculitis (NUV) and hypocomplementemic urticarial vasculitis (HUV). Patients with HUV (UV with associated complement activation) are much more likely to have systemic manifestations compared to patients with NUV, and many of these systemic features resemble those of systemic lupus erythematosus (SLE). These systemic manifestations include angioedema, glomerulonephritis, abdominal pain, and obstructive lung disease. It has been speculated that a continuum exists between UV, HUV, and hypocomplementemic urticarial vasculitis syndrome (HUVS), with urticaria and minimal vasculitis at one end of the spectrum and urticaria with marked systemic vasculitis and hypocomplementemia at the other end. One study, however, followed patients for over 20 years, and found that patients did not move or progress down this continuum with time, suggesting that if a continuum does exist between these disease entities, progression from one disease subtype to another is rare. HUVS was rst described in 1971, when a patient with hypocomplementemia and hypergammaglobulinemia was labeled as having an unusual SLE-related syndrome. Later a similar presentation in patients was described as hypocomplementemia with cutaneous vasculitis, and UV with associated hypocomplementemia was noted to be a more serious and systemic form of UV. In 1980, patients with urticarial vasculitis and hypocomplementemia were categorized as having a hypocomplementemic urticarial vasculitis syndrome (HUVS), which has also been called McDufe syndrome. Since then, more than 100 cases have been described, and this syndrome is now widely recognized as HUVS. When rst discovered, much of its nature, causes, and associated disease were unknown. The term syndrome was used to characterize patients with HUV who had variable organ system involvement. The term syndrome, however, may be inappropriate for HUVS based on the way syndrome has been dened in medical terminology, and hypocomplementemic urticarial vasculitis disease may be a more appropriate name for HUVS.

298 HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME

HUVS is a relatively rare disease, more common in women (6080% of cases), with a peak incidence in the fth decade (average age at diagnosis being 43). HUVS is less common in the pediatric population, but has been described as more severe in these patients. Although rare, HUVS should be considered in children with glomerulonephritis, urticarial rash, arthralgias or arthritis, and pulmonary disease.

Etiology
HUVS is an autoimmune disease of unclear etiology. Although more common in females, HUVS has not been associated with other genetic factors. A description of HUVS in identical twins has showed variation in disease severity and time of onset, suggesting HUVS as a disease entity requiring and inuenced by both genetic and environmental factors. HUVS is characterized by anti-C1q antibodies and low complement levels as well as manifestations of systemic inammatory vasculitis. Whether anti-C1q antibodies contribute to the pathogenesis of HUVS or SLE is not fully understood. It is hypothesized that anti-C1q antibodies contribute to organ dysfunction (especially renal) by forming complexes with C1q, dsDNA, and anti-dsDNA antibodies. These autoimmune complexes could then become less soluble and deposit in the membranes of vascular beds, giving rise to an inammatory autoimmune disease with unknown environmental exacerbating factors. These antibodies may also represent an epiphenomenon of HUVS as something commonly seen during the course of the disease, but not necessarily associated with the disease.

HUVS has been associated with viral infections, HenochScho nlein purpura, SLE, Sjo grens syndrome, IgM paraproteinemia (Schnitzlers syndrome), and malignancies. There have been various case reports describing the association of HUVS and malignancy and recently, the association with a diffuse large B cell lymphoma was noted. This recent nding is particularly interesting in that complement deciencies are known to precede the development of B cell lymphomas. It is proposed that HUVS may cause an underlying stimulation of B cells, much like that seen in mixed cryoglobulinemia, eventually leading to B cell overgrowth/expansion and lymphoma. If this proposed association is true, close follow-up of patients with HUVS would be needed due to the possible development of non-Hodgkins lymphoma. As previously mentioned, HUVS also shares many features characteristic of SLE, and can have renal involvement virtually indistinguishable from SLE nephritis, even histologically. Because of the many similarities between these two disease entities, some authors have suggested HUVS as being an SLErelated syndrome, and that a spectrum of diseases may occur ranging from SLE to HUVS (Table 1).

Pathology
Obtaining a biopsy of an active lesion remains the gold standard for diagnosing UV. Leukocytoclastic vasculitis (LCV) is what is typically seen histopathologically. The vasculitis involved in UV is most commonly described as a venulitis, affecting the postcapillary venules, and is considered to be a type III

Table 1 Causes and associations of UV and HUVS Category 1. 2. 3. 4. 5. 6. No cause Infections Hematologic diseases Connective tissue diseases Immune complex diseases Therapeutics and supplements Examples Idiopathic (most common) Hepatitis B, hepatitis C, infectious mononucleosis, Lyme disease Leukemia, lymphoma, polycythemia rubra vera, idiopathic thrombocytopenia purpura grens syndrome SLE, Sjo Serum sickness Diltiazem, potassium iodide, cimetidine, procarbazine, uoxetine, butylhydroxytoluene rin vaccine, (preservative in chewing gum), methotrexate, etanercept, cocaine, BacilleGue procainamide, dexfenuramine (and other centrally acting appetite suppressants), butylated hydroxyanisole, and certain herbs C3 deciencies, C4 deciencies, C3 nephritic factor activity IgM monoclonal gammopathy (Schnitzlers syndrome), IgG gammopathy (paraproteinemia), IgD gammopathy, IgA gammopathy, cryoglobulinemia Cold exposure (cold urticaria), sun exposure (solar urticaria), exercise-induced urticaria Hodgkins disease, malignant teratoma, paraneoplastic B cell non-Hodgkins lymphoma, IgA myeloma, metastatic adenocarcinoma of the colon Inammatory bowel disease, striae distensae of pregnancy, Cogans syndrome, amyloidosis (MuckleWells syndrome)

7. Complement involvement 8. Immunoglobulin abnormalities 9. Physical conditions 10. Malignancy 11. Other

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 299

hypersensitivity reaction. The minimal criteria used to characterize an urticarial syndrome as UV include leukocytoclasis or brin deposits within the lesion, whether or not blood cell extravasation exists. The exact definition of LCV remains ambiguous. LCV has many features including injury and swelling of endothelial cells (usually of the venules), extravasation of red blood cells, fragmentation of leukocytes with nuclear debris (leukocytoclasis), brin deposits in and around the venules, and a perivascular inltrate composed mostly of neutrophils. A predominance of neutrophils in the inltrate is characteristic of HUV. Leukocytoclasis and brinoid deposits have been deemed as the most important aspects of LCV because they represent direct signs of vessel damage. Inammation is seen within and around capillaries and postcapillary venules, with an inltrate composed mostly of neutrophils and occasional lymphocytes and eosinophils. Leukocytoclasis, dened as the fragmentation of neutrophils resulting in scattered nuclear fragments or nuclear dust, is frequently seen in the inltrate. Erythrocytes may extravasate, and can be seen perivascularly as well as in the interstitium. Swelling of the endothelial cells also occurs, and may be so extensive as to occlude the lumen of affected vessels. Finally, brinoid degeneration of the endothelial cells occurs, characterized by a blunting of the vascular outline due to edema and brin deposits.

Clinical Features
The most common presentation of HUVS clinically is the urticarial wheal. These cutaneous manifestations are usually persistent, painful, and tender, and sometimes have a burning sensation. As noted in the major criteria for the diagnosis of HUVS, these urticarial lesions must be persistent and present for at least 6 months. When the urticarial wheals go away, they characteristically leave purpura or hyperpigmentation as residual effects. Other cutaneous

manifestations include livedo reticularis, an erythema multiform-like eruption, angioedema, and rarely bullae or Raynauds phenomenon. When approaching patients with the possibility of HUVS, clinicians should rst establish the diagnosis of UV, and then look for systemic involvement and hypocomplementemia characteristic of HUVS. The rst step in the investigation involves a skin biopsy of a lesion for histologic examination. Histologic examination includes a hematoxylin and eosin stain of the biopsy specimen, to see if the criteria for vasculitis are fullled. Direct immunouorescence usually demonstrates immune complexes, complement, and brin deposits in the basement membrane. Hypocomplementemia can then be quantied, in terms of reduced levels of C1, C2, C3, and C4. These components represent factors from the classical complement pathway, and can result in reduced levels of CH50/100. With respect to the C1 component of complement, the C1q levels are almost exclusively reduced more significantly than levels of C1r and C1s (the other components of C1). The diagnosis of HUVS involves major, minor, and exclusion criteria. For the diagnosis of HUVS, the patient must have both major criteria, at least two minor criteria, and none of the exclusion criteria (Table 2). Once the diagnosis of HUVS has been established the astute clinician should be aware of and evaluate for systemic involvement (Table 3). Many systemic associations have been linked to HUVS, some of which are capable of causing a great deal of morbidity and mortality. A chest X-ray and pulmonary function tests may be appropriate for suspected pulmonary involvement, and an echocardiogram may give light to cardiac abnormalities. Infections, certain drugs, immunoglobulin abnormalities, and hematologic diseases have been linked to the development of HUVS, and should be considered as possible coexisting entities (see Table 1). Angioedema occurs in as many as 50% of patients with HUVS, frequently involving the lips, tongue,

Table 2 Diagnostic criteria for HUVS Level of criteria 1. Major criteria 2. Minor criteria Specifics Urticaria for more than 6 months and hypocomplementemia Venulitis of the dermis (established via biopsy), arthralgia or arthritis, mild glomerulonephritis, uveitis or episcleritis, recurrent abdominal pain, and a positive C1q precipitin test by immunodiffusion with an associated suppressed C1q level Significant cryoglobulinemia (cryocrit 41%), an elevated anti-DNA antibody level, a high titer of antinuclear antibodies, hepatitis B antigenemia, depressed C1 esterase inhibitor level, or an inherited complement deciency Needed for the diagnosis The patient must have both major criteria The patient must have at least two of the minor criteria

3. Exclusion criteria

The patient cannot have any of the exclusion criteria

300 HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME

Table 3 Approach to diagnosis of HUVS Step of investigation 1. Conrm the diagnosis of UV 2. Conrm the diagnosis of HUVS 3. Evaluate for systemic disease Laboratory data or study modality employed Biopsy of early lesions, histopathologic evaluation for vasculitis Conrm hypocomplementemia (serum complement levels: C1q, C3, C4, and CH50) and apply the diagnostic criteria Complete blood count, erythrocyte sedimentation rate, urea, urinalysis, chest X-ray, liver function tests, circulating immune complexes, electrolytes (including creatinine to evaluate for renal involvement), immunoglobulins, cryoglobulin Echocardiogram, bronchial lavage, pulmonary function tests, joint and skeletal X-rays Antinuclear antibodies, anti-DNA antibody, anti-SSA(Ro)/SSB(La) antibodies, Borrelia antibody titer, antiphospholipid antibodies, cryoglobulins, C1q precipitin, C3 nephritic factor, hepatitis B and C serology, monospot test for EpsteinBarr virus infection

4. Special tests that may be ordered according to the history 5. Other tests for suspected associations

periorbital tissues, and hands, but rarely life-threatening. Reversible tracheal stenosis has also been seen. Musculoskeletal complaints include arthritis and arthralgias, seen in nearly all patients (incidence as high as 75%). Jaccouds arthropathy, a condition paralleling rheumatoid arthritis (RA) clinically, has been seen in a number of patients with HUVS, as well as patients with SLE, scleroderma, Sjo grens syndrome, mixed connective tissue disease, acquired immuno-deciency syndrome (AIDS), mycosis fungoides, and angioimmunoblastic lymphadenopathy. In patients with HUVS, Jaccouds arthropathy has been associated with severe acute noninfectious endocarditis with supercial brinoid necrosis, and brin deposition in the cardiac valves, causing a cardiac valvulopathy, which in many instances requires cardiac valve replacement. Pulmonary involvement is seen up to 50% of the time in patients with HUVS, with pleuritis and chronic obstructive pulmonary disease (COPD) being common presentations. Lung biopsies in these patients demonstrate a leukocytoclasitic vasculitis characteristic of the disease. Lung involvement in HUVS patients is a frequent cause of morbidity and mortality, associated with respiratory failure and death. The mechanism of development of COPD in these patients is unclear; however, it is proposed that there is a local release of elastase by the neutrophils present, inducing a proteolytic lung destructive process. In addition, cigarette smoking may increase the number of neutrophils present as well as decrease the antiproteolytic activity of a1-antitrypsin, exacerbating the lung destructive process. Like C1q, surfactant apoproteins contain a collagen-like region thought to be a potential site of anti-C1q antibody cross-reactivity, proposed to be a potential cause of surfactant dysfunction and lung disease in these patients. Most patients with lung involvement are concomitant smokers. Although smoking is considered to be a contributing factor in these patients with lung disease, many have disease out of proportion to the number of years

smoked, and lung disease has been seen in patients in their 30s and 40s with no history of tobacco use. Cigarette smoking appears to be a risk factor for fatal lung disease, and is thought to potentiate the proposed neutrophil-mediated elastase release in areas of vasculitis within the lung, leading to breakdown of lung architecture and emphysema. Renal involvement is seen in 50% of patients, with proteinuria and hematuria occurring 2030% of the time. Renal involvement is generally mild; however, there have been a few instances of end-stage renal failure requiring dialysis and even kidney transplant in patients with HUVS. In most cases of renal involvement, deposits of immunoglobulins and complement have been recognized suggesting an immune-mediated component, or immune-complex type nephritis. Anti-C1q antibodies have been associated with renal involvement in both SLE and HUVS, making it difcult to distinguish between these two entities. In fact, it is virtually impossible to differentiate histologically between SLE- and HUVSrelated nephritis; however, the renal prognosis is better in patients with HUVS, and steroid therapy seems to be very effective in these patients. It has recently been suggested that monitoring the C1q levels could be an important tool in predicting renal ares. Gastrointestinal symptoms including abdominal pain, nausea, vomiting, and diarrhea are seen in approximately 1730% of patients. Ocular involvement is rare, and usually involves the uveal tract. Other ocular manifestations include conjunctivitis and episcleritis. More rare systemic manifestations of HUVS include pericarditis and cardiac tamponade, constitutional symptoms (fever, fatigue, malaise), myositis, dermal ulcerations, Raynauds phenomenon, digital infarction, dermatographism, livedo reticularis, serpiginous choroidopathy with vision loss, hypothyroidism, pseudotumor cerebri, peripheral neuropathy, lower cranial nerve palsies, and transverse myelitis.

HYPOCOMPLEMENTEMIC URTICARIAL VASCULITIS SYNDROME 301

Pathogenesis
By definition, HUVS is associated with low complement levels. Autoantibodies to C1q with a subsequent decreased C1q level is seen almost exclusively in patients with HUVS, and such levels may be predictive of severity. These autoantibodies are referred to as C1q precipitins, identied as IgG antibodies directed toward the collagen-like, Fc-dependent portion of C1q. C1q antibodies and low levels of C1q may also be seen in SLE, RA, Feltys or Sjo grens syndrome, glomerulonephritis, and cryoglobulinemia. Occasionally, depressed C3, C4, C5, and hemolytic complement levels (CH50) are also seen. Female sex, interstitial neutrophilia, and the presence of a lupus band on direct immunouorescence studies of skin biopsy specimens have been shown to predict hypocomplementemia. In addition, the erythrocyte sedimentation rate (ESR) is usually elevated, and 50% of the time patients have positive antinuclear antibody (ANA) titers. Patients do not usually have anti-dsDNA or anti-Smith (anti-Sm) antibodies.

Animal Models
There are currently no known available animal models of HUVS.

Management and Current Therapy

There is no definite therapy for HUVS that works all the time. Multiple treatments have been tried with attempts to manage the underlying causes, if one is identied (Table 4). Some therapies include nonsteroidal anti-inammatory drugs (NSAIDs) (indomethacin) and antihistamines for joint pain and pruritus. Moderate to high doses of glucocorticoids have proven effective; however, due to the multiple complications associated with long-term therapy, other regimens have been used including antimalarial and immunosuppressive therapy with dapsone,

colchicine, hydroxychloroquine, azathioprine, methotrexate and cyclophosphamide, mycophenolate mofetil (MMF) and intravenous immunoglobulin (IVIG). MMF is a relatively new immunosuppressant agent, recently shown to be efcacious as maintenance therapy in a couple of patients with HUV. In a recent report, another patient resistant to MMF and IVIG had a rapid response to rituximab. Hydroxychloroquine is an antimalarial sometimes used as therapy in RA and HUVS. Although the mechanism of action is unclear, dramatic responses to therapy have been reported in the literature in patients resistant to other therapeutic modalities. Dapsone is a relatively nontoxic drug compared to long-term corticosteroid or cytotoxic therapy, and sustained remissions in HUVS patients treated with dapsone have been described. Dapsone suppresses neutrophil-mediated inammation and has been proposed as a rst-line agent in HUVS therapy due to its high therapeutic index, ease of administration, low cost, and persistent benecial effects. Recently, the addition of pentoxyphylline to dapsone therapy was found to be helpful in inducing remission in HUV. Additionally, the use of dapsone followed by colchicine caused clearing of disease in one patient. Due to the proposed neutrophil involvement in HUVS-associated lung disease, dapsone may be an ideal drug in the early stages in efforts to arrest lung damage. Cyclosporin A (CyA) primarily suppresses T cell activation and has been used to treat a number of glomerular diseases associated with nephrotic syndrome. Recently CyA was found to be useful in a patient with HUVS-associated nephrotic syndrome. Although the great majority of patients with HUVS have multi-organ involvement, the prognosis still remains good, with few patients ever dying as a direct result of the disease. Some systemic complications, however, especially pulmonary (COPD) and laryngeal edema can be the source of great morbidity and at times mortality.

Table 4 Approaches to therapy for HUVS Symptom treated 1. Pruritus 2. Cutaneous manifestations (urticaria) 3. Systemic manifestations 4. Severe systemic disease or refractory to other therapies 5. Proposed combination therapy, also employed in refractory disease Therapeutic agent usually effective Antihistamines Dapsone, hydroxychloroquine, colchicine, indomethacine, and corticosteroids Dapsone, corticosteroids, and interferon alpha Azathioprine, methotrexate, mycophenolate mofetil, to cyclophosphamide, cyclosporin, and plasma exchange Cyclophosphamide and dexamethasome pulse therapy, interferon alpha and ribavirin (and occasionally doxepin), prednisone and azathioprine, dapsone and pentoxifylline, dapsone and corticosteroids, and prednisone and low-dose gold therapy

302 HYPOXIA AND HYPOXEMIA See also: Asthma: Overview. Bronchiectasis. Bronchiolitis. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1Antitrypsin Deciency; Emphysema, General; Smoking Cessation. Histamine. Panbronchiolitis. Signs of Respiratory Disease: Breathing Patterns. Symptoms of Respiratory Disease: Dyspnea. Vasculitis: Overview.
Falk DK (1984) Pulmonary disease in idiopathic urticarial vasculitis. Journal of the American Academy of Dermatology 1: 346352. Gammon WR and Wheeler CE (1979) Urticarial vasculitis: report of a case and review of the literature. Archives of Dermatology 115: 7680. Ghamra Z and Stoller JK (2003) Basilar hyperlucency in a patient with emphysema due to hypocomplementemic urticarial vasculitis syndrome. Respiratory Care 48: 697699. Jones MD, Tsou E, Lack E, and Cupps TR (1990) Pulmonary disease in systemic urticarial vasculitis: the role of bronchoalveolar lavage. American Journal of medicine 88: 431434. Mehregan DR, Hall MJ, and Gibson LE (1992) Urticarial vasculitis: a histopathologic and clinical review of 76 cases. Journal of the American Academy of Dermatology 26: 441448. Monroe EW (1999) Urticarial vasculitis: an updated review. Journal of the American Academy of Dermatology 5: 8895. ODonnell B and Black AK (1995) Urticarial vasculitis. International Angiology 14: 166174. Schwartz HR, McDufe FC, Black LF, Schroeter AL, and Conn DL (1982) Hypocomplementemic urticarial vasculitis: association with chronic obstructive pulmonary disease. Mayo Clinic Proceedings 57: 231238. Venzor J, Lee WL, and Huston DP (2002) Urticarial vasculitis. Clinical Reviews in Allergy and Immunology 23: 201216. Wisnieski JJ (2000) Urticarial vasculitis. Current Opinion in Rheumatology l2: 2431. Wisnieski JJ, Baer An, Christensen J, et al. (1995) Hypocomplementemic urticarial vasculitis syndrome: clinical and serologic ndings in 18 patients. Medicine (Baltimore) 74: 2441.

Further Reading
Berg RE, Kantor GR, and Bergfeld WF (1988) Urticarial vasculitis. International Journal of Dermatology 27: 468472. Berg RE, Kantor GR, and Bergfeld WF (1988) Urticarial vasculitis in adults. International Journal of Dermatology 27: 504505. Black AK (1999) Urticarial vasculitis. Clinics in Dermatology 17: 565569. Chen HJ and Bloch KJ (2001) Hypocomplementemic urticarial vasculitis, Jaccouds arthropathy, valvular heart disease, and reversible tracheal sienosis: a surfeit of syndromes. Journal of Rheumatology 28: 383386. Davis MDP and Brewer JD (2004) Urticarial vasculitis and hypocomplementemic urticarial vasculitis syndrome. Immunology and Allergy Clinics of North America 24: 183213. Davis MDP, Daoud MS, Kirby B, Gibson LE, and Rogers RS (1998) Clinicopathologic correlation of hypocomplementemic and normocomplementemic urticarial vasculitis. Journal of the American Academy of Dermatology 38: 899905.

HYPOXIA AND HYPOXEMIA

G Gutierrez, George Washington University, Washington, DC, USA
& 2006 Elsevier Ltd. All rights reserved.

may lead to hypoxia, a condition of low tissue O2 concentration.

Normal Physiological Processes Abstract

Hypoxemia and hypoxia refer to conditions of decreased oxygen concentration in arterial blood and in the peripheral tissues, respectively, that may be experienced by individuals exposed to extreme environmental conditions (high altitude), accidental conditions (diving accidents, near drowning, and carbon monoxide poisoning), or with diseases that affect the transport of oxygen to the tissues. If untreated, hypoxemia and hypoxia may lead to organ dysfunction and eventually to death. Anaerobic sources of energy help supplement faltering aerobic energy production during hypoxia. Treatment of hypoxemia and hypoxia should be directed at the resolution of the precipitating cause.

Prokaryotic cells derive their energy from glycolysis, a process that oxidizes and splits the six-carbon glucose molecule into two three-carbon pyruvate molecules (Figure 1). The energy released during glycolysis is conserved by the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) and by the reduction of nicotinamide adenine dinucleotide (NAD ) to NADH. Glycolysis would stop if it lacks supply of NAD . Higher organisms regenerate NAD by reducing pyruvate to lactate, allowing glycolysis to continue indenitely: Glucose 2 ADP 2 Pi-2 lactate 2 ATP where Pi denotes inorganic phosphate. The hydrolysis of ATP provides the energy required to power vital cellular processes, such as protein synthesis, locomotion, and membrane-associated

Description
Hypoxemia, a condition that literally means low blood O2 concentration, results from conditions or diseases that impede the transfer of O2 from the lungs into arterial blood (Table 1). Hypoxemia

HYPOXIA AND HYPOXEMIA 303

Table 1 Causes of hypoxemia, hypoxia, and dysoxia Hypoxemia High altitude Enclosed spaces with low PO2 Near drowning Airway obstruction Hypoventilation Central hypoventilation Neuromuscular disorders Hypoventilationobesity syndrome Ventilationperfusion mismatch Adult respiratory distress syndrome Alveolar hemorrhage Asthma/obstructive pulmonary disease Interstitial lung disease Lung contusion Pneumonia Pneumothorax Pulmonary hypertension Pulmonary and intracardiac vascular shunts Pulmonary emboli thrombus and fat Heart failure Anemia, including sickle cell anemia Carbon monoxide intoxication Hemoglobinopathies Methemoglobinemia Hypoxia Peripheral embolic phenomena Diffuse intravascular coagulation Shock cardiogenic, hypovolemic, and septic Dysoxia Pyruvate dehydrogenase inhibition Mitochondrial enzymes and cytochrome chain (a,a3) inhibition NAD depletion by poly-ADP ribose polymerase (PARP-1) activation Cyanide with uncoupling of oxidative phosphorylation

until transferred to oxygen, the nal electron acceptor. The energy liberated by the mitochondrial electron cascade is coupled to the partly understood mechanism of oxidative phosphorylation, producing 36 ATP molecules per glucose molecule. The abundant cellular energy provided by aerobic metabolism allowed eukaryotic cells to form complex multicellular organisms. Growth, however, was limited by the diffusion of O2 from the environment. Species adapted to prevailing ecological conditions by developing organs to carry O2 from the surrounding media to all tissue cells. Animal O2 transport systems consist of heme pigments to reversibly bind O2 (hemoglobin), specialized cells to enfold but not consume the potentially toxic O2 (erythrocytes), organs to promote O2 diffusion from the environment into blood (lungs or gills), and the hydraulic pumps and circulatory conduits needed to convey O2-containing cells to and from the tissues (the heart and blood vessels). Air reaches the alveolar spaces following a negative pressure gradient during inspiration. Alveolar O2 partial pressure (PAO2) may be estimated from the alveolar air equation PA O2 FI O2 Patm Pwater Pa CO2 FI O2 1 FI O2 =RQ mmHg where FIO2 is the inspired O2 fraction (0.21 for air), Patm is the atmospheric pressure, Pwater is the water vapor pressure, PaCO2 is the arterial blood CO2 partial pressure, and RQ is the respiratory quotient. When breathing air at sea level, this equation approximates to PA O2 150 Pa CO2 =0:8 mmHg Oxygen diffuses from the alveoli into pulmonary capillary blood, where it dissolves in plasma or binds to hemoglobin. The alveolararterial gradient is the difference between alveolar and arterial PO2 A a PA O2 Pa O2 . Ventilation perfusion inequality, shunt, or (rarely) diffusion limitation of oxygen can widen the Aa gradient across the blood gas barrier. The sigmoid-shaped oxyhemoglobin dissociation curve (ODC) denes the relationship between plasma PO2 and fractional hemoglobin O2 saturation (SO2). The concentration or O2 content of blood is the sum of O2 bound to hemoglobin and O2 dissolved in plasma: O2 content 13:9 SO2 Hb 0:031 PO2 ml O2 per liter of blood

ionic pumps: 2 ATP-2 ADP 2 Pi 2 H energy Combining the previous equations yields the general expression for glycolysis: Glucose-2 lactate 2 H energy The emergence of mitochondria, remarkable organelles capable of handling oxygen safely, provided the energy required for the evolution of eukaryotic cells. Mitochondria are thought to be descendants from symbiotic bacteria since they contain their own DNA and replicate independently. Mitochondria generate vast quantities of energy from O2, generating CO2 and water as by-products. In the mitochondria, pyruvate is oxidized to acetylCoA, which enters the tricarboxylic acid cycle, producing CO2 and electrons. These electrons move down a cascade of progressively lower energy states

304 HYPOXIA AND HYPOXEMIA

Glycolysis Fatty acids Amino acids Glucose ADP + Pi NAD+ Lactate

NADH Pyruvate Cytosol Mitochondrion e CO2 Acetyl-CoA Citrate Oxaloacetate e e e CO2 Tricarboxylic acid cycle

ATP

CO2 e

2H + 1/2 O2

Transfer of electrons coupled to oxidative phosphorylation ADP + Pi ATP

H2O
Figure 1 Glycolysis and oxidative phosphorylation.

where the term 13.9 is the O2 carrying capacity of hemoglobin, [Hb] is the concentration of hemoglobin in blood (g l 1), and the term 0.031 is the solubility of O2 in plasma for PO2 in mmHg. Arterial blood delivers oxygen and metabolic substrate to the tissues in accordance to local energy requirements. Higher level control of the circulation is aimed primarily at counteracting gravitational effects by stimulation of arteriolar a and b receptors. The microvasculature the vast array of terminal arterioles, capillaries, and venules regulates intraorgan blood ow through vasomotion, rhythmic oscillations in vascular tone. Accurate measurements of regional O2 delivery are difcult to obtain. A rst approximation to the adequacy of cellular O2 transport may be obtained by calculating the systemic rates of O2 delivery (DO2) and O2 uptake (VO2), DO2 cardiac output O2 content ml O2 per minute

Systemic O2 uptake (VO2) may be measured directly from the expired gases as V O2 FI O2 1 FE O2 FE CO2 =1 FI O2 FE O2 VE ml O2 per minute where FIO2 and FEO2 denote the inspired and expired fractional O2 concentration, respectively; FECO2 is the fractional concentration of expired CO2; and VE is the expired minute volume. A practical problem with this expression is that small inaccuracies in measuring FIO2 at values 40.60 may produce large errors in VO2. Systemic VO2 may also be calculated with the aid of Ficks principle as V O2 cardiac output arterial O2 content mixed venous O2 content ml O2 per minute This calculation requires the insertion of a pulmonary artery catheter to gain access to pulmonary

HYPOXIA AND HYPOXEMIA 305

artery blood. Ficks principle underestimates total VO2 since it does not account for pulmonary VO2. The latter can be substantial in severe pneumonia or in adult respiratory distress syndrome. The ratio of tissue CO2 production (VCO2) to VO2 is the respiratory quotient, RQ V CO2 =V O2 The type of substrate consumed determines the value of RQ, being 0.7 for free fatty acids and 1.0 for glucose. The O2 extraction ratio (ERO2), the fraction of DO2 taken up by the tissues, is another useful parameter: ERO2 V O2 =DO2 ERO2 is approximately 2025% at rest, increasing to 50% or greater with exercise or during cardiac failure at rest. ERO2 values o15% are suggestive of functional peripheral shunting, as might occur during sepsis. The DO2VO2 relationship (Figure 2) is the subject of great clinical interest. Experimental preparations subjected to decreases in DO2 initially show little variation in VO2 resulting from microvascular responses that increase ERO2. Decreases in VO2 occur below a critical DO2 because cellular O2 supply cannot keep up with the rate of aerobic metabolism. Anaerobic processes now must be recruited to complement the faltering mitochondrial supply of ATP. This condition, if protracted, may lead to the death of the animal. Measurements of DO2 and VO2 from critically ill individuals uniformly show a straight line, one lacking a critical DO2 dening the threshold for anaerobic metabolism. Failure to account for the passage

of time in the DO2VO2 relationship may explain the difference in ndings between clinical and experimental data. Animal experiments usually take a few hours, during which time DO2 is relentlessly lowered. Clinical measures are gathered over a longer period, typically several days, and no efforts are made to decrease DO2. During this time, the patients metabolic rate is bound to change, leading to variations in DO2 in response to changes in VO2, not the reverse. By considering time as an additional dimension, the two-dimensional DO2VO2 curve becomes a surface whose contour varies in concert with the patients clinical condition. Figure 3 illustrates the temporal evolution of a hypothetical DO2VO2 relationship. In this example, the persons condition worsens with time as increases in DO2 become less effective in their ability to increase VO2. The gure also shows that samples of DO2 and VO2 taken at different times (red circles) may project as a straight line if viewed from a two-dimensional perspective.

Physiological Processes in Respiratory Diseases

Diseases that affect the lungs will impair O2 diffusion, widen the Aa gradient, and produce hypoxemia, dened by a Pa O2 o60 mmHg or Sa O2 o90% . An acute response to hypoxemia is hyperventilation, resulting in decreased PaCO2 and, according to the alveolar air equation, a modest increase in PAO2. A more protracted response to hypoxemia is polycythemia produced by the release of erythropoietin by the kidneys. The arterial blood O2 content is a more precise index of hypoxemia than either PaO2 or SaO2. Decreases in [Hb] or conditions that interfere with hemoglobin O2 binding will diminish arterial blood O2 concentration but may not affect PaO2 or SaO2. Shown in Figure 4 are the O2 content curves for blood with Hb 150 g l1 , a normal value, and Hb 100 g l1 , a moderate degree of anemia. The O2 content at Pa O2 100 mmHg for the anemic case corresponds to a Pa O2 35 mmHg in the nonanemic case. Decreases in cardiac output, [Hb], and SaO2 are quantitatively similar in lowering DO2, but they elicit different systemic responses. Perhaps as an evolutionary response to blood loss, humans appear to tolerate decreases in [Hb] better than equivalent reductions in cardiac output or SO2. As long as circulating blood volume is maintained, humans tolerate acute decreases in [Hb] to 50% of normal values (i.e., 70 g l1 ) with remarkable ease. On the other hand, decreases in SaO2 or cardiac output up to 50% of normal values are likely to prove fatal.

Systemic oxygen uptake (V O2)

Critical D O2 Anaerobic region

Aerobic region

Systemic oxygen delivery (D O2)

Figure 2 Schematic D O2V O2 relationship derived from experimental studies.

306 HYPOXIA AND HYPOXEMIA

O2 consumption

de liv er y

Tim

Figure 3 Three-dimensional representation of the DO2VO2 relationship as a function of time. Darker regions represent conditions likely to produce hypoxemia and hypoxia. In this hypothetical example, the ability of the individual to increase VO2 in response to increases in DO2 decreases with time. This is perhaps the result of regional microcirculatory alterations or of cellular apoptosis. The red circles represent samples obtained at different times during the patients illness. When projected to a two-dimensional perspective, the DO2VO2 relationship traced by these samples will appear as a straight line.

200 O2 content (ml O2 per liter of blood) [Hb] = 150 g l1 150 [Hb] = 100 g l1

100

50

0 0 20 40 60 80 100 PO2 (mmHg)

Figure 4 Blood O2 content curves for hemoglobin concentrations of 150 g l 1 (normal) and 100 g l 1 (anemia). The O2 content of fully saturated anemic blood corresponds to the O2 content attained at PO2 35 mmHg for normal blood.

Considerable debate exists on the benets of therapeutic increases in DO2 during critical illness. One strategy used to augment DO2 in critically ill patients is to increase arterial blood O2 content by raising

either [Hb] or SaO2. This approach is problematic due to the slow process of de novo hemoglobin generation. Moreover, blood transfusions can be detrimental to some critically ill patients. Similarly, significant increases in arterial O2 content by increasing FIO2 are nearly impossible to attain, given the at portion of the ODC (unless the patient is taken to a hyperbaric chamber). A more common strategy used when increasing DO2 is to increase cardiac output produced by the infusion of inotropic agents, such as dobutamine. Randomized trials of protocol-driven increases in DO2 have not demonstrated improved survival. However, studies have shown decreases in mortality when resuscitation is initiated early in the course of the patients illness. Hypoxia refers to decreases in tissue oxygenation capable of limiting aerobic metabolism. Measures of venous SO2 and lactate concentration can detect individual organ hypoxia. On the other hand, systemic measures of mixed venous SO2 and arterial lactate concentration are unreliable indices of regional tissue hypoxia. Other, less practical techniques to measure intracellular oxygen concentration include surface

HYPOXIA AND HYPOXEMIA 307

Table 2 Anaerobic sources of ATP Glycolysis Glucose 2 ADP 2 Pi - 2 lactate 2 ATP Creatine kinase reaction PCr ADP H - Cr ATP Adenylate kinase reaction (myokinase) ADP ADP - AMP ATP

Hemorrhage. Arterial Blood Gases. Asthma: Acute Exacerbations. Carbon Monoxide. Diffusion of Gases. Diving. Endotoxins. Erythrocytes. Exercise Physiology. Hemoglobin. High Altitude, Physiology and Diseases. Hyperbaric Oxygen Therapy. Interstitial Lung Disease: Overview. Oxygen Therapy. OxygenHemoglobin Dissociation Curve. Peripheral Gas Exchange. Pulmonary Circulation. Pulmonary Edema. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts. Sleep Disorders: Hypoventilation. Vascular Disease. Ventilation, Perfusion Matching. Ventilation: Overview.

polarographic electrodes, oxygen microelectrodes, myoglobin cryospectrophotometry, uorescence techniques, and electron paramagnetic resonance oximetry. These methods provide little information on intramitochondrial PO2, a difcult parameter to measure with a magnitude o0.1 mmHg. Moreover, there may be instances in which mitochondrial oxygen may be plentiful but the cellular bioenergetic machinery is impaired and cannot use it a condition termed cytopathic hypoxia. Dysoxia is dened as an imbalance between the rates of aerobic energy production and cellular energy utilization. Dysoxia can be inferred by measuring metabolites involved in the anaerobic generation of ATP. There are three metabolic sources of anaerobic ATP: glycolysis, the creatine kinase reaction, and the adenylate kinase reaction (Table 2). As mentioned previously, glycolysis can be monitored by measures of blood lactate concentration, although clinical interpretation of hyperlactatemia can sometimes be challenging. The creatine kinase reaction occurs in selected organs (skeletal muscle, heart, and brain) and may be monitored by magnetic resonance spectroscopy. The adenylate kinase reaction occurs in most cells, and the products of AMP degradation, xanthine and hypoxanthine, may be monitored using high-pressure liquid chromatography. A more practical method to estimate regional dysoxia is to measure tissue CO2 concentration. Tissue acidosis occurs in hypoxia since the major anaerobic sources of ATP production, glycolysis and the adenylate kinase reaction, do not recycle H produced by the hydrolysis of ATP. Buffering of H by bicarbonate liberates CO2, a gas that can be measured with relative ease using gastric or sublingual tonometry.
See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Alveolar

Further Reading
Connett RJ, Honig CR, Gayeski TEJ, and Brooks GA (1990) Dening hypoxia: a systems view of VO2, glycolysis, energetics, and intracellular PO2. Journal of Applied Physiology 68: 842883. Fink M (2002) Cytopathic hypoxia. Critical Care 6: 491499. Gattinoni L, Brazzi L, Pelosi P, et al. (1995) A trial of goal-oriented hemodynamic therapy in critically ill patients. SvO2 Collaborative Group. New England Journal of Medicine 333: 10251032. Gladden LB (2004) Lactate metabolism: a new paradigm for the third millennium. Journal of Physiology 558: 530. Gutierrez G (2004) A mathematical model of tissueblood carbon dioxide exchange during hypoxia. American Journal of Respiratory and Critical Care Medicine 169: 525533. Gutierrez G, Wulf-Gutierrez ME, and Reines HD (2004) Monitoring oxygen transport and tissue oxygenation. Current Opinion in Anesthesiology 17: 107117. Lopez-Barneo J, del Toro R, Levitsky KL, Chiara MD, and OrtegaSaenz P (2004) Regulation of oxygen sensing by ion channels. Journal of Applied Physiology 96: 11871195. Masson N and Ratcliffe PJ (2003) HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O2 levels. Journal of Cell Science 116: 30413049. Nelson DL and Cox MM (eds.) (2004) Lehninger Principles of Biochemistry, 4th edn. New York: Worth. Nilsson H and Aalkjr C (2003) Vasomotion: mechanisms and physiological importance. Molecular Interventions 3: 7989. Rivers E, Nguyen B, Havstad S, et al. (2001) Early Goal-Directed Therapy Collaborative Group. Early goal-directed therapy in the treatment of severe sepsis and septic shock. New England Journal of Medicine 345: 13681377. Robin ED (1977) Special report: dysoxia. Abnormal tissue oxygen utilization. Archives of Internal Medicine 137: 905910. Vincent JL and De Backer D (2004) Oxygen transport the oxygen delivery controversy. Intensive Care Medicine 30: 19901996. Walsh TS (2003) Recent advances in gas exchange measurement in intensive care patients. British Journal of Anaesthesiology 91: 120131. West JB (2004) A century of pulmonary gas exchange. American Journal of Respiratory and Critical Care Medicine 169: 897902.

I
IDIOPATHIC PULMONARY HEMOSIDEROSIS
O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Idiopathic pulmonary hemosiderosis (IPH) is a rare disease of unknown etiology, characterized by recurrent episodes of diffuse alveolar hemorrhage and sideropenic anemia. IPH occurs most commonly in children. During an acute episode, a constellation of cough, dyspnea, and hemoptysis with alveolar inltrates and worsening anemia should raise the suspicion for intrapulmonary bleeding. Sputum, bronchoalveolar lavage (BAL), and eventually lung tissue examination show numerous hemosiderin-laden macrophages in the alveoli, without evidence of capillaritis/vasculitis, granulomatous inammation, deposition of immunoglobulins, or immune complexes. Glucocorticoids and other immunosuppressive drugs seem to be effective during IPH exacerbations and, possibly, also during the remission phase. The significant improvement in morbidity and mortality of IPH during the past several decades is probably due to the long-term use of immunosuppressive therapy.

had large amounts of hemosiderin in the lungs. The clinical triad of hemoptysis, pulmonary opacities, and anemia with no other identied cause was considered diagnostic if the lung biopsy excluded vasculitic processes, granulomatous diseases, or immune depositions. In the past, many IPH cases may have been misdiagnosed, especially without a lung biopsy and before the advent of newer diagnostic tools and disease markers (e.g., antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), antiglomerular basement membrane (anti-GBM) antibodies, immunouorescence studies). Approximately 80% of cases have the onset during childhood, most of them during the rst decade of life. In adults there are two categories of IPH: primary-pediatric cases followed into adulthood and primary adult onset. Gender distribution appears to be balanced in childhood-onset IPH, and is slightly skewed towards a male predominance in adult onset. The incidence is estimated to be between 0.24 and 1.23 cases per million children per year.

Introduction
Pulmonary hemosiderosis represents a disease characterized by iron accumulation in the local macrophages, as a consequence of bleeding and subsequent erythrocyte phagocytosis (Table 1). Hemorrhage into the lower respiratory tract can be a severe, lifethreatening condition. Conditions associated with diffuse alveolar hemorrhage (DAH) are very diverse and the diagnosis of idiopathic pulmonary hemosiderosis (IPH) is rendered only after meticulous exclusion of other conditions (see Alveolar Hemorrhage). DAH is characterized by hemoptysis, dyspnea, alveolar opacities on chest radiograph, and various degrees of anemia. Following a hemorrhagic episode, the alveolar macrophages convert the hemoglobins iron into hemosiderin within 72 h. Since the hemosiderin-laden macrophages reside for up to 48 weeks in the lungs, the term pulmonary hemosiderosis should be reserved for persistent or recurrent intra-alveolar bleeding beyond an 8-week interval. Virchow initially described the entity of IPH in 1864 as brown lung induration. In 1931, Ceelen published the autopsy ndings in two children who

Diagnosis
IPH is a diagnosis of exclusion. The clinical presentation varies significantly from acute, fulminant hemoptysis, to chronic cough and dyspnea, repetitive hemoptysis, fatigue, or only asymptomatic anemia. In adults the respiratory symptoms tend to be more pronounced, while in children failure to thrive and anemia (and less often hemoptysis) can be the presenting ndings. The clinical course waxes and wanes between two phases, alternating in an unpredictable fashion: 1. Acute phase, corresponding to active DAH episodes, manifested by cough, dyspnea, hemoptysis, and, sometimes, respiratory failure. Almost 100% of adults experience hemoptysis during the disease course. The physical examination ranges from normal to that consistent with chronic anemia. 2. Chronic phase, which is characterized by a slow resolution of previous symptoms, with or without treatment. Although the physical exam may be normal, there are usually signs of chronic

310 IDIOPATHIC PULMONARY HEMOSIDEROSIS

Table 1 Dictionary of terms Term Iron Etymology Lat. Ferrum Definition Element, atomic weight 55.847, atomic number 26, specic gravity 7.874 (201C), valence 2, 3, 4, or 6; by weight, the fourth most abundant element on earth, making up the crust. It is found in nature as hematite (Fe2O3), taconite or common ore. It is a vital component of plants and animal organisms, and appears in hemoglobin Intracellular storage form of iron, containing a complex of ferric hydroxides, polysaccharides, proteins and iron 433% by weight, which stains blue with Prussian stain (Perls method) Focal or general increase in tissue iron stores, especially in local macrophages Deposition of abnormal amounts of hemosiderin in the lungs, especially in the alveolar macrophages and interstitium, which may be due to various conditions (congestive heart failure, valvulopathies, vasculitides, etc.) Repeated, sudden attacks of dyspnea, hemoptysis, diffuse alveolar bleeding, and anemia; seen mostly in children, but also in adults (also called Heiner Ceelen disease) of unknown cause Disorder due to deposition of hemosiderin in the parenchymal cells, causing tissue damage of the liver, pancreas, heart, and pituitary Pneumoconiosis siderotica, a form of pneumoconiosis due to the presence of iron dust, usually serious when combined with silica exposure (silicosiderosis) Hemosiderosis Hyperferremia Macrophage laden with phagocytosed iron-containing particles

Hemosiderin

Gr. Hemo, blood Gr. Sideros, iron

Hemosiderosis

As above

Pulmonary B

As above

Idiopatic pulmonary B

As above

Hemochromatosis

Gr. Hemo, blood Gr. Chromatosis, staining Gr. Sideros, iron Gr. -osis, condition

Siderosis

Siderophage

Gr. Sideros Gr. Phagos, ingestion Gr. Sideros, iron Gr. Phyllos, loving

Siderophilin

Non-heme iron-binding protein, also called transferrin (in vertebrate blood), which serves as the major circulatory transporter of iron

B, hemoriderosis. Modied from Ioachimescu O, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12(1): 1625, with permission from Lippincott Williams & Wilkins.

illness: pallor, emaciation, failure to thrive, and hepatosplenomegaly. In those with advanced brosis, cyanosis, bilateral crackles and clubbing, and signs of cor pulmonale may be present.
Pulmonary Function Tests

Pulmonary function tests show variable degrees of ventilatory restrictive defects; no obstructive defects should be seen. The diffusing lung capacity for CO (DLCO) is low or low-normal during the chronic phase and elevated in the acute phase, as a manifestation of high CO uptake during active DAH. Arterial blood gas analysis may reveal hypoxemia or, rarely, hypercapneic respiratory failure.
Laboratory Features

Other causes of DAH must be ruled out (see Alveolar Hemorrhage). The laboratory investigation reveals

variable degrees of anemia, without any quantitative or qualitative platelet defects, liver or kidney disease, coagulopathies, hemorrhagic diatheses, or any inammatory syndromes. The sideropenic (transferrin saturation less than 40%), microcytic (mean corpuscular volume (MCV) o 80 ) anemia is the direct consequence of recurrent DAH. Plasma ferritin concentration is normal or elevated due to alveolar macrophage synthesis and releases into the circulation. Bone marrow biopsy typically shows low iron stores. Although not a sensitive test, sputum examination may demonstrate intra-alveolar bleeding (erythrocytes and hemosiderin-laden macrophages) by hematoxylin-eosin and Prussian blue (Perls) stains. Bronchoalveolar lavage (BAL) from involved areas has conceivably a higher diagnostic yield. The predominant cellular types are the alveolar macrophages, siderophages, and occasionally neutrophils and eosinophils.

IDIOPATHIC PULMONARY HEMOSIDEROSIS 311 Imaging Studies

The radiographic features of IPH vary in relation to the clinical phase. During the acute phase, the radiographs show diffuse alveolar opacities, predominantly in the dependant lung elds, with corresponding ground-glass attenuation or frank consolidations on the high-resolution computed axial tomography (CAT) scan (Figure 1). During the remission phase, the

alveolar opacities tend to resorb and an interstitial reticular or micronodular pattern of opacities may occur in the same areas, with variable degrees of traction bronchiectasis and, rarely, honeycombing. Technetium 99m (99mTc) or chromium 51 (51Cr) perfusion scans may demonstrate intra-alveolar bleeding, as opposed to a pneumonic process or alveolar edema, but their clinical utility has not been validated.

Lung Biopsy

Figure 1 CAT scan of the chest of a patient with IPH, showing ne bilateral nodules, ne subpleural reticulations, and discrete areas of ground-glass attenuation, the latter probably due to recent alveolar hemorrhage.

Transbronchial lung biopsy from involved areas is the initial diagnostic procedure of choice. Larger and multiple lobe samples can be obtained by videoassisted thoracoscopic surgery (VATS) or open lung biopsy. The lung biopsy can exclude specifically any granulomas, as seen in fungal, mycobacterial or other infections, in sarcoidosis, berylliosis, foreign-body reactions, Wegeners granulomatosis, ChurgStrauss syndrome, microscopic polyangiitis, or isolated pauciimmune pulmonary capillaritis. In IPH, in addition to light microscopy (Figure 2), immunouorescence or immunohistochemistry stains are needed to evaluate for immunoglobulin or immune complex depositions. Iron stains, such as Prussian blue (Perls stain), typically show numerous siderophages in alveolar and distal airways (Figure 3), as in other diseases characterized by DAH. The hemosiderotic lungs have a macroscopic appearance, called by Virchow as brown induration. On light microscopy, the alveolar walls are thickened, type 2 pneumocytes are hyperplastic, and the interstitium contains mild to moderate amounts

Figure 2 Hematoxylin-eosin stain of a hemosiderotic lung specimen, showing non-specic interstitial inltration, scattered areas of collagen deposition, and significant alveolar hemorrhage. Magnication 100. Courtesy of Dr C Farver.

312 IDIOPATHIC PULMONARY HEMOSIDEROSIS

Figure 3 Perls (Prussian blue) stain of the same lung biopsy specimen as in Figure 2, showing multiple intra-alveolar and interstitial siderophages. Magnication 200. Courtesy of Dr C Farver.

of collagen. In addition, three other features are important:

*

presence of intact or minimally fragmented erythrocytes in the distal airways and alveoli, as a reection of recent or active DAH; multiple hemosiderin-laden macrophages (siderophages) in the alveolar spaces and interstitium, as an expression of subacute/chronic or recurrent intrapulmonary bleeding; and absence of any of the following: focal or diffuse smooth muscle cell proliferations, vascular malformations, malignancy, pulmonary infarcts, capillaritis/vasculitis, granulomatous inammation, or infectious agents.

Useful ancillary tests are immunohistochemistry and immunouorescence to exclude any intrapulmonary immunoglobulin or immune complex depositions. Electron microscopy (not mandatory for diagnosis) may reveal swelling of the alveolar cells, with minimal thickenings and localized disruptions of the basement membranes, but no electron-dense deposits in the alveolar basement membrane.

Pathogenesis
The etiology of IPH remains largely unknown. There are several hypotheses that try to explain the occurrence of the structural lesions of the alveolar-endothelial membrane:
* *

Genetic theory. Familial clustering of IPH has been described, suggesting either a heritable trait

or a genetic predisposition to the inuence of as yet unidentied environmental agents. So far, no specic genetic linkages or polymorphisms have been reported. Environmental theory. A series of outbreaks of alveolar hemorrhage among infants living in water-damaged houses in Cleveland and Chicago led to an extensive investigation of possible infectious or mycotoxigenic pathogenesis. Environmental mold/fungi, especially Stachybotrys atra, were thought to be associated with the development of the process. However, both the association with the exposure to molds, that is, S. atra, and the diagnosis of IPH have been questioned by a Centers for Disease Control and Prevention (CDC) review. Other molds, such as Alternaria, Aspergillus, Penicillium, and Trichoderma spp., have also been implicated, but a clear relationship with IPH has not been established. Trichotecens, fungal toxins with potent protein synthesis inhibitory capacity, have been shown to impede the angiogenesis underneath the rapidly forming alveolar membranes of infants and create in the acinar region an area susceptible to bleeding. Other authors proposed an association between IPH and exposure to insecticides, but this theory has not been veried. Autoimmune theory. An autoimmune pathogenesis has been suggested by the demonstration of circulating immune complexes. Immunohistochemical examinations of lung tissue in IPH generally have not supported an immune pathogenesis. Interestingly, approximately 25% of the patients who

IDIOPATHIC PULMONARY HEMOSIDEROSIS 313

survive more than 10 years with this disease subsequently develop some form of autoimmune disease. Furthermore, the treatment of choice is immunosuppressive, which is another observation in support of an immunopathogenetic mechanism for this disease. Allergic theory. Several children with IPH had detectable plasma antibodies (precipitins and IgE) against cows milk, which has led to the hypothesis of a systemic allergy/hypersensitivity reaction to milk components, although these ndings have not been reproduced. Another interesting association is between IPH and celiac sprue. To date, there are more than 10 case reports of celiac disease (celiac sprue) with IPH, in whom the lung disease went into complete remission after institution of a gluten-free diet. Metabolic theory. In IPH, iron metabolism appears abnormal. In DAH, the alveolar macrophages accumulate acutely large amounts of iron, which quickly saturate intracytoplasmic ferritin, and then the free iron launches the sequence of cellular oxidative injury. Later, the iron derived from the metabolism of heme is found as trapped iron in hemosiderin (at 502000 times normal values) and, to a lesser extent, bound to other proteins (ferritin, etc.). Furthermore, in contrast to other macrophages, alveolar macrophages are less able to process and release iron. Within the macrophages, iron is removed from hemoglobin in an essential step catalyzed by heme-oxygenase (HO), which is subject to regulation. Heme-oxygenase 1 (HO-1) is induced by a variety of cell stressors, including various cytokines. The limited capacity of alveolar macrophages to metabolize hemoglobin may be related to its lack of HO, an enzyme that can be induced following the phagocytosis of alveolar erythrocytes. The exact relationship between HO and iron metabolism deserves further study.

inammation. Different animal models have been used (rabbits, hamsters, etc.) to study the effect of neutrophil elastase administration; the significance of DAH, diffuse alveolar damage (DAD) and emphysema caused by the elastase, and the interrelation with neutrophils recruitment process is still unclear.

Current Therapy
There are no controlled studies and no IPH registries, so the management of this disease is the result of observations on a relatively small number of patients, which are frequently lost in follow-up over the years. A number of measures have been tried, as follows:
*

The total amount of iron in the alveolar macrophages can be quantied using cytochemical (Perls stain), colorimetric (ferrozine stain), or particleinduced X-ray emission (PIXE) techniques. It is as yet unclear if the metabolic arrest within the alveolar macrophages occurs at the level of HO involved in the degradation of hemoglobin in the endoplasmic reticulum, at the level of ferroportin 1, ceruloplasmin, or hephaestin (or other proteins involved in the iron ion egress from the macrophages), or at a different site.

Splenectomy, without significant results (there is no evidence of hypersplenism in hemosiderosis, as opposed to hemochromatosis). Inhaled corticosteroids, with insufcient experience to date. Systemic glucocorticoids, commonly started during episodes of DAH, with apparent good control and apparent impact on mortality, but with unclear effect on the chronic phase. We recommend a starting dose of 1 mg kg 1 day 1 prednisolone or equivalent for a couple of months, at which time the alveolar inltrates tend to resorb, and then a slow taper over the next months, if symptoms do not recur. The majority of patients seem to respond favorably to long-term treatment with oral corticosteroids, with decreased number of IPH exacerbations and, possibly, a decline in brogenesis. Any attempt to stop the treatment portends a high risk of recurrence. However, in children and adolescents, the long-term treatment can be problematic because of side effects. Other immunosuppressive agents that have been tried with variable results include: azathioprine, hydroxychloroquine, cyclophosphamide, and methotrexate. Among them, azathioprine or hydroxychloroquine in combination with steroids are the most popular regimens. Singlelung transplantation has been reported in two cases. Unfortunately, both cases had a clinical course complicated by IPH recurrence in the transplanted lungs.

Animal Model
In animals, stachybotryomycotoxicosis leads to alveolar hemorrhage associated with significant

The response to therapy can be assessed during the episodes of DAH by improvement in symptoms, anemia, pulmonary opacities, and the return of DLCO to baseline, and on a long-term basis by reduction in the number and severity of hemorrhagic episodes and the progression of interstitial lung disease (as assessed by the decline of DLCO).

314 IMMUNOGLOBULINS

Prognosis
The lack of prospective studies or large clinical registries makes the evaluation of short/long-term prognosis and the benets of therapy very difcult. Overall, it appears that children and adolescents have a more severe course and prognosis, while adults have a more protracted course, with milder symptoms and more favorable prognosis. The most frequent cause of death is acute respiratory failure accompanying massive DAH, or chronic respiratory failure and cor pulmonale due to severe pulmonary brosis. The mean survival after the onset of symptoms is approximately 2.53 years (range 3 months to 10.5 years). In a small series of pediatric patients the 5-year survival rate was 86%, while in another retrospective analysis of 15 children with IPH followed for a mean of 17 years, 80% of the patients had a benign course. The better outcome might have been related to the more prolonged and sustained immunosuppressive therapy in these series. With improved immunosuppressive and corticosteroid therapy, most IPH patients tend to survive to childbearing age, so that exacerbations of pre-existing disease during pregnancy are more likely to occur. During pregnancy, both corticosteroids and azathioprine may be used in severe IPH ares, which tend to occur during the last trimester. There seems to be a general agreement that untreated disease is more deleterious than the side effects of the immunosuppressive agents, for both fetal and maternal health (at least with corticosteroids).
See also: Alveolar Hemorrhage. Bronchoalveolar Lavage. Corticosteroids: Therapy. Granulomatosis: Wegeners Disease. Lung Imaging. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpastures Syndrome. Vasculitis: Overview.

Further Reading
Centers for Disease Control and Prevention (1994) Acute pulmonary hemorrhage/hemosiderosis among infants Cleveland,

January 1993November 1994. MMWR. Morbidity and Mortality Weekly Report 43: 881883. Centers for Disease Control and Prevention (2004) Investigation of acute idiopathic pulmonary hemorrhage among infants Massachusetts, December 2002June 2003. MMWR. Morbidity and Mortality Weekly Report 53: 817820. Calabrese F, Giacometti C, Rea F, et al. (2002) Recurrence of idiopathic pulmonary hemosiderosis in a young adult patient after bilateral single-lung transplantation. Transplantation 74: 1643. Chryssanthopoulos C, Cassimos C, and Panagiotidou C (1983) Prognostic criteria in idiopathic pulmonary hemosiderosis in children. European Journal of Pediatrics 140: 123125. Helman DL, Sullivan A, Kariya ST, et al. (2003) Management of idiopathic pulmonary haemosiderosis in pregnancy: report of two cases. Respirology 8: 398. Ioachimescu OC, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12(1): 1625. Ioachimescu OC, Sieber S, and Kotch A (2004) Idiopathic pulmonary haemosiderosis revisited. European Respiratory Journal 24: 162170. Jarvis BB (2003) Stachybotrys chartarum: a fungus for our time. Phytochemistry 64: 5360. Kiper N, Gocmen A, Ozcelik U, Dilber E, and Anadol D (1999) Long-term clinical course of patients with idiopathic pulmonary hemosiderosis (19791994): prolonged survival with low-dose corticosteroid therapy. Pediatric Pulmonology 27: 180184. Kjellman B, Elinder G, Garwicz S, and Svan H (1984) Idiopathic pulmonary haemosiderosis in Swedish children. Acta Paediatrica Scandinavica 73: 584588. Le Clainche L, Le Bourgeois M, Fauroux B, et al. (2000) Longterm outcome of idiopathic pulmonary hemosiderosis in children. Medicine (Baltimore) 79: 318326. Milman N and Pedersen FM (1998) Idiopathic pulmonary haemosiderosis. Epidemiology, pathogenic aspects and diagnosis. Respiratory Medicine 92: 902907. Ohga S, Takahashi K, Miyazaki S, Kato H, and Ueda K (1995) Idiopathic pulmonary haemosiderosis in Japan: 39 possible cases from a survey questionnaire. European Journal of Pediatrics 154: 994995. Saeed MM, Woo MS, MacLaughlin EF, Margetis MF, and Keens TG (1999) Prognosis in pediatric idiopathic pulmonary hemosiderosis. Chest 116: 721725. Soergel K and Sommers SC (1962) Idiopathic pulmonary hemosiderosis and related syndromes. American Journal of Medicine 32: 499511.

IMMUNOGLOBULINS
A August, The Pennsylvania State University, University Park, PA, USA
& 2006 Elsevier Ltd. All rights reserved. or foreign material. These proteins are responsible for humoral immunity (or immunity found in serum) against pathogens, toxins, or in some pathological conditions, autoimmune diseases. Immunoglobulins are made by rearrangement of multiple gene segments in two separate genes, and are generally premade prior to exposure to pathogens. There are ve main types or classes of immunoglobulins, all of which have two functions, antigen binding and effector function, via their Fc regions. These classes include m, d, g, e, and a. The effector functions of immunoglobulins

Abstract
Immunoglobulins or antibodies are proteins secreted by B cells, specialized cells in the immune system, and interact with antigens

IMMUNOGLOBULINS
include the ability to activate the complement pathway as well as mast cells, macrophages, neutrophils, and natural killer cells via specic Fc receptors on these cells. Specic classes of immunoglobulins have been demonstrated to have roles in the development of different respiratory diseases, including transfusionrelated acute lung injury, pulmonary complications arising from systemic lupus erythematosus, and asthma, acting via various effector functions to induce pathology.

315

Introduction
Immunoglobulins (or antibodies) were rst discovered in serum responsible for humoral immunity (i.e., binding to foreign material or antigens). Subsequent analysis indicated that these proteins had two main functions, antigen binding and effector function (in this case, xation of complement). Immunoglobulins were later found to be part of the gamma globulins in serum, hence the name immune globulins or immunoglobulins. In serum, antibodies are a heterogeneous mixture with different recognition specicities, and so attempts to characterize them at the molecular level were initially not successful. A major breakthrough came with that realization that multiple myeloma patients carried tumors derived
Ag binding

from B cells that secrete antibodies, thus providing a source of antibodies with a single specicity. Purication and sequencing studies demonstrated that antibodies are homodimers with up to ve domains that fold in a particular manner, now referred to as immunoglobulin domains or folds (Figure 1). Subsequent cloning of the immunoglobulin genes illustrated that these proteins are actually assembled from two different gene products, a light chain and a heavy chain (Figures 1 and 2). Furthermore, it was shown that these genes undergo rearrangements prior to being assembled into functional proteins.

Structure
Immunoglobulins are homodimers of two light chains and two heavy chains. Each light chain has a single variable domain that is unique to each immunoglobulin and a constant domain that is one of two isotypes, k or l . Each heavy chain also has a single V or variable domain that is unique and a constant region that is made up of three or four constant domains. The constant region identies the immunoglobulin isotype or class as m (IgM), d (IgD),

Hinge region Fc lgG lgD lgE

J-chain J-chain

lgA

lgM

Figure 1 Structure of immunoglobulin isotypes. Immunoglobulins are symmetrical dimers with two light chains and two heavy chains. Darker ovals represent variable domains of light and heavy chains. These form the antigen-binding domains (one variable domain from each chain) and are also referred to as Fab regions. Lighter ovals represent the constant domains of the light and heavy chains. IgG, IgD, and IgA have hinge regions and three constant domains. IgE and IgM lack hinge regions and have an extra constant domain. IgA can exist as a monomer (left) or a dimer (right) held together by the J-chain or secretory component. IgM is secreted as a pentamer held together by the J-chain. The antigen-binding regions and Fc portions of the antibody are indicated.

316 IMMUNOGLOBULINS
VLn JLn CL DNA

Rearranged DNA (a) (AAAA)n mRNA

VHn

DHn

JHn

Alternate splicing (AAAA)n & (AAAA)n 2 (b) Isotype switch

Figure 2 Structure of immunoglobulin loci. (a) Generic structure of the light-chain locus. l and k light-chain loci are similar. The darker boxes represent variable (V) segments and the lighter box represents joining (J) segments. The constant domain is shown as the lightest box. Broken lines represent large distances between segments. Light-chain DNA is rearranged in developing B cells to place a V segment next to a J segment. The intervening DNA is deleted. The resulting DNA is then transcribed into RNA, which is spliced to make mRNA encoding the light-chain proteins. (b) Structure of the heavy-chain locus. Heavy-chain DNA is rearranged in developing B cells to place V, D, and J segments next to each other. This DNA is transcribed and alternatively spliced to make mRNA for IgM and IgD carrying the same VDJ segments. In mature B cells, isotype switching can occur in this example, to make IgE by placing the rearranged VDJ segments upstream of the e constant domains. The intervening DNA is deleted.

g (IgG), e (IgE), or a (IgA) (Figure 1). In many cases, subtypes of the different isotypes exist, such that there are four subclasses of IgG and two types of IgA in humans, and these are unique genes. There are also allotypes of immunoglobulin isotypes, which usually represent amino acid differences in the constant domains between individuals. An immunoglobulin molecule is thus a symmetrical dimer of two light chains and two heavy chains, with the light- and heavy-chain V domains folding to form the antigenbinding site (Figure 1). Immunoglobulins are assembled from the products of two separate genes light chains encoded on chromosomes 22 and 2 (l and k light chains) and heavy chains encoded on chromosome 14 in the human. These loci have multiple segments of DNA, which are brought together during B cell development to form a functional immunoglobulin. The light chains have multiple V segments, Vk or Vl, that are organized next to each other in the locus (Figure 2). These segments are followed by J or joining segments, which are followed by a segment that encodes for the constant domain of the k or l. These V and J segments are brought together during B cell development to make a functional light chain, as described

later. The heavy-chain locus is similar, except that it has more V segments, diversity or D segments that are not found in the light chains, and J segments upstream of the constant segments. The constant segments encode those of the m, d, g, e, and a isotypes. These V, D, J, and constant segments are brought together during B cell development to make a functional heavy chain via a process that is carried out by the RAG proteins. The variable domain forms part of the antigen-binding site of the immunoglobulin molecule, and the D and J segments form the most variable region of this domain the antigen contact site. This junction between the VD and J segments is a spot for insertion of extra nucleotides by the enzyme terminal deoxynucleotide transferase (TdT), which increases the diversity of immunoglobulins. Two other regions found in all variable domains are also hypervariable. Together, these three regions are called hypervariable regions 1, 2, and 3 or complementary determining regions (CDRs), and they form the antigen contact sites, with the hypervariable or CDR3 region being the most variable. The light-chain genes are rearranged in a similar way but are usually less variable because they lack the D segments and do not have extra added nucleotides.

IMMUNOGLOBULINS

317

The light-chain variable domains also have three CDR regions, with the third formed by the junction of the V segment and the J segment. Upon the formation of a functional light-chain protein, the light and heavy chains fold together to form the functional immunoglobulin molecule.
Structure of Immunoglobulin Isotypes

IgD, IgG, and IgE isotypes have 2 antigen-binding sites; however, IgM exists as a monomer on the surface of the B cell and as a pentamer with 10 antigen binding sites when secreted. IgA can exist either as a monomer (with 2 antigen-binding sites) or as a dimer (with 4 antigen-binding sites). IgM and IgA multimers are held together by a J chain, which is not encoded within the immunoglobulin locus (Figure 1 and Table 1). The constant domains or Fc regions of immunoglobulins control the effector functions of the molecule. This differs based on the isotype of the molecule, as discussed later. IgD, IgA, and IgG have a proline and disulde bond-rich hinge region in their constant domain that provides exibility within the molecule for access to antigens. These isotypes have three constant domains (CH13). In contrast, IgM and IgE do not have a hinge region but have an extra constant domain that may provide a similar function as these molecules (CH14). Although these domains are highly homologous and are all members of the immunoglobulin superfamily, they differ, are encoded by unique genes, and are responsible for the different effector functions of these molecules.
Table 1 Cytokines that induce Ig isotype switch Ig isotypea IgM Structure Part of BCR, secreted form is a pentamer and interacts with J chain Part of BCR, secreted form is a monomer Monomer Monomer Monomer or dimer, the latter form interacts with J chain Monomer Monomer Monomer Monomer or dimer, the latter form interacts with J chain Cytokines inducing isotype switching b N/A

IgM and IgD, both of which carry the same variable domains and associate with the same light chains, are also expressed as part of the B cell receptor (BCR). Both of these events (expression of a surface form of immunoglobulin and expression of the two isotypes with the same variable domains) are accomplished by alternative splicing to generate m and d heavy chains, both of which contain a hydrophobic membrane-spanning region that allows anchoring in the plasma membrane. Thus, these two isotypes expressed on the surface of B cells both carry the same antigen-binding specicity. The ve different isotypes of immunoglobulins are generated by a process called isotype- or class-switching, which rearranges the DNA of the VDJ segment of the heavy chain, placing it upstream of the desired isotype. For example, B cells undergoing class-switch to generate IgE will rearrange the VDJ segment DNAs of their immunoglobulins, deleting the genes for m , d, g, and a1, and place this VDJ region upstream of the e heavy-chain segments (Figure 2). These cells will then make IgE that has the same antigenic specicity of the original immunoglobulin.
Immunoglobulins Undergo Somatic Hypermutation

Antibody responses increase in afnity during the course of an immune response, a process referred to as afnity maturation. This occurs when the variable regions of the rearranged heavy chain undergo somatic mutations. These somatic mutations are selected by antigen such that hot spots within the CDR1, -2, and -3 of the V region exhibit the most changes. This process does not change the overall structure of the antibody but results in select changes in the antigen-binding pockets, increasing the afnity of the antibody for the antigen.

Regulation of Production and Activity

N/A IFN-g IFN-g TGF-b

IgD IgG3 IgG1 IgA1

Regulation of Immunoglobulin Secretion

IgG2 IgG4 IgE IgA2

IL-2, IFN-g, TGF-b IL-4 IL-4 TGF-b

Ig Isotypes are listed in the order they are found in the human Ig heavy-chain locus. b Cytokines also require CD40 signals via CD40L-mediated help from T cells. BCR, B-cell receptor; N/A, not applicable.

Immunoglobulins are made and secreted only by B cells. B cells are activated upon binding of specic antigen to their BCRs, and in the presence of Thelper (CD4 ) cell help in the form of cytokines and cellcell contact, generally via CD40CD40L interactions complete differentiation to plasma cells (T-dependent antigens). Plasma cells switch most of the immunoglobulin heavy-chain molecules (by alternative splicing of the primary RNA transcript) to molecules that lack the membrane-spanning domain, allowing them to be secreted. Plasma cells are antibody factories and the majority of their cellular physiology and processes are geared to synthesizing and secreting antibody, which they can

318 IMMUNOGLOBULINS

do at the rate of approximately 120 000 molecules 1 min 1 cell. The nature of the antibodies and the level of the different isotypes generally depend on the nature of the immune response as well as the anatomical location being analyzed. Plasma cells secreting IgA and some IgE are generally found in lymph nodes draining the respiratory system or in the lungs. Plasma cells have a relatively short half-life, so antibody secretion is not thought to be further regulated. Plasma cells can live for 4 or 5 days and have to be replenished by newly differentiating B cells. However, the different isotypes may have much longer half-lives in serum. In some cases, B cells can be activated by antigens that are T-cell independent; in these cases, T cell help is not required (T-independent antigen). Under these conditions, B cells can differentiate into plasma cells and secrete antibodies.
Regulation of Immunoglobulin Isotype Switching

CD40L-mediated T cell help and interleukin (IL)-4, a T-helper-2 (Th2) type cytokine, B cells will undergo class-switch to make IgE or IgG4. Thus, in diseases in which there is a predominant Th2 response, such as allergic asthma, high levels of circulating IgE are usually observed. Transforming growth factor beta (TGF-b), on the other hand, induces class-switch to IgA, usually found in mucosal surfaces, including the respiratory system and gut. Signals that induce classswitch initially induce the opening of the locus being switched, resulting in the rearrangement of the VDJ segment being used by this antibody to the desired isotype segments by a mechanism that is not clear but that involves the DNA repair machinery of the cell as well as the enzyme activation-induced cytosine deaminase (AID), the latter being inducible by IL-4 and CD40 signals (Figure 2). Due to the requirement for T cell help, T-dependent antigens are usually the only antigens that can induce class-switching.
Regulation of Immunoglobulin Somatic Hypermutation

Immunoglobulin isotype- or class-switching is regulated by the nature of the immune response and thus the nature of cytokines being made by other cells responding, particularly cytokines and cellcell contact from CD4 T cells. Activated B cells can undergo class-switch to change the isotype of the antibody in the presence of T cell help. The specic isotype that is switched depends on the cytokine milieu (Table 1). For example, in the presence of
Table 2 Serum concentration and function of Ig isotypes Ig isotype Serum concentration (mg/dl); half-life (days) 0.0750.3; 5 0.0030.01; 3 0.1; 8 Function

Immunoglobulins also undergo somatic hypermutation during the immune response, which usually results in an overall increase in the afnity of the
Ag binding

Fc
Part of the BCR; activates complement Part of the BCR Opsonization; activates complement; ADCC by NK cells Opsonization; activates complement; ADCC by NK cells Opsonization; activates complement; ADCC by NK cells Crosses mucosal surfaces and found in the respiratory system Opsonization; weakly activates complement; ADCC by NK cells Mast cells and basophil activation Crosses mucosal surfaces and found in the respiratory system

IgM IgD IgG3

Fc receptors on neutrophils, macrophages mast cells, basophils NK cells

Complement activation (lgM > lgG3 > lgG1 > lgG2) Anaphylatoxins MAC

ADCC, degranulation phagocytosis

IgG1

0.9; 23

Fc receptors on epithelial cells

IgG3

0.1; 8

Transport across mucous membranes

Figure 3 Functions of immunoglobulins. Immunoglobulins bind to antigen via their N-terminal variable domains, leading to neutralization or clearance of antigen. The Fc is referred to as the effector domain, and it interacts with various Fc receptors on different cell types, resulting in cell activation, degranulation, or induction of antibody-dependent cytotoxicity (ADCC), depending on the cell type. Alternatively, depending on the isotype, the Fc region can activate the classical complement pathway, leading to the production of anaphylatoxins as well as assembly of the membrane attack complex (MAC) on cells, bacteria, or viruses. Certain isotypes also can interact with Fc receptors on epithelial cells, resulting in transport across mucous membranes, including the respiratory system, and into milk.

IgA1

0.3; 6

IgG2

0.3; 23

IgE IgA2

0.000 03; 2.5 0.05; 6

BCR, B-cell receptor; ADCC, antibody-dependent cytotoxicity; NK, natural killer cells.

IMMUNOGLOBULINS

319

antibody response. This process results in genetic changes in the variable regions of the heavy chain of immunoglobulins. Like isotype switching, this process is dependent on T cell help, cytokines, as well as cellcell contact, and although the contributions of specic cytokines are not clear, CD40CD40L signals contribute to this process. Again, similar to isotype switching, this process is dependent on DNA repair machinery of the cell as well as AID.

immunoglobulins with regard to complement activation and Fc receptor binding (Figure 3).

Receptors
Immunoglobulins serve as receptors for antigen, either as soluble secreted proteins or as part of the BCR (IgM and IgD). However, a number of cells carry receptors for different immunoglobulin isotypes that allow these cells to respond in an antigenspecic manner via these Fc receptors (FcRs). Other cells carry these receptors in order to transport specic isotypes across membranes (e.g., PIgR transports IgM and IgA across mucous membranes). Table 3 summarizes the various FcRs for the different immunoglobulin isotypes, where they are expressed, and some of their functions.

Biological Function
Immunoglobulins have two functional domains the N-terminal regions with variable domains for antigen binding and the C-terminal isotype-specic domains for effector functions. These effector functions differ based on the isotype of the antibody but fall into two main types complement activation and Fc receptor binding. In general, the different immunoglobulin isotopes have different activities in activating the classical pathway of complement, such that IgM4IgG34IgG14IgG2. Table 2 summarizes the main functions of the different isotypes of
Table 3 Ig receptors, expression pattern, and function Ig isotype IgM IgD IgG3 Receptor PIgR None identied FcRg I (CD64, high afnity) FcRg II (CD32, intermediate afnity) FcRg III (CD16, low Afnity) FcRn FcRg I (CD64, high afnity) FcRg II (CD32, intermediate afnity) FcRg III (CD16, low afnity) FcRn FcaR (serum IgA, CD89) PolyIgR (mucosal IgA, PIgR) FcRg II (CD32, intermediate afnity) FcRg III (CD16, low afnity) FcRn FceRI (high afnity) FceRII (CD23, low afnity) FcRg II (CD32, intermediate afnity) FcRg III (CD16, low afnity) FcRn FcaR (serum IgA, CD89) PolyIgR (mucosal IgA, PIgR) Expression

Immunoglobulins in Respiratory Diseases

Deciencies in IgG2, IgG3, and IgG4 have been associated with increased respiratory infections. There is also a high correlation between increased levels of IgE

Function Transport IgM, IgA across mucous membranes N/A ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance Transport into milk to neonates ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance Transport into milk to neonates Antigen uptake and clearance Transport across mucous membranes ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance Transport into milk to neonates Activation of mast and basophils Unknown ADCC, antigen uptake and clearance ADCC, antigen uptake and clearance Transport into milk to neonates Antigen uptake and clearance Transport IgM, IgA across mucous membranes

Epithelial cells lining mucous membranes N/A Mono, Macs Mono, Macs, Neuts, B Macs, Neuts, NK Mono, Macs Mono, Macs, Neuts, B Macs, Neuts, NK Placental tissue and gut epithelia of neonates Neuts, Eos, T Epithelial cells lining mucous membranes Mono, Macs, Neuts, B Macs, Neuts, NK Mast, Baso Neuts, Eos, T Mono, Macs, Neuts, B Macs, Neuts, NK Neuts, Eos, T Epithelial cells lining mucous membranes

IgG1

IgA1

IgG2

IgE IgG2

IgA2

PIgR, poly Ig receptor; Mono, monocytes; Macs, macrophages; Neuts, neutrophils; Eos, eosinophils; NK, natural killer cells; T, T cells; B, B cells.

320 INFANT RESPIRATORY DISTRESS SYNDROME

and allergic asthma. High levels of allergen-specic IgE can result in binding of allergen to IgE, and interaction of this complex with the FceRI on mast cells and basophils leads to degranulation and release of pharmacological agents, including histamine, serotonin, proteoglycans, prostaglandins, leukotrienes, mast cell tryptase, as well as cytokines and chemokines. These agents have effects on the epithelial and smooth muscle cells in the lung and contribute to the development of goblet cell hyperplasia, mucous production, tissue remodeling, and airway hyperresponsiveness. Therapies based on blocking the interaction between IgE and the FceRI are currently being used for patients with allergies or asthma. rhumAb-E25 (omalizumab) is a humanized monoclonal antibody against the Fc region of IgE and is indicated for patients who have moderate to severe allergic asthma and those with seasonal allergic disease accompanying asthma and allergic rhinitis. A prominent role for immunoglobulin has also been demonstrated for transfusion-related acute lung injury (TRALI). The recognition of antigens expressed by neutrophils and granulocytes by immunoglobulins in transfused plasma can result in large-scale activation of these cells. These cells release products that lead to damage to the lung, resulting in bilateral pulmonary edema seen in TRALI. This lung damage has also been observed in rare cases following allogeneic bone marrow transplantation. Similar roles for immunoglobulins have been suggested for the presence of pulmonary injury in some patients with complications arising from systemic lupus erythematosus.
See also: Allergy: Overview. Asthma: Overview. Chymase and Tryptase. Complement. Histamine.

Interleukins: IL-4. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; Pulmonary Macrophages. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
DAmato G, Liccardi G, Noschese P, et al. (2004) Anti-IgE monoclonal antibody (omalizumab) in the treatment of atopic asthma and allergic respiratory diseases. Current Drug Targets Inammation and Allergy 3: 227229. Davies DR and Metzger H (1983) Structural basis of antibody function. Annual Review of Immunology 1: 87115. Finkelman FD, Holmes J, Katona IM, et al. (1990) Lymphokine control of in vivo immunoglobulin isotype selection. Annual Review of Immunology 8: 303333. Ghetie V and Ward ES (2000) Multiple roles for the major histocompatibility complex class I-related receptor FcRn. Annual Review of Immunology 18: 739766. Goldsby RA, Kindt TJ, Osborne BA, and Kuby J (2003) Immunology, 5th edn. New York: WH Freeman. Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE and the basis of allergic disease. Annual Review of Immunology 21: 579628. Heyman B (2000) Regulation of antibody responses via antibodies, complement, and Fc receptors. Annual Review of Immunology 18: 709737. Honjo T (1983) Immunoglobulin genes. Annual Review of Immunology 1: 499528. Honjo T, Kinoshita K, and Muramatsu M (2002) Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annual Review of Immunology 20: 165196. Kinet J-P (1999) The high-afnity IgE receptor (FceRI). Annual Review of Immunology 17: 931972. Looney MR, Gropper MA, and Matthay MA (2004) Transfusionrelated acute lung injury: a review. Chest 126: 249258. Mostov KE (1994) Transepithelial transport of immunoglobulins. Annual Review of Immunology 12: 6384. Ravetch JV and Bolland S (2001) IgG Fc receptors. Annual Review of Immunology 19: 275290.

INFANT RESPIRATORY DISTRESS SYNDROME

P A Dargaville, Royal Hobart Hospital, Hobart, TAS, Australia
& 2006 Elsevier Ltd. All rights reserved. airspace opacication. Histologically, the lack of surfactant results in collapse of primordial airspaces, epithelial disruption, and proteinaceous exudation with the formation of hyaline membranes. The functional consequence of surfactant deciency is poor lung volume at end expiration, with unfavorable pulmonary mechanics during inspiration. The pathogenesis of IRDS involves a deciency of all elements of pulmonary surfactant, in particular dipalmitoyl phosphatidylcholine, along with surfactant inhibition and a global immaturity of lung architecture commensurate with the degree of prematurity. The disease is in part preventable by the administration of antenatal glucocorticoids; the use of these agents is associated with a 50% reduction in the risk of IRDS, and a decreased mortality. Other than general intensive care measures and respiratory support, specic treatment for IRDS is available in the form of

Abstract
Infant respiratory distress syndrome (IRDS) is a disease of the premature infant caused by a deciency of pulmonary surfactant. Its incidence is inversely proportional to gestational age, with a heightened risk following Cesarean delivery without labor, and after perinatal asphyxia. Infants with IRDS have an early onset of a symptom complex including tachypnea, grunt, and retractions, associated with a requirement for supplemental oxygen. Chest radiograph shows loss of lung volume and diffuse

INFANT RESPIRATORY DISTRESS SYNDROME 321

exogenous surfactant administered directly into the trachea. Both natural and synthetic surfactant preparations are used, and have been seen to result in a 240% reduction in mortality, and a decrease in pneumothorax.

Introduction
Infant respiratory distress syndrome (IRDS) (synonyms: hyaline membrane disease, idiopathic respiratory distress syndrome) is a life-threatening neonatal lung disease occurring in premature infants. It is, in essence, the clinical and morphological expression of an immature lung forced to adapt to extrauterine life. IRDS is the single most common indication for applying assisted ventilation in the newborn period, and is a significant contributor to the relatively high mortality noted amongst preterm infants. The burden associated with IRDS in neonatal units worldwide is largely a consequence of the high prevalence of preterm birth; in the developed world around 6% of infants are liveborn before 37 weeks gestation, and somewhat more than 1% before 32 weeks gestation. These proportions have been rising in many countries in the past two decades, and when combined with the increasing proportion of preterm infants to whom life-sustaining intensive care therapy is being offered, the result is a larger than ever population of preterm infants that require treatment for IRDS and other complications of prematurity.
Epidemiology of IRDS

premature birth, with both the incidence of developing IRDS, and the risk of dying of the condition, being inversely related to length of gestation (Figure 1). Many other factors combine to modulate the risk and severity of IRDS at a given gestation (Table 1). Antenatal factors that most commonly lead to an increase in IRDS risk are birth asphyxia and Cesarean delivery without antecedent labor. Male gender is associated not only with an increased incidence, but also with an increased mortality risk. Several antenatal factors, including fetal growth restriction, prolonged rupture of membranes, and illicit drug exposure, are seen as relative protective factors. Additionally, negroid ethnicity lends some protection against IRDS. Postnatally, the adequacy of resuscitative measures, in particular positive pressure ventilation and avoidance of hypothermia, have a bearing on the subsequent risk and severity of IRDS.

Table 1 Factors modulating risk of IRDS at a given gestation Factors increasing IRDS risk Perinatal asphyxia Cesarean delivery without labor Male gender Maternal diabetes mellitusa Second of twins Familial risk Inadequate resuscitation Hypothermia
a

Factors decreasing IRDS risk Prolonged rupture of membranesa Fetal growth restrictiona Maternal illicit drug use Negroid ethnicity

IRDS occurs worldwide, sparing no population or ethnic groups. The disease is inextricably linked to

Not all available data support the assertion.

100

80

Incidence (%)

60 IRDS 40

20

0 3738 25 26 27 28 29 30 31 32 33 34 Gestational age (weeks) 35 36 3940

Figure 1 Relationship between gestational age and IRDS. Plot of the incidence of IRDS at each gestation. Reproduced from Robertson PA, Sniderman SH, Laros RK Jr, et al. (1992) Neonatal morbidity according to gestational age and birth weight from ve tertiary care centers in the United States, 1983 through 1986. American Journal of Obstetrics and Gynecology 166: 1629, with permission from Elsevier.

322 INFANT RESPIRATORY DISTRESS SYNDROME Historical Perspective

IRDS has been recognized as a distinct clinical entity occurring in the preterm infant only since the early 1950s. Prior to this, the recorded observations on neonatal lung disease were dominated by pathologic descriptions of hyaline membranes occurring in a host of neonatal lung diseases. Even with the recognition of IRDS as a distinct entity, the preoccupation with eosinophilic hyaline membranes persisted, and gave the disease its original name (hyaline membrane disease) and also a spurious causation (inux of eosinophilic material via aspiration of amniotic uid). Only with the understanding that hyaline membranes were derived from plasma protein came the recognition that these eosinophilic red herrings were not the cause of IRDS, but merely a reection of disruption of the alveolocapillary barrier. The seminal paper of Avery and Mead in 1959, in which lung efuent from preterm infants dying of IRDS was demonstrated to have poor surface activity, established the primacy of surfactant deciency in the pathogenesis of IRDS. A wealth of research followed in the decades thereafter, with the result that the biology and ontogeny of surfactant were elucidated, the precise nature of the surfactant abnormalities in IRDS was established, and exogenous surfactant replacement evolved from a distant hope to a therapeutic reality.

Figure 2 Pathology of IRDS. Lung section from a preterm infant dying of IRDS. Strongly eosinophilic hyaline membranes are seen at the airspace margins, with lightly stained proteinaceous exudate lling the remainder of several airspaces. Note the primitive lung architecture, and the highly cellular stroma.

Etiology
The primary cause of IRDS is a lack of pulmonary surfactant in the airspaces of the preterm infant. There is both deciency and dysfunction of surfactant in IRDS, and, in addition, global immaturity of the preterm respiratory system. The complex interplay of these factors is further discussed in section Pathogenesis below.

Pathology
The gross ndings at autopsy in IRDS are of large areas of atelectasis and congestion, particularly in the dependent areas, giving the lung a liver-like appearance. The distensibility of the lung is markedly reduced. Histologically, the appearances vary with age, with the earliest features being distal airspace collapse accompanied by epithelial necrosis and sloughing within the pathologically distended terminal bronchioles. These changes, present within 12 h of birth, are followed beyond 3 h by the appearance of hyaline membranes, generally within the terminal and respiratory bronchioles and extending into the primordial alveolar ducts (Figure 2). The hyaline membranes are eosinophilic linear aggregations of

plasma protein in which may be encased nuclear debris from sloughed pneumocytes. They occur as a direct result of disruption of the alveolocapillary barrier, and are not specic to IRDS, being found in other diseases of the newborn lung (e.g., meconium aspiration syndrome), and in lung injury beyond the newborn period (e.g., acute respiratory distress syndrome). The putative mechanism by which epithelial damage occurs is uneven distribution of transpulmonary pressure, generated by the infants own respiratory efforts, by positive pressure ventilation, or both. The resultant shear forces damage the lining epithelium and associated endothelium, leading to an inux of proteinaceous material some of which coalesces into hyaline membranes. By 24 h, inammatory cells (mostly macrophages with some neutrophils) appear in the airway lumen and interstitium, and debris from hyaline membranes is prominent within intraluminal macrophages by 4872 h. The epithelium begins to regenerate after 48 h, with the initial appearance of thick epithelial squames. Around this time, surfactant synthesis and secretion begins to occur. By 7 days, infants recovering from IRDS show resorption of hyaline membranes and further epithelial regeneration. Those still ventilated will show a mixture of ongoing damage and regeneration, ultimately with the development of brosis and scarring characteristic of neonatal chronic lung disease, also known as bronchopulmonary dysplasia. In the extremely preterm infant (o28 weeks gestation), these ndings will be accompanied by evidence of a failure of ex utero maturation, with arrest of alveolarization and secondary crest formation.

Clinical Features
Hallmarks of the clinical diagnosis of IRDS are the early onset (before 4 h) of a symptom complex

INFANT RESPIRATORY DISTRESS SYNDROME 323

Table 2 Clinical features of IRDS Pulmonary Tachypnea Retractions Grunting Cyanosis without supplemental oxygen Apnea Extrapulmonary Hypotension Oliguria Hypotonia Ileus Exaggerated neonatal jaundice

characterized by tachypnea, retractions, grunting, and cyanosis without supplemental oxygen (Table 2). Tachypnea has historically been dened as a respiratory rate above 60 breaths/min, although it is clear that extremely preterm infants may respire at rates of 7080 min 1 in the absence of parenchymal lung disease. Retractions indicate poor lung compliance, and refer to the suprasternal, intercostal, and subcostal skin recession noted during inspiration, and also to the recession of the highly compliant sternum and anterior chest wall in the preterm infant. Grunting is the expiratory noise generated by forced exhalation through a partially closed glottis, and is a reex mechanism to preserve the volume of the diseased lung in expiration. The clinical features of neonatal respiratory distress are by no means specic for IRDS, and the context in which they occur may be of more help in securing a clinical diagnosis, with the likelihood of IRDS being greatest at lower gestations (Figure 1), and in the presence of other risk factors (Table 1). Table 3 shows a differential diagnosis of respiratory distress in the newborn infant. Several extrapulmonary features are regularly encountered in IRDS, including hypotension, hypotonia, ileus, and oliguria with impaired capacity to excrete a water load (Table 2). These abnormalities are seen to resolve in parallel with restoration of lung function; a diuresis may in fact herald the onset of respiratory improvement. The natural course of IRDS, without assisted ventilation, is characterized by progressive deterioration in the rst 2448 h, highest mortality in the rst 3 days, and a recovery period in survivors beginning at approximately 72 h. The introduction of prenatal glucocorticoid therapy, assisted ventilation, and exogenous surfactant therapy has considerably changed the natural history and outcome of IRDS (see section Management and current therapy below).
Radiology

distinguish IRDS from a legion of other causes of neonatal respiratory compromise (Table 3), and give an impression of disease severity. The response to treatment and the development of complications can be ascertained on sequential radiographs. At their most prominent, the radiographic features of IRDS are striking and distinct (Figure 3). The lung elds show diffuse reticulogranular opacication, which may on occasions be more prominent on the right side, and in the lower zones. Anatomically, the radiolucent granules in the opacied lung represent distended terminal bronchioles surrounded by microatelectasis. The volume of the lung elds may be considerably reduced. With dense parenchymal opacication, there is loss of normal contours, including the cardiac silhouette and the diaphragms, and the development of air bronchograms. The radiographic appearances in IRDS take some hours to evolve, and are significantly moderated by the application of positive airway pressure, and by the administration of exogenous surfactant. IRDS in its natural form shows progressive parenchymal opacication over the rst 4872 h, followed by a gradual clearing of the chest radiograph, coincident with the resolution of surfactant deciency. Respiratory complications of IRDS may be apparent radiographically from early in the course of the disease, including pulmonary interstitial emphysema (Figure 4), pneumothorax (Figure 5), and other air leaks.
Lung Function Testing

Lung volumes and pulmonary mechanics The lack of surfactant and resultant high surface tension within uid-lined airspaces leads to low lung volume in expiration, and unfavorable pulmonary mechanics on inspiration (Table 4). Functional residual capacity is much reduced, and with the higher proportion of atelectatic lung units comes a higher alveolararterial oxygen difference. Lung compliance is poor, and improves gradually to normal values as the disease resolves. As a result, work of breathing is increased, but breathing efciency is decreased given the highly compliant chest wall and suboptimal diaphragmatic conguration of the premature infant. Arterial blood gas analysis Hypoxemia of some degree is universal in IRDS, and is usually accompanied by respiratory acidosis with elevated PaCO2. Where supplemental oxygen is being administered the disturbance of oxygenation is best quantied using measures such as the alveolararterial oxygen difference (see Table 4). These correlate well with radiographic severity and other indices of pulmonary mechanics.

Chest radiograph is an indispensable tool in the diagnosis and management of IRDS, and should be performed in any infant with respiratory distress, regardless of gestation. The image thus obtained will

Table 3 Differential diagnosis of respiratory distress in the newborna Condition IRDS Causea Primary surfactant deciency and lung immaturity Affects term or preterm infants Preterm Usual time of symptom onset o6 h Distinguishing features, comments Tachypnea, retractions, and grunting, with a significant requirement for supplemental oxygen, associated with respiratory acidosis; diffuse reticulogranular opacication radiographically Minimal oxygen requirement but persistent need for respiratory support, often for many weeks Transient relatively mild respiratory distress, rarely needing assisted ventilation; interstitial edema radiographically Meconium-stained infant with moderate or severe respiratory distress often with pulmonary hypertension; patchy opacities and regional hyperination radiographically Usually focal changes radiographically, but group B streptococcal pneumonia can mimic IRDS; nosocomial pneumonia may complicate the course of other neonatal lung diseases, especially if prolonged intubation is necessary May cause acute cardiorespiratory deterioration; small pneumothoraces can be detected on up to 1% of chest radiographs after delivery Bell-shaped chest and/or high diaphragms radiographically; risk of early pneumothorax

Pulmonary insufciency of prematurity Transient tachypnea of the newborn

Global immaturity of the respiratory system Retained fetal lung uid

Extremely preterm

o6 h o6 h

Both (especially following elective Cesarean section) Term

Meconium aspiration syndrome

Perinatal inhalation of meconium causing airway obstruction and pneumonitis

o6 h

Neonatal pneumonia

Transplacental, transtracheal, or hematogenous acquisition of pathogenic organisms

Both

Transplacental: o6 h; others: variable

Spontaneous pneumothorax

Gas in the pleural space

Both

o6 h if isolated; at any time if associated with positive pressure ventilation o6 h

Pulmonary hypoplasia

(a) oligohydramnios (b) neuromuscular or skeletal anomalies (c) developmental lung anomalies or cysts

Both

Pulmonary hemorrhage

Hemorrhagic pulmonary edema

Both

Usually o24 h

Congenital diaphragmatic hernia

Posterolateral diaphragmatic defect (usually left-sided) resulting in abdominal contents occupying hemithorax in fetal life; associated pulmonary hypoplasia

Term

o6 h

Pulmonary hypertension

Increased pulmonary vascular resistance

Term or occasionally preterm

o6 h

Cyanotic congenital heart disease Acyanotic congenital heart disease

Obligatory right-to-left shunt or mixing lesion Obligatory left-to-right shunt or left ventricular outow obstruction

Term

Term

Variable depending on ductal closure and/or degree of mixing 424 h

Airway obstruction

Anatomical / functional obstruction at any level Tissue ischemia related to perinatal asphyxia

Term

Variable

Lactic acidosis
a

Both

o6 h

Interstitial or patchy changes radiographically; can complicate the respiratory disease of an extremely preterm infant (especially after surfactant therapy), or a term infant with meconium aspiration syndrome and/or asphyxia Scaphoid abdomen and bowel sounds in hemithorax, often associated with prominent evidence of pulmonary hypertension; chest radiograph shows malpositioned abdominal organs associated with hypoplastic ipsilateral and contralateral lung Marked hypoxemia with pre/postductal difference in oxygenation, prominent right ventricular impulse and triscuspid incompetence; pulmonary hypertension often accompanies other parenchymal lung diseases, especially in the full-term infant Hypoxemia not overcome by oxygen therapy; often effortless tachypnea with normal or low PaCO2 Left ventricular outow obstruction may remain clinically silent until closure of the ductus arteriosus; left-to-right shunt lesions become symptomatic once pulmonary vascular resistance falls in the rst weeks or months Usually audible stridor or other airway noise associated with retractions; hypoxemia is a late and ominous sign Tachypnea often with minimal retraction but prominent pallor

Most common causes are listed; see Further reading section for more information on these and other rarer causes of neonatal respiratory distress.

326 INFANT RESPIRATORY DISTRESS SYNDROME

Pathogenesis
Biochemical Immaturity in IRDS

Figure 3 Radiographic appearance of IRDS. Chest radiograph of a preterm infant at 28 weeks gestation on day 1 of life showing moderately severe IRDS. Note the relatively uniform reticulogranular opacity in the lung elds, with loss of normal contours related to microatelectasis. Air bronchograms are noted bilaterally.

Pulmonary surfactant Deciency of pulmonary surfactant is the fundamental cause of IRDS. Surfactant is a mixture of phospholipid and protein vital for the normal function of the lungs, and instrumental in smoothing the passage from intrauterine to extrauterine life. The major phospholipid component, dipalmitoyl phosphatidylcholine (DPPC), forms a monolayer on the alveolar surface which lowers surface tension, thereby maintaining alveolar volume in expiration, and conferring favorable pulmonary mechanics during inspiration from low lung volume. Several other components of surfactant assist in the formation of the DPPC monolayer, including other phospholipids (e.g., phosphatidylglycerol), and surfactant-associated proteins A, B, and C (SP-A, SP-B, SP-C). All elements of surfactant are synthesized, stored, secreted, and recycled by the alveolar type II cell.

Figure 4 Radiographic appearance of pulmonary interstitial emphysema. Chest radiographs of separate preterm infants with pulmonary interstitial emphysema: (a) taken at 12 h of life shows rounded lucencies within the opacied lung, representing pathologically distended airways proximal to collapsed primordial alveoli; (b) is from an infant at 1 month with marked cystic emphysematous change in the right lung, with midline shift and volume loss on the left side.

Figure 5 Radiographic appearance of pneumothorax. Chest radiographs in a 29-week gestation infant. At 18 h of age (a) there is a left pneumothorax under some tension, with marked loss of ipsilateral lung volume. Chest radiograph 1 h later (b) shows that the extrapleural air in the left hemithorax has been removed by an intercostal drain tube (arrow). The infant also has an endotracheal tube. A pneumothorax is now present on the right side.

INFANT RESPIRATORY DISTRESS SYNDROME 327

Table 4 Lung function in IRDS Parameter Tidal volume Functional residual capacity Compliance of the lung Alveolararterial oxygen differencea PaCO2
a

IRDS 46 ml kg 1 1020 ml kg 1 body weight 0.20.5 ml cmH2O 1 kg 1 Elevated (up to 650 mmHg) Elevated

Preterm infant with normal lungs Similar 30 ml kg 1 12 ml cmH2O 1 kg 1 4060 mmHg 3545 mmHg

Alveolararterial oxygen difference FiO2 Pbarometric PH2 O P aCO2 =R P aO2 .

Surfactant ontogeny: the basis for biochemical immaturity IRDS Natures parsimony has dictated that during fetal life, the elements of surfactant only appear in the lung when there is a reasonable chance of survival ex utero. There is a natural delay in the development of the surfactant system, with phospholipid-containing lamellar bodies rst recognizable within type II cells at around 24 weeks gestation, and surfactant phospholipids only appearing in trace amounts in amniotic uid before 30 weeks of gestation. Beyond 30 weeks, there is a progressive rise in the ratio of PC (lecithin) to sphingomyelin (i.e., the L : S ratio) in amniotic uid, reecting gradual increase in secretion of surfactant from the type II cell and efux from the lung in fetal lung uid. A mature L : S ratio of 2 or above (an excellent predictor of surfactant sufciency and absence of IRDS in postnatal life) is only noted close to full term in a nonstressed pregnancy. In fetal life, the regulation of the surfactant system is under the control of many competing inuences, but particularly the levels of circulating hormones. The concentration of endogenous glucocorticoids is a fundamental determinant of the state of the surfactant system in the fetus, impacting upon the synthesis of many elements of surfactant, as well as many other facets of lung maturation. Upregulation of several key synthetic enzymes, most particularly choline phosphate cytidylyl transferase, and fatty acid synthetase, leads to a marked increase in surfactant phospholipid production. These glucocorticoid effects have been successfully exploited in the form of antenatal steroid therapy in the setting of threatened premature delivery (see section Treatment below). Levels of other hormones also impact upon fetal surfactant maturation. Circulating catecholamines promote surfactant synthesis in a similar way to glucocorticoids. On the other hand, both androgens and insulin depress cytidylyl transferase activity, contributing to the higher incidence of IRDS in males, and infants of diabetic mothers, respectively. In the days prior to birth, there are considerable changes in the fetal lung in preparation for extrauterine life. The normal secretion of lung uid slows, and synthesis of surfactant elements increases. Additional maturation occurs during labor, as catecholamine and

glucocorticoid concentrations surge. Postnatally, as the airliquid interface is formed, there is a rapid increase in the size of the surfactant pool, with establishment of an intra-alveolar surfactant compartment which evolves rapidly in the rst 24 h. The importance of this nal maturation process is demonstrated by the increased risk of IRDS in the infant delivered by Cesarean section without labor. On the basis of the above, it might be expected that many more infants less than 36 weeks would suffer from the effects of surfactant deciency. The fact that many premature infants (even some much less than 30 weeks) do not have IRDS is an indication of the effective escape route that nature can apply to the lungs of the preterm infant when delivery is imminent the rapid inducibility of surfactant synthesis in response to stress, mediated by endogenous catecholamines and glucocorticoids. Surfactant deciency in IRDS All elements of the surfactant system have been found to be decient in IRDS. Based on studies of phosphatidylcholine (PC) content, the size of the surfactant pool is markedly reduced in preterm animals with IRDS (e.g., for sheep, 20 mmol PC kg 1 in RDS vs. 100 mmol PC kg 1 in the normal newborn). In human preterm infants with IRDS surfactant pool size has been estimated at 10% of that at full term. Estimated pool size correlates well with measures of lung dysfunction, for example, lung compliance. In addition to an overall reduction in phospholipid content, the ratio of DPPC to total PC in lung uid is also considerably reduced in IRDS. At the outset, the DPPC : PC ratio may be 0.3 or less (compared to a normal value of 40.5), and has been noted to normalize during recovery. Estimates of alveolar PC and DPPC concentration from lung lavage samples are lower in IRDS than normal controls, and correlate well with other measures of lung dysfunction, in particular oxygenation. Concentrations of other surfactant-associated phospholipids are also reduced in IRDS. Phosphatidylglycerol is regularly absent in the rst days of life in lavage uid from preterm infants with respiratory distress, and is seen to appear during the recovery phase. Delay in the appearance of phosphatidylglycerol in the lung

328 INFANT RESPIRATORY DISTRESS SYNDROME

has been particularly noted in infants born to diabetic mothers, and may contribute to the higher risk of IRDS in this group. Reduced concentration of surfactant proteins has also been noted in lung uid from infants with IRDS. Content of SP-A in lavage uid is low at the height of disease, and increases in parallel with recovery in the rst week of life. SP-A concentration shows a lesser or no increase in infants who ultimately do not survive, or go on to develop chronic lung disease. Similarly, concentrations of the surfactant proteolipids SP-B and SP-C are measurably low in lavage uid from infants with IRDS. Inhibition of surfactant function In addition to surfactant deciency, there is evidence that considerable inhibition of surfactant function occurs in IRDS. At equimolar DPPC concentration, lung uid from aficted infants shows impaired surface-active properties. Putative mechanisms by which surfactant function is impaired include sequestration of surfactant in hyaline membranes, and inhibition both by plasma proteins and edema uid. Proteinaceous inhibitors of surfactant function are found at high levels in the airspaces in IRDS.
Structural and Cellular Immaturity in IRDS

biochemical immaturity in around 2% of infants born at 38 weeks gestation by Cesarean section without labor, and on the other as prolonged respiratory insufciency and susceptibility to ventilatorinduced damage in the structurally immature lung of an infant born at 25 weeks gestation needing no supplemental oxygen from the outset. The biochemical and structural immaturity of the preterm lung not only contributes to the pathogenesis of IRDS, but also renders the lung susceptible to structural damage as both the infant and the clinician attempt to overcome the respiratory insufciency. A critical event here is the pathological distension of primitive airspaces related to abnormally high transpulmonary pressure, generated by the infants extreme respiratory efforts and/or the application of positive pressure to the airways by the clinician. Rupture of these distended airspaces heralds several important complications of IRDS, pulmonary interstitial emphysema and pneumothorax. More globally, the initiation of a process of epithelial damage and repair is a key step in the pathogenesis of chronic lung disease, a relatively frequent complication in the preterm infant with IRDS (see below).

Animal Models
Other factors apart from surfactant deciency contribute to the risk and severity of IRDS in the premature infant. Whilst survival in the extrauterine environment is possible around 2224 weeks gestation, the lung at this stage is approaching the end of the canalicular phase of lung development, with a cellular interstitium and primitive air spaces with a thick bloodair barrier. The pulmonary epithelium is undifferentiated, relatively cuboidal, and susceptible to shear forces, rendering the lung unable to cope with superadded surfactant deciency, and uniquely vulnerable to injury from positive pressure ventilation. A number of extraparenchymal factors, such as the compliance of the chest wall, the function of the diaphragm, and the maturity of the neural respiratory control centers, profoundly inuence the capacity of the premature infant to sustain an air-breathing existence. These factors all contribute to the severity of respiratory dysfunction in IRDS. There may be a considerable disparity between the structural and biochemical maturity of the lung in a newborn preterm infant, related not only to inherent differences in ontogeny of these elements, but also to a difference in the capacity of the lung to rapidly mature in response to environmental and therapeutic agents. This disparity manifests on the one hand as severe but transient respiratory distress related to Animal models of IRDS aim to replicate all elements of the biochemical immaturity seen in the preterm human lung, along with a concomitant degree of structural and cellular immaturity. Once delivered, the animal should manifest the characteristic clinical and physiological disturbances of IRDS, and yet be salvageable with standard neonatal intensive care so as to allow prolonged study and evaluation of specic postnatal therapies. Over more than four decades, a number of mammalian models of IRDS have been extensively studied (Table 5), and have yielded much information about the biology of surfactant, the pathophysiology of IRDS, and the efcacy of exogenous surfactant preparations. Each model has limitations, chief amongst which is that the unique combination of circumstances that result in the birth of a preterm infant are difcult to reproduce experimentally. Additionally, the balance of structural, cellular, and biochemical immaturity noted at any given gestation in the human infant is somewhat different in each of the animal species.

Management and Current Therapy

Prevention of IRDS

Given the relationship between prematurity and IRDS, avoidance of preterm delivery is a logical way to reduce the incidence of IRDS. This said, the

INFANT RESPIRATORY DISTRESS SYNDROME 329

Table 5 Animal models of IRDSa Species Rabbit Lamb Term gestation (days) 31 147 Preterm gestation studied (days) 27 120130 Preterm weight 30 g 23 kg Comment Small size precludes intricate postnatal testing of lung function, or prolonged survival Morphological development in advance of surfactant ontogeny; prolonged survival and creation of a model of chronic lung disease is possible Survival for several days postnatally is possible with respiratory support Prolonged survival and creation of a model of chronic lung disease is possible Targeted disruption of SP-B gene causes fatal respiratory failure in the newborn period, with global atelectasisb

Rhesus monkey Baboon Mouse lacking SP-B

160 179 1821

127131 134147 Studied at or near full gestation

250350 g 500700 g 12 g (at full gestation)

a Animals at any age may also be rendered surfactant decient by repeated saline lavage. This model is not included in the above table as the resultant lung injury has more features in common with acute respiratory distress syndrome than with IRDS, and is not accompanied by structural and cellular immaturity. b A human homolog exists, in which lack of functional SP-B leads to respiratory failure at birth in infants at or near full term.

rates of preterm birth are in fact rising in the developed world, fueled by a combination of factors including advanced maternal age, multiple pregnancy, and uptake of assisted conception. Further, there is an increased preparedness in obstetric practice to deliver extremely preterm infants if the health of mother or fetus appears likely to be compromised by continuing the pregnancy. Ablation of uterine contractions using tocolytic drugs does have a role in spontaneous preterm labor, if only to allow sufcient time for glucocorticoids administered to the mother to have an effect on the fetus. Accepting that preterm birth is often inevitable, the incidence and severity of subsequent IRDS can be reduced by careful obstetric management, including acceleration of fetal maturation with glucocorticoids, avoidance of traumatic delivery and/or intrapartum asphyxia, and application of appropriate resuscitation to the infant immediately after birth. Antenatal glucocorticoid therapy The impact of antenatal glucocorticoids on human fetal lung maturation was rst documented in 1972, and this therapy is now widely applied in pregnancies threatening to end prematurely. Pooled data from randomized controlled trials of antenatal glucocorticoids show a 50% reduction in risk of IRDS, a lesser severity of IRDS, and a lower mortality. The risks of intracranial hemorrhage and periventricular leukomalacia are also reduced. The benets of glucocorticoids are apparent at all gestations below 32 weeks, with a maximal effect if delivery occurred between 24 h and 7 days after administration. Antenatal glucocorticoid therapy is not associated with an increase in maternal, fetal, or neonatal infection, and appears to

be safe even in the context of premature membrane rupture. The agents used are betamethasone (12 mg intramuscularly, two doses 24 h apart) and dexamethasone (6 mg intramuscularly, four doses 12 h apart), with the former resulting in the best neurological outcome for the premature infant. Other agents that have been administered antenatally for induction of fetal lung maturation include thyrotropin-releasing hormone and ambroxol, with no conclusive evidence of benet.
Postnatal Management

The management of the preterm infant with IRDS includes supportive care to overcome respiratory failure and other organ dysfunction, and specic therapy with exogenous surfactant to counter the primary surfactant deciency (Table 6). The importance of general supportive measures such as adequate delivery room resuscitation, temperature control, and circulatory stabilization cannot be overestimated, and improvements in these aspects of care led to a marked increase in survival for the preterm infant well before the advent of exogenous surfactant.
Specic Therapy for IRDS

Respiratory support Oxygen therapy, continuous positive airway pressure (CPAP), and positive pressure ventilation are the mainstays of conventional therapy to support the respiratory system in a preterm infant with IRDS. Current evidence favors the following approach in the early management of preterm infants with IRDS: 1. Judicious use of oxygen therapy, targeting arterial oxygen saturation values no higher than 95%

330 INFANT RESPIRATORY DISTRESS SYNDROME

Table 6 Management of IRDS Antenatal preventative therapy Postnatal therapy Supportive measures Tocolysis for spontaneous preterm labor Antenatal glucocorticoid administration Avoidance of birth trauma and intrapartum asphyxia Resuscitation Temperature control Intravenous uid therapy, avoiding uid overload Supplemental oxygen Nasal or nasopharyngeal continuous positive airway pressure Intubation and positive pressure ventilationa Circulatory support with volume and/or inotropes Drug therapy Exogenous surfactant therapy Diuretics Indomethacin (or ibuprofen) for prevention or treatment of patent ductus arteriosus Antibiotics if perinatal infection suspected

Multiple ventilatory modes are used in preterm infants with IRDS (see Goldsmith and Karotkin).

(corresponding to a PaO2 value of around 60 mmHg) so as to lower the risk of retinopathy of prematurity. The lower limits of the safe range of oxygenation for the preterm neonate remain to be determined; an oxygen saturation of at least 88% (PaO2 around 45 mmHg) is currently deemed acceptable in most neonatal intensive care units. 2. Intubation and positive pressure ventilation for preterm infants with: (a) significant respiratory failure (PaCO24 60 mmHg) and arterial pHo7.2) unresponsive to lesser measures, (b) marked hypoxemia (failure to maintain oxygenation in the acceptable range despite an FiO2 of 0.6 or more), and (c) a single apneic episode requiring positive pressure ventilation, or multiple lesser episodes causing physiological disturbance. 3. Early administration of exogenous surfactant (see below) for any intubated preterm infant with an FiO240.25, unless there is clinical suspicion of a diagnosis other than IRDS. 4. Nasal CPAP via short binasal prongs as primary therapy for infants with lesser respiratory dysfunction, and after extubation of those initially managed with an endotracheal tube. There remains uncertainty (and controversy) regarding: 1. The question of routine intubation of preterm infants o29 weeks gestation in the delivery room, given that many such infants can be effectively supported with nasal CPAP unless apneic. 2. The value of intubation solely to administer surfactant in infants where nasal CPAP is to be used as primary therapy.

3. The indications for intubation of a preterm infant on nasal continuous positive airway pressure. 4. The most appropriate mode of mechanical ventilation to use in preterm infants with IRDS (including the place of high-frequency oscillatory ventilation). 5. The value of synchronized ventilation. 6. The safe limits of oxygenation in the preterm infant. Exogenous surfactant therapy Exogenous surfactant preparations available for clinical use may be categorized as natural (derived from mammalian lung tissue) or synthetic (Table 7). The animal products are lipid extracts of either lung mince or alveolar lavage uid. In lung mince surfactants, the phospholipid prole of the lipid extract is modied, either by purication or fortication, to more closely replicate that of natural surfactant. All natural surfactants contain the surfactant proteolipids SP-B and SP-C (which coisolate with lipids in chloroformmethanol extraction), but there is significantly less SP-B in surfactant preparations derived from lung mince. Second-generation synthetic surfactants contain no surfactant proteins, and are simply phospholipid preparations to which other components are added to facilitate surface adsorption. These have now been superseded by thirdgeneration surfactants having surfactant proteolipid activity, the efcacy of which appears similar to natural surfactants in comparative trials. The dose of surfactant phospholipid used for treatment of IRDS (50200 mg kg 1) has been chosen to replenish the decient lung with an amount of phospholipid equivalent to that present in the normal newborn lung (100 mg kg 1). This dose is far in excess of the estimated amount of phospholipid required to form a surfactant lm over the entire respiratory epithelium (around 3 mg kg 1), but must

Table 7 Commercially available exogenous surfactant preparations Trade name (generic name) Product derivation Phospholipid content (% dry weight) 84% Phospholipid SP-B content per mg composition (% phospholipid total phospholipid) (%wt./wt.) SP-C content per mg phospholipid (%wt./wt.) Phospholipid concentration in fullstrength preparation (mg ml 1) 25 Usual phospholipid dose in IRDS (mg kg 1)

Natural surfactants: lung mince extracts Survanta Cow lung mince extract (beractant) fortied with DPPC, palmitic acid, and tripalmitin Curosurf (poractant) Pig lung mince extract, chromatographically puried by removal of neutral lipids Cow lung mince extract fortied with DPPC, palmitic acid, and tripalmitin

PC 83%a DPPC 59% PG 3.2% PC 73%a DPPC 41%a PG 1.2% PC 83%a DPPC 59% PG 3.2%

0.1%a

2.1%a

100

99%

0.3%a

1.3%

80

100200

Surfacten (surfactenTA)

84%

0.1%a

2.1%

25b

100

Natural surfactants: lung lavage extracts Alveofact Cow lung lavage extract (bovactant) bLES (bovine lung extract surfactant) Infasurf (calfactant) Cow lung lavage extract, acetone precipitated Calf lung lavage extract

90%

98%

91%

Synthetic surfactantsc: 2nd generation Exosurf Synthetic phospholipid mixture (colfoscleril supplemented with tyloxapol palmitate) and hexadecanol ALEC Synthetic phospholipid (pumactant) mixture Synthetic surfactantsc: 3rd generation Surfaxin Synthetic phospholipid (lucinactant) mixture supplemented with KL4 peptide (sinapultide) Venticute (rSP-C surfactant)
a b

PC 84% DPPC 33% PG 9% PC 82% DPPC 46% PG 12% PC 74% DPPC 46% PG 3.6% DPPC 100%

1.2%a

1.7%

42

50100

2% Combined

27

135

1.2%a

1.3%

35

105

84%

Nil

Nil

13.5b

67.5

100%

PC 75% DPPC 75% PG 25% PC 75% DPPC 75% PG 25% PC69% DPPC 69% PG 31%

Nil

Nil

12.5b

100

82%

KL4 3%

Nil

30

175

Synthetic phospholipid mixture supplemented with recombinant SP-C (lusupultide)

92%

Nil

rSP-C 2%

46b

50

Value is an average of several estimates. After reconstitution of powder with aqueous vehicle. c First-generation synthetic surfactants were suspensions of pure DPPC, without any agents added to promote spreading. These were found to be ineffective in treating IRDS in trials conducted in the 1960s. Reprinted with modication from Dargaville PA and Mills JF (2005) Surfactant therapy for meconium aspiration syndrome: current status. Drugs 65 (in press), with permission.

332 INFANT RESPIRATORY DISTRESS SYNDROME

compensate for uneven distribution and protein inhibition, as well as primary phospholipid deciency. A number of different methods of surfactant instillation have been devised, including delivery via a side-port connector attached to the endotracheal tube, or via a separate catheter inserted through the endotracheal tube. The latter method appears to have been more widely adopted in clinical practice. Surfactant has also been given as a bolus into the pharynx, and as an aerosol, and has even been instilled into the amniotic uid near the face of the fetus, gaining access to the lung during fetal respiration. These alternative methods of delivery remain in the province of research. Since the rst report of successful use of exogenous surfactant in human infants in 1980, more than 30 randomized controlled trials of surfactant treatment have been conducted. Pooled data from these trials show that mortality from IRDS is considerably reduced (odds ratio 0.590.73) after treatment with either natural or synthetic surfactant preparations (Figure 6). The effect is consistent, whether the drug is given prophylactically, or as a rescue therapy when
Natural surfactant prophylaxis Mortality Pneumothorax PDA CLD IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (a) Odds ratio Synthetic surfactant prophylaxis Mortality Pneumothorax PDA CLD IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (c) Odds ratio

the features of IRDS have become established. Risk of air leak is also consistently lower in surfactanttreated infants, with the odds ratio for pneumothorax being around 0.35 for natural surfactant, and 0.6 for synthetic surfactants. Where tested, a more favorable outcome has been noted in infants given two doses of surfactant, rather than one. There appears to be minimal gain from a third and fourth surfactant dose. The timing of surfactant therapy is of considerable importance, with most trials showing benet in early treatment, given either in the delivery room or shortly after arrival and stabilization in the neonatal intensive care unit. Administration of surfactant beyond 24 h of life is associated with a higher risk of treatment failure, presumably related to the progression of lung injury and accumulation of proteinaceous exudate in the alveolar space. A number of comparative trials of different surfactant preparations have been conducted. Those comparing natural with second generation synthetic surfactants have highlighted the shortcomings of the latter, in particular delayed clinical response and
Natural surfactant treatment Mortality Pneumothorax PDA CLD IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (b) Odds ratio

Synthetic surfactant treatment Mortality Pneumothorax PDA CLD IVH 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 (d) Odds ratio

Figure 6 Meta-analysis of clinical trials of IRDS. Meta-analysis of exogenous surfactant treatment in IRDS, showing odds ratios and 95% condence intervals for relevant outcomes in (a) eight trials (involving 988 infants) of natural surfactant administered in the delivery room; (b) 12 trials (1600 infants) of natural surfactant for treatment of established IRDS; (c) seven trials (1560 infants) of synthetic surfactant administered in the delivery room; and (d) ve trials (2352 infants) of synthetic surfactant for treatment of IRDS. Mortality is dened as death prior to day 29. PDA, patent ductus arteriosus; CLD, chronic lung disease, dened as the need for oxygen therapy at 28 days; IVH, all grades of intraventricular hemorrhage. Data from the Cochrane library.

INFANT RESPIRATORY DISTRESS SYNDROME 333

greater risk of pneumothorax. More recent comparative trials have generally demonstrated equivalence between natural and third-generation synthetic surfactants. In the developed world, exogenous surfactant is widely used in the treatment of IRDS, with the relatively high cost of the drug being offset by its known benets. Surfactant usage is inversely related to gestation, being administered to 7090% of infants at 2427 weeks gestation, and 3060% of those born at 2831 weeks. There are considerable regional differences in surfactant administration, governed largely by delivery room intubation practices, and the readiness to use nasal CPAP as a primary therapy for IRDS. Diuretic therapy Infants with IRDS are oliguric and susceptible to water overload, and in addition have a significant pulmonary edema. Their response to diuretic therapy is, however, modest at best, and the small gain in pulmonary function appears more than offset by an increased risk of patent ductus arteriosus.
Complications of IRDS

Complications of IRDS (Table 8) occur in relation to (1) the surfactant-decient lung, (2) the general insult visited upon the preterm organism, and (3) the treatment applied. Some are consequences of all three. The most notable acute respiratory complication is air leak, in particular pulmonary interstitial emphysema and pneumothorax. Pulmonary interstitial emphysema is a common accompaniment of severe IRDS treated with positive pressure ventilation (Figure 4). It is a consequence of bronchiolar rupture and tracking of gas into the interstitium. At its most aggressive, multiple gas-lled cysts form within the interstitium, leading to local gas trapping and atelectasis in lesser affected regions, markedly compromising gas exchange. Management includes downward positioning of affected areas, minimization of positive pressure excursions (including the use of high-frequency ventilation), and selective intubation of the lesser-affected lung. Pneumothorax occurs when interstitial gas enters the pleural space via a breach in the visceral pleura (Figure 5). If under
Table 8 Complications of IRDS Respiratory Pulmonary interstitial emphysema Pneumothorax Pneumomediastinum Nosocomial pneumonia Chronic lung disease/ bronchopulmonary dysplasia Cardiovascular Patent ductus arteriosus Poor left ventricular function Pulmonary hypertension

tension, there may be dramatic cardiorespiratory compromise, with hypotension, bradycardia, and hypoxemia. Pneumothorax occurs in around 5% of nonventilated infants with IRDS, 1015% of those receiving nasal CPAP, and between 10% and 30% of cases requiring intubation and ventilation. Its successful treatment relies on early clinical and radiographic recognition, and, if necessary, expeditious drainage of intrapleural gas via needling and intercostal drain insertion. Presence of pneumothorax increases mortality in IRDS, and also increases risk of intracranial hemorrhage. Patency of the ductus arteriosus is a regular feature of IRDS, particularly in the extremely preterm infant. At the height of parenchymal lung disease, whilst pulmonary vascular resistance remains elevated, there may be bidirectional shunt with little net ow through the ductus. As lung disease resolves, however, the resultant fall in vascular resistance and pulmonary arterial pressure can lead to a left-to-right ductal shunt of significant degree. The combination of increased pulmonary blood ow and pulmonary edema that results from this shunt causes a delay in recovery of respiratory function, and a prolonged need for assisted ventilation. Treatment with indomethacin (or ibuprofen) affects closure of the ductus in most instances; recalcitrant or recurrent cases may require surgical ligation. Chronic lung disease (CLD), also known as bronchopulmonary dysplasia, is a common complication of IRDS, occurring in 1030% of all infants less than 32 weeks, and 3060% of those less than 28 weeks. The pathogenesis of CLD is multifaceted, complex and continually evolving; the key features are outlined in Table 9. With the recognition of the contribution of barotrauma and volutrauma to CLD has come renements in the application of assisted ventilation to preterm infants, such that gross brous dysplasia and squamous metaplasia is now less common, whereas lung dysmaturity and arrest of alveolarization has become more common, particularly in infants at gestations of less than 28 weeks. With the increased survival at low gestation, care of infants with CLD now places a considerable burden on hospital resources. Treatment for such infants

Neurological Intraventricular hemorrhage Periventricular hemorrhage Periventricular leukomalacia

Other Necrotizing enterocolitis Systemic infection Retinopathy of prematurity

334 INFANT RESPIRATORY DISTRESS SYNDROME

Table 9 Features of neonatal chronic lung disease Definitions Need for any form of respiratory support (including oxygen) at 36 weeks postconceptional age in a preterm infant born at less than 32 weeks gestation Oxygen dependency (7 abnormal chest radiograph) at 36 weeks postconceptional age in a preterm infant born at less than 32 weeks gestation Oxygen dependency at 28 days with an abnormal chest X-ray Risk factors Maternal chorioamnionitis Lower gestation and birth weight Increasing severity of IRDSa Pathology Airways Loss of bronchial and bronchiolar epithelium with broproliferative response and squamous metaplasia; obliterative bronchiolitis in severe cases Interstitium/respiratory unit Interstitial brosis, smooth muscle proliferation and type II cell metaplasia In extremely preterm infants: large, poorly septated primitive airspaces with minimal inammation or brosis Vasculature Smooth muscle hypertrophy and adventitial brosis In extremely preterm infants: arrested vascular development

Associated pulmonary hypoplasia Oxygen therapy Male gender

Ventilation with high positive pressure and high tidal volume Patent ductus arteriosus Fluid overload Infection

Chronic lung disease can occur in preterm infants with minimal early IRDS, including those receiving no initial ventilatory support.

includes supplemental oxygen therapy, nasal CPAP, inhaled bronchodilators and/or glucocorticoids, and diuretic therapy. Systemic glucocorticoid therapy has been linked to poor neurodevelopmental outcome, in particular cerebral palsy, and is now infrequently used in CLD. Preterm infants with IRDS are at risk of serious neurological complications, in particular intracranial hemorrhage and injury to the periventricular white matter. There is a clear association between severity of IRDS and the incidence and severity of intra- and periventricular hemorrhage and periventricular leukomalacia. Hemorrhage within the ventricular system in the preterm neonate begins in the highly vascular germinal matrix, and may remain in the subependymal region, or rupture into the ventricular system itself. Venous infarction in the adjacent brain parenchyma may then occur. The developing white matter tracts in the internal capsule may also be injured; in this case, systemic hypotension and a pressure-passive cerebral circulation are causally implicated.
See also: Alveolar Surface Mechanics. Breathing: Breathing in the Newborn. Bronchiolitis. Bronchopulmonary Dysplasia. Fluid Balance in the Lung. Pediatric Aspiration Syndromes. Pediatric Pulmonary Diseases. Pulmonary Function Testing in Infants.

Further Reading
Askin FB (1988) Pulmonary disorders in the neonate infant and child. In: Thurlbeck WM (ed.) Pathology of the Lung, pp. 123146. New York: Thieme.

Comroe JH (1977) Premature science and immature lungs. Parts I III. American Review of Respiratory Disease 116: 127135, 311323, 497518. Dargaville PA and Mills JF (2005) Surfactant therapy for meconium aspiration syndrome: current status. Drugs 65 (in press). Farrell PM and Avery ME (1975) Hyaline membrane disease. American Review of Respiratory Disease 111: 657688. Goerke J and Clements JA (1986) Alveolar surface tension and lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of Physiology, Section 3, The Respiratory System, pp. 247261. Bethesda: American Physiological Society. Goldsmith JP and Karotkin EH (eds.) (2003) Assisted Ventilation of the Neonate. Philadelphia: WB Saunders. Greenough A and Milner AD (eds.) (2003) Neonatal Respiratory Disorders, 2nd edn. London: Hodder Arnold. Jobe AH (1998) Pathophysiology of respiratory distress syndrome and surfactant metabolism. In: Polin RA and Fox WW (eds.) Fetal and Neonatal Physiology, pp. 12991314. Philadelphia: WB Saunders. Jobe AH (1999) The new BPD: an arrest of lung development. Pediatric Research 46: 641643. Martinez AM, Dargaville PA, and Taeusch HW (2000) Epidemiology of bronchopulmonary dysplasia: clinical risk factors and associated clinical conditions. In: Bland RD and Coalson JJ (eds.) Chronic Lung Disease of Early Infancy, pp. 2040. New York: Dekker. Robertson B and Halliday HL (1998) Principles of surfactant replacement. Biochimica et Biophysica Acta 1408: 346361. Robertson PA, Sniderman SH, Laros RK Jr, et al. (1992) Neonatal morbidity according to gestational age and birth weight from tertiary care centers in the United States, 1983 through 1986. American Journal of Obstetrics and Gynecology 166: 1629. Slattery MM and Morrison JJ (2002) Preterm delivery. Lancet 360: 14891497. Tooley WH (1977) Hyaline membrane disease: telling it like it was. American Review of Respiratory Disease 115: 1928.

INHIBITION OF DIFFERENTIATION (ID) PROTEINS 335

INHIBITION OF DIFFERENTIATION (ID) PROTEINS

D Morse, University of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

bHLH heterodimer

CANNTG (a) E-box

Gene transcription

Abstract
Inhibition of differentiation (Id) proteins were originally described as negative regulators of basic helix-loop-helix transcription factors that play key roles in cell differentiation and proliferation. As more is learned about the regulation of Id proteins, their interactions, and downstream effects, it is becoming evident that Id proteins have more complex activities than originally suspected. Id proteins have now been shown to both induce apoptosis and sustain cell survival, to promote angiogenesis and tumorigenesis, to regulate lymphocyte development and perhaps to regulate matrix deposition. Here, we summarize and discuss the functions of Id proteins as they relate to lung biology.

Id protein No gene transcription

CANNTG (b) E-box

Introduction
Among the many factors known to regulate transcription and thereby fundamentally control lung development, function, and pathology, the inhibition of differentiation (Id) proteins are a relatively new addition. Originally described in 1990 as dominant negative antagonists of the basic helix-loop-helix (bHLH) transcription factors, a growing number of biological functions are now known to be under the inuence of Id proteins. Definition of specic roles of Id proteins in the lung is, for the most part, lacking. Nevertheless, the functions that they play in cell growth and differentiation, cancer development, immune function, and angiogenesis ensure that these newcomers will establish themselves in the canon of learning required for a sound understanding of lung biology.

Figure 1 Effect of Id proteins on gene transcription by bHLH transcription factors. (a) In the absence of Id proteins, bHLH proteins form dimers and bind DNA with their basic regions to activate transcription. (b) In the presence of Id proteins, dimers form between Id and bHLH proteins. Because Id proteins lack the basic region needed for DNA binding, the resulting dimers cannot initiate transcription and gene expression is inhibited.

possess the dening HLH sequence without the adjacent basic region necessary for DNA binding (Figure 1). These proteins are thus capable of binding to other HLH transcription factors and preventing their participation in gene transcription. The term Id refers to the ability of these proteins to both inhibit DNA binding and, given the control of HLH transcription factors over cell fate, cell differentiation.

Regulation of Activity
The expression of Id proteins, like other early response genes, is rapidly induced by stimulation with growth factors. Regulation of expression at the promoter level is incompletely understood, but Smad proteins, Egr1, Sp1, and ATF3, have been shown to affect promoter activity. Intracellular levels are also regulated through the ubiquitin-proteasome degradation pathway, resulting in half-lives for the Id proteins in the range of 2060 min. Heterodimerization with other bHLH proteins modulates the rate of degradation of Id proteins and thus can significantly extend their half-lives.

Structure
The family of helixloophelix (HLH) transcription factors contains over 200 members that function broadly as regulators of developmental processes. Common to all members of the family is a highly conserved HLH region containing two amphipathic a helices separated by a shorter intervening loop. The HLH domain allows these proteins to homo- or heterodimerize, a function that is necessary for their activity. Adjacent to this sequence is a basic region capable of binding to DNA elements containing the canonical E-box sequence, CANNTG. Members of the class of HLH transcription factors known as Id proteins

Biological Functions
Well-established functions of the HLH family of proteins in multicellular organisms include regulation of cell cycle, lineage commitment, and cell differentiation. The HLH transcriptional regulators

336 INHIBITION OF DIFFERENTIATION (ID) PROTEINS

and their inhibitors are expressed throughout phylogeny from yeast to humans. Four vertebrate Id proteins have been identied and are designated Id1 to Id4. Despite shared homology in their HLH regions and similar binding afnities for bHLH transcription factors, the four Id family members differ in their expression patterns during embryogenesis, and their known biological functions overlap only partially. The sections that follow introduce the reader to those functions beyond embryonic development that are currently being explored for these four proteins.
Cell Cycle Control

Cellular differentiation is in many cases accompanied by cell-cycle arrest, and by virtue of their role in cell differentiation, it is reasonable to postulate that the Id proteins may be involved in cell-cycle regulation. Indeed, the expression level of Id genes is high in proliferating cells, and the expression of Id1, Id2, and Id3 transcripts increases rapidly following mitogenic stimulation of serum-starved human broblasts. After stimulation with serum or growth factors, there are two peaks of expression of Id1 and Id2, in early and late G1. Inhibition of Id protein synthesis by antisense oligonucleotides prevents the progression of G0-arrested broblasts into the cell cycle, providing further evidence that the Id proteins exert control over cell proliferation. The mechanism of this effect is incompletely understood, but a number of recent studies
Mitogen

have elucidated interactions between Id proteins and other cell-cycle regulatory proteins. Id2 and Id3 are now known to be phosphorylated in late G1 by cyclin-dependent kinase 2 (cdk2), although the consequence of this phosphorylation is not clear. Id2 has been shown to antagonize retinoblastoma (Rb) protein and other Rb family proteins through direct protein protein interaction via its HLH domain (Figure 2). Negative regulation of growth-inhibitory proteins such as p21 and p16 by Id1 has been reported by a number of investigators; this presumably occurs via suppression of bHLH transcriptional activity, and leads to cell-cycle progression into the S-phase. Taken together, current evidence suggests that each Id family member is responsible for unique effects on growth regulation, and that the effect of each protein depends upon its specic binding partner and probably upon its state of phosphorylation. Ongoing work in this area of research should lead to a better understanding of the mechanism of action of each Id family protein in cell-cycle control.
Tumorigenesis

Given the evidence that Id proteins function as proliferative factors for a wide variety of cell types and regulate cell differentiation, it is natural to hypothesize that Id might also play a role in the development of cancer. There is a wealth of evidence, mainly gleaned from tissue immunostaining, that Id protein

Id Id Proliferation E2F Id Rb Rb 1 E2F

2 bHLH Id

cdk P Id 3

CANNTG E-box

p16 p21

Cell cycle arrest

Figure 2 Tentative scheme for the role of Id1 in cell-cycle regulation. 1. Id2 binds and antagonizes unphosphorylated forms of the Rb family proteins, which causes the release of E2F. Once E2F has dissociated, it activates genes involved in cell-cycle progression. 2. Id proteins antagonize basic helixloophelix (bHLH) proteins that regulate the expression of target genes, including p21 and p16. Expression of p21 and p16 is thus decreased, and their inhibitory effect on cell-cycle progression is lost. 3. During late G1 phase, cyclindependent kinases (cdk) phosphorylate Id2 and Id3, which may affect the avidity or specicity of their binding to bHLH proteins. Rb protein, retinoblastoma protein.

INHIBITION OF DIFFERENTIATION (ID) PROTEINS 337

levels are elevated in multiple types of human tumors. Ablation of Id protein expression in carcinoma cells leads to reduced proliferation and sometimes to inhibition of their malignant phenotype, indicating that these cells may depend upon Id expression for unrestricted proliferation. Ectopic expression of Id proteins in a variety of cell lines enhances cell proliferation and decreases sensitivity to growth factor depletion; in some cases it confers features of transformation, such as the ability for a broblast cell line to grow on soft agar. Despite abundant evidence implicating Id protein expression in tumorigenesis, there have been no mutations of Id genes reported in human cancers. It appears that Id proteins are downstream targets of altered oncogenes that affect Id expression via a wide variety of cellsignaling pathways.
Angiogenesis

Lymphopoiesis

In order to examine the role of Id proteins in differentiation, a number of groups of investigators have generated mice with targeted disruption of Id1, Id2, or Id3. Mice lacking either Id2 or Id3 exhibit hematopoietic abnormalities, but these mice otherwise appear to develop normally. On the assumption that the Id proteins have overlapping functions, Benezra and colleagues examined the effects of inactivating two members of the family, Id1 and Id3 simultaneously. The deletion of all alleles for Id1 and Id3 resulted in embryonic lethality, but the preservation of one single allele for Id1 allowed for normal development of adult mice. These mice were unique in their response to inoculation with primary tumor cells: they were resistant to tumor growth and metastasis. This elucidated a previously unsuspected function of the Id protein family as key mediators of tumor neovascularization. The mechanism by which the Id proteins promote angiogenesis in this setting is not known, but loss of Id function has been shown to lead to a decrease in vascular endothelial growth factor (VEGF) expression, and the expression of matrix metalloproteinases has been shown to vary with Id protein expression. Since this discovery, thrombospondin-1 has been identied as the transcriptional target of Id1, and a probable effector of its role in angiogenesis. One of the intriguing aspects of the newly discovered role of Id proteins in angiogenesis is that the blood vessel formation associated with tumor growth and metastasis is more sensitive to a decrease in Id protein levels than is the angiogenesis required to support fetal development. This may provide further insight into mechanisms of angiogenesis and make the Id proteins a potential target for anti-angiogenic therapy.

The bHLH class of transcription factors is crucial to lymphocyte development and function, and Id proteins play important roles in this process. Id2decient mice are defective in the production of natural killer (NK) cells, and lack Peyers patches and peripheral lymph nodes. Given that in the absence of activity of the bHLH transcription factor E2A, NK cell development is enhanced, it seems likely that the differential dose of the bHLH transcription factor E2A and Id2 in early thymocyte differentiation decides lineage commitment. Id3-null mutant mice exhibit defects in positive selection of both MHC class I- and MHC class II-restricted thymocytes, while Id1-null mutant mice do not appear to have defects in lymphocyte development or function. It is evident from the observations made in these mutant mice that the Id proteins act upon a number of stages of lymphocyte development, but the signaling mechanisms involved have yet to be fully dened. There is some evidence implicating both the mitogenactivated protein-kinase cascade and the transforming growth factor beta (TGF-b)/SMAD signaling cascade in Id3 activation, and current studies should further clarify these relationships.
Apoptosis

A number of proteins share a dual ability to stimulate proliferation of cells and induce cell death, and the Id proteins are such Janus-faced molecules. Id2 has been shown to augment apoptosis in two cell lines by a mechanism that is completely separate from its ability to dimerize with bHLH transcription factors. The N-terminal region of Id2 alone is sufcient to bring about cell death via increased expression of a known modulator of programmed cell death, Bax. Id3 has also been shown to induce apoptosis of lymphocyte progenitors following TGF-b1 stimulation, although in this case the effect was assumed to be due to modulation of bHLH transcription factor activity. As with other functions of Id, it is perplexing that these proteins can share similar functions and yet employ disparate mechanisms of action; understanding these mechanistic differences will be crucial to clarifying the role of Id proteins in cell death.
Fibrosis

Recent evidence suggests that Id1 may play a role in regulating the elaboration of extracellular matrix by broblasts (Figure 3). As in lymphocytes, broblast Id proteins levels are increased by stimulation with TGF-b1, a key mediator of brosis. Chambers and colleagues have shown that when human fetal and adult lung broblasts are stimulated with TGF-b1,

338 INHIBITION OF DIFFERENTIATION (ID) PROTEINS

TGF- 1

Id1 Metalloproteinases Matrix production Myofibroblast differentiation

Figure 3 Hypothetical scheme for the role of Id1 in TGF-b1-mediated broblast behavior. Increased TGF-b1 levels lead to the transition of broblasts towards a myobroblast phenotype with increased a-smooth muscle actin expression and matrix formation. TGFb1 also increases intracellular levels of Id1, which may act to suppress matrix formation and a-smooth muscle actin expression and increase matrix turnover.

Id1 expression increases in a time course typical for immediate-early response genes. The functional implication of this increased Id1 expression is not well understood, but differences in matrix expression between wild-type broblasts and broblasts decient in the gene for Id1 have been reported in two different experimental settings. Additionally, Id1 immunostaining is increased in the lung brotic lesions within the parenchyma of bleomycin-treated rats, suggesting a relationship between Id1 expression in vivo and lung brosis. Interestingly, the effect of TGF-b on Id1 expression in broblasts is opposite to that in epithelial cells, where Id1, Id2, and Id3 are all rapidly repressed by treatment with TGF-b1. As with other functions of Id proteins, their role in brosis will require clarication by further investigations.

Id Proteins in Respiratory Diseases

The adaptive responses of the lung to various injuries and pathologies depend upon a coordinated balanced

cell proliferation, differentiation, and death. The Id proteins play a key role in these cellular behaviors, but to date there is little known about their inuence in specic lung diseases. There is mounting evidence that Id1 is a downstream effector of TGF-b1 effects in lung brosis, and that Id protein expression can alter the behavior of lung tumors. Nevertheless, as Chairman Mao famously replied when asked his thoughts on the impact of the French revolution, It is too early to tell to what extent improved understanding of Id protein function will expand our grasp of lung biology. The discovery of Id proteins is relatively recent, and there is much yet to be learned about how they are regulated and how they integrate into the multiple systems that they are known to inuence. The currently known functions of Id proteins are relevant to the study of lung development, lung tumors, diseases involving the adaptive and innate immune responses, and interstitial lung diseases, and so a conservative guess would predict that we will be hearing more about Id proteins in the future.

INSULIN-LIKE GROWTH FACTORS 339 See also: Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Colony Stimulating Factors. Fibroblasts. Pulmonary Fibrosis. Tumors, Malignant: Overview.
dominant negative helix loop helix factor Id3. Journal of Experimental Medicine 186: 15971602. Jen Y, Manova K, and Benezra R (1997) Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis. Developmental Dynamics 208: 92106. Langlands K, Yin X, Anand G, and Prochownik EV (1997) Differential interaction of Id proteins with basic helixloophelix transcription factors. Journal of Biological Chemistry 272: 1978519793. Lasorella A, Uo T, and Iavarone A (2001) Id proteins at the cross-road of development and cancer. Oncogene 20: 8326 8333. Lyden D, Young AZ, Zagzag D, et al. (1999) Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401: 670677. Norton JD (2000) ID helixloophelix proteins in cell growth, differentiation and tumorigenesis. Journal of Cell Science 113: 38973905. Pan L, Sato S, Frederick JP, Sun XH, and Zhuang Y (1999) Impaired immune responses and B-cell proliferation in mice lacking the Id3 gene. Molecular and Cellular Biology 19(9): 5969 5980. Rivera R and Murre C (2001) The regulation and function of the Id proteins in lymphocyte development. Oncogene 20(58): 83088316.

Further Reading
Atchley W and Fitch W (1997) A natural classication of the basic helixloophelix class of transcription factors. Proceedings of the National Academy of Sciences, USA 94: 51725176. Benezra R, Davis R, Lockshon D, Turner D, and Weintraub H (1990) The protein ID a negative regulator of helix loophelix DNA binding proteins. Cell 61: 4959. Chambers RC, Leoni P, Kaminski N, Laurent GJ, and Heller RA (2003) Global expression proling of broblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. American Journal of Pathology 162(2): 533546. Florio M, Hernandez MC, Yang H, et al. (1998) Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helixloophelix factors. Molecular and Cellular Biology 18(9): 54355444. Heemskert MH, Blom B, Nolan G, et al. (1997) Inhibition of T cell and promotion natural killer cell development by the

INSULIN-LIKE GROWTH FACTORS

B W Winston, A Ni, and R C Arora, University of Calgary, Calgary, AB, Canada
& 2006 Elsevier Ltd. All rights reserved.

Introduction
Insulin-like growth factors (IGFs) (formerly known as somatomedans) are serum peptides with insulinlike activities. The two principal ligands are IGF-I and IGF-II. Unlike other peptide hormones, which are by and large stored within the intracellular space, IGFs circulate throughout the body at concentrations approximately 1000 times higher than those of most peptide hormones. Tissue levels of IGFs, in general, are at lower levels than those present in serum and the IGF levels are still in excess of the level required for maximal cellular stimulation. IGF-I, produced primarily by hepatocytes, is the predominant form of circulating IGF. The existence of these hormones was rst proposed by Salmon and Daughaday in 1957, due to the observation that impaired cartilage growth in growth hormone (GH)-decient rats could be rapidly repaired by the administration of GH in vivo, but the cartilage growth activity in culture media could not be restored by its addition in vitro. It was therefore hypothesized that there were other factors in the serum that mediated GH activity and acted directly

Abstract
Insulin-like growth factors, IGF-I and IGF-II, are single-chain polypeptides with significant homology to proinsulin. Mature IGF-I peptide is derived from two classes of prepro-IGF-I peptides after posttranslational processing. The majority of circulating IGF-I molecules bind to IGF binding protein-3 and an acid-labile subunit to form a ternary complex. The majority of biological functions of IGFs are mediated by the IGF-I receptor, a tyrosine kinase membrane receptor. Both IGF-I and IGF-II play important roles in development and growth by promoting mitogenesis, cell differentiation, and prevention of cellular apoptosis. IGF-I is the most active peptide at the postnatal stage, while IGF-II is principally expressed and exerts the majority of its actions prenatally. Owing to their strong mitogenic and anti apoptotic effects, IGFs, when present at excessive levels, have the potential to contribute to carcinogenesis. An increasing body of evidence suggests that IGFs are involved in a number of respiratory diseases. The elevated circulating IGF-I level, for example, has been positively related to the risk of lung cancer. Local IGF-I level is elevated in the lungs of patients with idiopathic pulmonary brosis, broproliferative acute respiratory distress syndrome, sarcoidosis, and other brotic lung disorders.

340 INSULIN-LIKE GROWTH FACTORS

on target tissues. They collectively called those unknown factors somatomedins. In 1978, Rinderknecht and Humbel isolated two peptides from Cohn plasmaprotein fractions. Their amino acid sequences shared about 70% identity. Since their tertiary structures are highly homologous to each other and to proinsulin, and their biological activities similar to those of insulin, they were named insulinlike growth factor-I and -II. It was later revealed that IGF-I and -II were identical to somatomedin C and somatomedin A, respectively. Both IGFs are crucial for the development and growth of bone, muscle, and other organs in mammals. They also exert insulin-like effects such as decreasing blood sugar levels and stimulating metabolism. A growing body of evidence indicates that IGF-I and IGF-II are also involved in the development of lung cancer and a number of other respiratory diseases such as IPF, sarcoidosis, and broproliferative acute respiratory distress syndrome (ARDS).

IGF Gene and Protein Structure

Gene Structure

with resultant multiple mRNA and peptide species. Only one of exon 1 or 2 can be used for each transcript. The exon 1- and exon 2-derived mRNAs are collectively denoted as class I and class II mRNA, respectively. Class I mRNA is in virtually every tissue except liver. On the other hand, class II mRNA is relatively high in liver. In humans, due to alternative splicing, there are a further two subtypes within class I and II transcripts. Therefore, there are in total four pre-pro-IGF-I peptides, all of which are processed posttranslationally, giving rise to a common mature (70 amino acids, 7.6 kDa) IGF-I peptide. Only the mature form of IGF-I exerts biological activity. The IGF-II gene consists of 10 exons with the mature peptide encoded principally by exons 79. The mature peptide is most commonly a 67 amino acid single-chain residue, although there are several human variants that are 6770 amino acids in length. The IGF-II gene also has complex gene regulation. Alternative splicing, multiple polyadenylation sites, and site-specic endonucleolytic cleavage of IGF-II RNAs can produce proteins of different molecular weights, which increase the complexity of gene transcription (Figure 2).
Protein Structure

The human IGF-I gene is a single copy, six-exon gene spanning approximately 70 kb on chromosome 12 (Figure 1). Both exon 1 and 2 contain promoter regions with multiple transcription start sites. The mechanism of IGF-I gene regulation is complex (Figure 1),
Class 1 5 Gene 1 2 3 Class 2

Both human mature IGF-I and IGF-II proteins are single-chain nonglycosylated peptides that are highly

3 4 5 Alternative transcription initiation and splicing 6

Class 1 or 2 mRNA 1 or 2

1 or 2

Pre-pro-IGF-IA peptide

E-region

Pre-pro-IGF-IB peptide

E-region

Signaling sequence

Signaling sequence Posttranslational processing

Mature IGF-I ( 70 amino acids, 7.6 kDa )

Figure 1 IGF-I gene, mRNA structure, and posttranslational processing. Multiple transcription initiation sites exist in both exon 1 and 2. Transcription initiated from any site in exon 1 gives rise to class 1 mRNA, while transcription initiated from any site in exon 2 gives rise to class 2 mRNA. The usage of exon 1 and exon 2 is mutually exclusive. Within each class, two species of mRNA exist due to alternative splicing. IGF-IA mRNA lacks exon 5 and IGF-IB mRNA lacks exon 6. The four species of IGF-I mRNA resulting from the combination of alternative transcription initiation and splicing produce four pre-pro-IGF-I peptides with different N-terminal signaling sequences and Cterminal E-regions. During posttranslational processing, both signaling sequence and E-region are cleaved by proteases, resulting in a common mature IGF-I peptide.

INSULIN-LIKE GROWTH FACTORS 341

Gene P1 1 2 3 P1 mRNAs 1 P2 4 Alternative transcription initiation and splicing P2 4 P3 6 P4 7 Posttranslational processing Pre-pro-IGF-II peptide Signaling sequence E-region Mature IGF-II (6770 amino acids) 8 9 10 8 9 10 5 8 9 10 8 9 10 2 3 8 9 10

P2 4 5

P3 6

P4 7 8 9 10

Figure 2 IGF-II gene, mRNA structure, and posttranslational processing. The IGF-II gene consists of 10 exons with alternate transcription initiated by four known promoters. The mature peptide is encoded principally by exons 79. Posttranslational processing results in a mature IGF-II peptide of varying size based on the promoter region used to initiate the transcription process.

conserved among both mammalian and nonmammalian species. They both consist of four contiguous domains (Figure 3): an N-terminal B-domain, a central C- and A-domain, and a C-terminal D-domain. This structure is similar to that of proinsulin, which consists of B-, C-, and A-domains. There is substantial amino acid sequence homology between IGFs and proinsulin in their B- and A-domains, but their C-domains contain little sequence identity. Unlike proinsulin, the C-domain in IGFs is not cleaved off during posttranslational processing.

Regulation of Production and Activity

Regulation of Production

Although liver is the primary organ producing circulating IGFs, most organs can also produce IGFs locally. The regulation of IGF-I and IGF-II production are quite different. IGF-I The production of IGF-I is regulated by GH, developmental stage, and nutrition status.

GH stimulates IGF-I production at a gene transcriptional level and increases the abundance of all species of IGF-I mRNA. The robust correlation between serum IGF-I level and GH level has led to the use of serum IGF-I measurement as a diagnostic method for detecting GH deciency. Although the main stimulatory effect of GH is on circulating IGFI, GH can also stimulate local IGF-I expression, such as in ovary and kidney. The regulatory effect of GH on IGF-I is seen only during postnatal life. Both circulating and local IGF-I levels in the fetus are unresponsive to GH. IGF-I levels vary widely during different developmental stages. In humans, prenatal IGF-I is expressed at relatively low level in multiple organs. After birth, serum IGF-I levels rise progressively and reach the level of normal adults before puberty. During puberty, there is a two- to threefold increase in serum IGF-I level, which correlates well with sex hormone levels. After puberty, serum IGF-I levels gradually fall to normal adult levels that are reached in the early thirties, and remains constant until approximately the age of 60, after which serum IGF-I levels begin to decline.

342 INSULIN-LIKE GROWTH FACTORS

C-terminal N-terminal D-domain S S S S

S S A-domain

B-domain

C-domain
Figure 3 Protein structure of IGFs. The protein structures of IGF-I and IGF-II are very similar. They both consist of four domains (B-, C-, A-, D-domains from N-terminal to C-terminal). The amino acid sequences of B- and A-domains are highly homologous with proinsulin, while C-domain contains little homology with proinsulin. D-domain is unique to IGFs and not found in proinsulin. There are two disulde bonds linking B- and A-domains, and a third disulde bond within A domain.

The nutrition status is another important factor that inuences IGF-I production. Malnutrition decreases serum IGF-I levels by reducing hepatic IGF-I production. The fact that GH administration to protein-deprived rats restores hepatic IGF-I mRNA to normal levels without changing hepatic and serum IGF-I protein levels, suggests that at least part of the regulation acts at a posttranscriptional stage. IGF-II In humans, IGF-II mRNA and protein levels are highest in fetal tissue, particularly in the liver. After birth, IGF-II levels decrease but do persist in neuronal and liver tissue. There are four known promoters (P1P4) of gene transcription (Figure 2). During fetal development, promoters P2P4 direct most of IGF-II transcription. P3, which produces a 6.0 kb mRNA transcript, is the most active. After birth, P1 functions as an adult-stage promoter that maintains IGF-II production in the liver. The most active form is a 7.5 kDa peptide. Promoters P2, P3, and P4 continue to initiate IGF-II gene expression in other tissues in the adult, but at much lower levels than P1-initiated expression in the liver.
Regulation of Activity

complexes, which stabilize mature IGFs and extend their half-life from about 10 min to several hours. The other ve IGFBPs can also bind to IGFs with high afnity to form other complexes. It has been shown that the insulin-like activities of IGFs, such as the hypoglycemic effect, are blocked by the formation of ternary complexes. Also, because this large complex is unable to get through the capillary barrier, it prevents bound IGFs from leaving the circulation, and therefore restricts the availability and activity of IGFs in tissues and organs. In vitro studies show that all six IGFBPs can inhibit IGF activities, primarily by attenuating the binding of IGFs to their receptors. IGFBP3 is the most efcient inhibitor of IGF activities, and is capable of reducing almost every effect of IGFs on cells. However, evidence suggests that IGFBPs also exhibit a stimulatory effect on IGF functions in some situations. The IGF-I-IGFBP3 binary complex has been shown to be able to enhance the effects of IGF-I in wound healing and bone growth in the rat and pig. Preincubation of bovine broblasts with IGFBP3 enhances the response of broblasts to subsequent IGF-I exposure. Similar enhancement has been seen in dephosphorylated-IGFBP1-treated broblasts. In the in vivo environment, multiple IGFBPs can interact with IGFs at the same time. Thus, the nal regulatory effect of IGFBPs on IGF activities results from the combined inhibitory and stimulating effects. Furthermore, the interactions between IGFs and IGFBPs are mutual, resulting in reciprocal regulation of both peptides. Taken together, IGFs and IGFBPs form an extremely complex regulatory network, much of which is still not elucidated.

Biological Function
IGF-I

The in vivo activity of IGFs is regulated by a family of proteins named IGF-binding proteins (IGFBP). Six IGF-binding proteins (denoted IGFBP16) have been identied to date, which act as a sophisticated regulatory system for IGF activity. About 80% of circulating mature IGF-I and -II bind to IGFBP3 and an acid-labile subunit (ALS) to form 150 kDa ternary

IGF-I is a pleiotropic peptide with a wide variety of physiological and cellular functions, one of which is controlling glucose metabolism. Free IGF-I peptide infusion can efciently decrease serum glucose level by stimulating peripheral glucose uptake as well as decreasing hepatic glucose production, resulting in hypoglycemia. In addition, unlike insulin, very high concentrations of IGF-I can also reduce circulating free fatty acid levels. A more important physiological function of IGF-I is mediating many of the effects of GH. GH controls IGF-I levels and its subsequent ability to enhance somatic growth. Studies on double transgenic mice with GH deciency and IGF-I overexpression show that IGF-I expression can completely correct the effects of GH deciency on body weight. Human GH insensitivity syndrome is a disease caused by GH

INSULIN-LIKE GROWTH FACTORS 343

receptor dysfunction. Whereas increasing serum GH level does not alter the clinical symptoms, administration of IGF-I to these patients restores the effects of GH resulting in an increase in nitrogen and phosphate retention and height growth velocity. In addition to its critical role in somatic growth, IGF-I also participates in the development and growth of multiple organs and tissues. IGF-I-decient mice suffer impaired fetal growth and development, with high perinatal death rates due to muscle hypoplasia and lung immaturity. IGF-I exerts these functions by stimulating cell proliferation, promoting cells to progress through G1 phase and continue through the cell cycle. A wide variety of cells demonstrate mitogenic and differentiation response to IGF-I. Complementary to its mitogenic activity, IGF-I is also capable of inhibiting apoptosis of certain cells. This antiapoptotic action has been well characterized in hematopoietic cells such as pre-B cells and bone marrow-derived mast cells. IGF-I can also promote differentiation of various cells. IGF-I increases the rate and extent of terminal differentiation of myoblasts in culture. IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment induces differentiation of pro-B cells into pre-B cells. IGF-I also promotes vitamin D3-induced promyeloid cell differentiation into a macrophage cell type. The strong mitogenic and antiapoptotic activities of IGF-I have the potential to contribute to carcinogenesis. A large amount of epidemiological data has provided evidence for a significant positive relationship between circulating IGF-I level and risk of several common cancers, such as breast, prostate, colon, and lung cancers. This relationship, however, is only an association and does not prove causation. Fifty per cent of IGF-I transgenic mice that specifically express IGF-I in the basal prostatic epithelium eventually develop small cell carcinomas or prostatic adenocarcinomas. In vitro studies have shown that IGF-I can stimulate growth of a variety of primary cancer cells and cancer cell lines.
IGF-II

substantially lower in the postnatal mouse as compared to the human. There has not been a syndrome of IGF-II deciency as such described in humans. IGF-II is the principal IGF peptide expressed in skeletal tissue. It contributes to the proliferation of myocytes during fetal development, and once a sufcient density of cells has been formed, an autocrine source of IGFs is produced to maintain survival and the differentiated state of the myocytes. In vitro, IGF-II has been shown to stimulate type I collagen synthesis and osteoblast proliferation; in vivo, it has been shown to increase bone formation. IGF-II, like IGF-I, is mitogenic and antiapoptotic for a number of cell lines. IGF-II has been shown to be upregulated during normal wound healing, specifically, during the nal part of the proliferative phase in granulation tissue formation. Similar to IGF-I, overexpression of the IGF-II gene has been frequently observed in both human and animal tumors and has been implicated in tumorigenesis. Abnormal expression of fetal IGF-II has been implicated in pathologic tissue development. For example, a switch in the regulation of IGF-II promoter usage has been shown to be involved in hepato cellular carcinoma. Similar observations have been made in a number of other tumorous tissues. There is also evidence that IGF-II may play a role in angiogenesis and it has been associated with the development of hemangiomas.

IGF Receptors
The biological activities of IGFs are mediated by two IGF cell-surface receptors, IGF-I receptor (IGF-IR) and IGF-II receptor (IGF-IIR), and by the insulin receptor (IR). The molecular structures of IGF-IR and IR are strikingly similar. They are both tetramers with two a-chains and two b-chains linked by disulde bonds (Figure 4). Both IGF-IR and IR belong to the classic type 2 group of the tyrosine kinase membrane receptor family. Both IGF-I and IGF-II peptides can bind to IGF-IR, and most of their physiological and cellular effects are mediated by IGF-IR. The binding of IGFs to IGF-IR induces a conformational change leading to autophosphorylation of three tyrosine residues in the cytoplasmic domain of the b-chain, which greatly enhances the tyrosine kinase activity of IGF-IR. Activated IGF-IR then phosphorylates a wide range of cytoplasmic adaptor proteins such as Src homology and collagen domain protein (SHC) and insulin receptor substrate (IRS), triggering downstream signaling pathways resulting in various altered cellular behaviors and functions such as proliferation, transformation, and inhibition of apoptosis (Figure 4).

IGF-II appears to be essential for normal embryonic development and, as such, IGF-II is thought to be a fetal growth factor. IGF-II is highly expressed in embryonic and neonatal tissues and promotes proliferation of many cell types primarily of fetal origin. In IGF-II knockout mice, fetal growth is significantly retarded. Postnatally, however, growth rates in IGF-II decient mice parallel the rates in normal mice, but attain only 60% of the normal size as catch-up growth does not occur in these animals. However, it should be noted that the levels of IGF-II are

344 INSULIN-LIKE GROWTH FACTORS

IGF-I IGF-II

IR

IGF-IR

IGF-IIR (M6PR)

Tyrosine kinase domain P IRS-1/2 SHC P

SOS/Grb-2

P110

P85

Ras-Raf-MAPK pathway

PI3 kinase pathway

Cell responses
Figure 4 Interaction between IGFs and IGF-IR, IGF-IIR, IR, and IGF-IR signaling pathway. Both IGF-I and IGF-II can interact with IGF-IR with high afnity and initiate intracellular signal transduction. IGF-I and IGF-II can also interact with IR in some circumstances, with much lower afnity. Only IGF-II can bind to IGF-IIR, which is identical to mannose-6-phosphate receptor. This binding does not induce any known intracellular signal transduction. The thickness of the arrows between IGFs and the receptors indicates the relative afnity of their interactions. The interaction between IGF-IR and its IGF ligands activates the b-chain tyrosine kinase domain by tyrosine residue phosphorylation. Activation of this tyrosine kinase domain phosphorylates adaptor proteins IRS-1/2 and SHC, both of which can interact with either son of sevenless (SOS) growth factor receptor-binding protein (Grb-2) complex or P85 and P110. SOS/Grb-2 complex then associates with Ras, resulting in membrane association and activation of Raf, followed by induction of mitogen-activated protein-kinase (MAPK) pathway. P85 and P110 activate phosphoinositide 3 kinase (PI3-K), which induces PI3 kinase pathway.

IGF-IIR is identical to the mannose-6-phosphate receptor. It is quite different from IGF-IR and IR (Figure 4). IGF-II, but not IGF-I, interacts with the IGF-IIR. IGF-IIR acts as a clearance receptor for IGFII, regulating the extracellular IGF-II levels, but has not been reported to mediate any IGF-II biological function. Several investigators have provided evidence that the IGF-IIR has a tumor suppression function. Genetic mutations of the gene allele transcripts for the receptor have been reported in a number of malignancies, such as hepatocarcinoma, melanoma, renal cell carcinoma, non-Hodgkins lymphoma, adrenocortical tumors, and aggressive breast cancer. Compared to the IGF-IR and the IGF-IIR, the IR exhibits much lower afnities to IGFs. The afnity of IGF-I-IR binding is about two orders of magnitude lower than that of IGF-IIGF-IR binding. Thus, only at pharmacological doses can IGF-I interact with the IR. IGF-II has been shown to bind to the IR at a low afnity during the early stages of development when IGF-IR expression is absent.

IGFs in Respiratory Diseases

The critical roles of IGFs in the prenatal and postnatal development of the respiratory system have been described above. Recent data has shown, however, that dysregulation of the IGF and IGFBP have been implicated in the pathogenesis of many respiratory diseases. As stated above, IGFs stimulate proliferation of a number of cell types, including broblasts. This interaction may have synergistic effects with other brogenic growth factors with the potential of inducing collagen synthesis. Local expression of IGF-I by alveolar macrophages has been well recognized since 1983, explaining why there is an alternative name for IGF-I: alveolar macrophage-derived growth factor (AMDGF). In a number of brotic lung disorders including idiopathic pulmonary brosis (IPF), broproliferative ARDS, and bleomycin-induced pulmonary brosis increased local IGF-I production by alveolar macrophages, epithelial cells, and interstitial

INSULIN-LIKE GROWTH FACTORS 345

macrophages has been suggested. In fact, an increased bronchoalveolar lavage (BAL) IGF-I level has been positively associated with disease severity. It has been found that the cytokine production in the lung shifts towards a T-helper-2 (Th2) prole in brotic lung disorders, and the Th2 cytokines interleukin (IL)-4 and IL-13 can stimulate the expression of IGFI by alveolar macrophages. IGF-IR expression in alveolar epithelial cells is also upregulated in response to enhanced levels of platelet-derived growth factor (PDGF) and basic broblast growth factor (bFGF) in patients with brotic lung disorders. These observations suggest a paracrine or autocrine effect of IGF-I may be operating in these disorders. While the involvement of IGF-I in brotic lung diseases is likely, its exact role in these diseases is largely unknown. It has been proposed that IGF-I can stimulate proliferation and differentiation of lung broblast, as well as extracellular matrix synthesis, and therefore may augment the severity of pulmonary broproliferation. Conversely, it has been suggested that apoptosis of alveolar epithelial cells, which is a key event to induce subsequent brogenesis, can be inhibited by IGF-I due to its strong antiapoptotic effect. Further studies are needed to elucidate the role of IGF-I in brotic lung diseases. IGFBPs have regulatory functions. IGFBPs exert both stimulatory and inhibitory effects on IGF-I-mediated actions and also exert IGF-independent effects themselves. Importantly, recent data has shown that both IGFBP-2 and -3 levels are increased in BAL uid taken from patients with idiopathic pulmonary brosis and in type II pneumocytes exposed to oxidant injury. It has also been demonstrated that IGFBP-3 may be induced by other inammatory mediators, such as transforming growth factor beta 1 (TGF-b1). In a recent experimental analysis of normal human adult lung tissue, both IGFBP-3 and -5 were shown to induce production and deposition of collagen and bronectin in primary adult lung broblasts. Thus, several IGFBPs may exert IGF-independent functions in the development of pulmonary brogenesis. Owing to its carcinogenic effects, IGF-I may also play a role in lung cancer. Population studies have shown a positive relationship between circulating IGF-I level and risk of lung cancer. Local expression of IGF-I has been found in various primary lung cancer cells in culture. Paradoxically, serum IGF-I levels are not raised in patients with advanced lung cancer. In some cases, IGF-I levels may even decrease due to malnutrition. Functional IGF-I-binding sites are also expressed in these primary cancer cells as well as lung cancer cell lines. In vitro studies on lung cancer cells possessing IGF-I-binding sites have shown that these cells exhibit enhanced mitogenesis,

invasive potential, and resistance to apoptosis in response to exogenous IGF-I. As the IGF-IR has been known to mediate the effects of IGF-I on tumor cells, anti-IGF-IR strategy is being evaluated as a potential therapy for cancer. A number of anti-IGFIR strategies, including dominant negative transgene, RNA interference, and antibodies have been shown to be very effective in preventing carcinogenesis as well as causing tumor regression in rodent models. While the IGF-IR has been deemed as a promising therapeutic target in cancers, the difculties in specic targeting and delivery need to be resolved before this strategy can be properly studied and used therapeutically.

Acknowledgment
BWW is supported by a scholarship from the Alberta Heritage Foundation for Medical Research.
See also: Acute Respiratory Distress Syndrome. Epidermal Growth Factors. Fibroblast Growth Factors. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Interleukins: IL-4; IL-13 Platelet-Derived Growth Factor. Systemic Disease: Sarcoidosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
Allen JT and Spiteri MA (2002) Growth factors in idiopathic pulmonary brosis: relative roles. Respiration Research 3(1): 113. Baserga R (1996) Controlling IGF-receptor function: a possible strategy for tumor therapy. Trends in Biotechnology 14(5): 150 152. Daughaday WH (2000) Growth hormone axis overview somatomedin hypothesis. Pediatric Nephrology 14(7): 537540. Daughaday WH and Rotwein P (1989) Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrine Reviews 10(1): 6891. Firth SM and Baxter RC (2002) Cellular actions of the insulinlike growth factor binding proteins. Endocrine Reviews 23(6): 824854. Jones JI and Clemmons DR (1995) Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews 16(1): 334. Khandwala HM, McCutcheon IE, Flyvbjerg A, et al. (2000) The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocrine Reviews 21(3): 215244. Pavelic K, Bukovic D, and Pavelic J (2002) The role of insulin-like growth factor 2 and its receptors in human tumors. Molecular Medicine 8(12): 771780. Phillips LS, Pao CI, and Villafuerte BC (1998) Molecular regulation of insulin-like growth factor-I and its principal binding protein, IGFBP-3. Progress in Nucleic Acid Research and Molecular Biology 60: 195265. Pilewski JM, Liv L, Henry AC, et al. (2005) Insulin-like growth factor binding proteins 3 and 5 are overexpressed in

346 INTERFERONS
idiopathic pulmonary brosis and contribute to extracellular matrix deposition. American Journal of Pathology 166: 399407. Rosenfeld RG and Roberts CT (1999) The IGF System: Molecular Biology, Physiology, and Clinical Applications. Totowa, NJ: Humana Press. Stewart CE and Rotwein P (1996) Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiological Reviews 76(4): 10051026. Van den Brande JL (1999) A personal view on the early history of the insulin-like growth factors. Hormone Research 51(supplement 3): 149175.

Integrins

see Adhesion, CellMatrix: Integrins.

INTERFERONS
J N Kline and K Kitagaki, University of Iowa, Iowa City, IA, USA
& 2006 Elsevier Ltd. All rights reserved.

identied as antiviral substances, they have subsequently been found to engender a variety of significant effects on tumor growth, immune function, and reproductive responses.

Abstract
Interferons (IFNs) are a family of cytokines that were rst identied almost half a century ago through their antiviral properties. IFNs not only have important antiviral effects but also have a role in antitumor and immunomodulatory responses. There are two major classes of IFNs: type I (IFN-a subtypes, IFN-b, etc.) and type II (IFN-g). Additional IFNs (IFN-like cytokines; IFN-l subtype) have recently been discovered, but they are not as well characterized. Type I and II IFNs use distinct but similar receptor systems. Research on these receptor systems has helped to provide a fundamental base to understand the function and signal pathways of other cytokines and their receptors. The biological effects of IFNs result primarily from the IFN-inducible proteins in responsive cells. Type I IFNs were the rst to be produced by recombinant DNA technology and used therapeutically for viral infections, cancers, and autoimmune diseases. IFN-g plays important roles in controlling diseases caused by intracellular bacteria, parasites, and fungi by induction of reactive oxidant species. It may also be important in modulating adaptive immune responses in the lung and participates in the pathogenesis of pulmonary diseases such as pulmonary brosis and asthma.

Structure
Type I IFNs are a closely related family of 165172 amino acid proteins that are produced by leukocytes (a subtypes), broblasts (b subtypes), lymphocytes (o subtypes), and ruminant embryos (t subtypes). Transcripts of type I IFNs lack introns. The IFN-a gene family resides on human chromosome 9. Excluding pseudogenes, 13 genes encode 12 IFN-a species and many allelic variants, which are single polypeptide chains of 165166 amino acids, with a molecular mass of approximately 20 kDa. There is only one form of IFN-b. It is encoded by a distinct gene located adjacent to the IFN-a locus in human chromosome 9 and contains a single polypeptide chain of 166 amino acids with a molecular mass of 20 kDa. The IFN-g gene is localized to human chromosome 12. It is 6 kb in size and contains 4 exons and 3 introns. Activation of the gene generates a 1.2 kb mRNA that encodes a 166 amino acid polypeptide. After proteolytic removal of the terminal 23 amino residues, a mature IFN-g consists of a 143 amino acid polypeptide with a predicted molecular mass of 17 kDa. IFN-g functions as a noncovalent homodimer.

Introduction
In 1957, Isaacs and Lindermann discovered a substance that was capable of protecting cells from viral infections. This was one of the rst cytokine bioresponses to be indentied, and Isaacs and Lindermann named the substance interferon (IFN). IFNs belong to the class II family of a-helical cytokines and constitute a multimember cytokine family (type I IFNs: IFN-a subtypes, -b, -e, -k, -o, -d, and -t; type II IFNs: IFN-g, IFN-like cytokines, IL-28A (IFN-l2), IL-28B (IFN-l3), IL-29 (IFN-l1), and limitin). Some of these members (IFN-d, IFN-t, and limitin) have not been isolated in humans. Although IFNs were initially

Regulation of Production and Activity

Production of IFNs is regulated by several transcription factors. One important class of these factors contains a DNA-binding motif and members are termed IFN regulatory factors (IRFs). There are at

INTERFERONS 347

least nine members: IRF-1 to IRF-9. IRF-4, -5, and -7 are primarily expressed in lymphoid cells, whereas others are ubiquitously expressed. Although a variety of cells can produce IFN-a/b in response to viral and bacterial stimuli, plasmacytoid dendritic cells are the most potent producers of type I IFNs. Recent studies demonstrate that the family of Toll-like receptors (TLRs) plays an important role in inducing IFN responses. Pathogen-associated molecular patterns (PAMPs) on a broad range of microbial products bind to TLRs; for example, dsRNA binds to TLR3, ssRNA to TLR7 (mouse) or TLR8 (human), lipopolysaccharide to TLR4, and prokaryotic unmethylated CpG-DNA to TLR9. These interactions lead to the activation of IRF-3, initiating transcription of type I IFNs. IFN-g is produced mainly by T cells (CD4 Th1 cells and CD8 cytotoxic lymphocytes) and natural killer cells in response to mitogenic and antigenic stimuli. Interleukin (IL)-12 and IL-18 induce IFN-g. Production of IFN-g is inhibited by transforming growth factor beta (TGF-b) and IL-10, which are important products of regulatory T cells and other regulatory cells.

JAK-STAT Pathway of IFN Signal Transduction

IFN-a/b signaling The monomer of IFN-a/b binds to the IFN-a receptor (IFNAR) complex, comprised of IFNAR1 and IFNAR2c. The receptor-associated JAKs, tyrosine kinase 2 (TYK2) and JAK1, are crossphosphorylated. STAT2 then binds to IFNAR2c and recruits STAT1. Both STAT1 and STAT2 are phosphorylated and detached, forming a heterodimer. The STAT1STAT2 heterodimer and IRF9 (p48) migrate to the nucleus, forming interferonstimulated gene factor 3 (ISGF3), and stimulate interferon-stimulated regulatory element (ISRE)-dependent transcription. IFN-l also utilizes STAT1 and STAT2, and stimulates ISRE-dependent transcription as well (see Figure 1). IFN-c signaling The dimeric IFN-g binds to two IFN-g receptor (IFNGR)-1 chains, after which two IFNGR2 are recruited into the ligandreceptor complex. This aggregation of receptor chains phosphorylates JAKs associated with IFNGR. Phosphorylation of JAK1 provides IFNGR1 with a docking site for STAT1. Once STAT1 is phosphorylated by JAK2, the STAT1 detaches from IFNGR1, forming a homodimer. The dimeric STAT1 migrates to the nucleus and stimulates gamma-activated sequence (GAS)-dependent transcription (see Figure 1).
Termination of IFN Signal Transduction

Biological Function
The biological responses to all IFNs are mediated by the numerous and pleiotropic proteins generated through induction of Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways.

Following the initiation of ISRE- and GAS-dependent transcription, mRNA for suppression of cytokine

IFN- /

IFN- IFNIFNGR1

IFNAR1

IFNAR2

IFNGR2

TYK2

JAK1 Phosphorylation

JAK2 JAK1 Phosphorylation

STAT2 STAT1 Nuclear translocation

Heterodimer IRF9

STAT1

Homodimer Nuclear translocation

Nuclear ISGF3 ISRE Gene transcription GAS Gene transcription

Figure 1 The IFN-stimulated JAK-STAT signaling pathways.

348 INTERFERONS

signaling 1 (SOCS-1) is synthesized over much the same time course as the mRNAs responsible for the IFN effects. SOCS-1 mRNA is translated into protein, which binds to JAK1, JAK2 and TYK2, and suppresses their tyrosine kinase activity, resulting in inhibition of JAK-STAT signal transduction. IFNinducible protein inhibitors of activated STAT1 (PIAS1) bind tyrosine-phosphorylated STAT1 and inhibit its transcription response.
Antiviral Effect of Type I IFN

20 ,50 -oligoadenylates (2-5A). 2-5A activates the 2-5A-dependent RNAase L (ribonuclease), resulting in extensive cleavage of mRNA. The Mx pathway IFN-inducible Mx protein interferes with viral replication, impairing the growth of inuenza and other negative-strand RNA viruses at the level of viral transcription and at other steps. It is believed to interfere with the trafcking or activity of viral polymerases.

Both type I and type II IFNs are essential aspects of the innate immune response, providing an early line of defense against viral infections. Type I IFNs induce antiviral and antiproliferative effects on target cells and also modulate immune responses by enhanced expression of major histocompatibility complex (MHC) molecules and cytokine receptors. Although IFNs are reported to inhibit a variety of steps of virus replication, including entry, transcription, translation, maturation, and release, the bestcharacterized IFN-induced antiviral pathways are the dsRNA-dependent protein kinase (PKR), the 2-5A system, and Mx protein pathways (see Figure 2). The PKR pathway IFN-inducible PKR is normally inactive but it autophosphorylates after binding to dsRNA in the presence of ATP. The phosphorylated (activated) PKR inactivates the initiation factor, eIF2, by phosphorylation of the alpha subunit (eIF2a), resulting in rapid inhibition of dsRNA translation. The 2-5A system IFN-inducible 2-5A synthetase is activated by dsRNA and then produces

Receptors
Although the exact composition of the active IFNAR is still controversial, the active IFNAR consists of at least two polypeptides, IFNAR1 and IFNAR2c. IFNAR1 is a 100 kDa ligand-binding subunit encoded by genes on human chromosome 21 and including a TYK2 binding site. IFNAR2c, a 125 kDa polypeptide encoded by a gene on human chromosome 21, also plays an important role in ligand binding and signal transduction, which is mediated by JAK1. There are three forms of the IFNAR2 chain. IFNAR2a is a soluble form of the extracellular domain of the IFNAR2 subunit. IFNAR2b is an alternatively spliced variant with a short cytoplasmic domain, which has dominant negative activity. The active IFNAR requires IFNAR2c. Active IFNGR consists of two subunits. IFNGR1 is a 90 kDa polypeptide encoded by a gene on human chromosome 6. It binds the ligand and JAK1, which plays an important role in signal transduction. IFNGR2 is a 62 kDa polypeptide encoded by a gene on human chromosome 21. Although it plays only a

The PKR pathway

The 2-5A system Induction by IFNs

The Mx pathway

PKR (inactive)

2-5A synthetase (inactive) Viral dsRNA

Mx protein

PKR (active)

2-5A synthetase (active) 2-5A

eIF-2 (active)

eIF-2 (inactive)

RNase L (inactive)

RNase L (active)

Inhibition of protein synthesis

Figure 2 Antiviral mechanisms of IFN action.

RNA cleavage

Transcription inhibition

INTERFERONS 349

minor role in ligand binding, it has a JAK2 binding site that is important for signaling. The IFN-l family utilizes IL-28R1 and IL-10R2 chains encoded by genes on human chromosome 1 and 21, respectively. Like IFNAR, they have JAK1 and TYK2 binding sites.

public health problem. Evaluation of the safety and efcacy of inhaled rIFN-g with antimycobacterial drugs is currently ongoing in a randomized, doubleblind, placebo-controlled phase II study.

Other Clinical Applications of Type I IFNs

IFN-a has been approved for the treatment of chronic hepatitis B and C as well as condyloma acuminata and labial and genital herpes. Recombinant human IFN-a2a and -a2b were rst approved for clinical use in the treatment of hairy cell leukemia and Kaposis sarcoma in many countries in the mid 1980s. Since then, they have been approved for the treatment of chronic myelogenous leukemia and metastatic malignant melanoma. Clinical trails continue for their use in other cancers such as renal cell cancer. IFN-b has been approved for the treatment of relapsing-remitting multiple sclerosis, although the mechanism of action still remains unclear.
See also: Allergy: Overview; Allergic Reactions. Asthma: Overview. CD14. Defense Systems. Dendritic Cells. Interstitial Lung Disease: Overview. Leukocytes: T cells. Toll-Like Receptors. Interleukins: IL-1 and IL-18; IL-12. Antiviral Agents.

IFN-c in Asthma and Other Respiratory Diseases

Airway hyperresponsiveness and reversible airow obstruction are characteristic features of asthma. Currently, asthma is recognized as chronic eosinophilic airway inammation that is mediated and modulated by CD4 T-helper (Th) cells. These T lymphocytes are divided into two populations, Th1 and Th2, based on their contrasting cytokine expression proles and distinct functional properties. Th2 cells secrete cytokines implicated in the pathogenesis of allergic disorders, particularly asthma. For example, IL-4 and IL-13 play a major role in promoting Th2 differentiation and IgE production. IL-5 is a critical factor for eosinophil maturation, chemotaxis, activation, and survival. On the other hand, Th1 cells produce IFN-g that promotes Th1 differentiation, while inhibiting Th2 cells and their cytokine production. Therefore, enhancement of Th1 cells and their cytokine production has been considered a potentially useful response to promote in the treatment of allergic disorders including asthma. This concept is supported by a large number of experimental studies. Hanin and colleagues reported in 1993 that subcutaneous injection of recombinant IFN-g (rIFN-g) results in clinical improvement along with a decrease in circulating eosinophil numbers in patients with atopic dermatitis compared with placebo-treated control patients in a double-blind trial. However, subcutaneously injected and nebulized rIFN-g was not significantly effective in patients with asthma, possibly due to the difculty in obtaining a sufciently high concentration locally in the airways. The persistent imbalance toward Th2 is also one of the major mechanisms responsible for the progression of pulmonary brosis. IFN-g regulates collagen deposition, probably through reduction of probrotic cytokines such as TGF-b. A recent clinical trail demonstrated that rIFN-g was effective in improving lung function in patients with idiopathic pulmonary brosis. A phase III clinical trail is ongoing in the US. Mycobacterial infections, especially multidrugresistant tuberculosis in the HIV-positive population, have recently been resurgent, becoming a serious

Further Reading
Antoniou KM, Ferdoutsis E, and Bouros D (2003) Interferons and their application in the diseases of the lung. Chest 123: 209216. Bach EA, Aguet M, and Schreiber RD (1997) The IFN gamma receptor: a paradigm for cytokine receptor signaling. Annual Review of Immunology 15: 563591. Barnes PJ (2004) New drugs for asthma. Nature Reviews: Drug Discovery 3: 831844. Boehm U, Klamp T, Groot M, and Howard JC (1997) Cellular responses to interferon-gamma. Annual Review of Immunology 15: 749795. Farrar MA and Schreiber RD (1993) The molecular cell biology of interferon-gamma and its receptor. Annual Review of Immunology 11: 571611. Hertzog PJ, ONeill LA, and Hamilton JA (2003) The interferon in TLR signalling: more than just antiviral. Trends in Immunology 24: 534539. Kalvakolanu DV (2003) Alternate interferon signaling pathways. Pharmacolgy and Therapeutics 100: 129. Katze MG, He Y, and Gale M Jr (2002) Viruses and interferon: a ght for supremacy. Nature Reviews: Immunology 2: 675687. Pestka S, Krause CD, Sarkar D, et al. (2004) Interleukin-10 and related cytokines and receptors. Annual Review of Immunology 22: 929979. Pestka S, Krause CD, and Walter MR (2004) Interferons, interferon-like cytokines, and their receptors. Immunological Review 202: 832.

350 INTERLEUKINS / IL-1 and IL-18

Ramana CV, Gil MP, Schreiber RD, and Stark GR (2002) Stat1-dependent and -independent pathways in IFN-gammadependent signaling. Trends in Immunology 23: 96101. Sen GC (2001) Viruses and interferons. Annual Review of Microbiology 55: 255281. Shtrichman R and Samuel CE (2001) The role of gamma interferon in antimicrobial immunity. Current Opinion in Microbiology 4: 251259. Stark GR, Kerr IM, Williams BR, Silverman RH, and Schreiber RD (1998) How cells respond to interferons. Annual Review of Biochemistry 67: 227264. Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Reviews: Immunology 3: 133146.

INTERLEUKINS
Contents

IL-1 and IL-18 IL-4 IL-5 IL-6 IL-7 IL-9 IL-10 IL-12 IL-13 IL-15 IL-16 IL-17 IL-23 and IL-27

IL-1 and IL-18

E Stylianou, University of Nottingham Medical School, Nottingham, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Although identied almost half a century apart, the cytokines interleukin-1 (IL-1) and interleukin-18 (IL-18) have marked structural and functional similarities. Research into these two molecules has significantly advanced knowledge of the molecular basis of innate immunity and inammatory disease. Both cytokines have similar intronexon arrangements and secondary structure, and are synthesized as precursor proteins that require cleavage by caspase 1. IL-1 and IL-18 bind different receptors but their receptor complexes and signaling pathways share similarities. This is reected in their distinct and overlapping functions in regulating inammatory and immune responses. Both cytokines have attracted huge interest as potential therapeutic targets but further research is required to determine whether blocking their effects would be efcacious in respiratory disease.

Introduction
Interleukin-1 (IL-1) was rst described in the 1940s as a heat-labile protein called endogenous pyrogen

that produced fever. Since then it has been puried as lymphocyte activating factor, thymocyte proliferation factor, and catabolin. The unifying term interleukin-1 was assigned to it in 1979. Considerable interest remains in this master cytokine as a key coordinator of immune and inammatory responses in almost every tissue. The term IL-1 refers to two distinct molecules IL-1a and IL-1b. The expression of these is regulated independently, but they bind to the same receptor and exert virtually identical biological effects. IL-1 markedly resembles its naturally occurring competitive inhibitor, the IL-1 receptor antagonist (IL-lra), IL-18 and six other members of the IL-1 family called IL-F5-10. Interleukin-18 (IL-18) is a 24 kDa nonglycosylated polypeptide that was rst reported as interferon gamma-inducing factor (IGIF) in 1995. It was found circulating during endotoxemia in Propionibacterium acnes-infected mice and induced interferon gamma (IFN-g) production by T cells and natural killer cells. In 1999 it was renamed interleukin-18. It has structural and functional similarities to IL-1 but uses a distinct receptor.

INTERLEUKINS / IL-1 and IL-18 351

Structure
The genomic structure and intronexon arrangement of the IL-1b, IL-1a, and IL-1ra loci suggests that they are derived from duplication of a common ancestral gene 350 Ma. All three of these genes map to human chromosome 2q. IL-18 and other IL-1 gene family members may also be descended from the same ancestral gene since they share a similar gene structure (Figure 1). Of the members of the IL-1 family, only IL-18 lies on a separate chromosome, 11q22.222.3. IL-18 and IL-1a/b consist of b sheets that are packed against each other to form a distinctive structure called a b-trefoil fold. IL-1b and IL-18 exhibit 28.6% amino acid similarity and 17% sequence identity. IL-1b and IL-1a are 23% identical.

Regulation of Production and Activity

Monocytes/macrophages are the best-studied sources of IL-18 and IL-1b and both can be synthesized by cells of hemopoietic and nonhemopoietic lineages. For example, IL-18 is synthesized by keratinocytes, osteoblasts, and adrenal cortex cells and IL-1a/b by T cells, endothelial cells, epithelial cells, and broblasts, amongst others. Human IL-1a and IL-1b are synthesized as 31 33 kDa, glycosylated precursor proteins. The structures of the 50 anking regions of the IL-18, IL-1a, IL-1b, and IL-1ra genes differ. The IL-1b and IL-1ra (but not the IL-1a) promoters have a typical TATA box but all three promoters contain regulatory elements for the nuclear factor kappa B (NF-kB) transcription factor family. IL1b and IL-1ra have binding sites for the transcription factors AP-1 and CREB. The promoters of IL-1a and IL-1b contain sites for the NF-IL6 family of transcription factors. Unusually, IL18 has two promoter regions but neither has a TATA box. The promoter upstream of exon 2 acts constitutively and the one upstream of exon 1 is inducible.

Both promoters have an NF-kB site which is important in the inducibility of IL-18 gene expression. IL-1b in human monocytes is synthesized extremely rapidly (within 15 min) and can induce its own synthesis, as can other cytokines, for example, tumor necrosis factor (TNF) that can enhance the stability of IL-1b mRNA. The 30 untranslated region contains elements (AUUUA and AU) that render the IL-1b mRNA relatively unstable whereas IL-18 mRNA is unusually stable due to the lack of similar motifs. IL-18 can be synthesized in response to stress-inducing stimuli (neurogenic and bacterial) but is constitutively expressed in human monocytes. Macrophages and keratinocytes store pro-IL-18 at high levels under normal conditions in contrast to little pro-IL-1b. IL-1a/b and IL-18 are enzymatically processed to generate active, mature cytokines. Only a small amount of precursor IL-1b (pro-IL-1b) is secreted. Most of pro-IL-1b is cytosolic; and some of it is retained in lysosomes associated with the microtubules. Most IL-18 also remains cytosolic until it is enzymatically cleaved and transported out of the cell. Neither IL-1b nor IL-18 contains a signal peptide and their precursors are cleaved by the interleukin-1b converting enzyme (ICE), now known as caspase 1. Other caspases can also cleave pro-IL-1b and pro-IL-18. In contrast to pro-IL-1b, pro-IL1a is fully active and it remains in the cytosol. It can be cleaved by calpain and is found in the membranes of monocytes and B cells. Unlike IL-1a/b or IL-18, pro-IL-1ra has a leader sequence, and is synthesized, processed, and secreted.

Receptors
The IL-1b and IL-18 receptors are members of the IL1 receptor (IL-1R) family. The type I IL-1 receptor (80 kDa) and the type II receptor (6068 kDa) are transmembrane glycoproteins. IL-1 receptors are found on all cells except red blood cells. IL-1a and

8844 210 Human IL-18 8 79

1371 12

3385 135

1335 134

4785 222 343

463 72 15 47

565 52

1990 202

547 165

1239 131

718 213 601

Human IL-1
Figure 1 Structure of the human IL-18 and IL-1b genes. Exons are represented by white boxes, 50 UTR (untranslated region) and 30 UTR are shown as shaded boxes and the size of these is given within the boxes. Single lines connecting boxes are introns and length of these is given in italics. Adapted from Huising et al. (2004) Developmental & Comparative Immunology 28: 395413, with permission.

352 INTERLEUKINS / IL-1 and IL-18

IL-1b each bind at subnanomolar concentrations to the type I IL-1 receptor. The latter has an extracellular, ligand-binding region that comprises three immunoglobulin-like domains. Its cytoplasmic domain is 213 amino acids in length and is homologous to the same regions in the other members of the IL-1R and Toll families. Binding of IL-1 leads to recruitment of the IL-1 accessory protein (IL-1RAP) which initiates signal transduction to activate target cells. The crystal structure of each of IL-1b and IL-1ra bound to the IL-1 receptor has been solved. IL-1ra can antagonize the IL-1 signaling pathway as it binds with slightly higher afnity to the type I receptor. It occupies the ligand-binding site, prevents IL-1a/b binding, and so does not recruit IL-1RAP. Binding of the type II receptor by IL-1b prevents it binding the type I receptor. Hence the type II receptor has been termed a decoy receptor. The type II receptor has a short cytoplasmic tail 29 amino acids in length which cannot transmit an intracellular signal. By internalizing it, it regulates IL-b levels for which it has a higher afnity than for IL-1a or IL-1ra. It can be cleaved to generate a soluble receptor which can also bind IL-1. The IL-18R complex is remarkably similar to the IL-IR complex. The IL-18R consists of two distinct but structurally related immunoglobulin-like domains that are members of the IL-1 receptor family: IL-18Ra and IL-18Rb. Secreted, mature IL-18 interacts with IL-18Ra. This complex heterodimerizes with the signal-transducing IL-18Rb accessory protein that facilitates a conformational change in the receptor to allow high-afnity binding of ligand. As with the IL-1R complex, both chains are required for signal transduction. Although the IL-18R does not

have a natural antagonist or soluble form, there is an IL-18 binding protein (IL-18bp) that binds IL-18 with similar afnity to the IL-18R complex. This modulates the activity of IL-18 during inammation. In addition to the two subunits in each of the IL-1R and IL-18R there are six other IL-1 receptor homologs and members of the IL-1R family which includes IL-1R type II. These are presumed capable of mediating biological responses but limited information is available regarding their ligands and function.

Biological Function
IL-1 exerts its effects via signaling events many of which are similar to those of IL-18. Both appear to use the adapter molecules myeloid differentiation primary response gene 88 (MyD88), interleukin 1 receptor-associated kinase (IRAK), and TNF receptor-associated factor 6 (TRAF 6), though it is unclear how many of the downstream responses are common to both cytokines. There is evidence for IL-1 and IL18 activation of JNK and p38 mitogen-activated protein kinase (MAPK) but there are conicting data as to whether IL-18 is a major activator of NF-kB. A principal function of IL-1 is to cause leukocyte accumulation by inducing adhesion receptors on vascular endothelium and to stimulate the production of a variety of chemokines, for example, IL-8 and monocyte chemoattractant protein-1 (Figure 2). IL-1 is a potent stimulator of hematopoiesis and the adaptive immune system and prepares the organism to deal with infection or injury. IL-1 is also required for the efcient synthesis of IFN-g, a key activator of macrophages. It also causes the production of other

Prostanoids, nitric oxide

Cytokine and chemokine synthesis

Adhesion molecule expression

Allergic reactions

Extracellular matrix turnover

IL-1

+IL-12

+/ IL-12 IFNproduction

IL-18

Neuroimmune responses, e.g., fever Cartilage and bone formation and remodeling

B and T lymphocyte activation

Acute phase protein synthesis

FasL expression and Fas-mediated apoptosis

Figure 2 Biological functions of IL-1b and IL-18. The main functions of each cytokine are shown to demonstrate that these have distinct as well as overlapping effects. Fas, an apoptosis-inducing receptor protein; FasL, ligand of the Fas receptor.

INTERLEUKINS / IL-1 and IL-18 353

cytokines, for example, TNF-a, IL-1 itself, along with prostanoids and nitric oxide. Another major role is to stimulate hepatic acute phase protein synthesis. IL-1 can also act as an accessory signal for lymphocyte activation. It can cause cartilage and bone resorption, bone formation, and insulin secretion, and regulates extracellular matrix turnover. There are diverse effects of this cytokine in the brain where IL-1 is the major endogenous pyrogen. A role for IL-1 in mediating hypophagia, slow-wave sleep, and neuroendocrine changes has been documented as is a role in neurodegeneration and neuronal cell death. IL-18 has a widespread though more restricted role than IL-1 in innate and adaptive immunity (Figure 2). It appears to act as an early-warning signal to the immune system of the presence of potentially dangerous pathogens. It acts synergistically with IL-12 to induce IFN-g production. IL-18 upregulates the cytolytic activity of lymphocytes and natural killer cells and promotes the development and differentiation of Th1 and Th2 cells. Like IL-1b, IL-18 is a key mediator in the pathogenesis of a variety of chronic inammatory disorders and can also induce the production of chemokines, for example, IL-8 and cytokines, and IL-1 and TNF-a. It can activate endothelial cells, upregulate adhesion molecule expression, and stimulate neutrophils, T cells, and dendritic cells, all pivotal events in immune and inammatory responses.

IL-1 and IL-18 in Respiratory Diseases

IL-1b is one of the major proinammatory cytokines along with IL-1ra that has been identied in bronchial alveolar lavage uids from patients with acute respiratory distress syndrome (ARDS). In patients the levels of IL-1b relative to its antagonist are elevated 10-fold. Both soluble type II IL-1R and shed type IL1R that can bind antagonists have also been detected in these patients indicating that the balance of production is tilted in favour of proinammatory activity in these conditions. Also, IL-1b has an important role in the injury of resident epithelial cells and endothelial cells and in the pathogenesis of both acute lung injury and acute respiratory distress syndrome. It is part of the complex network of cytokines that mediate, amplify, and perpetuate lung injury. Biopsy studies have demonstrated that there is increased expression of IL-1b and other proinammatory and Th2 type cytokines in this disease. IL-1b is one of the cytokines that regulate the inammatory response of the lower airways that cause the remodeling and structural alterations of the airway in asthma. IL-18 exacerbates a number of severe inammatory diseases. Its aberrant expression with IL-12 is linked to Th1-related inammatory diseases, for

example, rheumatoid arthritis. Data from transgenic mice studies indicate that IL-18 can inuence Th2related responses in asthma and allergic rhinitis in which elevated levels of the cytokine have been correlated with disease severity. Keratinocyte-specic caspase 1 transgenic mice have a phenotype linked with Th2 type responses. Increased levels of IL-18 in these mice cause the appearance of IL-4 and IL-13 in the airways and skin in an immunoglobulin E (IgE)dependent manner. IL-18 in the presence of antigen transforms Th1 cells into cells that can produce cytokines and chemokines specic for Th1 and Th2 responses, for example, in bronchial asthma. In this way IL-18 directly induces allergic inammatory responses relevant to allergic rhinitis and asthma. Interestingly, common single nucleotide polymorphisms (SNPs) associated with allergic rhinitis have been identied in the human il-18 gene. One SNP in promoter is strongly associated with total IgE and specic sentisitization to common antigens, for example, pollen and dust mites, but the effect of this SNP on IL-18 gene expression is unknown. Anti-inammatory agents that target IL-1 and IL18 are required for therapeutic benet and are in clinical development. Anti-IL-1 strategies have been efcacious in ameliorating a variety of inammatory diseases, for example, rheumatoid arthritis, in preclinical studies. Neutralization of IL-1b through employment of a recombinant form of the IL-1ra called anakinra has decreased disease activity. However, the efcacy of anti-IL-1 and anti-1L-18 strategies in respiratory diseases is unknown. In animal models, blockade of IL-1 (and TNF) has been successful in limiting lung injury associated with sepsis and respiratory distress syndrome. Recent evidence indicates that in patients with severe disease, anti-Th1 cytokines (e.g., TNF-a) may be highly effective. However, the complexity of the signaling events involved makes the selection of appropriate targets difcult and suggests that a combination of anticytokine therapies might be required. Further research must address the problem of non-specic side effects associated with such strategies and increase understanding of inammatory signaling pathways to provide new therapeutic targets for respiratory diseases.
See also: Acute Respiratory Distress Syndrome. Allergy: Allergic Rhinitis. Asthma: Overview. Bronchoalveolar Lavage. Matrix Metalloproteinases.

Further Reading
Dinarello CA (1997) Interleukin-1. Cytokine and Growth Factor Reviews 8: 253265. Dinarello CA (1999) Interleukin-18. Methods 19: 121132.

354 INTERLEUKINS / IL-4

Dinarello CA (2004) Therapeutic strategies to reduce IL-1 activity in treating local and systemic inammation. Current Opinion in Pharmacology 4: 378385. Fantuzzi G and Dinarello CA (1999) Interleukin-18 and Interleukin-1b: two cytokine substrates for ICE (caspase-1). Journal of Clinical Immunology 19: 111. Huising, et al. (2004) Developmental & Comparative Immunology 28: 395413. Kato Z, Jee JG, Shikano H, et al. (2003) The structure and binding mode of interleukin-18. Nature Structural Biology 10: 966971. Lee J-K, Kim S-H, Lewis EC, et al. (2004) Differences in signaling pathways by IL-1b and IL-18. Proceedings of the National Academy of Sciences USA 101: 88158820. Sims JE (2002) IL-1 and IL-18 receptors, and their extended family. Current Opinions in Immunology 14: 117122. Stylianou E and Saklatvala J (1998) Interleukin-1. International Journal of Biochemistry and Cell Biology 30: 10751079. Tone M, Thompson SAJ, Tone Y, et al. (1997) Regulation of IL-18 (IFN-g-inducing factor) gene expression. Journal of Immunology 159: 61566163. Towne JE, Garka KE, Renshaw BR, et al. (2004) Interleukin (IL)1F6, IL-1F8, and IL-1F9 signal through IL-1Rrp2 and IL-1RAcP to activate the pathway leading to NF-kB and MAPKs. Journal of Biological Chemistry 279: 1367713688. Tsutui H, Yoshimoto T, Hayashi N, et al. (2004) Induction of allergic inammation by interleukin-18 in experimental animal models. Immunological Reviews 202: 115138. Ushio S, Namba M, Okura T, et al. (1996) Cloning of the cDNA for human IFN-g-inducing factor, expression in the Escherichia coli, and studies on the biologic activities of the protein. Journal of Immunology 156: 42744279. Vigers GPA, Anderson LJ, Caffes P, and Brandhuber BJ (1997) Crystal structure of the type-1 interleukin-1 receptor complexed with interleukin-1b. Nature 386: 190194. Vigers GPA, Caffes P, Evans RJ, et al. (1994) X-ray structure of resolution. Journal of interleukin-1 receptor antagonist at 2.0-A Biological Chemistry 269: 1287412879. Yoshimoto T, Mizutani H, Tstsui H, et al. (2000) IL-18 induction of IgE: dependence on CD4 T cells, IL-1 and STAT 6. Nature Immunology 1: 132137. tissue repair. All these same actions, however, could also be detrimental to the host in some circumstances, by antagonizing interferon gamma (IFN-g)-driven TH1 responses or by synergizing with IL-13 to foster tissue brosis. IL-4 is a ligand for the IL4Ra chain, which can form two distinct signaling complexes, by pairing either with the gamma common chain shared with specic receptors for other four a-helix bundle family cytokines, or with IL-13Ra1, which also binds IL-13. Polymorphisms of IL-4 or of the IL-4Ra chain have been associated with increased severity of asthma.

Introduction
As the central immunoregulatory cytokine responsible for initiating type 2 immune responses, interleukin 4 (IL-4) is of great importance to atopy, allergic responses including asthma, and pulmonary brosis. IL-4 is a glycoprotein produced by a limited spectrum of hematopoietic cell types, including CD4 TH2 lymphocytes, mast cells, and basophils. IL-4 binds to specic cell-surface receptor complexes expressed on a wide variety of immune and parenchymal cell types. The pleiotropic actions of IL-4 include crucial roles in IgE production and polarization of T cells to the TH2 cytokine phenotype, as well as driving production of mucus and extracellular matrix proteins, while counteracting inammation driven by type 1 responses. IL-4 was rst identied in 1982 based on its ability to induce activated murine B cells to proliferate and secrete IgG1. This bioassay explains its original historical name, B-cell stimulatory factor-1 (BSF-1). The cDNA encoding murine IL-4 was cloned virtually simultaneously by groups in the US and Japan in 1996. Human IL-4 and the IL-4Ra chain were both cloned soon thereafter.

Structure

IL-4
J L Curtis, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

IL-4 Protein

Abstract
Interleukin 4 (IL-4) is the prototypic immunoregulatory cytokine responsible for initiating allergic responses. IL-4 is a small (1519 kDa) single-chain glycoprotein that is a member of the four a-helix bundle family of cytokines. It is produced chiey by activated TH2 lymphocytes, mast cells, and basophils. IL-4 is uniquely required to polarize uncommitted T cells to a type 2 phenotype, characterized by secretion of IL-4 itself, plus IL-5, IL-9, IL-10, and IL-13. IL-4 also induces B cells to switch their immunoglobulin production to IgE secretion. Through a broad range of actions on many other hematopoietic and parenchymal cell types, IL-4 drives immune responses towards antibody production, reduced proinammatory cytokine elaboration, and

The cDNA for human IL-4 encodes a single open reading frame of 153 amino acids which is processed to a mature protein of 129 amino acids. Full-length human IL-4 contains three disulde bridges and two predicted N-linked glycosylation sites. Consistent with this prediction, expression of IL-4 in mammalian cells produces proteins of 15, 18, and 19 kDa, corresponding to un-, mono-, and di-glycosylated isoforms. There is no cross-reactivity between murine IL-4 and human IL-4. The three-dimensional structure of IL-4 has been solved using both nuclear magnetic resonance (NMR) and X-ray crystallography. The expressed IL-4 protein has a compact globular conguration similar to that of IL-3, macrophage colony stimulating factor (M-CSF), and granulocyte-macrophage

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colony-stimulating factor (GM-CSF). Like those cytokines, IL-4s principal structural feature is a four a-helix bundle, consisting of helices arranged in a left-handed antiparallel bundle that are connected by peptide loops.
IL-4 Gene

surface receptors with classical natural killer (NK) cells; however, NKT cells display a restricted T-cell antigen repertoire and depend on the major histocompatibility complex (MHC) class I-like molecule CD1d for development and activation. Secretion of IL-4 has also been reported by g/d T cells, eosinophils, and alveolar macrophages.

The human IL-4 gene (Gene ID 3565) is located on the long arm of chromosome 5 (5q31.1), at the centromeric end of a cluster of cytokine genes that also contains the genes for IL-3, IL-5, IL-9, IL-13, and GM-CSF. The IL-4 gene is approximately 10 kb in size, and consists of four exons (encoding 45, 16, 59, and 33 amino acids, respectively) and three introns (Figure 1). A variant IL-4 mRNA transcript, called IL-4d2, occurs naturally and is found in greater abundance in bronchoalveolar lavage cells and thymocytes. The IL-4d2 transcript lacks 48 base pairs (bp) due to alternative splicing of exon 2. This change deletes one disulde bridge and part of a peptide loop. IL-4d2 has no T-cell proliferative capacity and in vitro antagonizes T-cell proliferation induced by full-size IL-4. A biological role for IL-4d2 is best established in the response to Mycobacterium tuberculosis, in which increased production of IL-4d2 is seen in healthy contacts (relative to unexposed control subjects) and following successful chemotherapy. Relatively increased production of IL-4d2 has also been postulated to reduce asthma susceptibility. What regulates the balance between full-length IL-4 and this apparent natural antagonist, an important subject for vaccine research, is unknown.

Biological Function
IL-4 acts on many cell types to polarize immune responses toward antibody production and away from cell-mediated cytotoxicity (Figure 2). The next eight sections outline direct effects of IL-4 on individual cell types, followed by a summary of the integrated effects of IL-4 on immune responses and of conclusions derived from transgenic mice that either overexpress or lack IL-4 or the IL-4Ra chain.
T-Cell Polarization

Regulation of Production and Activity

IL-4 is not secreted constitutively, but is induced on cellular activation via transcriptional regulation. Principal sources are CD4 TH2 lymphocytes, mast cells, and basophils. Early in immune responses, NKT cells are also a key source of IL-4. NKT cells are an innate cell type that shares expression of some
5 45 16 Human IL-4 59 33 3

Probably the most important action of IL-4 is to induce differentiation of uncommitted T cells to the type 2 phenotype. T cells of this phenotype (called TH2 cells if CD4 and TC2 cells if CD8 ) produces IL-4 itself, plus IL-5, IL-9, IL-10, and IL-13. This differentiative effect of IL-4 is mediated by induction of the fate-determining transcription factor GATA-3. IL-4 further promotes the survival and proliferation of TH2 cells, by acting as an autocrine growth factor. Additionally, IL-4 antagonizes development of the TH1 subset, by blocking interferon gamma (IFN-g) production that would lead to TH1 commitment, and by inhibiting IL-2-induced proliferation needed to sustain TH1 and TC1 cells. Unlike many other actions of IL-4, these inductive effects to TH2 polarization cannot be replaced by IL-13.
B-Cell Differentiation

IL-4 enhances the proliferation of activated B cells and potently induces them to class-switch their immunoglobulin production to IgE and IgG4 (IgE and IgG1 in mice). IL-4 also increases expression of several receptors on B cells, including surface IgM, class II MHC antigens, CD40, and CD23, the lowafnity receptor for IgE (FceRII). By downregulating FcgRII (CD32), IL-4 counteracts the inhibitory effect of immune complexes on B-cell activation. IL-4 also induces B cells to secrete IL-6 and tumor necrosis factor alpha (TNF-a).
Mononuclear Phagocytes

44 16 Murine IL-4

51

29

Figure 1 Diagram of IL-4 gene structure in humans and mice. For exons, translated sequences are indicated in white, and untranslated sequence in blue. Adapted from data in the National Center for Biotechnology Information.

IL-4 inhibits macrophage production of proinammatory cytokines, including TNF-a, IL-1, and IL-6, in part via transcriptional repression. IL-4 also enhances

356 INTERLEUKINS / IL-4

APC

Naive T cell IL-4 IL-4

B cell

M Eosinophil

NKT cell

TH2 cell IL-4

Chemotaxis survival

IL-4 (and other TH 2 cytokines) IL-4 IL-4 Endothelial cells Mast cell VCAM E-selectin Fibroblasts Epithelial cells

Basophil Chemotaxis survival

Chemotaxis, proliferation Mucus production ICAM-1, collagen, fibronectin expression

Figure 2 IL-4 exerts pleiotropic actions on hematopoietic and parenchymal cell types. The antigen-presenting cell (APC) delivers appropriate signals (including antigen in the context of MHC and costimulation), activating the naive T cell (yellow starburst). After activation, the naive T cell becomes a TH2 cell via exposure to IL-4. This exposure could come either from an NKT cell, an innate immune cell type that rapidly makes IL-4 on stimulation, or from the T cell itself, via a stochastic process following T-cell activation. Once polarized, the TH2 cell produces IL-4 (and the additional type 2 cytokines IL-13, IL-5, IL-6, and IL-9). Cell types are shown in black text and IL-4 effects in red text; M, macrophage. This diagram highlights the known actions of IL-4, but during in vivo immune responses many of these actions synergize with those of other type 2 cytokines, especially IL-13.

production of molecules that counteract these cytokines, such as IL-1R antagonist and the decoy receptor IL-1RII. In monocytes, IL-4 decreases expression of surface and soluble p75 TNF receptors. Additionally, IL-4 upregulates CD13, a cell-surface aminopeptidase which inactivates inammatory peptides. IL-4 upregulates macrophage expression of MHC class II antigens LFA-1 and CD23, while decreasing CD14 and all three FcgRs. IL-4 also induces formation of multinucleated giant cells, and suppresses macrophage apoptosis. Macrophage production of superoxide and prostaglandin E2 (PGE2), and induction of cyclooxygenase activity are all profoundly inhibited by IL-4. Furthermore, IL-4 switches eiscosanoid production in both macrophages and dendritic cells by downregulating 5-lipoxygenase and upregulating 15-lipoxygenase.
Granulocytes

Mast Cells

IL-4 induces mast cells to proliferate and increase surface expression of FceRI, the receptor that triggers release of granular products such as histamine and tryptase on binding of IgE. IL-4 also regulates expression by mast cells of leukotriene C4 synthase.
Epithelial Cells

IL-4 induces goblet cell metaplasia in murine models and mucin gene activation in mice and cultured human airway epithelial cells.
Vascular Endothelial Cells

Acting early in development of bone marrow progenitors, IL-4 favors granulopoiesis over production of monocytemacrophages. Synergizing with IL-3, IL-4 drives basophil production. IL-4 is a chemoattractant for eosinophils and neutrophils, and induces eosinophils to produce TH2 cytokines.

Acting together with TNF-a, IL-4 alters adhesion molecule expression on vascular endothelial cells, by inducing vascular cell adhesion molecule 1 (VCAM1) and suppressing E-selectin. IL-4 also counteracts the procoagulant effect of lipopolysaccharides (LPS), IL-6, and TNF-a.
Fibroblasts

IL-4 exerts several actions on broblasts that have the net effect of increasing brosis. IL-4 induces broblast chemotaxis and proliferation. It increases

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intercellular adhesion molecule 1 (ICAM-1) expression and synthesis of such extracellular matrix proteins as bronectin and types I and II collagen.
Integrated Effects of IL-4

capsulatum that is normally mediated by development of a type 1 immune response. However, IL-4 does not cause a universally defective host response, as these transgenic mice show accelerated clearance of Pseudomonas aeruginosa.

Collectively, IL-4-induced changes enhance the capacity of B cells to survive and present antigen to T cells. IL-4 reduces many of the inammatory effects of TH1 responses, by suppressing production of IFN-g, IL-1, and TNF-a, and by reducing the responsiveness of macrophages to those cytokines. Induction of VCAM and suppression of E-selectin on endothelial cells by IL-4 favors recruitment of Th2 cells, basophils, and eosinophils over neutrophils. Inhibiting IL-4 activity via neutralizing antibody against the cytokine or IL-4Ra chain, or by gene targeting either of these molecules, diminishes TH2 responses. This nding substantiates the central role of IL-4 in T-cell differentiation, but also does not exclude the possibility that the biological effects seen in these experimental models may derive from effects of other TH2 cytokines. In mice, such treatments ablate the protective effect of previous infection with two nematode parasites, and has a striking effect on murine models of allergic airways disease. Overexpression of IL-4 in the lungs of transgenic mice impairs the elimination of Histoplasma

Receptors
The reason for the overlapping effects of IL-4 and IL13 can best be appreciated by understanding the cell surface receptors that bind these two related cytokines (Figure 3). IL-4 binds to the IL-4Ra chain with high afnity (KdB20300 pM). The IL-4Ra chain can then dimerize with either of two chains: (1) the common gamma subunit (gc), which is shared by receptors for multiple members of the four a-helix bundle family of cytokines, to form the type I receptor complex; or (2) with low afnity to the IL-13Ra1 chain, to form the type II receptor complex. By contrast, IL13 rst binds with high afnity to the IL-13Ra1 chain, which then dimerizes with the IL-4Ra chain to form a complex identical to the type II receptor. IL-13 is unable to bind the IL-4Ra chain directly. Both type I and type II receptor complexes transduce signals; how the cell distinguishes between IL-4 and IL-13 bound to type II receptor complexes is currently unclear. Type I receptor complexes predominate on hematopoietic cells, while type II

Type I receptor complex IL-4

Type II receptor complex IL-13 OR

c Chain

IL-4R chain

IL-13R 1 chain

IL-13R 2 chain

Jak3

Jak1

Jak1

Tyk2

STAT3 STAT6

(a)

(b)

(c)

Figure 3 IL-4 and IL-13 receptors. The IL-4Ra chain can pair with either of two transmembrane receptors. (a) Pairing of the IL-4Ra chain with the gc chain in response to IL-4 forms the type I receptor complex. This complex is associated with Jak3 and Jak1 (members of the Janns tyrosine kinase family), and leads to activation of signal transducer and activator of transcription 6 (STAT6). (b) Pairing of the IL-4Ra chain with the IL-13Ra1 chain in response to IL-4 or IL-13 forms the type II receptor complex. This complex is associated with Jak3 and Tyk2, and leads to activation of STAT3. Thus, IL-4 can bind to either the type I receptor complex (a) or the type II receptor complex (b), and in either case, can activate receptor-associated kinases to induce STAT activation. By contrast, IL-13 cannot bind to the type I receptor complex, but does signal via the type II receptor complex. (c) IL-3 can also bind to the monomeric IL-13Ra2 chain, a decoy receptor.

358 INTERLEUKINS / IL-4

receptor complexes are expressed on both hematopoietic cells and nonhematopoietic cells, including airway epithelium. Expression of IL-4Ra chain on lymphocytes is upregulated by activation (e.g., by LPS or anti-IgM stimulation of B cells or by antigenstimulation of T cells). IL-4 itself also upregulates expression of the IL-4Ra chain, setting the stage for a positive feedback loop. In addition to the cDNA encoding the full-length IL-4Ra chain, cDNAs for two shorter forms have been isolated. One encodes a membrane-bound form lacking a cytoplasmic domain, predicted to be a decoy receptor. The other encodes a soluble form of the receptor, which has been found to be secreted and to block the ability of IL-4 to induce T-cell proliferation in vitro. Neither IL-4Ra nor gc has enzymatic activity. Instead, binding of IL-4 recruits and activates a pair of Janus family tyrosine kinases (Jak-1 to IL-4Ra, Jak-3 to gc, Tyk2 to IL-13Ra) (Figure 3). The Jak kinases rst transphorphorylate and activate each other. Next, they phosphorylate tyrosines in the cytoplasmic domain of IL-4Ra (or IL-13Ra), providing docking sites for several signaling molecules. Among these are the latent transcription factors STAT6 (which binds to phosphorylated IL-4Ra), and STAT3 (which binds to phosphorylated IL-13Ra1). Phosphorylation induces STAT6 monomers to dimerize, migrate to the nucleus, and bind to specic sites in the promoters of many genes to mediate most effects of IL-4.

Supporting this generalization, soluble IL-4 receptor that inhibited allergen-induced IgE production in preclinical models has been administered by inhalation in two small trials in asthmatic patients. The modest clinical efcacy seen in these short trials awaits conrmation in larger, longer studies.
See also: Adhesion, CellMatrix: Focal Contacts and Signaling. Allergy: Allergic Reactions. Apoptosis. Asthma: Extrinsic/Intrinsic. Chymase and Tryptase. Colony Stimulating Factors. Dendritic Cells. Dust Mite. Extracellular Matrix: Collagens. Fibroblasts. Histamine. Immunoglobulins. Interferons. Interleukins: IL-1 and IL-18; IL-5; IL-6; IL-9; IL-13. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Mucins. Mucus. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens); Parasitic; Viral. Pulmonary Fibrosis. Signal Transduction. Smooth Muscle Cells: Airway. Transcription Factors: Overview. Transgenic Models.

Further Reading
Barnes PJ (2002) Cytokine modulators as novel therapies for asthma. Annual Review of Pharmacology and Toxicology 42: 8198. Beghe B, Barton S, Rorke S, et al. (2003) Polymorphisms in the interleukin-4 and interleukin-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population. Clinical and Experimental Allergy 33: 11111117. Chatila TA (2004) Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends in Molecular Medicine 10: 493 499. Chiu BC, Freeman CM, Stolberg VR, et al. (2003) Cytokine chemokine networks in experimental mycobacterial and schistosomal pulmonary granuloma formation. American Journal of Respiratory Cell and Molecular Biology 29: 106116. Finkelman FD and Urban JF Jr (2001) The other side of the coin: the protective role of the TH2 cytokines. Journal of Allergy and Clinical Immunology 107: 772780. Gordon S (2003) Alternative activation of macrophages. Nature Reviews: Immunology 3: 2335. Jakubzick C, Kunkel SL, Puri RK, and Hogaboam CM (2004) Therapeutic targeting of IL-4- and IL-13-responsive cells in pulmonary brosis. Immunologic Research 30: 339349. Kronenberg M and Gapin L (2002) The unconventional lifestyle of NKT cells. Nature Reviews: Immunology 2: 557568. Martin JG, Suzuki M, Ramos-Barbon D, and Isogai S (2004) T cell cytokines: animal models. Paediatric Respiratory Reviews 5(supplement A): S47S51. Mueller TD, Zhang JL, Sebald W, and Duschl A (2002) Structure, binding, and antagonists in the IL4-/IL-13 receptor system. Biochimica et Biophysica Acta 1592: 237250. Nelms K, Keegan AD, Zamorano J, Ryan JJ, and Paul WE (1999) The IL-4 receptor: signaling mechanisms and biologic functions. Annual Review of Immunology 17: 701738. OByrne PM, Inman MD, and Adelroth E (2004) Reassessing the Th2 cytokine basis of asthma. Trends in Pharmacological Sciences 25: 244248.

IL-4 in Respiratory Diseases

Polymorphisms of the IL-4 gene have been associated with severity of atopic asthma in several ethnic groups. Two naturally occurring polymorphisms of the IL-4Ra chain have been implicated in atopy. One of these, substitution of Val50 by isoleucine, enhances signal transduction resulting in increased IgE production. The exact importance of IL-4 in respiratory diseases remains to be determined. The unique role of IL-4 polarization of uncommitted T cells to the TH2 phenotype argues that it should play a key role in allergies and host defense against pathogens. However, virtually all other actions of IL-4 can be replaced by other TH2 cytokines, especially IL-13, which appears to be more crucial as a mediator of immune responses in tissues outside organized lymph nodes. Mast cells, basophils, and eosinophils have recently been shown to be capable of producing IL-13 transcripts in an IL-4Ra- and STAT6-independent fashion.

INTERLEUKINS / IL-5
Steinke JW (2004) Anti-interleukin-4 therapy. Immunology and Allergy Clinics of North America 24: 599614. Wills-Karp M (1999) Immunologic basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255281. Zhu J, Guo L, Watson CJ, Hu-Li J, and Paul WE (2001) Stat6 is necessary and sufcient for IL-4s role in Th2 differentiation and cell expansion. Journal of Immunology 166: 72767281.

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Relevant Website
www.ncbi.nim.nih.gov National Center for Biotechnology Information.

IL-5
M L Moore and R S Peebles Jr, Vanderbilt University School of Medicine, Nashville, TN, USA
& 2006 Elsevier Ltd. All rights reserved.

eosinophil differentiation activity, and it was renamed IL-5. DNA sequencing revealed that IL-5 was identical to B-cell growth factor II (BCGF-II). By 1987, complementary DNA (cDNA) clones for mouse and human IgA-enhancing factor (IgA-EF), eosinophil colony-stimulating factor (Eo-CSF), and human eosinophil differentiation factor (EDF) were all found to encode for the same molecule, IL-5. In 1988, it was demonstrated that recombinant hIL-5 stimulates the differentiation, proliferation, recruitment, survival, and activity of mouse eosinophils. Since then, a body of evidence has rmly established the requirement for IL-5 in airway eosinophilia. Mortality and morbidity associated with asthma has increased over the last two decades, prompting research aimed at elucidating the role of IL-5 and eosinophils in this disease.

Structure
IL-5 is a member of the helical bundle family of cytokines, along with IL-3, IL-4, and granulocytemacrophage colony-stimulating factor (GM-CSF). hIL-5 and mIL-5 contain 114 and 113 amino acids, respectively, and they are 70% identical. IL-5 is a disulde-linked, homodimeric protein with an apparent molecular weight of 4560 kDa; it is heavily glycosylated. Each monomer of IL-5 consists of four a-helical domains with a small antiparallel b-sheet between each of the helices. The two monomers are interdigitating, such that the C-terminal end and helix of each polypeptide interacts with the N-terminal three helices of the other polypeptide (Figure 1). The formation of an interdigitating homodimer is necessary for the function of IL-5. Among the short chain helical bundle cytokines, dimerization is unique to IL-5.
N

Abstract
Interleukin-5 (IL-5) is a cytokine secreted principally by activated helper T cells of the T-helper-2 (Th2) subset. IL-5 is a short chain helical bundle cytokine structurally similar to IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor. The IL-5 gene is tightly linked with the IL-4 and IL-13 genes in a Th2 cytokine gene cluster on chromosome 5q, and the expression of these genes is coordinately regulated. The bioogy of IL-5 has been best studied in the context of asthma in humans and allergic airway inammation in mouse models. IL-5 directly mediates eosinophilic inammation by stimulating the differentiation, proliferation, recruitment, survival, and activity of eosinophils. Eosinophils release granule proteins and leukotrienes that are hypothesized to contribute to airway hyperreactivity and epithelial damage in allergic asthma. However, recent studies indicate that the immunopathogenic role of IL-5 and eosinophils in asthma is not clear.

Introduction
Interleukin-5 (IL-5) is a cytokine synthesized and secreted mainly by activated CD4 helper T cells of the T-helper-2 (Th2) subset, and also by mast cells and eosinophils. The effector functions of human IL5 (hIL-5) are principally associated with a single cell type, the eosinophil. In mice, IL-5 stimulates B cells in addition to eosinophils. One hundred years after eosinophils were rst associated with symptomatic asthma, molecular cloning enabled the identication of IL-5 as a cytokine that stimulates the functions of eosinophils. In the 1970s and 1980s, soluble factors produced by T cells cultured in vitro were puried biochemically and named according to their biological activities. Mouse IL-5 (mIL-5) was originally identied as T-cellreplacing factor (TRF). TRF was shown to have

N
Figure 1 View of the homodimeric IL-5 protein along the dimer twofold axis. IL-5 consists of two interdigitating polypeptides and contains two helical bundle domains. Each helix is represented by a cylinder. There are four helices per polypeptide (shaded or unshaded) with a small antiparallel b-sheet (represented by a solid or dashed line) connecting each of the helices. The C-terminal end and helix of each polypeptide interacts with the N-terminal three helices of the other polypeptide. Dimerization is required for function.

360 INTERLEUKINS / IL-5

Regulation of IL-5 Synthesis

hIL-5 is located on chromosome 5 and mIL-5 is located on chromosome 11. In both species, the IL-5 gene is tightly linked to the genes for IL-4 and IL-13, and these three genes are contained in a Th2 cytokine gene cluster that spans 140 kb. Coding and noncoding regions within the Th2 cytokine gene cluster are conserved between mouse and man. The IL-5 gene is also linked to the IL-3 and GM-CSF genes, which are approximately 500 kb from IL-5 in both mouse and man. In humans, the region containing the Th2 cytokine cluster and the genes for IL-3, GM-CSF, IL-9, and broblast growth factor 1 is called the chromosome 5q cytokine cluster. The genes for IL-4, IL-5, and IL-13 are coordinately expressed and regulated. Evidence suggests that chromatin remodeling mechanisms, such as histone modication and DNA methylation, are an integral part of transcriptional regulation of the Th2 cytokine cluster. In differentiating Th2 cells, ligation of the T-cell receptor results in activation of the transcription factors signal transducer and activator of transcription 6 (STAT6) and GATA-3, which direct chromatin remodeling and specifically stimulate transcription from the Th2 cytokine cluster. Mast cells, another source of IL-5, do not express STAT6 or GATA-3. However, mast cells and Th2 cells contain the same DNase I hypersensitivity (HS) sites at this locus. HS sites mark accessible chromatin and reect noncoding regions of DNA that act as docking sites for chromatin remodeling and transcriptional protein complexes. Thus, Th2 cells and mast cells may regulate IL-5 transcription via similar chromatin remodeling pathways but distinct signaling pathways. Elements regulating IL-5 synthesis are not fully dened. The proximal 50 IL-5 promoter contains conserved sequence motifs also found in the promoters of the IL-3, IL-4, and GM-CSF genes. However, like the IL-3, IL-4, and GM-CSF promoters, the IL-5 promoter alone cannot reproduce physiological expression patterns. A number of distal DNA elements that regulate IL-5 transcription have been dened, including a 6.2 kb upstream enhancer with potential GATA-3 binding sites. Cytokines that stimulate the differentiation and activation of Th2 cells can promote IL-5 production, including IL-2, IL-4, and IL-13. IL-2 is produced by T cells and is mainly an autocrine T-cell growth factor. The IL-4 and IL-13 receptors have a common subunit (IL-4Ra) that activates STAT6, resulting in transcription of many genes, including IL-4, IL-5, and IL-13. Prostaglandin E2 (PGE2) can increase or decrease IL-5 expression in T cells in vitro, depending on the culture conditions. Cytokines that can

suppress IL-5 production via inhibition of Th2 cells include interferon (IFN)-a, IFN-g, and IL-12. The cytokine transforming growth factor beta (TGF-b) can directly block the antiapoptotic effect of IL-5 on eosinophils and inhibit eosinophil differentiation.

Biological Function
IL-5 promotes the differentiation of eosinophil precursor cells in the bone marrow, the recruitment of eosinophils from the bone marrow to tissues, the activation of tissue eosinophils, and eosinophil survival (Figure 2). Eosinophils are bone marrow-derived granulocytes that are important for defense against helminthic parasites (ukes, tapeworms, and roundworms) but contribute to pathogenesis in allergic diseases. IL-5 is the predominant hematopoietic growth factor for eosinophils, stimulating colony formation by CD34 myeloid progenitor cells in the bone marrow and their development into mature eosinophils. The transcription factors CCAAT/enhancer binding protein (C/EBP) is required for eosinophil and neutrophil maturation, whereas GATA-1 specifically promotes the terminal differentiation of eosinophils.
Bone marrow CD34+

IL-5

CCR3 Blood

EO

PAF-R

CD11b IL-5 Lung parenchyma Leukotrienes, ECP, EPO, MBP, EDN, PAF, cytokines

EO

IL-5 Apoptosis

Figure 2 IL-5 promotes the maturation, recruitment, activation, and survival of eosinophils. IL-5 is the predominant growth factor for eosinophils, stimulating the differentiation of CD34 myeloid progenitor cells in the bone marrow into mature eosinophils (EO). IL-5 mediates eosinophil recruitment by upregulating the cell surface expression of the adhesion molecule CD11b and by upregulating the expression of PAF-R and CCR3, the receptors for PAF and eotaxin, respectively. IL-5 promotes eosinophil activation by stimulating the production of leukotrienes, granular proteins (ECP, EPO, MBP, and EDN), PAF, and cytokines; IL-5 induces eosinophil degranulation. IL-5 promotes the survival of eosinophils by inhibiting apoptosis.

INTERLEUKINS / IL-5

361

IL-5 mediates eosinophil recruitment by a variety of mechanisms. The direct chemotactic potential of IL-5 for eosinophils is modest. However, IL-5 upregulates the expression of the adhesion molecule CD11b on the surface of eosinophils, thereby facilitating recruitment. IL-5 also increases the expression of the platelet-activating factor (PAF) receptor on eosinophils. PAF is a known chemotactic factor for eosinophils. A potent inducer of eosinophil recruitment is the chemokine eotaxin, which functions cooperatively with IL-5 to mediate eosinophil extravasation from the blood to the lung parenchyma. IL-5 also primes eosinophils for chemotaxis mediated by the chemokines IL-8 and regulated upon activation normally T cell expressed and secreted (RANTES). Cells of the promyelocytic leukemia cell line HL-60 can be induced to differentiate into eosinophils by treatment with butyric acid. In this model of eosinophil development, addition of IL-5 to the culture media increases expression of CCR3, the receptor for eotaxin. IL-5 augments eosinophil activation and cytotoxicity. Eosinophils release a number of effector molecules, including the eosinophil granule proteins eosinophilic cationic protein (ECP), major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), as well as leukotrienes, PGE2, a number of cytokines (IL-2, IL-3, IL-4, IL-5, GM-CSF, IL-6, INF-g, IL-12, and TGF-b), and reactive oxygen intermediates, such as hydrogen peroxide and hydroxy radicals. The granule proteins released by activated eosinophils are highly cytotoxic to cells, including helminthic cells, in the case of parasitic infections, and mammalian airway epithelial cells, in the case of asthma. mRNAs for the eosinophil granule proteins are among the most abundant transcripts in IL-5-differentiated umbilical cord precursor cells, with MBP being the most abundant mRNA. IL-5 stimulates eosinophil production of leukotrienes, superoxide, and cytokines. In addition, IL-5 promotes eosinophil degranulation. PAF may play a role in IL-5-mediated eosinophil activation since antagonists of PAF inhibit IL-5-induced eosinophil superoxide production and degranulation. In addition to IL-5, IL-3, and GM-CSF can stimulate eosinophil effector functions. Mature eosinophils express approximately 1000 receptors per cell for IL-3, IL-5, and GM-CSF. IL-5 and GM-CSF promote eosinophil survival by inhibiting apoptosis. Mature eosinophils undergo apoptosis in vitro and in vivo if these cytokines are absent from the milieu. Mitochondria play an important role in regulating apoptosis, in addition to facilitating cellular respiration. Interestingly, human peripheral blood eosinophils contain few

mitochondria, and eosinophil mitochondria are decient in cellular respiration but maintain the ability to induce apoptosis via release of cytochrome c. Recent studies suggest that IL-5 inhibits eosinophil apoptosis by signaling through the Janus kinase (JAK)/STAT pathway to prevent the translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondrial membrane, thereby inhibiting Bax-induced cytochrome c release.

IL-5 Receptor
Receptors for the cytokines IL-5, IL-3, and GM-CSF consist of a cytokine-specic a receptor subunit and a common bc subunit. The a subunit of the IL-5 receptor (IL-5Ra) binds to its ligand with low afnity. Association of the bc subunit with the a subunit results in a high-afnity receptor. Both a and bc subunits contain intracellular domains, but the bc subunit plays a more significant role in signal transduction. When IL-5 binds to the IL-5Ra/bc receptor, receptor-mediated signal transduction is initiated. The juxtamembranous protein kinases JAK-2 and Lyn are physically associated with the bc subunit of the IL-5 receptor at basal conditions. Receptor dimerization leads to a cascade of tyrosine phosphorylation and activation of downstream targets, such as STAT1a and STAT5 transcription factors in the JAK/STAT pathway, G proteins of the mitogen-activated protein-kinase (MAPK) pathway, phosphatidylinositol-3 kinase (PI3 kinase), and the tyrosine kinases Syk and Fyn. Recent studies have begun to determine which signaling molecules and pathways are involved in specic IL-5-mediated functions. Lyn and Syk are important for IL-5-mediated inhibition of apoptosis, whereas Raf-1 kinase of the MAPK pathway is important for eosinophil degranulation.

IL-5 in Respiratory Diseases

IL-5 is important for respiratory diseases that involve eosinophilic inammation, such as nasal polyps, eosinophilic pneumonias, hypereosinophilic syndrome, and, especially, asthma. Asthma is an inammatory disease of the lung characterized by reversible airway obstruction, airway hyperresponsiveness (AHR), pulmonary eosinophilia, and excessive airway mucus production. Allergic asthma is a Th2 cell-dependent disease. Th2 cells mediate asthmatic inammation via secretion of the cytokines IL-4, IL-5, and IL-13. Allergen inhalation leads to antigen uptake by antigen-presenting cells, particularly dendritic cells, which reside in the airway epithelium and migrate to draining

362 INTERLEUKINS / IL-5

lymph nodes of the lung to present antigen to CD4 T cells. In asthmatic inammation, a Th2 immune response ensues that can initiate two phases of pathogenesis. The immediate brochospastic phase occurs within minutes and is characterized by IgEmediated mast cell activation and degranulation. Mast cells, the principal effector cells of the immediate phase, release histamine and leukotrienes that contribute to acute bronchoconstriction. The late phase occurs 36 h after inhalation and can persist for several days without therapy. The eosinophil is hypothesized to be the principal effector cell of the allergen-induced pulmonary late-phase reaction. Th2 cells and mast cells produce IL-5, resulting in eosinophil maturation, recruitment to the lung, and activation (Figure 2). Activated eosinophils release granular proteins (discussed above) and leukotrienes, which are thought to contribute to AHR and airway epithelial damage in asthma. The relationship between IL-5 and eosinophilia is clear. In anti-IL-5 antibody-treated, IL-5 gene knockout, and IL-5 transgenic mice it has been demonstrated that IL-5 is necessary and sufcient for the development of eosinophilia in allergic airway inammation. In asthma patients, the causal relationship between IL-5 production in the airways and the development of eosinophilia is also well established. However, the relationship between IL-5 and AHR is not clear. In mice, there is a body of evidence indicating that IL-5 contributes to AHR. IL-5 gene knockout mice and mice treated with anti-IL-5 antibodies exhibit reduced AHR in allergen challenge models. IL-5 transgenic mice that constitutively express IL-5 in the lung epithelium spontaneously develop AHR. Mice that express the diphtheria toxin A chain under control of the EPO promoter, resulting in the complete absence of eosinophils, do not develop AHR upon allergen challenge. However, some groups have reported that depletion of eosinophils by anti-IL-5 antibody treatment had no effect on AHR. Also, GATA-1-decient mice, which lack eosinophils, exhibit AHR upon allergen challenge. Treatment of mice with anti-IL-13 antibody reduces AHR without affecting the number of eosinophils in the lung. Therefore, the role of IL-5 in mouse models of allergic lung inammation remains controversial. In humans, the role of IL-5 in asthma pathogenesis is also controversial. Eosinophil levels correlate with asthma disease kinetics and severity. Effective corticosteroid therapy is associated with reduced eosinophilia. Independent investigators have found that susceptibility to asthma maps to the human chromosome 5q cytokine cluster. Depletion of IL-5 in humans has provided important clues for the role of this cytokine and eosinophils in asthma pathogenesis. In asthmatic

patients, anti-IL-5 did not alter blood or bone marrow CD34 myeloid progenitor cells, but reduced the number of eosinophils in the bone marrow by 70%, blood eosinophils by 85%, and ECP concentrations by two-thirds, without affecting the composition of T-cell subsets or T-cell cytokine production. In another study of allergic asthmatics, anti-IL-5 decreased airway eosinophils by 55% without having an appreciable effect on bronchial mucosal staining of MBP. In this report, anti-IL-5 had no effect on airway responsiveness, forced expiratory volume in 1 s, or peak ow measurements. Additionally, anti-IL-5 had no effect on the allergen-induced late-phase response in a different group of allergic asthmatics. However, anti-IL-5 has reduced the bronchial expression of proteins that are associated with airway wall remodeling in patients with allergic asthma. Therefore, these data would suggest that the current route, dose, and duration of anti-IL-5 given in human studies does not affect pulmonary function, but may have an effect on airway remodeling. In conclusion, IL-5 is a critical eosinophil differentiation, recruitment, and activation factor. However, the role of this cytokine in human diseases such as asthma remains to be clearly dened.
See also: Allergy: Overview. Apoptosis. Asthma: Overview. CD11/18. Chemokines. Chemokines, CC: RANTES (CCL5). Chemokines, CXC: IL-8. Dendritic Cells. Gene Regulation. Genetics: Gene Association Studies. Histamine. Interferons. Interleukins: IL-9; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells. Lipid Mediators: Overview; Leukotrienes. Mucus. Platelet-Derived Growth Factor. Signal Transduction. Systemic Disease: Eosinophilic Lung Diseases. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Transgenic Models. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Abbas AK, Lichtman AH, and Pober JS (2003) Cellular and Molecular Immunology, 5th edn., pp. 243274. Philadelphia: Saunders. Adachi T and Alam R (1998) The mechanism of IL-5 signal transduction. American Journal of Physiology 275: C623C633. Ansel KM, Lee DU, and Rao A (2003) An epigenetic view of helper T cell differentiation. Nature Immunology 4: 616623. Borish L and Rosenwasser LJ (2003) Cytokines in allergic inammation. In: Adkinson NF Jr, Yunginger JW, Busse WW, et al. (eds.) Middletons Allergy Principles & Practice, 6th edn., pp. 135157. Philadelphia: Mosby. Foster PS, Mould AW, Yang M, et al. (2001) Elemental signals regulating eosinophil accumulation in the lung. Immunological Reviews 179: 173181. Gleich GJ (2000) Mechanisms of eosinophil-associated inammation. Journal of Allergy and Clinical Immunology 105: 651663. Gleich GJ, Adolphson CR, and Leiferman KM (1993) The biology of the eosinophilic leukocyte. Annual Review of Medicine 44: 85101.

INTERLEUKINS / IL-6
Hamelmann E and Gelfand EW (2001) IL-5-induced airway eosinophilia the key to asthma? Immunological Reviews 179: 182191. Lalani T, Simmons RK, and Ahmed AR (1999) The biology of IL-5 in health and disease. Annals of Allergy and Asthma Immunology 82: 317333. Renauld JC (2001) New insights into the role of cytokines in asthma. Journal of Clinical Pathology 54: 577589. Viola JPB and Rao A (1999) Molecular regulation of cytokine gene expression during the immune response. Journal of Clinical Immunology 19: 98108. Wills-Karp M (1999) Immunological basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255281. Wills-Karp M and Karp CL (2004) Eosinophils in asthma: remodeling a tangled tale. Science 305: 17261728.

363

IL-6 was originally discovered as a cytokine that stimulated B lymphocytes to become plasma cells and was rst called B-cell stimulatory factor in the early 1980s. It has subsequently been found to be one of the most broad-ranging cytokines there are in terms of its effector prole. In addition to stimulating lymphocytes it affects osteoclasts, neural cells, keratinocytes, renal cancer cells, hepatocytes, and many other cell types. Its clinical effects range from both inammatory to anti-inammatory, making it one of the most complex cytokines currently known.

Structure

IL-6
N Ward, Brown University, Providence, RI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-6 (IL-6) is a pleiotropic cytokine that is generally produced at sites of inammation. It is a member of a family of cytokines classied as IL-6-type cytokines which also includes IL-11, leukemia inhibitory factor, cardiotrophin-1, oncostatin M, and ciliary neurotrophic factor based on the overlapping effector proles of these cytokines and their shared use of gp130 as the b-subunit in their multimeric receptor complexes. IL-6 induces fever, activates B and T lymphocytes, and stimulates hepatocytes to produce acute phase proteins. IL-6 is produced early in most forms of inammation by many different cell types and so it plays a role in almost all forms of lung disease that involve infection or inammation. Its levels in serum and lung uid have been shown to correlate with some clinical outcomes in the acute respiratory distress syndrome but other than that, little is known about its role in specic diseases. It is not currently thought to play a significant role in asthma or allergic lung diseases. Despite often being classied as a proinammatory cytokine, recent studies have demonstrated that IL-6 also may have potent antiinammatory and protective properties. These include the ability to inhibit the production of tumor necrosis factor (TNF), IL-1, and MIP-2; decrease neutrophil sequestration; increase levels of IL-1 receptor antagonist (IL-1ra) and TNF-soluble receptor (TNFsr); stimulate the production of metalloproteinase inhibitors; reduce intracellular superoxide production; reduce tissue matrix degradation; and inhibit cellular apoptosis.

IL-6 is produced by a single gene but exists in many different forms based on posttranslational modication. The IL-6 gene has been localized to the p21 region of chromosome 7. It is extremely similar to the gene coding G-CSF and is thought to perhaps have arisen from a common ancestor. The primary gene product is 212 amino acids long but is cleaved to 184 amino acids within the cell. This peptide is then further modied to a nal protein that can range from 17 to 84 kDa in size. The different subtypes of IL-6 produced tend to be tissue specic with monocytes characteristically producing a 24 kDa species and endothelial cells producing a 45 kDa form.

Regulation of Production and Activity

IL-6 is produced by wide variety of cell types including monocytes, lymphocytes, broblasts, endothelial cells, mesangial cells, and keratinocytes. Its transcription is stimulated by a variety of mediators including tumor necrosis factor alpha (TNF-a), IL-1, and transforming growth factor beta. Viruses and endotoxin are also known to stimulate the production of IL-6. Studies have shown that different cell types are more or less sensitive to different stimuli. For example, in alveolar macrophages endotoxin is a potent stimulator of IL-6 but produces little in response to IL-1. Fibroblasts produce little IL-6 to endotoxin but are heavily stimulated by IL-1. Glucocorticoids are known to inhibit the production of IL-6 by binding to the IL-6 promoter site on DNA. There is evidence that glucocorticoids also inhibit IL6 production by decreasing mRNA stability.

Introduction
Interleukin-6 (IL-6) is a pleiotropic cytokine that is generally produced at sites of tissue inammation. It is part of a family of cytokines classied as IL-6type cytokines. IL-6 type cytokines have in common overlapping effector proles and their shared use of gp130 as the b-subunit in their multimeric receptor complexes. This family of cytokines also includes IL-11, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF).

Receptors
IL-6 binds to an 80 kDa receptor protein, called IL6Ra, which has both extracellular and intracellular domains. The IL-6/IL-6Ra complex then interacts with and binds to the 130 kDa signal transducing molecule, gp130, which together with IL-6Ra forms the functional IL-6 receptor. The gpl30 subunit is

364 INTERLEUKINS / IL-6

also used by a variety of related cytokines with some similar functions such as IL-11, LIF, CT-1, OSM, and CNTF. IL-6 receptors are found on a diverse array of cells including hepatocytes, lymphocytes and other myeloid cells, keratinocytes, various nervous system cells, hematopoietic stem cells, and many other tissue epithelial and endothelial cells.

Interestingly, megakaryocytes both produce IL-6 and have IL-6 receptors on their surface. Other functions of IL-6 include effects on the stimulation of adrenal axis through stimulation of ACTH production and effects on skin keratinocytes which both express IL-6 receptors as well as produce IL-6.

IL-6 in Respiratory Disease Biological Functions

Stimulator of Acute Phase Proteins

One of the most important functions of IL-6 appears to be its role as a stimulator of the acute phase proteins, a heterogeneous collection of proteins generally produced by the liver during inammation and major stresses which act to limit the proinammatory response. This is one of the reasons IL-6 is now classied as having both pro-and anti-inammatory properties in vivo. When administered exogenously IL-6 stimulates fever as well as production of the acute phase proteins. Some of these proteins are produced in response to IL-6 alone and others need costimulation with IL-1. Glucocorticoids may positively interact with this system as well.
Stimulator of B Cells and T Cells

The ability of IL-6 to stimulate B lymphocytes has been known since its discovery and was thought initially to be its main function. While IL-4 and IL-5 are more important cytokines for the proliferation, activation, and maturation of B cells, IL-6 appears to be essential for the later phase of activation leading to production and secretion of antibodies. IL-6 stimulates B lymphocytes to produce immunoglobulins IgG, IgM, and IgA. IL-6 has important roles in the activation of T lymphocytes as well yet acts in the early stages of Tcell activation, in contrast to its role in B-cell activation. IL-6 induces T-cell proliferation and increases the sensitivity of T cells to the stimulatory factor IL-2. Interestingly, IL-6 is also produced by some activated T cells. Finally, IL-6 is a stimulatory factor for cytotoxic T cells and lymphokine-activated killer cells.
Other Functions

IL-6 has a broad array of other functions that have been discovered over the last several decades. Most notably, IL-6 plays a role in hematopoiesis. It can stimulate the growth of stem cells and has several noted effects on the maturation and survival of myeloid cells. IL-6 also has potent effects on thrombopoiesis. It is a stimulator of platelet production and mice will actually increase their serum IL-6 levels in response to articially induced thrombocytopenia.

As a mediator of the inammatory response, IL-6 plays a prominent role in many systemic diseases which can affect the lung including (but not limited to) transplant rejection, human immunodeciency virus, rheumatoid arthritis, systemic lupus erythematosus, and other vasculitic and chronic inammatory diseases. It is also an important mediator of disease in several hematologic malignancies such as leukemia, lymphoma, and multiple myeloma. Its most active role in the lung, however, is in states of acute inammation such as sepsis, bacterial pneumonia, and trauma. IL-6 is produced by almost all different types of lung cells including endothelial cells, airway epithelial cells, broblasts, and, most notably, alveolar macrophages. IL-6 is thought to play a role in all types of acute lung inammation through its stimulatory effects on lymphocytes and the acute phase response. IL-6 is also a modulator of both the proinammatory cytokines IL-1 and TNF. IL-6 has been used extensively as a marker of inammation in systemic infectious diseases such as sepsis and in lung-specic conditions such as acute respiratory distress syndrome (ARDS). IL-6 levels in both brochoalveolar lavage uid and serum have been shown to rise early in the development of ARDS and accurately track with the clinical course of the disease. Several studies have linked early and/or late levels of IL-6 with clinical outcomes in ARDS such as mortality. It is important to note, however, that while IL-6 is often used as a marker of the proinammatory state it may not be acting as a proinammatory mediator. As previously mentioned, IL-6 can have many antiinammatory effects such as the stimulation of acute phase proteins. In one study of ARDS in mice, the lack of the IL-6 gene dramatically increased the inammatory response and prolonged tissue leukocytosis in response to lung injury. Giving IL-6 to these mice reversed the effect. Other studies have shown IL-6 to inhibit the production of TNF, IL-1, and MIP-2, increase levels of IL-1 receptor antagonist (IL-1ra) and TNF-soluble receptor (TNFsr), stimulate the production of metalloproteinase inhibitors, reduce intracellular superoxide production, reduce tissue matrix degradation, and inhibit cellular apoptosis.

INTERLEUKINS / IL-7 See also: Acute Respiratory Distress Syndrome. Interleukins: IL-1 and IL-18. Tumor Necrosis Factor Alpha (TNF-a).

365

Introduction
IL-7 was originally identied as a stromal factor that could support the growth of murine B-cell progenitors in long-term bone marrow cultures, and subsequently was shown to play critical roles in the development of T and B cells in mice and T cells in humans. IL-7 exerts its effects, both as a growth and survival factor. Although mouse IL-7 cannot stimulate human pre-B cells, human IL-7 has activity on murine cells.

Further Reading
Barnes PJ (2001) Cytokine modulators as novel therapies for airway disease. European Respiratory Journal 34(supplement): 67s77s. Chung KF (2001) Cytokines in chronic obstructive pulmonary disease. European Respiratory Journal 34(supplement): 50s59s. Dreyfuss D, Ricard JD, and Saumon G (2003) On the physiologic and clinical relevance of lung-borne cytokines during ventilatorinduced lung injury. American Journal of Respiratory and Critical Care Medicine 167(11): 14671471. Hirano T (1992) Interleukin-6 and its relation to inammation and disease. Clinical Immunology and Immunopathology 62(1 Pt 2): S60S65. Hirano T (1992) The biology of interleukin-6. Chemical Immunology 51: 153180. Martin TR (1999) Lung cytokines and ARDS: Roger S. Mitchell Lecture. Chest 116(1 supplement): 2S8S. Parsons PE, Eisner MD, Thompson BT, et al. (2005) Lower tidal volume ventilation and plasma cytokine markers of inammation in patients with acute lung injury. Critical Care Medicine 33(1): 16. discussion 230232. Ranieri VM, Suter PM, Tortorella C, et al. (1999) Effect of mechanical ventilation on inammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. Journal of the American Medical Association 282(1): 5461. Strieter RM and Kunkel SL (1994) Acute lung injury: the role of cytokines in the elicitation of neutrophils. Journal of Investigative Medicine 42(4): 640651. Toews GB (2001) Cytokines and the lung. European Respiratory Journal 34(supplement): 3s17s. Ward NS, Waxman AB, Homer RJ, et al. (2000) Interleukin-6-induced protection in hyperoxic acute lung injury. American Journal of Respiratory Cell and Molecular Biology 22(5): 535542. Xing Z, Gauldie J, Cox G, et al. (1998) IL-6 is an antiinammatory cytokine required for controlling local or systemic acute inammatory responses. The Journal of Clinical Investigation 101(2): 311320. Zitnik RJ, Elias JA (1993) Interleukin-6 and the Lung. In: Kelley J (ed.) Cytokines of the Lung, vol. 1, pp. New York: Dekker.

Structure
IL-7 is a type I cytokine that has a typical four-a helical bundle structure with an up-up-down-down topology. A three-dimensional model of human IL-7 was constructed by using the X-ray crystal structure of IL-4 as a template and superimposing the IL-7 helices onto those of IL-4. The model was completed by incorporating the disulde bond assignments as determined by MALDI spectroscopy and site-directed mutagenesis. The IL-7 gene is located on human chromosome 8q1213 and murine chromosome 3, and the human and mouse genes share about 80% homology within their protein-coding regions. Alternatively spliced isoforms of IL-7 have been described, the predominant active isoform being 25 kDa. The increased apparent molecular weight over the size of the predicted amino acid sequence (17.4 kDa) is due to glycosylation.

Regulation of Production and Activity

Regulation of the IL-7 gene is not well understood, with no apparent upstream TATA or CAAT sequences typical of inducible genes. Unlike other cytokines whose receptors utilize the common cytokine receptor g chain, gc, the production of IL-7 is constitutive rather than inducible, and there is an ambient, low level in the serum. Its production in bone marrow is downregulated by transforming growth factor beta (TGF-b), and may be positively regulated by the transcription factor C/EBPb, given that C/EBPb-decient bone marrow cells have decreased production of IL-7. IL-7 is produced by fetal liver cells and stromal tissues in bone marrow, thymic epithelial cells, and intestinal epithelial cells. Its production is tightly regulated during ontogeny, with peak production in fetal development, accompanied by massive thymic expansion, and a substantial decrease in the aging thymus.

IL-7
W J Leonard and H-H Xue, National Heart, Lung, and Blood Institute, Bethesda, MD, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-7 (IL-7) is a stroma-derived cytokine that is indispensable for thymopoiesis in mice and humans. IL-7 also plays a pivotal role in the generation of memory CD4 and CD8 cells, and in controlling the numbers of na ve and memory T cells. Its combined effects of increasing thymic output and enhancing homeostatic expansion of T cells make IL-7 a potentially ideal agent to restore immune competence in patients with lymphopenia.

Receptors
The IL-7 receptor (IL-7R) complex consists of the IL7Ra chain and gc. IL-7Ra is also utilized by another

366 INTERLEUKINS / IL-7

cytokine, thymic stromal lymphopoietin (TSLP) (Figure 1), whereas gc is shared by other cytokines including IL-2, IL-4, IL-9, IL-15, and IL-21. The binding of IL-7 to its receptor complex activates multiple signal cascades, including the Janus family tyrosine kinase (Jak)-signal transducer and activator of transcription (STAT) pathway, with Jak1, Jak3, Stat5a, and Stat5b being primarily activated, and the phosphatidyl inositol 3-kinase (PI3-K) pathway. The intracellular domain of IL-7Ra is 195 amino acids long and lacks intrinsic kinase activity but instead provides docking sites for signaling molecules. An 8 amino acid motif termed Box1 in the membrane proximal region is conserved among the type I cytokine receptor family members and in IL-7Ra, this motif mediates the recruitment of Jak1. Tyrosine 449 in the cytoplasmic domain is part of a YXXM motif that mediates the docking of both Stat5 proteins and the p85-binding subunit of PI3-K. IL-7Ra is dynamically regulated during thymic development and the T-cell immune response. The most immature thymocytes are double negative cells that lack expression of CD4 and CD8 (CD4 CD8 ) but express IL-7Ra. These cells then downregulate expression of IL-7Ra as they mature to CD4 CD8 double-positive thymocytes. IL-7Ra is highly expressed in mature CD4 or CD8 single positive thymocytes and remains so in peripheral resting T cells. Upon exposure to antigens, na ve T cells become activated, undergo clonal expansion, and differentiate into effector T cells that express much lower IL-7Ra. IL-2 is a cytokine produced by activated T cells; it decreases expression of IL-7Ra via a PI3-K/Akt-dependent mechanism. The low IL-7Ra expression on effector T cells is thus maintained by IL-2. When the antigen is

cleared and the immune response subsides, memory T cells to the antigen are formed and IL-7Ra expression is regained on these cells. During B-cell development, IL-7Ra is expressed on early B-cell precursors and pro-B cells, with its expression progressively downregulating as B cells mature.

Biological Functions
The phenotypes manifested in human genetic deciencies and gene-targeted mouse strains have demonstrated an absolute requirement of IL-7 signaling for lymphopoiesis. Ablation of IL-7 or IL-7Ra gene expression in mice results in a severe decrease in thymocytes and peripheral ab T cells. In the most severe cases, T-cell development is blocked at the double negative (CD4 CD8 ) stage. B-cell development in these mice is also impaired, showing a block at CD43 pro-B cells or earlier. Interestingly, genetic deciencies in the IL-7Ra gene in humans result in severe combined immunodeciency (SCID) with a T B NK phenotype, indicating that in humans the IL-7/IL-7R system is essential for T-cell development, but not for B-cell maturation (see Table 1 for the major actions of IL-7). So far, more than 20 SCID patients manifesting a T B NK phenotype have been found to have mutations in the IL-7Ra gene. These mutations are clustered in the rst ve exons, resulting in diminished expression of IL-7Ra, impaired IL-7 binding to the receptor, or in presumably perturbed receptor structure and/or function. In addition to its role in ab T-cell development, IL-7 has a nonredundant role in the development of gd T cells, as indicated by their complete absence in IL-7 or IL-7Ra-decient mice. The important role of IL-7 in ab T-, gd T-, and B-cell development is reected in its contribution to variable diversity joining (V(D)J) rearrangements in T-cell receptor b chain (TCRb), TCRg, and immunoglobulin heavy chain (IgH) loci. IL-7 contributes to the regulation of accessibility of these loci to the V(D)J recombinase. For the TCRg locus, IL-7 increases histone acetylation, resulting in an open chromatin structure. Besides its effects on developing lymphocytes, IL-7 provides survival signals to naive T cells in the periphery and to memory T cells. For na ve T cells,
Table 1 Major biological action of IL-7

IL-7

TSLP

TSLPR
c

Human

Mouse Yes Yes Yes Yes

IL-7R
Figure 1 IL-7Ra is a shared receptor component for IL-7 and TSLP. The functional IL-7 receptor is IL-7Ra gc whereas the functional TSLP receptor is IL-7Ra TSLPR. TSLPR is the TSLP-binding protein.

T-cell development B-cell development T-cell homeostasis Memory T-cell generation and maintenance

Yes No Yes Unkown

INTERLEUKINS / IL-7

367

survival also requires contact with self-peptide major histocompatibility complexes (MHCs). The survival effect of IL-7 has been attributed at least in part to its upregulation of antiapoptotic protein Bcl-2, and the down-modulation of pro-apoptotic proteins Bad and Bax, given that a Bcl-2 transgene rescued and Bax deciency partially corrected T lymphopoiesis in IL-7Radecient mice. Thus, IL-7 plays key roles in the homeostatic maintenance of na ve T cells, and because of its limited availability, IL-7 also contributes to control the size of T-lymphocyte pool in the periphery. In athymic mice and patients with T-cell depletion (e.g., postchemotherapy), changes in the immune system milieu activate peripheral mechanisms to restore T-cell numbers in the host, a process known as peripheral homeostatic expansion. When transferred to an IL-7-decient lymphopenic host, normal T cells undergo little if any homeostatic proliferation, indicating a key role of IL-7 in this process. IL-7 contributes to both the generation and efcient maintenance of antigen-specic memory T cells, which are essential for long-lasting immunological protection. IL-7 promotes the transition of CD4 effector T cells to persistent memory cells. In the absence of IL-7, the generation of memory CD4 T cells is greatly impaired in both lymphoid and nonlymphoid compartments, in spite of normal CD4 T-cell responses upon antigen challenge. The selective expression of IL-7Ra on the CD8 effector T cells (especially CD8aa cells) is a prerequisite for the generation of memory CD8 T cells; IL-7 signals are required for the survival of CD8 memory T-cell precursors also. Memory T cells maintain their numbers for long periods after antigen exposure through homeostatic proliferation. Along with IL-15, IL-7 contributes to the maintenance of memory CD8 T cells; in lymphopenic hosts especially, IL-7 can compensate for the absence of IL-15. Whereas memory CD8 T cells persist in the absence of MHC-I ligand, homeostasis of memory CD4 T cells requires both IL-7 and MHC-II-derived TCR signals. IL-7 is also a potent modulator of immune responses. First, IL-7 acts as a potent co-stimulatory signal for T-cell activation by enhancing proliferation and cytokine production. It not only can substantially increase the number of antigen-specic T cells that respond to stimuli with high afnity for the TCR, but it can also convert an otherwise nonreactive stimulus to a reactive one when TCR/peptide interactions are weak. Second, in human peripheral blood mononuclear cells, IL-7 can enhance the generation and function of cytolytic T lymphocytes (CTLs), which substantially contribute to cell-mediated immune responses. In addition, IL-7 induces lymphokineactivated killer activity and the cytotoxicity of

CD56 NK cells. Moreover, IL-7 can synergistically act with IL-21 to expand CD8 T cells in the absence of TCR stimulation, accompanied by increased interferon g expression. Third, IL-7 is a co-factor for the development of lymphoid dendritic cells in the thymus and for the differentiation of monocytic dendritic cells from myeloid precursors. Several lines of IL-7 transgenic mice have been described. When placed under the control of an MHC-II promoter, IL-7 overexpression resulted in a 30-fold increase in peripheral T and B cells without affecting thymic cellularity or CD4/CD8 ratios. Similarly, expression of IL-7 under control of an immunoglobulin k light chain promoter resulted in an increase in immature and mature B cells, and all mature T-cell subsets, but did not lead to an increase in CD4 CD8 double-negative T cells. On the other hand, when driven by the IgH promoter and enhancer, IL-7 overproduction caused a severe perturbation in thymic development with a profound decrease in DP thymocytes, a marked increase in mature T cells, and a lymphoproliferative disorder in the periphery characterized by T- and B-cell lymphomas. However, in transgenic mice in which IL-7Ra expression is driven by the human CD2 promoter, T cells developed normally but total thymocyte numbers decreased as a function of age, consistent with decreased IL-7 production in aging thymus. The biological actions of IL-7 are coordinately regulated by the concentration of available IL-7 and its expression levels, and cell-type specic expression of IL-7Ra. The expression pattern of IL-7Ra during T-cell development and after antigen stimulation corresponds to the actions of IL-7. As IL-7 is a limited resource during thymopoiesis, the downregulation of IL-7Ra on CD4 CD8 double-positive thymocytes minimizes competition with CD4 CD8 double-negative thymocytes for local IL-7, and allows the elimination of autoreactive T cells by the process of negative selection. IL-7Ra is expressed at a high level on na ve T cells, allowing their receipt of survival signals from IL-7, thus maintaining T-cell homeostasis. The diminished expression of IL-7Ra on effector T cells may make the cells more susceptible to apoptosis during the contraction phase of immune response, whereas the selective expression of IL-7Ra on effector T cells can facilitate the transition to CD4 or CD8 memory T cells. The expression of IL-7Ra, at a level higher than that on na ve T cells, contributes to the maintenance of these CD4 and CD8 memory T-cell pools by supporting both survival and homeostatic division. The expression of IL-7Ra in multipotential hematopoietic progenitors, early B-cell precursors, and pro-B cells is modulated by an Ets factor, PU.1,

368 INTERLEUKINS / IL-9

through an Ets-binding motif in the promoter region of IL-7Ra gene. PU.1 is also expressed in immature thymocytes, but is downregulated as T cells mature. The regulation of IL-7Ra in T cells is regulated through the same Ets-binding motif, but by a different Ets factor, GA-binding protein.

Transcription Factors: Overview; PU.1. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading
Alpdogan O and van den Brink MRM (2005) IL-7 and IL-15: therapeutic cytokines for immunodeciency. Trends in Immunology 26: 5664. Beq S, Delfraissy JF, and Theze J (2004) Interleukin-7 (IL-7): immune function, involvement in the pathogenesis of HIV infection and therapeutic potential. European Cytokine Network 15: 279289. Bradley LM, Haynes L, and Swain SL (2005) IL-7 maintaining T-cell memory and achieving homeostasis. Trends in Immunology 26: 172176. Cosenza L, Rosenbach A, White JV, Murphy JR, and Smith T (2000) Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation. Protein Science 9: 916926. DeKoter RP, Lee HJ, and Singh H (2002) PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16: 297309. Fry TJ and Mackall C (2002) Interleukin-7: from bench to clinic. Blood 99: 38923905. Hofmeister R, Khaled AR, Benbernou N, et al. (1999) Interleukin7: physiological roles and mechanisms of action. Cytokine Growth Factor Review 10: 4160. Kroemer RT, Kroncke R, Gerdes J, and Richards WG (1998) Comparison of the 3D models of four different human IL-7 isoforms with human and murine IL-7. Protein Engineering 11: 3140. Leonard WJ (2003) Type I cytokines and interferons and their receptors. In: Paul WE (ed.) Fundamental Immunology, 5th edn., pp. 701747. Philadelphia: Lippincott Williams & Wilkins. Leonard WJ (2005) Interleukin-7 deciency in rheumatoid arthritis. Arthritis Research & Therapy 7: 4243. Namen AE, Lupton S, Hjerrild K, et al. (1988) Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 333: 571573. Park LS, Friend DJ, Schmierer AE, Dower SK, and Namen AE (1990) Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line. Journal of Experimental Medicine 171: 10731089. Xue HH, Bollenbacher J, Rovella V, et al. (2004) GA binding protein regulates interleukin 7 receptor alpha-chain gene expression in T cells. Nature Immunology 5: 10361044. Zeng R, Spolski R, Finkelstein SE, et al. (2005) Synergy of IL-21 and IL-15 in regulating CD8 T cell expansion and function. Journal of Experimental Medicine 201: 139148.

IL-7 and Clinical Implications

IL-7 acts as a potent modulator of T-cell immune reconstitution, and is able to restore immune competence through its combined effects on increased thymic output and enhanced homeostatic expansion of T cells. The biological actions of IL-7 and preclinical data provide a strong rationale for its potential clinical efcacy. Chemotherapy of cancer patients, lympho-depleting therapy of autoimmune diseases such as rheumatoid arthritis, and transplantation of allogeneic bone marrow grafts are all accompanied by therapy-induced lymphopenia. IL-7 could be an effective agent in augmenting immune recovery and helping to optimize immune therapies. IL-7 may also enhance de novo T-cell generation after highly active antiretroviral treatment in human immunodeciency virus patients. Pulmonary brosis is a devastating disease that is characterized by a perturbed balance between the synthesis and breakdown of the extracellular matrix. TGF-b contributes to the pathogenesis of pulmonary brosis by enhancing synthesis and deposition of extracellular matrix components including collagen. Fibroblasts from patients with pulmonary brosis express IL-7Ra, and treatment of these cells with IL7 significantly decreases the production of TGF-b. Additionally, IL-7 can potently inhibit the actions of TGF-b by (1) the upregulation of Smad7, which inhibits TGF-b-mediated phosphorylation of Smad2/3, (2) the inhibition of TGF-b-induced activation and protein kinase C-d, and (3) the suppression of TGFb-induced collagen synthesis. These effects indicate that IL-7 is a potential antibrotic agent. In mouse models, when IL-7 is administered systemically or its production is induced along with co-stimulatory molecules within a tumor microenvironment through gene therapy, it can enhance antitumor response. Such effects are mediated by multiple mechanisms including the expansion and maintenance of T cells expressing TCRs with high afnity for tumor antigens and the recruitment of low-afnity T cells to the site of tumor. Translation of these ndings to clinical use awaits further preclinical tests.
See also: Apoptosis. Dendritic Cells. Gene Regulation. Human Immunodeciency Virus. Leukocytes: T cells. Pulmonary Fibrosis. Signal Transduction.

IL-9
A Dasvarma and A N J McKenzie, Laboratory of Molecular Biology, Cambridge, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-9 (IL-9) was originally cloned as a growth factor for certain mouse T helper cell clones. It is a 3040 kDa glycoprotein produced by activated T cells, with a variety of roles

INTERLEUKINS / IL-9
including T-cell stimulation, mast cell proliferation and differentiation, erythroid differentiation, Ig production, protection against parasite infection, and neuronal differentiation. Its deregulated expression has been implicated in a variety of disorders, including asthma and lymphomagenesis.

369

Introduction
Uyttenhove and colleagues identied interleukin-9 (IL-9), originally called p40, in 1988, as a growth factor for T cell clones. A collection of T helper cell lines was established from lymph nodes of antigenprimed mice. Cells were cultured in the presence of antigen and maintained via regular feeding with antigen and irradiated splenic antigen-presenting cells. Two clones derived from these cells proliferated in response to their own conditioned medium without the aid of antigen and feeder cells. The supernatant was obtained after concanalavin A (Con A) stimulation, indicating that the growth factor was associated with stimulated, not resting T cells. The growth factor eluted as a symmetrical peak in the region of 3040 kDa, leading to its designation as p40. Murine p40 was cloned and characterized via screening of a cDNA library, from a T helper cell clone that produced large amounts of p40 postConA stimulation. The cDNA encoding the human homolog of mouse p40 was identied by expression cloning of hematopoietic growth factors stimulating the proliferation of a megakaryoblastic leukemic cell line.

The distinctive activity of p40 from other immune growth factors was also conrmed by its inability to stimulate cell lines dependent on IL-3, IL-5, or IL-6. In addition to T cells, p40 was also found to be able to stimulate bone-marrow-derived mast cell lines (BMMC): recombinant p40 supported the growth of BMMC lines sensitive to mast cell-growth enhancing activity, induced IL-6 secretion, and enhanced the survival of primary mast cell cultures. p40 was renamed IL-9 in light of the fact that it was able to support the growth of several different immune cell types.

Structure
Analysis of genomic clones of IL-9 revealed that both human and mouse p40 genes consist of ve exons and four introns spread over approximately 4 kb of DNA (Figure 1(a)). Both the mouse and human genes are highly similar with significant identity in the 50 untranslated region (50 UTR) and coding regions. The human gene is 3.58 kb in length, whereas the mouse gene is 2.97 kb. The human gene is located on chromosome 5, at cytoband 5q31.1, whereas the mouse gene is located at cytoband 13B2. In humans the gene is located in a region containing genes encoding various cytokines and immune-related molecules, including IL-3, IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (Figure 1(b)). This region is commonly deleted in patients with acquired 5q abnormalities, leading to

2 3

114 1 (a)

36 33 2 3 4

132

120 5

120

IL-3 GM-CSF IL-5

IL-13 5q31.1

IL-4

IL-9

131.4 Mb

135.3

(b)

Human chromosome 5

Figure 1 (a) Genomic organization of the human and mouse IL-9 genes. Exons are represented as numbered boxes, with the empty region on exon 1 representing the 50 UTR. Exon sizes in base pairs are represented by numbers in italics. (b) Location of the IL-9 gene on human chromosome 5 in relation to other cytokine genes.

370 INTERLEUKINS / IL-9

hematologic disorders, although this does not necessarily implicate any of the individual cytokines in the pathologies. The IL-9 protein is a cationic cysteine-rich polypeptide that exists as a secreted monomer, with four N-linked glycosylation sites. Both the mouse and human proteins are 144 amino acids in length, with a signal peptide of 18 residues and a molecular weight of approximately 16 kDa.

activate the T cell to proliferate and produce more IL-9, along with other roles.

Receptors
IL-9 binds to the IL-9 receptor (IL-9R), which is a 54 kDa polypeptide associated with 10 kDa N-linked sugars, spanning the cell membrane. The receptor peptide is 468 and 521 amino acids in length, in humans and mice respectively, and belongs to the hematopoeitin receptor superfamily. In mice, a signal peptide exists between residues 15 and 40; there is a transmembrane domain between residues 271 and 291 and an extracellular domain, which comprises 233 amino acids with two potential N-linked glycosylation sites (Figure 2). The receptor is constitutively bound to Janus kinase 1 (JAK1), which allows induction of several different signaling pathways, leading to transcription of further genes. The region required for JAK binding in the IL-9 receptor is the Box1 motif, a proline-rich region close to the transmembrane domain. The IL-9 specic chain can bind IL-9 with high afnity, but cannot mediate any signal on its own. The functional IL-9 receptor consists of the IL-9R described above and the common cytokine receptor g

Cellular Synthesis
IL-9 is synthesized by activated T cells, which participate in an adaptive immune response. T cells develop in the thymus, after which they enter the bloodstream and circulate to peripheral lymphoid organs. Following exposure to specic antigens, the T cells develop into armed effector T cells which are induced to proliferate and differentiate into cells that contribute to the elimination of pathogens. T cells fall into two classes, CD4 or CD8 T cells. CD4 T cells undergo differentiation into either T-helpertype-1 (Th1) or T-helper-type-2 (Th2) cells. Th2 cells produce IL-9 in response to pathogen exposure. Following synthesis in the endoplasmic reticulum and posttranslational modications, IL-9 molecules are secreted and act in an autocrine manner to further

Extracellular domain c Chain Hematopoietic receptor consensus sequence

Cell membrane

Cytoplasmic domain

Box1 motif

Transmembrane domain

Figure 2 Structure of the IL-9R chain of the IL-9 receptor, which comprises the IL-9R chain and the gc chain.

INTERLEUKINS / IL-9

371

chain, gc, which is a shared component of receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. While the IL9R binds JAK1, the gc chain binds JAK3.

Table 1 Roles of IL-9 in various cell types and processes Biological process Hematopoiesis Cells/systems affected Erythroid and myeloid precursors Mast cells B cells T cells Th2 and other cytokines Tumorigenesis T lymphoma Hodgkins lymphoma Pulmonary response/ asthma Mast cells Effect Clonogenic differentiation

Biological Function and Mode of Action

To exert its effects, IL-9 rst binds to its receptor (IL-9R). As described previously, the receptor comprises two separate chains, the IL-9 specic receptor and the gc chain. Upon receptor ligation, a change in receptor conformation leads to auto and/or transphosphorylation of JAK kinases, phosphorylation of the receptor, and activation of one of three different pathways involved in mediation of IL-9 intracellular signaling: the signal transducer and activator of transcription (STAT) pathway, the PI3/ IRS-1/2 pathway, and the mitogen-activated protein(MAP) kinase pathway, all of which are involved in cell differentiation, proliferation, and cell death and development. The STAT pathway involves signal transduction and transcription factors 1, 3, and 5. Upon ligand binding to the IL-9 receptor, the residue tyrosine 407 in the IL-9R chain is phosphorylated and serves as a docking site for the SH2 domain of the STAT transcription factors. The STAT factors are then phosphorylated, inducing their dimerization, migration to the nucleus, and binding to specic DNA sites in the promoter of cytokine-regulated genes. The PI3 pathway is mediated by phosphatidylinositol 3-kinase (PI3K), belonging to a family of lipid and serine/threonine kinases. Various cytokines including IL-9 recruit PI3K to the plasma membrane where it phosphorylates phosphoinositides, which function as important second messengers for intracellular signaling in several processes. The IL-9R cannot recruit PI3K directly owing to a lack of PI3K binding sites; however, the Box1 domain and a downstream region are required for insulin receptor substrate 2 (IRS-2) activation, which leads to PI3K activation via its regulatory subunit, p85. IL-9 induces IRS-2 phosphorylation and association with the PI3K p85 subunit, and cell proliferation is induced. The IL9-induced MAP kinase pathway is not as prominent in IL-9 signaling as the other two pathways, and is restricted to only certain cell types. The signaling cascade is linked to IL-9R through the adaptor protein, Shc. Shc phosphorylation is thought to lead to activation of rat sarcoma (RAS) GTPases, and the mitogenic cascade is mediated further through the phosphorylation and activation of Raf-1 and Map/Erk kinase 1/2 (MEK1/2) extracellular signal-regualted kinases 1 and 2 (ERK1 2) leads to activation of the MAP kinase pathway. IL-9 affects a variety of cell types (Table 1). It is produced by Th2 cells and acts in an autocrine manner to stimulate the proliferation of T cells as
Immunity

Proliferation and differentiation Ig production Proliferation and differentiation Increased expression

Survival and proliferation Survival and proliferation Proliferation

Goblet cells Th2 cytokines Other Neurones

Proliferation Increased expression Neuronal precursor differentiation

part of the humoral immune response. The existence of specic high-afnity receptors for IL-9 has been demonstrated in T cells, mast cells, macrophages, and tumor T cells. IL-9 is also involved in T-cell stimulation, erythroid differentiation, antibody production, and neuronal differentiation. Blockade of the IL-9R a-chain resulted in a severe reduction in the number of human thymocytes, with a major decrease in the number of cells maturing from thymic precursor cells, illustrating that IL-9R signaling is essential in early T-lymphoid development. Injection of mice with T cells transfected with IL-9 led to widespread T-cell proliferation in vivo, illustrating IL-9s role as a T cell growth factor. In IL-9 lung-specic transgenic mice, the Th2 cytokines IL-4, IL-5, and IL-13 (Figure 3) were expressed in response to IL-9, demonstrating its role in potentiating a Th2 response. IL-9 induces mast cell proliferation and differentiation and stimulates expression of mast cell proteases. In IL-9-decient mice, a decrease in mastocytosis was observed, demonstrating a specic role for IL-9 in mast cell proliferation. It is also required for an efcient mast cell-mediated immune response against intestinal parasites such as Trichuris muris. IL-9 has also been implicated in IL-4-induced IgG1 and IgE production in B cells. IL-9 has also been demonstrated to have an antiapoptotic role in that it was shown to protect a murine thymic lymphoma cell line from apoptosis

372 INTERLEUKINS / IL-9

Mast cell hyperplasia

Airway eosinophilia

Increased Th2 cytokine expression, including IL-4, IL-5, IL-13 Increased IL-9

Goblet cell hyperplasia Mucus hyperproduction

Figure 3 IL-9 overexpression, both systemically and lung-specifically, has been shown to lead to various asthmatic symptoms, including, (a) increased mast cell hyperplasia, proliferation, and differentiation; (b) increased eosinophil and lymphocyte inltration; (c) increased cytokine expression; and (d) goblet cell hyperplasia and mucus hyperproduction.

induced by dexamethasone and maintained cellular proliferation in its presence.

Regulation of IL-9 Activity

IL-9 signal attenuation (and hence modulation of its activity) occurs mainly through the activity of the suppressors of cytokine signaling (SOCS) or cytokine-inducible SH2-containing proteins (CIS), which comprise a family of signaling inhibitors. Expression of these inhibitors is induced by several cytokines and negatively regulates signal transduction. In a T-cell lymphoma line, IL-9 induced the rapid expression of CIS, SOCS-2, and SOCS-3. STAT activation was shown to be required for CIS/SOCS induction using IL-9R mutants. Each of these inhibitor proteins was used to transiently transfect a cell line with demonstrated IL-9 and STAT activity, with only SOCS3 being able to inhibit IL-9-induced signal transduction as well as downstream effects such as STAT activation, gene induction, and antiapoptotic activity.

IL-9 in Respiratory and Other Diseases

The rst association of IL-9 with disease pathology was made through the observation of elevated IL-9 expression in human malignant lymphomas, with lymph nodes from patients with Hodgkins disease (HD) and large cell anaplastic lymphoma (LCAL) constitutively producing IL-9. Using patient-derived cell lines, elevated IL-9 expression was demonstrated in two of six cases of LCAL and 6 of 13 cases of HD.

Increased levels of IL-9 were detected in the sera from 18 out of 44 HD patients but not in the sera from healthy controls. IL-9 mRNA was also detected in 42% of HD cases via Northern blot analysis. In vitro, IL-9 induces the proliferation of murine T lymphoma and myeloid leukemia cells: in one of the studies, IL-9 significantly stimulated the proliferation of 26 of 45 mouse thymic lymphoma cell lines, induced by mutagen or X-ray treatment. IL-9 transgenic mice were found to develop thymic lymphomas spontaneously 39 months after birth, implicating IL-9 as a potential tumorigenic factor. In another study, more than 85% of IL-9 transgenic mice were found to have a severe lymphoproliferative disorder resulting in highly enlarged lymphoid tissues and lymphocyte inltration into other tissues. Various studies have demonstrated a link between asthma and IL-9. It was originally designated as a risk factor for asthma since its expression was markedly reduced in bronchial hyporesponsive mice. IL-9 has been shown to stimulate goblet cell proliferation in differentiating primary cultures of human airway epithelia, and has also been implicated in repair of injured epithelia, both characteristics of asthma. A significant difference in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. The proportion of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects. Mice overexpressing IL-9, either systemically

INTERLEUKINS / IL-10 373

or lung-specifically, developed more severe asthmatic symptoms than wild-type littermates. IL-9 expression in the lungs of such mice was shown to cause various asthmatic symptoms, including airway inammation, mast cell hyperplasia, mucus hyperproduction, eosinophil and lymphocyte inltration into lung tissues, and bronchial hyperresponsiveness (Figure 3). Mice decient in IL-9 have decreased goblet cell proliferation and mast cell proliferation in response to a pulmonary challenge, illustrating IL-9s essential role in the rapid onset of these two phenomena in a pulmonary Th2 response.
See also: Histamine. Interleukins: IL-4; IL-5; IL-13; IL16. Leukocytes: T cells.

illness-induced immunosuppression and matrix deposition/ remodeling in interstitial lung disease. Thus, IL-10 is clearly a complex molecule with potential therapeutic uses in modulating inammation, but its salutary anti-inammatory effects must be weighed against its potentially stimulatory effects on cytotoxic activity and adverse impacts on host defense and brosis.

Introduction
Interleukin-10 (IL-10) is an immunomodulatory cytokine with pleiotrophic activities. When initially discovered in 1989, IL-10 was named cytokine synthesis inhibitory factor because of its ability to inhibit the production of proinammatory cytokines, including interferon gamma (IFN-g), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-a) by various cell types. Subsequently, a viral homolog of IL-10 was identied in EpsteinBarr virus (EBV), which was believed to facilitate immune evasion, thereby enabling EBV to survive in the host and promote the development of B cell lymphoma. While the predominant role of IL-10 is to suppress inammation, this cytokine also possesses immunostimulatory properties for certain cell types, including cytotoxic T cells and natural killer (NK) cells. Thus, it is evident that this molecule displays a complex array of biological activities effects that are dependent upon the timing of its release, cellular targets, and the immune context within which this cytokine is acting.

Further Reading
Benczik M and Gaffen SL (2004) The interleukin (IL)-2 family cytokines: survival and proliferation signalling pathways in t lymphocytes. Immunological Investigations 33(2): 109142. Demoulin JB and Renauld JC (1998) Interleukin 9 and its receptor: an overview of structure and function. International Reviews of Immunology 16: 345364. Knoops L and Renauld JC (2004) IL-9 and its receptor: from signal transduction to tumorigenesis. Growth Factors 22(4): 207215. Skinnider BF and Mak TW (2002) The role of cytokines in classical Hodgkin lymphoma. Blood 99(2): 42834297. Uyttenhove C, Simpson RJ, and Van Snick J (1988) Functional and structural characterization of p40, a mouse glycoprotein with t-cell growth factor activity. Proceedings of National Academy of Sciences 85: 69346938.

Structure
The IL-10 gene locus spans approximately 2 kb and resides on chromosome 1 in humans and mice. The IL10 gene is composed of ve exons, with sequences that are involved in transcriptional regulation (e.g., binding sites for transcriptional factors nuclear factor kappa B (NF-kB) and AP-1; see Figure 1). No splice variants are known to exist. The IL-10 protein is synthesized as a monomer composed of six a-helices, with a molecular weight of 18.5 kDa, but mainly exists in its biologically active homodimeric form. Murine and human IL-10 share approximately 72% sequence homology, with limited crossreactivity as described below. At low protein concentrations, the IL-10 dimer is relatively unstable. Its activity may therefore be curtailed by this instability, in that IL-10 may only exert its effects locally where the concentrations of secreted protein are high. The IL-10 interdomain angle is approximately 901, which reduces the number of hydrophobic interdomain interactions, rendering this cytokine relatively hydrophilic.

IL-10
T J Standiford and J C Deng, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-10 (IL-10) is an immunomodulatory cytokine that regulates important aspects of innate and cell-mediated immunity. It is synthesized by hematopoietic and nonhematopoietic cells, including macrophages and epithelial cells within the lung. The biologically active form of IL-10 is a homodimer that binds to the IL-10 receptor, which is expressed on leukocytes as well as structural cells. The primary function of this cytokine is to downregulate inammation via inhibition of cytokine production and other inammatory mediators. IL-10 is secreted in response to acute inammation and under conditions that promote tolerance or anergy. The absence of IL-10 has been implicated in the pathogenesis of uncontrolled inammation during the development of acute respiratory distress syndrome, chronic Pseudomonas colonization in cystic brosis, and allergic airways disease. However, IL-10 also possesses immunostimulatory properties for cytotoxic T cells and natural killer cells. Furthermore, IL-10 has been implicated as a mediator of critical

Regulation of Production and Activity

IL-10 is produced by a variety of cell types, including CD8 and CD4 T cells (e.g., T-helper-2 (Th2)

374 INTERLEUKINS / IL-10

TATA I 2+4 200 AP-1 binding 10+4 1084 NF- B like reg 1531 1535 1563 1795 2680 II III 2739 3045 3197 4205 IV 4270 5892 V 6686

Figure 1 Schematic depicting human IL-10 gene structure and putative regulatory elements.

cells, regulatory T cells), gd-T cells, NK cells, NK T cells, B cells, dendritic cells, eosinophils, mast cells, and monocytic cells (e.g., microglia, monocytes, macrophages). Macrophages are the major source of IL-10, although regulatory T cells are now recognized as an important subpopulation of T cells that release IL-10. Nonleukocyte cell populations, including epithelial cells, keratinocytes, and melanoma cells, also express IL-10. In the lung, human alveolar macrophages (AMs), bronchial epithelial cells, and alveolar epithelial cells have been shown to express IL-10 mRNA and protein. In contrast, murine resident AMs secrete minimal amounts of IL-10, although these cells do express IL-10 receptors. Macrophages produce IL-10 in response to inammatory or infectious stimuli, including lipopolysaccharide, TNF-a, and catecholamines. High levels of IL-10 are also produced under conditions that induce anergy and tolerance, such as repeated antigen stimulation. In these contexts, IL-10 likely provides negative feedback to dampen the magnitude of immune responses. Interestingly, several viruses, such as EBV, produce IL-10 or IL-10-like molecules (vIL-10). The genomes of other viruses, including members of the herpesvirus family, poxvirus, and primate cytomegaloviruses (CMV), also contain homologs of the IL-10 gene. Many vIL-10 proteins share extensive sequence homology with human IL-10. Viral IL-10 appears to engage the same IL-10 receptor in the host and is capable of producing some of the same biological effects as host-derived IL-10. Given the anti-inammatory properties of IL-10, vIL-10 likely confers a survival advantage for these viruses by suppressing antiviral host immune responses.

Biological Function
IL-10 is a critical regulator of innate and cellmediated immune responses. The main cellular targets of IL-10 are lymphocytes and antigen-presenting cells. In early studies, IL-10 was found to inhibit cytokine synthesis by T cells, which was attributed to its ability to inhibit antigen presentation by macrophages and dendritic cells. Subsequent studies have demonstrated that IL-10 can suppress cytokine production directly by inhibiting inhibitor kappa B (IkB) kinase activation and blocking NF-kB DNA

binding, thereby inhibiting NF-kB activity. IL-10 also inhibits inammatory cytokine signaling by activating suppressors of cytokine signaling (SOCS)-1 and SOCS-3, which suppress Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cytokine signaling pathways. Importantly, IL-10 appears to be a potent inhibitor of type-1 immune responses. Mice decient in IL-10 (IL-10 / ) have augmented Th1 responses, as evidenced by the enhanced ability of these animals to clear both bacterial and fungal pathogens. IL-10 also suppresses the production of IL-12 and interferon-inducible protein-10 (IP-10) and counteracts the effects of IFN-g, all of which are important type 1-associated cytokines. In activated monocytes and macrophages, IL-10 inhibits the synthesis of many inammatory mediators, including cytokines and reactive nitrogen species instrumental to the generation of protective innate and cell-mediated immune responses in the lung. Conversely, IL-10 directly stimulates the expression of IL-1 receptor antagonist and soluble TNF receptors, molecules that antagonize the effects of selected proinammatory cytokines. IL-10 has similar anti-inammatory effects on neutrophils. In addition, IL-10 impairs the ability of monocytes and macrophages to present antigen and activate T cells by decreasing expression of MHC class II molecules, CD52 (ICAM-1), and the costimulatory molecules CD80 and CD86. Furthermore, IL-10 inhibits monocyte differentiation into dendritic cells, further suppressing the antigen-presenting capabilities of the innate immune system. Finally, IL-10 enhances the phagocytic ability of monocytes and macrophages, but suppresses microbicidal activity of these cells. In addition to prominent inhibitory effects on monocyte/macrophage effector cell functions, IL-10 also exerts suppressive effects on selected T-cell activities. For instance, IL-10 inhibits mitogen-induced proliferation and cytokine production (e.g., IL-2, IFN-g) by CD4 T cells. IL-10 also promotes the development of regulatory CD4 T cells, cells that are instrumental in the development of immune tolerance. Inhibitory effects of IL-10 on CD8 T cells are less pronounced and, under certain conditions, IL-10 can even activate CD8 T-cell populations, including cytotoxic T cells. Likewise, IL-10 can stimulate NK cells to produce IFN-g, TNF-a, and granulocytemacrophage colony-stimulating factor (GM-CSF).

INTERLEUKINS / IL-10 375

Innate immunity Differentiation Inflammatory cytokine secretion

Acquired immunity Inflammatory cytokine production Mitogen-induced proliferation Th0 Th2 phenotype

Dendritic cell

Monocyte

Th cell

Phagocytosis Microbicidal activity

Inflammatory cytokine secretion Antigen Macrophage presentation Treg cell IL-10

Treg phenotype development

Inflammatory cytokine secretion Neutrophil Cytokine production Cytoxicity NK cell Structural cells Cytokine production Collagen production MMP production B cell Tc cell
YY Y

Cytotoxic activity Proliferation

YY

Proliferation Immunoglobulin secretion

Epithelial cell fibroblast

Figure 2 Overview depicting the biological effects of IL-10. Treg, regulatory T cell; Tc, cytotoxic T cell.

IL-10 also stimulates mast cell growth, B-lymphocyte proliferation, and immunoglobulin secretion. IL-10 exerts diverse effects on functions of structural cells. Similar to effects in macrophages, IL-10 inhibits the expression of inammatory cytokines from various parenchymal and stromal cell populations, including alveolar and airway epithelial cells and broblasts. This cytokine also appears to modulate matrix production and remodeling. In isolated broblast populations, IL-10 generally inhibits collagen and bronectin expression. Effects on the release of matrix metalloproteinase proteins (MMPs) involved in matrix remodeling are varied, as IL-10 stimulates MMP-1 and stomelysin release, whereas MMP-9 expression is diminished in the presence of IL-10. Therefore, given the complexity of IL-10 effects on immune and structural cell responses, it is more accurate to consider IL-10 as an immunomodulatory cytokine, rather than simply an immunosuppressive molecule. A schematic depicting the biological effects of IL-10 is shown in Figure 2.

most hematopoietic cells and a few nonhematopoietic cell types (e.g., broblasts, epithelial cells), IL10R1 binds ligand and has a predicted molecular weight of 90120 kDa. IL-10R1 binds both cellular and viral IL-10 proteins, but its afnity for vIL-10 is approximately 1000-fold less than cell-derived IL10. The IL-10R1 receptor exhibits some species specicity. For example, murine IL-10R1 binds both human and murine IL-10, whereas the human receptor binds only human IL-10. IL-10R2 initiates signal transduction upon IL-10 binding via the JAK/ STAT pathway, using the Jak1 and Tyk3 kinases and STAT3 transcription factor. Expressed on most cell types, IL-10R2 also complexes with the IL-22, IL-28A, IL-28B, and IL-29 receptors. Although no splice variants have been found for either IL-10R1 or IL-10R2 mRNA, it is plausible that these variants exist. In particular, the removal of exon 6 or 7 could result in the formation of a soluble IL-10 receptor. An engineered soluble IL-10R has displayed antagonist activity.

Receptors
The IL-10 receptor complex is composed of four transmembrane chains, two of which comprise IL10R1 and two of which form IL-10R2. Expressed on

IL-10 in Respiratory Disease States

Acute Lung Injury

IL-10 is produced by many cells of the lung in respiratory disease states, although the exact role of this

376 INTERLEUKINS / IL-10

cytokine in disease pathogenesis is unclear. In general, IL-10 appears to play a pivotal role in controlling the magnitude of inammatory responses during either systemic or pulmonary inammation. In the gut, for example, mice lacking the IL-10 gene (IL-10 / ) develop spontaneous enterocolitis that mimics inammatory bowel disease due to the unchecked immune responses to endogenous bowel ora. Induction of sepsis (either due to endotoxin administration or cecal ligation and puncture) results in the systemic production of IL-10, and blockade of IL-10 during the septic response dramatically enhances sepsis-induced inammatory cytokine release, tissue injury, and mortality. Similarly, IL-10 / mice have prolonged inammation following acute intrapulmonary challenge with cytotoxic strains of Pseudomonas aeruginosa. Conversely, endotoxin-induced tissue injury in animal models of sepsis can be substantially reduced by the administration of recombinant IL-10. IL-10 is expressed in the airspace of patients with acute respiratory distress syndrome (ARDS), and lower levels of IL-10 in bronchoalveolar lavage uid have been associated with increased mortality in these patients. These data suggest that insufcient IL-10 responses may contribute to enhanced and/or persistent lung inammation in ARDS, which in turn may lead to adverse clinical outcomes.
Lung Host Defense

Airways Disease

While the endogenous production of IL-10 is required in order to appropriately compartmentalize inammatory responses, the IL-10-mediated suppression of cytokines involved in innate and acquired immunity can substantially impair antimicrobial host defense mechanisms. For instance, virulent bacterial pathogens such as Klebsiella pneumoniae and Streptococcus pneumoniae require a robust innate immune response for effective clearance from the lung, and the endogenous production of IL-10 significantly impairs host antibacterial immunity against these respiratory pathogens. IL-10 also suppresses cellmediated responses against intracellular pathogens of the lung, including fungi and Mycobacterium spp. Moreover, IL-10 appears to be a major mediator of leukocyte deactivation that occurs in states of critical illness, most notably sepsis. In particular, the release of IL-10 during the septic response causes alveolar macrophage dysfunction and resultant impairment of pulmonary host defenses. Collectively, these observations illustrate the need for a balanced IL-10 response in order to limit inammation while preserving sufcient host immune function against microbial challenge.

Mounting evidence suggests that IL-10 plays a fundamental role in shaping immune responses within the airway. Studies in animal models demonstrate that IL-10 promotes the development of tolerance to inhaled allergen challenge, resulting in decreased eosinophilic inammation and airway hyperreactivity. The mechanism of protection is due to suppression of type 2 cytokine release (e.g., IL-4, IL-5, and IL-13) and resultant attenuation of allergic airways inammation. In allergic diseases, including asthma, the severity of disease appears to be inversely correlated with local IL-10 levels. The expression of IL-10 is elevated in some patients with asthma, with further upregulation by allergen challenge, perhaps released in order to counterbalance the actions of type 2 cytokines. This cytokine also participates in control of airway inammation in response to infectious challenge of the respiratory tract. For example, prolonged and more severe airway inammation is observed in IL-10-decient mice during chronic endobronchial Pseudomonas infection, as compared to wild-type animals, and insufcient IL-10 responses may account for the persistent airway inammation seen in patients with cystic brosis. Indeed, patients with cystic brosis have lower levels of airway epithelial cell-derived IL-10, as compared to healthy subjects. Similarly, in patients with chronic obstructive pulmonary disease (COPD), IL-10 levels in sputum are diminished compared to healthy controls. As IL-10 has been shown to inhibit MMP-9 release, reductions in IL-10 observed in COPD patients may promote a lung microenvironment characterized by accelerated parenchymal destruction. Taking into account the dominant anti-inammatory properties of IL-10, exogenous administration of this cytokine has been tested and found to be benecial in several immune-mediated diseases, including inammatory bowel disease, rheumatoid arthritis, and psoriasis. Given the putative roles that IL-10 plays in sepsis and sepsis-induced lung injury, allergic airways disease, and COPD, it is possible that IL-10 may be a therapeutic target in the development of immunomodulatory therapies for these diseases. The potential benecial effects of IL-10 immunotherapy must be reconciled with the less desirable effects on immune host defenses and selective T and NK cell activation.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-12. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: T cells; Pulmonary Macrophages.

INTERLEUKINS / IL-12 377

Further Reading
Asadullah K, Sterry W, and Volk HD (2003) Interleukin-10 therapy review of a new approach. Pharmacological Reviews 55(2): 241269. Barbarin V, Arras M, Misson P, et al. (2004) Characterization of the effect of interleukin-10 on silica-induced lung brosis in mice. American Journal of Respiratory Cell and Molecular Biology 31: 7885. Barnes PJ (2003) Cytokine-directed therapies for the treatment of chronic airway diseases. Cytokine and Growth Factor Reviews 14: 511522. Bergeron A, Soler P, Kambouchner M, et al. (2003) Cytokine proles in idiopathic pulmonary brosis suggest an important role for TGF-b and IL-10. European Respiratory Journal 22: 6976. Chmiel JF, Konstan MW, Saadane A, et al. (2002) Prolonged inammatory response to acute Pseudomonas challenge in interleukin-10 knockout mice. American Journal of Respiratory and Critical Care Medicine 165: 11761181. Conti P, Kempuraj D, Kandere K, et al. (2003) IL-10, an inammatory/inhibitory cytokine, but not always. Immunology Letters 86: 123129. Goodman RB, Pugin J, Lee JS, and Matthay MA (2003) Cytokinemediated inammation in acute lung injury. Cytokine and Growth Factor Reviews 14: 523535. Hawrylowicz CM and OGarra A (2005) Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma. Nature Reviews: Immunology 5(4): 271283. Kay AB (2003) Immunomodulation in asthma: mechanisms and possible pitfalls. Current Opinion in Pharmacology 3: 220226. Kim JM, Brannan CL, Copeland NG, et al. (1992) Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. Journal of Immunology 148: 36183623. Lynch EL, Little FF, Wilson KC, Center DM, and Cruikshank WW (2003) Immunomodulatory cytokines in asthmatic inammation. Cytokine and Growth Factor Reviews 14: 489502. Mocellin S, Panelli MC, Wang E, Nagorsen D, and Marincola FM (2003) The dual role of IL-10. Trends in Immunology 24(1): 3643. Moore KW, Malefyt RD, Coffman RL, and OGarra A (2001) Interleukin-10 and the interleukin-10 receptor. Annual Review of Immunology 19: 683765. Oberholzer A, Oberholzer C, and Moldawer LL (2002) Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inammatory drug. Critical Care Medicine 30(supplement 1): S58S63. Oltmanns U, Schmidt B, Hoernig S, Witt C, and John M (2003) Increased spontaneous interleukin-10 release from alveolar macrophages in active pulmonary sarcoidosis. Experimental Lung Research 29: 315328. Pestka S, Krause CD, Sarkar D, et al. (2004) Interleukin-10 and related cytokines and receptors. Annual Review of Immunology 22: 929979.

proteins, termed the IL-12 related cytokines. The IL-12 related cytokines are generated from ve independently regulated genes that homodimerize and heterodimerize to form seven secreted family members. These proteins have overlapping as well as distinct biologic properties and this article focuses specifically on IL12, p80, and p40. IL-12, a disulde-linked heterodimer composed of a p40 and p35 subunit, has been identied as an immune cell stimulator that promotes differentiation and proliferation of T cells and enhances the production of interferon gamma. Human studies and murine experimental models have implicated IL-12 in the regulation of multiple immunologic processes including regulation of CD4 and CD8 T-cell differentiation, host defense, autoimmunity, tumor cell immune recognition, alloimmunity, and host response to vaccine administration. p80 is a disulde-linked p40 homodimer that can function as a competitive antagonist of IL-12 and a macrophage chemoattractant. Specic topics discussed in this article include the structure, regulation, function, receptor utilization, and role of IL-12 related cytokines in respiratory disease.

Introduction
IL-12 is a heterodimeric cytokine with broad immunoregulatory properties that belongs to a larger family of related proteins called the IL-12 related cytokines. IL-12 has been identied as an immune cell stimulator that promotes differentiation and proliferation of T cells and enhances the production of interferon gamma (IFN-g). Since its discovery, IL-12 has been implicated in the regulation of multiple immunologic processes that include regulation of CD4 and CD8 T-cell differentiation, host defense, autoimmunity, tumor cell immune recognition, alloimmunity, and host response to vaccine administration. Molecular cloning revealed that IL-12 was a heterodimeric cytokine composed of two disulde-linked glycoproteins. These individual subunits were named p40 and p35 according to their electrophoretic molecular weight. A p40 homolog named EpsteinBarr virus-induced gene 3 (EBI3), and two p35 homologs named p19 and p28 have been identied. These ve independently regulated genes encode the proteins that comprise the IL-12 related cytokines. Due to monomer secretion as well as homodimeric and heterodimeric partnering, the IL-12 family currently consists of seven secreted proteins: IL-12 (a p40/p35 heterodimer), p80 (a p40/p40 homodimer), p40 (a p40 monomer), EBI3/p35 (an EBI3/p35 heterodimer), EBI3 (an EBI3 monomer), IL-23 (a p40/p19 heterodimer), and IL-27 (EBI3/p28 heterodimer) (Table 1). These family members have overlapping as well as distinct biologic properties. This article focuses specifically on the IL-12, p80, and p40 family members.

IL-12
M J Walter, Washington University School of Medicine, St Louis, MO, USA
& 2006 Elsevier Ltd. All rights reserved.

Structure of IL-12 Abstract

Interleukin-12 (IL-12) is a heterodimeric cytokine with broad immunoregulatory properties that belongs to a larger family of

The human p40 gene was originally termed IL-12B and is located within a cytokine gene cluster on chromosome 5q31. The gene encodes a 328 amino

378 INTERLEUKINS / IL-12

Table 1 Characteristics of IL-12 related cytokines IL-12 member IL-12 p80 p40 IL-23 EBI3 EBI3/p35 IL-27
a b

Protein components p35 and p40 p40 and p40 p40 p19 and p40 EBI3 EBI3 and p35 EBI3 and p28

Receptors IL-12Rb1 and IL-12Rb2 IL-12Rb1 IL-12Rb1 IL-12Rb1 and IL-23R ND ND TCCR and gp130

Receptor afnity (humana/mouseb) 4-7/2-3 20-70/4 400-700/25-100 ND ND ND ND

Biologic functions Proliferation and differentiation of T cells IFN-g secretion from T/NK cells IL-12 antagonist Macrophage chemotaxis ND Proliferation of memory T cells IFN-g, IL-17A, IL-17F secretion from T cell ND ND Proliferation of naive T cells IFN-g secretion from T cells

Inhibitory concentration (IC) 50% (ng ml 1) in KIT225/K6 cells. IC 50% (ng ml 1) in mouse concavalin A blasts. ND, not determined. EBI3, EpsteinBarr virus-induced gene 3; IFN-g, interferon gamma; IL, interleukin; IL-12Rb1, IL-12 receptor b1; NK, natural killer; TCCR, T-cell cytokine receptor; gp130, glycoprotein 130.

acid polypeptide which contains a 22 amino acid signal peptide, four potential N-glycosylation sites, four intramolecular disulde bonds, one free cysteine, and one cysteine that forms a disulde bond with p35. The p40 protein shares homology with the extracellular domain of the hematopoietic cytokine receptor family and is most closely related to the IL-6 receptor a-subunit. Although related to this receptor family, the p40 protein does not contain a transmembrane domain and membrane-bound p40 has not been identied. Murine p40 is located on chromosome 11 in a cytokine gene cluster that is equivalent to the human locus and is 70% homologous to human p40. The human p35 gene was originally named IL-12A and is located on chromosome 3. The gene encodes a 253 ammo acid with two separate initiation codons. Initiation at the second methionine results in a 22 amino acid hydrophobic region that is characteristic of a signal peptide, whereas initiation at the rst methionine results in a longer but less hydrophobic region that may serve to anchor p35 to the cell membrane. The p35 protein is an a-helix-rich structure that contains three potential N-glycosylation sites, three intramolecular disulde bonds, and one cysteine that forms a disulde bond with the p40 protein. p35 does not have any sequence homology with p40 but does share high homology with IL-6 and granulocyte colony stimulating factor (G-CSF). Murine p35 is located on chromosome 3 and is 67% homologous to human p35.

Regulation of IL-12 Production

Numerous cell types can produce IL-12 and many stimuli are involved in the regulation of its production.

IL-12 production has been found in dendritic cells, monocytes, macrophages, neutrophils, keratinocytes, and airway epithelial cells. These cell types are integral members of the innate immune response, suggesting a critical role for IL-12 in this arm of the immune system. Secretion depends on simultaneous expression of both p40 and p35 subunits, and cell-specic differences in the expression of p40, p35, and IL-12 have been noted. Cytokines, microbiologic products, and costimulatory protein ligation induce p40 gene transcription and the secretion of IL-12 correlates closely with p40 induction. p35 expression is constitutive and regulated through transcriptional and translational mechanisms; however, p35 monomer is not secreted. Although initial reports indicated p35 expression was not enhanced by inammatory cytokines, recent reports demonstrate IFN-g priming can result in lipopolysaccharide (LPS)-dependent interferon regulator factor (IRF)-1-driven p35 gene transcription. While a large number of mediators are involved in activating IL-12 expression, an equally large number of inammatory cytokines, chemoattractants, and G-protein-coupled receptors have been shown to inhibit its production (Table 2). A p40 monomer can dimerize with p35 to form IL-12, but it can also be secreted as a p40 monomer, homodimerize with itself to form p80, or heterodimerize with p19 to form IL-23. Many stimuli that induce p40 result in the secretion of large molar excesses of p40 monomer and p80 compared to IL-12. The cellular mechanisms that control p40 monomer secretion and the disulde linking of p40 with either p35, p40, or p19 are not known. The cellspecic responses to the wide variety of activating and inhibitory mediators and the ability of p40 to couple with different partners likely provides the

INTERLEUKINS / IL-12 379

Table 2 Regulation of IL-12 secretion IL-12 activators IFN-g IL-4 (o24 h) IL-13 (o24 h) Bacterial DNA CpG oligonucleotides Lipopolysaccharide Brucella Viruses Double-stranded RNA Fungi Intracellular parasites Toxoplasma CD40 ligation IL-12 inhibitors IFN-a IFN-b IL-4 (424 h) IL-13 (424 h) IL-10 Transforming growth factor beta Monocyte chemoattractant protein 1-4 C5a FMLP Prostaglandin E2

fMLP, formylmethionylleucylphenylaline.

immune system with numerous regulatory checkpoints to nely control the temporal and spatial concentrations of the IL-12 related cytokines.

Biologic Function of IL-12, p80, and p40

IL-12 evokes potent immunoregulatory properties by promoting CD4 T-helper-1 (Th1) cell differentiation, inhibiting CD4 Th2 cell differentiation and enhancing immune cell production of IFN-g. IL-12 is required for the development of an appropriate Th1 response to some, but not all, microbial pathogens. For example, IL-12 is required for the development of a Th1 response to Leishmania major, Mycobacterium tuberculosis, Histoplasma capsulatum, Salmonella, and Klebsiella pneumoniae. By contrast, IL-12 is not required for the Th1 response to lymphocytic choriomeningitis virus, vesicular stomatitis virus, mouse hepatitis virus, and mouse parainuenza virus. In the context of a murine Th1 response to pathogens, exogenously administered recombinant IL-12 can provide protection or cause enhanced inammation depending on the microbial challenge and administration regimens. Additional studies have demonstrated the inuence of IL-12 during a Th2 immunologic response. For example, in mice recombinant IL-12 administration concurrent with allergen sensitization or challenge prevented airway hyperreactivity, eosinophil recruitment, and IL-4 and IL-5 production while it increased IFN-g levels. In addition, IL-12 blockade with neutralizing antibody at the time of allergen challenge allowed for the development of a Th2 response in an otherwise resistant mouse strain and increased the Th2 response in a sensitive strain. Ovalbumin sensitized and challenged p40 decient mice develop an enhanced Th2 response with increased airway, peripheral blood and bone marrow eosinophils and

increased IL-4 and IgE concentrations, although this effect is not found in all studies. p80 has an afnity for IL-12Rb1 that is comparable to IL-12. Originally, p80 was found to be a competitive antagonist for IL-12 binding, but recent experiments indicate p80 has distinct functions that are independent from the other IL-12 related cytokines. As an IL-12 antagonist, p80 can effectively block in vitro IL-12-dependent IFN-g production, proliferation of lymphocytes, and natural killer (NK) cell activation. Similarly, exogenously administered p80 can inhibit murine endotoxin-induced IFN-g production and decrease mortality. Transgenic mice with p40 and p80 overexpression in the liver exhibit a blunted Th1 immune response to Plasmodium berghei, and selective overexpression of p40 and p80 in the basal epithelial cells of the skin generates an inammatory skin lesion resembling eczema. Recombinant p80, but not IL-12 or p40, signals through IL-12Rb1 to cause macrophage chemotaxis and can induce inducible nitric-oxide synthetase expression and activate nuclear factor kappa B (NF-kB) in isolated mouse peritoneal macrophages and microglial cells. Although p40 monomer is secreted, its afnity for IL-12Rb1 is at least 100-fold less than IL-12 and a biologic role for this protein has not been identied.

IL-12 Receptors
The high-afnity receptor for human IL-12 is composed of two independently regulated type I transmembrane glycoproteins referred to as IL-12 receptor b1 (IL-12Rb1) and IL-12Rb2. Both of these receptors belong to the hematopoietin receptor superfamily and share homology with gpl30, G-CSF, and leukemia-inhibitory factor receptors. The human IL-12Rb1 gene is located on chromosome 19pl3 and encodes a 662 amino acid polypeptide with a 24 amino acid signal peptide, a 516 amino acid extracellular domain, a 31 amino acid transmembrane domain, and a 91 amino acid cytoplasmic tail. IL12Rb1 expression is conned to immune cells and is found on T cells, NK cells, B cells, dendritic cells, and macrophages. The extracellular cytokine-binding domain interacts with the p40 subunit of IL-12 and the cytoplasmic portion interacts with the Janus kinase family member tyrosine kinase 2 (Tyk2). The mouse IL-12Rb1 gene encodes a 738 amino acid polypeptide with 54% homology to the human at the amino acid level and has identical protein interactions with p40 and Tyk2. The human IL-12Rb2 receptor is located on chromosome 1p31 and encodes a 862 amino acid polypeptide with a 27 amino acid signal peptide, a 595 amino acid extracellular domain, a 24 amino

380 INTERLEUKINS / IL-12

IL-12 p40 p35 p40 p80 p40 p40 p40

IL-12R 1 Tyk2

IL-12R 2 Jak2

IL-12R 1 Tyk2

IL-12R 1 Tyk2

Unknown Stat4 Stat4-P NF- B activation chemotaxis

Figure 1 Signal transduction pathways for IL-12 related cytokines. IL-12 binds to IL-12Rb1 and IL-12Rb2 to activate the transcription factor Stat4. p80 binds to IL-12Rb1 to activate NF-kB and chemotaxis. p40 binds to IL-12b1 with very low afnity and activation of intracellular signaling has not been described. IL-12, interleukin-12; IL-12Rb1, IL-12 receptor b1; IL-12Rb2, IL-12 receptor b2; Jak2, Janus kinase 2; NFkB, nuclear factor kappa B; Stat4, signal transducer and activator of transcription-4; Stat4-P, phosphorylated Stat4; Tyk2, tyrosine kinase 2.

acid transmembrane domain, and a 216 amino acid cytoplasmic tail. The extracellular region binds to the p35 subunit of IL-12 and the cytoplasmic tail associates with Janus kinase 2 (Jak2) and contains three conserved tyrosines that are required for intracellular signaling. The binding of IL-12 to the receptor chains initiates subsequent tyrosine phosphorylation of the receptor associated tyrosine kinases (Tyk2 and Jak2, respectively), IL-12Rb2, and the transcription factors signal transducer and activator of transcription-4 (Stat4), Stat3, Stat1, and Stat5. Although IL-12 has been reported to activate multiple members of the Stat family as well as mitogen-activated protein kinase (MAPK), NF-kB, and Lck, the predominant signaling molecule appears to be Stat4 (Figure 1). IL-12Rb2 is absent on resting immune cells, and the induction of IL-12Rb2 expression confers the ability to respond to IL-12. The mouse IL-12Rb2 gene encodes a 875 amino acid polypeptide with 68% homology to the human at the amino acid level.

IL-12-Related Cytokines in Respiratory Disease

Identication of individuals with susceptibility to mycobacterial disease has led to a description of a human syndrome known as Mendelian susceptibility to mycobacterial disease. Molecular characterization of these individuals has identied 73 patients from 21 countries that are completely decient in p40 (n l9) or IL-12Rb1 (n 54). Since p40 and IL-12Rb1 are required for the function of IL-12, IL-23, p80, and p40 monomers, these individuals are decient in all of these IL-12 related cytokines. Collectively, these individuals

display an increased susceptibility to primary infection with environmental mycobacteria, live Bacille Calmette-Guerin (BCG), and Salmonella. Follow-up of these individuals noted essentially no recurrence of mycobacterial disease, whereas secondary infection with Salmonella did occur. Penetrance for this infectious phenotype is low because 18% of probands failed to develop BCG infection after vaccination, and affected siblings often do not develop these spontaneous infections. This group of individuals demonstrates no reported increased susceptibility to viral, Grampositive, Gram-negative, protozoa, or fungal infections (one case of Nocardia asteroides and Paracoccidioides braziliensis) suggesting a redundant role for these IL-12-related cytokines in these infections. IL-12-related cytokines have also been implicated in asthma. In one report, bronchoalveolar lavage uid from human subjects with asthma had low protein levels of IL-12 but increased amounts of p80. The increased p80 expression was independent of concurrent inhaled glucocorticoids and associated with increased accumulation of macrophages. In contrast, others have found decreased p40 mRNA in mild asthmatics at baseline, and in the steroidsensitive subjects these levels increased to normal after oral glucocorticoid administration. In this study the protein levels of IL-12 family members were not determined so the relative contribution of IL-12 and p80 was not dened. Lastly, in an attempt to modulate asthma, four escalating doses of weekly subcutaneous IL-12 were administered to mild asthmatic subjects. This treatment decreased peripheral blood and sputum eosinophilia but failed to alter airway reactivity to histamine or inhaled antigen.

INTERLEUKINS / IL-12 381

Table 3 Associations between respiratory disease and polymorphisms in the IL-12 gene family Disease Asthma Gene p40 Authors (year) Morahan et al. (2002) Khoo et al. (2004) Noguchi et al. (2001) Randolf et al. (2004) Population Australia Australia Japan USA Polymorphisma IL-12B promoter IL-12B promoter Ser226Asn 1188A/C IL-12B promoter IL-12B 1025 anking IL-12B 2508 intron IL-12B 4237 intron IL-12B 4496 intron IL-12B 6402 intron IL-12B 13478 30 UTR None detected in promoter (1883 bp) None detected in promoter (1579 bp) IL-12B 30 UTR Promoter Intron 2 Intron 4 Exon 5 30 UTR Q214R, M365T, G378R haplotype 111A-T 2C-T 378G-C 409 21C-A 467G-A 641A-G 1094 T-C 1791 46 T-C 1989 34C-T 1188 (A/C) 30 UTR Association Heterozygosity with sever asthma None None None None None None Positive None Positive Positive None None None Positive Positive Positive None Positive Positive Positive Positive None None None None None None None None

Birkisson et al. (2004) p35 Tuberculosis p40 Birkisson et al. (2004) Ma et al. (2003) Tso et al. (2004)

Iceland Iceland USA China

IL-12Rb1

Akahoshi et al. (2003) Remus et al. (2004)

Japan Morocco

Idiopathic pulmonary brosis

p40

Latsi et al. (2003)

UK

a Nomenclature as reported in manuscript. 30 UTR, 30 untranslated region of gene.

The p40 gene is located within a cytokine gene cluster on chromosome 5q31-33. This region of chromosome 5 has been associated with numerous nonrespiratory and respiratory human diseases in population-based linkage analysis studies. Specic polymorphisms in the p40 gene have been associated with type I diabetes, atopic dermatitis, psoriasis, multiple sclerosis, malaria, and hepatitis C viral infection in some but not all studies. No association with p40 polymorphisms and celiac disease, aphthous stomatitis, Crohns disease, or Behcets disease has been reported. Additional studies have been performed to identify associations between polymorphisms in the IL-12-related cytokines and respiratory diseases such as asthma, tuberculosis, and idiopathic pulmonary brosis (Table 3).
See also: Allergy: Overview. Asthma: Overview. Genetics: Gene Association Studies. Interferons.

Interleukins: IL-23 and IL-27. Leukocytes: Monocytes; T cells; Pulmonary Macrophages.

Further Reading
Abdi K (2002) IL-12: the role of p40 versus p75. Scandinavian Journal of Immunology 56: 111. Devergne O, Hummel M, Koeppen H, et al. (1996) A novel interleukin-12 p40 related protein induced by latent EpsteinBarr virus infection in B lymphocytes. Journal of Virology 70: 11431153. Fieschi C and Casanova JL (2003) The role of interleukin-12 in human infectious diseases: only a faint signature. European Journal of Immunology 33: 14611464. Gately MK, Renzetti LM, Magram J, et al. (1998) The interleukin12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annual Review of Immunology 16: 495521. Ha SJ, Lee CH, Lee SB, et al. (1999) A novel function of IL-12 p40 as a chemotactic molecule for macrophages. Journal of Immunology 163: 29022908. Kobayashi M, Fitz L, Ryan M, et al. (1989) Identication and purication of natural killer cell stimulatory factor (NKSF), a

382 INTERLEUKINS / IL-13

cytokine with multiple biologic effects on human lymphocytes. Journal of Experimental Medicine 170: 827845. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12 p40 to form a cytokine, IL-23, with biologic activities similar as well as distinct from IL-12. Immunity 13: 715 725. Panz S, Timans JC, Cheung J, et al. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4( ) T cells. Immunity 16: 779790. Russell TD, Yan Q, Fan G, et al. (2003) IL-12 p40 homodimerdependent macrophage chemotaxis and respiratory viral inammation are mediated through IL-12 receptor beta. Journal of Immunology 171: 68666874. Trinchieri G (2003) Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nature Reviews: Immunology 3: 133146. Trinchieri G, Panz S, and Kastelein RA (2003) The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses. Immunity 19: 641644. van de Vosse E, Lichtenauer-Kaligis EG, van Dissel JT, and Ottenhoff TH (2003) Genetic variations in the interleukin-12/interleukin-23 receptor (beta1) chain, and implications for IL-12 and IL-23 receptor structure and function. Immunogenetics 54: 817829. Walter MJ, Kajiwara N, Karanja P, Castro M, and Holtzman MJ (2001) IL-12 p40 production by barrier epithelial cells during airway inammation. Journal of Experimental Medicine 193: 339351. Watford WT, Moriguchi M, Morinobu A, and OShea JJ (2003) The biology of IL-12: coordinating innate and adaptive immune responses. Cytokine & Growth Factor Reviews 14: 361368. Wolf SF, Temple PA, Kobayashi M, et al. (1991) Cloning of cDNA for natural killer cell stimulatory factor, a heterodimer cytokine with multiple biologic effects on T and natural killer cells. Journal of Immunology 146: 30743081. allergic inammatory cells, induces airway obstruction through direct effects on target airway cells, and is associated with airway brosis. In addition to its proinammatory role, IL-13 also induces matrix metalloproteinases of the airway that promote resolution of allergic inammation.

Introduction
Originally described as a secreted polypeptide, p600, and a product of the newly described T-helper-2 (Th2) cells in 1989, interleukin-13 (IL-13) was subsequently shown to be homologous to IL-4, a cytokine described several years earlier. Indeed, the marked structural and functional similarities between IL-4 and IL-13 led to the belief among many investigators that IL-13 was functionally redundant. Like IL-4, IL-13 was shown to inhibit activation of monocytes and stimulate immunoglobulin E (IgE) secretion from CD40-activated human B cells. IL-13 also induces expression of the low-afnity IgE receptor, CD23, and major histocompatibility class II molecules on hematopoietic cells, as does IL-4. Almost 10 years after its discovery, the more important role of IL-13 as a potent effector cytokine during allergic inammatory reactions nally came to light. Today, IL-13 is recognized as a key mediator of allergic effector responses and disease rather than as a growth and differentiation factor for hematopoietic cells, a role that is best dened by IL-4. The biology of IL-4 and IL-13 overlaps further with respect to the molecules that they share as part of their receptor complexes. Despite such insight, a complete molecular explanation for the distinct functions of IL-4 and IL-13 remains an important and elusive goal.

IL-13
D B Corry and F Kheradmand, Baylor College of Medicine, Houston, TX, USA
& 2006 Elsevier Ltd. All rights reserved.

Structure
Both human and mouse IL-13 are located in a cluster of immunologically important genes in the distal arms of chromosomes 5q 23-31 and 11q, respectively, and encode a polypeptide of approximately 12 400 Da. The IL-4 gene is located 12 kb from the IL-13 gene and both are transcribed in an opposite direction to all other genes in the region. Human IL-13 complementary DNA (cDNA) and polypeptides are 66% and 58% homologous with the mouse cytokine, respectively, and human IL-13 is between 20% and 25% identical with human IL-4. These ndings suggest that IL-4 and IL-13 have descended from the same ancestral gene. IL-13 exists in two alternatively spliced forms, differing only by the addition or not of a single glycine residue.

Abstract
Interleukin-13 (IL-13) is a cytokine of T-helper-2 cells that is an important mediator of allergic inammation and disease. In addition to effects on immune cells that are similar to those of the closely related cytokine IL-4, IL-13 is more importantly implicated as a central mediator of the physiologic changes induced by allergic inammation in many tissues. IL-13 induces its effects through a multisubunit receptor that includes the a-chain of the IL-4 receptor (IL-4Ra), which is also part of the IL-4 receptor, and at least one of two known IL-13-specic binding chains. Most of the biological effects of IL-13, like those of IL-4, are linked to a single transcription factor signal transducer and activator of transcription 6. Depending on the organ involved, IL-13 plays either benecial or deleterious roles in immunity. IL13 is required for the expulsion of a variety of parasites from the gut. Conversely, IL-13 mediates granulomatous disease, which causes dysfunction of many organs in the setting of allergic inammation, and may suppress benecial immune functions of macrophages. In the lung, IL-13 coordinates the recruitment of

Production and Activity

Like most hematopoietin cytokines (the interleukins), production of IL-13 is usually regulated by

INTERLEUKINS / IL-13 383

transcription, the major exception being the preformed IL-13 that is released as part of the granule contents of activated eosinophils and mast cells. Other cells that are reported to make IL-13 include monocytes/macrophages, basophils, and natural killer cells. However, the principal source of IL-13 during allergic inammation is probably the T cell, especially Th2 cells. Posttranslational modications to IL-13 such as glycosylation, although documented, do not contribute to activity; other modications such as phosphorylation, sulfation, and proteolytic cleavage have not been described. IL-13 is secreted during activation of allergic effector cells in response to a wide variety of stimuli. Perhaps best described in this regard are Th2 cells, which release IL-13 simultaneously with other Th2 cytokines following antigen stimulation. The close proximity of the IL-4 and IL-13 genes suggested a shared regulatory pathway and, indeed, the promoters for IL-13 and IL-4, although not identical, are activated by many of the same transcription factors (activator protein 1 (AP-1), nuclear factor kappa B (NF-kB), signal transducer and activator of transcription 6 (STAT6), GATA3). Additional regulatory elements are critical to the production of these cytokines, including a 400 bp noncoding genomic element that coordinately regulates IL-4, IL-5, and IL-13 production and which lies in the intergenic region between the IL-4 and IL-13 loci, termed central nervous system (CNS)-1. How organisms such as parasitic worms of the gut and common allergens such as pollens, dust mites, and fungi elicit IL-4 and IL-13 responses is not fully understood. However, these allergenic agents all have strong proteinase activity, which at least for some fungi is required to elicit strong Th2 responses and production of IL-4 and IL-13 in mice.

required to expel the offending organisms or their products. For example, expulsion from the gut of a variety of murine helminths requires IL-13 secreted by Th2 cells. IL-13 induces several changes in the gut that create a hostile environment for the parasite, including enhanced contractions and hypersecretion of glycoprotein from gut epithelial cells, which ultimately lead to detachment of the organism from the gut wall and their removal. The eggs of the parasite Schistosoma may lodge in a variety of organs including the gut wall, liver, lung, and even the CNS, inducing the formation of granulomas under the control of IL-13. In this case, the eventual result is organ damage and often profound or even fatal disease, not resolution of the infection. An emerging concept is that IL-13 may antagonize Th1 responses that are required to resolve intracellular infections. In this immune dysregulated context, marked by the recruitment of aberrantly large numbers of Th2 cells, IL-13 inhibits the ability of macrophages to destroy intracellular pathogens such as Leishmania major and probably other organisms as well.

Receptors
The IL-13 receptor remains the least understood of all the interleukins. The a-chain of the IL-4 receptor (IL-4Ra) is the principal signaling chain for both the IL-4 and IL-13 receptors. However, like all hematopoietin cytokine receptors, the IL-4 and IL-13 receptors are comprised of several molecules (Figure 1). In addition to IL-4Ra, the IL-4 receptor includes the g-chain of the IL-2 receptor (IL-2Rg), forming the type I IL-4 receptor. The type1 IL-4 receptor is, however, probably incapable of signaling with IL-13 as the ligand neither binds IL-4Ra nor IL-2Rg to IL-13. Therefore, the IL-13 receptor must include at least one of two known IL-13 binding chains, IL13Ra1 or IL-13Ra2; the requirement of IL-2Rg for IL-13 signaling remains controversial. However, current evidence based on IL-13Ra2-decient mice indicates that IL-13Ra2, which actually binds to IL13 with much greater afnity compared to IL-13Ra1, is a decoy receptor that serves to downregulate IL-13 responses. Thus, current evidence indicates that the principal IL-13 receptor consists of IL-13Ra1 together with IL-4Ra, with or without IL-2Rg. IL-4 can also signal through this receptor, which is referred to as the type II IL-4 receptor. Activation of the IL-13 receptor induces recruitment of two of the four known cytoplasmic tyrosine kinases of the Janus kinase family (Jaks) to the receptor complex. While the usage of Jaks is typically highly predictable among cytokine receptors, IL-13 appears to be capable of recruiting different combinations of Jaks

Biological Function
The functions of IL-13 overlap considerably with those of IL-4, especially with regard to changes induced on hematopoietic cells, but these effects are probably less important given the more potent role of IL-4. Thus, although IL-13 can induce IgE secretion from activated human B cells, deletion of IL-13 from mice does not markedly affect either Th2 cell development or antigen-specic IgE responses induced by potent allergens. In comparison, deletion of IL-4 abrogates these responses. Instead of a lymphoid cytokine, IL-13 acts as an important molecular bridge linking allergic inammatory cells to the nonimmune cells in contact with them and drastically altering their function. IL-13 specifically induces physiologic changes in parasitized organs that are

384 INTERLEUKINS / IL-13

IL-4 receptor IL-4R IL-4R

IL-13 receptor IL-13R 1 IL-13R 2

IL-2R STAT6 Allergic disease

Figure 1 The IL-4 and IL-13 receptors. The IL-4 and IL-13 receptors share the IL-4Ra subunit, but also have their unique components. The IL-2Rg subunit is only found as part of the IL-4 receptor, whereas IL-13Ra1 is thought to be the principal unique subunit of the IL-13 receptor. IL-13Ra2 may function as a decoy receptor that inhibits IL-13 signaling (dashed line). IL-4 may also signal through the combination of IL-4Ra and IL-13Ra1. Both IL-4 and IL-13 activate the transcription factor STAT6, which in turn regulates most aspects of allergic inammation and disease.

Goblet cell metaplasia Macrophage intracytoplasmic killing

IL-13

Airway hyperresponsiveness Enhanced secretion of: Proallergic chemokines Matrix metalloproteinases Glycoproteins Th2 cell

Subepithelial fibrosis

{
Stimulatory role

Inhibitory role

Figure 2 Pulmonary effects of IL-13. Although the functions of IL-13 overlap with those of IL-4, these are less important compared to IL-13-mediated physiological effects during allergic inammation. These include prominent changes of the airway epithelium and smooth muscle resulting in airway hyperresponsiveness, goblet cell metaplasia, and subepithelial brosis. Th2 cell-derived IL-13 further enhances secretion of airway glycoproteins, matrix metalloproteinases, and proallergic chemokines. Under some conditions, IL-13 may compromise the ability of macrophages to destroy intracellular pathogens.

depending on the exact tissue expressing the IL-13 receptor. Regardless of which Jaks are recruited, they phosphorylate STAT6. There is now widespread agreement that STAT6 is the principal transcription factor activated by both the IL-4 and IL-13 receptors,

although the IL-4 receptor (through IL-2Rg) may also activate additional factors of the insulin response substrate (IRS) family. STAT6 in turn activates most if not all genes induced by IL-13 and is therefore responsible for most biological effects of IL-13.

INTERLEUKINS / IL-15 385

IL-13 in Respiratory Diseases

IL-13 induces many features of allergic lung disease, including airway hyperresponsiveness, goblet cell metaplasia, and glycoprotein hypersecretion, which all contribute to airway obstruction (Figure 2). IL-4 contributes to these physiologic changes, but is less important than IL-13 as established by IL-4- and IL-13-decient mice, mice decient in both of these cytokines, and wild-type mice in which either IL-4 or IL-13 was inhibited using specic neutralizing agents. IL-13 also induces secretion of chemokines that are required for recruitment of allergic effector cells to the lung. Studies of STAT6 transgenic mice suggest the interesting possibility that IL-13 signaling occurring only through the airway epithelium is required for most of these effects. The chronic presence of IL-13 in the airway is further associated with subepithelial collagen deposition, widespread deposits of Charcot-Leyden-like crystals, and early mortality, indicating that IL-13 is an important mediator of airway disease during both acute and chronic allergen challenge. While no studies have as yet directly implicated IL-13 in the control of human diseases, many polymorphisms in the IL-13 gene have been shown to confer an enhanced risk of atopic respiratory diseases such as asthma. Although IL-13 is associated primarily with the induction of airway disease it also has anti-inammatory properties. Airway matrix metalloproteinases (MMPs), such as MMP2 and MMP9, are required to establish chemokine gradients across the airway epithelium that serve to induce egression of effete parenchymal inammatory cells into the airway lumen where they are then cleared. Among other factors, IL-13 induces these MMPs as part of a mechanism that protects against excessive allergic inammation that predisposes to asphyxiation.
See also: Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview. Chemokines, CC: TARC (CCL17). Chymase and Tryptase. Dust Mite. Eotaxins. Epithelial Cells: Type II Cells. Interleukins: IL-4; IL-5; IL-9. Lipid Mediators: Overview. Matrix Metalloproteinases. Mucus. Transcription Factors: Overview. Transgenic Models.

intestinal nematode parasites. Immunology Reviews 201: 139155. Jiang H, Harris MB, and Rothman P (2000) IL-4/IL-13 signaling beyond JAK/STAT. Journal of Allergy and Clinical Immunology 105: 10631070. Murata T, Taguchi J, Puri RK, and Mohri H (1999) Sharing of receptor subunits and signal transduction pathway between the IL-4 and IL-13 receptor system. International Journal of Hematology 69: 1320. Murphy KM, Ouyang W, Farrar JD, et al. (2000) Signaling and transcription in T helper development. Annual Review of Immunology 18: 451494. Townley RG and Horiba M (2003) Airway hyperresponsiveness: a story of mice and men and cytokines. Clinical Review of Allergy and Immunology 24: 85110. Wills-Karp M (1999) Immunologic basis of antigen-induced airway hyperresponsiveness. Annual Review of Immunology 17: 255281. Wynn TA (2003) IL-13 effector functions. Annual Review of Immunology 21: 425456.

IL-15
Q Hamid and I Ito, McGill University, Montreal, QC, Canada S Muro, Kyoto University, Kyoto, Japan
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-15 (IL-15) is a 1415 kDa protein that shares many biological activities with IL-2. IL-15 binds and signals via a trimetric receptor consisting of a common gamma chain, IL2Rb and IL-15Ra. Both IL-15 and IL-15Ra, which are essential for the high-afnity binding, have a much broader tissue distribution than IL-2/IL-2Ra. Multiple complex posttranscriptional regulatory mechanisms tightly control IL-15 expression. These ndings suggest that IL-15 has pleiotropic functions in immune and nonimmune systems. It is possible that IL-15 has a role in respiratory diseases such as sarcoidosis and tuberculosis through its role in Th1-driven-inammation.

Introduction
Interleukin-15 (IL-15) was rst isolated from cell culture supernatants of a simian kidney epithelial cell line in 1994 by Grabstein and co-workers as a novel T-cell growth factor whose biological activity is similar to IL-2. At the same time, Burton and co-workers showed that interleukin-T (IL-T), a lymphokine secreted by a human T-cell lymphotrophic virus-1 (HTLV-1)-associated leukemia cell line, stimulates T cells and lymphokine-activated cells. This was subsequently proven to be identical to IL-15. IL-15 is a 1415 kDa protein of 114 amino acids (AAs), and belongs to the four a-helix bundle cytokine family, which includes IL-2, IL-3, IL-6, IL-7, IL-21, granulocyte-colony stimulating

Further Reading
Corry DB (1999) IL-13 in allergy: home at last. Current Opinion in Immunology 11: 610614. Corry DB and Kheradmand F (2002) Biology and therapeutic potential of the interleukin-4/interleukin-13 signaling pathway in asthma. American Journal of Respiratory Medicine 1: 185193. Finkelman FD, Shea-Donohue T, Morris SC, et al. (2004) Interleukin-4- and interleukin-13-mediated host protection against

386 INTERLEUKINS / IL-15

factor (G-CSF) and granulocyte-macrophage colonystimulating factor (GM-CSF). IL-15 mRNA is widely expressed in monocytes, macrophages, bone marrow stroma, and thymic epithelium, as well as by epithelial cells in the kidney, skin, and intestines. IL-15 binds to a receptor complex that consists of its own IL-15Ra, IL-2Rb and the gc chain. IL-2 and IL-15 share the same beta and gamma subunits, both of which are common subunits for signal transduction. IL-15 mediates functions similar to IL-2 in vitro. However, the IL-15Ra shows broader tissue distribution compared to IL-2Ra. Studies using mice with targeted disruptions of the IL-15 and IL-15Ra genes has demonstrated that IL-15 and IL-2 play different functions in vivo.

Structure
The IL-15 gene is located on human chromosome 4q31 and the central region of mouse chromosome 8. The genomic structures of human and murine IL-15 are similar, with a length of 34 kb or more, and contain nine exons (7 coding exons). IL-15 is highly conserved between the two species (97% identity comparing human and simian, 73% identity comparing human with murine). The mature IL-15

protein consists of 114 AAs with the molecular weight of 1415 kDa, and is encoded by exons 58 (Figure 1). IL-15 protein contains two disulde bonds at positions C42C88 that are identical to IL-2 at C35 C85. There are two N-linked glycosylation sites at the C-terminus of the IL-15 protein, at N79 and N112. The nucleotide and protein sequences of IL-15 show little homology with IL-2. However, the intron/ exon structure (4 exons and 3 introns) is similar to that of the IL-2 gene and that of other four a-helix bundle cytokines. Therefore, the mature gene and protein structure of all members of the four a-helix bundle cytokine family appear similar. The originally identied human IL-15, long-signaling peptide (LSP) IL-15, contains a 50 untranslated region (UTR) of at least 316 bp, and a 486 bp open reading frame; and a 30 UTR of at least 400 bp, encoding a precursor IL-15 with an unusually long 48 AA leader peptide and a 114 AA mature protein. Recently, an additional sequence, exon 4A, has been found between exons 4 and 5 that encodes an alternative, shorter leader peptide (Figure 1). SSP-IL-15 lacks exons 1 and 2 of the original cDNA, and instead contains 119 nucleotides (nt) within intron 4 resulting in a smaller spliced mRNA product (Figure 1). This

Human IL-15 gene Exon (bp) 91 125 Intron (kp) ? 1 2 99/11

>10 0.59

98 119 35/51
0.49 0.75 5.8

45
1.9

137
2.8

112/397

4A

LSP-IL-15 classical 5 UTR LSP IL-15 3 UTR

IL-15 mRNA isoforms 5 UTR

SSP-IL-15 alternative SSP IL-15 3 UTR

1 2 3 4 5 6 7

8 IL-15 Precursor proteins

In2 3 4 4A 5 6 7

LSP (48AA) N 3,4,5,

IL-15 (114AA) C 5,6,7,8 N 3,4

SSP (21AA) C N 4A,5

IL-15 (114AA) C 5,6,7,8

IL-15 Mature protein N 5,6,7,8

Figure 1 Human IL-15 gene, mRNA, and protein structure. The human IL-15 locus consists of nine exons and eight introns and is located on chromosome 4q31. Two IL-15 mRNA isoforms have been described, the classical LSP and alternative SSP, with both encoding an identical IL-15 mature protein of 114 AAs (see text for details). Translational start sites (3) and stopcodons (F) are indicated; In2 indicates intron 2. Reproduced from Fehniger TA and Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood 2001; 97: 1432. Copyright American Society of Hematology, used with permission.

INTERLEUKINS / IL-15 387

exonic sequence, intron 4A, encodes three premature stopcodons and a novel downstream ATG translation start site. As a consequence, the translated product contains a 21 AA short-signal peptide. Thus, there are two isoforms of IL-15: LSP form (48 AAs) that is secreted from the cell, and a shortsignaling peptide (SSP) form (21 AAs) that remains intracellular and localized to nonendoplasmic regions in both cytoplasmic and nuclear compartments (see below). Although the signal sequences are different, both IL-15 isoforms encode an identical mature IL-15 protein in human and mouse.

identied at 348 to 336 relative to the cap site of the murine IL-15 promoter. The observation that IRF-1 / mice lack inducible IL-15 expression and natural killer (NK) cells suggests that IRF-induced IL-15 expression is essential for the NK cell development in the bone marrow.
Translation, Intracellular Trafcking, and Secretion

Regulation of Production and Activity

The two IL-15 mRNA isoforms contain significant variance in their leader peptides (Figure 1). These differences in leader peptides are thought to determine the intracellular localization and potential secretion of the associated mature protein. LSP IL-15 is targeted to the secretory pathway (endoplasmic reticulum (ER)/ Golgi apparatus), whereas SSP IL-15 appears to be restricted to the cytoplasm and nucleus. The distribution of IL-15 mRNA is different between LSP IL-15 and SSP IL-15. LSP IL-15 is shown to be expressed in the heart, thymus, lung, liver, kidney, skeletal muscle, and placenta. On the other hand, SSP IL-15 mRNA is detected in the heart, thymus, appendix, and testis. The biological significance of this differential expression is still unclear.

Regulation of IL-15 Gene Expression

IL-15 transcription, translation, and secretion are shown to be regulated through multiple complex mechanisms and the most important stage of IL-15 regulation appears to be posttranscriptional.
Control of Transcription

Multiple startcodons (AUGs) in the 50 UTR, the unusual LSP and SSP, and a negative regulator near the C-terminus of the precursor proteins, are shown to regulate IL-15 mRNA translation into the IL-15 precursor protein. LSP IL-15 was shown to have a lower translational efciency than SSP IL-15. The LSPIL-15 50 UTR is relatively long (more than 316 nt in humans) and contains multiple (12 in humans) AUGs upstream of the actual translation start site. These multiple AUGs have been shown to reduce translational efciency. The expression of SSP IL-15 was shown to be localized to the cytoplasm and nucleus, whereas LSP IL-15 was within the ER/Golgi apparatus. Thus, SSP IL-15 seems to be translated efciently but remains intracellular, whereas the translation of LSP IL-15 is less efcient but the protein is transported to the ER/ Golgi pathway and is secreted from the cell at low levels. It is suggested that a signal in the carboxyl portion of the mature IL-15 protein may act as a retention signal, resulting in low secretion. Transgenic mice that overexpress IL-15, as a result of elimination of these posttranscriptional regulations, developed early expansions in NK and CD8 T cells, followed by a fatal lymphocytic leukemia, suggesting that the tight regulation of IL-15 is critical for the homeostasis of immune system.

Biological Function
IL-15 shares a number of biological activities with IL-2, including stimulation of the proliferation of activated CD4 , CD8 , as well as the gd subset of T cells. However, in general, IL-15 has antiapoptotic actions to activated T cells and inhibits IL-2-mediated activation-induced cell death. IL-15 stimulates proliferation and persistence of memory CD8 T cells whereas IL-2 inhibits these functions. IL-15 also stimulates the proliferation of NK cells and acts as a co-stimulator with IL-12 to facilitate the production of interferon gamma (IFN-g) and tumor necrosis factor-a by these cells. The coexistence of IL-15 and IL-12 is also shown to increase the IFN-g production from T cells. However, IL-15 cannot substitute for IL-2 in the differentiation of

Mouse and human IL-15 promoter regions contain transcription factor-binding motifs including aINF-2, NFIL-6, g-IRE, myb, Gc factor (GCF), and NF-kB. The NF-kB site, located at 75 to 65 relative to the transcription start site of the human IL-15 promoter, was shown to be important for HTLV-1 tax protein-induced IL-15 mRNA upregulation and for lipopolysaccharide (LPS)-induced IL-15 gene expression in murine macrophages. The 50 deletion of region 201 and 141 in the human IL-15 promoter region was shown to result in a dramatic increase in IL-15 promoter activity, suggesting that this region contains an unidentied site responsible for negative regulation of IL-15 expression. An essential interferon regulatory factor (IRF)-E consensus-binding site was

388 INTERLEUKINS / IL-15

CD4 T cells into a Th2 type phenotype in the presence of IL-4, suggesting that IL-15 has the potential to skew the immunological response in favor of Th1 rather than a Th2 response. On the other hand, IL-15 promotes the production of IL-5 in human T helper cells, and stimulates and induces proliferation of mast cells through a distinct, unidentied receptor. IL-15 has been shown to induce the production of IL-8 in human neutrophils and to stimulate the induction of cytolytic effector cells such as cytotoxic T cells and lymphokineactivated killer cells. IL-15 also acts as a potent T-cell chemoattractant. Thus, IL-15 seems to play an important role in innate and acquired immunity. Human monocyte/macrophage cell lines and primary human monocytes have been shown to express constitutively bioactive IL-15 protein on their cell-surface membrane. IL-15 protein is also detected in human peripheral blood mononuclear cells by immunohistochemistry, Western analysis, and ow cytometry. These IL-15 membrane expressions are shown to be upregulated by stimulation with IFN-g, suggesting that IL-15 could exert a biological effect while being undetectable in culture supernatant. Further study should elucidate the functional signicance of constitutive cell-surface expression of IL-15. Furthermore, IL-15 induces the proliferation and synthesis of immunoglobulin by human tonsillar B cells activated by CD40 ligand or an immobilized antibody to IgM. IL-15 has been shown to take effect on nonimmune cells. For example, skeletal muscle cells express IL-15 mRNA and its receptor, and IL-15 induces skeletal muscle ber hypertrophy. Other nonimmunological systems such as the kidney, lung, liver, epithelial cell lines, and activated endothelial cells are also shown to express mRNAs for IL-15 and its receptor.

Receptor and Signal Transduction

Like the IL-2 receptor, the IL-15 receptor is also composed of three subunits: a, b, and g (Figure 2). The b and g subunits of the IL-15 receptor are shared with IL-2. In contrast to the restricted expression of IL-2Ra to subsets of lymphocytes, IL-15Ra expression is far more diverse. The expression of IL-15Ra has been reported in lymphoid and myeloid cells as well as nonhematopoietic cells: CD8C T cells (na ve and activated), NK, NKT and g/d T cells, monocytes, macrophages, dendritic cells, neutrophils, endothelial cells, kidney epithelial cells, colonic epithelial cells, brain microglial cells, and skeletal muscle cells. In addition, both receptors are inducible and a recent study indicates that both IL-2 and IL-15 increase

IL-2Ra mRNA and protein expression, while IL-15 selectively causes IL-2Ra expression on the cell surface. Thus, different regulation of cell-surface expression of IL-2Ra and IL-15Ra may contribute to the distinct functions of IL-2Ra and IL-15Ra in vivo. Although isolated IL-15Ra has high afnity for IL15 (KdB10 pM), IL-15Ra alone has been reported to exert no signal transduction. However, recent evidence suggests that IL-15 may indeed mediate certain intracellular signals. IL-15Ra has been shown to interact with tumor necrosis factor receptorassociated factor 2 (TRAF2) and further investigation is necessary to clarify the biological significance of IL-15Ra-mediated signals. So far, only IL-2 and IL-15 have been shown to utilize the IL-2/15Rb. The IL-2Rg, also referred to as the common gamma chain (gc), is shared by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. IL-15Ra, IL-2/15Rb, and gc constitute a complete heterotrimeric high-afnity complex which exerts signals through IL-2/15Rb and gc, identical to the IL2R after the ligand binding. In the absence of IL-15Ra, the heterodimeric IL-2/15Rbgc can bind to IL-15 with intermediate afnity (KdB1 nM) with signal transduction. It has been shown that mast cells respond to IL-15 with unique high-afnity binding receptor (60 65 kDa, IL-15RX) that does not include the identied IL-2/15Rb, gc, or IL-15Ra, and transduces different intracellular signals compared to IL-15Rabgc. As IL-2 and IL-15 share common signaling components (IL-2/15Rbgc), the interaction of IL-2 or IL-15 with their respective receptors leads to similar signaling events such as the activation of the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway. IL-2/15Rb is associated with Jak1 and the gc is associated with Jak3, resulting in STAT3 and STAT5 phosphorylation respectively, following ligation with IL-15 (Figure 2). The stimulation of IL-2/15R complexes are also shown to activate the src-related tyrosine kinases, induce Bcl-2, and stimulate the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway that ultimately results in fos/jun activation. The IL-2/15Rb also activates the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway.

IL-15 in Respiratory Diseases

It has been shown that alveolar macrophages isolated from bronchoalveolar lavage (BAL) of patients with active sarcoidosis and tuberculosis produce increased levels of IL-15 compared to normal subjects. BAL cells from individuals with granulomatous sarcoid lesions show a marked increase in the numbers of CD4 lymphocytes, which have been shown to

INTERLEUKINS / IL-15 389

IL-15

IL-2

Kd 1011 M

Kd 1011 M

Kd 109 M

Kd 1011 M

Kd 108 M

(a)

Intermediate affinity IL-2R or IL-15R

S A

Jak1 Jak3

No signal

(b)

P (Syk) (Src) Shc STAT3 Grb2/SOS STAT5a/b Ras Raf PI-3K MAPK Akt

Figure 2 (a) Schematic gure of the IL-15 and IL-2 receptor complexes. The high-afnity, heterotrimeric IL-2 receptor is composed of the IL-2a, IL-2/15Rb and gc, while the intermediate-afnity receptor is made up of the heterodimer of IL-2/15Rb and gc. The isolated IL-2Ra binds IL-2 with low-afnity. The high-afnity heterotrimeric IL-15 receptor is comprised of IL-15Ra, IL-2/15Rb and gc, and similar to IL-2, IL-15 binds to the heterodimeric IL-2/15Rbgc with intermediate-afnity. In contrast to IL-2Ra, the isolated IL-15Ra alone has high-afnity for IL-15. Blue ovals represent conserved cysteine bonds, while the WXSWS motif shared between hematopoietin receptor members is depicted as red ovals. (b) Signaling cascade from the IL-15 and IL-2 receptor complexes. Signals from the IL-2/15Rb and gc are shared between the IL-15 and IL-2 receptor complexes through the signaling pathways shown. Recent studies have shown that the IL-15Ra cytoplasmic tail binds to TRAF2, preventing its interaction with the pro-apoptotic cascade members and may increase Bcl2. IL-2Ra does not mediate any known signals. The serine-rich (S), acidic (A), and proline-rich (P) portions of the IL-2/15Rb cytoplasmic domain are indicated, as well as tyrosine residues (Y). Reproduced from Fehniger TA, Cooper MA, and Caligiuri MA (2002) Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine & Growth Factor Reviews 13: 169183, with permission from Elsevier.

390 INTERLEUKINS / IL-16

IL-12 Mph/DC IL-15

Memory CD8+ T cell NKT cell IFN-

Persistence

Th1 response ?

NK cell

Sarcoidosis tuberculosis

Figure 3 Possible role in the pathogenesis of respiratory diseases. IL-15 stimulates proliferation of NK cells, NKT cells, and memoryphenotype CD8 T cells in concert with IL-12. Memory-phenotype CD8 T cells are maintained by IL-15. These cells produce IFN-g, which leads to Th1 response seen in respiratory diseases such as sarcoidosis and tuberculosis.

proliferate in response to IL-15. Therefore, IL-15 may contribute to the development of T-cell alveolitis in this disease. Its expression is also increased in bronchial biopsies from patients with sarcoidosis and tuberculosis patients and, to a lesser extent in patients with chronic bronchitis, compared to asthmatics and normal control subjects. Thus, it is suggested that IL-15 is involved in Th1-dominant inammation in respiratory diseases. (Figure 3). To date, administration of IL-15 has not been used in clinical trials for disease therapy. In mice, IL-15 administration has been shown to enhance T- and NK-cell function resulting in increased antitumor activity in mice, and IL-15 was less toxic than IL-2. Thus IL-15 is one of the candidates for antitumor immunotherapy. IL-15 is shown to be involved in the chronic inammation in rheumatoid arthritis and clinical intervention studies on IL-15 blockade in inammatory synovitis are ongoing.
See also: Interferons. Leukocytes: T cells. Signal Transduction. Systemic Disease: Sarcoidosis.

Musso T, Calosso L, Zucca M, et al. (1999) Human monocytes constitutively express membranebound, biologically active, and interferon-gamma-upregulated interleukin-15. Blood 93: 35313539. Waldmann TA, Dubois S, and Tagaya Y (2001) Contrasting roles of IL-2 and IL-15 in the life and death of lymphocytes: implications for immunotherapy. Immunity 14: 105110. Waldmann TA and Tagaya Y (1999) The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens. Annual Review of Immunology 17: 1949.

IL-16
F F Little, W W Cruikshank, and D M Center, Pulmonary Center, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-16 (IL-16) is a pleotropic cytokine that is produced by immune cells and epithelial cells. The secreted C-terminal product of caspase-3 cleavage is a PDZ-domain-containing protein whose terminal 16 amino acids bind the D4 domain of CD4, its major receptor. IL-16 elicits monocyte, eosinophil, and T-cell chemoattraction in vitro that is preferential for TH1 cells, yet also anergizes T cells resulting in downregulation of antigen recognition and T-cell activation. In addition, IL-16 has direct effects on the generation of tolerogenic dentritic cells and is a cofactor in mast cell development. The dominant T-cell immunomodulatory effect in vivo appears to be downregulation of TH2-mediated allergic inammation.

Further Reading
Agostini C, Trentin L, Facco M, et al. (1996) Role of IL-15, IL-2, and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis. Journal of Immunology 157: 910918. Alpdogan O and van den Brink MR (2005) IL-7 and IL-15: therapeutic cytokines for immunodeciency. Trends in Immunology 26: 5664. Fehniger TA and Caligiuri MA (2001) Interleukin 15: biology and relevance to human disease. Blood 97: 1432. Fehniger TA, Cooper MA, and Caligiuri MA (2002) Interleukin-2 and interleukin-15: immunotherapy for cancer. Cytokine & Growth Factor Reviews 13: 169183. Giri JG, Kumaki S, Ahdieh M, et al. (1995) Identication and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO Journal 114: 36543663. Muro S, Taha R, Tsicopoulos A, et al. (2001) Expression of IL-15 in inammatory pulmonary diseases. Journal of Allergy and Clinical Immunology 108: 970975.

Introduction
Interleukin-16 (IL-16) was originally described as a T-cell chemoattractant (lymphocyte chemoattractant factor (LCF)) found in the supernatants of mitogen-stimulated peripheral blood mononuclear cells (PBMCs). IL-16 is synthesized by varied immune and

INTERLEUKINS / IL-16 391

parenchymal cells, including CD4 and CD8 T cells, eosinophils, macrophage/dendritic cells, mast cells, bronchial epithelium, and broblasts. IL-16 has two major in vitro effects on CD4 T cells: chemoattraction, preferentially of TH1 cells, and inhibition of CD3/T-cell receptor mediated activation, preferentially of TH2 cells. The binding site of IL-16 on CD4 maps to the sequence in the D4 domain of CD4 that is responsible for autodimerization, supporting a molecular explanation for the interference of IL-16 on CD4 T-cell activation seen in vitro. As the most studied and primary receptor for IL-16 is CD4, its therapeutic potential in HIV infection and allergic lung disease has been investigated in detail.

CK2 cdc2 NLS PDZ PDZ

D/S PDZ RRKS... C-terminal: secreted

N-terminal Pro-IL-16

Figure 1 Protein structure of IL-16. Leukocyte IL-16 is synthesized as a ca. 80 kd precursor which is cleaved at Asp510-Ser511 by caspase-3, releasing the C-terminal peptide whose mechanism of secretion is unknown. This protein has a PDZ domain in the proximal end and the RRKS (RRTS in the mouse) CD4-binding site in the distal 16 amino acids. Also depicted is the dual phosphorylation-regulated nuclear localization sequence (CcN motif) of pro-IL-16, which includes a casein-kinase 2 substrate site, cdc2 kinase substrate site, and bipartite nuclear localization sequence (NLS).

IL-16 Gene Structure, Expression, and Protein Product

The IL-16 gene is a single copy gene located on human chromosome 15.26.13, and on chromosome 7 D2D3 in the mouse. The entire gene stretches over 20 kb and encodes for two forms of the precursor molecule, synthesized by alternative splicing. Neuronal 1322 and 1332 amino acid precursor forms (in mouse and human, respectively) are expressed exclusively in the brain and have an unknown number of exons. The non-neuronal (leukocyte) form of the human IL-16 gene contains seven exons, of which the rst is noncoding, and results in a 2.6 kb mRNA species. The promoter is TATA-less, yet it contains two CAAT box-like motifs and three GA-binding protein transcription factor binding site consensus sequences which are functional in T cells, one of which lies in the 50 untranslated region (UTR). Leukocyte IL-16 is a 631 (human) and 624 (mouse) amino acid protein whose major biologic function relevant to pulmonary inammation resides in the 121/118 amino acid C-terminal, secreted end of the molecule. The N-terminal product of cleavage (by caspase-3, see next section) is a PSD-95, dlg, Z01 homology (PDZ) domain-containing protein which localizes to the nucleus (Figure 1). In addition to two PDZ domains, this B60 kDa N-terminal product of caspase-3 cleavage has a dual phosphorylationregulated nuclear localization sequence permitting nuclear translocation, where it plays a role in regulating the levels of p27KIP1 and G0/G1 arrest of T cells. The structure of the secreted C-terminal inteleukin, IL-16, is unusual as it is a functional extracellular protein that contains a PDZ domain. There is low homology between the originally described PDZ proteins and IL-16; NMR spectroscopy has revealed that the core GLGF region in IL-16 is partially occluded by a tryptophan residue. Yeast-2

hybrid experiments using lymphocyte and splenocyte cDNA libraries have not identied an intracellular binding partner for this PDZ domain. While its mechanism is unclear, autoaggregation of IL-16 monomers is required for bioactivity and occurs intracellularly before secretion. Contained within the distal 16 amino acids is a core 4 amino acid sequence (arg-arg-lys-ser (RRKS) in humans, arg-arg-thr-ser (RRTS) in the mouse) which is required for CD4binding ability and consequent T-cell chemotactic and immunomodulatory properties. There is a high degree of functional and sequence homology of IL-16 from all species (e.g., human, simian, and murine). Both murine and simian IL-16 are chemotactic to human T cells, and monoclonal antibodies generated to secreted human IL-16 can be used to neutralize the bioactivity as well as to afnity purify murine IL-16. The conservation across species of protein structure and biologic actions of IL-16 implies a conserved evolutionary function and bears on the application of animal models to understanding IL-16s biologic effects in humans.

Regulation and Cellular Synthesis of IL-16

While the transcriptional regulation of IL-16 has not been fully characterized, the nature of IL-16 message production in different cell types has been examined in detail. CD4 and CD8 T cells, eosinophils, mast cells, and dendritic cells constitutively generate IL-16 mRNA. By contrast, nonimmune cells such as airway epithelial cells and broblasts must be induced to transcribe IL-16 message. Full-length leukocyte IL-16 is synthesized as a 631 amino acid precursor that is cleaved into two fragments by caspase-3. This occurs readily without other associated apoptotic events. With the notable exception of CD8 T cells, caspase-3 activation is a major regulator of IL-16 release in all cells that constitutively express mRNA and protein. CD8 T cells express constitutively active caspase-3, and store cleaved

392 INTERLEUKINS / IL-16

bioactive IL-16 that is released within hours following stimulation by histamine or serotonin via H2 or S2 receptors. While only one functional caspase-3 cleavage site has been denitively identied in ProIL-16 from T cells, further proteolytic processing has been suggested in dendritic cells. It is likely that the stimuli responsible for IL-16 release in non-T-cells, such as broblasts, result in new transcription, translation, and posttranslational cleavage by activated caspase-3. The mechanism of secretion has not been identied.

IL-16 Receptor: Relationship between IL-16 and CD4

The ability of IL-16 to function as a chemotactic stimulus for CD4 T cells led to an investigation of the interaction between IL-16 and cell surface CD4. All cells with a well-described effector response to IL16 express surface CD4, including T lymphocytes, eosinophils, dendritic cells, and neuronal cells. IL-16 has a distinct binding site on CD4 from other CD4 ligands, including HIV gp120 and major histocompatibility class II antigens. T-cell migration to IL-16 can be inhibited by coincubation with monomeric Fab fragments directed against CD4, and murine Tcell hybridomas transfected with CD4 gain migratory function to IL-16 which is eliminated by various mutations of CD4. Similarly, CD4 expression on CD8 T cells following anti-CD3/anti-CD28 costimulation confers IL-16 responsiveness, which is blocked by antibodies to CD4. While it appears that alternate receptors for IL-16 exist on monocytes and dendritic cells, the evidence for a predominant IL-16-CD4 ligandreceptor relationship in T cells is compelling. Following ligation of CD4 by IL-16, activation of the CD4-associated src-related tyrosine kinase p56lck occurs followed by rises in intracellular Ca2 , IP3, and activation of PI-3 kinase. This rst response is associated with autocatalytic activity of p56lck with tyrosine phosphorylation and translocation of protein kinase-C to the membrane. While the catalytic activity of p56lck is not necessary to mediate T-cell chemoattraction, an adaptor function of p56lck coupling to PI-3 Kinase via the SH3 domain of p56lck is required. Point mutations in key intracytoplasmic regions of CD4 necessary for CD4 p56lck association abrogate the T-cell chemotactic response to IL16. Cells expressing these mutations retain migratory responses to other chemoattractants. The chemotactic response also appears independent of the rise in Ca2 and IP3 turnover. It is likely that these two latter signals and the enzymatic activity of p56lck are required for IL-16-induced expression of CD25; however, this has not been proved. These ndings

imply that there are multiple CD4-associated signals induced by IL-16 that lead separately to lymphocyte migration and selective gene expression. The precise binding sites on CD4 for IL-16 have been mapped by peptide inhibition and CD4 mutagenesis studies, and are comprised of two six amino acid stretches that form a groove in the tertiary structure of the D4 region. This sequence of the D4 domain of CD4, closest to the cell membrane, is responsible for autoaggregation that facilitates activation of the TCR/CD3 antigen recognition complex. The presence of CCR5, expressed primarily on TH1 cells, appears to contribute to IL-16 binding and likely accounts for the preferential migratory response of these cells. The mechanism by which CCR5 increases IL-16 binding to CD4 has not been identied as yet.

Biologic Effects of IL-16

This section briey discusses the in vitro and in vivo effects of IL-16 (Figure 2).
Chemotactic Activity of IL-16

CD4-mediated chemoattraction A variety of target cells for IL-16 stimulation have been identied. IL-16 was initially described as a chemoattractant specically for CD4 T cells. Recent evidence has shown that IL-16 is preferentially chemoattractant to TH1 cells, suggesting a contributory role in the downregulation of TH2-cell-mediated lung disease such as allergic asthma. IL-16 is also a potent chemoattractant for all peripheral immune cells expressing CD4, including CD4 monocytes, eosinophils, and dendritic cells. In vitro studies have indicated that for lymphocytes and eosinophils, the ED50 (half maximal effective dose) for recombinant IL-16 is 1011 M, which is consistent with other reported chemoattractants such as regulated on activation, normal T-cell expressed and secreted (RANTES); (CCL5) and monocyte chemoattractant protein-1 (MCP-1); (CCL2). Indirect effects of IL-16 on chemoattraction via chemokine receptors Indications that the CD4/C-C chemokine receptor 5 (CCR5) receptor complex is more than a docking site for human immunodeciency-1 (HIV-1) binding and internalization were suggested by the observation that HIV-1 gp120 activates intracellular signaling of both CD4 and CCR5. This concept was furthered by the observation that ligation of either CD4 or CCR5 by their natural ligands results in reciprocal receptor cross-desensitization. IL-16 treatment of CD4 T cells results in transient inhibition of macrophage

INTERLEUKINS / IL-16 393

Lung Anergy TH1 cell influx ? Treg activity Chemokine receptor desensitization ?Chemoattraction CD4+ and CD8+ T cells Eosinophil IL-16 DC tolerization

Epithelium

Mast cell

Expansion

Bone marrow Expansion of pre-B cells ? IL-16 Progenitor cell Expansion

Figure 2 Major cellular sources and biologic effects of IL-16 in the lung and bone marrow (refer to text for details). In the lung, there are varied sources of IL-16, some of which require stimulation to synthesize and secrete IL-16 (epithelial cells), while others constitutively synthesize IL-16 (T lymphocytes, eosinophils, mast cells). There are consequent direct effects on T cells and eosinophils as outlined. The effects on dendritic cells and mast cells have yet to be explicitly demonstrated. The source of IL-16 in the bone marrow is unknown, but stimulates CD4 progenitor cells to expand in number and percentage of pre-B cells and markedly increase the production of chymase and tryptase in mast cells.

inammatory protein 1b (CCL4) induced chemoattraction, indicating that an IL-16CD4 association desensitizes CCR5 to stimulation by a natural CCR5 ligand. Reciprocal receptor cross-desensitization is also observed: CCL4 treatment of the same cells prevents IL-16-induced migration. This autoregulatory effect is not due to steric inhibition nor changes in surface receptor expression; CCR5 desensitization by IL-16 requires intact signal transduction by CD4. Both of these effects are likely to be relevant to the nding that rIL-16 inhibits HIV-1 replication, but not infectivity, in peripheral blood mononuclear cells. In addition, ligation of CCR5 can induce phosphorylation of CD4-associated p56lck. Similarly, the presence of CCR5 contributes not only to IL-16/CD4 binding, but to CD4 signaling as well. T cells from CCR5 / mice have a markedly diminished migratory response to IL-16. CCR5 signaling likely contributes to this response as pertussis toxin treatment of CCR5 cells reduces the migratory response to IL-16 to levels noted in CCR5 / cells. The previously noted preferential migration of TH1 cells to IL-16 noted in these experiments has direct implications on the role of IL-16 in inammatory lung disease; the enhanced migration of TH1 cells over TH2 cells is not due to differences in surface CD4 expression, but due to the co-receptor function of

CCR5. The receptor cross-desensitization phenomenon is not unique to CCR5. IL-16/CD4 signaling also desensitizes C-X-C chemokine receptor 4 (CXCR4) response to stimulation with its ligand stromal derived factor 1a (CXCL12). IL-16/CD4 signaling has no effect on migration induced by CCL2/CCR2b, eotaxin (CCL11)/CCR3, or macrophage-derived chemokine (CCL22)/CCR4.
Modulation of the Primary Immune Response

Downregulation of antigen-mediated T-cell activation The location of IL-16s binding site in the D4 region of CD4 bears directly on its ability to inhibit aspects of CD3-T-cell receptor (TCR)-mediated Tcell activation by antigen or that induced by a mixed lymphocyte reaction (MLR) (Figure 3). In both humans and mice, 1 h rIL-16 pretreatment of responder cells inhibits the MLR by 50%; this inhibition is reversible by cotreatment with either anti-IL-16 antibody or recombinant soluble CD4. These ndings are consistent with the report that activation of T-cells by antigen or anti-CD3 is inhibited by IL-16 pretreatment. Furthermore, treatment of antigen-specic murine T cells with IL-16 during antigen stimulation reduces production of the TH2 cytokines IL-4 and IL-5 without affecting release of interferon gamma (IFN-g) or IL-10. It is noteworthy that the T-cell

394 INTERLEUKINS / IL-16

D1 D2 CD4 D3 IL-16 D4 D4 D3

D1 D2

DC Ag-MHC II

DC Ag-MHC II CD4 (undimerized)

CD4 (autodimerized) IL-16

p56lck

TCR/CD3

TCR/CD3

p56lck

p56lck phosphorylation CD25 induction TH1-cell chemoattraction

Antigen-driven activation TH2 cytokine production Augmented allergic response

Alteration of CD4-TCR/CD3 interaction Anergy Downregulation of allergic response

Figure 3 Putative mechanism of IL-16-mediated immunomodulation in CD4 T-lymphocyte activation. In the absence of antigen, IL-16 oligomers bind CD4 in the D4 region causing a complex steric relationship that leads to coupling and autophosphorylation of p56lck (see text (IL-16 receptor) for details), with subsequent induction of CD25 and preferential TH1-cell chemotaxis (left). In the presence of antigen/MHC II (center), TCR/CD3-mediated cell activation is facilitated by dimerized CD4 of a different steric conformation than that bound by IL-16, leading to antigen-driven proliferation and cytokine production that augments the allergic response. In the presence of IL-16 (right), CD4 dimerization and facilitation of TCR/CD3 activation by antigen/MHC II is altered, causing anergy and modulation of antigen-driven responses.

chemoattractant and immunomodulatory effects of IL-16 occur at equimolar concentrations. Thus, as a consequence of being bound by IL-16, CD4 can function to transduce signals sufcient to induce Tcell chemotaxis and expression of IL-2Ra and b while CD4s facilitation of antigen recognition function is inhibited. Altered phenotype of T cells and dendritic cells IL16 treatment of T cells increases surface expression of human leukocyte antigen-DR (HLA-DR), suggesting that IL-16 contributes to facilitation of antigen recognition by T cells. Four-week culture of T cells in the presence of IL-2 demonstrates increased surface expression of surface CD25, CD122, and cell proliferation, providing a competence growth function for IL-16. While often a marker of activated T cells, the increased surface expression of CD25 attributable to IL-16 could reect induction of a regulatory phenotype, though this hypothesis has not been directly tested. This concept is supported by the nding that a tolerogenic dendritic cell (DC) phenotype is induced by culturing CD34 hematopoetic progenitor cells with IL-16 in addition to standard DC generating factors. These DCs tolerize cocultured T cells in a manner that persists upon restimulation with cognate immunogenic DCs. This may have a human in vivo correlate in that monocyte-derived dendritic cells from normal individuals store and secrete IL-16, while similarly derived cells

from individuals with atopic dermatitis have impaired synthesis and secretion of IL-16.
Cofactor in Hematopoetic Cell Development into Pre-B and Mast Cells

The reversal of declining levels of recombinationactivating gene (RAG) mRNA hematopoetic cells in old and nude mice by supernatants of activated T cells is attributable to the presence of IL-16 as the major factor. IL-16 not only increased RAG mRNA but also increased the numbers and percentages of bone marrow pre-B cells. Related developmental effects have been observed in umbilical cord and peripheral blood cell expansion into mast cells. After 3 weeks of culture in the presence of c-kit ligand, IL-16 induced a 20-fold increase in tryptase and chymase in CD4 hematopoetic progenitor-derived mast cells from normal individuals. Along with its role as a competence growth factor for T cells noted earlier, these experiments demonstrate that the IL-16/CD4 interaction is relevant in hematopoetic progenitor cells of varied lineages as it is in mature T cells.
Role of IL-16 in Lung Diseases

The role of IL-16 in lung disease is incompletely dened. Its role as a putative modulator of the allergic immune response in the lung is supported by its attenuation of airway reactivity and eosinophilia in a mouse model of allergy airway inammation. In humans and mice, IL-16 is readily detected in allergic

INTERLEUKINS / IL-17 395

bronchial epithelium and in the bronchoalveolar lavage after allergen or histamine challenge. In concert with well described in vitro immunomodulation of Tcell activation by antigen, it is postulated that IL-16 acts as a natural regulator of T-cell-mediated inammation by its direct interaction with CD4.
See also: Allergy: Overview. Asthma: Overview. Chemokines. Chemokines, CC: RANTES (CCL5). Chymase and Tryptase. Dendritic Cells. Interleukins: IL-4; IL-5; IL-9. Leukocytes: T cells. Macrophage Inammatory Protein. Stem Cells.

IL-17
tuve , and Q Hamid, S Lajoie-Kadoch, S Le ller, McGill University, Montreal, QC, Canada Z Mu
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-17 (IL-17) has become the focus of many recent studies, specifically with respect to its roles in host defense and the increasing evidence supporting its involvement in lung pathology. This cytokine has a unique structure containing an atypical cystine knot and is the founding member of the IL-17 family of cytokines that shares a high degree of structural homology. IL-17s biological effects relate to its ability to induce the release of various inammatory mediators following binding to its receptor IL-17R. Since it is produced by activated T lymphocytes, this cytokine is thought to play a critical role at the interface between innate and adaptive immunity. The most notable property of IL-17 is its ability to promote neutrophilic inammation via the release of specic chemoattractants and activators. Neutrophil activation by IL-17 leads to the production of cytotoxic mediators such as free oxygen radicals and elastase with resultant tissue injury. Thus, elevated concentrations of IL-17, as recorded in several inammatory conditions of the airways, implicate this cytokine in the onset, maintenance, and progression of many respiratory diseases.

Further Reading
Center DM, Kornfeld H, Ryan TC, and Cruikshank WW (2000) Interleukin 16: implications for CD4 functions and HIV-1 progression. Immunology Today 21(6): 273280. Cruikshank WW, Center DM, Nisar N, et al. (1994) Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression. Proceedings of the National Academy of Sciences, USA 91(11): 5109 5113. Cruikshank WW, Lim K, Theodore AC, et al. (1996) IL-16 inhibition of CD3-dependent lymphocyte activation and proliferation. Journal of Immunology 157(12): 52405248. de Bie JJ, Jonker EH, Henricks PA, et al. (2002) Exogenous interleukin-16 inhibits antigen-induced airway hyper-reactivity, eosinophilia and Th2-type cytokine production in mice. Clinical and Experimental Allergy 32(11): 16511658. Kornfeld H and Cruikshank WW (2001) Prospects for IL-16 in the treatment of AIDS. Expert Opinion on Biological Therapy 1(3): 425432. Kurschner C and Yuzaki M (1999) Neuronal interleukin-16 (NIL16): a dual function PDZ domain protein. Journal of Neuroscience 19(18): 77707780. Little FF and Cruikshank WW (2004) Interleukin-16 and peptide derivatives as immunomodulatory therapy in allergic lung disease. Expert Opinion on Biological Therapy 4(6): 837846. Liu Y, Cruikshank WW, OLoughlin T, et al. (1999) Identication of a CD4 domain required for interleukin-16 binding and lymphocyte activation. Journal of Biological Chemistry 274(33): 2338723395. Lynch EA, Heijens CA, Horst NF, Center DM, and Cruikshank WW (2003) Cutting edge: IL-16/CD4 preferentially induces Th1 cell migration: requirement of CCR5. Journal of Immunology 171(10): 49654968. Mashikian MV, Ryan TC, Seman A, et al. (1999) Reciprocal desensitization of CCR5 and CD4 is mediated by IL-16 and macrophage-inammatory protein-1 beta, respectively. Journal of Immunology 163(6): 31233130. Muhlhahn P, Zweckstetter M, Georgescu J, et al. (1998) Structure of interleukin 16 resembles a PDZ domain with an occluded peptide binding site. Nature Structural Biology 5(8): 682686. Nicoll J, Cruikshank WW, Brazer W, et al. (1999) Identication of domains in IL-16 critical for biological activity. Journal of Immunology 163(4): 18271832. Rand TH, Cruikshank WW, Center DM, and Weller PF (1991) CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. Journal of Experimental Medicine 173(6): 15211528.

Introduction
Interleukin-17 (IL-17), also referred to as IL-17A, is the prototype for a relatively novel, six-membered family of cytokines, including IL-17B, IL-17C, IL17D, IL-17E (IL-25), and IL-17F. These additional cytokines have been identied based on their sequence homology with IL-17, and appear to share proinammatory properties among the family to an extent related to their degree of sequence similarity, as assessed by phylogenic analysis (Figure 1). Indeed, the most homologous members IL-17A and IL-17F have very similar biological effects. IL-17E is the most divergent member sequence-wise and displays a distinct, Th2, activity in vivo. Secreted by activated T lymphocytes, IL-17 is a pleiotropic proinammatory cytokine playing a major part in the neutrophilic airway inammation common to many respiratory disorders. In particular, IL-17 biology is strongly associated with the recruitment of neutrophils via stimulation of the release of specic cytokines and chemokines acting on these granulocytes. In line with this, IL-17 administration into the airways of mice promotes the local release of neutrophil mediators. IL-17 was cloned in 1993 using a library of murineactivated T-cell hybridoma cDNA. The resulting protein was rst named cytotoxic T-lymphocyteassociated antigen (CTLA) 8 and was found to have strong sequence homology with the open reading frame 13 of the T-lymphotrophic virus Herpesvirus

396 INTERLEUKINS / IL-17

IL-17A IL-17F IL-17B IL-17D IL-17C IL-17E
Figure 1 Dendogram representing the degree of sequence similarly and evolutionary relationship among members of the IL-17 family of cytokines. Analysis was performed using the Clustal W method.

1 5 1

2 2

3 3 4.25 kb IL-17 gene

5 5- UTR

3 468 bp ORF 3- UTR

1.86 kb IL-17 mRNA

Amino acid (aa) #

155 aa IL-17 protein 19 1 Protein region Signal peptide Exon sequences Intron sequences 155

Legend Gene mRNA: 3-UTR element ShawKamen sequence (AUUUA)

Figure 2 IL-17 gene, mRNA, and protein structure.

saimiri. Messenger RNA encoding IL-17 was then identied in human peripheral blood T cells and its expression was seen to increase significantly upon Tcell activation. Mapped to chromosome 6p12, human IL-17 shares a 6070% sequence homology with its rodent counterparts and has a highly unique structure when compared to all other identied interleukins.

Structure
IL-17 is synthesized as a monomer of 155 amino acids, with a molecular weight ranging from 15 to 22 kDa (Figure 2). Secreted as a disulde-linked homodimeric glycoprotein, IL-17 has a unique structure and is highly conserved throughout vertebrate evolution. The structural homology reported between rodent and human species is found most notably at sites of glycosylation and suggests a critical role for this cytokine in the mammalian immune system.

The IL-17 family of cytokines is characterized by the presence of a cystine knot conformation. This structural motif resembles the canonical cystine knot-fold formed by six cysteine residues which is typical of the growth factor superfamily. In the case of the IL-17 group of related cytokines, only four cysteines are involved in this three-dimensional (3D) structure and they have been shown to form disulde bridges within IL-17 chains and to be involved in the ultimate formation of the dimeric cytokine.

Regulation of Production and Activity

Human IL-17 mRNA is predictably unstable, as the 30 untranslated region contains many ShawKamen sequences characterized by the presence of AU enriched motifs (50 -AUUUA-30 ) Figure 2. These motifs, common to several other cytokines, growth factors, and oncogenes, are characterized by the instability which they confer to mRNA. Both CD4 and CD8

INTERLEUKINS / IL-17 397

Chemotaxis (through IL-8) NO, elastase IL-8 IL-6 GM-CSF CXCL1 CXCL6 Epithelium Neutrophils

IL-1 TNFIL-6 IL-10 IL-12 IL-1R COX-2 PGE2

Monocytes/ macrophages

Sources of IL-17 Eosinophil CD4+ T cell

PMA Ionomycin CD8+ T cell Neutrophil IL-15 IL-23

Endothelium G-CSF IL-8 IL-6 NO

Fibroblasts IL-6 G-CSF PGE2

Figure 3 Cellular sources and targets for IL-17.

T cells, mainly of the memory phenotype, have been identied as major sources of IL-17. This cytokine is not easily characterized as either a Th1 or Th2 cell type and its cellular distribution resembles that reported for the proinammatory cytokines tumor necrosis factor alpha (TNF-a) or IL-1b. However, it is not a T-cell-specic cytokine since it is also synthesized by neutrophils and eosinophils. Little is known so far about the regulation of IL-17 synthesis. High levels of this cytokine can be induced in vitro in human T cells and peripheral blood mononuclear cells, following their stimulation with a combination of the mitogen, phorbol myristate acetate, and the calcium ionophore, ionomycin. It is also induced by IL-15 in human CD4 T lymphocytes and this nding is complemented by data obtained from murine studies which point to IL-15 as a key

cytokine for the induction of IL-17 in lymphocytes and neutrophils. IL-23 is yet another important cytokine implicated in the activation of IL-17. Produced by dendritic cells, IL-23 is upregulated during infection and promotes the release of IL-17 by CD4 and CD8 T cells. Importantly, IL-23 drives the differentiation of a specialized subset of Th cells, namely ThIL-17, that coexpresses IL-17 and TNF-a (Figure 3).

Biological Function
Cytokine and Chemokine Induction

When exogenously expressed in mouse airways, IL-17 promotes local production of chemokines belonging to the CXC group and alveolar neutrophilia. However, it does not directly elicit neutrophil

398 INTERLEUKINS / IL-17

chemotaxis, but rather induces neutrophil chemotactic activity in a number of structural cells. IL-17 upregulates CXC chemokine production in many types of airway structural cells in vitro, such as epithelial and smooth muscle cells as well as broblasts. This arises primarily through up-regulation of the chemokine IL-8/CXCL8, a strong neutrophil chemoattractant. IL-17 is also known to increase the production of the chemokines CXCL1/GROa; CXCL6/GCP-2 is known to act on neutrophils in vitro (Figure 3).
Interaction with Inammatory Mediators

Immune Response

The proinammatory effects of IL-17 most likely relate to its ability to induce TNF-a and IL-1b production by human monocytes. Importantly, IL-17 also synergizes with these mediators to induce high levels of chemokines such as IL-8 and CXCL1 that act on neutrophils. This synergism is also responsible for increased levels of growth factors such as granulocytecolony stimulating factor (G-CSF) and granulocyte macrophage-CSF (GM-CSF) in human bronchial epithelial cells, venous endothelial cells, and broblasts. IL-17 also acts concomitantly with the Th1 cytokine IFN-g, and with the Th2 cytokines IL-4 and IL-13, to upregulate IL-8 production (Figure 3). Other inammatory mediators upregulated by IL17 include IL-6, IL-10, IL-12, IL-1 receptor antagonist, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). IL-17 also likely contributes to the in situ production of oxygen radicals through inducible nitric oxide synthase (iNOS) and the resultant increase in nitric oxide (NO) release as shown in stromal cells. These observations underline IL-17s role as a potent inammatory mediator of the inammatory process in both Th1 and Th2 settings. IL-17 may also favor neutrophil migration to the sites of inammation since it amplies IFN-g-mediated increases in intercellular adhesion molecule-1 (ICAM-1) in broblasts and human bronchial epithelial cells. In addition, IL-17 increases neutrophil survival, a process involved in cell accumulation in the tissues. The production of G-CSF and GM-CSF, two growth factors that are known to inhibit neutrophil death by apoptosis, is upregulated in human airway epithelial cells. It is through this increase in growth factors as well as in stem cell factor, that IL-17 enhances the capacity of broblasts to sustain the proliferation of CD34 hematopoietic progenitors and promotes their subsequent differentiation into neutrophils. In line with these properties, transgenic mice that systemically overexpress IL-17 are characterized by enhanced granulopoiesis and massive peripheral neutrophilia.

IL-17 is produced early during airway allergen challenge in antigen-sensitized mice and inhibition of endogenous IL-17 is seen to decrease neutrophil numbers at the expense of an exacerbation of eosinophilic inammation linked to higher expression of IL-5. These observations suggest a role for IL17 in the balanced production and accumulation of granulocytes in tissues. However, the exact function of IL-17 in airway antigen challenge requires further definition, as it has also been shown to contribute to allergic inammation and the development of non-specic airway hyperresponsiveness in mice. As described above, IL-17 can activate on cells specialized in the recognition of pathogens and antigen presentation (innate immunity) leading to subsequent generation of specic (adaptive) immune responses. Although IL-17 has not been shown to interact directly with the biology of the pathogen receptor Toll-like receptor (TLR) 4, several reports have proposed a link between this cytokine and the activation of innate immune systems. IL-17 is induced in mice upon infection with endotoxin and the Gram-negative bacteria Klebsiella pneumoniae. IL-17 knockout mice are indeed documented to have impaired clearance of this microorganism in the lung. This phenomenon is associated with a lack of G-CSF production and the absence of neutrophils in the bronchoalveolar lavage. IL-17-mediated host defense may also involve the production of the antimicrobial peptide b-defensin-2, as well as macrophage inammatory protein-3 (MIP-3). The importance of IL-17 in antigen response may also involve its capacity to promote dendritic cell differentiation, as well as its ability to activate T cells in allergic models of hapten-specic hypersensitivity responses in mice.
Tissue Remodeling

IL-17 plays a critical role in tissue remodeling in bone homeostasis and inammatory joint disease by enhancing the expression of enzymes involved in matrix degradation, such as matrix metalloproteinases (MMPs)-1,-3, -9, and -13 in osteoblasts, synoviocytes, chondrocytes, and macrophages. It may also promote bone degradation via the induction of the receptor activator of NF-kB ligand (RANKL) in osteoblasts. IL-17 has been implicated in tissue brosis through induction of IL-11 and IL-6 production by cultured airway broblasts. Additionally, this cytokine promotes angiogenesis by acting on endothelial cell growth and favoring neovascularization and production of angiogenic factors such as vascular endothelial growth factor, PGE1 and PGE2 and NO in lung broblasts and endothelial cells.

INTERLEUKINS / IL-17 399

Receptor
Structure

IL-17 in Respiratory Diseases

IL-17 is present at low levels in the airways under physiological conditions. Apart from T cells, eosinophils within airways represent an important source of this cytokine during allergic inammation, especially in asthma where these granulocytes show increased intracellular levels of IL-17. In line with this observation, IL-17 levels are increased in the bronchoalveolar lavage and the bronchial submucosa. IL-17 is similarly upregulated in induced sputum of individuals with asthma, as compared to controls and subjects with chronic obstructive pulmonary disease. Furthermore, these elevated cytokine levels were found to be associated with the extent of non-specic reactivity of the airways, and correlated with PC20 values in response to metacholine challenge. Higher expression of IL-17 was detected in bronchial biopsies from subjects with moderate-to-severe asthma, as compared to mild asthmatics and control donors. This suggests that IL-17 is involved in the more severe forms of asthma which are characterized by neutrophilic inammation. In this study, importantly, IL-17 levels observed in moderate-to-severe asthma were decreased by a 2-week course of oral corticosteroids. Little data are available to date in relation to the presence of IL-17 in other respiratory diseases. IL-17 expression is increased in both nasal polyps of subjects with chronic hyperplastic sinusitis and induced sputum of subjects with chronic bronchitis. In healthy human volunteers challenged by exposure to inhalation of swine dust, IL-17 levels were noted to be elevated within the bronchoalveolar lavage coinciding with the development of severe neutrophilic lung inammation. This effect is likely to be related to IL-17s property to amplify cellular responses to proinammatory mediators, such as TNF-a and IL-1b. In this context, IL-17 may thus represent a tissue-specic target for pharmacotherapies aimed at resolving airway inammation. It is therefore expected that pharmacotherapy that directly targets IL-17 will ensure more specicity and produce fewer side effects than current anti-inammatory drugs or anti-IL-1b/TNF-a antibody treatments. Neutralizing antibodies against IL-17 have been tested in mice and can effectively reduce the inux of neutrophils into the lung. However, such treatments are also seen to aggravate symptoms in mice suffering from colitis. Therefore, given the critical role of IL-17 and neutrophils in host defense, anti-IL-17 therapy would most likely require selective, tissue-restricted use.
See also: Chemokines, CXC: IL-8. Interleukins: IL-15; IL-23 and IL-27. Leukocytes: Neutrophils; T cells.

IL-17 receptor (IL-17R) is expressed by a wide range of human cell types. The array of cells includes epithelial, smooth muscle and vascular endothelial cells, broblasts, marrow mesenchymal stem cells, as well as cells from the immune system, that is, B and T lymphocytes and myelomonocytic cells. IL-17R is transcribed from multiple exons, giving rise to a protein that displays a molecular weight of 130 kDa. IL-17R is a type I receptor, that is, a single-pass transmembrane protein containing an extracellular amino terminus. Experiments involving blockage of IL-17R with antibodies were seen to inhibit the release of IL-6 upon IL-17 induction, indicating that IL-17R is integral to the IL-17-mediated response. Notable however is IL-17s weak afnity for its receptor, with a Ka value that ranges from only 2 107 to 2 108 M 1. In view of the low concentrations of IL-17 needed to induce biological reactions in vivo, it is hypothesized that IL-17 binds to additional receptors.
Signal Transduction

The signal transduction involved in IL-17-induced cytokine and chemokine production involves activation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated (ERK) and c-Jun terminal kinase (JNK) pathways. MAPK activation is indeed the central pathway mediating IL-17-induced IL-6 and IL-8 production by human bronchial epithelial cells. The synergy observed between IL-17 and other proinammatory cytokines is likely to occur at the mRNA level, in a process involving mRNA stabilization and/or increased transcription. The transcription factor NF-kB appears to have a widespread role in the mediation of cellular responses to IL-17. Activation of MAPKs and NF-kB is required for the induction of the anti-microbial peptide b-defensin-2 as well as iNOS and COX-2 by IL-17 in airway epithelial cells. The adaptor molecule TNF-associated factor (TRAF) 6, rather than TRAF2, is required for IL-17R-mediated activation of NF-kB and JNK and subsequent production of IL-6 and ICAM-1 in broblasts. IL-17-mediated production of inammatory mediators in broblasts can display corticosteroid sensitivity similar to that reported for IL-8, CXCL1, and CXCL6. However, the intracellular pathways involved in cytokine production by IL-17 are probably cell type specic since IL-8 production by IL-17 is not inhibited by corticosteroids in bronchial epithelial and smooth muscle cells.

400 INTERLEUKINS / IL-23 and IL-27

Further Reading
Aggarwal S and Gurney AL (2002) IL-17: prototype member of an emerging cytokine family. Journal of Leukocyte Biology 71(1): 18. Awane M, Andres PG, Li DJ, and Reinecker HC (1999) NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells. Journal of Immunology 162(9): 53375344. Barczyk A, Pierzchala W, and Sozanska E (2003) Interleukin-17 in sputum correlates with airway hyperresponsiveness to metacholine. Respiratory Medicine 97(6): 726733. Chakir J, Shannon J, Molet S, et al. (2003) Airway remodelingassociated mediators in moderate to severe asthma: effect of steroids on TGF-beta, IL-11, IL-17, and type I and type III collagen expression. Journal of Allergy and Clinical Immunology 111(6): 12931298. Hellings PW, Kasran A, Liu Z, et al. (2003) Interleukin-17 orchestrates the granulocyte inux into airways after allergen inhalation in a mouse model of allergic asthma. American Journal of Respiratory Cell and Molecular Biology 28(1): 4250. Kolls JK and Linden A (2004) Interleukin-17 family members and inammation. Immunity 21(4): 467476. Laan M, Cui ZH, Hoshino H, et al. (1999) Neutrophil recruitment by human IL-17 via C-X-C chemokine release in the airways. Journal of Immunology 162(4): 23472352. Linden A and Adachi M (2002) Neutrophilic airway inammation and IL-17. Allergy 57(9): 769775. Miljkovic D and Trajkovic V (2004) Inducible nitric oxide synthase activation by interleukin-17. Cytokine & Growth Factor Reviews 15: 2132. Molet S, Hamid Q, Davoine F, et al. (2001) IL-17 is increased in asthmatic airways and induces human bronchial broblasts to produce cytokines. Journal of Allergy and Clinical Immunology 108(3): 430438. Moseley TA, Haudenschild DR, Rose L, and Reddi AH (2003) Interleukin-17 family and IL-17 receptors. Cytokine & Growth Factor Reviews 14(2): 155174. Prause O, Laan M, Lotvall J, and Linden A (2003) Pharmacological modulation of interleukin-17-induced GCP-2-, GRO-alphaand interleukin-8 release in human bronchial epithelial cells. European Journal of Pharmacology 462(13): 193198. Yao Z, Spriggs MK, Derry JM, et al. (1997) Molecular characterization of the human interleukin (IL)-17 receptor. Cytokine 9(11): 794800. Ye P, Garvey PB, Zhang P, et al. (2001) Interleukin-17 and lung host defense against Klebsiella pneumoniae infection. American Journal of Respiratory Cell and Molecular Biology 25(3): 335340.

cytokines. Structurally, both are closely related to IL-12; however, they are functionally distinct. In response to Gramnegative infection in the airway, the most prominent result of IL-23 activation is the induction of IL-17 as part of a proinammatory axis critical for host defense. Lipopolysaccharide ligation of TLR4 induces IL-23 production by antigen-presenting cells, most prominently myeloid dendritic cells and macrophages. IL-23 stimulates memory CD4 T cells to produce IL-17 resulting in neutrophilic inltration. IL-23 has also been implicated in autoimmune responses, specifically in arthritis and experimental autoimmune encephalitis. The antitumorigenic and antimetastatic properties of IL-23 have also been characterized and, in contrast to those of IL-12, rely on CD8 T cells to mediate the effects; IL-23 activation also induces a memory response to tumors. While IL-27 also primarily induces a T-helper-1 response to infection and is produced by antigen-presenting cells in response to infectious triggers, it acts primarily on naive CD4 T cells, augmenting the actions of IL-12 by inducing high levels of IFN-g. IL-27 also exhibits antitumorigenic properties that are similar in magnitude to those of IL-23 and IL-12. In contrast to IL-23, IL-27 suppresses IL-17 and thus may downregulate autoimmunity.

Introduction
In 2000, Oppmann et al. described a novel factor, which they called p19 or IL-23p19. This member of the IL-6 family of cytokines was noted to have sequence homology with the p35 subunit of IL-12 and was also biologically inactive unless associated with the p40 subunit of IL-12, a soluble cytokine receptor. The p19/p40 heterodimer was named IL-23 and was shown to be produced by activated dendritic cells. Acting primarily on memory CD4 T cells, as well as on CD8 T cells, IL-23 triggers the production of IL-17, a proinammatory cytokine that is critical for neutrophil recruitment in infection by Gram-negative organisms. The identication of IL-23 also helped to explain inconsistencies in the IL-12 literature resulting from differences in the phenotype of p35 / and p40 / mice in experimental models of infection and autoimmunity. Concurrent with investigations dening the role of IL-23 in linking the innate and adaptive immune response to infection, studies demonstrated that IL-23 plays a significant role in the autoimmune response, specifically in experimental autoimmune encephalomyelitis and collagen-induced arthritis. Other groups have also demonstrated that IL-23 has significant activity in the inhibition of growth and metastasis of tumors. In 2002, IL-27 was described. Again, the structural and functional similarities to IL-12 are notable. IL-27 is a heterodimer that consists of p28, an IL12p35-related polypeptide, and EBI3, an IL-12p40related protein. IL-27 is also produced by activated antigen-presenting cells and stimulates naive T-cell subsets, exhibiting synergy with IL-12 to induce

Relevant Website
http://www.ncbi.nlm.nih.gov IL-17 in NCBI database.

IL-23 and IL-27

J K Kolls and P J Dubin, Childrens Hospital of Pittsburgh, Pittsburgh, PA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Interleukin-23 (IL-23) and IL-27 are recently identied heterodimer cytokines with components related to the IL-6 family of

INTERLEUKINS / IL-23 and IL-27 401

interferon gamma (IFN-g) production. Though structurally IL-12, IL-23, and IL-27 are closely related, there are distinct differences in their functions; in some cases the cytokines act synergistically, in others they act antagonistically.

Table 1 Cytokine components of IL-12, IL-23, and IL-27: IL-12p35, IL-23p19, and IL-27p28 are unique type I cytokine, IL-6 subfamily subunits; IL-12p40 and EBI13 are soluble type I receptors Cytokine Heterodimer constituents Type I cytokine Unique IL-6 subfamily subunit IL-12 IL-23 IL-27 p35 p19 p28 Type I receptors Soluble receptors IL-12p40 IL-12p40 EBI3

Structure
IL-23 is comprised of covalently linked p19 and IL-12p40 subunits. The p19 subunit is unique to IL23 and has been classied as a member of the type I cytokines, specifically the IL-6 family of cytokines, based on its sequence homology and a-helical structure. Encoded on chromosome 10 in mouse and chromosome 12 in humans, it appears to be most closely related structurally to the p35 subunit of IL-12, though neither the human nor the mouse p19 exhibit N-glycosylation sites. Both proteins also contain ve cysteines and the p19/p40 complex is linked by disulde bonds. Mouse and human p19 are 196 and 189 amino acids in length, respectively, and there is 70% homology between the two. The IL-12p40 subunit is a type I receptor, more specifically a member of the group 3 soluble receptors. Though it complexes with the p35 subunit of IL-12 and IL-23p19, p40 can also be secreted as a monomer or homodimer. In mice, p40 or (p40)2 has been noted to inhibit IL-12 function; this effect is less prominent when studying human IL-12 and IL-12p40. Of note, this effect was studied using antiIL-12p40 neutralizing antibodies and the effects of the IL-23p19/p40 heterodimer were not controlled for in these experiments. There are a number of parallels between the structures of IL-27 and IL-23. IL-27 is also a heterodimeric cytokine, which consists of a unique p28 subunit that complexes with EBI3, a group 3 soluble cytokine receptor. The p28 subunit, also a member of the IL-6 family of cytokines, is coded on human chromosome 16 and mouse chromosome 7, ultimately forming mature proteins of 24.5 and 23.6 kDa, respectively. Though neither mouse nor human p28 are heavily glycosylated, both exhibit differences in N- and O-glycosylation. Both also exhibit a highly negatively charged loop region, which has not been described in any of the other IL-6 subfamily cytokines. The EBI3, or EpsteinBarr virus-induced gene3, is a group 3 soluble receptor that has roughly 30% homology to IL-12p40. It was initially identied in EBV-infected B lymphocytes, but is also expressed in monocytes and macrophages. EBI3 does not exclusively dimerize with p28; dimerization with IL-12p35 has been documented but is of unclear significance (Table 1).

Regulation of Production and Activity

Though IL-23p19 contains a signal peptide, it is not secreted from mouse or human dendritic cells in appreciable amounts and does not exhibit activity as a single molecule. As a result, coexpression with a variety of nonsignaling group 3 soluble receptors was studied (including EBI3); and only the coexpression of IL-23p19 and IL-12p40 led to detection of enhanced p19 secretion and co-immunoprecipitation of the p19 subunit as a heterodimer. It is also worth noting that because IL-12p40 is found as part of the IL-12 heterodimer, as a monomer (p40) and as a homodimer (p40)2, there is usually detectable IL-12p40 in the absence of IL-23 induction. As a result, IL-23 activity is dependent on IL-23p19 induction, in the presence of IL-12p40 production. Also, though p40 monomer and homodimers inhibit IL-12 activity, it is not clear if the same holds true for IL-23. Because IL-12 and IL-23 are structurally similar and there is a history of apparent inconsistencies in the IL-12 literature pertaining to phenotypic differences of mice decient in IL-12p40 versus IL-12p35, controls for the effect of both of these cytokines are requisite in current studies of IL-23 and IL-12. The initiating trigger for IL-23 production in infection is activation of antigen-presenting cells (dendritic cells or macrophages); lipopolysaccharide (LPS) ligation of TLR4 is the primary trigger for this, though other bacterial components such as other lipopeptides (TLR2) and unmethylated CpG DNA (TLR9) augment this response. The most prominent target of IL-23 is the memory CD4 T cell creating an IL-23/IL-17 proinammatory axis; however, IL-23 receptors are also expressed on subpopulations of antigen-presenting cells suggesting that this is an additional route of IL-23 regulation. Assessment of IL-27 regulation again demonstrates striking parallels between IL-23 and IL-27. That IL27 activity requires coexpression of p28 and EBI3 was determined by coexpressing p28 with other soluble receptors including IL-12p40, CLF-1, and soluble IL-11 receptor. Biological activity occurs only

402 INTERLEUKINS / IL-23 and IL-27

when the heterodimeric form is present. IL-27 production is induced by LPS activation of antigenpresenting cells within 36 h of stimulation in vitro. This time frame is shorter than that required for IL12 production and similar to that observed for IL-23.

IL-23 and IL-27 Receptors

Just as there are many parallels between IL-12, IL23, and IL-27 there are striking similarities among their receptors. While the IL-12 receptor is a heterodimer consisting of IL-12Rb1 and IL-12Rb2 chains, the IL-23 receptor consists of the 12Rb1 chain and an IL-23-specic chain, IL-23R. The IL-23R chain is encoded on human chromosome 1, upstream of the IL-12Rb2. In addition, the extracellular domains of the IL-23R chain are all related to corresponding domains of IL-12Rb2, containing signal sequence, an N-terminal Ig-like domain, and two cytokine receptor domains. The major difference is that IL-23R does not contain three transmembrane-proximal bronectin type III domains found in the IL-12Rb2 chain. There are also potential N-linked glycosylation sites in IL-23R, which are not found in the IL12Rb2 chain. Whether a cell responds to IL-12 or IL-23 depends on the expression of an IL-12Rb1/IL12Rb2 or an IL-23R/IL-12Rb1 receptor complex. The IL-23R chain does not complex with IL-12Rb2 and functionality of the intact IL-23R complex was demonstrated with binding studies as well as cell proliferation studies. The transmembrane domain of IL-23R contains a WQPWS sequence that, in mice, is repeated in a 20 amino acid duplication not found in human IL-23R. This WQPWS is similar to the WSXWS motif observed in other cytokine receptors. The cytoplasmic domain of the human IL-23R chain contains seven tyrosines, six of which are conserved in the mouse IL23R. These tyrosines dene potential binding sites for major components of the intracellular signaling cascades including Src homology 2 domain, signal transducer and activator of transcription (STAT) 1,
IL-23 p40 p19 IL-12R 1 II-23R IL-12 p40 p35 IL-12R 1 IL-12R 2 gp130

STAT3, and STAT4. Though the structure does not clearly predict this, there is also an observed association with Janus kinase (Jak) 2. The differences between the IL-12 b2 chain and IL-23R chain correlated with differences in STAT activation: most notably, IL-12 ligation preferentially activates STAT4 and IL-23 STAT3, and STAT3/ STAT4 dimers; and IL-23s weaker activation of STAT4 correlates with significantly lower IFN-g induction. Though the mechanism is unknown, IL23 ligation on T cells, specifically memory T cells, results in the production of IL-17 A and F. The IL-27 receptor consists of WSX-1 or alternately, the T-cell cytokine receptor (TCCR) and gp130. Though both components are members of the group 2, type I receptors or IL-6 subfamily cytokine receptors, WSX-1/TCCR had previously been identied and its ligand was unknown; gp130 is a promiscuous receptor that binds IL-6 and IL-11 among others (Figure 1 and Table 2). Both WSX-1/TCCR and gp130 are coexpressed on T and B cells, natural killer (NK) cells, monocytes, dendritic cells, mast cells, and endothelial cells. In T cells, Jak1, STAT1, STAT3, STAT4, and STAT5 are phosphorylated, while in monocytes and mast cells STAT3 is phosphorylated.

Biological Function
Studies of the biological function of IL-23 and IL27 preceded or were conducted concurrently with
Table 2 Cytokine receptor components of IL-12, IL-23, and IL27 receptors: all subunits are members of the type I cytokine receptor family; IL-12Rb2, IL-23R, and WSX-1/TCCR are unique components for the IL-12, IL-23, and IL-27 receptors, respectively; IL-12Rb1 and gp130 subunits are promiscuous Cytokine receptor Heterodimer constituents Type I receptors IL-12R IL-23R complex IL-27R IL-12Rb2 IL-23R WSX-1/TCCR Type I receptors Promiscuous/common IL-12Rb1 IL-12Rb1 gp130

IL-27 EBI3 p28 WSX-1

Figure 1 IL-12, IL-23, and IL-27 cytokine and receptor complexes. The IL-23 cytokine consists of a heterodimer of IL-12p40 and IL-23p19, which is similar in structure to the IL-12p35 subunit. IL-12 is comprised of the IL-12p40 subunit and a p35 subunit. IL-27 consists of EBI3 and p28. IL-12p40 and EBI3 are group 3 soluble cytokine receptors. IL-23p19, p35, and p28 are members of the IL-6 subfamily of type I cytokines. The IL-23 receptor consists of the IL-12Rb1 and IL-23R subunits. The IL-12 receptor consists of the IL-12Rb1 and IL-12Rb2 subunits. The IL-12Rb2 and IL-23R subunits are structurally similar. The IL-27 receptor consists of a gp130 and WSX-1 subunit.

INTERLEUKINS / IL-23 and IL-27 403

studies of IL-23 and IL-27 receptors. The earliest studies by Weikowski et al. compared the effects of ubiquitous, liver-specic, and bone-marrow-specic constitutive expression of IL-23p19 in vivo. With constitutive, ubiquitous expression, the affected mice exhibited runting, shortened life span, infertility, and systemic inammatory changes. Specifically, levels of IL-1, tumor necrosis factor alpha (TNF-a), and the number of circulating neutrophils were increased (IL-17 levels were not reported as this interaction was not known at the time). Similar problems were observed with bone-marrow-specic, but not liverspecic overexpression; this difference was attributed to the lack of p40 expression in the liver and suggested that the main mediator of IL-23 production was hematopoietic cells. Other studies conrmed that stimulated antigen-presenting cells were the primary source of IL-23. After the identication of the IL-23 receptor, the pattern of IL-23 receptor expression was also utilized to study IL-23 function; IL-23 receptor mRNA was present primarily in T-helper-1 (Th1) and Th2 cells and NK cells. p40 / and p35 / mice initially developed to study IL-12 function were also utilized early on and it was not until the development of p19 / mice that the direct analysis of IL-23 function could be performed. Some of these studies demonstrated that IL-23 played an important role in

regulating the production of CXC chemokines (a subfamily of the SCY chemokines), particularly CXCL1 (KC and MIP-2), to a variety of infectious agents, the most prominent pulmonary pathogens being extracellular Gram-negative bacteria such as Klebsiella pneumoniae. These effects were mediated by IL-23 induction of IL-17, a key regulator of the CXC chemokines KC, MIP-2, and CXCL1 and CXCL5 (LIX), as well as G-CSF (Figure 2). IL-23 has also been shown to play a key role in autoimmune encephalitis and arthritis, though a role for IL-23 in autoimmune pulmonary manifestations has not yet been demonstrated. In the model of autoimmune encephalitis, IL-23 generates a unique population of CD4 T cells termed ThIL-17 cells, which coexpress IL-17A, IL-17F, IL-6, and TNF and upon adoptive transfer are the encephalopathogenic T-cell populations. In contrast, classic Th1 cells grown in the presence of IL-12, which produced IFN-g upon antigen restimulation, were largely devoid of IL-17 production and failed to confer encephalitis upon adoptive transfer. More recently, IL-23 has been shown to have antitumor properties. Studies of IL-23 antitumorigenic and antimetastatic properties demonstrated that IL23 induced long-term regression of cancers similar to that of IL-12 transduced tumors; in addition, IL-23 induced a memory response to tumor rechallenge,

TLR4/TLR2/TLR9 Pathogen

IL-23 + CD8+ T cell + IL-17 CD4+ T cell

Vascular endothelium

Fibroblasts

Airway epithelium

ICAM-1, CXCL8, GM-CSF

CXCL1, 5, 8

IL-6, CXCL1, CXCL5, CXCL8, G-CSF, GM-CSF

Figure 2 IL-23 its proximate targets and downstream effects. On TLR stimulation by a pathogenic stimulus, the antigen-presenting cell is stimulated to produce IL-23. IL-23 activation of CD4 and CD8 T cells results in IL-17 production. IL-17 stimulates the vascular endothelium, broblasts, and the airway epithelium to produce CXC chemokines and G-CSF, GM-CSF, and IL-6.

404 INTERLEUKINS / IL-23 and IL-27

Pathogen

IL-27 + Mast CD8+ T cell CD4+ T cell NK NK CD4+ T cell CD8+ T cell Monocyte

IFN-

IL-4/IL-5

IFN-

Degranulation

IL-6/TNF-

Figure 3 IL-27 its targets and downstream effects. The antigen-presenting cell produces IL-27 in response to stimulation by a pathogenic antigen. IL-27 has both pro- and anti-inammatory effects on CD8 , CD4 , and natural killer (NK) cells as well as antiinammatory effects on mast cells and monocytes. IL-27 is involved most prominently in IFN-g, IL-4, and IL-5 regulation. It blocks degranulation of mast cells and downregulates IL-6 and TNF-a production by monocytes.

which was mediated through CD8 T cells. Other studies also have shown that CD40 ligand expression on lung tumors in mice activates the immune response, upregulating p19 and p40 at a transcriptional level, with resulting regression of the tumors. Currently, the main function of IL-27 appears to be stimulation of naive CD4 T cells and production of IFN-g (Figure 3). Though IL-27 does promote an early Th1 response, it is not absolutely required, working synergistically with IL-12 for the development of a Th1 response. IL-27 promotes IFN-g by STAT1-dependent and STAT1-independent induction of T-bet. T-bet is a transcription factor that also induces expression of IL-12Rb2 on effector cells, allowing increased responsiveness to IL-12. The IL27R also activates STAT4, which acts to polarize Th1 effector cells. As with many other IL-6 family cytokines, IL-27 exhibits both proinammatory and anti-inammatory properties. In toxoplasmosis, WSX-1 / mice develop immune-mediated liver necrosis, implying that the presence of IL-27 signaling plays an inhibitory role in the immune response. The mechanisms for this are currently unknown and similar anti-inammatory roles have not been specically described for IL-27 in the lung; of note,

however, WSX-1-decient mice do develop more severe lung pathology and succumb to infection, in spite of a lower organism burden.

IL-23 and IL-27 in Respiratory Diseases

To date, IL-23 has been studied in murine models of Gram-negative (Klebsiella) and mycobacterial pulmonary infection as well as lung tumors. Though studies have not dened the role of IL-23 in human lung diseases, the key differences between IL-12 and IL-23 are the ability of IL-23 to induce IL-17 production through activation of memory CD4 and, possibly, CD8 T cells, as well as the induction of a memory response to IL-23-sensitive tumors. It appears that optimal IL-23 is required for optimal IL17 production, which subsequently regulates CXC chemokines and G-CSF production. Though IL-27 has been even less well studied, experimental models demonstrate that it plays a significant role in Listeria monocytogenes and mycobacterial infection by inducing Th1 immune mechanisms.
See also: Acute Respiratory Distress Syndrome. Chemokines. Defense Systems. Dendritic Cells. Interferons. Interleukins: IL-12. Leukocytes: T cells; Pulmonary Macrophages. Pneumonia: Community

INTERSTITIAL LUNG DISEASE / Overview 405 Acquired Pneumonia, Bacterial and Other Common Pathogens. Toll-Like Receptors. Transgenic Models.
Langrish CL, Mckenzie BS, Wilson NJ, et al. (2004) IL-12 and IL-23: master regulators of innate and adaptive immunity. Immunology Reviews 2002: 96105. Lankford CS and Frucht DM (2003) A unique role for IL-23 in promoting cellular immunity. Journal of Leukocyte Biology 73(1): 4956. Oppmann B, Lesley R, Blom B, et al. (2000) Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar to as well as distinct from IL-12. Immunity 13(5): 715725. Parham C, Chirica M, Timans J, et al. (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. Journal of Immunology 168(11): 56995708. Park H, Li Z, Yang XO, et al. (2005) A distinct lineage of CD4 T cells regulates inammation by producing IL-17. Nature Immunology 6(11): 11331141. Panz S, Hibbert L, Mattson J, et al. (2004) WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27. Journal of Immunology 172(4): 22252231. Panz S, Timans JC, Cheung J, et al. (2002) IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4( ) T cells. Immunity 16(6): 779790. Villarino A, Hibbert L, Lieberman L, et al. (2003) The IL-27R (WSX-1) is required to suppress T cell hyperactivity during infection. Immunity 19(5): 645655. Villarino A, Huang E, and Hunter CA (2004) Understanding the pro- and anti-inammatory properties of IL-27. Journal of Immunology 173(2): 715720. Wiekowski MT, Leach MW, Evans EW, et al. (2001) Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inammation, runting, infertility, and premature death. Journal of Immunology 166(12): 75637570.

Further Reading
Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, and Gurney AL (2003) Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. Journal of Biological Chemistry 278(3): 19101914. Brombacher F, Kastelein R, and Gottfried A (2003) Novel IL-12 family members shed light on the orchestration of Th1 responses. Trends in Immunology 24(4): 207212. Cooper AM, Kipnis A, Turner J, et al. (2002) Mice lacking bioactive IL-12 can generate protective, antigen-specic cellular responses to mycobacterial infection only if the IL-12 p40 subunit is present. Journal of Immunology 168(3): 13221327. Ghilardi N, Kljavin N, Chen Q, et al. (2004) Compromised humoral and delayed-type hypersensitivity responses in IL-23decient mice. Journal of Immunology 172(5): 28272833. Happel KI, Zheng M, Young E, et al. (2003) Cutting edge: roles of Toll-like receptor 4 and IL-23 in IL-17 expression in response to Klebsiella pneumoniae infection. Journal of Immunology 170(9): 44324436. Harrington LE, Hatton RD, Mangan PR, et al. (2005) Interleukin17-producing CD4 effector cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nature Immunology 6(11): 11231132. Hunter CA (2005) New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions. Nature Reviews: Immunology 5(7): 521531. Kolls JK and Linden A (2004) Interleukin-17 family members and inammation. Immunity 21(4): 467476.

INTERSTITIAL LUNG DISEASE

Contents

Overview Alveolar Proteinosis Amyloidosis Cryptogenic Organizing Pneumonia Hypersensitivity Pneumonitis Idiopathic Pulmonary Fibrosis Lymphangioleiomyomatosis

Overview
K K Brown and G P Cosgrove, University of Colorado Health Sciences Center, Denver, CO, USA
& 2006 Elsevier Ltd. All rights reserved.

clinical features, the mechanism of disease, outcome, type, or even availability of therapy for each specic disease often differs dramatically. An accurate diagnosis is therefore essential for the determination of prognosis and appropriate management. This article provides a framework for the classication and evaluation of patients with ILD.

Abstract
Comprised of over 150 named disorders, interstitial lung disease (ILD) is a complex area of pulmonary medicine for generalists and experts alike. While many of the disorders share common

Introduction
The interstitial lung disease (ILD) is a heterogeneous group of disorders that adversely affects the

406 INTERSTITIAL LUNG DISEASE / Overview

pulmonary parenchyma. They are classied together due to similar clinical characteristics (Table 1). A more appropriate term for this group of disorders would be the diffuse parenchymal lung diseases since the entire lung, not just the interstitium, may be involved. For the purposes of this article, these terms can be considered synonymous. Pulmonary infections can mimic ILD, and while they may share
Table 1 Clinical classication of interstitial lung disease (with examples) Systemic diseases Collagen vascular diseases Scleroderma Rheumatoid arthritis grens syndrome Sjo Polymyositis/dermatomyositis Immunodeciency Common variable immunodeciency Exposures Avocational Hypersensitivity pneumonitis Hot tub lung Bird breeders lung Compost lung Environmental Hypersensitivity pneumonitis Farmers lung Environmental molds Occupational Asbestosis Berylliosis Silicosis Popcorn workers lung Drug-induced lung disease Amiodarone Methotrexate Nitrofurantoin Genetic Familial interstitial pneumonia HermanskyPudlack syndrome Surfactant protein-C mutation Idiopathic Idiopathic interstitial pneumonias Idiopathic pulmonary brosis Non-specic interstitial pneumonia Cryptogenic organizing pneumonia Desquamative interstitial pneumonia/respiratory bronchiolitis interstitial pneumonia Lymphoid interstitial pneumonia Acute interstitial pneumonia Primary disorders Sarcoidosis Pulmonary Langerhans cell histiocytosis Pulmonary alveolar proteinosis Small vessel vasculitides of the lung Wegeners granulomatosis ChurgStrauss syndrome Microscopic polyarteritis

clinical and radiographic features, they will not be considered a form of ILD herein. The focus of this article is to provide a framework for the physician to think about the evaluation and classication of the patient with ILD.

Etiology
The sheer number and variety of clinical entities that fall under the heading of ILDs (greater than 150 have been described) make any generalizations regarding etiology only marginally helpful. While many of the dened disorders share clinical, radiographic, and in some cases even pathologic features, the mechanism of disease, prognosis, type of therapy, and expected response for each specic disease often differs drastically, making identication of the specic etiology essential. Categorization on the basis of an etiologic scheme leads to the following groupings: those disorders associated with systemic disease; those associated with inhaled or systemic exposures (medication, drug, avocations, occupations, environmental, and accidental); those associated with heritable genetic traits (familial interstitial pneumonia); and those that are currently idiopathic, offers a potentially useful approach to initial evaluation. There are approximately 100 000 hospital admissions that result from acute or chronic ILDs. The prevalence has been estimated to be 80.9 per 100 000 in males and 67.2 per 100 000 in females with the respective incidence of 31.5 per 100 000 and 26.1 per 100 000. Of those patients, idiopathic pulmonary brosis (IPF) accounted for 45% of the cases, making it the most common ILD (see Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis). The basic mechanisms of disease for the majority of these disorders have yet to be determined. A generalized hypothesis suggests an initial injury to the epithelium (and less frequently to the endothelium), with a subsequent pathologic repair response, with varying degrees of cellular inammation, extracellular matrix deposition (brosis), and architectural distortion. In progressive brotic disease, it has been suggested that a process of disordered and incomplete wound healing in response to recurrent epithelial injury occurs. Known causes of lung injury leading to cellular inltration or lung brosis include: inhalation of environmental inorganic particles (such as silica), or organic antigens (such as avian antigens as occurs in hypersensitivity pneumonitis); vascular dissemination of an injurious agent to the lung (as occurs in drug-induced or some collagen vascular disease-associated ILDs). In several primary diseases such as lymphangioleiomyomatosis, amyloidosis, and lymphangitic carcinomatosis, rather than excessive

INTERSTITIAL LUNG DISEASE / Overview 407

deposition of extracellular matrix or inltration with inammatory cells, accumulations of smooth muscle cells, amyloid brils, and malignant cells occur, respectively.

Clinical Features
ILDs, although diverse in etiology, generally present with similar symptoms and ndings. The value of a thorough medical history and examination cannot be overemphasized and will often identify both the underlying diagnosis and its etiology. Dyspnea on exertion, a nonproductive cough, or an abnormal chest radiograph are the most common presenting features. The duration of symptoms and the rate of their progression differentiate the acute (days to weeks) and subacute (weeks to months) from the much more common chronic (months to years) ILDs (see Table 2). Patients should be specifically questioned about the presence of wheezing, hemoptysis, or chest pain, as the presence of these more unusual symptoms helps pare the differential diagnostic possibilities and may suggest an airwaycentered process (e.g., bronchiolitis), diffuse alveolar hemorrhage, or those diseases that often present with pleural involvement (e.g., rheumatoid arthritis) or pneumothoraces (e.g., lymphangioleiomyomatosis). A detailed past medical history and review of systems should be obtained to identify prior or evolving systemic diseases that may be associated with ILDs. For example, a subset of patients can develop parenchymal lung disease prior to the onset of the characteristic systemic manifestations of collagen vascular disease; adult onset Raynauds phenomenon, sicca symptoms, or dysphagia in the setting of an ILD would raise this concern. Inammatory bowel diseases may manifest with ILD. Therefore, chronic diarrhea or abdominal complaints should be queried. Neurologic symptoms, such as polydypsia and polyuria, may be extrapulmonary manifestations of sarcoidosis or pulmonary Langerhans cell histiocytosis. Vasculitides, such as Wegeners granulomatosis or ChurgStrauss syndrome, often present with systemic manifestations such as hematuria, mononeuritis multiplex, and ILD.
Table 2 Acute or subacute interstitial lung diseases Acute interstitial pneumonia Idiopathic Collagen vascular disease-associated ILD Acute eosinophilic pneumonia Cryptogenic organizing pneumonia Drug-induced ILD Diffuse alveolar hemorrhage Hypersensitivity pneumonitis

Current and previously used medications, including over-the-counter and naturopathic medications as well as nutritional supplements, should be reviewed to identify any potential agents predisposing patients to ILD. Specic examples are prior exposure to amiodarone in patients with dysrhythmias, nitrofurantoin prophylaxis in those with recurrent urinary tract infections, or chemotherapy such as bleomycin in those with remote malignancies such as lymphomas. All medications should be considered in the differential diagnoses of ILD, especially when there is a clear temporal association with the onset of symptoms. An exhaustive investigation into clinically signicant environmental, occupational, avocational, or accidental exposures should be performed in all patients with ILD. In the case of hypersensitivity pneumonitis, the immunologic response and lung injury is driven by continued exposure (see Interstitial Lung Disease: Hypersensitivity Pneumonitis). To avoid persistent and/or progressive lung disease, antigen avoidance with abatement of the exposure is almost always needed. Patients should be questioned about prior or ongoing exposure to birds (an avocation highly associated with hypersensitivity pneumonitis), hot tubs, or other damp environments (potential exposure to molds and fungi). While it is important to document a patients previous and current occupations, it is just as important to identify the specic duties performed, as general job titles often fail to alert one to clinically pertinent exposures. Heritable interstitial lung disease is more common than previously appreciated, and a detailed family medical history serves to identify potential disorders such as tuberous sclerosis, familial interstitial pneumonia, sarcoidosis, or HermanskyPudlak syndrome. Siblings or a rst-degree relative with documented ILD are important historical keys. As with most pulmonary diseases, quantifying the extent of current or former tobacco use is important. There are several smoking-related ILDs that should be considered in a current smoker or individual with significant passive exposure: respiratory bronchiolitis ILD (RB-ILD), desquamative interstitial pneumonitis (DIP), pulmonary Langerhans cell histiocytosis, and Goodpastures syndrome with diffuse alveolar hemorrhage. In all of these diseases, active tobacco use is associated with the development of the disease and/ or increased disease activity. On physical examination, resting tachypnea may exist depending on the severity of disease. Clubbing, while most commonly found in patients with IPF, may be seen in other chronic brotic ILDs such as asbestosis, silicosis, or chronic or brotic

408 INTERSTITIAL LUNG DISEASE / Overview

hypersensitivity pneumonitis. Bibasilar, mid-late-inspiratory crackles are a hallmark of most brosing ILDs. A mid-inspiratory wheeze or squeak may be heard in airway-centered ILD such as the bronchiolitides. Alternatively, patients with granulomatous lung disease such as sarcoidosis or berylliosis may have few if any demonstrable ndings on auscultation, despite significant physiologic or radiographic abnormalities. An increased second heart sound, a holosystolic murmur in the tricuspid area, or overt cor pulmonale with ascites and lower extremity edema suggests pulmonary hypertension. Extrathoracic signs of collagen vascular diseases, such as xerostomia, xero-ophthalmia, synovitis with deforming arthritis, sclerodactyly, telangectasias, or proximal muscle weakness should be specifically sought.

Physiology
Pulmonary physiologic abnormalities are present in the majority of patients at the time of presentation and may be restrictive, obstructive, or mixed. An isolated abnormality in gas exchange, as indicated by an isolated decrease in carbon monoxide diffusing capacity (DLCO) or exercise-induced desaturation as measured by pulse oximetry, may exist particularly early in the course or with milder ILD. Pulmonary physiology serves both as a guide to the diagnosis and as a way to grade the severity of impairment, which may be important in determining prognosis and response to therapy. Occasionally, pulmonary function testing may lack the sensitivity to detect clinically significant disease, especially in early disease. In some diseases, physiological ndings may under-represent the extent of disease, as is the case in some patients with hypersensitivity pneumonitis or mixed disease with emphysema and ILD. In those patients in which ILD is clinically a concern but the pulmonary physiology is normal, formal cardiopulmonary exercise testing should be considered to more accurately quantify ventilatory, cardiovascular, and gas exchange abnormalities.

In general, plain chest radiographic abnormalities can be classied as alveolar-lling, interstitial, or mixed disorders. Alveolar-lling disorders often result in poorly dened coalescent opacities and consolidation with air bronchograms, as occurs in alveolar proteinosis or cryptogenic organizing pneumonia (COP). Interstitial opacities denote either reticular (linear) or nodular opacities, typically the result of expansion of the interstitial compartment with extracellular matrix, inammatory or malignant cells, or smooth muscle proliferation. In most disorders, a combination of reticular and nodular opacities occurs. High-resolution computed tomography (HRCT) is the most sensitive and accurate radiographic study in the evaluation of ILD and should be considered a standard piece of the evaluation. Specic abnormal radiographic features (such as ground-glass opacities or honeycomb cysts) can be combined into an overall radiographic pattern, and this pattern of disease almost always gives a strong indication of the underlying histopathology, serving to suggest a small group of clinical disorders or even a specic disease (see Table 3 and Figure 1). Broadly speaking, abnormal HRCT features in ILD can be classied as parenchymal opacication (ground-glass attenuation and consolidation), lines (reticular lines and septal thickening), cysts (isolated
Table 3 High-resolution computed tomographic features in ILD Abnormal feature Parenchymal opacication Ground-glass opacities Consolidation Diseases (e.g.) NSIP DIP COP Eosinophilic pneumonia

Lines Reticular Septal thickening

IPF Asbestosis Sarcoid Lymphangitic cancer

Cysts Isolated

Radiology
An abnormal plain chest radiograph is a common initial clue to the presence of ILD. While certain characteristic patterns may suggest specic disorders (e.g., chronic eosinophilic pneumonia), the plain radiograph is almost never diagnostic by itself, and up to 10% of patients with clinically significant disease may have a normal plain radiograph at presentation, as is the case with some patients with desquamative interstitial pneumonitis, sarcoidosis, or hypersensitivity pneumonitis.
Honeycombing

Lymphangioleiomyomatosis PLCH IPF Other brosing lung disease

Nodules Ground-glass Soft tissue Calcic Traction bronchiectasis Mosaic pattern Air trapping

Hypersensitivity pneumonitis Sarcoidosis/berylliosis Remote granulomatous infection IPF Other brosing lung disease Bronchiolitis Bronchiolitis

INTERSTITIAL LUNG DISEASE / Overview 409

Figure 1 High-resolution computed tomograms demonstrating the radiographic features commonly present in ILD. (a) Dense alveolar consolidations with air bronchograms in a patient with pulmonary lymphoma. (b) Reticular opacities with traction bronchiectasis in a patient with idiopathic non-specic interstitial pneumonitis. (c) Subpleural honeycombing, traction bronchiectasis, and reticular opacities in a patient with IPF. (d) Ground-glass opacities with associated pulmonary cysts in a patient with desquamative interstitial pneumonia.

Table 4 Diagnostic utility of bronchoalveolar lavage in the correct clinical context Often diagnostic Alveolar proteinosis Alveolar hemorrhage Eosinophilic pneumonia Bronchoalveolar cell carcinoma Certain infections Often useful Hypersensitivity pneumonitis IPF Often useful when combined with transbronchial biopsy Sarcoidosis/beryliosis COP PLCH

Table 5 Probability of denitive histologic diagnosis on transbronchial biopsy Often diagnostic Sarcoidosis/beryliosis Hypersensitivity pneumonitis Eosinophilic pneumonia Lymphangitic malignancy Alveolar proteinosis Certain infections Occasionally diagnostic Vasculitis Amyloidosis PLCH Lymphangioleiomyomatosis Never diagnostic Usual interstitial pneumonitis Organizing pneumonia Non-specic interstitial pneumonia DIP/RBILD LIP Bronchiolitis

and honeycomb), nodules (soft tissue, ground-glass and calcic density), traction bronchiectasis, mosaic pattern, and air trapping. By combining these features with their distribution, a differential diagnosis can be

410 INTERSTITIAL LUNG DISEASE / Overview

Figure 2 Photomicrographs of histologic patterns that may occur with different interstitial lung diseases. (a, b) Dense alveolar consolidation with areas of intraluminal brin plugs termed Mason bodies (mb) in a patient with cryptogenic organizing pneumonia. (c, d) Homogeneous septal expansion with lymphoplasmocytic inltration in a patient with idiopathic non-specic interstitial pneumonia. (e, f) Heterogeneous brosing process with septal expansion, honeycombing, and broblastic foci (ff) directly adjacent to relatively normal alveoli in a patient with IPF/UIP. (g, h) Peribronchiolar inammation and septal thickening are accompanied by multinucleated gaint cells (arrow) and septal noncaseasting granulomas in a patient with hypersensitivity pneumonitis. Original magnications: (a, c, e, g) 40; (b, d, f, h) 200. Photomicrographs courtesy of C D Cool.

generated. For example, the combination of peripheral predominant, basal predominant reticular lines, honeycombing, and traction bronchiectasis with minimal ground-glass opacity and in the absence of other abnormal features can provide a highly condent

diagnosis of pathologic usual interstitial pneumonia (UIP). Abnormal CT ndings outside of the abnormal lung parenchyma such as pleural thickening with calcication, esophageal dilation, and mediastinal lymphadenopathy may add to the discriminative

INTERSTITIAL LUNG DISEASE / Overview 411

ability of the scan and suggest disorders such as asbestos-related lung disease, collagen vascular disorders such as scleroderma, and sarcoidosis. However, while a particular radiographic pattern may suggest a small group of disorders, or even a specic disorder, it must be viewed within the entire clinical context using the comprehensive history, examination, physiology, and pathologic data to allow for an accurate clinical diagnosis.

lung injury. It remains the most common model used today to investigate the mechanisms underlying interstitial lung disease. A newer model has been developed in which transforming growth factor beta1 is overexpressed within the lung and appears to result in abnormalities more representative of the progressive brotic response noted in the human diseases.

Management and Current Therapy

The management of patients with ILD is critically dependent upon the underlying etiology. Diseasespecic treatment is addressed in each individual disease article. In all patients, regardless of available therapy, supplemental oxygen should be utilized in those with hypoxemia at rest, with activity, or nocturnally to avoid the secondary development of right heart disease. Pulmonary rehabilitation should be considered in all patients to maximize their functional status, and the use of vaccination to decrease the frequency of respiratory infections is strongly encouraged. In those with chronic, progressive disorders, psychosocial support in the form of local or national support groups is often very helpful.

Pathology
In those in whom a condent diagnosis cannot be obtained by clinical, physiologic, radiographic, and laboratory testing, additional invasive or semi-invasive testing is often necessary. Bronchoscopy can be used to visually inspect the airway, perform bronchoalveolar lavage (BAL), and perform endobronchial and transbronchial lung biopsies. BAL is rarely diagnostic (Table 4), must be interpreted within the clinical context, and generally serves to rule out or lower the likelihood of competing diagnoses. Lung biopsy is often required to provide a denitive pathologic diagnosis and can be performed via a bronchoscope (transbronchial biopsy) and/or via a surgical route (video-assisted thoracoscopic (VATS) or open procedure). Transbronchial and endobronchial biopsies are small, focal, and limited in their ability to provide a pathologic diagnosis. However, they can be performed on an outpatient basis and can be diagnostic in the correct clinical settings (see Table 5). Surgical lung biopsies are performed in those patients in whom a condent clinico-radiographic diagnosis cannot be obtained and transbronchial biopsies are thought to be of limited usefulness or demonstrate non-specic ndings. The histopathologic features identied on surgical lung biopsy samples lead to an accurate diagnosis in greater than 90% of patients (see Figure 2). The morbidity and mortality of surgical lung biopsies has significantly decreased with the advent of the VATS approach. In light of the dramatic differences in prognosis between several ILDs that may mimic one another, a surgical lung biopsy is recommended when a condent diagnosis is unattainable in its absence, or a denitive diagnosis is required.

Acknowledgment
This work is supported by the NIH (NHLBI SCOR HL67671). Dr Cosgrove is a Parker B Francis fellow in Pulmonary Research.
See also: Interstitial Lung Disease: Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis.

Further Reading
American Thoracic Society Committee of the Scientic Assembly on Environmental and Occupational Health. (1997) Adverse effects of crystalline silica exposure. American Journal of Respiratory and Critical Care Medicine 155(2): 761768. American Thoracic Society (2000) Idiopathic pulmonary brosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). American Journal of Respiratory and Critical Care Medicine 161(2 Pt 1): 646664. American Thoracic Society/European Respiratory Society (2002) American Thoracic Society/European Respiratory Society international multidisciplinary consensus classication of the idiopathic interstitial pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. American Journal of Respiratory and Critical Care Medicine 165(2): 277304. American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Other Granulomatous Disorders (1999) Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other

Animal Models
One of the major limitations to understanding the pathobiology of ILD is the lack of an appropriate model to mimic the overwhelming majority of ILD. Bleomycin- or paraquat-induced pulmonary brosis serves as a model of brosing lung disease but is likely more representative of acute toxic or drug-induced

412 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis

Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee, February 1999. American Journal of Respiratory and Critical Care Medicine 160(2): 736755. Akira M, Hara H, and Sakatani M (1999) Interstitial lung disease in association with polymyositisdermatomyositis: long-term follow-up CT evaluation in seven patients. Radiology 210(2): 333338. Bodolay E, Szekanecz Z, De ve nyi K, et al. (2005) Evaluation of interstitial lung disease in mixed connective tissue disease (MCTD). Rheumatology (Oxford) 44(5): 656661. Coultas DB, Zumwalt RE, Black WC, and Sobonya RE (1994) The epidemiology of interstitial lung diseases. American Journal of Respiratory and Critical Care Medicine 150(4): 967972. Fan LL, Kozinetz CA, Deterding RR, and Brugman SM (1998) Evaluation of a diagnostic approach to pediatric interstitial lung disease. Pediatrics 101(1 Pt 1): 8285. Guidotti TL, Miller A, Christiani D, et al. (2004) Diagnosis and initial management of nonmalignant diseases related to asbestos. American Journal of Respiratory and Critical Care Medicine 170(6): 691715. Lacasse Y, Selman M, Costabel U, et al. (2003) HP Study Group. Clinical diagnosis of hypersensitivity pneumonitis. American Journal of Respiratory and Critical Care Medicine 168(8): 952 958. Raghu G and Brown KK (2004) Interstitial lung disease: clinical evaluation and keys to an accurate diagnosis. Clinics in Chest Medicine 25(3): 409419, v. Research and Scholarship (1999) Respiratory health hazards in agriculture. American Journal of Respiratory and Critical Care Medicine 158(5 Pt 2): S1S76. Schwarz MI, King TE, and Raghu G (2003) Approach to the evaluation and diagnosis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, pp. 130. ON: Blackwell. Selman M (2004) Hypersensitivity pneumonitis: a multifaceted deceiving disorder. Clinics in Chest Medicine 25(3): 531547, vi. Strimlan CV (1997) Interstitial lung disease and primary Sjogrens syndrome: clinical-pathological evaluation and response to treatment. American Journal of Respiratory and Critical Care Medicine 155(4): 1489. previously healthy individuals, who present with several months of dyspnea and impaired pulmonary gas transfer of variable severity. Milky appearing, lipid-rich uid recovered at bronchoalveolar lavage is typical. Lung biopsy, when performed, shows minimally inamed alveoli uniformly lled with amorphous eosinophilic material that on electron microscopy contains characteristic lamellar bodies. Current concepts of pathogenesis of acquired PAP are that the excess surfactant results from impaired clearance by alveolar macrophages, as a result of inhibition by specic polyclonal autoantibodies of trophic physiological signals from the endogenous cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). Current therapy consists of whole-lung lavage under general anesthesia, repeated as guided by clinical status. Some adult patients also respond to the repeated administration of recombinant human GM-CSF.

Introduction
Pulmonary alveolar proteinosis (PAP), rst recognized in 1958, is a rare but distinctive condition characterized by the widespread intra-alveolar accumulation of excess surfactant with associated lung dysfunction of variable severity. The most common form (490%) is that acquired by previously healthy adults without any familial predisposition, but congenital and secondary forms also exist. Cases of acquired PAP have been described from all populated continents, without any clear racial or geographic predisposition. The median age of adult patients is B40 years, with more than 70% being smokers prior to diagnosis. Men are affected twice as commonly as women, but this may be confounded by a globally greater proportion of men who smoke, as the genders are equally affected among nonsmoking patients. PAP in infants is a pathogenetically separate process to that in adults with a distinct histopathology, resulting from one of several identied genetic disorders. Even the most common adult acquired form of PAP is rare, with an estimated annual incidence of 0.36 cases per million, and a prevalence of 3.70 cases per million.

Alveolar Proteinosis
J J Presneill, The Royal Melbourne Hospital, Melbourne, VIC, Australia K Nakata, Niigata University Medical and Dental Hospital, Niigata, Japan J F Seymour, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
& 2006 Elsevier Ltd. All rights reserved.

Etiology
More than 90% of cases are acquired by apparently previously healthy adults. This disease was previously termed idiopathic PAP and is now regarded as an acquired autoimmune disease based upon the recognition of high-afnity polyclonal autoantibodies that specifically eliminate physiologically important granulocyte-macrophage colony-stimulating factor (GM-CSF) activity in the lungs of essentially all cases so far examined. In contrast, congenital PAP (2% of PAP cases) results from various genetic disorders of surfactant proteins B and C, or the common signal transducing b-chain of the GM-CSF

Abstract
Pulmonary alveolar proteinosis (PAP) is a rare clinical syndrome with three currently recognized forms: congenital, secondary, and acquired. More than 90% of cases are an acquired autoimmune disorder. In PAP excess accumulation of surfactant leads to bilateral airspace opacities on chest radiograph and extensive alveolar densities with thickened interlobular septa on computed tomography lung scan. PAP typically aficts

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 413

Figure 1 Lung biopsy appearance of acquired PAP, showing alveoli lled with eosinophilic lipoproteinaceous material with otherwise relatively preserved lung architecture (hematoxylineosin, 40). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215235, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Figure 2 Cytological preparation of BAL showing alveolar macrophages containing abundant PAS-positive material in a background of PAS-positive granular lipoproteinaceous material (PAS, 400). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215235, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

receptor shared by interleukin-3 (IL-3) and IL-5 (bc). This congenital form of PAP has a very poor prognosis and is optimally managed with lung transplantation (in contrast to acquired PAP, which has recurred in transplanted lungs). PAP occurs occasionally as a secondary process associated with a range of underlying disorders affecting the number and function of alveolar macrophages, most commonly myeloid hematopoietic malignancies, but also other hematopoietic and immunodeciency diseases, acute silicosis and other inhalation syndromes, and the rare genetic disorder lysinuric protein intolerance. At least in the case of myeloid malignancies, secondary PAP may resolve following therapies that restore normal hematopoiesis.

500 nm
Figure 3 Ultrastructural appearance of BAL uid showing cellular debris and characteristic concentrically laminated phospholipid lamellar bodies. Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215235, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Pathology
Lung biopsies in affected adults show the characteristic appearance of minimally inamed alveoli with preserved normal architecture lled with foamy macrophages amongst granular acellular eosinophilic, periodic acid-Schiff positive lipoproteinaceous material (Figure 1). In some cases the diagnosis may be established less invasively by bronchoalveolar lavage that typically returns milky appearing uid with a microscopic appearance identical to that described above (Figure 2). Electron microscopy can conrm the diagnosis in difcult cases by demonstrating the presence of characteristic concentrically laminated phospholipid lamellar bodies (Figure 3).

Clinical Features
In adults, the usual presentation of PAP is slowly progressive dyspnea of several months duration, sometimes with minimally productive cough or fatigue and occasional weight loss. A minority of patients is minimally symptomatic. Fever and hemoptysis are uncommon, and suggest complicating secondary infection, most commonly with Nocardia spp. Physical signs on examination are often inconspicuous, but crackles are audible in up to 50% of patients. Bilateral patchy, nodular, or conuent

414 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis

clinically distinctive, PAP may be mistaken initially for any of the numerous infective, inammatory, inltrative, or neoplastic causes of bilateral pulmonary inltrates.

Pathogenesis and Animal Models

Surfactant Catabolism in Human PAP: Defect in Surfactant Clearance

Figure 4 Thin-section, high-resolution computed tomography scan of the thorax showing features typical of acquired PAP. Extensive opacity involves most of the lungs. The areas of ground-glass opacity demonstrate thickening of the interlobular septa leading to the characteristic crazy paving appearance typical of PAP. Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215235, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

airspace opacities on chest radiology are revealed by computed tomographic scans to be due to widespread airspace consolidation with thickened interlobular and intralobular septa (crazy paving appearance) (Figure 4). The mean (SD) arterial oxygen tension at the time of diagnosis in a large group of reported cases was 58.6 (15.8) mmHg. Pulmonary function tests typically show a relatively mild restrictive ventilatory defect that contrasts with a moderate or severe impairment in pulmonary gas transfer. The most common biochemical abnormality is a slight to moderate elevation in serum levels of lactate dehydrogenase, serial measurement of which can be used as a useful index of disease activity. Although

The initial report of Rosen et al. recognized that the eosinophilic material accumulating within alveoli in the lung of patients with PAP was rich in lipids, proteins, and some carbohydrate, with similar biochemical composition and electron microscopic appearance to pulmonary surfactant. Surfactant, composed of approximately 90% lipids, 10% proteins, and a small amount of carbohydrates, plays an important role in reducing surface tension at the alveolar wall airliquidtissue interface thus preventing alveolar collapse and transudation of capillary uid. Surfactant proteins comprising SP-A, SP-B, SP-C, and SP-D contribute to the surface-active properties and support the host defense functions of alveolar macrophages. Surfactant lipids and proteins are synthesized and partially recycled by alveolar type II cells, with catabolism in both alveolar type II cells and alveolar macrophages. Both production and degradation of surfactant materials have been extensively evaluated in PAP, with impaired clearance being the predominant cause of the increased alveolar pool size. Markedly elevated levels of these surfactant proteins are found in the blood of all patients with PAP, but these ndings are not specic for PAP and can occur in other interstitial lung diseases, although usually at lower levels.

Figure 5 Surfactant homeostasis and impaired surfactant catabolism in pulmonary alveolar proteinosis. GM-CSF has a critical role in surfactant homeostasis in the normal lung (a). Interruption of GM-CSF signaling in the lung results in pulmonary alveolar proteinosis (b). Surfactant lipids and surfactant proteins A, B, C, and D are produced by alveolar type II epithelial cells (solid black arrows). Surfactant protein precursors are processed in the Golgi network. Some (surfactant proteins B and C) are then assembled, along with surfactant phospholipids, in lamellar bodies; surfactant proteins A and D are secreted by other secretory vesicles. After exocytosis into the alveolar surface liquid, the lamellar bodies assemble into surfactant structures known as tubular myelin as well as into large and small aggregates. Phospholipids from extracellular surfactant structures form continuous monolayers and multilayers of phospholipids that line the alveolar spaces and airways, with their polar heads oriented toward the liquid and their acyl chains toward the air. The large aggregates, extracellular lamellar bodies, and tubular myelin all have surface-active properties. Normally (a) surfactant is inactivated by mechanical and biologic processes and converted into small, surface-inactive aggregates. Approximately 7080% of the small aggregates are taken up by alveolar type II cells, transported to phagolysosomes, and reused (green arrows) or catabolized (red arrows). Alveolar macrophages internalize and catabolize the remaining surfactant pool, a process critically dependent on GM-CSF. Although GM-CSF stimulates lung growth and causes alveolar type II epithelial-cell hyperplasia, a potential role for GM-CSF in surfactant recycling by these cells has not been dened (dashed black arrows). In pulmonary alveolar proteinosis (b), interruption of GM-CSF signaling (small red bar) in the alveolar macrophage for example, by targeted ablation of the gene encoding GM-CSF or its receptor in mice or, presumably, by neutralizing anti-GM-CSF autoantibodies in humans impairs the catabolism of surfactant by alveolar macrophages without impairing its uptake. This results in the intracellular buildup of membrane-bound, concentrically laminated surfactant aggregates. Progressive expansion of the extracellular surfactant pool and accumulation of cellular debris due to the impaired catabolism eventually cause lling of the alveoli, thus reducing the size of the available gas-exchange surface and eventually leading to the clinical syndrome. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 25272539, with permission. Copyright & 2003 Massachusetts Medical Society. All rights reserved.

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 415 GM-CSF Plays a Critical Role in Surfactant Homeostasis in the Lung

Gene-targeted mice lacking either GM-CSF or bc develop PAP, indicating that lack of GM-CSF bioactivity leads to reduced surfactant clearance. Local expression of GM-CSF in airway epithelial cells or

inhalation of GM-CSF in GM-CSF-knockout mice corrected PAP. Surfactant metabolism studies in GMCSF-knockout mice have demonstrated normal levels of surfactant protein mRNA and normal secretion rates, but grossly impaired clearance of surfactant phospholipids and SP-A by alveolar macrophages.

Lamellar body

Surfactant

Phospholipid monolayer Alveolar space

Alveolar type II epithelial cell ?

Small aggregate Liquid Air

GM-CSF

Alveolar type I epithelial cell Alveolar macrophage (a)

Phagolysosome

Surfactant

Diminished airspace Surfactant inclusions

Liquid

Neutralizing anti-GM-CSF autoantibody

Surfactant aggregates

Cellular debris

Foamy alveolar macrophage (b)

416 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis Dysfunction of Alveolar Macrophages in Patients with PAP and Gene-Targeted Mice Impaired Differentiation of Alveolar Macrophages in PAP and GM-CSF-Knockout Mice: Role of PU.1

The abnormal accumulation of surfactant materials in both patients and murine PAP models establishes that a defect in surfactant catabolism by alveolar macrophages is causative (Figure 5). Functional defects in the macrophages from patients with PAP include impaired chemotaxis, adhesion, phagocytosis, microbicidal activity, and phagolysosome fusion. In GM-CSF-knockout mice, alveolar macrophages have similar functional abnormalities in cell adhesion, cell-surface pathogen recognition receptor expression, phagocytosis, endocytosis, superoxide production, microbial killing, cytokine secretion, and catabolism of both surfactant phospholipids and proteins. It is likely that alveolar macrophages from patients with PAP will share this spectrum of functional defects.
Surfactant homeostasis Pathogen receptor GM-CSF GM-CSF receptor Surfactant Bacteria

The functional abnormalities in alveolar macro phages suggest a defect in maturation and, indeed, alveolar macrophages from patients with PAP are ultrastructurally immature. This is caused by an absence of PU.1, an ets-family transcription factor that normally leads to the terminal differentiation of alveolar macrophages. In the physiological state, bioactive GM-CSF in the lung leads to upregulation of PU.1 in alveolar macrophages. Thus, it appears that PU.1 mediates a number of the biological effects of GM-CSF in alveolar macrophages (Figure 6). Acquired PAP is a manifestation of functional GM-CSF-deciency caused by an autoantibody against GM-CSF. It had long been recognized that a factor in the bronchoalveolar lavage uid and sera from patients
Adaptive immunity Tumor necrosis factor Type 1 helper T cell InterferonIL-18 T cell

Innate immunity

B cell IL-12 Natural killer cell Increased PU.1 Phagolysosome Nucleus Alveolar macrophage Cell adhesion Fc receptor Antibody Filopodium

Figure 6 GM-CSF in modulating the function of alveolar macrophages in mice. In vivo, pulmonary GM-CSF stimulates an increase in the level of PU.1, a transcription factor, in alveolar macrophages in the lung. In vitro, alveolar macrophages from knockout mice without the GM-CSF gene have a number of functional defects, including defects in cellular adhesion, catabolism of surfactant proteins and surfactant lipids, expression of pathogen-associated molecular pattern receptors (e.g., Toll-like receptors and the mannose receptor), Toll-like-receptor signaling, phagocytosis of pathogens (bacteria, fungi, and viruses), intracellular killing of bacteria (independent of uptake), pathogen-stimulated secretion of cytokines (tumor necrosis factor, IL-12, and IL-18), and Fc receptor-mediated phagocytosis. Cytoskeletal organization is abnormal and may in part account for defects in phagocytosis. The inability of alveolar macrophages to release IL-12 and IL-18 severely impairs the interferon response to pulmonary infection, thus impairing an important molecular connection between innate and adaptive immunity in the lung. Retroviral vector-mediated, constitutive expression of PU.1 in alveolar macrophages from GM/ mice corrects all these defects, suggesting that GM-CSF stimulates terminal differentiation of the macrophages through the global transcription factor PU.1. The purple arrows represent the functions of PU.1 that are affected by the absence of GM-CSF. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 25272539, with permission.

INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis 417

300 Serum anti-GM-CSF autoantibody level (g ml1)

250

200

150

100

50

0 Acquired Congenital Secondary Pulmonary alveolar proteinosis

Figure 7 Presence of autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) within the bronchoalveolar lavage uid and serum of patients with acquired pulmonary alveolar proteinosis. The autoantibodies are not present in persons with congenital or secondary pulmonary alveolar proteinosis or other lung diseases or in normal persons. Each point represents an individual patient. Some data are taken from Kitamura T, Uchida K, Tanaka N, et al. (2000) Serological diagnosis of idiopathic pulmonary alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 162: 658662. Reproduced from Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349(26): 25272539, with permission.

Other lung diseases

Normal

with PAP inhibited the mitogen-stimulated proliferation of normal monocytes. In 1999, very high levels of a high-afnity neutralizing IgG autoantibody against GM-CSF were identied in the bronchoalveolar lavage uid and sera of patients with acquired PAP from many geographic regions. This autoantibody specifically inhibited GM-CSF-induced cell growth in vitro. Various assays for this autoantibody have identied patients specifically with acquired PAP (as opposed to congenital or secondary forms) with high sensitivity and specicity, making this a very efcient screening and diagnostic test (Figure 7). It is likely that the autoantibody against GM-CSF obliterates the bioactivity of GM-CSF in vivo, resulting in impaired alveolar macrophage maturation, functional impairment, and surfactant accumulation. This pathogenetic model is supported by the observation that patients with acquired PAP do not develop the expected peripheral blood neutrophilia after systemic administration of exogenous GM-CSF, despite the absence of mutations in either the GMCSF or GM-CSF receptor genes, and normal antigenic levels of GM-CSF in bronchoalveolar lavage uid.

Management and Current Therapy

After a diagnosis of PAP is established, therapy directed at improving gas exchange may not immediately be

required. Commonly used and broadly accepted criteria for the institution of therapy are: at least moderately severe hypoxia (arterial PO2 p65 mmHg), persistent and functionally significant respiratory symptoms attributable to PAP, and rarely progressive systemic deterioration (weight loss, repeated respiratory infections, etc.). However, in all patients, it is mandatory that the supervising physician be vigilant in surveillance for opportunistic infections, both respiratory and systemic. While the prognosis for adult patients with acquired PAP is very good and has improved in recent years (predicted 5-year disease specic survival of B90%), approximately 20% of deaths in patients with PAP are due to infection. Although there are anecdotal cases claiming improvements in respiratory function with corticosteroids in the older literature, there is currently no role for the use of these agents in patients with uncomplicated PAP. Approximately 30% of patients with PAP will have minimal or mild symptoms and have stable or minimally uctuating disease severity and never require therapy, and B10% of patients will have some degree of spontaneous improvement in their PAP with diminished symptoms and functional improvement. These patients have an extremely good prognosis; however, it is not known if the underlying disease process has truly resolved. For the remaining patients who require therapy, the standard approach to treatment for the last

418 INTERSTITIAL LUNG DISEASE / Alveolar Proteinosis

40 years has been whole lung lavage. This treatment is based on the revolutionary studies of Ram rezRivera in the 1960s at the Veterans Administration Hospital in Baltimore. He used repeated segmental ooding through a percutaneous transtracheal endobronchial catheter in awake patients. Over the decades, the original procedure has been sequentially rened through the routine use of general anesthesia, increased lavage volumes, the recognition that saline is safer and as efcacious as other lavage uids, the additional benet of concomitant chest percussion, and the successful completion of bilateral sequential whole lung lavage in the same treatment session. Whole lung lavage remains the current standard of care for patients with PAP, and although the procedure is complex to perform and the associated physiologic changes are quite profound, it is usually well tolerated when performed by an experienced team. However, it does require a prolonged general anesthetic and has associated morbidity and very rarely mortality. Unfortunately, there are no established response criteria for therapeutic lavage. However, significant clinical, physiological, and radiological improvements

are usually seen after the initial therapeutic lavage in 8090% of patients. The median duration of symptomatic benet for patients is B15 months, with 2030% of patients remaining free of recurrent symptoms beyond 3 years. Recent single-center reports have suggested meticulous attention to lavage technique in highly experienced centers may achieve more durable benets. A review of published cases described a mean improvement in arterial PO2 of 20 mmHg, a 31 mmHg improvement in (Aa)DO2 and a 0.5 l improvement in vital capacity, and a 4.4 ml mmHg 1 min 1 improvement in DLCO (corresponding to B30% improvement in percent predicted). Some degree of physiological improvement is usually seen within a few days postlavage, making this therapy the preferred approach for severely hypoxic patients. The other treatment option is the therapeutic use of GM-CSF. This has mostly been used subcutaneously starting at a dose of 5 mg kg 1 day 1, with some patients requiring dose escalation to achieve responses, and less commonly as a nebulized inhalation. Unlike whole lung lavage, responses to GM-CSF are generally slow to be manifest, with

60

(Aa)DO gradient (mmHg)

40

20

0 0 14 28 42 56 70 84 98 Time on treatment (days)

Figure 8 Kinetics of improvement in (A a)DO2 for patients responding to their rst effective course of GM-CSF therapy (5 mg kg 1 day 1 for ve patients and 20 mg kg 1 day 1 for one patient). Each individual responding patient is represented by a separate color, with the patient responding only at the higher dose of GM-CSF represented by a broken line with open circles. The patient represented by a line with solid circles had only one baseline ABG evaluation. For other patients, pretreatment values are shown as mean 7 SEM for all ABG analyses performed within 28 days of commencing therapy (in two cases SEM are contained within symbols). Reproduced from Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166(2): 215235, Ofcial Journal of the American Thoracic Society, & American Thoracic Society, with permission.

INTERSTITIAL LUNG DISEASE / Amyloidosis 419

improvements in (Aa)DO2 not evident until 46 weeks, with maximal improvements not achieved until 610 weeks of therapy (Figure 8). Subcutaneous GM-CSF treatment is easily delivered but expensive, with few patients ceasing treatment due to adverse effects, usually rash or local reaction at the injection site. Indeed, patients with PAP also appear to have a lower incidence of adverse effects with GM-CSF than anticipated. The presence of GM-CSF-neutralizing antibodies likely results in a reduced effective biologic dose. The likelihood of response to GM-CSF, predominantly among patients no longer deriving benet from whole lung lavage, is 4050%, with the magnitude of physiological improvement among responders similar to that seen with lavage. Given the detection of GM-CSF antibodies in all of the treated patients, it is not apparent why therapeutic responses are variable. Although limited data are available, the effective antibody titer, reecting total GM-CSF-neutralizing capacity, may be lower in responders. The sequential application of increasing doses seeking evidence of biologic activity of GM-CSF, such as peripheral blood eosinophilia, may identify appropriate dose ranges for further evaluation. While the subcutaneous route was used in most patients, based on its proven safety and efcacy in other settings, recent animal work suggests that inhaled GM-CSF may have greater pulmonary effects, but conversely may not ameliorate any extrapulmonary aspects of the disease. Although aerosolized GM-CSF is well tolerated in humans, and this route of GM-CSF delivery has been successfully used in a small number of patients with acquired PAP, the issues of pharmacokinetics and reliable drug delivery remain unresolved.
See also: Autoantibodies. Leukocytes: Pulmonary Macrophages. Surfactant: Overview.

factor in patients with acquired pulmonary alveolar proteinosis. Blood 92: 26572667. Seymour JF and Presneill JJ (2002) Pulmonary alveolar proteinosis: progress in the rst 44 years. American Journal of Respiratory and Critical Care Medicine 166: 215235. Seymour JF and Presneill JJ (2004) Pulmonary alveolar proteinosis. What is the role of GM-CSF in disease pathogenesis and treatment? Treatments in Respiratory Medicine 3: 229234. Seymour JF, Presneill JJ, Schoch OD, et al. (2001) Therapeutic efcacy of granulocyte-macrophage colony-stimulating factor in patients with idiopathic acquired alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 163: 524 531. Shah PL, Hansell D, Lawson PR, Reid KB, and Morgan C (2000) Pulmonary alveolar proteinosis: clinical aspects and current concepts on pathogenesis. Thorax 55: 6777. Shibata Y, Berclaz P-Y, Chroneos Z, et al. (2001) GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1. Immunity 15: 557567. Trapnell BC and Whitsett JA (2002) GM-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annual Review of Physiology 64: 775802. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349: 2527 2539. Uchida K, Nakata K, Trapnell BC, et al. (2004) High-afnity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 103: 1089 1098. Yoshida M, Ikegami M, Reed JA, Chroneos ZC, and Whitsett JA (2001) GM-CSF regulates surfactant protein-A and lipid catabolism by alveolar macrophages. American Journal of Physiology. Lung Cellular and Molecular Physiology 280: L379L386.

Amyloidosis
J P Lynch III, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA H D Tazelaar, Mayo Clinic Arizona, Scottsdale, AZ, USA
& 2006 Elsevier Ltd. All rights reserved.

Further Reading
Beccaria M, Luisetti M, Rodi G, et al. (2004) Long-term durable benet after whole lung lavage in pulmonary alveolar proteinosis. European Respiratory Journal 23: 526532. Dranoff G, Crawford AD, Sadelain M, et al. (1994) Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264: 713716. Goldstein LS, Kavuru MS, Curtis-McCarthy P, et al. (1998) Pulmonary alveolar proteinosis: clinical features and outcomes. Chest 114: 13571362. Kitamura T, Uchida K, Tanaka N, et al. (2000) Serological diagnosis of idiopathic pulmonary alveolar proteinosis. American Journal of Respiratory and Critical Care Medicine 162: 658 662. Presneill JJ, Nakata K, Inoue Y, and Seymour JF (2004) Pulmonary alveolar proteinosis. Clinics in Chest Medicine 25: 593613. Seymour JF, Begley CG, Dirksen U, et al. (1998) Attenuated hematopoietic response to granulocyte-macrophage colony-stimulating

Abstract
Amyloidosis is a heterogeneous group of diseases characterized by deposition of amyloid protein, a brillar insoluble crystalline material, in the extracellular matrix of involved tissues. Amyloidosis can be localized or systemic; both inherited and acquired forms exist. Four major categories of systemic amyloidosis include primary or immunoglobulin light-chain (AL) disease, secondary or amyloid protein A (AA) disease, familial or mutant transthyretin (ATTR) disease, and dialysis-associated or b-microglobulin (b2-M) disease. Primary amyloidosis is associated with deposition of monoclonal k or l immunoglobulin light chains (amyloid AL) and can be idiopathic or associated with plasma cell dyscrasias. Secondary amyloidosis results from excessive production of serum amyloid A (SAA), an acute phase protein released in response to inammation. Amyloid AA may complicate diverse chronic inammatory conditions. Hereditary or familial amyloidosis comprises 4% of cases of amyloidosis.

420 INTERSTITIAL LUNG DISEASE / Amyloidosis

Most cases are due to mutations in the transthyretin gene. In this article, we discuss the salient clinical features of the various forms of amyloidosis, and emphasize specic complications of respiratory tract involvement. Specic types of intrathoracic involvement in AL amyloidosis include tracheobronchial amyloidosis, diffuse interstitial or alveolarseptal disease, nodular disease (amyloidomas), intrathoracic adenopathy, and pleural disease.

Introduction
Amyloidosis is a heterogeneous group of diseases characterized by deposition of amyloid protein, a brillar insoluble crystalline material, in the extracellular matrix of involved tissues. Amyloidosis can be localized or systemic; both inherited and acquired forms exist. Classication of amyloidosis is based on different subunit proteins. Nomenclature includes an A for amyloid and a letter indicating the respective proteins involved. The four major categories of systemic amyloidosis include (1) primary or immunoglobulin light-chain (AL) disease, (2) secondary or amyloid protein A (AA) disease, (3) familial or mutant transthyretin (ATTR) disease, and (4) dialysis-associated or b-microglobulin (b2M) disease. Additional diseases associated with amyloid deposition include Alzheimers disease (b-amyloid precursor protein), type II diabetes (islet amyloid polypeptide), spongiform encephalopathies, and medullary carcinoma of the thyroid and other malignancies. These latter entities are beyond the scope of this article.
Pathogenesis

Figure 1 Nodular amyloidosis stained with Congo-red and examined under polarized light showing apple-green birefringence.

Amyloid is an insoluble b-pleated brillar glycoprotein that forms rigid brils that are resistant to proteolysis. Deposits of amyloidosis in extracellular spaces disrupt tissue architecture and function and can impair organ function by space-occupying effects. Amyloid brils may be cytotoxic and promote apoptosis. Amyloid deposits are unusually stable, but can regress when the supply of bril precursors is reduced. Certain glycosaminoglycans (GAGs) are invariably associated with the brils. Although the pathogenesis of amyloidosis has not been elucidated, serum amyloid protein (SAP) and GAGs may be important. In SAP knockout mice, experimentally-induced AA amyloid is attenuated. Little is known regarding individual susceptibility to amyloidosis or why specic amyloid proteins preferentially affect specic organs.
Histopathology

Figure 2 Transmission electron microscopic imaging comparing collagen (upper left) with a diameter of 20100 nm to amyloid . brils (lower right) with a mean diameter of 100 A

Congo red-associated birefringence after oxidation in permanganate solution, whereas AL amyloid retains this feature. Amyloid also stains with sulfated alcian blue. Electron microscopic examination of amyloid reveals a dominant (95%) brillar component (Figure 2), with distinct periodicity. A glycoprotein component, derived from soluble amyloid protein (SAP), is also present. More than 15 types of amyloid, which share common morphological features, are recognized. Immunohistochemical techniques to detect k and l light chains, amyloid-associated protein, or transthyretin (TTR) can be done to classify the bril type. Negative stains for serum amyloid A (SAA) protein and TTR exclude AA-type and TTRtype amyloidosis, but stains for k and l chains are positive in only 60% of cases of AL amyloidosis.

Amyloid is a homogeneous amorphous eosinophilic material that binds the dye Congo red, appearing orange-red by conventional light microscopy (Figure 1). Under polarized light microscopy, amyloid exhibits apple-green birefringence. AA amyloid loses its

Primary (AL) Amyloidosis

Primary (AL) amyloidosis is associated with deposition of monoclonal k and l immunoglobulin light

INTERSTITIAL LUNG DISEASE / Amyloidosis 421

chains (amyloid AL) and can be idiopathic or associated with plasma cell dyscrasias (e.g., multiple myeloma). Both, systemic and localized, forms of AL exist. AL amyloidosis is the most common type of amyloidosis, but only 12753200 new cases are diagnosed annually in the United States.
Clinical Features

Clinical manifestations of AL amyloidosis are protean. Amyloidosis can affect any organ, but predominant sites include kidney, heart, peripheral nervous system, gastrointestinal (GI) tract, joints, the tongue, spleen, liver, and upper respiratory tract. Initial symptoms are often fatigue and weight loss. Specic symptoms related to amyloidosis develop, affecting specic target organs. Predominant features include kidney failure, congestive heart failure (CHF), and peripheral neuropathy. Renal amyloid may result in nephrotic syndrome and varying degrees of renal insufciency. Amyloid deposits in the myocardium or pericardium may lead to conduction disturbances, restrictive cardiomyopathy, and CHF. Autonomonic and sensory neuropathies and carpal tunnel syndrome are relatively common but the central nervous system is not involved in AL amyloidosis. Amyloidosis may affect any site in the GI tract; hemorrhage, ulcerations, obstruction, diarrhea, or malabsorption caused by bowel involvement may occur. Rectal biopsy may reveal amyloid deposits in systemic AL amyloidosis. Hepatomegaly is common in AL amyloidosis but splenomegaly occurs in o5% of cases. However, splenic dysfunction (i.e., the presence of HowellJolly bodies in peripheral blood smear) occurs in 24% of cases. Macroglossia, a classical feature of amyloidosis, occurs in 1520% of patients. Inltration of thyroid or adrenal glands may result in hypothyroidism or hypoadrenalism, respectively. Skin ndings include waxy thickening and easy bruising. Amyloidosis may also be associated with a bleeding diathesis, due to factor X deciency as well as amyloid involving blood vessels. Clinically significant pulmonary involvement is rare in other forms of amyloidosis, but occurs in 520% of patients with AL amyloidosis. Localized AL amyloidosis most often affects the upper respiratory, urogenital, or GI tracts, skin, or orbit. In such cases, the amyloidogenic light chains are produced by clones of lymphocytes or plasma cells localized in proximity to the amyloid deposits. Diagnostic evaluation of AL amyloidosis requires an assessment of the extent and nature of organ involvement. Biopsies of affected site(s) may establish the diagnosis. Biopsies of subcutaneous abdominal fat or bone marrow are positive in 485% of patients

with systemic AL amyloidosis. Examination of bone marrow aspirates and biopsies is critical to determine whether plasma cell dyscrasias or myeloma is present. Serum and urine light chains should be measured to assess for monoclonality. Twenty fourhour urine should be obtained to rule out nephrotic syndrome or renal involvement. Radionuclide scintiscans using 123I-labeled SAP component provide a macroscopic whole body survey that may assess anatomical distribution. SAP scans are expensive and not available in most centers. Electrocardiograms and echocardiograms should be performed to assess cardiac involvement, which is the major determinant of prognosis in AL amyloidosis. 2D echocardiographic features of cardiac involvement include septal thickening, concentrically thickened left ventricle, normal-to-small ventricular cavity, reduced ejection fraction (EF), high left-sided pressures (a restrictive lling pattern), and a speckled pattern. A clinical clue to cardiac amyloidosis is marked worsening of heart failure after treatment with calcium channel blockers. Serial studies of urine and serum light chains may be invaluable to assess the course of the disease.
Pathogenesis

The brils in systemic AL amyloidosis are derived from circulating monoclonal light chains produced by clonal plasma cell dyscrasias. In localized forms of AL amyloidosis, the light chains are produced by a clone of lymphoplasmacytes localized in proximity to the amyloid deposits. Clinical expression of the disease is determined by the extent and anatomic distribution of amyloid deposits.
Management and Current Therapy

Prognosis of AL amyloidosis is determined by extrapulmonary organ involvement. Mean survival in untreated AL amyloidosis is 12 years. Most deaths are due to cardiac, renal, or neurological causes. Multisystem involvement portends a dismal prognosis. Optimal therapy for AL amyloidosis is not clear. Alkylating agents (particularly melphelan) plus prednisone are appropriate for amyloidosis associated with plasma cell dyscrasias (e.g., multiple myeloma). For idiopathic AL amyloidosis, 25% of patients respond to melphelan/prednisone but only modest gains in survival (810 month improvement) are achieved. Colchicine and a-interferon are ineffective. High-dose chemotherapy followed by peripheral blood stem cell transplantation (SCT) achieves remissions in 65% of patients, and is the most promising therapy. However, late relapses may occur and data are limited regarding long-term survival.

422 INTERSTITIAL LUNG DISEASE / Amyloidosis

Anecdotal responses were reported with thalidomide; dimethyl sulfoxide (DMSO); vincristin, adriamycin, and dexamethasone (VAD); and 40 -iodo40 -deoxy-doxorubicin (a new anthracycline), but data are sparse. Novel agents that stabilize amyloid precursor proteins, inhibit bril formation, or enhance bril degradation are currently under development. Candidate drugs include GAG mimetics and SAP-binding inhibitors. In addition, treatment needs to be directed towards the organ involved. Treatment of specic complications is important.

Etiology

Most cases of hereditary amyloidosis are due to mutations in the TTR gene found on chromosome 18 (autosomal dominant with high penetrance). More than 50 mutations of TTR have been identied in familial amyloidosis; however, mutations of apolipoprotein A-1, gelsolin, brinogen A a, and lysozyme also lead to amyloidosis.
Clinical Features

Secondary Amyloidosis (Amyloid AA)

Secondary amyloidosis (amyloid AA) results from excessive production and organ-deposition of amyloid formed from SAA, an acute phase protein released in response to inammation. There are several SAA proteins in existence. In humans, AA amyloid deposits consist of fragments of at least ve different molecular forms.
Etiology

The onset of familial amyloidosis is usually after age 50. Most common features include peripheral neuropathy (480%), autonomic neuropathy (470%), cardiomyopathy (450%), carpal tunnel syndrome (440%), and nephropathy (o20%). Macroglossia does not occur. Lung involvement is rare (o4%). Biopsies or aspirates of subcutaneous fat or bone marrow are the standard tests to diagnose amyloidosis. Immunohistochemical stains to detect TTRderived amyloid conrm the diagnosis. Molecular genetic studies of blood can determine the specic mutant TTR.
Management and Current Therapy

Amyloid AA occurs in 15% of patients with diverse chronic inammatory conditions, for example, osteomyelitis or chronic infections (i.e., tuberculosis, bronchiectasis, leprosy, malaria); familial Mediterranean fever; connective tissue disease; and inammatory bowel disease. Secondary amyloidosis may complicate renal cell carcinoma or other malignancies. Most cases (but not all) are AA amyloid. Given the marked reduction of chronic infectious diseases in developed countries, AA amyloidosis is exceptionally rare.
Clinical Features

The onset of AA amyloidosis ranges from several months to many decades after acquiring the underlying disorder. Principal target organs include kidneys (91%), GI tract (22%), liver (5%), nerves (3%), and lymph nodes (2%). Lung involvement is rare (o1%) in AA amyloidosis. Survival in AA amyloidosis is inuenced primarily by renal involvement. Renal failure is associated with a mean survival of 11 months, compared to 57 months among patients with normal renal function. In contrast to AL amyloidosis, cardiac involvement is rare in AA amyloidosis. As in ATTR amyloidosis, macroglossia is not a feature of AA amyloidosis.

Survival among patients with ATTR amyloidosis varies with the specic mutation, but median survival rates approximate 47 years after diagnosis. The most common causes of death in ATTR amyloidosis are progressive CHF or sudden cardiac death and inanition due to chronic autonomic neuropathy. In one study, median duration of survival was 3.4 years among ATTR patients with cardiomyopathy and 7 years without cardiomyopathy. For patients with familial amyloidosis, treatment of specic complications (e.g., orthostatic hypotension, endocrine insufciency, and cardiac arrhythmias) is critical. Since TTR is synthesized in the liver, liver transplantation is the optimal therapy for ATTR amyloidosis. Liver transplantation may prevent further deposition of amyloid, and may effect regression of amyloid deposits and symptoms. Combined heart and liver transplantation has been successful in a few patients.

Other Rare Causes of Amyloidosis

Deposition of wild-type TTR amyloid protein may be found with advanced age (senile amyloidosis), usually as an incidental nding. Autopsy studies detected senile pulmonary amyloidosis in approximately 10% of patients older than 80 years of age and in 50% of patients older than 90 years of age. These deposits are usually incidental ndings and are not associated with specic symptoms. Other rare

Hereditary Amyloidosis
Hereditary or familial amyloidosis comprises 4% of cases of amyloidosis.

INTERSTITIAL LUNG DISEASE / Amyloidosis 423

sources of amyloidosis include b2-microglobulin in chronic renal failure patients on dialysis, b-protein amyloid in the cerebral senile plaques of Alzheimers disease, procalcitonin in medullary carcinoma of the thyroid, and islet cell amyloid polypeptide in the pancreas in patients with type II diabetes.

Pulmonary Involvement in Amyloidosis

Clinically significant pulmonary involvement occurs in o1020% of patients with AL amyloidosis, but at necropsy, amyloid deposits in pulmonary vessels are present in up to 90% of patients. Lung involvement is rare in AA and ATTR amyloidosis, and, when present, is usually an incidental nding (e.g., diffuse alveolar septal opacities without clinical symptoms). In the following sections, the specic forms of pulmonary amyloidosis (principally due to AL amyloid) will be discussed. Five types of extravascular lung involvement are seen in AL amyloidosis (systemic or localized). These include (1) diffuse interstitial or alveolarseptal disease, (2) nodular disease, (3) intrathoracic adenopathy, (4) pleural disease, and (5) diaphragmatic deposition and miscellaneous sites. As was previously noted, pulmonary involvement is rarely lethal in AL amyloidosis.
Diffuse Interstitial (AlveolarSeptal) Amyloidosis

Diffuse parenchymal (alveolarseptal) amyloidosis is an uncommon complication of systemic or localized AL amyloidosis (Figure 3). Deposits of AL amyloid in the alveolar septae and interstitium may be associated with reticulonodular or micronodular lesions, with or without pleural effusions, on chest radiographs. Chest computed tomographic (CT) scans typically demonstrate reticular opacities, interlobular septal thickening, small, 24 mm nodules in the subpleural regions, honeycomb change, and groundglass opacities. Physiological aberrations include reduced lung volumes, decreased diffusing capacity for carbon monoxide (DLCO), and widened alveolar arterial O2 gradient. Pathologically, pulmonary vascular involvement is relatively common in this form of amyloid (Figure 4), but clinically significant pulmonary hypertension is rare. Pulmonary involvement is rarely severe, and most patients with alveolarseptal amyloidosis do not have specic pulmonary symptoms. The prognosis is dictated by the cardiac status. In one study of AL patients with cardiac failure (i.e., left ventricular EF o40%), median survival was only 12 weeks, compared to 16 months with alveolarseptal inltrates and normal cardiac function.

Figure 3 (a)Alveolarseptal amyloid stained with sulfated alcian blue in which the amyloid appears bright green. (b) Without the special stain, it is very difcult to appreciate the amyloid even at a higher microscopic power.

Nodular Amyloid Lesions (Amyloidomas)

Localized pulmonary amyloid nodules (amyloidomas) most commonly reect a localized abnormal immune response of bronchus-associated lymphoid tissue (BALT), but rarely complicate systemic AL amyloidosis, orthotopic liver transplantation, IgA nephropathy, or systemic light-chain deposition disease. Histopathological features of nodular amyloidosis Grossly, nodules exhibit gray-yellow translucence on a cut section (Figure 5). Histologically, irregular, amorphous, and predominantly acellular masses of amyloid replace the lung parenchyma (Figure 6) and are frequently surrounded by a lymphoplasmacytic inltrate (Figure 7) that is usually light-chain restricted. AL amyloid may be found in vessels and interstitium adjacent to the nodules. Fibrosis, calcication, ossication, giant cells, and mononuclear inammatory cells may be present.

424 INTERSTITIAL LUNG DISEASE / Amyloidosis

Figure 4 Vascular amyloid deposition highlighted by sulfated alcian blue stain.

Figure 6 Low-power photomicrograph from an amyloidoma. Note irregularly shaped deposits of amorphous eosinophilic amyloid.

Figure 7 High-power photomicrograph showing an amyloidoma associated with a marked lymphoplasmacytic inltrate. Figure 5 Surgically excised nodule of amyloid showing yellow homogeneous cut section.

tracheobronchial amyloidosis (TBA) (discussed later). Amyloid-inltrated nodes may calcify.

Pleural Effusions Complicating AL Amyloidosis

Clinical features Chest radiographs or CT scans demonstrate single or multiple nodular densities, ranging in size from 0.4 to 15 cm, with a proclivity for the lower lobes. Calcication is present in 2550% of lesions. Metaplastic bone or cartilage formation may be present. Cavitation is rare. The nodules usually grow slowly over the years. Most patients are asymptomatic but cough or hemoptysis may occur. Pulmonary function is usually normal. Management and current therapy Prognosis of nodular amyloid is generally excellent. Since systemic disease is lacking, chemotherapy is not necessary. Surgical resection is advised for solitary symptomatic lesions.
Intrathoracic Lymphadenopathy

Pleural effusions in primary AL amyloidosis may reect CHF, nephrotic syndrome, or amyloid deposits along the pleural surface. Transudative effusions suggest CHF or nephrotic syndrome; hemorrhagic, exudative effusions suggest direct deposits along the pleural surface. Rarely, chylous pleural effusions occur in patients with extensive amyloid burden.
Rare Respiratory Tract Complications of AL Amyloidosis

Hilar and mediastinal adenopathy due to amyloid deposition occurs in 8% of patients with AL amyloidosis and may occur in AA amyloidosis or

Rare manifestations of AL amyloidosis include respiratory muscle weakness (due to diaphragmatic amyloid), obstructive sleep apnea (secondary to involvement of the tongue), recurrent laryngeal nerve palsy, hoarseness or stridor due to amyloid deposits in the supraglottic larynx, and pulmonary marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) type.

INTERSTITIAL LUNG DISEASE / Amyloidosis 425 Primary Pulmonary Amyloidosis

Primary pulmonary amyloidosis (PPA) refers to amyloidosis that is conned to the lungs and associated structures (e.g., tracheobronchial tree, pleura, hilar, or mediastinal lymph nodes). Investigators from the Mayo Clinic reported 55 patients with PPA from 1980 to 1993. Forms of amyloidosis included AL amyloidosis (n 35), AA amyloid (n 2), localized pulmonary amyloid (n 11), senile amyloid (n 6), and familial (n 1). A review of 126 cases of localized AL amyloid identied three major forms of pulmonary disease namely (1) tracheobronchial disease (53%), (2) nodular opacities (44%), and (3) diffuse opacities (3%). Multinodular parenchymal amyloidosis associated with thin-walled pulmonary cysts has been described in patients with Sjogrens syndrome (Table 1).
Tracheobronchial Amyloidosis

TBA may be the only site of involvement, with submucosal amyloid deposits in the airways ranging from the larynx to the distal airways. It is rare; a review of the literature in 2002 found fewer than 140 published cases of TBA. Even at large referral centers, less than 12 cases per year of TBA are diagnosed. Etiology Most cases of TBA are due to excessive deposition of light chains, produced from localized collections of plasma cells or BALT. Clinical features Narrowing or involvement of the nasopharynx, sinuses, larynx, trachea, or bronchi may give rise to symptoms such as hoarseness, wheezing, dyspnea, hemoptysis, cough, atelectasis,

recurrent pneumonia, or chronic infections. In TBA, the lung parenchyma is spared. Bronchoscopic features of TBA include single or multifocal submucosal plaques or nodules (Figure 8) and/or varying degrees of stenosis of the trachea or bronchi. Some cases are associated with tracheobronchopathia osteoplastica, a rare disorder characterized by calcied or cartilaginous submucosal nodules within the tracheobronchial tree. Amyloidosis involving the trachea may cause severe (even fatal) airow obstruction. Pulmonary function tests (PFTs) in patients with proximal tracheal stenosis reveal truncation of peak inspiratory and expiratory ow rates (consistent with xed upper airway obstruction (UAO)). Involvement of distal trachea or bronchi may manifest in air trapping; ow rates may be normal or obstructed. The conguration of ow-volume loops can usually distinguish UAO from distal stenoses. Spirometry is invaluable in detecting progressive airow obstruction. Spiral CT scans are useful to quantitatively assess the degree and sites of airway narrowing, and follow the course of the disease. CT scans demonstrate tracheal- or bronchial-wall thickening, nodules, or plaques, irregularly narrowed airway lumens, and heterotopic tracheal-wall calcications in patients with long-standing TBA. Postobstructive features comprise atelectasis, bronchiectasis, and consolidation, or hyperination. Management and current therapy The course of TBA is usually slowly progressive; one-third of patients with proximal or severe mid-airway disease die from progressive respiratory insufciency within 7 to 12 years after diagnosis. Symptomatic foci involving trachea, larynx, or bronchi require intermittent bronchoscopic dilatation or debridement, surgical resection, laser therapy, tracheostomy, or tracheal resection. Recurrence is frequent, and stenosis of airways may ensue. Unfortunately, repetitive

Table 1 Pulmonary and TBA Radiographic features Single or multiple nodular or mass lesions (0.415 cm) Tracheobronchial amyloidosis: airway wall thickening, irregularly narrowed airway lumens, heterotopic tracheal wall calcications Diffuse interstitial, reticular, or micronodular lesions Hilar or mediastinal lymphadenopathy Pleural effusions Histologic features Plaques, nodules, interstitial or vascular deposits amyloid protein Congo red ( ); green birefringence under polarized light microscopy Submucosal nodules of amyloid with or without calcication (tracheobronchial tree) Therapy Localized pulmonary amyloidosis: surgical resection or no treatment

Figure 8 Tracheal amyloid. The submucosa is lled with deposits of amyloid.

426 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

bronchoscopy or laser treatments denude airways and promote collagen deposition. In rare cases, tracheostomy is required. External beam radiation therapy has been used, with anecdotal successes, for severe airow obstruction. Chemotherapeutic agents, colchicine, or corticosteroids are of doubtful efcacy. Because TBA is localized, systemic agents should generally be avoided.
See also: Stem Cells. Surfactant: Surfactant Proteins B and C (SP-B and SP-C).

in patients with amyloidosis in the respiratory tract. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 19: 134142. Thompson PJ and Citron KM (1983) Amyloidosis and the lower respiratory tract. Thorax 38: 8487. Utz JP, Swensen SJ, and Gertz MA (1996) Pulmonary amyloidosis. The Mayo Clinic Experience from 19801993. Annals of Internal Medicine 124: 407413.

Cryptogenic Organizing Pneumonia

J-F Cordier, Claude Bernard University, Lyon, France
& 2006 Elsevier Ltd. All rights reserved.

Further Reading
Bergethon PR, Sabin TD, Lewis D, et al. (1996) Improvement in the polyneuropathy associated with familial amyloid polyneuropathy after liver transplantation. Neurology 47: 951955. Berk JL, ORegan A, and Skinner M (2002) Pulmonary and tracheobronchial amyloidosis. Seminars in Respiratory Critical Care Medicine 23: 155166. Capizzi SA, Betancourt E, and Prakash UB (2000) Tracheobronchial amyloidosis. Mayo Clinic Proceedings 75: 11481152. Comenzo FL, Vosburgh E, Falk RH, et al. (1998) Doseintensive melphalan with blood stem-cell support for the treatment of AL (amyloid light-chain) amyloidosis: survival and responses in 25 patients. Blood 191: 36623670. Cordier JF, Loire R, and Brune J (1986) Amyloidosis of the lower respiratory tract. Clinical and pathologic features in a series of 21 patients. Chest 90: 827831. Falk RH, Comenzo RL, and Skinner M (1997) The systemic amyloidoses: current approaches to diagnosis and treatment. The New England Journal of Medicine 337: 898909. Gertz MA, Kyle RA, and Thibodeau SN (1992) Familial amyloidosis: a study of 52 North American-born patients examined during a 30-year period. Mayo Clinic Proceedings 67: 428440. Gillmore JD and Hawkins PN (1999) Amyloidosis and the respiratory tract. Thorax 54: 444451. Hawkins PN, Lavender JP, and Pepys MB (1990) Evaluation of systemic amyloidosis by scintigraphy with 123I-labelled serum amyloid P component. The New England Journal of Medicine 323: 508513. Hui HN, Koss MN, Hochholzer L, et al. (1986) Amyloidosis presenting in the lower respiratory tract: clinicopathologic, radiologic, immunohistochemical, and histochemical studies on 48 cases. Archives of Pathology & Laboratory Medicine 110: 212 218. Khoor A, Myers JL, Tazelaar HD, and Kurtini PJ (2004) Amyloidlike pulmonary nodules, including localized light-chain deposition: clinicopathologic analysis of three cases. American Journal of Clinical Pathology 121: 200204. Kim HY, Im JG, Song KS, et al. (1999) Localized amyloidosis of the respiratory system: CT features. Journal of Computer Assisted Tomography 23: 627631. Kyle RA, Gertz MA, Greipp PR, et al. (1997) A trial of three regimens for primary amyloidosis: colchicine alone, melphalan and prednisone, and melphalan, prednisone and colchicine. The New England Journal of Medicine 336: 12021207. ORegan A, Fenlon HM, Beamis JF Jr, et al. (2000) Tracheobronchial amyloidosis: the Boston University experience from 1984 to 1999. Medicine (Baltimore) 79: 6979. Shah PL, Gillmore JD, Copley SJ, et al. (2002) The importance of complete screening for amyloid bril type and systemic disease

Abstract
Cryptogenic organizing pneumonia (formerly called idiopathic bronchiolitis obliterans organizing pneumonia) is an inltrative lung disease of unknown cause dened by a pathologic pattern of organizing pneumonia consisting of buds of granulation tissue in the distal airspaces. Symptoms include mild dyspnea, fever, and weight loss. Sparse crackles are heard at auscultation over involved areas. Imaging features typically consist of multiple patchy subpleural alveolar opacities, often containing an air bronchogram, and sometimes migratory. Although many causes of organizing pneumonia have been reported (especially infection, and drug-induced reaction) or its occurrence in specic contexts (especially in the connective tissue diseases), it is more often cryptogenic (idiopathic). Improvement is obtained within a few days with corticosteroid treatment, but relapses are common after reducing or stopping treatment.

Introduction
Cryptogenic organizing pneumonia (COP) is a rare distinct idiopathic interstitial pneumonia included in the American Thoracic Society (ATS)/European Respiratory Society (ERS) international multidisciplinary consensus classication of the idiopathic interstitial pneumonias. Organizing pneumonia was historically described as a process of nonresolving infectious pneumonia. The four successive macroscopic stages of acute lobar pneumonia were described by Laennec at the beginning of the nineteenth century. In the rst stage the affected lobe is congested and heavy. The following stage of red hepatization is characterized by a red and dense lobe (as the normal liver). Then gray hepatization takes place with the lobe becoming gray and airless. Finally, in favorable cases, the pneumonia resolves with normalization of the lobe. Failure of the latter process characterizes nonresolving pneumonia. By the end of the nineteenth and the beginning of the twentieth century the usual cause of acute lobar pneumonia was identied as being an infection due to Streptococcus pneumoniae, and the four

INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia 427

macroscopic stages described by Laennec were further studied histopathologically. The sequential stages of pneumonia were characterized by the following lesions: intra-alveolar edema with many pneumococci, polymorphonuclear leukocytes, and erythrocytes; brin intra-alveolar exudates; liquefaction of brin exudates; and eventual resolution of pneumonia without significant sequelae. Nevertheless, before antibiotics were available a number of patients died from pneumococcal pneumonia, including nonresolving pneumonia. Pathologists observed that nonresolving pneumonia was characterized by a process of intra-alveolar organization of the inammatory brinous exudates with a nal characteristic lesion consisting of granulation tissue (as seen in wound healing) formed by intra-alveolar buds of broblasts embedded in connective matrix. Pathologists later considered for a long time that the histopathological nding of the organizing pneumonia pattern was just a witness of a previous nonresolving infection with little clinical significance. However, this pathologic pattern and the concept of organizing pneumonia were revisited two decades ago when it was matched with characteristic clinical imaging features, thus allowing the identication of a clinical syndrome of unknown origin called COP.

pneumophila. Other bacteria as well as viruses, fungi, and parasites have been reported to induce organizing pneumonia (Table 1). Drug-induced organizing pneumonia (Table 2) represents one of the histopathological pulmonary patterns secondary to drug reactions which also comprise diffuse alveolar damage, eosinophilic pneumonia, alveolar hemorrhage, etc. Drug-induced organizing pneumonia is often difcult to diagnose especially when the disorder for which the drug has been prescribed may by itself be associated with organizing pneumonia (e.g., connective tissue disease or immunopathological processes associated with transplantation). The eventual diagnosis of druginduced organizing pneumonia often remains elusive because corticosteroid treatment is generally given to hasten improvement after drug withdrawal. Iatrogenic organizing pneumonia after radiation therapy for breast cancer is distinct from radiation lung injury which is conned to the radiation eld. It differs from the latter by the involvement of the nonirradiated parts of the lungs and complete improvement under corticosteroid treatment without significant sequelae. It develops within 36 months after radiotherapy. Migration of the pulmonary opacities on chest imaging occurs in a majority of patients, before treatment or at relapse.
Table 2 Drugs causing drug-induced organizing pneumonia

Etiology
By definition, the cause of COP is unknown. The nal diagnosis is thus made only once the known causes of organizing pneumonia have been carefully excluded. Organizing pneumonia may also be diagnosed in specic contexts or conditions of unknown etiology, such as the connective tissue diseases. Furthermore several causes or specic associated conditions may contribute to the development of organizing pneumonia. As mentioned above, bacterial infection was the rst established cause of organizing pneumonia. The main bacteria causing organizing pneumonia are probably Streptococcus pneumoniae and Legionella
Table 1 Infectious agents causing organizing pneumonia Bacteria Streptococcus pneumoniae Legionella pneumophila Others Burkholderia cepacia Chlamydia pneumoniae Coxiella burnetii Mycoplasma pneumoniae Nocardia asteroides Pseudomonas aeruginosa Serratia marcescens Staphylococcus aureus Viruses Adenovirus Cytomegalovirus Herpesvirus Human immunodeciency virus Inuenza virus Parainuenza virus Human herpesvirus-7 Respiratory syncytial virus Parasites Plasmodium vivax Dirolaria immitis Fungi Cryptococcus neoformans Penicillium janthinellum Pneumocystis jiroveci Acebutolol 5-Aminosalicylic acid Amiodarone Amphotericin B Bleomycin Busulphan Carbamazepine Chlorambucil Doxorubicin Fluvastatin Gold salts Interferon alpha Interferon beta 1a Mesalazine Methotrexate Minocycline Nilutamide Nitrofurantoin Phenytoin Sirolimus Sotalol Sulfasalazine Tacrolimus Ticlopidine Transtuzumab L-Tryptophan

428 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

The organizing pneumonia histopathological pattern has been described in association with the connective tissue diseases as well as those of nonspecic interstitial pneumonia (NSIP) and of usual interstitial pneumonia. It may develop in the course of a previously diagnosed connective tissue disease, or it may precede the clinical systemic manifestations, or be contemporaneous with these. Organizing pneumonia is especially common in dermatomyositis/polymyositis. The muscular and/or cutaneous involvement may manifest only when corticosteroid treatment given for organizing pneumonia is decreased or stopped. Organizing pneumonia has also been reported in rheumatoid arthritis, but is much more rarely seen in the other connective tissue diseases (systemic lupus erythematosus, scleroderma, Sjo gren syndrome). Organizing pneumonia has increasingly been described in patients after lung transplantation or allogenie bone marrow graft, as a process distinct from the constrictive bronchiolitis with airow obstruction common in these conditions. Organizing pneumonia is often associated with acute rejection after lung transplant, and it is correlated with chronic bronchiolitis obliterans. After allogenic bone marrow graft organizing pneumonia may be associated with graft-versus-host disease. Although organizing pneumonia is likely a process of rejection, or graft-versus-host disease in the respective context of lung transplantation and bone marrow graft, it may also result from infection, aspiration pneumonitis, drug-induced reaction, or from several associated processes. Case reports or short series have described organizing pneumonia occurring in various settings susceptible to induce an inammatory pulmonary process (Table 3).

Table 3 Organizing pneumonia of undetermined cause in specic contexts Connective tissue disorders Polymyositis/ dermatomyositis Rheumatoid arthritis Bone marrow graft and transplantation Bone marrow graft Lung transplantation Liver transplantation Miscellaneous

Myelodysplasia Leukemia Myeloproliferative disorders Lung cancer Sarcoidosis Ulcerative colitis Crohns disease Sweets syndrome Polymyalgia rheumatica Common variable immunodeciency Wegeners granulomatosis Polyarteritis nodosa Thyroid disease Cystic brosis Coronary artery bypass graft surgery Spice processing

Pathology
The histopathologic pattern of organizing pneumonia is dened by the presence of buds of granulation tissue (consisting of broblasts and myobroblasts embedded in a loose connective matrix) within the lumen of the distal airspaces (i.e., the alveoli, the alveolar ducts, and possibly the bronchioles). This pattern has also been called bronchiolitis obliterans organizing pneumonia (BOOP). However, since organizing pneumonia is the characteristic and predominant pathologic pattern with bronchiolitis being only an accessory nding, the present international terminology is organizing pneumonia which further avoids confusion with the different types of bronchiolitis obliterans (especially bronchiolitis obliterans with airow obstruction).
Figure 1 Histopathological pattern of organizing pneumonia with buds of granulation tissue within the alveoli.

The histopathologic diagnosis of the pattern of organizing pneumonia rst necessitates the nding of the characteristic buds of granulation tissue in the distal airspaces especially the alveoli (Figure 1). These may extend from one alveolus to the adjacent one through the pores of Kohn thus forming buttery-shaped gures. The process is patchy, and usually the lesions are all the same age. The different stages of organization (from brinoid inammatory cell clusters to mature brotic buds) may be found on the

INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia 429

same biopsy specimen but the lesions are usually temporally uniform. The lesions are bronchiolocentric and distal to the bronchioles. Cellular inammatory interstitial inltration is usually present but it is mild or moderate. Foamy macrophages are commonly present in the airspaces. In order to make a diagnosis of COP the pattern of organizing pneumonia must clearly be the major type of lesion and not just an accessory nding of another interstitial pneumonia as NSIP, especially cellular or cellular and brotic, where intra-alveolar buds are commonly found. Negative ndings of the organizing pneumonia pattern include the lack of prominent interstitial brosis, the lack of broblastic foci, granulomas, hyaline membranes, vasculitis, and eosinophilia. Other processes where organizing pneumonia may be present and even prominent must be excluded, as the BOOP variant of Wegeners granulomatosis. Although lung specimens obtained by transbronchial lung biopsy may show the characteristic intraalveolar and occasionally intrabronchiolar buds of granulation tissue, it does not allow the exclusion of other possible specic lesions or etiologic agents (such as uncommon infections, neoplasia, vasculitis) which may be demonstrated on larger lung specimens (including with the help of special stains or microbiological analysis of lung tissue). Videothoracoscopic lung biopsy is currently the preferred technique both to establish the histopathologic diagnosis of organizing pneumonia and to exclude associated lesions specic for other disorders.

Physical examination is non-specic. Some ne crackles are often heard over affected pulmonary areas but there are no wheezes and no diffuse velcro rales. Finger clubbing is not present.
Imaging

The chest X-ray and especially high-resolution computed tomography (HRCT) ndings have a major role in the diagnosis and further dene the main characteristic types of COP. The most typical imaging features of COP (classical COP) comprise patchy alveolar opacities on chest X-ray, usually bilateral (Figure 2), asymmetric, and predominating in the lower lung zones. These may migrate in the lungs within a few days (with the ensuing necessity of making a chest X-ray within the 2 days preceding a lung biopsy to ensure it is done in an affected area). On HRCT, the density of the opacities varies from ground glass to consolidation (Figure 3). Their size ranges from a few

Clinical Features
COP affects men and women equally (a female predominance is found in some series). It usually occurs at the age of 5060 years, and more commonly in nonsmokers or ex-smokers.
Clinical Manifestations
Figure 2 Typical COP with bilateral alveolar opacities on chest X-ray.

Often preceded by a u-like syndrome, symptoms generally develop subacutely over a few weeks with mild and persistent fever, anorexia, weight loss, sweats, nonproductive cough, and mild dyspnea. Chest pain, hemoptysis, and bronchorrhea are uncommon. The initial diagnosis being usually that of an infectious pulmonary disease, most patients receive antibiotics which are not efcient, thus leading to consideration of another diagnosis. Diagnosis of COP is usually obtained only after about 612 weeks. Occasionally symptoms are more severe and some patients present with features of acute respiratory distress syndrome (ARDS).

Figure 3 Typical COP with patchy alveolar opacities on HRCT (with an air bronchogram on the left).

430 INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia

centimeters to an entire lobe. They are generally peripheral, but they may predominate in a peribronchovascular location (bronchocentric pattern). Sometimes these opacities may present as large nodules or masses often containing an air bronchogram. The clinical syndrome together with the above typical imaging features must evoke the possible diagnosis of COP to the clinician. Based on the nding of the characteristic features of patchy subpleural and/ or peribronchovascular airspace consolidation often associated with areas of ground glass attenuation, COP is diagnosed correctly in about 80% of cases in patients with idiopathic interstitial pneumonia. The second common clinical imaging presentation of COP consists of solitary focal COP presenting as a solitary nodule or mass, often located in the upper lobes. Some patients are asymptomatic, with both diagnosis and cure obtained by surgical excision of the lesion suspected to correspond to lung cancer (intense tracer uptake may occur on 18F uorodeoxyglucose positron emission tomography in organizing pneumonia). Other patients present with persistent fever and inammatory biologic abnormalities despite antibiotics. Hemoptysis and cavitation of the mass may be present. The third distinct and more confusing clinical imaging presentation of COP is that of diffuse inltrative lung disease with interstitial opacities on HRCT often associated with superimposed small alveolar opacities (Figure 4). Although there is no honeycombing initially, some reticular subpleural opacities and eventually honeycombing may later develop. Histopathologically this type of COP is usually associated with more interstitial mononuclear inammation and overlaps with NSIP especially cellular with foci of intra-alveolar buds of granulation tissue.

Many other less characteristic imaging features of COP have been occasionally reported, including small nodular opacities, irregular linear opacities, bronchial wall thickening, dilatation pneumatocele, and multiple cavitary lung nodules. The micronodular pattern centered on the bronchioles may correspond to bronchiolitis with peribronchiolar organizing. Band-like areas of consolidation in the subpleural area (paralleling the chest wall) may be observed especially during healing on corticosteroid treatment (a nding also present in idiopathic chronic eosinophilic pneumonia). Central ground glass opacity and surrounding airspace consolidation of crescent or ring shape (reversed halo sign) may be found in some patients, usually in association with other types of opacity.
Laboratory Findings

There is no laboratory nding really helpful in the diagnosis of COP. An increased level of blood leukocytes with an increase in the proportion of neutrophils is usual. The erythrocyte sedimentation rate and C-reactive protein level are increased. Occasionally antinuclear antibodies and rheumatoid factor are present (usually at a low titer) in the absence of patent connective tissue disease. Gammaglutamyl transferase and alkaline phosphatase are mildly increased.
Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) differential cell count is helpful especially when showing a mixed pattern with an increase in lymphocytes (about 2545% with a decreased CD4 /CD8 ratio in the majority of cases), neutrophils (about 10%), and eosinophils (about 5%). Foamy macrophages are often present, and interestingly some mast cells and plasma cells may be found in some patients. In addition to providing arguments for the diagnosis of COP the BAL helps in excluding causes of organizing pneumonia as infectious processes. It also helps in the differential diagnosis (eosinophilia pneumonia, lymphoma, bronchioloalveolar carcinoma).
Lung Function Tests

Figure 4 COP with an inltrative pattern on HRCT.

Lung function tests in patients with COP usually show a mild (or moderate) restrictive pattern. Airow obstruction is not typically present, although it may be found in smoker patients with underlying chronic obstructive pulmonary disease. The CO transfer factor is reduced, but the CO transfer coefcient is usually normal. Mild hypoxemia is usually present. More severe hypoxemia may occur in patients with severe, rapidly

INTERSTITIAL LUNG DISEASE / Cryptogenic Organizing Pneumonia 431

progressive diffuse COP; in patients with an underlying respiratory disease (e.g., emphysema); or in patients with a right-to-left shunt at the alveolar level demonstrated by the presence of an increased alveolararterial oxygen gradient while breathing 100% oxygen together with negative contrast echocardiography.

Pathogenesis
COP is a quite original model of pulmonary inammatory and brosing disease because of the usual complete disappareance of the intra-alveolar buds of connective matrix with corticosteroid treatment. This is in marked contrast with the other brosing interstitial lung diseases, especially idiopathic pulmonary brosis, where dense and mutilating brosis relentlessly progresses despite all therapeutic attempts. The dynamic histopathological features offer some insight on the mechanisms of organization, with the individualization of a sequence of distinct processes. Alveolar epithelial cells are the rst target of injury. Pneumocytes undergo necrosis thus leaving the epithelial basal laminae denuded. Whereas cell necrosis is intense the basal laminae remain well preserved although some gaps are present. The striking difference with the diffuse alveolar damage seen in ARDS (or acute interstitial pneumonia) is the lack of hyaline membranes. Inammatory cells are present in the interstitium. The endothelial cells are not markedly damaged. Fibrinoid inammatory cell clusters appearing within the alveolar lumen represent the rst stage of the process of organization. Inammatory cells (especially mononuclear cells) are associated with bands of brin. Then brin is fragmented and inammatory cells are less numerous as broblasts colonize the brin deposits after they have entered the alveolar lumen through gaps in the basal laminae. The proliferation of broblasts is illustrated by the presence of mitotic gures. The broblasts further undergo phenotypic modulation whereby they acquire intracellular laments characteristic of myobroblasts. The parallel re-epithelialization of the basal laminae is probably a key to avoid the interstitial incorporation of the intra-alveolar connective matrix into the alveolar interstitium. The nal stage of organization is that of the typical mature brotic buds. At this stage usually no more brin is present and only few inammatory cells may persist within the buds. Alternating circular layers of broblast/ solidus myobroblasts and connective matrix deposits compose the mature buds. Whereas in usual interstitial pneumonia (the pathologic pattern of idiopathic pulmonary brosis) the connective matrix

consists predominantly of dense bundles of collagen I, the loose connective matrix of the buds in COP comprises collagen III and bronectin in addition to collagen I, together with glycoproteins and other collagen types. Intra-alveolar bud capillarization is prominent (with vascular endothelial growth factor and broblast growth factor widely expressed in the buds). This organization of the intra-alveolar buds shares many features with the granulation tissue developing during cutaneous wound healing. Interestingly both conditions represent young reversible brosis capable of complete healing without significant brotic scarring. The biopathological interpretation of the above morphological processes is largely inferred from human or experimental studies of early diffuse alveolar damage. The formation of brin with the alveolar lumen results from the leakage of plasma coagulation proteins as a consequence of epithelialendothelial alveolar damage, with clotting resulting from an intra-alveolar inbalance between the coagulation and brinolytic cascades. The coagulation and brinolysis factors and inhibitors including plasminogen activator inhibitor 1 have a complex role in the process of brogenesis. Tissue remodeling further necessitates the cleaving of the connective matrix components by matrix metalloproteinases which may play a major role in the disappearance of the buds. The main factors for the parallel disappearance of the broblasts and myobroblasts remain a mystery. This contrasts with the brosing and mutilating role attributed to the broblast foci (which morphologically resemble the buds) in usual interstitial pneumonia.

Animal Models
Only few experimental animal studies have been conducted in organizing pneumonia. The early stage of alveolar damage may be studied in monkeys receiving paraquat or in rats after intratracheal instillation of bleomycin, but the most interesting animal model of organizing pneumonia used intranasal inoculation of reovirus at various titers in CBA/J mice. This model especially demonstrated the possible role of genetic factors as the lesions develop in CBA/J strains of mice but not in other strains. Furthermore, whereas moderate titer of reovirus induces organizing pneumonia, higher titers determine the development of diffuse alveolar damage. Interestingly only intraluminal alveolar lesions produced by moderate doses of reovirus are improved by corticosteroids. Thus this animal model suggests that both genetic factors and the degree of severity of the initial alveolar injury determine the evolution toward

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either nonreversible diffuse alveolar damage or organizing pneumonia capable of improvement with corticosteroids.

Management and Current Therapy

The main initial prerequisite for management is the condence of diagnosis, both positive and etiologic. The differential diagnosis of typical COP is listed in Table 4. The current history and investigations usually exclude these alternative diagnoses rather easily. The diagnosis of COP on transbronchial biopsy ndings is reliable only in patients with the typical clinicoradiological prole of COP and a careful follow-up so that any unusual evolution may be detected soon and a surgical lung biopsy done (allowing for example a diagnosis of pulmonary lymphoma having initially responded to corticosteroid treatment). The diagnosis of COP without any histopathologic conrmation is hazardous and may be accepted only in rare cases by an experienced clinician. Videothoracoscopic lung biopsy is the reference diagnostic procedure. It is a safe procedure which allows a definite diagnosis of COP and allows the recognition of other disorders requiring specic treatment (as vasculitis). It is also useful in convincing patients reluctant to prolonged corticosteroid treatment to accept it. The etiologic diagnosis of organizing pneumonia always need to be very carefully examined to disclose any definite cause (especially drugs) or underlying disorder (as paucisymptomatic connective tissue disease). When the etiologic diagnosis is negative, the diagnosis of cryptogenic (idiopathic) organizing pneumonia may be definitely accepted. Although occasional improvement of typical COP has been reported spontaneously or after treatment with antibiotics, corticosteroids remain the current reference treatment. The response to corticosteroids is very rapid. The clinical manifestations
Table 4 Differential diagnosis of typical COP presenting with multiple patchy alveolar opacities Infectious pneumonia Aspiration pneumonia and aspiration pneumonitis Pulmonary thromboembolism Bronchioloalveolar carcinoma Primary pulmonary lymphoma Chronic eosinophilic pneumonia Exogeneous lipid pneumonia Sarcoidosis Alveolar lipoproteinosis Alveolar hemorrhage Wegeners granulomatosis

improve within 48 h, and complete resolution of the pulmonary opacities on imaging is obtained within a few days or weeks without significant functional or imaging sequelae. Although the efciency of corticosteroids is well established, the best dose and duration of treatment have not been determined. High doses (1 mg kg 1 day 1) of prednisone for 13 months, then 40 mg day 1 for 3 months, then 10 mg daily (or 20 mg every other day) for 1 year, have been proposed. We use lower doses and shorter duration of treatment, with 0.75 mg kg 1 day 1 of prednisone for 4 weeks, then 0.5 mg kg 1 day 1 for 6 weeks, then 20 mg day 1 for 6 weeks, then 5 mg day 1 for 6 weeks. Although the response to corticosteroids is excellent, a proportion of patients (about 1560%) experience a relapse of COP, and several relapses for some of them. About two-thirds of patients still receive corticosteroids (at a mean dose of about 10 mg day 1 prednisone) when they experience relapse. Relapse on more than 20 mg day 1 prednisone is rare and must lead to reconsider the diagnosis of COP (especially in patients with disease diagnosed without surgical biopsy). The treatment of relapses may use moderate doses of prednisone (about 20 mg day 1). The predictors of multiple relapses occurrence comprise delayed treatment and mild cholestasis (elevated gamma-glutamyl transferase and alkaline phosphatase). Relapses are not associated with increased mortality nor increased long-term functional morbidity. Treatment aims at obtaining an optimal balance between efciency and the adverse effects of corticosteroids. The prognosis of COP treated with corticosteroids is excellent in the vast majority of cases. Patients with severe progressive disease not responding to corticosteroids often have overlapping conditions with pathologic features of organizing diffuse alveolar damage or acute brinous and organizing pneumonia.
See also: Acute Respiratory Distress Syndrome. Bronchoalveolar Lavage. Coagulation Cascade: Overview. Corticosteroids: Glucocorticoid Receptors; Therapy. Drug-Induced Pulmonary Disease. Extracellular Matrix: Matricellular Proteins; Degradation by Proteases. Fibrinolysis: Overview. Fibroblasts. Interstitial Lung Disease: Overview. Lung Imaging. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Myobroblasts. Permeability of the BloodGas Barrier. Pneumonia: Overview and Epidemiology. Polymyositis and Dermatomyositis. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Radiation-Induced Pulmonary Disease. Surgery: Transplantation.

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Further Reading
American Thoracic Society/European Respiratory Society (2002) Classication of the idiopathic interstitial pneumonias: international multidisciplinary consensus. American Journal of Respiratory and Critical Care Medicine 165: 277304. Beasley MB, Franks TJ, Galvin JR, Gochuico B, and Travis WD (2002) Acute brinous and organizing pneumonia: a histological pattern of lung injury and possible variant of diffuse alveolar damage. Archives of Pathology & Laboratory Medicine 126: 10641070. Colby TV (1992) Pathologic aspects of bronchiolitis obliterans organizing pneumonia. Chest 102: 38S43S. Cordier JF, Loire R, and Brune J (1989) Idiopathic bronchiolitis obliterans organizing pneumonia: definition of characteristic clinical proles in a series of 16 patients. Chest 96: 9991004. Cordier JF, Peyrol S, and Loire R (1994) Bronchiolitis obliterans organizing pneumonia as a model of inammatory lung disease. In: Epler GR (ed.) Diseases of the Bronchioles, pp. 313345. New York: Raven Press. Crestani B, Valeyre D, Roden S, et al. (1998) Bronchiolitis obliterans organizing pneumonia syndrome primed by radiation therapy to the breast. American Journal of Respiratory and Critical Care Medicine 158: 19291935. Davison AG, Heard BE, McAllister WAC, and Turner-Warwick ME (1983) Cryptogenic organizing pneumonitis. Quarterly Journal of Medicine 52: 382394. Epler GR, Colby TV, McLoud TC, Carrington CB, and Gaensler EA (1985) Bronchiolitis obliterans organizing pneumonia. New England Journal of Medicine 312: 152158. Johkoh T, Muller NL, Cartier Y, et al. (1999) Idiopathic interstitial pneumonias: diagnostic accuracy of thin-section CT in 129 patients. Radiology 211: 555560. Lazor R, Vandevenne A, Pelletier A, et al. (2000) Cryptogenic organizing pneumonia: characteristics of relapses in a series of 48 patients. American Journal of Respiratory and Critical Care Medicine 162: 571577. Lohr RH, Boland BJ, Douglas WW, et al. (1997) Organizing pneumonia: features and prognosis of cryptogenic, secondary, and focal variants. Archives of Internal Medicine 157: 13231329. Lynch DA, Travis WD, Mu ller NL, et al. (2005) Idiopathic interstitial pneumonias: CT features. Radiology 236: 1021. Oikonomou A and Hansell DM (2002) Organizing pneumonia: the many morphological faces. European Radiology 12: 14861496. Raghu G (ed.) (2004) Interstitial lung disease: idiopathic interstitial pneumonia. Clinics in Chest Medicine 25: 621782. Travis WD, Colby TV, Koss MN, et al. (2002) Atlas of Nontumor pathology: Non-Neoplastic Disorders of the Lower Respiratory Tract. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology.

in which the repeated inhalation of certain nely dispersed antigens, mainly organic particles or low-molecular-weight chemicals, provokes a hypersensitivity reaction with granulomatous inammation in the distal bronchioles and alveoli of susceptible subjects. The disease has three kinds of clinical presentation acute, subacute, or chronic depending on the frequency and intensity of exposure to the antigen and the duration and clinical course of the disease. The diagnosis of HP is often made on the basis of clinical history of exposure with resulting respiratory symptoms, but the denitive diagnosis requires a constellation of clinical, radiologic, laboratory, and pathologic ndings. The complete avoidance of exposure to the inciting antigen may result in a spontaneous resolution of the symptoms, especially for the acute form; the chronic progressive form may improve, stabilize, or, rarely, evolve to brosis and respiratory insufciency even though the patient completely avoids re-exposure to the inciting antigen. Corticosteroid therapy can significantly and quickly reduce acute symptoms; however, the long-term benecial effects on arresting disease progression in subacute and chronic HP remain to be denitively established.

Introduction
Hypersensitivity pneumonitis (HP), also called extrinsic allergic alveolitis, is a group of immunologically mediated lung diseases in which the repeated inhalation of certain nely dispersed antigens, mainly organic particles or low-molecular-weight chemicals, provokes a hypersensitivity reaction with granulomatous inammation in the distal bronchioles and alveoli of susceptible subjects. In 1713, B. Ramazzini from Carpi, one of the fathers of occupational medicine, described a grain harvester disease in De Morbis Articum Diatriba. In 1932, J. Campbell described the clinical features of an acute illness in a farmer following work with hay and coined the term Farmers lung. Subsequently, a large number of etiologic agents have been recognized and new causes continue to be recognized. The disease has three general forms of clinical presentation: acute, subacute, or chronic. These presentations depend mainly on the intensity of exposure to the antigen and frequency and the duration and clinical course of the disease. The diagnosis of HP is often considered on the basis of history of exposure to an antigen known to cause the illness. However, denitive diagnosis requires a constellation of clinical, radiologic, laboratory, and pathologic ndings.

Hypersensitivity Pneumonitis
V Poletti, Ospedale GB Morgagni, Forli, Italy M Zompatori, University of Parma, Parma, Italy P Vailati, Ospedale GB Morgagni, Forli, Italy M Chilosi, University of Verona, Verona, Italy
& 2006 Elsevier Ltd. All rights reserved.

Etiology
Agents implicated as causes of HP are numerous and can be found in different occupations, hobbies, and environments (Table 1). Causative antigens include microbes, animal and plant proteins, and organic and inorganic low-molecular-weight particles.

Abstract
Hypersensitivity pneumonitis (HP), also called extrinsic allergic alveolitis, is a group of immunologically mediated lung diseases

434 INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis

Table 1 Causative agents of hypersensitivity pneumonitisa Agent Fungal and bacterial Thermophilic actinomycetes Saccharopolyspora rectivirgula Thermoactinomyces vulgaris Thermoactinomyces sacchari Bacillus subtilis enzimes Aspergillus clavatus Aspergillus versicolor Aspergillus species Aspergillus fumigatus Penicillium casei, A. Clavatus Chrysonilia sitophila Penicillium chrysogenum Cryptostroma corticale Aureobasidium pullulans Aureobasidium species Alternaria species Merulius lacrymans Botrytis cinerea Trichosporon cutaneum Cephalosporium Mucor stolonifer Candida albicans Mycobacterium avium-intracellulare Pseudomonas uorescens Amoebae Naegleria gruberi, Acanthamoeba Animals Avian proteins Rat proteins Gerbil proteins Animal fur protein Ox and pork protein Mollusk shell protein Fish Wheat weevil Silkworm larvae proteins Plants Soybean Coffee Lycoperdon species Chemicals Isocyanates Anhydrides Sodium diazobenzene sulfate Copper sulfate Pyrethrum Phtalyc anhydride Metals Cobalt Beryllium Unknown Moldy typesetting water Cloth wrappings of mummies Cereal grain Source Moldy plant materials Moldy hay Moldy hay, compost Sugarcane residue Detergent enzymes Moldy grains Animal bedding Tobacco mold Esparto dust Cheese mold Moldy cork Moldy wood dust Moldy maple bark Moldy sequoia dust Contaminated water Wood or wood pulp Rotten wood Grape mold Mold in Japanese homes Sewage Paprika Saxophone mouthpiece Contaminated water Aerosolized metalworking uids Disease Farmers lung Farmers lung, mushroom workers lung, composters lung Bagassosis Detergent workers lung Malt workers lung Doghouse disease Tobacco workers lung Stipatosis Cheese washers lung Suberosis Woodworkers lung Maple bark strippers lung Sequoiosis Sauna takers disease Woodworkers lung Dry rot lung etlase lung Wine growers lung or Spa Summer-type HP Sewage workers lung Paprika splitters lung Sax lung Hot tub lung Machine operators lung

Contaminated water

Humidier lung

Bird excreta, blood, or feather Rat urine or serum Gerbil Animal fur Pituitary snuff Mollusk shell dust Fishmeal dust Flour Silkworm larvae

Bird breeders lung, bird fanciers lung Rodent handlers lung Gerbil keepers lung Furriers lung Pituitary snuff takers lung Oyster shell lung Fishmeal workers lung Millers lung Sericulturists lung

Soybean hulls Coffee bean dust Puffballs

Soybean workers lung Coffee workers lung Lycoperdonosis

Paints, plastics Plastics Laboratory reagent Bordeaux mixture Insecticides Heated epoxy resin

Paint renishers lung Chemical workers lung, plastic workers lung, epoxy workers lung Paulis reagent lung Vineyard sprayers lung Insecticide lung Epoxy resin lung

Hard metal lung disease Berylliosis Bible printers lung Coptic lung (mummy handlers lung) Grain measurers lung

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Table 1 (Continued ) Agent Source Coffee bean dust Contaminated tap water Tea plants Sea snails shell Aerosolized endotoxin from pool water sprays and fountains
a

Disease Coffee workers lung Tap water lung Tea growers lung Mollusk shell HP Swimming pool workers lung

The more frequent causative agents are listed in bold type. Adapted from Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661670.

However, only a few of these are responsible for the majority of cases of HP. The diagnosis of HP and consequently the avoidance of exposure to HP caused by rare or unknown inciting antigens can be very difcult to conrm. The more common clinical entities include farmers lung, bird breeders lung, mushroom workers disease, and air-conditioner lung, but the prevalence of the different forms of HP diagnosed varies greatly within and between countries as well as with seasonal changes; in Japan, summer-type hypersensitivity pneumonitis due to Trichosporon cutaneum mold seems to account for the majority of cases of HP (74.4%).

Pathology
Although the earliest pathologic changes in HP are not well dened, lung changes seem to be characterized by a centrilobular interstitial and alveolar inammation and edema, epithelial alveolar necrosis, and acute bronchiolitis. Subacute HP shows typical histopathologic features that may affect the airblood barrier and peripheral airways conguring a bronchiolocentric interstitial granulomatous pneumonitis. The characteristic triad of (1) patchy interstitial inltrate consisting of lymphocytes, mainly CD8 , plasma cells, and monocytes with peribronchiolar accentuation, (2) small, loosely formed, and poorly circumscribed non-necrotizing granulomas, and (3) intra-alveolar buds of granulation tissue is considered diagnostic (Figures 1 and 2). Ancillary ndings are intra-alveolar accumulation of foamy macrophages and peribronchiolar lymphoid follicle hyperplasia. Rarely, histology may be less specic: patchy interstitial cellular pneumonitis, dense lymphoid interstitial inltrate, and organizing pneumonia have been reported as the only or predominant feature. The chronic phase (Figure 3) may maintain some histopathologic features suggesting the diagnosis of HP, such as a moderate lymphocytic inltrate or poorly formed granulomas, but mostly non-specic extensive interstitial brosis and honeycombing in the upper lobes may be found. Typically,
Figure 1 HP, subacute form. Open lung biopsy: centrilobular patchy interstitial pneumonitis and lymphoid follicular hyperplasia are evident (H&E, low power).

Figure 2 HP, subacute form. Loose non-necrotizing granuloma (H&E, high power).

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exposure and the symptoms experienced during every episode of exacerbation tend to become progressively more severe and long-lasting.
Chronic HP

Figure 3 HP, chronic form. Interstitial brosis with exuberant bronchiolar epitelization in the centrilobular zone (H&E, midpower).

bronchiolar epithelium extending from the centrilobular site to the surrounding alveoli are evident (so-called lambertosis).

The chronic form of HP may develop from unrecognized and untreated subacute HP or begin with very mild progressive symptoms mimicking chronic bronchitis. Typically, patients present with cough, mucus production, exertional dyspnea, progressive weight loss and anorexia, malaise, and, sometimes, low-grade fever. Finger clubbing and chest crackles are uncommon. The symptoms are not clearly related to each antigen exposure. Chronic HP may be progressive and irreversible, with or without continued exposure to the inciting antigen. The illness may lead to chronic respiratory failure. The development of pulmonary brosis is associated with older age, greater restrictive lung physiology, and diminished survival. Strict definitions of acute, subacute, and chronic stages of HP have not been generally agreed on. It has been proposed that HP be described as recently diagnosed, recurrent or progressive, or residual disease.

Diagnostic Approach Clinical Features

Acute HP

Acute HP is generally subsequent to heavy exposure to the inciting antigen and is probably the easiest form to recognize. The classical symptoms of presentation are fever, chills, malaise, nausea, scarcely productive cough, dyspnea, and chest tightness arising 46 h after exposure and reaching a peak intensity between 12 and 24 h after exposure. Spontaneous resolution generally occurs within 48 h after cessation of exposure. Physical examination may identify diffuse ne inspiratory rales associated with tachypnea.
Subacute HP

The diagnosis of HP has most often relied on a high index of suspicion and on an array of non-specic clinical symptoms and signs developed in an appropriate setting, with demonstration of interstitial markings on chest radiographs, serum precipitating antibodies against offending antigens, characteristic bronchoalveolar lavage (BAL) ndings, and a granulomatous reaction on lung biopsies (Table 2). One study used a stepwise linear regression model to evaluate the importance of different clinical, laboratory, and radiologic variables in the diagnosis of HP. This study identied six significant predictors of HP:
* * * * * *

The distinction between acute and subacute HP is often difcult. The subacute form generally has the same symptoms (dyspnea on effort, cough, and lowgrade fever) and signs as the acute form but with a more gradual period of development. As in the acute form, during exposure-free periods patients are asymptomatic. Individuals who have recurrent episodes of subacute HP over a period of months to years are at high risk of developing chronic HP; in this case, the subsequent episodes are elicited by a lower dose

exposure to a known offending antigen; positive precipitating antibodies; recurrent episodes of symptoms; inspiratory crackles on physical examination; symptoms occurring 48 h after exposure; and weight loss.

When all six predictors were evident, the probability of HP was 98%. Chronic HP may mimic idiopathic pulmonary brosis or the advanced brotic stage of any inltrative diffuse lung disease. In these cases, environmental or laboratory-controlled challenge test with the suspected antigen and surgical lung biopsy are usually necessary to conrm the diagnosis.

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Table 2 Diagnostic criteria for hypersensitivity pneumonitisa Major criteria 1. History of symptoms compatible with hypersensitivity pneumonitis that appear or worsen within hours after antigen exposure 2. Conrmation of exposure to the offending agent by history, investigation of the environment, serum precipitin test, and/or bronchoalveolar lavage uid antibody 3. Compatible changes on chest radiography or high-resolution computed tomography of the chest 4. Bronchoalveolar lavage uid lymphocytosis, if bronchoalveolar lavage performed 5. Compatible histologic changes, if lung biopsy performed 6. Positive natural challenge (reproduction of symptoms and laboratory abnormalities after exposure to the suspected environment) or by controlled inhalational challenge Minor criteria 1. Basilar crackles 2. Decreased diffusion capacity 3. Arterial hypoxemia, either at rest or with exercise
a The diagnosis is considered conrmed if the patient fullls four of the major criteria and at least two of the minor criteria and if all other diseases with similar symptoms and signs have been excluded. Although these criteria provide useful guidelines with regard to the diagnosis of the disease, strict clinical application of the criteria appears unwarranted. From Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661670.

Inhalation challenge If antibodies cannot be detected, the patient may undergo an inhalation challenge with two modalities through exposure to the environment of the supposed antigen or through inhalation of the puried antigen through a hand nebulizer for a short time (approximately 10 min) in a clinical laboratory setting. Many parameters are monitored during the 24 h after challenge, such as clinical symptoms including cough, dyspnea, chills, fever, and malaise; leukocyte counts; C-reactive protein; arterial blood gas analyses; pulmonary function tests; chest radiographs; and high-resolution computed tomography (HRCT). Postchallenge monitoring is time-consuming but may be helpful in determining a potential diagnosis, and, fortunately, the reactions induced are usually well tolerated. Some authors do not recommend the performance of inhalation challenge because of the risk that it will induce progressive disease and because of the absence of standardized antigen preparations. Pulmonary function tests Pulmonary function tests in all forms of HP may show a restrictive or mixed ventilatory deficit, impaired diffusion capacity, decreased compliance, and exercise-induced hypoxemia. A resting hypoxemia and hypocapnia may also be found. Bronchospasm and bronchial hyperreactivity are sometimes found in acute cases. BAL The typical BAL prole is characterized by a three- to vefold increase in cell count, with high lymphocytes rates ranging between 20% and 70% and often higher than 50%, with a predominance of CD3 CD8 cells and a consequent CD4 /CD8 ratio usually o1 (in Western countries the CD4/CD8 ratio is 43 in only approximately 3% of cases). Lymphocytes show the morphology of activation with irregular nuclei, evident cytoplasm, and, in a minority, small azurophilic intracytoplasmatic granules (large granular lymphocytes). Immunophenotypical analysis shows that there is also an increase in CD16 CD3 (NK cells) and CD3 cells expressing the IL-2 receptor (CD25 cells) and HLA-DR antigens. Plasma cells and mast cells (better seen using Alcian staining) are also present. This BAL prole is not diagnostic by itself because it may also be observed in subjects exposed to antigens but without clinical evidence of the disease; however, this BAL prole may be helpful to exclude other interstitial disorders, such as idiopathic pulmonary brosis (IPF) and sarcoidosis. A different pattern with high neutrophilic rates can be found if the procedure is performed within 48 h after acute exposure. Neutrophil also seems to play a major role in lung damage and brosis; a positive

Routine Laboratory Tests

Especially during acute exacerbation, moderately elevated leukocyte count, lymphopenia, and increased erythrocyte sedimentation rate and C-reactive protein, immunoglobulins, and immune complexes are found. Peripheral eosinophil count and serum IgE concentration are generally normal. Serum precipitins The presence of serum precipitins is useful for identifying exposure to a potential causative antigen. The level of serum precipitins against many antigens is rarely helpful in the diagnosis of HP. Serum precipitins are classically assessed by radial diffusion (Ouchterlony) analysis, although enzyme-linked immunosorbent assay, which is more sensitive, is increasingly being used. The analysis of serum precipitating IgG antibodies is not diagnostic but requires a more complex analysis of clinical, radiologic, and immunologic data. In fact, as many as 4050% of asymptomatic exposed people may have precipitins in their serum. Another problem is that the absence of serum precipitins does not rule out HP; negative results are quite frequent both for the wide variety of causative antigen and for the possibility to test precipitins with different laboratory methods with different sensitivity and specicity for any single causative antigen. Skin tests are not helpful in the diagnosis of HP and are not routinely used.

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correlation between the percentage of lung neutrophils and the percentage of lung brosis has been demonstrated, and in advanced disease a neutrophilic rate 45% is common, often accompanied by mild eosinophilia and an increase in the CD4 / CD8 ratio. Transbronchial biopsy The role of transbronchial biopsy in HP diagnosis seems to be limited. The characteristic histopathologic triad may also be discernable in tiny specimens; however, it should probably be performed in patients with intermediate pretest probability of HP.
Imaging Findings

The radiological features of HP are similar, regardless of the responsible agent, and can be divided into three stages: acute, subacute, and chronic. Different features may coexist in the same patient. Chest X-ray can be normal, especially between acute episodes, and can demonstrate diffuse opacities during relapses, but plain radiography is not sensitive or specic (abnormalities detected in less than 50% of proven cases). On the other hand, HRCT is abnormal in nearly 100% of patients with HP and can suggest the correct diagnosis in the appropriate clinical setting, particularly when the patient is exposed to recognized organic antigens. The acute phase of HP, poorly documented in the literature, is characterized by diffuse, bilateral areas of consolidation or ground glass density, predominating in the mid- to lower zones, with sparing of the costophrenic angles (Figure 4). In the subacute phase (Figure 5), HRCT shows diffuse ground glass opacities in a panlobular distribution (5290% of cases) and/or small, poorly dened centrilobular nodules less than 5 mm in diameter (4076%). These indistinct, peribronchiolar nodules should suggest the diagnosis of HP but are

not pathognomonic. They can also be found in other entities (e.g., respiratory bronchiolitis). In the axial plane, the distribution may be diffuse or patchy. The HRCT ndings are generally most evident in the parahilar and basal regions. Small, thinwalled cystic spaces containing air have been reported in a small percentage of patients with subacute HP. Lobular areas of decreased attenuation and expiratory air trapping are common (5075% and up to 92% in patients with dynamic inspiratoryexpiratory HRCT). Air trapping is particularly common within the areas of ground glass density and represents small airways obstruction. Its detection is regularly enhanced and sometimes only demonstrated by performing expiratory scans. In the chronic phase (Figure 6), HRCT shows signs of brosis, characterized by inter- and intralobular septal thickening, honeycombing, traction

Figure 5 Subacute HP; HRCT features (see text for details).

Figure 4 Acute HP; HRCT features (see text for details).

Figure 6 Chronic HP; HRCT features (see text for details).

INTERSTITIAL LUNG DISEASE / Hypersensitivity Pneumonitis 439

bronchiectasis, and volume loss. The presentation can overlap that of sarcoidosis or usual interstitial pneumonitis (UIP)/IPF, but in case of HP, lesions are more patchy and predominantly distributed to midlung regions, with relative sparing of apices and bases, and need not be subpleural. In some cases of chronic HP, however, lower lobe predominance can be found.

Pathogenesis
The pathogenesis of HP involves both type III and type IV hypersensitivity reactions. A genetic predisposition to the development of HP has also been identied. It has been shown that pigeon fanciers with HP have an increased frequency of HLA-DRB1*1305 and HLA-DQB1*0501 alleles, a decreased frequency of the HLA-RB1*0802 allele, and an increased frequency of the TNF-2(-308) polymorphism of the tumor necrosis factor alpha (TNF-a) promoter gene. Type III hypersensitivity seems to characterize the acute phase of HP alveolitis, which is mainly neutrophilic. The immunologic process is probably triggered, in a susceptible host, by the formation of immune complexes that activate the complement cascade and macrophages that secrete chemokines and cytokines (MCP-1, MIP-1a, IL-1a, IL-6, and CXCL8/IL-8) responsible for the neutrophilic recruitment and migration, release of toxic oxygen metabolites and proteinases, the differentiation of B cells into plasma cells, and maturation of CD8 cells into cytotoxic cells. TNF-a may play a role in the induction of the adhesion molecules L-selectin and Eselectin and alveolar damage. Respiratory epithelial cells and mast cells are other actors in this phase. If antigenic stimulation persists, this early phase is followed by an inux of mononuclear cells in the lung, with type IV cell-mediated delayed hypersensitivity assuming a pivotal role in the immunopathogenetic events. Particularly alveolar recruitment and/ local proliferation of suppressor/cytotoxic CD8 lymphocytes and macrophages, associated with increased production of IL-1, TNF-a, IL-12, IL-15, and IL-18 and with a reduction in IL-10 production, increased production of interferon gamma (IFN-g), and elaboration by dendritic cells of CC chemokine-1 interact to promote granulomatous inammation. Oligoclonal expansion of T cells that express homologous or identical complementary-determining region 3 has been documented in blood and lung cells, indicating that the immune reaction that occurs at the lung level gives rise to a systemic reaction. Although several chemokines contribute to the development of the characteristic HP cellular inltrate

and granulomas, the specic biologic steps leading to transformation of lymphocytes and macrophages in organized granulomas are unknown. A particularly intriguing question is why only a small percentage of individuals exposed to the different antigens develop the disease. Cofactors are probably needed to render patients hypersensitive to environmental antigens: viral infection, bacterial endotoxin, or fungal glucans have been proposed as possible factors enhancing host susceptibility to the antigen. Using polymerase chain reaction techniques, common respiratory viruses have also been demonstrated in the lower airways of patients with an acute episode of HP, and both respiratory syncytial and Sendai virus increase the expression of HP in mice. Convincing evidence indicates that active cigarette smoking protects against the development of HP. The pathogenetic events leading to abnormal extracellular remodeling and progression to brosis seem to involve neutrophils, endothelin-1 upregulation, and transforming growth factor-b overproduction.

Animal Models
Many animal models of HP have been performed using a wide variety of animal species (rabbits, guinea pigs, mice, primate, and calves); the most common route of administration is via intratracheal instillation of various antigens (Saccharopolydpora rectivirgula, Penicillium notatum, bat feces, tubercle bacilli antigens, Corynebacterium parvum, and many others). These studies were useful to understand the different roles of macrophages, T cells, mast cells, cytokines and chemokines (mainly IFN-g, IL-12, IL-10, and IL-4), and vascular adhesion molecules (E-selectin, P-selectin, and vascular cell adhesion molecule-1) in HP. Obviously, these animal models have important limitations, such as the administration of large amounts of antigenic material as an intratracheal bolus, the difculties in producing a granulomatous reaction and ongoing brosis, and the numerous obvious differences between experimental animals and humans. The development of the inammatory reactions and the formation of granuloma and brosis scars derive from a complicated sequence of tissue and cellular events that still needs better characterization.

Treatment
The treatment of HP is not well standardized and the most common recommendations are based on studies of farmers lung and pigeon breeders lung. Avoidance of antigen exposure and corticosteroid therapy are the key elements in the treatment of HP.

440 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis Avoidance of Exposure Interleukins: IL-1 and IL-18; IL-6; IL-10; IL-12; IL-15. Interstitial Lung Disease: Overview; Alveolar Proteinosis; Cryptogenic Organizing Pneumonia; Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Neutrophils. Pulmonary Fibrosis. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a).

The complete avoidance of exposure to the inciting antigen may result in spontaneous resolution of the symptoms, especially for the acute form. The chronic progressive form may improve, stabilize, or, rarely, evolve to brosis and respiratory insufciency even though the patient completely avoids re-exposure to the inciting antigen. Patients with progressive disease should strongly be recommended to avoid the causative environment. After an acute rst episode the risk of recurrence is unclear and drastic measures to avoid inciting antigens may not always be indicated. In many cases, the source of exposure (birds and domestic yeast) can easily be removed, but if occupational exposure is involved, an initial attempt to avoid the antigens heaviest areas of exposure and the use of preventative measures should be tried. Dust and pollen masks with lters, airstream helmets, and ventilated helmets with a supply of fresh air are increasingly efcient means of purifying inhaled air and, in association with mechanization of the feeding process on farms, industrial hygiene, and alterations in forced-air ventilatory systems may permit adequate control of the disease.
Corticosteroid Therapy and Other Immunomodulating Drugs

Further Reading
Agostini C, Trentin L, et al. (2004) New aspects of hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 378382. Bourke SJ, Dalphin JC, et al. (2001) Hypersensitivity pneumonitis: current concepts. European Respiratory Journal 32: 81S92S. King TE Jr (2005) Classication and clinical manifestations of hypersensitivity pneumonitis (extrinsic allergic alveolitis). King TE Jr (2005) Diagnosis of hypersensitivity pneumonitis (extrinsic allergic alveolitis). Up To Date. King TE Jr (2005) Treatment and prognosis of hypersensitivity pneumonitis (extrinsic allergic alveolitis). Lacasse Y, Selman M, et al. (2003) Clinical diagnosis of hypersensitivity pneumonitis. American Journal of Respiratory and Critical Care Medicine 168: 952958. Mohr LC (2004) Hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 401411. Patel AM, Ryu JH, and Reed CE (2001) Hypersensitivity pneumonitis: current concepts and future questions. Journal of Allergy and Clinical Immunology 108: 661670. Schuyler M (2004) Hypersensitivity pneumonitis. In: Crapo JD, Glassroth J, Karlinsky J, and King TE Jr (eds.) Baums Textbook of Pulmonary Diseases, pp. 481498. Philadelphia: Lippincott Williams & Wilkins. Selman M (2004) Hypersensitivity pneumonitis: a multifaceted deceiving disorder. Clinics in Chest Medicine 25: 531547. Travis WD, Colby TV, et al. (2001) Hypersensitivity pneumonitis. In: Non-Neoplastic Disorders of the Lower Respiratory Tract, pp. 115122. Washington, DC: Armed Forces Institute of Pathology/American Registry of Pathology.

Although there are no controlled clinical trials regarding the treatment of chronic HP, it is recommended that patients with severe acute or progressive chronic HP be treated with a trial of corticosteroids. The recommended therapy is usually a starting dose of 0.5 1 mg kg 1day 1 of prednisone (maximum, 60 mg) for 24 weeks, followed by a slow tapering of the dosage until suspension or the minimal maintenance dose, if required, is reached. Corticosteroid therapy can significantly and quickly reduce acute symptoms; however, the long-term benecial effects on arresting disease progression in subacute and chronic HP remain to be denitively established. There is also concern that steroid-treated patients are more likely to relapse than those who are untreated. Little experience has been reported on the use of systemic immune modulators (azathioprine and cyclophosphamide) in the treatment of severe refractory HP.
See also: Bronchiectasis. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chemokines. Chemokines, CC: MCP-1 (CCL2)MCP-5 (CCL12). Chemokines, CXC: IL-8. Corticosteroids: Glucocorticoid Receptors; Therapy. Dendritic Cells. Endothelins. Endotoxins. Environmental Pollutants: Overview; Particulate and Dust Pollution, Inorganic and Organic Compounds. Genetics: Overview. Infant Respiratory Distress Syndrome. Interferons.

Relevant Websites
http://germop.univ-lyon1.fr Group of Studies and Research on the Orphan Diseases Pulmonary. www.uptodate.com UpToDate is specifically designed to answer the clinical questions that arise in daily practice and to do so quickly and easily so that it can be used right at the print of case.

Idiopathic Pulmonary Fibrosis

F J Martinez, C M Hogaboam, E S Choi, G B Toews, K R Flaherty, and V J Thannickal, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Idiopathic pulmonary brosis (IPF) is a clinical syndrome of unknown cause affecting primarily middle aged or elderly patients. IPF follows an inexorably progressive and, usually, fatal

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 441

course. It is associated with the histopathological pattern of usual interstitial pneumonia on surgical lung biopsy (SLB), showing heterogeneous distribution of broblastic foci interspersed with normal-appearing lung and minimal inammation. Clinical features include progressive dyspnea with/without dry cough, end-expiratory crackles on lung auscultation, and digital clubbing in up to half of all patients. Interstitial (brotic) lung diseases associated with environmental or drug exposures and connective tissue diseases must be excluded. Pulmonary function testing typically shows restrictive physiology with gas exchange abnormalities. High-resolution computed tomography (HRCT) classically demonstrates subpleural and lower lobe-predominant interstitial/septal thickening in a reticular pattern and honeycomb change. SLB is recommended when the clinical and/or HRCT ndings are atypical. Current concepts on pathogenesis of IPF suggest an aberrant host response to alveolar epithelial injury, although the etiology of the injury remains unknown. Dysregulated repair involving altered communication between the injured epithelium and broblasts/myobroblasts (in broblastic foci) plays a dominant role in disease pathogenesis. Traditional therapies with steroids and potent immunosuppressive agents have been disappointing. Newer antibrotic agents are currently under investigation.

inconclusive. Cigarette smoking has been reported to be an independent risk factor for the development of IPF; however, prognosis in established cases of IPF appears to be paradoxically improved in active smokers. Familial IPF accounts for less than 3% of IPF patients. Recent evidence suggests that some families harbor mutations in the prosurfactant protein-C (proSP-C) gene expressed in alveolar epithelial cells; this alters protein processing and results in epithelial injury. Further studies are required to determine if germline/somatic mutations of this or other proteins may account for injury of alveolar epithelial cells. Gene polymorphisms involving transforming growth factor beta-1 (TGF-b1), tumor necrosis factor alpha, interleukin-1-receptor antagonist, interleukin-6, and major histocompatibility complex loci among others have been associated with increased risk or progression of IPF.

Pathology Introduction
Idiopathic pulmonary brosis (IPF) is a clinical radiologicalpathological syndrome that belongs to a group of enigmatic diffuse parenchymal lung disorders known as the idiopathic interstitial pneumonias (IIPs). In 1975, Liebow rst proposed a classication of the IIPs into ve distinct histopathological categories that were subsequently lumped into a single IPF disease category in the 1980s up until the late 1990s. More recently, these disorders have been reclassied in a new classication scheme that was adopted, by consensus, jointly by the American Thoracic Society and the European Respiratory Society in 2002. Although overlapping histopathological patterns of IIPs are seen in the individual patient with IPF, classication of patients into as distinctive a group as is currently possible will allow for more meaningful studies of prognoses and responses to emerging drug therapies for these disorders. The key histologic features of the usual interstitial pneumonia (UIP) pattern have been well dened and include architectural remodeling, brosis often with honeycombing, scattered broblastic foci, patchy distribution, and involvement of the periphery of the acinus (Figure 1). A cardinal feature is the bilateral but heterogeneous (patchy) involvement, with a predilection for the lower lobes and peripheral (subpleural) regions. A key feature of this temporal heterogeneity is the presence of dense acellular collagen and broblastic foci (aggregates of proliferating broblasts and myobroblasts). Fibroblastic foci develop in areas of lung injury and are associated with active collagen synthesis, suggesting that brosis is active. Although broblastic foci are not pathognomonic, they are necessary for the histologic diagnosis of UIP. The extent of interstitial inammation is usually mild to moderate, patchy, and characterized by a septal inltrate of lymphocytes, plasma cells, and histiocytes associated with hyperplastic type II pneumocytes. Additional features include smooth muscle hypertrophy, reactive metaplasia and hyperplasia of type II pneumocytes, mucostasis, secondary pulmonary hypertensive changes, traction bronchiectasis and bronchiolectasis, reduced airspace volume, and destroyed and distorted alveolar architecture. Subpleural fat and metaplastic bone are noted in severe disease. Without areas of relatively normal lung in the surgical specimen, the presence of other interstitial disorders may be difcult for the pathologist to exclude and the UIP histological pattern may be difcult to recognize. Recently, several groups have conrmed that in some patients a UIP pattern may be seen in one specimen from a surgical

Etiology
The etiology of IPF is not known. Based on the observations that the disease is limited to the lungs and that alveolar epithelial cells (AECs) viewed under electron microcopy appear to be injured, potential causative agents that directly target these cells have been postulated. Respirable environmental agents and occupational exposures to inorganic/organic dusts have been proposed, but none have been clearly linked to IPF. Studies of latent viral infections, particularly EpsteinBarr virus (EBV) and cytomegalovirus (CMV), as risk factors in IPF have been

442 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

Figure 1 (a) Lower power image illustrating heterogeneous (patchy) involvement with a predilection for the peripheral (subpleural) regions. (b) Higher power image illustrating a key histological feature of UIP, the presence of dense acellular collagen and broblastic foci (aggregates of proliferating broblasts and myobroblasts).

lung biopsy (SLB), while specimens from a second or third lobe of lung exhibit other histologic patterns. Importantly, these patients exhibit the survival characteristics of idiopathic UIP or IPF. The histologic approach to differential diagnosis includes a careful search for histologic clues to potential causes such as asbestos bodies, infectious agents, or other exogenous agents. The differential diagnosis of the UIP histologic pattern includes other IIPs including brosing non-specic interstitial pneumonia (NSIP), desquamative interstitial pneumonia (DIP), organizing pneumonia (OP), and diffuse alveolar damage (DAD). Lesions that can be histologically similar (but not identical) to UIP include asbestosis, collagen vascular disease, brosing hypersensitivity pneumonitis, radiation pneumonitis, and HermanskyPudlak syndrome. Temporal heterogeneity is a central histologic feature that may aid in distinguishing UIP from other IIPs, as non-UIP IIPs are generally temporally uniform. In addition, broblastic foci, which are prominent in UIP, are rarely numerous in DIP, respiratory bronchiolitis interstitial lung disease (RBILD), or NSIP. Although broblastic foci are present in acute interstitial pneumonia, this disorder is diffuse and temporally uniform, in contrast to UIP. Furthermore, inammatory cells are generally more conspicuous in other IIPs. Intraalveolar macrophages may be seen in UIP, but are not a dominant histologic feature, while dense intraalveolar or peribronchiolar alveolar macrophages suggest DIP or RBILD. Nevertheless, recent work by a group of expert pathologists suggests that the differential diagnosis of UIP remains difcult, particularly in separating UIP from cases of brosing NSIP.

Clinical Features
The typical clinical features of IPF include dry cough, exertional dyspnea, and end-inspiratory velcro-like

rales on physical examination. Exertional dyspnea is one of the most common symptoms and progresses inexorably over months to years. A dry, hacking cough may be the initial symptom, and is often paroxysmal. Bibasilar end-inspiratory velcro rales are present in the majority of patients with IPF, while clubbing is present in a smaller proportion of patients. Cyanosis and cor pulmonale are features noted in advanced clinical disease. The physical examination is likely most useful in identifying extrapulmonary signs; extrapulmonary involvement does not occur in IPF and, if present, suggests an alternative diagnosis. Laboratory abnormalities are non-specic and may include an elevated erythrocyte sedimentation rate, rheumatoid factor, or circulating antinuclear antibodies (ANA). In general, serologic studies do not correlate with the extent or activity of the disease and have limited prognostic value. However, some investigators have suggested that serum levels of surfactant protein (SP)-A and SP-D correlate with the extent of alveolitis, and may be predictive of longterm survival. The physiologic abnormalities in IPF patients characteristically include reduced lung volumes (e.g., vital capacity (VC), and total lung capacity (TLC)), normal or increased expiratory ow rates, increased forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) ratio, and a reduced diffusing capacity for carbon monoxide (DLCO). Hypoxemia or increased alveolar-arterial oxygen difference (PA-aO2 ) may be present, which is generally accentuated by exercise. Cardiopulmonary exercise testing (CPET) generally documents hypoxemia or widened PA-aO2 . Additional ndings may include decreased oxygen consumption VO2 , increased physiologic dead space, increased minute ventilation for the level of VO2 , high respiratory rate/low tidal volume respiratory pattern, and a decreased O2 pulse. The

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 443

impairment in gas exchange (DLCO) and oxygenation are likely the earliest abnormalities. Although a restrictive defect with reduced FVC and TLC is characteristic of IPF, lung volumes may be normal if emphysema coexists. In this setting, DLCO and oxygenation are disproportionately reduced. In general, physiological parameters provide only a rough estimate of disease severity, with the DLCO correlating better with the extent of disease on high-resolution computed tomography (HRCT) scans. Chest radiographs generally exhibit diffuse, bilateral reticular opacities, with a predilection for basilar and peripheral (subpleural) regions (Figure 2). As disease progresses, lung volumes decrease. HRCT scans, using 12 mm thin sections, are superior to conventional radiographs in depicting the characteristics, extent, and distribution of parenchymal inltration. The three major HRCT patterns include ground glass opacity (GGO), reticular pattern, and honeycomb cysts. The characteristic HRCT features of IPF include a basilar, subpleural predominance of abnormality, patchy involvement with large areas of spared lung parenchyma, coarse reticular or linear opacities (intralobular and interlobular septal lines), honeycomb cysts, and traction bronchiectasis or bronchiolectasis (Figure 3). More than focal areas of GGO (i.e., ill-dened, hazy zones of increased alveolar attenuation) are unusual and should suggest an alternative diagnosis (e.g., DIP, NSIP, or hypersensitivity pneumonia (HP)). Honeycomb cysts are the key feature in ensuring a radiographic diagnosis of IPF. Zones of emphysema (usually upper lobe predominant) may be present in smokers; these cysts are generally distinguished from honeycombing by the lack of well-dened walls but may make radiographic diagnosis difcult.

Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) has been quite valuable in the evaluation of some ILDs, although its value in the diagnosis of IPF appears to be limited. Increases in polymorphonuclear leukocytes (PMNs), mast cells, alveolar macrophages, eosinophils, and various cytokines have been noted in BAL uid from IPF patients, although such measurements are rarely diagnostic or clinically valuable. Radionuclide scans have been used to evaluate numerous ILDs. Increased intrapulmonary uptake of gallium 67 citrate had been touted to be a marker of alveolitis, although its diagnostic and prognostic value in IPF has been disappointing. Increased clearance of technetium 99m-diethylenetriamine penta-acetate aerosol, a marker of increased lung permeability, is accelerated in IPF, which some investigators have argued may have prognostic value. Unfortunately, this technique has not become widely utilized. The onset of IPF is indolent, but progresses inexorably over months to years. Spontaneous remissions do not occur, although some patients stabilize for long periods of time; this can sometimes occur after an initial decline. Most patients die of respiratory failure within 38 years of onset of symptoms, with a mean survival of several years. Other causes of death include cardiac failure, pulmonary embolism (due to immobility or cor pulmonale), pulmonary infections, and cerebrovascular accidents. Lung cancer occurs in 613% of patients with IPF. Acute exacerbation of IPF, also known as the accelerated form of IPF, is characterized by acute progression of dyspnea over 1 month or less, in association with new, diffuse opacities on chest radiography, worsening hypoxemia, and rapid development of respiratory failure in the absence of infection or alternative diagnoses.

Figure 2 (a) Chest radiograph of a patient with mild to moderate IPF. Note the basilar predominance of reticular ndings. (b) Chest radiograph of a patient with more advanced IPF. Note the reduction in lung volumes and diffuse, bilateral interstitial or reticulonodular pattern of inltration.

444 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

Figure 3 (a, b) HRCT images of a patient with moderately severe UIP proven using SLB. Upper and lower lung zone reticular inltrates are very subtle; basilar predominant, subpleural honeycomb cysts are noted. (c, d) HRCT images of a patient with advanced IPF. Note the predominately peripheral honeycomb change.

Pathologic examination typically shows acute alveolar injury with or without hyaline membrane formation. While the exact incidence of this acute syndrome is unknown, one recent series noted that approximately half of patients with mild to moderate IPF who died of an IPF-related cause appeared to follow such a pattern of accelerated disease. As such, rapid respiratory decompensation in patients with mild to moderate IPF is likely more common than previously suspected.

Pathogenesis
The pathogenesis of IPF is complex. It is, perhaps, best understood as an abnormal host tissue response to injury. The inciting event appears to be injury to the alveolar epithelial cells (AECs), although the precise nature of the injury is not known. Host responses to tissue injury, in general, typically involve both inammation and repair (Figure 4). UIP represents a dysregulated repair response in which there is

persistent activation of broblasts/myobroblasts in broblastic foci, a dening lesion in IPF. Dysregulated communication between AECs and mesenchymal cells are likely central to the ongoing, unresolving repair process that culminates in brosis. AECs appear to be in a state of dysrepair with an inability of these cells to reconstitute the alveolar wall. Exaggerated proliferation of AECs is observed in bronchoalveolar junctions; whereas epithelial cells directly overlying broblastic foci appear to be undergoing cell death. It is not known if a defect exists in the ability of replicating type II cells to differentiate into type I AECs or if there is impairment in the migratory capacity of these cells. Fibroblast/myobroblast-secreted factors, such as angiotensin peptides and oxidants, may contribute to AEC injury by paracrine mechanisms. Fas ligand and mitochondria-mediated apoptosis of AECs in the lungs of IPF patients have also been reported. In contrast to AECs, broblasts/myobroblasts in broblastic foci are highly activated and resistant to

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 445

Lung injury Age Genetic factors Environmental factors Nature of injury - etiologic agent - recurrent vs. single - endothelial vs. epithelial

Histopathologic pattern DIP RB-ILD LIP COP NSIP AIP UIP

Inflammation

Fibrosis

Figure 4 The histopathological spectrum of IIPs represents varied tissue reaction with varying degrees of inammation and brosis. This reaction pattern probably depends on multiple factors, including age, genetic susceptibility, environmental factors, and perhaps the nature of the injurious agent. DIP, desquamative interstitial pneumonia; RB-ILD, respiratory bronchiolitis-associated interstitial lung disease; LIP, lymphocytic interstitial pneumonia; COP, cryptogenic organizing pneumonia; NSIP, non-specic interstitial pneumonia; AIP, acute interstitial pneumonia; UIP, usual interstitial pneumonia. Reproduced from Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary brosis. Annual Review of Medicine 55: 395417.

apoptosis. Myobroblasts are an intriguing group of cells that play a central role in connective tissue remodeling in the lung as well as other tissues. They are cells with contractile and synthetic properties that may account, in large part, for the restrictive physiology and gas-exchange abnormalities in IPF. The probrotic cytokine TGF-b1 plays a central role in the differentiation, activation, and survival of myobroblasts. Other pathogenetic mechanisms that play significant roles in different phases of the disease include: imbalances in the chemokine/cytokine milieu, angiogenesis, impaired brinolysis, reduced matrix degradative capacity, and oxidative stress (Figure 5) (see Pulmonary Fibrosis for details). There has been recent interest in bone-marrowderived cells in both the regeneration of injured alveolar epithelium (stem cells) as well as a source of broblasts and myobroblasts in brotic lesions. Most of these studies have been performed in animal models and whether similar mechanisms are important in human disease awaits further study. The recruitment of repair cells to the lung in response to injury is likely to involve small molecular weight peptides known as chemokines. Several groups have examined the expression of chemokines in SLB and other samples from IIP and non-IIP patient populations. The published data have provided a snapshot
Age Genetic factors Extrinsic factors

Repetitive alveolar epithelial injury

Oxidative stress

Profibrotic cytokines

TIMPsMMPs imbalance Fibrosis

Altered alveolar microenvironment

Chemokines

Impaired fibrinolysis

Eicosanoid imbalance

Dysregulated repair Loss of epithelial cells Accumulation of mesenchymal cells

Figure 5 Progressive brosis results from dynamic alterations in the alveolar microenvironment eventually promoting the loss of alveolar epithelial cells and accumulation of activated broblasts/myobroblasts. Such alterations include: the presence or activation of probrotic cytokines, growth factors, and chemokines; eicosanoid imbalance with increased production of probrotic leukotrienes and deciency in prostaglandin E2; impaired brinolysis; overproduction of tissue inhibitors of matrix metalloproteinases (TIMPs) relative to matrix metalloproteinases (MMPs); and a state of elevated oxidative stress. Reproduced from Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary brosis. Annual Review of Medicine 55: 395417.

446 INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis

of the various chemokines that may contribute to the pathology of IIP, although the understanding of the sequence of events that lead to changes in chemokine synthesis is limited.

Animal Models
An animal model of pulmonary brosis that recapitulates the cardinal pathologic and physiologic features of human IPF has been difcult to establish. The most commonly utilized model of pulmonary brosis relies on a single injurious insult with intratracheal bleomycin administered to rodents (mice or rats). Bleomycin injury in the lungs of these animals is typically followed by an acute inammatory response and brosis that is typically centered around peribronchiolar structures. The brotic response to bleomycin is self-limited and does not lead to the classical restrictive physiology of human IPF. A large number of studies using this model, however, has provided significant insights into basic mechanisms of host inammatory and repair responses. Newer animal models that are better at reproducing the progressive nature of the brotic response have been studied. A number of groups have utilized an active (mutant) form of TGF-b1 expressed either by an inducible promoter in Clara cells of transgenic mice, by adenovirus-mediated gene transfer to rat lung, or by epithelial cell-specic expression of the mutant gene in mice lung explants. Nonrodent mammalian species may offer better models of human IPF; a recent report suggests the development of UIP-like histopathological lesions in spontaneous feline pulmonary brosis.

Management and Current Therapy

The general approach to the patient with a suspected IIP is to identify the most likely diagnosis in an expeditious fashion; this is particularly important with regard to a diagnosis of IPF, given its particularly ominous prognosis. The American Thoracic Society (ATS) and European Respiratory Society (ERS) have recently recommended an integrated clinical, radiologic, and pathologic approach to the diagnosis of diffuse parenchymal lung disease. This integrated approach includes a comprehensive assessment of clinical features, physical examination ndings, selected laboratory studies, imaging studies, and, in selected patients, transbronchial biopsy or SLB. We have conrmed that such an iterative approach maximizes interobserver agreement among expert clinicians, radiologists and pathologists. A careful history is required in the initial evaluation with attention to identifying diagnostic and prognostic clinical

features. These include gender, age, the presence of comorbid diseases, medication exposures, and identication of potential exposures at home or work. For example, it is particularly important to identify the presence of a collagen vascular illness, as collagen vascular diseases associated with histologic UIP exhibit an improved prognosis. A multicenter, case-control study identied several occupational exposures that were associated with an increased likelihood of IPF, including farming, working with livestock, hairdressing, metal dust work, raising birds, stone cutting/polishing, and vegetable dust/animal dust exposure. Significant exposure to asbestos is crucial to identify, as asbestosis may present with a clinical picture remarkably similar to IPF but is associated with a longer, more benign clinical course. Exposure to potential antigens can result in HP; advanced cases may clinically mimic IPF. Cigarette smoking has been implicated in the development of IPF and may be associated with improved survival. Familial types of pulmonary brosis are well described, making a careful family history crucial in the evaluation of patients with suspected IPF. Routine laboratory tests and specic serologic studies may be useful in the exclusion of alternative diagnoses, particularly HP and an associated collagen vascular illness. Imaging studies have assumed a pre-eminent role in the evaluation of suspected IPF. HRCT is particularly likely to be diagnostic in patients with IPF. As such, numerous investigators have conrmed that typical HRCT ndings of IPF are associated with a high positive predictive value for a histological diagnosis of UIP in the appropriate clinical setting. In addition, recent work conrms that patients with these typical HRCT features of IPF (all of whom exhibited typical histological features of UIP) exhibited the worst survival. The importance of SLB in the diagnosis of IPF has been conrmed by numerous investigative groups. In the setting of a nonstereotypical HRCT, identication of histological UIP at SLB is associated with a significantly worse prognosis. Importantly, three groups have documented that histologic ndings of NSIP and UIP are frequently noted in multiple lobes (and even in the same lobe) of patients undergoing SLB; two of these groups have conrmed that the presence of histologic UIP in any sample is associated with a much worse survival. The totality of these data has led to the diagnostic algorithm depicted in Figure 6. Current therapy for IPF is limited. Traditional pharmacotherapy has involved prolonged courses of high-dose corticosteroids with or without the addition of cytotoxic agents such as azathioprine or cyclophosphamide. Although some patients appear to improve or stabilize with this approach, the overall

INTERSTITIAL LUNG DISEASE / Idiopathic Pulmonary Fibrosis 447

Patient with suspected ILD

Hx, PE, CXR, PFT, Labs

Dx likely by bronch? No HRCT

Yes

Is bronch diagnostic? No

Yes

Stop

Hx and HRCT consistent with IPF

Hx and HRCT Dx of other ILD

Suspected other ILD

Atypical clinical or CT features of IPF

Stop

Stop

Dx likely by bronch?

Yes

Is bronch diagnostic?

Yes

No

No

Stop

VATS

UIP

NSIP

RBILD

DIP

DAD

OP

LIP

Non IIP

Figure 6 A potential algorithm for the evaluation of a patient with a diffuse parenchymal lung disease. Hx, history; PE, physical examination; CXR, chest radiograph; PFT, pulmonary function test; bronch, bronchoscopy; LIP, lymphocytic interstitial pneumonitis. Reproduced from Martinez FJ and Flaherty (2004) Diagnosis of interstitial lung disease. Pulmonary and Critical Care Update 18: Lesson 3.

effectiveness of such therapies is disappointing. The assessment of anti-inammatory therapy for IPF is further complicated by the fact that previous trials were underpowered, often unblinded, lacked placebo/ controls, and involved variable dosage regimens and endpoints. Moreover, these studies were performed prior to the recent ATS/ERS reclassication of the IIPs. It is suspected that many of the patients that responded to immunosuppressive therapy in early clinical trials of presumed IPF may have had other forms of IIP such as NSIP. Newer therapeutic approaches that are designed to target brogenic responses per se have received greater attention. Recently, a large phase III clinical trial has been completed using interferon gamma, an immunomodulatory T-helper-1 (Th1) cell cytokine. In this double-blind study of 330 IPF patients randomized to receive subcutaneous interferon gamma (200 mg three times weekly) or placebo, no significant

difference was noted in the primary end point of progression-free survival, dened as the time to disease progression or death. Moreover, no differences were observed in any of the pulmonary physiologic parameters, dyspnea, or quality-of-life measures. Overall, survival in interferon-gamma-treated and placebo groups was not statistically different (10% and 17%, respectively, p 0:08). In post hoc explorative subgroup analyses, patients with less severe disease (baseline FVC above the median, 62% of predicted) and a treatment adherent cohort (patients receiving at least 80% of scheduled doses) appeared to have a survival advantage in the interferon-gamma-treated group. Another prospective, randomized trial (International Study of Survival Outcomes in Idiopathic Pulmonary Fibrosis with Interferon-gamma 1b, INSPIRE trial, sponsored by Intermune Pharmaceuticals) is now underway to test the validity of these ndings in patients

448 INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

with mild-moderate disease (baseline FVCX55% of predicted). Other novel antibrotic agents in various stages of clinical development include a small molecule inhibitor of broblast activation (Pirfenidones), endothelin-1 antagonists (Bosentans), a soluble tumor necrosis factor-receptor fusion protein (Etanercept), leukotriene inhibitors (Zileutons), a copper-chelating anti-angiogenic compound (Tetrathiomolybdate), monoclonal antibodies against TGF-b1 and connective tissue growth factor, a protein kinase inhibitor (Gleevacs), and the glutathione-antioxidant precursor N-acetylcysteine (NAC). It is hoped that one or more of these approaches will be more effective than conventional therapy solely with immunosuppressive agents. It is unlikely that a single approach will be more effective than combination therapy given the heterogeneity of the pathologic lesions found in individual patients with IPF. Thus, accurate clinical, and perhaps biological, phenotyping will be necessary to individualize therapy for maximal benet.
See also: Interstitial Lung Disease: Overview, Leukocytes: Pulmonary Macrophages, Matrix Metalloproteinase Inhibitors, Matrix Metalloproteinases, Pulmonary Fibrosis.

Further Reading
Ambrosini V, Cancellieri A, Chilosi M, et al. (2003) Acute exacerbations of idiopathic pulmonary brosis: report of a series. European Respiratory Journal 22: 821826. American Thoracic Society (2000) Idiopathic pulmonary brosis: diagnosis and treatment. International consensus statement. American Journal of Respiratory and Critical Care Medicine 161: 646664. American Thoracic Society/European Respiratory Society (2002) American Thoracic Society/European Respiratory Society international multidisciplinary consensus classication of the idiopathic interstitial pneumonias. American Journal of Respiratory and Critical Care Medicine 165: 277304. Azuma A, Nukiwa T, Tsuboi E, et al. (2005) Double blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary brosis. American Journal of Respiratory and Critical Care Medicine 171: 10401047. Charbeneau RP and Peters-Golden M (2005) Eicosanoids: mediators and therapeutic targets in brotic lung disease. Clinical Science (London) 108: 479491. Collard HR and King TE Jr (2003) Demystifying idiopathic interstitial pneumonia. Archives of Internal Medicine 163: 1719. Egan JJ, Martinez FJ, Wells AU, and Williams T (2005) Lung function estimates in idiopathic pulmonary brosis: the potential for a simple classication. Thorax 60: 270273. Flaherty KR, King TE Jr, Raghu G, et al. (2004) Idiopathic interstitial pneumonia: what is the effect of a multi-disciplinary approach to diagnosis? American Journal of Respiratory and Critical Care Medicine 170: 904910. Flaherty KR, Thwaite EL, Kazerooni EA, et al. (2003) Radiological versus histological diagnosis in UIP and NSIP: survival implications. Thorax 58: 143148.

Hunninghake GW, Lynch DA, Galvin JR, et al. (2003) Radiologic ndings are strongly associated with a pathologic diagnosis of usual interstitial pneumonia. Chest 124: 12151223. Hunninghake GW, Zimmerman MB, Schwartz DA, et al. (2001) Utility of a lung biopsy for the diagnosis of idiopathic pulmonary brosis. American Journal of Respiratory and Critical Care Medicine 164: 193196. Katzenstein ALA and Myers JL (1998) Idiopathic pulmonary brosis. Clinical relevance of pathologic classication. American Journal of Respiratory and Critical Care Medicine 157: 13011315. Lynch JP III, Wurfel M, Flaherty K, et al. (2001) Usual interstitial pneumonia. Seminars in Respiratory and Critical Care Medicine 22: 357385. Martinez FJ and Flaherty KR (2004) Diagnosis of interstitial lung disease. Pulmonary and Critical Care Update 18: Lesson 3. Martinez FJ, Safrin S, Weycker D, et al. (2005) The IPF Study Group. Clinical course of patients with idiopathic pulmonary brosis. Annals of Internal Medicine 142: 963967. Nicholson AG, Addis BJ, Bharucha H, et al. (2004) Inter-observer variation between pathologists in diffuse parenchymal lung disease. Thorax 59: 500505. Raghu G, Brown KK, Bradford WZ, et al., for the Idiopathic Pulmonary Fibrosis Study Group (2004) A placebo-controlled trial of interferon gamma-1b in patients with idiopathic pulmonary brosis. New England Journal of Medicine 350: 125133. Selman M, King TE Jr, and Pardo A (2001) Idiopathic pulmonary brosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Annals of Internal Medicine 134: 136151. Selman M, Thannickal VJ, Pardo A, et al. (2004) Idiopathic pulmonary brosis: pathogenesis and therapeutic approaches. Drugs 64(4): 405430. Thannickal VJ, Flaherty KR, Martinez FJ, and Lynch JP III (2004) Idiopathic pulmonary brosis: emerging concepts on pharmacotherapy. Expert Opinion in Pharmacotherapy 5(8): 16711686. Thannickal VJ, Toews GB, White ES, Lynch JP III, and Martinez FJ (2004) Mechanisms of pulmonary brosis. Annual Review of Medicine 55: 395417. Thomas AQ, Lane K, Phillips J III, et al. (2002) Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecic interstitial pneumonitis in one kindred. American Journal of Respiratory and Critical Care Medicine 165: 13221328.

Lymphangioleiomyomatosis
J Moss, W K Steagall, and A Taveira-DaSilva, National Institutes of Health, Bethesda, MD, USA
Published by Elsevier Ltd.

Abstract
Lymphangioleiomyomatosis (LAM), a rare multisystem disease affecting women, is characterized by cystic lung disease and abdominal tumors (e.g., angiomyolipomas (AMLs) and lymphangioleiomyomas). Proliferation of abnormal smooth musclelike cells (LAM cells) is observed throughout the lung, along axial lymphatics in the thorax and abdomen, and in AMLs. Patients can present with progressive dyspnea, recurrent pneumothoraces, hemoptysis, chylous pleural effusions, and/ or ascites. Diagnosis may be made by lung biopsy or by clinical and roentgenographic data. LAM occurs sporadically or in

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

association with tuberous sclerosis complex (TSC), an autosomal dominant disorder with variable penetrance caused by mutations in the tumor suppressor genes TSC1 and/or TSC2. LAM cells from sporadic LAM patients contain TSC mutations. TSC1 encodes hamartin, a protein with a role in cell adhesion; TSC2 encodes tuberin, a protein involved in cell-cycle progression and control of cell growth and proliferation, in part, through its regulation of mammalian target of rapamycin (mTOR). The natural course of LAM is highly variable. Therapy includes hormonal manipulations, bronchodilators, supplemental oxygen, and lung transplantation. Clinical trials are under way to study the effects of rapamycin, an inhibitor of mTOR.

449

one-third of female TSC patients have some degree of cystic involvement in their lungs. TSC is caused by mutations in either the TSC1 or TSC2 genes and may be characterized by hamartomatous lesions, seizures, and mental retardation. There are more than 500 known patients with sporadic LAM in North America. Tuberous sclerosis-associated LAM was rst described in 1918 by Lutembacher. Sporadic LAM was rst described in 1937 by von Stossel.

Etiology Introduction
Lymphangioleiomyomatosis (LAM), a rare multisystem disorder affecting primarily middle-aged women, is characterized by cystic lung lesions, involvement of the axial lymphatics, and abdominal tumors (e.g., angiomyolipomas (AMLs)). A hallmark of the disease is the proliferation of abnormal smooth muscle-like cells (LAM cells), which leads to the formation of thin-walled cysts in the lungs and uid-lled cystic structures in the axial lymphatics (i.e., lymphangioleiomyomas). AMLs are composed of LAM cells and adipocytes, intermixed with incompletely developed vascular structures. LAM occurs sporadically or in association with tuberous sclerosis complex (TSC), an autosomal dominant disorder with variable penetrance. Approximately LAM cells from patients with sporadic LAM contain TSC2 mutations. Identical mutations in TSC2 have been detected in lung lesions and AMLs from the same patient with sporadic LAM, and in the donor lung transplanted in a LAM patient. These data suggest that LAM cells can metastasize. Because 50% of sporadic LAM cases do not have demonstrable AMLs, another primary source of LAM cells may exist, perhaps in the lymphatic system. There is no evidence for involvement of infectious, carcinogenic, or inammatory agents in LAM pathogenesis.

Pathology
Lung lesions are characterized by nodules of LAM cells in the interstitium and walls of cysts (Figure 1).

Figure 1 Lung pathology in LAM. Thin-walled cysts (a) and nodules of various sizes (b) are seen in involved lung (H&E, original magnication 50). (c) Bronchiolitis: deposition of mucus in the bronchial lumen, focal goblet cell hyperplasia, and moderate inltration of inammatory cells, including eosinophils, in the wall of a bronchiole. Note the inltrates of smooth muscle cells around the bronchiole (H&E, original magnication 160).

450 INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

Figure 2 Immunohistochemical reactivity of LAM cells. Immunoperoxidase method and counterstaining with hematoxylin. (A and B) aSmooth muscle actin. Most LAM cells show strong reactivity (a). Vascular smooth muscle cells are also strongly positive (arrow). Most of the LAM cells in the thin walls of the cysts are also strongly reactive (b) (original magnication 250 for each). (c) HMB-45. Positive cells are mainly distributed in the periphery of LAM nodules (original magnication 250). (d) HMB-45. Higher magnication view of tissue shown in (c). A strong granular reaction is present in large epithelioid LAM cells adjacent to epithelial cells covering LAM nodules (original magnication 1000).

Bronchiolitis and lung hemorrhage may also be present. The LAM nodules, covered with hyperplastic type II pneumocytes, contain spindle-shaped cells in the center and epithelioid-like cells at the periphery. Both types of cells react with antibodies against smooth muscle cell-specic antigens. The epithelioid cells are more likely to react with HMB-45, which is a monoclonal antibody that recognizes gp100, a premelanosomal protein (Figure 2); the spindle-shaped cells are more likely than epithelioid cells to react with anti-PCNA antibodies, consistent with a more proliferative state. LAM lesions contain damaged elastic bers and collagen brils. Matrix metalloproteinases (MMPs), including MMP-2 (gelatinase A), MMP-1, MMP-9 (gelatinase B), and MMP-14, are associated with the lesions. Decreased expression of tissue inhibitor of metalloproteinase (TIMP)-3, an inhibitor of some MMPs, has been reported, leading to the hypothesis that the destructive lesions are due to an imbalance between proteases and their inhibitors.

decade of life. In a large cohort of LAM patients, the average age of patients at the time of diagnosis was 41 years; it was estimated that these patients may have had the disease for approximately 5 years prior to the diagnosis. Patients with LAM present with progressive dyspnea, recurrent pneumothoraces, chylous pleural effusions, hemoptysis, and ascites. Less frequently, the rst manifestation of LAM is an acute hemorrhage into an AML requiring surgical intervention. In 80% of cases, spontaneous pneumothorax and pleural effusions are the events leading to the diagnosis.
Roentgenographic Findings

Chest X-rays may be normal or show pleural effusions or pneumothorax. Chest computed tomography (CT) shows uniquely circular, thin-walled cysts distributed throughout the lungs (Figure 3). Other abnormalities seen on chest CT include adenopathy, pleural effusions, or pericardial effusions. Abdominal CT scans may show renal masses (i.e., AMLs), lymphangioleiomyomas, and ascites (Figure 3).
Pulmonary Function Abnormalities

Clinical Features
Clinical Manifestations

LAM occurs in both pre- and postmenopausal women; it has been observed in patients in the eighth

Reduction of single-breath diffusing capacity (DLCO) and airow obstruction, causing a decrease in forced expiratory volume in 1 s (FEV1), are the main

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

451

Figure 3 CT scans of four patients with LAM. (a) The CT scan of a patient with severe disease (CT scan grade 3) with a predominance of large cysts. (b) Another case of severe LAM in which there are numerous small cysts distributed throughout the lungs. (c) Multiple angiomyolipomas of the left kidney in a patient who had already undergone a right nephrectomy for treatment of a bleeding angiomyolipoma. (d) A large lymphangioleiomyoma located posteriorly, between the tip of the spleen and the kidney.

abnormalities. Both are seen in more than half of patients. Some patients have decreased DLCO with preservation of FEV1, whereas others have predominantly airow obstruction. Restriction is uncommon except in patients who have undergone extensive pleurodesis. Normal spirometry is seen in early stages of LAM or in patients who have mild lung disease, such as those patients with TSC who are screened for LAM.
Diagnosis

also be isolated from blood and other body uids for genetic analysis.
Natural Course: Measures of Disease Severity

The diagnosis of LAM can be made by lung biopsy or by clinical and roentgenographic data. The latter criteria consist of a history of recurrent pneumothoraces and/or chylothorax, chylous ascites, abdominal lymphangioleiomyomas and/or AMLs, and a chest CT scan showing multiple thin-walled cysts throughout the lungs. In patients with TSC, the presence of lung cysts is usually sufcient to establish a diagnosis. In patients without TSC, the presence of lung cysts in the absence of extrapulmonary disease is not diagnostic of LAM. In those cases, tissue biopsy may be necessary to conrm the diagnosis. LAM cells can

The natural course of LAM is highly variable. Severity of disease may be assessed by lung biopsy (LAM histology score), pulmonary function testing, high-resolution chest CT scans (CT grade), and cardiopulmonary exercise testing. The LAM histologic score (LHS), based on the extent of replacement of lung tissue by cystic lesions and inltration by LAM cells (LHS-1, less than 25%; LHS-2, 2550%; LHS-3, more than 50%), correlates with time to transplantation or death. The severity of disease can also be graded on CT scans by estimating the amount of the lung involved with LAM (grade 0, no involvement; grade 1, less than 30% involved; grade 2, 3060% involved; grade 3, more than 60% involved). DLCO and, to a lesser extent, FEV1 correlate with LHS scores and CT grade of severity. Cardiopulmonary exercise testing with measurement of maximal oxygen uptake (VO2 max) is another method of assessing disease severity in LAM. VO2

452 INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

100 *

Percent predicted

membrane proteins through the amino termini, thus bridging the plasma membrane and cortical actin laments. Hamartin also binds neurolament L in cortical neurons, perhaps inuencing neuronal migration. Hamartin regulates Rho activity; the absence of hamartin leads to loss of focal adhesions, detachment from substrate, and cell rounding.
TSC2

50

**

**

**

The TSC2 gene, containing 41 exons, is located on chromosome 16 (16p13.3) and is postulated to have roles in pathways that control cell growth and proliferation. Cell cycle Tuberin is a negative regulator of cellcycle progression because loss of tuberin function shortens the G1 phase of the cell cycle (Figure 5). Tuberin stabilizes levels of p27KIP1, a cyclindependent kinase inhibitor, and thus inhibits cellcycle progression. With mutation of tuberin, p27 is unstable and mislocated to the cytoplasm, allowing the cell cycle to progress. cyclin-dependent kinase-1 (CDK1) and cyclins A and B1 have been shown to interact with the hamartintuberin complex. Cell growth and proliferation Tuberin is sensitive to at least two types of signals that are important for growth: mitogens and energy status. Mitogenic stimuli activate phosphatidylinositide-3-OH kinase, which activates Akt, leading to phosphorylation and inhibition of tuberin, thus relieving the negative regulation of Ras homolog enriched in brain (Rheb), a GTP-binding protein, and mammalian target of rapamycin (mTOR), resulting in cell growth and proliferation (Figure 5). Tuberin is also a substrate of AMP-activated kinase, which is activated by high levels of AMP, indicative of a low-energy state. This phosphorylation activates tuberin and enforces the inhibition of cell growth. Transcriptional activation Tuberin can interact with members of the retinoid X receptor family, and it has different effects on transcription depending on which family member is bound. Tuberin facilitates transcription of genes controlled by the vitamin D receptor and peroxisome proliferator-activated receptor, but it negatively regulates transcription of genes controlled by the glucocorticoid receptor. Rab5 and endocytosis Tuberin interacts with rabaptin-5, a protein that also associates with Rab5. Tuberin acts as a Rab5-GAP (GTPase-activating protein), inuencing a critical step in docking and

VO2 max

DLCO

FEV1

Figure 4 VO2 max, DLCO, and FEV1, according to CT scan grade of disease severity. Severity grade 1, white bars; severity grade 2, solid bars; severity grade 3, cross-hatched bars. *Significantly different by ANOVA (p o0:001) from severity score 1 patients. **Significantly different (p o0:001) from severity scores 1 and 2.

max correlates with DLCO and FEV1, CT scans, use of oxygen, and LHS scores (Figure 4). DLCO is the single best predictor of VO2 max and exerciseinduced hypoxemia. Exercise-induced hypoxemia, however, may occur in patients with near normal DLCO and FEV1.

Pathogenesis
TSC1 and TSC2 are tumor suppressor genes, and, consistent with Knudsons two-hit hypothesis of tumor development, loss of heterozygosity of TSC2 has been detected in LAM lesions from lung and kidney. In vivo, 130 kDa hamartin, the protein product of the TSC1 gene, and 198 kDa tuberin, that of the TSC2 gene, form a 450 kDa complex that is primarily localized in the cytosol, with a small fraction associated with the cytoskeleton or membranes. Complex formation is important for the stability of both proteins: tuberin is a cytosolic chaperone of hamartin, and hamartin stabilizes tuberin by preventing its proteasomal degradation.
TSC1

The TSC1 gene maps to chromosome 9 (9q34) and contains 23 exons. Hamartin is generated from exons 323. It has limited similarity with other known proteins and is postulated to play a role in cell adhesion by binding activated ezrinradixinmoesin (ERM) proteins. These proteins bind lamentous actin through their carboxy termini and integral

INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

Mitogens AMP Akt PI3K

453

AMPK

TSC1/2

hemangiosarcomas of the spleen, leiomyomas/ leiomyosarcomas of the uterus, pituitary adenoma, and brain tumors. TSC1 or TSC2 knockout mice are embryonically lethal, whereas the heterozygous mice develop kidney cystadenomas and liver hemangiomas. As seen in the human disease, the TSC1 heterozygous phenotype seems somewhat milder than that of the TSC2 heterozygote.

Rheb

p27

Management and Therapy

Hormonal Therapy
Cell cycle

mTOR

4E-BP1

S6K1

eIF-4e

S6

Because estrogens are implicated in the pathogenesis of LAM, antiestrogen therapy, especially progesterone, had been widely used as treatment. However, a retrospective study of a large group of patients found no significant difference in rates of decline in FEV1 and DLCO between patients treated with intramuscular or oral progesterone and untreated patients.
Bronchodilators

Cell growth, proliferation

Figure 5 Model of TSC1/2 pathways. The TSC1/2 complex controls cell-cycle progression and cell growth and proliferation. Tuberin stabilizes levels of p27KIP1, a cyclin-dependent kinase inhibitor, and thus inhibits cell-cycle progression. Tuberin also has Rheb GAP (GTPase-activating protein) activity, which leads to inactivation of Rheb by conversion of Rheb-GTP to RhebGDP. Rheb controls mTOR, which regulates the phosphorylation of at least two substrates: 4E-BP1, relieving the inhibition of eIF4E and allowing cap-dependent translation, and S6K1, resulting in the phosphorylation of S6 and 50 TOP (terminal oligopyrimidine tract) translation. TSC1/2 complex is sensitive to at least two types of signals: mitogens and energy status. Mitogenic stimulation activates phosphatidylinositide-3-OH kinase (PI3K), which activates Akt serine/threonine kinase, leading to phosphorylation and inhibition of tuberin and thus resulting in increased translation and cell growth and proliferation. A state of low cellular energy is signaled by increased AMP, which activates AMP-activated kinase (AMPK). AMPK phosphorylates and, in this case, activates tuberin, slowing translation and cell growth and proliferation.

Approximately 25% of LAM patients respond to bronchodilators with an increase in airow. Treatment with these drugs is recommended for those who show a positive response.
Pneumothorax

Uncomplicated pneumothorax should be treated by closed chest tube drainage. If the lung does not expand or if the pneumothorax recurs, pleurodesis is recommended. Talc pleurodesis is most effective but is also more likely to result in compromise of lung volumes.
Chylothorax

fusion during endocytosis. Loss of tuberin function may increase Rab5-GTP, leading to an increased rate of uid-phase endocytosis, possibly resulting in improper targeting of proteins.

Animal Models
There is no animal model for pulmonary LAM. However, there are models of other effects of TSC1 or TSC2 gene loss. The Eker rat has a germline retrotransposon insertion in TSC2. The homozygous mutant dies at approximately 13 days of fetal life. Renal carcinomas develop in heterozygous rats, with the potential for development of hemangiomas/

Repeated thoracentesis may lead to progressive malnutrition and infectious complications. Talc pleurodesis by video-assisted thorascopic surgery may be effective if the rate of chyle generation is reduced. To minimize the volume of chyle generated, the patient should be placed on a fat-free total parenteral nutrition regime prior to, during, and after surgery. Additional treatments to be tried are medroxyprogesterone injections and octreotide. No treatment has proven consistently effective. Pleuroperitoneal shunts have also been tried.
Ascites and Edema

Ascites, peripheral edema, and compression of the bladder and other viscera by large lymphangioleiomyomata may cause severe symptomatology, including pain, obstipation, and urinary frequency. A low-fat diet, diuretics, and medroxyprogesterone have

454 INTERSTITIAL LUNG DISEASE / Lymphangioleiomyomatosis

not proven effective. A peritoneal venous shunt may be considered for most severe cases when the ascites is disabling and causing mechanical/nutritional problems.
Oxygen Therapy

and Yellow Nail Syndrome. Pneumothorax. Smooth Muscle Cells: Airway. Tuberous Sclerosis.

Further Reading
Bittman I, Rolf B, Amann G, et al. (2003) Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis. Human Pathology 34: 9598. Chu SC, Horiba K, Usuki J, et al. (1999) Comprehensive evaluation of 35 patients with lymphangioleiomyomatosis. Chest 115: 10411052. Costello LC, Hartman TE, and Ryu JH (2000) High frequency of pulmonary lymphangioleiomyomatosis in women with tuberous sclerosis complex. Mayo Clinic Proceedings 75: 591594. Franz DN, Brody A, Meyer C, et al. (2001) Mutational and radiographic analysis of pulmonary disease consistent with lymphangioleiomyomatosis and micronodular pneumocyte hyperplasia in women with tuberous sclerosis. American Journal of Respiratory and Critical Care Medicine 164: 661668. Henske EP (2003) Metastasis of benign tumor cells in tuberous sclerosis complex. Genes, Chromosomes and Cancer 38: 376 381. Karbowniczek M, Astrinidis A, Balsara BR, et al. (2003) Recurrent lymphangioleiomyomatosis after transplantation. American Journal of Respiratory and Critical Care Medicine 167: 976982. Kitaichi M, Nishimura K, Harumi I, et al. (1995) Pulmonary lymphangioleiomyomatosis: a report of 46 patients including a clinicopathologic study of prognostic factors. American Journal of Respiratory and Critical Care Medicine 151: 527533. Krymskaya V (2003) Tumour suppressors hamartin and tuberin: intracellular signalling. Cellular Signalling 15: 729739. Li Y, Corradetti MN, Inoki K, and Guan K (2004) TSC2: lling the GAP in the mTOR signaling pathway. Trends in Biochemical Sciences 29: 3238. Matsui K, Takeda K, Yu Z, et al. (2001) Prognostic significance of pulmonary lymphangioleiomyomatosis histologic score. American Journal of Surgical Pathology 25: 479484. Moss J, Avila NA, Barnes PM, et al. (2001) Prevalence and clinical characteristics of lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex. American Journal of Respiratory and Critical Care Medicine 163: 669671. Pechet TT, Meyers BF, Guthrie TJ, et al. (2004) Lung transplantation for lymphangioleiomyomatosis. Journal of Heart and Lung Transplantation 23: 301308. Sullivan EJ (1998) Lymphangioleiomyomatosis. A review. Chest 114: 16891703. Taveira-DaSilva AM, Hedin CJ, Stylianou MP, et al. (2001) Reversible airow obstruction, proliferation of abnormal smooth muscle cells and impairment of gas exchange as predictors of outcome in lymphangioleiomyomatosis. American Journal of Respiratory and Critical Care Medicine 164: 10721076. Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. (2003) Maximal oxygen uptake and severity of disease in lymphangioleiomyomatosis. American Journal of Respiratory and Critical Care Medicine 168: 14271431. Taveira-DaSilva AM, Stylianou MP, Hedin CJ, et al. (2004) Decline in lung function in patients with lymphangioleiomyomatosis treated with or without progesterone. Chest 126: 18671874. Urban TJ, Lazor R, Lacronique J, et al. (1999) Pulmonary lymphangioleiomyomatosis. A study of 69 patients. Medicine 78: 321 337. Yeung RS (2003) Multiple roles of the tuberous sclerosis complex genes. Genes, Chromosomes and Cancer 38: 368375.

Since exercise-induced hypoxemia occurs in patients with near normal DLCO and FEV1, exercise oximetry should be considered to determine the need for supplemental oxygen.
Transplantation

Patients with severe LAM in whom DLCO and/or FEV1 have decreased to levels under 40% of predicted should be referred to a transplantation center for evaluation. The survival rate of LAM patients undergoing lung transplantation has been reported to be slightly better than that seen in other lung transplant patients (69 versus 53%). However, this report referred to a small number of patients, and the difference was not statistically significant. Operative morbidity, because of prior pleurodesis, tends to be higher. In addition, recurrence of LAM in the allograft has been reported. However, the clinical implications of LAM cells seeding in the allograft lung appear to be minor, and this is not a contraindication to lung transplantation.
Rapamycin

Rapamycin is a microbial macrolide that inhibits mTOR. It is a Food and Drug Administrationapproved drug that is used for immunosuppression after organ transplantation. Clinical trials to study the effect of rapamycin on growth of AMLs are under way. Studies in Eker rats showed a decrease in the size of kidney tumors with rapamycin. However, active disease was still detected after 2 months of therapy. It was not clear if this was residual disease or new tumors.
Osteoporosis

Osteopenia and osteoporosis occur in 70% of patients with LAM. It is recommended that LAM patients undergo periodic evaluation of bone density and those with osteoporosis be treated with calcium, supplementary vitamin D, and bisphosphonates. In view of the rapid deterioration of bone density after lung transplantation, early initiation of therapy in LAM patients with severe lung disease and osteopenia is recommended.
See also: Lung Imaging. Matrix Metalloproteinase Inhibitors. Matrix Metalloproteinases. Oxygen Therapy. Pleural Effusions: Chylothorax, Psuedochylothorax, LAM,

ION TRANSPORT / Overview 455

ION TRANSPORT
Contents

Overview Calcium Channels Chloride Channels ENaC (Epithelial Sodium Channel) Potassium Channels

Overview
D C Eaton and L Jain, Emory University School of Medicine, Atlanta, GA, USA
& 2006 Elsevier Ltd. All rights reserved.

Fibrosis Transmembrane Conductance Regulator (CFTR) Gene), and acute respiratory distress syndrome (see Acute Respiratory Distress Syndrome).

Abstract
Efcient gas exchange in the lungs depends on precise regulation of the amount of uid in the thin liquid layer lining the alveolar epithelium. Studies in vivo and in isolated lungs have shown that uid can be transported from the alveolar into the interstitial spaces, and that this process can be inhibited by the addition of amiloride, an Na channel inhibitor, to the alveolar space. These results suggested that alveolar epithelial cells are the major sites of Na transport and uid absorption in the adult lung. The alveolar epithelium, which covers more than 99% of the large internal surface area of the lung (100150 m2 in humans), is composed of two distinct cell types, alveolar type I (T1) and type II (T2) cells. T2 cells, which cover only 25% of the internal surface area of the lung, are cuboidal cells that secrete pulmonary surfactant. T2 cells contain ion channels, including several forms of the amiloride-sensitive epithelial sodium channel (ENaC), and a chloride channel, the cystic brosis transmembrane regulator (CFTR). T1 cells are large squamous cells whose thin cytoplasmic extensions cover 495% of the internal surface area of the lung. T1 cells contain functional ENaC, pimozide-sensitive, cyclic nucleotide-gated, nonselective cation channels, and K channels, and appear to contain some CFTR. Besides these ion channels, the apical membrane of most airway epithelia contains several different other Cl channels. The Ca2 -activated Cl-channel (CLCA) is another anion channel at the apical membrane of airway epithelia that responds acutely to increases in intracellular calcium and is involved in transepithelial uid transport. In some airway cells, there are also volume-regulated Cl-channels (ClC family of channels) that respond to changes in cell volume to alter chloride transport.

The Role of Airway Epithelium in Fluid Clearance

Epithelial cells line the compartments of the body and separate the interior compartments (cells, blood, and interstitial uid) from compartments that are contiguous with the outside world (GI tract, urinary space, and, particularly important for this text, airways). Epithelial tissues have specifically evolved to move substances from one of these compartments to the other using ATP energy in the process. It is the active movement or transport of ions across the airway epithelia that drives airway uid clearance. Fluid appears in the airways because of the passive movement of uid driven by hydrostatic pressure out of the pulmonary capillaries across the airway epithelium. The amount secreted is relatively constant (because the pulmonary capillary pressure and permeability of the epithelium are relatively constant) except under pathological conditions when either the pulmonary blood pressure may be abnormally elevated (see Pulmonary Circulation) or the permeability of the epithelium may be increased by inammation or disease (see Acute Respiratory Distress Syndrome). The amount of uid on the airway surface, therefore, represents a balance between the rate at which uid is passively secreted and the rate at which it is actively reabsorbed. Since uid absorption depends upon the rate of ion transport, regulation of the ion transport provides the mechanism by which the amount of airway uid is regulated.

Introduction
Normal lung function is critically dependent upon maintaining a thin layer of uid on the cell surfaces of the airway, but this uid layer is especially critical in the alveoli where abnormalities in lung uid balance can lead to substantial pathology including pulmonary edema (see Fluid Balance in the Lung), a propensity to develop airway infections (see Cystic Fibrosis: Cystic

A General Mechanism for Airway Epithelial Ion Transport: The Transcellular Route
Epithelial transport requires that epithelial cells are polarized, that is, the proteins present in the apical

456 ION TRANSPORT / Overview

Airway lumen Sodium permeability pathways

Interstitium Pulmonary capillary Step 4 K+ Anions Water Aquaporins Anions Water Step 3 Basolateral membrane Tight junctions Lateral space Step 1

Na+ K+ Step 2 Anions Water

Na+

ATP

Na+

Apical membrane

Figure 1 A schematic of generalized airway epithelium salt and water transport. Transport can be viewed as a four step process. Step 1 is the active extrusion of sodium via (Na,K)-ATPase from the cell to the interstitium. This creates a low concentration of sodium within the cell so that sodium moves downhill from the lumen to cell interior via a variety of sodium-permeable ion channels. The consequence of this transcellular sodium movement is separation of charge (excess Na on the interstitial side) that promotes step 2, the movement of anions to balance the positive charge. The accumulation of sodium and anions in the interstitial space produces an osmotic gradient from lumen to interstitium that promotes water movement (step 3). Finally, the accumulation of salt and water in the interstitium promotes the bulk ow of solute and water into the pulmonary capillaries (step 4). The low intracellular sodium generated by the (Na,K)-ATPase also plays a role in establishing the energy gradient necessary for the transport of other ions.

(airway facing) and the basolateral (blood facing) membrane are not the same. This polarization can promote net ux of sodium from lumen to inte rstitium. Figure 1 shows the morphology of a generalized airway epithelium in which salt and water transport can be viewed as a four-step process. Step 1 is the active extrusion of sodium via (Na,K)-ATPase from the cell to the interstitium. This creates a low concentration of sodium within the cell so that sodium moves downhill from the lumen to cell interior via a variety of sodium-permeable ion channels (see Ion Transport: ENaC (Epithelial Sodium Channel)). The consequence of this transcellular sodium movement is separation of charge (excess Na on the interstitial side) that promotes step 2, the movement of anions to balance the positive charge (see Ion Transport: Chloride Channels). The accumulation of sodium and anions in the interstitial space produces an osmotic gradient from lumen to interstitium that promotes water movement (step 3). Finally, the accumulation of salt and water in the interstitium promotes the bulk ow of solute and water into the pulmonary capillaries (step 4). The low intracellular sodium generated by the (Na,K)-ATPase also plays a role in establishing the energy gradient necessary for the transport of other ions (see Ion Transport: Calcium Channels; Potassium Channels). The general point is that the transcellular

route involves crossing two membranes apical and basolateral and the transporters in the two membranes are different. Despite the initial requirement for the energy-dependent removal from airway epithelial cells of sodium by the (Na,K)-ATPase at the basolateral membrane, the ion channels lining the airway lumen constitute the rate-limiting step for both secretion and absorption (see Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels).

The Paracellular Route

In the absence of any other process, as water follows sodium and its anions across the epithelium, the uid volume remaining in the airway lumen would be reduced. Therefore, any solute that has not been specifically transported via the transcellular route would become more concentrated. As the luminal concentration rises, this would generate a concentration gradient across the tight junctions between lumen and interstitium. In addition, if the tight junctions are permeable to the substance in question, then the substance will diffuse from lumen to interstitium. This does happen for some ions such as sodium, potassium, chloride, calcium, and magnesium. The amounts reabsorbed depend on the permeability of

ION TRANSPORT / Overview 457

the tight junctions. Since ions are driven not only by concentration gradients, but also by voltage gradients, the transepithelial voltage also plays a role. The lumen of airway epithelium is negative relative to the interstitium and enhances paracellular anion absorption and reduces cation absorption. Because of the paracellular transport pathway, ion transport across airway epithelium can only establish a small concentration gradient: any significant lowering of luminal concentration relative to the interstitium results in a leak back into the lumen as fast as the substance is transported out. The epithelium has a nite passive permeability to the substance, usually through the tight junctions, such that a large concentration gradient between the interstitium and lumen results in a large passive ux. Consider the case of sodium. In the alveolar epithelium, the tight junctions are quite permeable to sodium. As sodium is transported into the interstitium, the interstitial concentration begins to rise. This rise creates a gradient that drives a ux of sodium out of the interstitium, both into the pulmonary capillaries, and back through the tight junctions into the lumen. Most of the sodium moves into the blood, accomplishing the goal of absorption, but some does leak back into the lumen. When the concentration of sodium reaches a high enough value, the concentration gradient between the interstitium and the lumen drives sodium back across the tight junctions as fast as it can be transported through the transcellular pathway from lumen to interstitium. At this point transport through paracellular and transcellular pathways is large, but net transport is zero: the system has established the largest gradient possible, its gradient limit. The leakier the epithelium, the lower is the gradient limit which is presumably why, when epithelial integrity is compromised, the epithelium has difculty in absorbing sufcient salt and uid to compensate for the large passive ux through the paracellular pathway. Of course, the same is true for other ions, in particular chloride. The transport of sodium generates a potential driving force to move chloride. However, there can be no substantial concentration gradient since the chloride will begin to leak back across the tight junctions as fast as it moves into the interstitium. As pointed out above, the gradient limit on the movement of ions establishes a balance between the volume of alveolar uid and salt and the interstitial volume of uid and salt. If salt and water absorption increases, the accumulation of isotonic uid in the interstitium will force salt and water across the tight junctions to almost exactly balance the increase in absorption. Thus, large changes in transport often produce only small changes in alveolar uid amounts. By the same token, increases in permeability of the tight junctions

with concomitant increase in secretion of salt and water can be compensated by increasing the rate of absorption (until the upper limit of the transport capacity of the apical ion channels and the basolateral (Na,K)-ATPase is reached after which alveolar ooding will occur) (see Acute Respiratory Distress Syndrome).

Ion Channels in Airway Epithelium

From the discussion above, it is clear that absorption and secretion of ions across the airway epithelium is responsible for maintaining the correct composition and volume of the airway surface liquid. Each of the major ion channels present in airway epithelial cells are dealt with in more depth in Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels.

Cation Channels
The amiloride-sensitive, epithelial Na channel (ENaC) is expressed at the apical membrane of all airway epithelial cells and is rate-limiting step in controlling the rate of transepithelial Na and uid absorption (see Ion Transport: ENaC (Epithelial Sodium Channel)). Besides ENaC, cyclic-nucleotidegated cation channels (CNGC) are widely expressed in airway (alveoli, trachea, bronchi, and bronchioles). This channel is stimulated by increases in intracellular cGMP. The channel is equally permeable to Na and K but apparently does still contribute to transepithelial sodium transport in airway epithelial cells. Blockers of cyclic- nucleotide-gated cation channels (l-cis-diltiazem and dichlorobenzamil) inhibit an amiloride-insensitive component of sodium and uid absorption in airways. Also, lung epithelial cells have several other types of cation channels whose molecular identity has been less well dened. The most common types are several species of nonselective cation channels that are stimulated by relatively high concentrations of intracellular calcium and are particularly prevalent in fetal lung epithelial cells. Potassium channels in airway epithelial cells mostly reside in the basolateral membrane where they form a pathway to allow exit of the potassium transported into the cell by the (Na,K)-ATPase. Many different types of potassium channels have been described (for a complete description, see Ion Transport: Potassium Channels). More surprising than the basolateral K channels are potassium channels on the apical surface of airway epithelial cells. There appear to be several members of the voltage-gated potassium channel (Kv) family present in T1 cells. Such channels offer the interesting prospect of Na

458 ION TRANSPORT / Overview

for K exchange rather than sodium chloride uptake. This would make these cells analogous to principal cells of the cortical collecting duct in the mammalian kidney and would offer yet another way to adjust the volume and ionic composition of the alveolar uid.

Anion Channels
There are also several different Cl channels in the apical membrane of most airway epithelia. The cystic brosis transport regulator (CFTR) is a member of the ATP-binding cassette (ABC) family of membrane transporters. CFTR is a Cl channel that is stimulated by increases in intracellular cAMP and is the channel that is responsible for basal Cl transport (either absorption or secretion depending upon the location and circumstances) in airway epithelia text (see Cystic Fibrosis: Overview). The Ca2 -activated Cl-channel (CLCA) is another anion channel at the apical membrane of airway epithelia that responds acutely to increases in intracellular calcium and is involved in transepithelial uid transport (see Ion Transport: Chloride Channels). There are at least four members of this family with widespread tissue distribution, but only CLCA2 is present in tracheal epithelial cells. In some airway cells, there are also volume-regulated Cl-channels (ClC family of channels) that respond to changes in cell volume to alter chloride transport (see Ion Transport: Chloride
Alveolus

Channels). ClC-2 is ubiquitously distributed including lung epithelial cells; ClC-3 is a volume-activated chloride channel that appears to be present in all cells including lung epithelial cells. ClC-6 and ClC-7 are also present in the airways but are not functionally expressed as membrane Cl channels, but rather as intracellular organelle channels contributing to acidication of endosomal compartments. Recently, a so-called maxi-Cl channel has been cloned and is present ubiquitously in airway epithelial cells. The physiological role of this channel is unclear since the conductance is so high that the activity of even one channel would ood epithelial cells with chloride. Some have suggested that its normal function is in the mitochondrial membrane, but that during apoptosis, the channel is translocated to the plasma membrane where it contributes to programmed cell death. Besides ion channels, airway cells also have water channels (members of the aquaporin family of proteins), with different members of the family expressed in different regions of the mammalian lung. These water channels mediate the osmotically induced uid movement in response to ion transport (see Aquaporins).

Summary
Ion channels are critical for normal uid balance in the lung. The large diversity of ion channels present
+ Cl ENaC Na Na+ ,K+ HSC

Cl CFTR

Paracellular Na+,K+,Cl CNGC Na+,K+ CNGC H2O Na+,K+ AQP5

C K els nn ha

Paracellular Na+,K+,Cl ENaC ENaC HSC NSC Na+ Na+,K+ H2O AQP5 Na+

CLC ENaC NSC T2 cell

Paracellular Na+,K+,Cl ENaC HSC Na+ K+

T1 cell

K+ K+ K Channels Interstitium

Cl

Na+

K+ Na,K-ATPase

K K+ Channels CLC (Na,K)-ATPase

Pulmonary capillary
Figure 2 Schematic depiction of alveolar epithelial ion transport. Na is absorbed from the apical surface of both T1 and T2 cells via ENaC (HSC and NSC channels). Electroneutrality is conserved with Cl movement through CFTR or CLC channels in T2 cells, and/or paracellularly through tight junctions. Na is transported from the basal surface of both cell types into the interstitial space by (Na ,K )ATPase. K may be transported from alveolar epithelial cells via K channels located on the apical surface of T1 cells or through potential basolateral K channels in T2I cells. Cyclic-nucleotide-gated channels (CNGC) are alternative pathway for the movement of Na that would be amiloride insensitive. CNG channels could also be a pathway for calcium entry into cells. If net ion transport is from the apical surface to the interstitium, an osmotic gradient would be created, which would in turn direct water transport in the same direction, either through aquaporins or by diffusion.

ION TRANSPORT / Calcium Channels 459

in airway epithelium means that there are multiple, redundant pathways for the control of uid balance. Abnormalities in ion channel function can strongly exacerbate the tendency for edema formation in sepsis or acute lung injury. A model for ion transport in airway is shown in Figure 2.

Schwiebert EM, Egan ME, Hwang TH, et al. (1995) CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 10631073. Strange K, Emma F, and Jackson PS (1996) Cellular and molecular physiology of volume-sensitive anion channels. American Journal of Physiology 270: C711C730.

Acknowledgments
This study was supported by R01 HL063306 to LJ; and R01 HL071621 and P01 DK061521 to DCE.
See also: Acute Respiratory Distress Syndrome. Apoptosis. Aquaporins. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Ion Transport: Calcium Channels; Chloride Channels; ENaC (Epithelial Sodium Channel); Potassium Channels. Pulmonary Circulation.

Calcium Channels
Y Marunaka and N Niisato, Kyoto Prefectural University of Medicine, Kyoto, Japan
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The Ca2 channels are classied into: (1) voltage-gated Ca2 channel, (2) inositol 1,4,5-triphosphate (IP3)-activated Ca2 channel, (3) ryanodine-receptor (RyRs) Ca2 -release channel, (4) store-operated Ca2 (Ca2 -release-activated Ca2 (CRAC)) channel, and (5) transient receptor potential (TRP)-related Ca2 channel. The cytosolic Ca2 has function as a regulator of various enzymes and plays an important role as an intracellular second messenger of various hormones in tissues such as the lung. Ca2 enters into the cytosolic space via several types of Ca2 channels such as voltage-gated Ca2 channels across the plasma membrane and is also released into the cytosolic space from intracellular organelles such as endoplasmic reticulum via Ca2 -release channels associated with IP3 and RyRs receptors. Ca2 also enters into the cytosolic space through CRAC channels triggered by emptying of intracellular Ca2 stores so-called capacitative Ca2 entry. The molecular basis of the CRAC channel would be the TRP-related Ca2 channel. These channels cooperatively regulate the cytosolic Ca2 concentration ([Ca2 ]c) in the lung. Regulation of the [Ca2 ]c maintains cell function in the lung such as contraction of smooth muscles, cytokine secretion, ion channel activity etc. On the other hand, disorders of these channels might cause pathogenesis of lung function such as lung edema.

Further Reading
Chinet TC, Gabriel SE, Penland CM, et al. (1997) CFTR-like chloride channels in non-ciliated bronchiolar epithelial (Clara) cells. Biochemical and Biophysical Research Communications 230: 470475. Frank J, Roux J, Kawakatsu H, et al. (2003) TGF-beta 1 decreases expression of the epithelial sodium channel alpha ENaC and alveolar epithelial vectorial sodium and uid transport via an ERK 1/2-dependent mechanism. Journal of Biological Chemistry 278(45): 4393943950. Giebisch G, Hebert SC, and Wang WH (2003) New aspects of renal potassium transport. Pugers Archives 446: 289297. Jentsch TJ, Friedrich T, Schriever A, and Yamada H (1999) The CLC chloride channel family. Pugers Archives 437: 783795. Knowles MR, Olivier K, Noone P, and Boucher RC (1995) Pharmacologic modulation of salt and water in the airway epithelium in cystic brosis. American Journal of Respiratory and Critical Care Medicine 151: 6569. Leroy C, Dagenais A, Berthiaume Y, and Brochiero E (2004) Molecular identity and function in transepithelial transport of K(ATP) channels in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 286: L1027L1037. Matalon S, Lazrak A, Jain L, and Eaton DC (2002) Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells. Journal of Applied Physiology 93: 18521859. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial uid transport and the resolution of pulmonary edema. Physiological Reviews 82: 569600. Matulef K and Zagotta WN (2003) Cyclic nucleotide-gated ion channels. Annual Review of Cell and Developmental Biology 19: 2344. OGrady SM and Lee SY (2003) Chloride and potassium channel function in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: 689700. Schwiebert EM, Cid-Soto LP, Stafford D, et al. (1998) Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic brosis airway cells. Proceedings of the National Academy of Sciences, USA 95: 38793884.

Introduction
Cytosolic Ca2 plays essential roles in respiratory tissues including airway epithelium, alveolar epithelium, endothelium of blood vessels, and smooth muscles. In the airway epithelium, the cytosolic Ca2 stimulates uid secretion via activation of Cl efux to prevent the airway epithelium from drying out. In the alveolar epithelium, the cytosolic Ca2 plays an important role in the regulation of the thin uid layer covering the epithelium via regulation of Na absorption. An increase in the cytosolic Ca2 concentration elevates the endothelial permeability to protein and other solutes, leading to lung edema. Further, smooth muscles contract as a result of an increase in the cytosolic Ca2 concentration. This leads to a decrease in the diameter of airways and blood vessels, resulting in an increase of resistance.

460 ION TRANSPORT / Calcium Channels

Thus, an increase in the cytosolic Ca2 concentration has various important roles in the function of respiratory tissues. This increase in the cytosolic Ca2 concentration is mediated by Ca2 inux from the extracellular space via Ca2 channels and also is caused by Ca2 release from intracellular organelles such as endoplasmic reticulum via Ca2 -release channels associated with the inositol 1,4,5-triphosphate (IP3) receptors and ryanodine receptors (RyRs). Table 1 summarizes the classication of Ca2 channels: (1) voltage-gated Ca2 channel; (2) inositol 1,4,5-triphosphate (IP3)-activated Ca2 channel; (3) ryanodine-receptor (RyRs) Ca2 -release channel; (4) store-operated Ca2 (Ca2 -releaseactivated Ca2 (CRAC)) channel; and (5) transient receptor potential (TRP)-related Ca2 channel.
Table 1 Classication of Ca2 channels Classication of channels Voltage-gated Ca
2

Voltage-Gated Ca2+ Channels

Ca2 channels at the plasma membrane are characterized, in general, as voltage-gated Ca2 channels (Table 2 and Figure 1).This means that the voltagegated Ca2 channel is activated by membrane depolarization, that is, open probability of the Ca2 channel is increased by depolarization of the plasma membrane. The voltage-gated Ca2 channels are classied as L, T, N, P/Q, and R types based on the current proles through the channels. These types of channels are, in general, located in skeletal muscles, heart muscles, and neurons. The voltage-gated Ca2 channel belongs to the superfamily of voltagegated cation channels including Na , K , and Ca2 channels. The voltage-gated cation channels have the

Function Ca2 inux into the cytoplasmic space from the extracellular space linking membrane depolarization Ca2 release to the cytoplasmic space from the ER at IP3 binding to this ligandgated Ca2 channel Ca2 release to the cytoplasmic space from the SR blocked by ryanodine binding to its receptor in the channel facing the L-type voltage-gated Ca2 channel located on the T tubules in skeletal muscles and activated by activation of L-type voltage-gated Ca2 channel Ca2 entry through store-operated Ca2 channels triggered by emptying of intracellular Ca2 stores; capacitative Ca2 entry TRP-related Ca2 channel reported as a candidate for the store-operated Ca2 channel (or Ca2 -release-activated Ca2 (CRAC) channel)

channels

IP3-activated Ca2 channel Ryanodine-receptor Ca2 -release channel

Store-operated Ca2 channel Transient receptor potential (TRP)-related Ca2 channel

ER, endoplasmic reticulum; SR, sarcoplasmic reticulum. Table 2 Properties and classication of voltage-gated Ca2 channels Channel type of property L Kinetics Long duration Voltage activation High threshold (4 30 mV) Pharmacology Blocked by DHPs Location Heart, skeletal, muscle, vascular smooth muscle, neuroendocrine Sinoatrial node of heart, brain neurons Presynaptic terminals, dendrites, cell bodies of neurons Cerebellar Purkinje cells, granule cells, and cell bodies of central neurons Cerebellar granule cells, neurons Function Link membrane depolarization to cytosolic Ca2 signaling Repetitive ring of action potentials Exocytotic neurotransmitter release Genes aS, aC, aD, aF

Transient

Low threshold (o 30 mV) High threshold (4 30 mV)

Blocked by DHPs to a less extent Insensitive to DHPs, blocked by o-conotoxin GVIA Insensitive to DHPs, blocked by o-agatoxin IVA Insensitive to DHPs, blocked by o-agatoxin IIIA

aG, aH, aI

Intermediate long duration

aB

P/Q

Intermediate long duration

High threshold (4 30 mV)

Exocytotic neurotransmitter release

aA

Intermediate

High threshold (4 30 mV)

Exocytotic neurotransmitter release

aE

DHP, 1,4-dihydropyridine.

ION TRANSPORT / Calcium Channels 461

2 1 s s ss

N Domain I Domain II Domain III 1 2 3 4 + 5p 6 Domain IV 1 2 3 4 + 5 p6

4 p 1 2 3 4 + 5 6 1 2 3 + 5 p6 N C N

C C

Figure 1 A membrane-folding model of voltage-gated Ca2 channels. A voltage-gated cation channel has the sequence of the S4 segments generating pore for ion permeation across the membrane, which is called an a1 subunit. A voltage-gated Ca2 channel is composed of a pseudo-oligomeric a1 subunit, as well as an extracellular a2 subunit, a cytoplasmic b subunit, and membrane-spanning g and d subunits. The a1 subunit consists of four internally homologous repeats (domains I, II, III, and IV) and this subunit forms an ionpermeable pore. Each domain contains an S1 through S6 transmembrane motif. Thus, the a1 subunit has 24 transmembrane domains in total. Data from Isom LL, De jongh KS, and Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12: 11831194.

sequence of the S4 segments generating pore for ion permeation across the membrane, the a1 subunit. The voltage-gated Ca2 channel is composed of a pseudo-oligomeric a1 subunit, as well as an extracellular a2 subunit, a cytoplasmic b subunit, and membrane-spanning g and d subunits. The a1 subunit consists of four internally homologous repeats: domains I, II, III, and IV. Each domain contains an S1 through S6 transmembrane motif. Thus, the a1 subunit has 24 transmembrane domains in total. Mammalian a1 subunits of the L-, T-, N-, P/Q-, and R- type Ca2 channels are encoded by distinct genes: a1S, a1C, a1D, and a1F genes for the L-type Ca2 channel; a1G, a1H, and a1I genes for the T-type Ca2 channel; a1B gene for the N-type Ca2 channel; a1A gene for the P/Q-type Ca2 channel; and a1E gene for the R-type Ca2 channel. In respiratory tissues, smooth muscles have an L-type Ca2 channel.
L-Type Ca2+ Channel

muscle, smooth muscle, neurons, uterus, and neuroendocrine cells. The L-type Ca2 channel is activated by protein kinase A via an increase of open probability, and plays a role in excitation-contraction coupling. The L-type Ca2 channels are classied as CaV1.1, CaV1.2, CaV1.3, and CaV1.4.
T-Type Ca2+ Channel

The L-type Ca2 channel is activated when the plasma membrane depolarizes (4 30 mV, high threshold). Furthermore, once the L-type Ca2 channel is activated, it maintains its activity over a long period of time. The L-type Ca2 channel is blocked by 1,4-dihydropyridine (1,4-DHP) such as nitrendipine via binding of 1,4-DHP to the channel, while Bay K 8644, an analog of 1,4-DHP, activates the channel by locking it into an open state. The L-type Ca2 channel is located in heart muscle, skeletal

The T-type Ca2 channel is activated when the plasma membrane depolarizes (o 30 mV; low threshold), and is inactivated over a more negative voltage range. When the T-type Ca2 channel is activated, it maintains its activity during a short time period (transient duration). These characteristics of the T-type Ca2 channel indicate that it functions at the initial time of action potential and produces repetitive ring. The T-type Ca2 channel is located in the sinoatrial (SA) node of heart and brain neurons. The T-type Ca2 channel is blocked by 1,4-dihydropyridines to a lesser extent than the L-type Ca2 channel. T-type Ca2 channels are classied as CaV3.1, CaV3.2, and CaV3.3 based on the amino acid sequence.
N-Type Ca2+ Channel

Similar to L-type Ca2 channels, N-type Ca2 channels are activated when the plasma membrane depolarizes (4 30 mV; high threshold). When the N-type Ca2 channel is activated, it maintains its activity over a relatively long time period (intermediate

462 ION TRANSPORT / Calcium Channels

to long duration). The N-type Ca2 channel is insensitive to 1,4-dihydropyridines, and is blocked by o-conotoxin GVIA. This type of Ca2 channel is located in presynaptic terminals, dendrites, and neuronal bodies, and is involved in exocytotic neurotransmitter release. The N-type Ca2 channel is classied as CaV2.2.
P/Q-Type Ca2+ Channel

through the IP3-activated Ca2 channel is terminated by dephosphorylation of IP3.

Ryanodine-Receptor Ca2+-Release Channel

The Ca2 -release channel is located in the sarcoplasmic reticulum of muscles. This type of Ca2 channel is called a ryanodine-receptor Ca2 -release channel, since the channel is blocked by ryanodine. The ryanodine-receptor Ca2 -release channel is a tetramer and each subunit has four transmembrane domains. This channel faces the L-type Ca2 channel located on the T tubules in skeletal muscles.

The P/Q-type Ca2 channel is activated when the plasma membrane depolarizes (4 30 mV; high threshold). When the P/Q-type Ca2 channel is activated, the channel maintains its activity during a relatively long time period (intermediate to long duration). The P/Q-type Ca2 channel is insensitive to 1,4-dihydropyridine, and is blocked by o-agatoxin IVA. This type of Ca2 channel is located in cerebellar Purkinje cells, granule cells, and cell bodies of central neurons. This channel plays a role in exocytotic neurotransmitter release. The P/Q-type Ca2 channel is classied as CaV2.1 based on the amino acid sequence.
R-Type Ca2+ Channel

Store-Operated Ca2+ Channels

Store-operated Ca2 channels play a role in regulating intracellular Ca2 . Emptying of intracellular Ca2 stores triggers the opening of store-operated Ca2 channels, and the Ca2 entry through these channels is known as capacitative Ca2 entry; hence, these channels are also called capacitative Ca2 entry (CCE) channels. Furthermore, the terms of Ca2 -release-activated Ca2 current (ICRAC)/ Ca2 -release-activated Ca2 (CRAC) channel are used to distinguish this Ca2 entry through storeoperated Ca2 channels from other Ca2 entry channels. Two models for activation of store-operated Ca2 channels (or CCE channels) are proposed. One is a direct conformational coupling between store-operated Ca2 channels and IP3-activated Ca2 channels. The other involves Ca2 -inux factor (CIF), that is, a diffusible messenger based on the fact that CIF is released when the intracellular Ca2 store is empty.

The R-type Ca2 channel is activated when the plasma membrane depolarizes (4 30 mV, high threshold). Once this channel is activated, it maintains its activity for a relatively long time period (intermediate duration), but less time than the T-, N-, and P/Q-type Ca2 channels. The N-type Ca2 channel is insensitive to 1,4-dihydropyridines, and is blocked by o-agatoxin IIIA. This type of Ca2 channel is located in cerebellar granule cells and neurons, and plays a role in exocytotic neurotransmitter release like the N-type Ca2 channel. The R-type Ca2 channel is classied as CaV2.3 based on the amino acid sequence.

IP3-Activated Ca2+ Channels

Phospholipase Cb stimulated by G-protein generates IP3 and diacylglycerol from phosphatidylinositol 4,5-biphosphate (PIP2). IP3 interacts with a receptor located in the membrane of endoplasmic reticulum (ER). The receptor is a ligand-gated Ca2 channel. When IP3 binds to the ligand-gated Ca2 channel, Ca2 is released to the cytoplasmic space from the ER. Therefore, this type of Ca2 channel is called an IP3-activated Ca2 channel. The IP3 receptor is a tetramer (approximately 260 kDa), and is phosphorylated by PKA, PKC, and Ca2 /calmodulin (CaM)-dependent protein kinase. Each subunit has six transmembrane domains. The Ca2 release

Transient Receptor Potential-Related Ca2+ Channel

This is a large class of channel subunits based on a modestly similar primary structure and permeability to monovalent cations and Ca2 . The TRP-related Ca2 channel is a candidate for the store-operated Ca2 channel (i.e., CRAC channel). This family is subclassied into three major subfamilies (TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid)), and four more distantly related subfamilies (TRPP, TRPML, TRPN, and TRPA). All TRP channels have six transmembrane regions with cytoplasmic N- and C-termini. In this section, only three major TRPC, TRPM, and TRPV are described. TRPC has seven members (TRPC1 through TRPC7), TRPM

ION TRANSPORT / Calcium Channels 463

has eight members (TRPM1 though TRPM8), and TRPV has six members (TRPV1 through TRPV6):
*

TRPC1: TRPC1 shows a nonselective cation current including Ca2 . The TRPC1 is activated by IP3 or thapsigargin to deplete intracellular Ca2 stores, and is blocked by Gd3 . The TRPC1 is widely expressed throughout the body. The TRPC1 is the intracellular Ca2 release channel (store-operated channel). TRPC2: TRPC2 is activated by diacylglycerol, but it is not conrmed whether the TRPC2 is activated by depletion of the intracellular Ca2 store. TRPC3, 6, and 7: TRPC3, TRPC6, and TRPC7 form nonselective cation channels. TRPC3, 6, and 7 are activated by diacylglycerol with no requirement for either IP3 or the IP3 receptor. TRPC3 is blocked by SKF96365, verapamil, La3 , Gd3 , and Ni2 . The differences among TRPC3, TRPC6, and TRPC7 are their ion selectivities. TRPC6 is Ca2 selective. TRPC6 is highly
Voltage-gated Ca2+ channel Ca2+

expressed in the lung tissues including pulmonary arteries. TRPC3 and TRPC7 have relatively low selectivity for Ca2 compared with TRPC6. TRPC4 and 5: TRPC4 and TRPC5 show Ca2 selective currents that are activated by either IP3 or thapsigargin-induced depletion of intracellular Ca2 stores. TRPC4 is activated by G-protein-coupled receptors. TRPC5 is reported as a receptor-operated cation channel. TRPM1: TRPM1 might elevate the intracellular Ca2 concentration by an unknown mechanism. TRPM2: TRPM2 is expressed in the brain, bone marrow, spleen, heart, leukocytes, liver, and lung. TRPM2 is activated by ADP-ribose, NAD, and H2O2. TRPM2 permeates not only monovalent cations such as Na , K , and Cs , but also Ca2 . TRPM3: TRPM3 elevates the basal level of intracellular Ca2 concentration. TRPM3 permeates not only monovalent cations such as Na and Cs , but also Ca2 and Mg2 . This channel is activated by extracellular hypotonicity.

In skeletal muscles L-type voltage-gated Ca2+ channel

Ca2+ Contraction of muscles

SR Ca2+

Ca2+-activated K+ channel K+ (+)

RyRs Ca2+-release channel (+) [Ca2+]c

Cl Ca2+-activated Cl channel

(+)

IP3-activated Ca2+ channel

Ca2+ Empty of Ca2+ Ca2+ ADP ATP Ca2+ (+)

Ca2+ -ATPase Na+

Na+/Ca2+ exchanger Ca2+ Store-operated Ca2+ channel (TRP-related Ca2+ channel)

Figure 2 Function of Ca2 channels in cells: regulation of cytosolic Ca2 concentration ([Ca2 ]c) by Ca2 channels and Ca2 transporters in smooth and skeletal muscles (see area indicated by skeletal muscle). IP3-activated Ca2 channel, inositol 1,4,5triphosphate (IP3)-activated Ca2 channel; RyRs Ca2 -release channel, ryanodine-receptor (RyRs) Ca2 -release channel; TRP-related Ca2 channel, transient receptor potential (TRP)-related Ca2 channel. ( ), stimulation or activation.

464 ION TRANSPORT / Calcium Channels

*

TRPM4: TRPM4 was initially reported to be a plasma protein of 1040 amino acid residues with Ca2 permeability: TRPM4a. Subsequently, a full-length TRPM4 (TRPM4b) was revealed that is activated by Ca2 but is Ca2 -impermeable. TRPM5: TRPM5 is reported to be a Ca2 -permeable, store depletion-activated ion channel. This channel is also regulated by a receptor-mediated mechanism that is coupled to phospholipase C activation. TRPM6: TRPM6 produces currents that are activated by a reduction in free Mg2 and Mg-ATP, and have identical permeability to Ca2 and Mg2 . TRPM6 mainly contributes to Mg2 balance in the body. TRPM7: TRPM7 forms a Ca2 -permeable, nonselective cation channel. The TRPM7-induced channel is activated by increasing intracellular ATP concentration, but is inhibited by high intracellular Mg2 and Mg-ATP. The ion selectivity of TRPM7-induced channels is Zn2 4Ni2 4Ba2 4Co2 4Mg2 4Mn2 4Sr2 4Cd2 4Ca2 . TRPM8: TRPM8 is responsible for cold-sensing mechanisms. It is activated by cooling cells such as dorsal root ganglia neurons p241C and by cooling agents such as menthol and icilin. TRPM8 is permeable to Ca2 .

TRPV1, 2, 3, and 4: TRPV1, TRPV2, TRPV3, and TRPV4 play a role in sensing of body temperature. The heat thresholds are 4431C in the TRPV1 channel, 4531C in the TRPV2 channel, 431391C in the TRPV3 channel, and 424331C in the TRPV4 channel. These channels are expressed widely in dorsal root ganglia neurons, hypothalamus, mechanosensory cochlear hair cells, and skin epidermal keratinocytes. TRPV5 and 6: TRPV5 is known as ECaC1, and TRPV6 as ECaC2. TRPV5 and TRPV6 have different characteristics from TRPV1, TRPV2, TRPV3, and TRPV4. They are highly Ca2 selective compared with other TRPVs. These channels are expressed in epithelia. The term ECaC (epithelial Ca2 channel) originates from the expression of the channel in epithelia. These channels are constitutively active.

Regulation of the Cytosolic Ca2+ Concentration

The cytosolic Ca2 concentration is regulated by the various types of Ca2 channels mentioned above and other ion transporters such as Na /Ca2 exchanger and Ca2 -ATPase (pump) in different ways depending on the type of cell or tissue in various organs. Figure 2 gives a summary of how the

Acute hypoxia

Chronic hypoxia

(+)

(Vascular smooth muscle)

(+)

(+) Expression of TRP-related Ca2+ channel in endothelial cell

(+) Expression of TRP-related Ca2+ channel in vascular smooth muscle

Voltage-gated Ca2+ channel

Store-operated Ca2+ channel (TRP-related Ca2+ channel)

[Ca2+]c

[Ca2+]c in endothelial cell

[Ca2+]c in vascular smooth muscle

Contraction of muscle

ET-1 in endothelial cell

Contraction of muscle

Pulmonary vascular resistance

IP3 in vascular smooth muscle (+)

Pulmonary vascular resistance

Pulmonary artery hypertension

IP3-activated Ca2+ channel in vascular smooth muscle

Pulmonary artery hypertension

Figure 3 Pathogenesis in abnormal regulation of Ca2 channels in respiratory system. A model scheme of pulmonary artery hypertension induced by acute and chronic hypoxia. ET-1, endothelin 1; IP3-activated Ca2 channel, inositol 1,4,5-triphosphate (IP3)-activated Ca2 channel; TRP-related Ca2 channel, transient receptor potential (TRP)-related Ca2 channel. ( ), stimulation or activation.

ION TRANSPORT / Chloride Channels 465

Ca2 channels described above and other Ca2 transporters regulate the cytosolic Ca2 concentration in smooth and skeletal muscles.

Role of Ca2+ Channels in Respiratory Diseases

Regulation of the cytosolic Ca2 concentration maintains cell function in the lung such as contraction of smooth and skeletal muscles, secretion of cytokine, and activity of ion channel (e.g., Ca2 -activated K and Cl channels), etc. On the other hand, disorders of these channels might cause defects in lung function. Genetic defects of Ca2 channels are known. LambertEaton syndrome, an autoimmune disorder that is most often seen in patients with small cell lung cancer, is known to be due to an impairment of presynaptic Ca2 channels at motor nerve terminals. Antibodies against presynaptic Ca2 channels reducing the channel activity (number) cause dysfunction of neurotransmitter release from the presynaptic terminals leading to pathogenesis of neuronal activity in patients. Abnormal environments also cause some diseases. For example, hypoxia causes pulmonary artery hypertension via elevation of cytosolic Ca2 concentration. Figure 3 shows a scheme of how acute and chronic hypoxia cause pulmonary artery hypertension. Acute hypoxia directly elevates the cytosolic Ca2 concentration in smooth muscle of the pulmonary artery by activating voltage-gated Ca2 channels and store-operated Ca2 channels (possibly TRP-related Ca2 channels), resulting in pulmonary artery hypertension (Figure 3). Chronic hypoxia also acts on endothelial cells of the pulmonary artery by elevating the cytosolic Ca2 concentration, which stimulates secretion of endothelin. Endothelin acts on its receptor in smooth muscle of the pulmonary artery, elevating the cytosolic Ca2 concentration of the smooth muscle, which causes pulmonary artery hypertension via an increase in the resistance of pulmonary artery resulting from Ca2 induced contraction of smooth muscle (Figure 3). The diseases mentioned above are just a few of a number of disorders of Ca2 channels; in other words, the Ca2 channel plays a key role in the maintenance of normal function of respiratory organs.
See also: Fluid Balance in the Lung. Ion Transport: Overview; Chloride Channels. Smooth Muscle Cells: Airway; Vascular.

Catterall WA, Striessnic J, Snutch TP, and Perez-Reyes E (2003) International Union of Pharmacology. XL. Compendium of voltage-gated ion channels: calcium channels. Pharmacological Reviews 55: 579581. Cortright DN and Szallasi A (2004) Biochemical pharmacology of the vanilloid receptor TRPV1. European Journal of Biochemistry 271: 18141819. Fleig A and Penner R (2004) The TRPM ion channel subfamily: molecular, biophysical and functional features. Trends in Pharmacological Sciences 25: 633639. Hoenderop JGJ, Nilius B, and Bindels RJM (2005) Calcium absorption across epithelia. Physiological Reviews 85: 373422. Isom LL, De jongh KS, and Catterall WA (1994) Auxiliary subunits of voltage-gated ion channels. Neuron 12: 11831194. McGeown JG (2004) Interactions between inositol 1,4,5-triphosphate receptors and ryanodine receptors in smooth muscle: one store or two? Cell Calcium 35: 613619. Mehta D, Bhattacharya J, Matthay MA, and Malik AB (2004) Integrated control of lung uid balance. American Journal of Physiology. Lung Cellular and Molecular Physiology 287: L1081L1090. Moczydlowski EG (2003) Electrophysiology of the cell membrane. In: Boron WF and Boulpaep EL (eds.) Medical Physiology, pp. 145171. Philadelphia: Saunders/Elsevier Science (USA). Moczydlowski EG (2003) Electrical excitability and action potentials. In: Boron WF and Boulpaep EL (eds.) Medical Physiology, pp. 172203. Philadelphia: Saunders/Elsevier Science (USA). ONeil RG and Brown RC (2003) The vanilloid receptor family of calcium-permeable channels: molecular integrators of microenvironmental stimuli. News in Physiological Sciences 18: 226231. Revens U, Wettwer E, and Hala O (2004) Pharmacological modulation of ion channels and transporters. Cell Calcium 35: 575582. Striessnig J, Hoda J-C, Koschak A, et al. (2004) L-type Ca2 channels in Ca2 channelopathies. Biochemical and Biophysical Research Communications 322: 13411346. Strock J and Diverse-Pierluissi MA (2004) Ca2 channels as integrators of G protein-mediated signaling in neurons. Molecular Pharmacology 66: 10711076. Thevenod F (2002) Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins. American Journal of Physiology. Cell Physiology 283: C651C672.

Chloride Channels
H R de Jonge and B C Tilly, Erasmus University Medical Center, Rotterdam, The Netherlands
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Chloride channels in the airways are especially abundant in the surface epithelium and gland compartments where they play an essential role in the transepithelial movement of electrolytes and water. The cystic brosis transmembrane conductance regulator, a member of the superfamily of ATP-binding cassette transport proteins, functions as a cyclic AMP- and cyclic GMP-activated chloride channel and is a major determinant of airway uid homeostasis by triple action: it inhibits isotonic NaCl and uid absorption by the ciliated cells through its interaction with epithelial sodium channels, it promotes uid secretion by serous cells in the submucosal glands, and it mediates b-adrenergic

Further Reading
Benham CD, Gunthorpe MJ, and Davis JB (2003) TRPV channels as temperature sensors. Cell Calcium 33: 479487.

466 ION TRANSPORT / Chloride Channels

stimulation of NaCl and uid absorption in the distal airways and alveolar compartment. Consequently, CF airways show a depletion of airway surface liquid resulting in impaired mucociliary clearance, and a propensity to pulmonary edema. Calcium-activated chloride channels are abundantly expressed in tracheal and bronchial epithelium. Upon activation by Ca2 mobilizing, purinergic, or cholinergic receptors, they may serve as compensatory chloride channels in CF. Their molecular nature is not yet elucidated. Putative molecular candidates include the CLCAs (hCLCA14). hCLCA1 (gob-5) is associated with mucin granule membranes and plays a key role in mucus overproduction and the pathogenesis of bronchial asthma. In addition, numerous other chloride channel species (e.g., CLCs, bestrophins, CLICs, and ORCCs) have been identied in the respiratory tract and are briey discussed in this review.

Introduction
Chloride channels form proteinaceous pores in plasma lemma and intracellular membranes that allow the passive diffusion of negatively charged ions along their electrochemical gradient. Their functions range from ion homeostasis to regulation of electrical excitability, cell volume, cell cycle and apoptosis, pH of intracellular organelles, and transepithelial ion transport. Whereas three molecularly distinct Cl channel families (cystic brosis transmembrane conductance regulator (CFTR), CLCs, ligand-gated g-aminobutyric acid (GABA) and glycine receptors) are structurally and functionally well characterized,
CFTR

other gene families are less well understood (e.g., calcium-activated chloride channels (CLCAs), bestrophins, chloride intracellular channels (CLICs)) or not yet identied at the molecular level (e.g., volume-regulated anion channel (VRAC), outwardly rectifying chloride channel (ORCC)). This review focuses on the role of Cl channels in the vectorial transport of salt and water across proximal airways and distal pulmonary epithelium and their involvement in the pathophysiology of cystic brosis and pulmonary edema. It also delineates a putative role in other airway functions, including the secretion of mucus and antimicrobial molecules, and in the pathogenesis of asthma.

Structure
CFTR emerged from the search for the cystic brosis locus and was the rst anion channel identied by expression cloning and reconstitution in lipid bilayers. It belongs to the superfamily of ATP-binding cassette (ABC) transporters for organic compounds but displays Cl conducting properties. Its unique regulatory domain (R) serves as a target for cAMPand cGMP-dependent protein kinases (Figure 1). The molecular nature of calcium-activated chloride channels (CaCCs) is not yet resolved. Putative
CLCA Cleavage site N

C N ATP NBD1 ATP R Pn NBD2 C

CBS1

N C CLC CBS2

N C C

Barttin

Bestrophin

Figure 1 Topology models of the major classes of chloride channels expressed in the respiratory tract. NBD1,2: nucleotide-binding domains; binding of ATP to NBD2 promotes NBD1NBD2 cassette heterodimerization and allows channel opening following multisite phosphorylation of the regulatory (R) domain by cAMP- and cGMP-dependent protein kinase. CBS1,2: conserved structural domain of unknown function (named after cystathione-b synthase). Barttin: needed as a b-subunit in ClC-K channel isoforms.

ION TRANSPORT / Chloride Channels 467

candidates belong to a new family of cloned membrane proteins, the CLCAs, including multiple human and murine homologs (hCLCA14; mCLCA16). Proteins of this family are characterized by sizes of 902943 amino acids, four or ve transmembrane regions, and a highly conserved pattern of ve cysteine residues in the extracellular amino-terminus. These proteins are processed by proteolytic cleavage into B90 and B35 kDa fragments (Figure 1). Although transfection studies and reconstitution studies in lipid bilayers suggest that some of the CLCA isoforms may represent genuine Ca2 -activated chloride channels, major discrepancies were found between functionally expressed CLCAs and native CaCC currents. At least in one example, this appeared due to the absence of an essential regulatory subunit. In other cases, it remains possible that CLCA proteins activate endogenous Cl channels rather than being channels themselves. In mammals, at least nine CLC genes have been discovered, encoding for ClCs17 and ClCs Ka/b. A topology model of monomeric CLC is depicted in Figure 1. From early electrophysiological studies, it has been proposed that these CLC channels have a so-called double barrel structure, for example, each functional unit has two chloride selective pores that are gated independently. High-resolution structures of two bacterial homologs of CLCs (EcClC from Escherichia coli and StClC from Salmonella typhimurium) have conrmed this model. Bestrophins were originally identied in Caenorhabditis elegans by their sequence homology as a distinct group of membrane proteins. Recent models of bestrophin (hBest14) suggest that the protein consists of four to six transmembrane domains (Figure 1) and that it is functional as a heterooligomer (tetra- or pentamer). The protein has a large cytoplasmic C-terminus containing consensus sequences for phosphorylation by cyclic AMP- and cyclic GMP-dependent protein kinases and for protein kinase C, as well as a binding site for the b-catalytic subunit of protein phosphatase 2A. The rst member of the CLIC family, p64, was isolated from bovine kidney microsomes and subsequently cloned from a kidney cDNA library. To date, six additional homologs genes have been identied, encoding for CLIC15 and parchorin. These ubiquitous proteins exist in both soluble and membrane forms, with a single putative transmembrane domain. Recently, the three-dimensional structure of CLIC1 has been elucidated. Surprisingly, the N-terminal domain resembles a so-called thioredoxin fold as is present in members of the superfamily of glutathione transferases.

In addition, several other proteins have been reported to function as putative anion selective channels, including ICln, a ubiquitously expressed soluble phosphoprotein that translocates to the membrane upon cell swelling; an outwardly rectifying chloride channel (ORCC), of unknown molecular identity; Mid-1-related chloride channel (MClC), an intracellular chloride channel containing four putative transmembrane domains and expressed moderately in the lung; and Tweety, a novel class of large conductance chloride channels (hTTY13), showing sequence homology with the BK-type of calcium-activated potassium channels and activated by cell swelling or a rise in intracellular Ca2 . However, current information about the expression and function of these channels in the respiratory tract is rudimentary or absent.

Expression and Regulation

The CFTR protein is localized in the apical membrane of epithelial cells including the ciliated cells in human nasal and bronchial epithelium, and the airway serous glandular cells (but not the goblet cells) (Figure 2). Low but functionally important expression is found in the Clara cells of the distal airways and in alveolar epithelia. CFTR activation is triggered primarily by cAMP signals evoked in response to b-adrenergic, VIPergic, and purinergic agonists. In addition, air-side activation of CFTR in Clara cells mediated through cGMP signals is triggered autocrinically by guanylin, a bioactive peptide stored in secretory granules underneath the apical membrane. CaCCs have been functionally identied in the apical membrane of many epithelial tissues, including tracheal and bronchial cells. The putative anion channel protein mCLCA3 (gob-5) is associated exclusively with mucin granule membranes of gastrointestinal and respiratory goblet cells (Figure 2). The mode of activation of CaCC, involving direct voltage-dependent binding of Ca2 to the channel or phosphorylation by the Ca2 /calmodulin-dependent protein kinase II, differs from CFTR channel gating. Activation of CaCC in epithelial cells requires agonist stimulation of G-protein-coupled receptors (e.g., purinergic and cholinergic) in the apical or basolateral membrane followed by intracellular Ca2 mobilization and/or Ca2 inux through transient receptor potential (TRP) or cyclic nucleotide-gated (CNG) cation channels. Whereas the expression of some CLCs is tissue specic, for example, ClC-1 and ClC-Ka/b are predominantly found in skeletal muscle and kidneys/inner ear, respectively, the other members of the family

468 ION TRANSPORT / Chloride Channels

Cilium

Apical membrane

ORCC

CFTR

ENaC Mucin Ca2+

CaCC

CIC-2 Tight junction

NO/cGMP PKG

cAMP PKA

Gob-5 (mCLCA3)

ER/golgi

Bestrophin

ORCC CIC-3/6 CLIC-1/3

CIC-2

Basolateral membrane

Figure 2 Subcellular localization of anion channels and sodium channels (ENaC) in airway epithelial cells. The direction of chloride and sodium ion movements is indicated by black or blue arrows, respectively. Signaling molecules and their interaction with the molecular targets are indicated in red.

are more ubiquitously expressed. In mammalian lung, the presence of at least six different CLC channels (ClC-27) has been reported, some of them being expressed only at specic stages during lung development. ClC-1, -2, -Ka, and -Kb are targeted towards the plasma membrane, whereas the other members are localized predominantly in intracellular compartments. With the exception of ClC-Ka and -Kb, which require barttin for activation (Figure 1), all CLCs channels are fully functional by themselves and do not need the presence of accessory proteins. The CLC chloride channels are opened and closed in a voltage-dependent manner. Changes in the extracellular Cl concentration as well as alterations of the pH modulate the activation kinetics. The presence of ClC-2 has been established in nasal polyps, at the luminal side of the trachea, at the apex of bronchial ciliated cells, in bronchioles, as well as type II alveolar cells. However, chloride current measurements in colonic epithelium suggest a predominantly basolateral localization of ClC-2 in polarized epithelia (Figure 2). ClC-2 mRNA is highest in fetal lung and drops rapidly after birth. In addition to transcriptional and translational regulation, ClC-2 is acutely activated in response to a low extracellular pH. Like ClC-2, the highly conserved ClC-3 is widely expressed in mammals. In the airways, ClC-3 expression has been found in tracheal, bronchial, and bronchiolar epithelia, as well as in the submucosal

glands of nasal tissue. The subcellular localization of ClC-3 is primarily intracellular, although upon overexpression some of the channels may trafc to the plasma membrane. Like ClC-2, expression of ClC-3 is developmentally regulated, although species differences may exist. In rat lung, the expression of ClC3 increases robustly after birth. In humans, however, ClC-3 expression is most prominent mid-gestation. At least three other members of the CLC family are expressed in the airways, ClC-4, -5, and -6, all of them primarily localized intracellularly. ClC-4 and -5 share a high-sequence homology and have similar functional properties, including their ability to function as Cl /H antiporters rather than as ion channels. The highest expression of ClC-5 was found in the kidneys and the intestine, but lower amounts were observed in tracheal epithelium and alveoli. In rat, ClC-5 expression in the lungs is most abundant during embryonic development, but significant levels are also present in neonatal and adult animals. Both ClC-4 and -6 are more ubiquitously expressed. Bestrophins are widely expressed throughout the animal kingdom. Four members of this family have been found in humans (hBest14). Bestrophin is localized in the basolateral membranes of airway epithelia and in the Calu-3 human airway serous cell line. Using hBest1- directed siRNA to achieve gene silencing, a prominent contribution of bestrophin to the basolateral anion conductance was demonstrated in this cell type. The conductance could be activated

ION TRANSPORT / Chloride Channels 469

by cyclic AMP- and NO/cyclic GMP-dependent signaling pathways. Moreover, several isoforms have been found to be activated by intracellular Ca2 . CLIC channels have a broad tissue distribution and are mainly expressed intracellularly. Despite the fact that these relatively small proteins contain only a single transmembrane domain and the observation that at least one of them, parchorin, exists predominantly in a soluble form, heterologous expression of several members resulted in an increase in the chloride conductance of intracellular vesicles. This, together with the relatively high expression of these proteins in tissues involved in water transport as well as the similarity of their C-termini with pore-forming bacterial proteins, led to the notion that they might function as (regulators of) intracellular chloride channels. Parchorin, a highly hydrophilic phosphoprotein named after its high expression in parietal cells and the choroid plexus, has been studied in lung. Expression of parchorin was found in the mucous epithelial cells of the trachea, as well as in the bronchioles and type II alveolar cells. Although parchorin is largely localized intracellularly, a translocation to the apical plasma membrane has been observed in response to cAMP, supporting a putative role in chloride and water transport. The ORCC is found in many different types of epithelial cells, including airway epithelial cells. Although its biophysical properties are well established, the molecular identity of the channel has not yet been resolved. ORCC has been found both in the apical and basolateral membranes of airway cells, but is regulated differently (Figure 2). In the apical membrane, the channel is activated by cAMP and CFTR, either directly or by a CFTR-dependent release of ATP. In the basolateral membrane, ORCC is stimulated by adenosine receptor activation. Exposure of lung epithelial cells to reactive oxygen species was found to inhibit ORCC activation.

Biological Function and Involvement in Respiratory Disease

The biological function and involvement in respiratory disease are discussed as follows (see Table 1).
Cystic Fibrosis Transmembrane Conductance Regulator

The apparent function of CFTR in airway ion and uid transport, and the consequences of its dysfunctioning in CF disease, differ in each airway compartment. In supercial epithelium, by concomitantly inhibiting Na absorption through the epithelial Na channels (ENaC) and promoting isotonic NaCl and uid secretion, CFTR regulates the thickness of the thin lm (B30 mm) of airway surface liquid (ASL). This ASL consists of a periciliary sol and a mucus gel that are propelled toward the mouth by coordinated ciliary beating. Consequently, CF airways show increased Na absorption driving increased absorption of Cl across non-CFTR shunt pathways and promoting uid absorption. This results in a depletion of ASL volume, dehydration of mucus, and a reduced mucociliary clearance of pathogens. Symptoms of CF disease could be mimicked in transgenic mice by overexpression of the b-subunit of ENaC channels in the bronchial compartment. The mechanism by which ENaC is upregulated in CF is not yet fully resolved. In submucosal glands, CFTR is essential as a mediator of VIPergic/cyclic AMP-mediated as well as cholinergic/calcium-mediated NaCl/NaHCO3 and uid secretion by the serous cells. It also promotes the secretion of other serous cell products, that is, antimicrobial, anti-inammatory, and antioxidant molecules involved in microbial growth inhibition, inhibition of serine protease-activation of ENaC channels, and protection against oxidative damage,

Table 1 Summary of key functions of chloride channels in the respiratory tract, and associated respiratory diseases Chloride channel CFTR Key function(s) in the respiratory tract Regulator of ASL volume (ciliated cells) Regulates secretion of serous cell products (salt, water, antimicrobial, anti-inammatory, antioxidant molecules) Promotes salt and uid absorption in alveoli Regulator of ASL volume (upregulated in CF) Synthesis, condensation, or secretion of mucins Fetal uid secretion Endosomal acidication, endocytosis Endosomal acidcation, endocytosis Cell volume regulation? Cellular detoxication? cAMP-triggered salt and water secretion? Coactivated with CFTR Respiratory disease Cystic brosis

CaCC hCLCA1/gob-5 CIC-2 CIC-3 CIC5 Bestrophin CLIC Parchorin ORCC

Pulmonary edema Cystic brosis Bronchial asthma, CF (?) Lung cyst formation Not known Not known Not known Not known Not known Cystic brosis

470 ION TRANSPORT / Chloride Channels

respectively. Loss of these functions in CF may contribute to a reduced ASL, mucus plugging, mucosal obstruction, and lung infection. In the distal airways and alveolar compartment, the lack of pathologic lung development in newborns with CF suggests that active chloride secretion and the production of lung uid in fetal pulmonary epithelium occurs through CFTR-independent pathways. Likewise, alveolar clearance of perinatal uid at the time of birth involves Na uptake through ENaC and Cl uptake through CFTR-independent chloride shunt pathways; this explains why the risk of acute respiratory failure at birth does not increase in CF patients and CF mice. However, in vivo studies in CF mice and ex vivo studies in human CF lungs have shown that CFTR is necessary for b-adrenergic, cyclic AMP-stimulated salt, and uid absorption that is important in minimizing pulmonary edema in septic and hypovolemic shock. Expression of human CFTR increased Na absorption and uid clearance in alveolar epithelium of normal rats and mice, and administration of isoproterenol or overexpression of a human b2-adrenergic receptor enhanced alveolar uid clearance in normal, but not in CF, mice. Whereas the cross-talk between the b2 receptor and the CFTR channel is mediated through physical interactions involving PDZ (postsynaptic density protein 95/disc large/Zonula occludens protein 1) domain-scaffholding proteins, the coupling between CFTR and ENaC is most plausibly electrogenic, that is, CFTR activation results in hyperpolarization and increases the driving force for Na uptake through the sodium channels.
Calcium-Activated Chloride Channels

including AHR and mucus overproduction, whereas adenovirus overexpression of gob-5 exacerbated the asthma phenotype. These observations indicate that mCLCA3/hCLCA1 plays a key role in mucus overproduction and the pathogenesis of murine and human asthma. Consequently, CLCA1 gene polymorphisms may be useful for predicting the susceptibility of chronic obstructive pulmonary disease, and may also contribute to the phenotypic variability of CF lung disease.
CLCs

Numerous physiological roles have been proposed including stabilization of the membrane potential (ClC-1), transepithelial ion transport (ClC-2, -Ka, -Kb), cell volume regulation (ClC-2, -3), and regulation of the intracellular pH and acidication of endosomes (ClC-2, -3, -5, -7). The pathophysiological importance of ClC members is exemplied by the ndings that myotonia congenita, Dents disease, osteopetrosis, and Bartters syndrome, all human genetic disorders, are due to mutations in CLC genes (ClC-1, ClC-5, ClC-7, and barttin, respectively).
ClC-2

The apparent downregulation of CaCCs by CFTR, presumably through interaction with the CFTR-R domain, and their upregulation under conditions of CFTR dysfunction, suggests a putative role of the CaCCs as compensatory chloride channels in CF and may at least in part explain the lack of airway disease in most CF mouse models. Apart from chloride transport, additional putative roles include the promotion of apoptosis (mCLCA1 and 2), cell adhesion of lung metastatic cells through b4 integrin-binding motifs (hCLCA2 and mCLCA1 and 5), and the synthesis, condensation, or secretion of mucins (mCLCA3 and its human ortholog, hCLCA1, gob-5). Gob-5 expression was induced specifically in murine lung tissue associated with airway hyperresponsiveness (AHR), and in patients with bronchial asthma. Moreover, intratracheal adenovirus expression of antisense gob-5 RNA into AHR mice suppressed the asthma phenotype,

It is now well established that ClC-2 is activated in the presence of a reduced extracellular pH. Because the alveolar pH of fetal lambs is acidic, it was suggested that ClC-2 had a role to play during fetal uid secretion. Indeed, using primary cultures of rat fetal lung cells, an increased chloride secretion was observed in the presence of an acidic luminal uid. In addition, inhibition of ClC-2 was found to disrupt the formation of lung cysts in fetal explants. Although these results suggest that ClC-2 is essential during lung morphogenesis, the lungs of ClC-2 knockout mice were found to develop normally.
ClC-3

Although a role for ClC-3 in the regulation of cell volume has been reported by several groups, its intracellular localization as well as the observation that ClC-3 channels are constitutively active, argues against the concept that ClC-3 may represent the volume-regulated anion channel. Accordingly, cellvolume regulation is not impaired in ClC-3 KO mice. In contrast, present evidence indicates that ClC-3 has an important function during endosomal acidication, acting as a shunt conductance for the vesicular proton ATPases, similar to ClC-5.
ClC-46

From these three isoforms, ClC-5 has been studied most intensively, due to the fact that mutations in

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 471

ClC-5 results in Dents disease, an X-linked hereditary kidney disease characterized by low-molecular-weight proteinuria, hypercalciuria, and hyperphosphaturia. ClC-5 is localized intracellularly at the luminal surface, associated with early or recycling endosomes, and, like ClC-3, is involved in vesicular acidication and receptor-mediated endocytosis. A possible role of ClC-4 and -6 in lung development and function remains to be established.
Bestrophins

Mutations in human bestrophin1 are responsible for Best disease, a dominant autosomal hereditary disorder, which leads to macula degeneration and a subsequent loss of vision. The bestrophins do not share any sequence homology with other groups of ion channels; however, early observations that the electroretinogram is altered in Best patients suggested that bestrophins could function as anion channels. Further studies, which included heterologs expression of cloned isoforms as well as the generation of specic mutations within the protein that alter the biophysical properties of the conductance, demonstrated that these proteins could function as bona de (parts of) anion channels. The function of bestrophin in the basolateral membrane of airway cells has not been not claried yet, but, in analogy to retinal pigmented epithelium, may be pertinent to cell-volume regulation.
Chloride Intracellular Channels

vesicular function. Journal of Clinical Investigation 115: 20392046. Mall M, Grubb BR, Harkema JR, ONeal WK, and Boucher RC (2004) Increased airway epithelial Na absorption produces cystic brosis-like lung disease in mice. Nature Medicine 10: 487493. Mutlu GM, Adir Y, Jameel M, et al. (2005) Interdependency of b-adrenergic receptors and CFTR in regulation of alveolar active transport. Circulation Research 96: 9991005. Nakanishi A, Morita S, Iwashita H, et al. (2001) Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proceedings of the National Academy of Sciences. USA 98: 51755180. Nilius B and Droogmans G (2003) Amazing chloride channels: an overview. Acta Physiologia Scandinavica 177: 119147. Rowe SM, Miller S, and Sorscher EJ (2005) Cystic brosis. New England Journal of Medicine 352: 19922001. Schwiebert EM, Cid-Soto LP, Stafford D, et al. (1998) Analysis of ClC-2 channels as an alternative pathway for chloride conduction in cystic brosis airway cells. Proceedings of the National Academy of Sciences USA 95: 38793884. Suzuki M and Mizuno A (2004) A novel human Cl channel family related to Drosophila ightless locus. Journal of Biological Chemistry 279: 2246122468.

ENaC (Epithelial Sodium Channel)

D C Eaton and L Jain, Emory University School of Medicine, Atlanta, GA, USA
& 2006 Elsevier Ltd. All rights reserved.

The nding of a thioredoxin fold in the CLIC structure suggests that CLIC proteins, in addition to their putative function as an anion channel, may serve a role in cellular detoxication.
See also: Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Ion Transport: Overview; Calcium Channels.

Abstract
Amiloride-sensitive sodium channels in the lung play an important role in lung uid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate uid layer on the surface of the alveoli. Both alveolar type 1 and 2 cells (T1 and T2) have several different amiloride-sensitive, sodiumpermeable channels in their apical membranes. This diversity appears to play a role in both normal lung physiology and pathological states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins designated a-, b-, and g-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung that has enormous exibility to alter the magnitude and regulation of salt and water transport. In lung, ENaC is regulated by many transmitter and hormonal agents. The mechanism of regulation depends upon which type of sodium channel is present, but involves controlling either the number of channels present in the apical membrane or the activity of individual channels.

Further Reading
Ashley RH (2003) Challenging accepted ion channel biology: p64 and the CLIC family of putative intracellular anion channel proteins. Molecular Membrane Biology 20: 111. Duta V, Szkotak AJ, Nahirney D, and Duszyk M (2004) The role of bestrophin in airway epithelial ion transport. FEBS Letters 577: 551554. Fang X, Fukuda N, Barbry P, Sartori C, Verkman AS, and Matthay MA (2002) Novel role for CFTR in uid absorption from the distal airspaces of the lung. Journal of General Physiology 119: 199207. Fuller CM and Benos DJ (2000) Ca2 -activated Cl channels: a newly emerging anion transport family. News in Physiological Sciences 15: 165171. Jentsch TJ, Maritzen T, and Zdebik AA (2005) Chloride channel diseases resulting from impaired transepithelial transport or

Introduction
Efcient gas exchange in the lungs depends on precise regulation of the amount of uid in the thin

472 ION TRANSPORT / ENaC (Epithelial Sodium Channel)

liquid layer lining the alveolar epithelium (see Fluid Balance in the Lung. Peripheral Gas Exchange). In airway epithelium, uid is transported from the airway lumen into the interstitial spaces, and this process can be substantially inhibited by the addition of amiloride, an epithelial Na channel (ENaC) inhibitor, to the alveolar space. While all parts of the airway have ENaC and absorb Na , the alveolar epithelium covers more than 99% of the large internal surface area of the lung (100150 m2 in humans) suggesting that alveolar epithelial cells are the major sites of Na transport and uid absorption in the adult lung. The alveolar epithelium is composed of two distinct cell types, alveolar type 1 (T1) and type 2 (T2) cells (see Epithelial Cells: Type I Cells; Type II Cells). T2 cells, which cover only 25% of the internal surface area of the lung, are cuboidal cells that secrete pulmonary surfactant (see Surfactant: Overview). T2 cells contain ion channels, including the amiloridesensitive ENaC and the cystic brosis transmembrane regulator (CFTR). T1 cells are large squamous cells whose thin cytoplasmic extensions cover 495% of the internal surface area of the lung; and, although less is known about these cells, they also appear to have a full complement of ion channels including ENaC and contribute to alveolar Na absorption.

Single-Channel Measurements from Alveolar Cells: A Family of AmilorideSensitive Channels

Because of the diversity of ENaC channels in airway epithelial cells, they are best examined using patch clamp methods to examine the properties of single ENaC channels. Single-channel studies have identied at least four different amiloride-blockable

channels with either high selectivity, moderate selectivity, or no selectivity for Na over K in the apical membranes of alveolar epithelial cells (Table 1). These channels differ not only in their ion selectivity, but also in their single-channel conductance and other characteristics; nevertheless, all appear to play some role in alveolar Na absorption. The channels can be grouped based on their different biophysical characteristics into four main categories: (1) highly Na selective cation (HSC) channels with a singlechannel conductance of 45 pS and Na /K selectivity of 440; (2) moderately selective cation (MSC) channels with a single-channel conductance of 89 pS and Na /K selectivity of 58; (3) a different moderately selective channel with similar Na / K selectivity but a much greater single-channel conductance of 56 pS; and (4) nonselective cation (NSC) channels with a single-channel conductance of 1924 pS and Na /K selectivity of about 1.5. The cloning of an epithelial Na channel consisting of three homologous subunits a, b, and g from rat colon provided a molecular definition of one class of Na channel. Expression studies of various combinations of the different ENaC subunits in heterologous expression systems showed that HSC channels were composed of a-, b-, and g-ENaC subunits (see Figure 1), and that NSC channels were composed of a subunits alone. The moderately selective Na channels were composed of the a ENaC subunit in combination with either b- or g ENaC (see Figure 2). Both T1 and T2 cells contain all types of ENaC channels: HSC, MSC, and NSC, although HSC and NSC are by far the most prevalent. The density of all forms of ENaC is similar in both T1 and T2 cells, but T1 cells have slightly more NSC than HSC channels per cell while the reverse is true for T2 cells.

Table 1 Comparison of different amiloride-blockable channels in epithelial apical membranes (including lung epithelial cells) Channel property Highly selective (HSC) 440 45 450 Moderately selective (type I; MSC) 58 89 700 Moderately selective (type II) 7 56 44000 Nonselective (NSC)

Na /K selectivity Unit conductance (pS) Amiloride sensitivity (K0.5 nM) Increased cAMP Increased cGMP

1.5 1924 2000

Increases N None

Increases Po None

None ?

Increases Po Decreases Po

In the table, Na /K selectivity is the ratio of Na permeability (PNa) to the K permeability (PK) determined from the reversal potentials of single channel currents. N is the channel density per unit area of membrane which is equivalent to the average number of channels per patch and Po is the open probability of a single channel dened as the ratio of amount of time the channel is open to the total recording time.

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 473

Extracellular loop

Pore?

M2 C-terminus N-terminus

M1

Figure 1 The structure of an ENaC channel consisting of three homologous subunits a, b, and g. The subunits consist of protein with short amino- and carboxy-terminal tails, two membrane spanning domains, M1 and M2, a putative pore region near the second membrane spanning domain, and a very large extracellular loop containing about 400 amino acids. Assembly of the channel appears to require four subunits, at least two of which must be the a-subunit (although the exact stoichiometry is controversial).

Unassembled ENaC subunits in membrane

Hypoxia, steroid depletion, or impermeable matrix

Steroid repletion and impermeable matrix

Normoxia, steroid repletion, and permeable matrix

NSC channel (20 pS): -ENaC only

MSC channel (10 pS): -, -, or -, -ENaC

HSC channel (6 pS): -, -, -ENaC

Figure 2 A schematic model for assembly and regulation of amiloride-sensitive sodium channels in the lung. Alveolar environment, particularly oxygen tension, steroid exposure, and alveolar distension, are likely to inuence assembly of ENaC subunits. Signal transduction pathways mediated by several protein kinases including A, G, and C regulate each of these channel types in different ways (see Figure 3). T1 and T2 cells with different channel types (and, therefore, different regulation) will have very different levels of sodium transport that will respond quite differently to hormonal and transmitter agents.

Regulation of ENaC in the Lung

Excessive accumulation of uid in the alveolar spaces frequently accompanies acute lung injury, and the failure of the lungs to rapidly clear this edema uid has been related to higher morbidity and mortality. Since alveolar uid clearance is driven by active transport of sodium and water across the epithelial

lining of air spaces (see Fluid Balance in the Lung), significant effort has focused on strategies to enhance this process particularly under pathological conditions. This has often times involved trying to enhance the activity of ENaC at the apical membrane of alveolar epithelial cells. ENaC in the airways is regulated by an enormous variety of agents (see Signal Transduction). These include transmitters

474 ION TRANSPORT / ENaC (Epithelial Sodium Channel)

ENaC Airway lumen Na+ P2YR D1R

Gi Inhibitory pathway PKC

Gq PIP2

Gs

PIP3 PI-3-K

cAMP

Stimulatory pathway PKA cAMP Na+ Gs Interstitium d2AR K+ (Na,K)-ATPase

GR GC

Gs

D2R

Figure 3 Major signaling pathways that regulate ENaC. For clarity, not all elements of the pathways are shown. A typical airway cell is shown with, starting from the top left, purinergic signaling mediated by P2Y receptors (P2YR). Purinergic signaling reduces ENaC activity by reducing the amount of membrane phosphatidylinositol-4,5-bis-phosphate (PIP2) by phospholipase C hydrolysis and by activating protein kinase C (PKC) which also inhibits ENaC. At the top right is the type 1 dopamine receptor (D1R) that promotes apical production of cyclic adenosine monophosphate (cAMP) and then, through a complicated signaling pathway that involves the small G protein, Rap1, stimulates phosphatidylinositol-3-kinase to form phosphatidylinositol-3,4,5-tris-phosphate (PIP3) that strongly stimulates ENaC. At the basolateral membrane is the type 2 dopamine receptor (D2R) that activates ENaC in a manner similar to the type receptor, but also activates the basolateral (Na,K)-ATPase. The basolateral membrane also contains b2-adrenergic receptors (b2-AR) that stimulate production of an alternative pool of cAMP that activates PKA to promote ENaC insertion in the apical membrane. Glucocorticoids (GC) bind to their receptor (GR) which when bound activates a complicated signaling pathway that requires gene expression but nally leads to activation of phosphatidylinositol-3-kinase to form PIP3 that strongly stimulates ENaC. Gs, Gi and Gq are G proteins that stimulate or inhibit adenylyl cyclase or stimulate phospholipase, respectively.

interacting with G-protein-coupled receptors (e.g., purinergic, adrenergic, and dopaminergic) (see GProtein-Coupled Receptors), circulating hormones (e.g., glucocorticoids, angiotensin, and eicosanoids), and chemokines (e.g., tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-b), and interleukins (ILs)) (see Chemokines) among others. The large number of regulatory agents attests to the fact that regulation of lung Na absorption is so critical for normal lung uid balance and function (see Fluid Balance in the Lung). Several agents have a particularly profound effect on Na reabsorption (summarized in Figure 3).

across T2 cells, can be upregulated by b-agonists suggests that these agents might be useful in limiting alveolar edema and decreasing morbidity and mortality in patients with acute lung injury. Activation of b2 receptors (see G-Protein-Coupled Receptors) on T2 cells stimulates adenylyl cyclase that, in turn, increases intracellular cyclic adenosine monophosphate (cAMP) levels and increases the number of HSC channels in the apical membrane and increases the activity of NSC channels already in the apical membrane. The effects of increased cAMP were totally blocked by the b-antagonist, propranolol, and by the protein kinase A (PKA) blocker, H89.

Regulation of ENaC by b-Adrenergic Agents

The demonstration that Na transport across the alveolar epithelium in vivo and ex vivo, as well as

Regulation of ENaC by Purinergic Agonists

The luminal surface of airway epithelial cells contains several different types of purinergic receptors.

ION TRANSPORT / ENaC (Epithelial Sodium Channel) 475

These include adenosine type 1 and 2a receptors (see G-Protein-Coupled Receptors). Adenosine at low concentrations (o100 nM) stimulates ENaC by increasing the open probability of single channels presumably by activating A1 receptors. At higher concentrations (41 mM), adenosine inhibits amiloridesensitive Na transport through A2a receptors. Besides adenosine receptors, there are several P2 purinergic receptors sensitive to ATP and UTP. The receptors are of both the P2Y metabotropic and P2X ligand-gated ion channel type. P2Y2 receptors (see G-Protein-Coupled Receptors) are sensitive to ATP and strongly inhibit ENaC through a protein kinase C (PKC)-dependent mechanism. The chloride channel, CFTR (see Cystic Fibrosis: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene), is often associated with release of ATP and some have suggested that ATP, acting through the P2Y2 receptor, allows for coordinated control of sodium and chloride absorption across the apical membrane of T2 cells. P2X receptors (probably P2X5) are, when activated by purines, ion channels themselves; however, they can also alter intracellular calcium and, therefore, would be expected to activate NSC channels and increase Na transport in alveolar epithelial cells.

Regulation of ENaC by Steroids

Lung epithelial cells contain both mineralocorticoid and glucocorticoid receptors (see Corticosteroids: Glucocorticoid Receptors), but since circulating levels of aldosterone are usually low compared to glucocorticoids, it is likely that the major steroids regulating lung ENaC are glucocorticoids. Glucocorticoids regulate sodium absorption by short- and long-term processes: an initial phase which increases transport four- to sixfold in the rst 26 h and a late phase which requires 1248 h and increases transport another three- to fourfold. A glucocorticoid, like other steroid hormones, enters target cells and binds to cytosolic, glucocorticoid receptor complexes. After some rearrangement, the glucocorticoid-bound receptor moves to the nucleus and acts as a DNAbinding protein which targets steroid response elements on genetic DNA. Binding to the response elements alters gene expression. Increases in Na transport can be measured within 1 h of exposure to glucocorticoids, and this increase is dependent on gene transcription and translation with the gene products generically referred to as steroid-induced proteins (SIPs). In the short term, glucocorticoids regulate sodium transport by inducing expression of the small G protein, K-Ras2A, and by subsequent K-Ras2A-induced activation of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K) to produce phosphotidylinositol-3,4,5-phosphate (see Signal Transduction) that ultimately increases the activity of individual ENaC channels. In the long term, glucocorticoids regulate sodium transport by inducing SIPs that alter trafcking, assembly, and degradation of ENaC, and thereby change the number of ENaC in the surface membrane. Glucocorticoids also apparently increase the number of HSC channels at the expense of NSC channels ensuring that the epithelium becomes highly selective for Na so that Na is preferentially absorbed by the lung epithelium by a mechanism that involves altering rates of channel insertion and degradation (see Figure 4).

Regulation of ENaC by Dopamine

Dopamine increases lung liquid clearance under basal conditions and in situations where edema accompanies lung injury. Alveolar epithelial cells appear to contain both type 1 and 2 dopamine receptors (D1 and D2) (see G-Protein-Coupled Receptors). Stimulation of D1 or D2 receptors on the basolateral surface of T2 cells activates (Na,K)-ATPase, but some of the increase in lung uid clearance is mediated by D1 activation of apical ENaC. Apical D1 receptors stimulate the production of cAMP which, through a complicated signaling pathway that involves the small G protein, Rp-1, but not PKA, nally increases the activity of individual ENaC proteins. The effect of dopamine on both ENaC and (Na,K)-ATPase suggests a coordinated response of both transporters to promote maximal increases in transport and alveolar uid clearance. The action of dopamine also demonstrates that signaling within alveolar cells is compartmentalized. Basolateral production of cAMP by b-adrenergic receptors produces a PKA-dependent increase in the number of ENaC in the apical membrane with little or no change in the activity of individual channels. On the other hand, the apical production of cAMP by D1 receptors produces a PKA-independent increase in the activity of individual channels with little or no change in the number of channels.

Regulation of ENaC by Inammatory Chemokines

Many inammatory cytokines reduce ENaC activity and, thus, exacerbate the pulmonary edema associated with sepsis. TNF-a, IL-1b, and TGF-b all reduce ENaC activity via mitogen-activated protein kinase signaling pathways (see Chemokines). Besides direct inhibition, IL-1b and TGF-b also interfere with glucocorticoid gene activation. Therefore, they not only reduce ENaC activity, but prevent glucocorticoid-mediated

476 ION TRANSPORT / ENaC (Epithelial Sodium Channel)

3

HSC channel c.d.i channel NSC channel c only channel

Ubiquitination 4 Recycling Retrieval

Insertion

6 5 Resorting Processing 7 Degradation ER d Trafficking

Proteasome c

Assembly 1 i

Figure 4 A schematic diagram of the assembly, trafcking, and degradation of ENaC with major components numbered. At (1) is the initial translation of separate ENaC subunits and accumulation in the ER pool. Only if the subunits are assembled into heteromeric complexes are they efciently transported out of the ER. (2) Trafcking through the Golgi prior to insertion into the membrane. There may be a substantial pool of assembled HSC channels in a subapical vesicular pool which can be stimulated to insert into the membrane in response to increases in cAMP (b adrenergic stimulation). (3) Aging of the channels which appears to involve Nedd4-mediated ubiquitination that leads to retrieval of channels (4) and resorting and separation of subunits (5) where the b and g subunits are destined for rapid proteasomal degradation (7), but the a subunits are resistant to degradation and may be recycled to the surface to form a-only NSC channels (6).

increases in ENaC activity and hamper the effectiveness of exogenously administered steroids.

ENaCs Role in Lung Disease

The effect of inammatory cytokines on ENaC implies that under certain pathological conditions, lung uid clearance could be compromised by reduced ENaC activity. Obviously, several pathological conditions can lead to an increase in TGF-b and, in these conditions, TGF-b will have many effects, of which two will be to increase alveolar epithelial permeability and inhibit ENaC. Thus, at the same time that excess uid enters the alveoli from the pulmonary capillaries because of the loss of epithelial barrier function, the ability of the epithelium to reabsorb uid is severely compromised due to reduction in ENaC activity (see Fluid Balance in the Lung). ENaC dysregulation has also been implicated in other lung pathologies. Recently, several investigators have shown that respiratory viruses can reduce ENaC activity when the viruses bind to the surfaces of lung epithelial cells. The consequence of this inhibition is to reduce alveolar uid clearance, thereby, promoting movement of excess uid containing viral

particles up the airways and out of the lungs which allows subsequent aerosol formation and transmission of the viruses. There are also genetic defects that lead to abnormalities of lung uid clearance some of which are due to ENaC dysfunction. As mentioned above, a-ENaC knockout mice die shortly after birth due to an inability to clear fetal uid from their lungs. In humans, a loss-of-function mutation in a-ENaC (pseudohypoaldosteronism type I) is not lethal, but does cause excessive lung uid production that can be cleared successfully, and does put individuals with the defect at risk for pulmonary infections. Cystic brosis (CF) is a defect in the gene for a chloride channel, but surprisingly is characterized by increased ENaC activity. This leads to reduced alveolar and airway uid depth that, in the upper airways, causes significant problems with the mucus accumulation characteristic of the disease. The defect in ENaC regulation associated with a CF suggests a tight regulation between sodium and chloride reabsorption in normal lung which is probably controlled by the CF gene product, CFTR. The genetic defect in CF disrupts this normal regulation and allows unregulated, high, levels of ENaC activity that disturbs uid balance (Cystic Fibrosis: Cystic Fibrosis

ION TRANSPORT / Potassium Channels 477

Transmembrane Conductance Regulator (CFTR) Gene). That there is extremely tight control of ENaC in the lung is underscored by another genetic defect, Liddles syndrome which is an ENaC gain-of-function mutation that leads to such excessive reabsorption in the kidney that individuals with the disorder have life-threatening hypertension. However, they have no lung symptoms suggesting that ENaC regulatory mechanisms in the lung are sufcient to maintain proper uid clearance.

Glucocorticoid Receptors. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. G-Protein-Coupled Receptors. Ion Transport: Overview; Calcium Channels; Chloride Channels; Potassium Channels. Lipid Mediators: Overview. Peripheral Gas Exchange. Proteasomes and Ubiquitin. Signal Transduction. Surfactant: Overview. Tumor Necrosis Factor Alpha (TNF-a). Vesicular Trafcking.

Further Reading Summary

It seems clear that ion channels that are members of the ENaC family of proteins play an important role in lung uid balance. Because the ENaC channel density/ patch is similar in T1 and T2 cells and because T1 cells cover 495% of the alveolar surface area, it seems likely that T1 cells may be responsible for the bulk of Na transport in the lung. In alveolar T1 and T2 cells, there are a variety of different amiloridesensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and pathological states. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of a-, b-, and g-ENaC subunits to form channels with different biophysical properties and different mechanisms for regulation. In particular, when only a-ENaC subunits are assembled together to form channels, the result is a 2128 pS nonselective cation channel which is sensitive to intracellular calcium and whose open probability is increased by cAMP. In marked contrast, when a-, b-, and g-ENaC subunits are assembled together, the result is a 46 pS channel that is highly selective for Na over K , is insensitive to intracellular calcium, and cAMP increases channel number with no effect on open probability. The diversity of channel expression and functional properties leads to epithelial tissue in the lung that has enormous exibility to alter the magnitude and regulation of salt and water transport. Better understanding of these pathways will help delineate the mechanisms that regulate alveolar uid balance and may provide the basis for developing strategies to prevent or treat the respiratory compromise associated with alveolar ooding.
Chen XJ, Eaton DC, and Jain L (2002) Beta-adrenergic regulation of amiloride-sensitive lung sodium channels. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: 609620. Eaton DC, Malik B, Saxena NC, Al Khalili OK, and Yue G (2001) Mechanisms of Aldosterones action on epithelial Na transport. Journal of Membrane Biology 184: 313319. Farman N, Talbot CR, Boucher RC, et al. (1997) Noncoordinated expression of a-, b-, and g-subunit mRNAs of epithelial Na channel along rat respiratory tract. American Journal of Physiology 272: C131C141. Firsov D, Gautschi I, Merillat AM, Rossier BC, and Schild L (1998) The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO Journal 17: 344352. Fukuda N, Jayr C, Lazrak A, et al. (2001) Mechanisms of TNF-a stimulation of amiloride-sensitive sodium transport across alveolar epithelium. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L1258L1265. Jain L, Chen XJ, Ramosevac S, Brown LA, and Eaton DC (2001) Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. American Journal of Physiology: Lung Cellular and Molecular Physiology 280: L646L658. Matalon S and OBrodovich HM (1999) Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annual Review of Physiology 61: 627661. Matalon S, Lazrak A, Jain L, and Eaton DC (2002) Invited review: biophysical properties of sodium channels in lung alveolar epithelial cells. Journal of Applied Physiology 93: 18521859. Rossier BC, Pradervand S, Schild L, and Hummler E (2002) Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annual Review of Physiology 64: 877897. Yue G, Malik B, Yue G, and Eaton DC (2002) Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates epithelial sodium channel activity in A6 cells. Journal of Biological Chemistry 277: 1196511969.

Potassium Channels
S M OGrady, University of Minnesota, St Paul, MN, USA
& 2006 Elsevier Ltd. All rights reserved.

Acknowledgments
This study was supported by R01 HL063306 to LJ; and by R01 HL071621 and P01 DK061521 to DCE.
See also: Acute Respiratory Distress Syndrome. Adenosine and Adenine Nucleotides. Adrenergic Receptors. Aquaporins. Chemokines. Corticosteroids:

Abstract
Airway and alveolar epithelial cells express a variety of K channels that are specifically localized to either the apical or

478 ION TRANSPORT / Potassium Channels

basolateral membrane depending upon their physiological function. These channels play important roles in maintenance of membrane potential, control of cell volume, regulation of cell proliferation, and support of transepithelial electrolyte and nutrient transport. K channels present in the apical membrane of alveolar epithelial cells, for example, facilitate K secretion, thus contributing to the elevated K concentration observed in alveolar uid. K channels localized to the basolateral membrane of airway epithelial cells contribute to transepithelial electrolyte and uid transport by sustaining the electrical driving force necessary for anion efux or cation inux and limit uctuations in intracellular K concentration associated with changes in Na K ATPase activity. Signaling molecules that control the absorptive and secretory functions of lung epithelia have been shown to regulate specic K channel subtypes through the mobilization of intracellular second messengers such as Ca2 and cAMP. In this review, the diversity of K channel expression and the functional role of these channels in transepithelial electrolyte and uid transport in lung epithelia are addressed.

K+ Channel Expression in Lung Epithelia

K channels are multimeric structures composed of pore-forming a-subunits and specic accessory proteins that serve to regulate the expression and
Table 1 K channel-related genes identied in lung epithelial cells Gene KCNQ1 Channel name Kv7.1;KvLQT1 Auxilary subunits KCNE2; KCNE3 Location Airways

gating properties of the channel. Approximately 80 K channel-related genes have been identied in the human genome. Airway epithelial cells express several distinct families of K channels, many of which have been previously identied in excitable cells. Table 1 presents a summary of K channel families and specic subfamilies known to be expressed in lung epithelia and their membrane localization. Multiple subtypes of voltage-gated (Kv) K channels (the Shaker-related channels, Kv1 4), the ether-a-go-go-related K channel, Kv7 (KCNQ channels), inwardly rectifying K channels (Kir), ATP-regulated K channels (KATP) and calcium-activated K channels (SK4/IK1) have been identied. In addition, several accessory K channel proteins are also expressed. Voltage-gated K channels possess six transmembrane domains and one pore region (6TM/1P) where both the N-terminal and C-terminal domains are located within the cytoplasm (Figure 1). The S4 segment contains several regularly spaced, positively charged amino acids that play a role in voltage sensing by the channel. The pore loop located between S5

Membrane Basolateral

Activators cAMP L364373 DIDS Niumic acid Ca2 1-EBIO Chlorzoxazone Voltage

Blockers Chromanol 293B Clofilium

KCNN4 KCNA1

KCa3.1;SK4; K1 Kv1.1

Calmodulin Kvb1; Kvb2

Airways Alveolus

Basolateral Apical

KCNA3

Kv1.3

Kvb

Alveolus

Apical

Voltage

KCNA4

Kv1.4

Kvb

Alveolus

Apical

Voltage

KCND1 KCND2

Kv4.1 Kv4.2

KChIP1 KChIP2

Alveolus Alveolus

? Apical

Voltage Voltage

KCND3

Kv4.3

KChIP2

Alveolus

Apical

Voltage

KCNS3 KCNMA1

Kv9.3 Slo; BK

Kv2 a-subunits BKb

Alveolus A549 cells

? ?

None Ca2 voltage

KCNJ2

Kir2.1

Kir2.2 a-subunits

Alveolus trachea

None

KCNJ8

Kir6.1

SUR2B

Alveolus

(r) basolateral (h) apical

Pinacidil YM934 Diazoxide nucleotide diphosphates

Clotrimazole Charybdotoxin TEA 4-AP Dendrotoxin Charybdotoxin 4-AP TEA 4-AP TEA Riluzole 4-AP TEA Arachidonic acid 4-AP Heteropodatoxins Bupivacane Nicotin 4-AP Hypoxia Iberiotoxin Charybdotoxin TEA Mg2 Polyamines CS Glibenclamide

ION TRANSPORT / Potassium Channels 479

NH2

6TM/1P G Y G S5 S6 P-X-P

S1

S2

+ + + S3 S4 + + +

COOH

T1

(a)

NH2

COOH

2TM/1P G Y/F G M2 M1

Kv

T1 NH2

COOH (c)

(b)

Figure 1 Topology of the two classes of K channel a-subunits present in lung epithelial cells. (a) Schematic diagram of a voltagegated K channel with six transmembrane domains and one pore domain (6TM/1P). The single transmembrane b-subunit (KCNE) is also represented. (b) Schematic diagram of an inwardly rectifying K channel a-subunit. (c) Tetramer organization of a voltage-gated K channel showing the orientation of the pore domains.

and S6 contains the selectivity lter sequence motif (YTxGYG). The pore of the channel is formed when four identical a-subunits (homotetramer) or four different a-subunits from the same subfamily (heterotetramer) interact to create the walls of the pore. In the Kv1 subfamily, the T1 region, located in the Nterminus preceding the rst transmembrane domain (S1), appears to be involved in tetramerization. In KCNQ1, however, sequence motifs (K535 to L650) within the C-terminus appear to be necessary for heterotetramer formation. The intermediate conductance, Ca2 -activated K channel KCa3.1 also belongs to the group of K channels that possess six transmembrane domains and one pore region. However, these channels differ from Kv channels in that they exhibit voltage-independent gating and bind calmodulin at the C-terminus. Calmodulin association with the a-subunits permits Ca2 binding and subsequent activation of the channel. In contrast to voltage-gated and KCa K channels, inwardly rectifying and ATP-regulated K channels

have a-subunits composed of two transmembrane domains and one pore region (2TM/1P). These channels lack the voltage-sensing S4 domain and thus do not exhibit strong voltage-dependent activation. The degree of inward rectication varies among subfamilies and depends upon the binding afnity of the channel for intracellular Mg2 or polyamines. Kir channels possess a selectivity lter sequence motif, G(Y/F)G, that is similar to Kv channels, making them significantly more selective for K over Na . As with Kv channels, functional Kir channels are formed by the interaction of four homologous or heterologous a-subunits within the same subfamily. The N-terminus also contains the T1 sequence motif (N96 to D184) found in certain Kv channel subtypes and appears to be involved in tetramer assembly. Auxiliary subunits are typically required for reconstitution of the physiological properties of K channels expressed in native tissues. In lung epithelial cells, several Kv channel b-subunits (Kvb) and at least

480 ION TRANSPORT / Potassium Channels

two K channel-interacting protein (KChIP) subfamilies are thought to associate with Shaker-related Kv channels in alveolar epithelial cells. The minimum K channel (minK; KCNE1) subunit has been shown to coassemble with Kv7.1 (KCNQ1) in airway epithelial cells and the sulfonourea receptor (SUR2B) coassembles with Kir6.1 to confer ATP regulation to this inwardly rectifying K channel in alveolar epithelial cells.

K+ Channel Function in Airway Epithelial Cells

K channel activation occurs in parallel with increases in anion secretion. Signaling molecules that increase intracellular Ca2 or cAMP increase anion channel activity in the apical membrane and activate specic K channel subtypes located in the basolateral membrane. A conceptual model illustrating the pathways involved in cAMP-dependent anion secretion is shown in Figure 2. The principal cAMPregulated anion channel is the cystic brosis transmembrane conductance regulator (CFTR). Cyclic AMP produces activation of protein kinase A and subsequent stimulation of apical CFTR. Associated with the increase in CFTR activity is activation of basolateral KCNQ1 K channels. The auxiliary subunit KCNE3, identied in certain airway epithelial cells, presumably coassembles with KCNQ1 to modify its functional activity. Studies in colonic epithelia have indicated that KCNE3 alters the gating properties of KCNQ1, rendering the channel constitutively active and voltage independent. Activation of KCNQ1/KCNE3 facilitates electrogenic Cl transport by increasing the electrical driving force
CI P PKA CI CFTR Na+

cAMP V AC ATP 2K+ ATP + 3Na+ NKCC KCNE3 K+ Na+ 2CI KCNQ1 K+ Na+

for Cl exit across the apical membrane. In addition, the recycling of K across the basolateral membrane prevents K accumulation within the cells and ensures that K depletion within the extracellular unstirred layer adjacent to the basolateral membrane does not occur so that Na K ATPase activity is not rate limited by K availability. The importance of KCNQ1 activation is highlighted by the fact that inhibition of these channels by chromanol compounds such as 293B, significantly inhibits cAMPstimulated anion secretion. A similar role for basolateral K channels holds for Ca2 -dependent anion secretion. Stimulation of apical P2Y receptors in bronchial epithelial cells or trachea, for example, leads to a rapid increase in intracellular [Ca2 ] that activates Ca2 -dependent Cl channels in the apical membrane and intermediate conductance, Ca2 -activated K channels (KCNN4) in the basolateral membrane. KCNN4, as mentioned earlier, forms a tight association with calmodulin at its C-terminus, which allows for Ca2 binding and subsequent regulation of channel function. Activation of KCNN4 hyperpolarizes the basolateral and apical membranes of the cell (the degree of apical membrane hyperpolarization depending on the ionic permeability of the paracellular junctions), thus offsetting the depolarization of the apical membrane produced by anion efux and sustaining the electrical driving force required for net anion secretion. It is worth noting that basolateral K channel activation can also increase electrogenic Na transport across airway epithelia. In tracheal epithelial cells (CFT1 cells) derived from cystic brosis (CF) patients, for example, K channel opening compounds such as chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) increase transepithelial Na absorption. Airway epithelia from most cystic brosis patients do not effectively trafc CFTR to the apical membrane and Na uptake by epithelial Na channels (ENaC) is elevated compared to non-CF individuals. Under these conditions chlorzoxazone or 1-EBIO stimulates Ca2 -activated K channels (presumably KCNN4), which can be blocked by the antifungal agent clotrimazole. The hyperpolarization produced by K exit from the cell compensates for the depolarization produced by Na uptake across the apical membrane, thus enabling the continued absorption of Na across the epithelium.

K+ Channel Function in Alveolar Epithelial cells

The K concentration in alveolar lining uid is greater than that of plasma, suggesting that the alveolar epithelium is capable of K secretion. Some

Figure 2 Model of cAMP-stimulated Cl secretion across the airway epithelium. AC, adenylyl cyclase; PKA, protein kinase A; NKCC, Na-K-2Cl cotransporter; CFTR, cystic brosis transmembrane conductance regulator.

ION TRANSPORT / Potassium Channels 481

K secretion presumably occurs through nonselective cation channels located in the apical membrane; however, immunocytochemical studies have shown that several Kv channel subfamilies are expressed in the apical membrane of adult alveolar epithelial cells. Given that Kv channel subfamilies gating is dependent on the membrane potential, their possible contribution to K secretion will depend on the apical membrane voltage. Analysis of the voltage dependence of activation and inactivation of alveolar cell Kv channels indicates that a measurable open probability exists between 40 and 10 mV, suggesting that these channels may contribute to K secretion if the apical membrane voltage lies within this range. Some Kv channel subtypes identied in alveolar epithelial cells have been shown to play a role in oxygen sensing in vascular smooth muscle and when expressed in HEK cells. For example, the electrically silent Kv9.3 a-subunit has been shown to associate with Kv2.1 to produce functional K channels that are oxygen sensitive. Moreover, Kv b-subunits also confer oxygen sensitivity to Kv4.2 channels expressed in HEK293 cells. In chronic hyperoxia, expression of Kv1.1, Kv4.3, and Kv9.3 is reduced in pulmonary artery smooth muscle cells. These observations suggest that certain Kv channels may function as part of an oxygen-sensing mechanism within the alveolar epithelium to detect changes in alveolar ventilation and oxygen diffusion. Rat and guinea pig adult alveolar epithelial cells are known to express Kir2.1, but the membrane localization of this inwardly rectifying K channel is not certain. Kir6.1 and its auxiliary subunit SUR2B have also been detected in adult rat alveolar epithelial cells. This ATP-regulated K channel is expressed in the basolateral membrane and is inhibited by glibenclamide. Treatment with the KATP channel opener 2-(3,4-dihydro-2,2-dimethyl-6nitro-2K-1, 4-benzoxazin-4-yl) pyridine N-oxide (YM934) increases alveolar liquid clearance in human lung. In cultured rat alveolar epithelial cells, the KATP channel opener penacidil increased Na and Cl transport by approximately 35%, consistent with the observation in human lung that activation of this KATP results in an increase in alveolar uid clearance. Two types of Ca2 -activated K channels have been identied in A549 cells, an epithelial cell line derived from human lung. The large conductance BK channel with a single channel conductance of 242 pS was activated by both increases in [Ca2 ] and membrane depolarization. BK channel blockers including Ba2 , TEA, and quinidine were all found to inhibit the large conductance channel in A549 cells. The intermediate conductance KCNN4 channel was also identied and shown to be activated by adenosine

and by nucleoside transport inhibitors and inhibited by clotrimazole. Attempts to detect mRNA for slo1, the ion-conducting subunit of BK and KCNN4, which encodes for the intermediate conductance channel in rat alveolar epithelial cells, were unsuccessful, thus the expression of these channels may vary among species.

Clinical Significance
Compounds that open basolateral K channels in airway epithelial cells may have therapeutic potential for the treatment of CF. At this time two lead compounds (1-EBIO and chlorzoxasone) have been identied and their effects on airway ion transport function characterized. As previously stated, these agents have been shown to increase electrogenic Cl secretion in CFTR-expressing epithelial cells by activating intermediate conductance Ca2 -dependent K channels. These K channels are activated following stimulation with Ca2 mobilizing agonists such as UTP. In airway epithelia from CF patients, apical P2Y receptor stimulation using stable analogs of UTP produces activation of Ca2 -dependent anion channels that provide an alternative pathway to that of CFTR for increasing net anion and uid secretion. Compounds related to 1-EBIO or chlorzoxasone could potentially augment P2Y receptor-mediated anion and uid secretion by enhancing the driving force for anion efux. However, as previously mentioned, airway epithelia from CF patients exhibit increased rates of ENaC-dependent Na absorption and increasing the basolateral membrane K conductance with 1-EBIO or chlorzoxasone was shown to stimulate Na absorption. Thus, the therapeutic benet of K channelactivating compounds in CF may probably depend on strategies that limit their effect on Na absorption. The identication of KATP channels in alveolar epithelia suggests that KATP channel openers related to YM934 or penacidil could have therapeutic potential in the treatment of conditions that cause lung edema. In human lung, YM934 was shown to increase K inux into the alveolar space. In contrast to the results in rat alveolar epithelial cells, this result suggested that the channels were localized to the apical membrane. Treatment with the KATP channel blocker, glibenclamide, inhibited the YM934-stimulated increase in uid clearance, indicating that these channels play a role in transalveolar electrolyte and uid absorption. However, it is worth noting that glibenclamide is an inhibitor of CFTR, which has been identied in alveolar and airway epithelial cells. Thus, it is possible that at least some of the effects of glibenclamide on uid clearance may have been due to CFTR inhibition.

482 ION TRANSPORT / Potassium Channels

Conclusions
Lung epithelial cells express a remarkable diversity of K channel genes and this list continues to expand. Many of these channels are present in excitable cells, but some have distinct physiological and pharmacologic properties in epithelial cells. Such differences appear to be the result of associations with auxiliary subunits that modify gating, cell surface expression, and pharmacological characteristics of the ion-conducting a subunits. Studies of ion transport function in airway and alveolar epithelial cells have denitively demonstrated that specic K channel subtypes are activated by signaling molecules that regulate anion secretion and that K efux through these channels is required to sustain the electrical driving force necessary for anion exit across the apical membrane. However, there are several K channel genes expressed in lung epithelia where a functional role is less certain. Additional studies will be necessary to elucidate the physiologic functions of these channels and any newly discovered K channel genes in the future.
See also: Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Epithelial Cells: Type I Cells; Type II Cells. Ion Transport: Overview; Chloride Channels; ENaC (Epithelial Sodium Channel).

Further Reading
Armstrong CM (2003) Voltage-gated K channels. Science STKE 10: 17.

Bleich M and Warth R (2000) The very small-conductance K channel KvLQT1 and epithelial function. Pugers Archives 440: 202206. Cowley EA and Linsdell P (2002) Characterization of basolateral K channels underlying anion secretion in the human airway cell line Calu-3. Journal of Physiology 538: 747757. Gutman GA and Chandy GK (2002) Potassium channels. In: Catterall WA, Chandy GK, and Gutman GA (eds.) The IUPHAR Compendium of Voltage-Gated Ion Channels, pp. 57190. Leeds: IUPHAR Media. Leroy C, Dagenais A, Berthiaume Y, and Brochiero E (2004) Molecular identity and function in transepithelial transport of KATP channels in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 286: L1027L1037. Mall M, Wissner A, Schreiber R, et al. (2000) Role of KvLQT1 in cyclic adenosine monophosphate-mediated Cl secretion in human airway epithelia. American Journal of Respiratory Cell and Molecular Biology 23: 283289. MacKinnon R (2003) Potassium channels. FEBS Letters 555: 6265. Mannhold R (2004) KATP channel openers: structureactivity relationships and therapeutic potential. Medical Research Reviews 24: 213266. OGrady SM and Lee SY (2003) Chloride and potassium channel function in alveolar epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 284: L689 L700. Schultz SG and Dubinski WP (2001) Sodium absorption, volume control and potassium channels: in tribute to a great biologist. Journal of Membrane Biology 184: 255261. Warth R (2003) Potassium channels in epithelial transport. Pugers Archives 446(5): 505513. Wu JV, Krouse ME, Rustagi A, Joo NS, and Wine JJ (2004) An inwardly rectifying potassium channel in apical membrane of Calu-3 cells. Journal of Biological Chemistry 279(45): 46558 46565.

K
KALLIKREINKININ CASCADE
K D Bhoola, The University of Western Australia, Perth, WA, Australia t Mu nchen, E Fink, Ludwig-Maximilians-Universita Munich, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Recent evidence increasingly supports the view that kinins exercise an important regulatory control in inammation and in the growth and proliferation of cancer cells. Kinins are formed by the serine proteases plasma and tissue kallikreins. In their functional capacity as proteases, the two enzymes release the vasoactive kinin peptides (bradykinin and lys-bradykinin (kallidin)) from multifunctional protein molecules called kininogens, of which two species have so far been described in humans. Plasma kallikrein is expressed in many epithelial and endocrine cells apart from hepatocytes. Tissue kallikrein is found in the endothelial and endocrine cells, and neutrophils. The kinins released by the kallikreins are considered to play a primary role as inammatory mediators (local hormones) by causing contraction of smooth muscles, dilation of arterioles, increasing permeability of the capillary membrane, and interacting with sensory nerve terminal transmitters to evoke pain. The various cellular actions of kinins are mediated by the activation of two specic receptors, B1 and B2. Both these seven-transmembrane receptors of the rhodopsin-like subfamily have been cloned, and are linked to specic Gaq=11 , Gas, Gbg, and Ga12,13 proteincoupled second messenger signaling systems. The development of kallikrein inhibitors as well as kinin receptor antagonists for use in immune-modulated disorders (e.g., asthma) and in tumors (namely lung cancer) may provide a new generation of drugs of therapeutic value in inammation and in carcinogenesis.

Werle and colleagues identied a new substance kallidin, which was shown to be the enzymatic product of kallikrein. Kallidin (now known as lysbradykinin) contracted isolated smooth muscle preparations and showed marked hypotensive activity. Only a few years later, bradykinin was discovered by William Beraldo working in Rocha e Silvas laboratory in Brazil. He showed that when trypsin or snake venom proteases were incubated with serum, a hypotensive substance was formed. Bradykinin was so called because it caused a slow sustained contraction of the isolated guinea pig ileum (brady slow and kinin movement). The kinin peptides, lys-bradykinin (decapeptide) and bradykinin (nanopeptide), show a similar structureactivity prole but differ in the potency of their cellular actions.

Kallikreins
Plasma Kallikrein

Historical Discovery of Kallikrein and Kinins

The rst observation on the kallikreinkinin cascade was made at the turn of the twentieth century, and in 1928 by the German surgeon E K Frey in Munich, who demonstrated the presence of a hypotensive molecule in the human urine and later from a pancreatic cyst. The observed hypotension was considered at that time to be a hormone (Kreislauf hormone) secreted by the pancreas into the circulation. It was called kallikrein after the Greek synonym for pancreas. In further studies kallikrein was identied in the blood, pancreas, and salivary glands. Subsequent pharmacological experiments of Eugene

Sites of synthesis and properties Plasma prekallikrein (PPK), the zymogen of the serine protease plasma kallikrein (PK, EC 3.4.21.34), is a singlechain glycoprotein which is the product of the gene KLKB1 located on chromosome 4q34q35. It had been accepted for several decades that PPK is synthesized only in the liver, but recently, expression of the mRNA of PPK has been proven in multiple tissues; hence, it is evident that the protein is also produced extrahepatically. Furthermore, in immunocytochemical studies PK/PPK has been specifically visualized in several human tissues such as pancreas, kidney, testis, stomach, and lung (unpublished data). Immunolocalization of PPK in the epithelial lining cells of the human bronchus is illustrated in Figure 1 and on the external surface membrane of the human neutrophil in Figure 2. The PPK synthesized in hepatocytes is secreted into the blood where it circulates at a concentration of 3550 mg l 1 mainly as a complex with high-molecular-weight kininogen (HMWK; H-kininogen). Two forms of PPK with different carbohydrate contents (Mr 85 and 88 kDa) exist in blood plasma. PPK is a single-chain protein with 619 amino acids. Conversion to active PK is achieved by cleavage of the

484 KALLIKREINKININ CASCADE

Bronchial epithelium

Figure 1 Visualization of the immunolabeled PPK on the epithelial cells of the human bronchus. Arrows indicate labeled epithelial cells; Positive label shown by the dark brown immunoprecipitated chromogen complex (DAB, diaminobenzidine); PPK, plasma prekallikrein. Magnication 20.

Figure 2 Confocal images of the immunolabeled plasma prekallikrein on the cell membrane of the human neutrophil. Intensity of the signal expressed in pixels (2500) is shown in the color llerchart. Reproduced from Henderson LM, Figueroa CD, Mu Esterl W, and Bhoola KD (1994) Assembly of contact phase factors on the surface of the human neutrophil membrane. Blood 84(2): 474482, with permission.

peptide bond Arg371Ile372 on negatively charged surfaces by activated factor XII (FXII, Hageman factor) with HMWK as cofactor. Alternatively, when PPK is linked to surface-bound HMWK on endothelial cells, it can be activated by prolylcarboxypeptidase. The two chains of active PK are held together by a disulde bond. The N-terminal heavy chain contains four tandemly arranged apple domains of 90 or 91 amino acids which are important for the interaction with proteins. The light chain contains the catalytic domain and is homologous to the members of the chymotrypsin family of peptidases. Biological activities Biological activities attributed to the PPK circulating in the blood are involved in the activation of neutrophils and C3 convertase of the alternative pathway, induction of the brinolytic cascade by converting the prourokinase plasminogen activator to an active molecule, and the release from

HMWK of bradykinin, which through specic G-protein-coupled receptor is linked to second messengers that regulate cellular events. The PPK in the blood originates essentially or totally from the liver; therefore, it is reasonable to conclude that the extrahepatically synthesized PPK has special functions at or near the site of its synthesis. Interestingly, novel biological roles of PK which t into the concept of such special local functions have been reported recently. PK and other proteases activate bradykinin B2 receptor directly, independent of kinin release. PK is required for adipogenesis; it mediates activation of a plasminogen cascade, which in turn promotes adipocyte differentiation. PK can activate prohepatocyte growth factor and thus may also regulate processes that involve the hepatocyte growth factor/c-Met signaling pathway. Impaired liver regeneration by circulating endotoxin after partial hepatectomy seems to be caused by a PK-mediated activation of latent transforming growth factor beta (TGF-b). Involvement in pathologic disorders The two protease zymogens, PPK and FXII, together with the nonenzymatic cofactor HMWK represent the components of the contact activation system. The contact system becomes activated when blood is exposed to negatively charged articial surfaces or, physiologically, when PPK, complexed with HMWK, binds to endothelial cells and is activated by prolylcarboxypeptidase or FXIIa bound to the cell surface (details of these processes are still unclear). Upon contact system activation, the two zymogens FXII and PPK are converted into the active enzymes. Subsequently, FXIIa activates both PPK and FXII, and PPK activates FXII. Thus, the result is an accumulation of PK and FXIIa. Spatial and temporal

KALLIKREINKININ CASCADE 485

control of their activities is provided mainly by the C1 inhibitor (C1INH). The active PK releases the nanopeptide bradykinin from HK which is the activator of kinin B2 receptor. By a carboxypeptidase, bradykinin can be transformed to des-Arg9-bradykinin, which is a kinin B1 receptor agonist. Bradykinin and des-Arg9-bradykinin are involved in various biological activities (cf. kinins). Control of the bradykinin plasma concentration is achieved, on the one hand, by the regulation of PK activity and, on the other hand, by the degradation of bradykinin to inactive peptides by peptidases (cf. kininases). Elevated plasma levels of bradykinin are thought to be co-responsible for anaphylactoid reactions with symptoms ranging from ushing, cough, edema, and dizziness to life-threatening reactions with severe bronchospasm, hypotension, and cardiorespiratory arrest. Such increased bradykinin plasma concentrations may be due to the following: 1. excess activation of PPK resulting from contact activation on articial surfaces (e.g., hemodialysis membranes, low density lipoprotein-apheresis with immobilized dextran sulfate, ltration of platelet concentrates with white cell-removal lters, colloid volume substitutes, and radiocontrast media); 2. normal PPK activation, but reduced bradykinin degradation because of angiotensin converting enzyme (ACE)-inhibitor treatment or decreased level(s) of kinin degrading enzymes, especially in combination with (1); and 3. insufcient regulation of PK activity due to reduced inhibitory activity of C1 inhibitor in hereditary or acquired angioedema.

are located in a distinct gene region on the human chromosome 19q13.3q13.4. TK genes (KLK) comprise ve exons and four introns, and are 410 kb in size. The differences in size are attributed to the introns, whereas the exons are highly conserved. The alignment of the genes commences with KLK1 KLK15KLK3KLK2 and then continues downstream with the gene spacing of 25 kb. Gene products The KLK genes encode for the pre pro form of serine proteases, which are synthesized bound to a 17-amino-acid-signal peptide. All of these gene products (except KLK1, which shows kininforming activity (kininogenase)) have extensive sequence similarity at DNA and protein levels. Phylogenetic analysis of the 15 gene products showed that hK1 (tissue kininogenase), hK2, and hK3 (PSA) reside on the same branch but have evolved away from a branch that links with trypsin and chymotrypsin B. The KLK1 gene product (hK1, tissue kininogenase, EC.3.4.21.35) is a single-chain acid glycoprotein of 238 amino acids with Ile at the amino-terminal and Ser at the carboxy-terminal, and has a molecular weight of 2545 kDa. By its catalytic triad of His41, Asp96, and Ser189, hK1 hydrolyzes one methionyl and one arginyl bond of domain 4 of its physiological substrates, L- and H-kininogens to release the decapeptide, kallidin. Cellular expression Expression of the TK genes is wide spread and tissue specic. The KLK1 gene is expressed predominantly in the salivary glands (Figure 3), pancreas, kidney (Figure 4), and lung. In the normal human lung, high mRNA expression for KLK1KLK12 genes has been reported. The expression of hK1 has also been demonstrated in many tissues (colon, endometrium and myometrium of the uterus, placenta and aminiotic uid, cardiac tissue extracts), bronchial lavage from asthmatics, and nasal secretions from patients with allergic rhinitis. In addition, tissue kininogenase has been identied in synovial uid from arthritic joints, and in the circulating and synovial uid neutrophils. KLK2 and KLK3 are expressed almost exclusively in the prostate. Similarly, KLK11 trypsin-like serine protease is expressed in many tissues, including the lung. Essentially, some of the genes show prostate-restricted expression (KLK2KLK4) or pancreas-restricted expression (KLK6KLK13). Lower expression is noted in kidney, ovary, testis, and salivary gland. KLK14 showed high expression in brain and bone marrow. Expression of TKs in cancer Recent reports implicate TK (KLK1) in the carcinogenic process arising

As a whole, due to its bradykinin-releasing activity PK is directly involved in such pathologic disorders which result from increased bradykinin levels in the circulation. On the other hand, partial or complete PPK deciency is not connected to any adverse clinical phenotype.
Tissue Kallikrein Family

Genomics and molecular structure Tissue kallikreins (TKs) belong to a closely related multigene family of 15 members, designated as KLK1KLK15, which enact different patterns of tissue-specic gene expression. Of the 15 gene members, KLK1 (hK1, kininogenase), KLK2 (hK2), and KLK3 (hK3, prostate-specic antigen (PSA)) show about 6070% homology, whereas KLK4KLK15 show about 30 40% homology. All 15 are clustered within a 320 kb region. They have several features in common. They

486 KALLIKREINKININ CASCADE

TK 200 TK 150 100 50 0

SD 10 m
Figure 3 Confocal image of the human salivary gland showing intensely labeled tissue kallikrein in the duct cells of the gland. Arrows pointing at immunolabeled TK in the DT; intensity of the signal expressed in pixels (2500) is shown in the color chart.

TK TK DT

Figure 4 Visualization of tissue kallikrein in granules within the connecting tubule cells of the human kidney. Positive label is shown by the dark brown DAB chromogen; arrows pointing at immunolabeled TK in the CT. Magnication 40.

from the induction of one or more genes. Expression of hK1 has been localized in a number of human carcinomas, namely esophageal, gastric, and renal carcinomas and astrocytomas. Such studies predict that the TK gene (KLK1) and its protein product (hK1) are expressed in lung tumors. Of the two kallikrein genes, KLK2 and KLK3 (PSA) present in the prostate, KLK2 is overexpressed in prostate cancer, with increasing expression, as the benign epithelium is transformed into high-grade intraepithelial prostatic neoplasia. Tissue analysis also shows high expression of the kallikreins in prostate (hK2, hK3, hK4, hK10, hK11, and hK15), breast (hK5 and hK10), testicular (hK10, hK11, and hK15), ovarian (hK10), and renal (hK1) cancers. Elevated circulating levels of tissue-specic kallikreins may prove to be of value as markers for lung carcinomas in which there may be induction of one or more kallikrein genes. Most kallikrein genes, for example, KLK2KLK7, KLK10KLK13, and KLK15, are upregulated by estrogens, whereas

KLK4 is downregulated in breast cancer and KLK11 (NES1) in breast and possibly other cancers. As cells become pleomorphic and the tumor invades surrounding parenchyma, the continuing upregulation of kallikrein genes increasingly assumes importance as prognostic indicator of advancing carcinogenesis and disease progression. Circulating levels of hKs may be of value in monitoring progression as well as therapeutic efcacy. Gene induction of TK and the subsequently formed kinins enhances the proliferation of tumor cells. As the carcinoma invades the surrounding parenchymal tissue, endothelial cells migrate into the tumor to form new blood vessels. The endothelial cells are attracted into the carcinoma by tumorkines and a number of promoters of angiogenesis which include vascular endothelial growth factor (VEGF), transforming growth factors, angiopoietins, and the proteins of the kallikreinkinin cascade. The role of TK (hK1) is illustrated in Figure 5 which shows the sequential

KALLIKREINKININ CASCADE 487

TK

TK (a) (b)

CP

(c)

(d)

Figure 5 Images of microvascular endothelial cells. The sequential events in the formation of new capillary blood vessels (a, b, c) are regulated by proteins of the kallikreinkinin cascade. Confocal images show immunolabeled tissue kallikrein (TK, white label see color chart) in the body and pseudopodia of the angiogenic endothelial cells as they progress toward forming a new capillary blood vessel. c1, is a phase contrast image; intensity of the signal expressed in pixels (2500) is shown in the color chart. (a) Reproduced from Plendl J, Snyman C, Naidoo S, Sawant S, Mahabeer R, and Bhoola KD (2000) Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells. Biological Chemistry 381:11031115, with permission from Walter de Gruyter GmbH & Co. K G. (b) Reproduced with kind permission of Springer Science and Business Media.

orientation of endothelial cells that are involved in the formation of new capillary blood vessel. Although there are no data on the functional consequences of kallikrein (hK1) expression in tumors, it can be postulated that this enzyme may have growth regulatory, extracellular matrix remodeling, and angiogenic effects in lung cancers and their metastasis. Functions biological role TKs are considered to process in vitro a variety of promolecules that may include matrix metalloproteinases, procollagenase, and progelatinase, and the processing of growth and peptide hormones. The potential activation of these proteinases by TKs suggests that it may play a role in endothelial and tumor cell migration, neutrophil diapedesis, leukocyte aggregation, angiogenesis, and lung tissue remodeling. The primary function of human tissue kininogenase (hK1) is the cleavage of L- or H-kininogen to release the decapeptide lysbradykinin (kallidin).

Kallikrein Inhibitors
1. The specic endogenous inhibitor of hK1 TK known as kallistatin has been puried and cloned.

Kallistatin also binds to elastase and chymotrypsin, but not to PK, urokinase, or collagenase. At the gene and protein level, kallistatins are members of the serpin (serine protease inhibitor) superfamily. The enzyme and inhibitor form a complex of 92 kDa, which is endocytosed by hepatocytes and cleared from the circulation. Human kallistatin and TK are colocalized in a variety of tissues. 2. A low-molecular-weight inhibitor of TK (CH2856) reduced eosinophilia in an animal model of allergic inammation, and further such peptide inhibitors of TK have also been designed. 3. Aprotinin (trasylol) is an in vitro inhibitor of both kallikreins, and the substitution of an amino acid has resulted in the synthesis of [Val 15] aprotinin which selectively binds PK and neutrophil elastase. 4. In addition, whereas the actions of hK1 are inhibited by a1 antitrypsin, PK is inhibited by a2 macroglobulin and C1 esterase inhibitor; the genetic reduction or absence of the latter is associated with angioneurotic edema. DX88, a novel recombinant-specic inhibitor of PPK, inhibits hereditary angioedema in mice, and is currently

488 KALLIKREINKININ CASCADE

undergoing clinical trials in patients with angioneurotic edema and also those undergoing cardiopulmonary bypass.

Kininogens
Genomics and Molecular Structure

In humans there are only two species of kininogens, H-kininogen (HMWK, HK) and L-kininogen (lowmolecular-weight kininogen (LMWK), LK). H-kininogen and L-kininogen are encoded by a gene located on chromosome 3q26-qter consisting of 11 exons. Distinct mRNAs for H-kininogen and L-kininogen result from alternative splicing. H-kininogen and L-kininogen share the coding regions of the rst nine exons, a part of exon 10 which transcribes the kinin sequence, and the rst 12 amino acids that follow the carboxy-terminal of the sequence. Exon 11 codes for the 4 kDa light chain of L-kininogen. The complete exon 10 possesses the full coding sequence for the 56 kDa light chain of Hkininogen. The kininogen molecule consists of an amino-acid-terminal heavy chain and a carboxy-terminal light chain, with the kinin moiety interleaved between the two domains. L-kininogen is a 66 kDa bglobulin with a plasma concentration of 60 mg ml 1 (2.4 mM) and an isoelectric point of 4.7, whereas H-kininogen is a 120 kDa a-globulin with a plasma concentration of 80 mg ml 1 (0.67 mM). The heavy chain is common to both H-kininogen and L-kininogen, but the light chain is unique to either H-kininogen or L-kininogen. The molecular structure is illustrated in Figure 6.
Cellular Expression

between the cells containing TK and its substrate, L-kininogen, suggests that kinins could be generated in the lumen of tubular structure of glandular organs, namely kidney, salivary gland, and bronchi. Specic, reversible, and saturable binding sites for kininogens have been reported on human neutrophils, platelets, and endothelial cells. On the surface membrane of the human neutrophil, H-kininogen is bound to PPK, the substrate and enzyme forming a unique complex.
Functions and Biological Role

The kininogens are present in extracellular uids and have been localized on human neutrophils, platelets, and endothelial cells, the collecting ducts of human kidney and sweat glands. The close relationship

D2 D1 D3

Although each domain of the complex molecule has one or more specic functions, the protein as a whole participates in several biological processes. The heavy chain consists of three domains (D1D3): domain 1 has a low-afnity Ca2 binding site, the function of which is unknown; domain 2 is the calpain inhibitory region; and domains 2 and 3 with the sequence GluValValAlaGly are potent cysteine protease inhibitors. Domain 4 contains the kinin moiety which is released from the kininogen molecule by the enzymic action of kininogenases. In addition, domain 4 has the ability to inhibit athrombin-induced platelet aggregation, and also serves as a cell-binding site for the parent molecule. The carboxy-terminal portion of the kinin moiety and the amino-terminal portion of the light chain participate as low-afnity binding sites to endothelial cells. More importantly, the domain 4 cell-binding region holds the kininogens in the appropriate conformation for optimal cell binding. The light chain of L-kininogen is 4 kDa and consists of one domain (D5) of unknown function. The light chain of H-kininogen is 56 kDa and consists of domains 5 (D5) and 6 (D6). Domain 5 contains two histidineand glycine-rich regions, one at its carboxy-terminal and the other on its amino-terminal. It serves as a cell-binding site on platelets, neutrophils, and endothelial cells. In addition, the histidine- and glycinerich regions have the ability to bind to anionic surfaces such as Zn2 and heparin. Domain 5 of Hkininogen (kininostatin) inhibits critical steps in the angiogenic process, namely endothelial cell proliferation and apoptosis. This nding emphasizes the pertinent role that is likely to be played by domain 5 in tumor angiogenesis. Domain 6 of H-kininogen has a PPK and FXI binding site.

D6 D5

D4

Kinin domain

Kinins
Cellular Actions of Kinins

H-kininogen
Figure 6 Molecular architecture of the kininogen molecule.

Kinins are most important primary mediators of inammation. Kinins express their functional effects by activating specic kinin receptors situated on the

KALLIKREINKININ CASCADE 489

surface membranes of many cell types. The kinin peptides are potent contractors of smooth muscle, cause arteriolar dilatation, and increase vascular permeability. They are powerful pain-producing substances, and they cause pain through two mechanisms: (1) by the direct stimulation of nociceptor bers (C and Ad), and (2) by the sensitization of sensory bers to physical and chemical stimuli. This algesic effect of BK is potentiated by thromboxanes and prostaglandins, and 50 -HT. It is postulated that the release of the kinin moiety from the kininogen molecule on the surface of the neutrophil by the enzymic action of kiningenases results in opening the junctions between the endothelial cells, and thus promotes the local diapedesis of neutrophils and cancer cells, as well as the extravasation of plasma. In addition, kinins stimulate the release of the proinammatory cytokines, interleukin (IL-1) and tumor necrosis factor (TNF), osteoclastic bone resorption, and stimulate the release of several second-generation mediators, for example, platelet activating factor, leucotrienes, prostaglandins, substance P, acetlycholine, and noradrenaline. Other biological activities of kinins include the ability to lower systemic blood pressure (the plasma concentration has been reported to be about 10 pg ml 1). Kinins also stimulate the secretion of renin from the kidney and the release of vasopressin from the neurohypophysis. In the nervous system BK is involved in the central regulation of blood pressure and increasing neuronal excitability.
Mitogenic Actions of Kinins

by kinin receptors, which have been identied in a number of human carcinomas. Kinin B2 receptors have been immunolocalized in squamous cell and papillary adenocarcinomas of the lung. Direct ligand binding studies have demonstrated kinin B2 receptors in squamous cell and adenocarcinomas and in normal lung membranes. The observation of increased kinin receptor expression due to oncogenic transformation gives further credence to a mitogenic role for kinins in tumor tissue. Sequential analysis of in vitro cultures of endothelial cells immunolabeled with TK (hK1) clearly unfolds the role that the kallikrein kinin protein components play in the formation of new blood vessels in the angiogenic processes linked to tumor invasiveness.

Kininases
The turnover of kinins depends on both the rate of formation and the rate of destruction. After kinins are formed, they are destroyed at variable rates in the circulation, extracellular uid space and in cells by the enzymatic action of peptidases. This family of enzymes is generally called kininases. There are two families of kininases: kininase I carboxypeptidases (KI) and kininase II peptidylpeptidases (KII). The KI family comprises kininase I-carboxypeptidase N (KICPN) and kininase I-carboxypeptidase M (KI-CPM) and the KII family includes kininase II-angiotensin I-converting enzyme (KII-ACE) and kininase II-neutral endopeptidase (KII-NEP). The enzymes which mainly cleave kinins are ACE (KII), CPN (KI), and NEP (KII).

Although kinins are considered to play a primary role in inammation, they are also mitogenic and regulate the proliferation of cancer cells. Several studies support the view that the kinin peptides stimulate DNA synthesis and thereby promote the process of tumorigenesis through their cell-differentiating and growth-promoting actions. Transformation of cells in vitro by the ras oncogene, which is mutated in a large number of human tumors, is associated with increased expression of kinin receptors. Kinins activate nuclear factor kappa B (NF-kB) through coupling with Gaq/11, which requires the induction of protein kinase C, and is independent of the TNF-a pathway. These ndings together with the known release of kinin imply that kinin peptides formed in the tumor parenchyma could enhance the process of tumorigenesis. The ability of kinins to induce cell division and proliferation of tumor cells enhances the spread of cancerous cells, and by increasing vascular permeability they initiate the metastatic migration of cancer cells. These carcinogenic effects are mediated

Kinin Receptors
Genomics and Molecular Structure

The kinins B1 and B2 are members of the superfamily of G-protein-coupled rhodopsin-like receptors characterized by seven-transmembrane regions connected by three extracellular and three intracellular loops, linked to second messenger signaling systems. The kinin binding site is located at the amino-terminal part of the third extracellular loop. Homology between the family members of the kinin receptors is most pronounced in the transmembrane regions, whereas the loop regions are more divergent in their sequences. The receptors have been mapped to chromosome 14q32, comprising more than 28 kb, and organized in three exons and two introns. The cDNA clone encoding a human B1 kinin receptor was isolated from a human embryonic lung broblast cDNA library by expression cloning. The mRNA encoding the B1 receptor is approximately 2 kb shorter than

490 KALLIKREINKININ CASCADE

that of the B2 receptor. The kinin B1 receptor has a predicted sequence of 353 amino acids, whereas the B2 receptor protein comprises 364 amino acids, is highly glycosylated, and exists in multiple isoforms at 69 kDa with an isoelectric point of pH 6.87.1. The two receptors show 36% protein sequence and 54% nucleotide homology.
Signal Transduction

The signal coupling of kinin receptors results in the activation of protein kinase C and tyrosine kinase pathways, coordinated with the activation of mitogen activated protein kinase (MAPK) and NF-kB. Kinin receptors also stimulate phosphatidylinositol hydrolysis in smooth muscle leading to mobilization of intracellular Ca2 , phospholipase C or phospholipase A, and appear to stimulate biosynthesis and release of prostaglandins. Stimulation of the kinin receptors on macrophages has been reported to release IL-1 and TNF. These bioactive peptides on signaling through the Gaq-linked receptor initiate the release of nitric oxide (EDRF) in endothelial cells. One attractive hypothesis is that on conversion of BK or lys-BK to the B1 agonist, the released carboxyterminal arginine may act as a substrate for eNOS (Figure 7).
Kinin B1 Receptors

receptor. In vitro, the B1 receptor is rapidly upregulated in experimental inammation, by noxious stimuli, bacterial products (endotoxins, lipopolysaccharide), and inammatory mediators, including proinammatory cytokines (IL-1b, IL-8, TNF-a). Several growth factors such as epidermal growth factor (EGF) and endothelial growth factor also induce a kinin B1 receptor response. There is an increase in the number of B1 binding sites in inamed or septic tissue. Normally, the B1 receptor is latent but appears to be rapidly upregulated in infections, carcinoma, and immune-modulated disorders, including rheumatoid arthritis, transplant rejection, and glomerulonephritis (Figure 8), and has been identied also in human brotic lung tissue. They also participate in mechanisms of hyperalgesia. The upregulation of the B1 receptors may be a mechanism of host defense.
Kinin B2 Receptors

The pharmacological activity of the B1 receptor is regulated by the carboxypeptidase metabolites; des-Arg9-BK and des-Arg10-kallidin, in contrast to bradykinin, which is essentially inactive at the B1

These receptors are ubiquitous and are expressed in most tissues. They are constitutively expressed in all mammalian tissues, and are activated specifically by bradykinin and lys-bradykinin. The B2 receptor accounts for the majority of the physiological actions of the kinins. Thus, the acute nociceptive and inammatory responses as well as the vasoactive properties elicited by the parent kinin molecules appear to be mediated through B2 receptors. In vivo, the B2 receptors have been implicated in hypotension, acute bronchoconstriction, and edema formation. Upregulation of the B2 receptors has been reported following

BK P M K R BK1 BK2 AA Gp GC cGMP Gp P G F S P

CPM GC

NO synthase

Arginase

Arginine

NO

Orn

Figure 7 Diagramatic illustration of the conversion of bradykinin to des-Arg9-bradykinin by a membrane carboxypeptidase M on the surface of cells that expresses both kinin receptors which are linked to cyclic GMP second messenger signaling pathway. MKBK, methly-lys-bradykinin; KBK, lys-bradykinin; RR, bradykinin; BK2, bradykinin B2 receptor; BK1, bradykinin B1 receptor; NO, nitric oxide; Gp, G protein; GC, guanyl cyclase.

KALLIKREINKININ CASCADE 491

100.00

DT DT

Kinin B1 receptors

Figure 8 Visualization of immunolabeled kinin B1 receptors on the renal tubule cells of the human kidney. Positive label is shown by the dark brown DAB chromogen; DT, distal tubules. Magnication 20.

myocardial infarction and in response to cytokines. TNF-a and IL-1b both induced a rapid and transient increase in B1 and B2 receptor mRNA expression in human embryonic lung broblasts, and IL-1b has been shown to increase the expression of B2 receptors in human deciduas derived cells. In addition, immunolabeling for B2 receptors has been observed on the astrocytic cells in patients with astrocytomas, and in esophageal, gastric, and renal carcinomas. A recent interesting observation with regard to bradykinin receptors is that PK and other serine proteases such as trypsin and cathepsin G are able to activate the B2 receptor. Thus, by a mechanism analogous to that whereby thrombin and trypsin activate the protease-activated receptors (PAR-1 and PAR-2, respectively), the B2 receptor may function as a novel serine-protease-activated receptor. However, kallikrein was able to activate a mutated B2 receptor missing the 19 N-terminal amino acids, suggesting a different mechanism of activation to that of the thrombin-activated PAR-1 receptor. The B1 receptor has not been investigated in this regard.

Kinin Receptor Antagonists

The understanding of the critical role the kinin receptor plays in health and disease has been greatly enhanced by the development of several antagonists. By definition, antagonists are compounds that bind to the receptors without invoking intrinsic activity. Over several decades kinin receptor antagonists have been developed comprising ve generations of synthetic molecules. The rst selective antagonist of the B1 receptor was des-Arg9 [leu8] bradykinin. Other antagonists such as des-Arg10 [leu9] lys-bradykinin (kallidin), with higher afnity and a longer duration of action, have been reported. The second-generation kinin B2 receptor antagonist, DArg[Hyp3, Thi5,

Dtic7, Oic8]bradykinin (Hoe 140 or icatibant), proved to be more potent than the rst-generation molecules with a longer in vitro lifetime and has been efcacious in clinical trials for allergic asthma and rhinitis. The recently synthesized kinin receptor antagonists are anticancer agents. Their mode of action is unique in that they inhibit the growth of cancer cells by a novel-agonist-based mechanism that involves silencing Gaq11 and stimulating Ga12,13. The most potent growth inhibitor of small-cell lung carcinoma, in this series of antagonists, is the N-terminal cross-linked molecule dimerized with suberimide [Darg-arg-pro-hyp (trans-4-hydroxy-L-proline)gly-Igl (alpha-2-indanylglycine)-ser-DIgl-Oic(octahydroindole-2-carboxylic acid)-arg]. Small mimetic (BKM-570, FSC-OC2Y-Atmp) antagonists are also potent inhibitors of the growth of lung cancer cell lines. Domain 5 (kininostatin) of H-kininogen, the endogenous substrate of PK, inhibits critical steps in the angiogenic process, namely endothelial cell proliferation and apoptosis. This nding further emphasizes the pertinent role that is being played by the kallikreinkinin proteins in new capillary blood vessel formation and in carcinogenesis.
Nonpeptide Kinin Antagonists

Several orally available nonpeptide kinin receptor antagonists, namely FR 173657, 165649, and 167344, have been developed. These have been found to be potent against B2 binding, with no effect on B1 binding. In vivo, they blocked human B2-receptor-mediated phosphatidylinositol hydrolysis, bradykinin-induced bronchoconstriction in guinea pigs, and carrageenin-induced paw edema. In addition, a nonpeptide antagonist for the B1 receptor (PS020990) has been developed recently.

492 KALLIKREINKININ CASCADE

KallikreinKinin Cascade in Lung Diseases

Cellular Expression and Mediator Release

The kinin peptides play an important role in the regulation of airway inammation. All of the components of the cascade kallikrein, kininogen, and free kinins have been detected in bronchial lavage uid of asthmatics but not in control subjects. Allergen challenge of asthmatics resulted in an increase of TK and kinin in the airways. Neutrophils, monocyte/ macrophages, and submucous glands are likely to be the sources of TK in the airways, whereas the kininogen substrates are probably derived from neutrophils and plasma transudate. When the bronchial epithelium is inamed, as is the case in asthma, kinins are capable of producing a marked and persistent contraction of bronchial smooth muscle; bradykinin is a potent bronchoconstrictor in asthmatics, but not in normal subjects. The release of prostanoids, thromboxane, substance P, and C-ber evoked axon reexes, and direct stimulation of kinin receptors has been implicated in the bronchoconstrictor actions of bradykinin. In addition to stimulating contraction of airway smooth muscle, bradykinin induces IL-8, VEGF, COX-2, and PGE2 production, and extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK dependent IL-6 expression by cultured airway smooth muscle cells. Airway hyperresponsiveness to bradykinin has recently been identied as a sensitive indicator of corticosteroid responsiveness in asthma. Bradykinin increases IL-8 generation in the airway epithelial cells by a mechanism dependent on COX2-derived prostanoids. Furthermore, bradykinin increases the expression of the IL-8 chemokine receptors, CXCR1 and CXCR2, in nasal tissue and epithelium of patients with allergic rhinitis. Since IL-8 is a potent neutrophil chemoattractant, bradykinin may contribute to neutrophilic inammation in asthma and chronic obstructive pulmonary disease by stimulating IL-8 production from airway epithelial cells. Other effects of bradykinin that may be important in the context of airway inammation are its ability to increase capillary permeability producing mucosal edema, and to enhance signal trafc in pulmonary nerves by stimulating the secretion of acetylcholine as well as neuropeptides. Bradykinin stimulates alveolar macrophages to release eosinophil chemotactic factors (predominantly leukotriene B4). The kinin-related link between neutrophils and eosinophils appears to be bidirectional in that eosinophilgranule-derived major basic protein increases airway hyperreactivity. Evidence of an eosinophilkinin link in airway inammation comes

from a study showing that in subjects with mild asthma, the magnitude of eosinophilic inammation of the airways was more strongly associated with responsiveness to inhaled bradykinin compared with methacholine. In asthmatics, pulmonary function gradually improved during 4 weeks of treatment with icatibant (Hoe 140, specic B2 receptor antagonist), suggesting that B2 receptor blockade may have anti-inammatory effects.

Signal Transduction

Most of the biological effects of bradykinin in human airways appear to be mediated by the constitutively expressed B2 receptor, which signals through p38, ERK-1/2, MAP kinases, protein kinase C, and NFkB. Furthermore, nasal challenge with the B1 receptor agonist lys-[des-Arg10]-bradykinin activated ERK in subjects with allergic rhinitis, but not in normal subjects, and additionally lys-[des-Arg10]-bradykinin activated the transcription factor AP-1 in human airway epithelial cells. However, the precise mechanisms by which kinins may inuence the pathogenesis of inammatory diseases such as asthma and chronic pulmonary obstructive disease are unknown.
See also: Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide.

Further Reading
Barnes PJ (1992) Bradykinin and asthma. Thorax 47: 979983. Bhoola KD, Figueroa CD, and Worthy K (1992) Bioregulation of kinins: kallikreins, kiniogens and kininases. Pharmacological Reviews 44: 180. Blaukat A (2003) Structure and signaling pathways of kinin receptors. Andrologia 35: 1723. Borgono CA and Diamandis EP (2004) The emerging roles of human tissue kallikreins in cancer. Nature Reviews: Cancer 4: 876890. Chan DC, Gera L, Stewart JM, et al. (2002) Bradykinin antagonist dimer, CU201, inhibits the growth of human lung cancer cell lines by a biased agonist mechanism. Proceedings of the National Academy of Sciences, USA 99: 46084613. Chan DC, Gera L, Stewart JM, et al. (2002) Bradykinin antagonist dimer, CU201, inhibits the growth of human cancer cell lines in vitro and in vivo and produces synergistic growth inhibition in combination with other antitumour agents. Clinical Cancer Research 8: 12801287. Clements J, Hooper J, Dong Y, and Harvey T (2001) The expanded human kallikrein (KLK) gene family: genomic organization, tissue-specic expression and potential functions. Biological Chemistry 382: 514. Clements JA, Willemsen NM, Myers SA, and Dong Y (2004) The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers. Critical Reviews in Clinical Laboratory Sciences 41: 265312.

KERATIN 493
Colman RW and Schmaier AH (1997) Contact system: a vascular biology modulator with anticoagulant, profibrinolytic, antiadhesive, and proinammatory attributes. Blood 90: 38193843. Cyr M, Eastlund T, Blais C, Rouleau JL, and Adam A (2001) Bradykinin metabolism and hypotensive transfusion reactions. Transfusion 41: 136150. Davis AE (2005) The pathophysiology of hereditary angioedema. Clinical Immunology 114: 39. Figueroa CD, Henderson LM, Kaufmann J, et al. (1992) Immunovisualization of high (HK) and low (LK) molecular weight kininogens on isolated human neutrophils. Blood 79: 754759. Figueroa CD, MacIver AG, and Bhoola KD (1989) Identication of a tissue kallikrein in human polymorphonuclear leucocytes. British Journal of Haematology 72: 321328. Guo Y-L, Wang S, and Colman RL (2001) Kininostatin an angiogenic inhibitor, inhibits proliferation and induces apoptosis of human endothelial cells. Arteriosclerosis, Thrombosis, and Vascular Biology 21: 14271433. Haasemann M, Figueroa CD, Henderson L, et al. (1994) Distribution of bradykinin B2 receptors in target cells of kinin action. Visualization of the receptor protein in A431 cells, neutrophils and kidney sections. Brazilian Journal of Medical and Biological Research 27: 17391756. Hecquet C, Becker RP, Tan FL, and Erdo s EG (2002) Kallikreins when activating bradykinin B-2 receptor induce its redistribution on plasma membrane. International Immunopharmacology 2: 17951806. Henderson LM, Figueroa CD, Muller-Esterl W, and Bhoola KD (1994) Assembly of contact-phase factors on the surface of the human neutrophil membrane. Blood 84: 474482. Hermann A, Arnhold M, Kresse H, Neth P, and Fink E (1999) Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme. Biological Chemistry 380: 10971102. Mahabeer R and Bhoola KD (2000) Kallikrein and kinin receptor genes. Pharmacology & Therapeutics 88: 7789. Marceau F, Hess FJ, and Bachvarov DR (1998) The B1 receptor for kinins. Pharmacological Reviews 50: 357386. Marceau F and Regoli D (2004) Bradykinin receptor ligands. Nature Reviews 3: 845847. Neth P, Arnhold M, Nitschko H, and Fink E (2001) The mRNAs of prekallikrein, factors XI and XII, and kininogen, components of the contact phase cascade are differentially expressed in multiple non-hepatic human tissues. Thrombosis and Haemostasis 85: 10431047. Peek M, Moran P, Mendoza N, Wickramasinghe D, and Kirchhofer D (2002) Unusual proteolytic activation of prohepatocyte growth factor by plasma kallikrein and coagulation factor XIa. Journal of Biological Chemistry 277: 4780447809. Plendl J, Snyman C, Naidoo S, Sawant S, Mahabeer R, and Bhoola KD (2000) Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells. Biological Chemistry 381: 11031115. Poblete MT, Garces G, Figueroa CD, and Bhoola KD (1993) Localization of immunoreactive tissue kallikrein in the seromucous glands of the human and guinea-pig respiratory tree. Histochemical Journal 25: 834839. Renne T, Dedio J, Meijers CM, Chung D, and Mu ller-Esterl W (1999) Mapping of the discontinuous H-kininogen binding site of plasma prekallikrein evidence for a critical role of apple domain-2. Journal of Biological Chemistry 274: 2577725784. Stuardo M, Gonzalez CB, Nualart F, et al. (2004) Stimulated human neutrophils form biologically active kinin peptides from high low molecular weight kininogens. Journal of Leukocyte Biology 75: 631640. Vidal MA, Astroza A, Matus CE, et al. (2005) Kinin B2 receptorcoupled signal transduction in human cultured keratinocytes. Journal of Investigative Dermatology 124: 178186.

KERATIN
S H Randell, University of North Carolina, Chapel Hill, NC, USA J L V Broers, Eindhoven University of Technology, Eindhoven, The Netherlands F C S Ramaekers, University of Maastricht, Maastricht, The Netherlands
& 2006 Elsevier Ltd. All rights reserved. role to maintain epithelial integrity. Emerging evidence indicates that keratin intermediate laments modulate additional cellular functions such as receptor-mediated signaling and cellular-protein targeting. Keratins are a dening feature of epithelial cells and the predictable expression pattern in each lung epithelial cell type underlies their use for cell identication. Since keratin fragments are released from tumors and in other pathological conditions affecting epithelial cells, they may also serve as disease markers. Importantly, immunostaining for keratin may assist the differential diagnosis of lung neoplasms.

Abstract
Keratins, represented by more than 50 genes and 20 known polypeptides, are amongst the most abundant proteins of lung epithelial cells. They comprise keratin intermediate laments which, together with microlaments and microtubules, form the cytoskeleton that dynamically maintains epithelial cell structure. The diverse keratin intermediate lament family evolved from a common ancestral gene and shares a common structural plan. Type I and II subtypes of keratin form polymers in an epithelial cell-type-specic manner, which is also inuenced by developmental stage, cellular differentiation, and pathophysiology. Mutations in keratins, while not resulting in a primary pulmonary phenotype, cause genetic diseases, establishing their

Introduction
Early electron micrographs demonstrated brous, detergent-resistant elements extending throughout the cytoplasm of metazoan cells. This network was subsequently termed the cytoskeleton. It is now known to consist of three polymeric structures microlaments (6 nm diameter), microtubules (23 nm diameter), and intermediate laments (10 nm diameter) that serve as an internal scaffold, maintaining cellular shape,

494 KERATIN
Table 1 Intermediate lament classes, genes, and expression patterns (a) Intermediate laments Class Type I Type I hair Type II Type II hair Type III Molecule K9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 23 KRTHA1-8 CHaAKRT K1, 2, 3, 4, 5, 6, 7, 8 Hb1-6, ChHb A, B C, D Vimentin Desmin GFAP Peripherin NF-L, M, H a-internexin Lamin A/C, B1, B2 Phakinin, lensin Cell type Epithelial Hair and nail Epithelial Epithelial Hair and nail Epithelial Fibro, endo, leuko Muscle Glia Peripheral neurons CNS neurons CNS neurons Nucleus, widespread Lens

Type IV Type V Other

(b) Keratin intermediate laments in normal adult lung Pair K4, K13 K5, K14 K8, K18 Distribution Occasional columnar cells Basal cells in pseudostratied epithelium Columnar cells in pseudostratied epithelium, cuboidal bronchiolar cells, and alveolar cells Distribution Columnar cells in pseudostratied epithelium, cuboidal bronchiolar cells, and alveolar cells Basal cells in pseudostratied epithelium Subset of columnar and basal cells in pseudostratied epithelium, cuboidal bronchiolar cells, and alveolar cells

Unpaired K7

K15 K19

Data from Broers JLV, de Leij L, Klein Rot M, et al. (1989) Expression of intermediate lament proteins in fetal and adult human lung tissues. Differentiation 40: 119128; Coulombe PA and Wong P (2004) Cytoplasmic intermediate laments revealed as dynamic and multipurpose scaffolds. Nature Cell Biology 6: 699706; Hesse M, Magin TM, and Weber K (2001) Genes for intermediate lament proteins and the draft sequences of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. Journal of Cell Science 114: 25692575; Oshima RG (2002) Apoptosis and keratin intermediate laments. Cell Death and Differentiation 9: 486492; and Porter RM and Lane EB (2003) Phenotypes, genotypes and their contribution to understanding keratin function. Trends in Genetics 19: 278285.

imparting structural integrity, and regulating motility. Microlaments and microtubules are evolutionarily conserved, similar in many different cell types within an organism, and undergo distinct and rapid processes of regulated, polar assembly and disassembly utilizing nucleotide hydrolysis. In contrast, intermediate laments are highly diverse, and distinct groups of intermediate laments are expressed in different cell types (Table 1). Keratin intermediate laments are a dening feature of epithelial cells and are visible as tonolament bundles in basal cells of the airway epithelium. Keratin proteins are subdivided into two groups and systematically numbered 123 (except 11 and 22) based on molecular weight and isoelectric point. Type I keratins (K9, 10, 1220, and 23) are large and acidic, while type II keratins (K18), are relatively smaller and basic. In addition to the epithelial (soft) keratins given

above, type I and II keratins are further subdivided into hair and nail (hard) type keratins. Unlike microlaments and microtubules, assembly of intermediate laments does not require nucleotide hydrolysis. Turnover is highly variable and is regulated by posttranslational modications including phosphorylation, ubiquitinylation, glycosylation, transglutamination, and protease cleavage. Furthermore, diversity in keratin stability is imparted by variable degrees of disulde bridging, progressively increasing in keratins of stratied epithelia of internal organs, epidermal keratinocytes, and hair and nail keratins, respectively.

Structure
The intermediate lament gene family ranks amongst the 100 largest in the human genome. All functional

KERATIN 495
Head DNA 1A L1 1B Rod Tail 2A L12 L2 2B

Type I/type II protein dimer

Staggered, antiparallel tetramer

Keratin fiber

Figure 1 Keratin structure and lament assembly. The tripartite keratin monomer consists of head, central rod, and tail domains. The a helical segments within the rod (1A, 1B, 2A, 2B) are connected by linker segments (L1, L12, L2). The cross-hatched areas represent the highly conserved domains that drive formation of coiled-coil heterodimers. Two heterodimers in a staggered, reverse orientation create tetramers that, in turn, form linear polymers. Keratin intermediate lament bers consist of four aligned octameric protolaments.

keratin genes are contained within clusters on chromosomes 12q13 and 17q21. There are numerous keratin pseudogenes and gene fragments, many related to keratins 8 and 18, which are spread amongst most human chromosomes. Alignment of the multiple DNA sequences of intermediate lament genes reveals a common tripartite structure. The keratin monomer consists of a conserved central a-helical section, the rod domain, anked by nonhelical head and tail domains of variable length and sequence (Figure 1). The highly conserved ends of the rod domain drive lament assembly via the formation of coiled-coil heterodimers. In turn, two dimers form tetramers and these associate in a staggered, reverse orientation to form linear polymers called protolaments. When a puried type I keratin and a puried type II keratin are mixed in vitro, they spontaneously form laments (Figure 2(a)). Although there may be variability among different intermediate lament types, mature bers apparently consist of four aligned octameric protolaments. Keratin laments in human airway epithelial cells in culture are illustrated in Figure 2(b).

Regulation of Keratin Expression

Keratin intermediate lament expression is inextricably linked to the formation of an epithelial sheet and to its type and differentiation state. A simple epithelium, made up of a single layer of polarized cells expressing keratins 8 and 18, rst appears during early embryogenesis. These keratins continue to be expressed in all simple epithelia, which may then acquire additional keratins characteristic of the particular epithelium. For example, the simple cuboidal bronchiolar epithelium and type II epithelial cells express keratins 7, 8, 18, and 19. Complex epithelia, such as the pseudostratied tracheobronchial epithelium, express characteristic keratin pairs and these pairs systematically vary according to cell type. Typically, keratins 5, 14, and 15 are present in the basal cells and gland myoepithelial cells, while keratins 7, 8, 18, and 19 predominate in columnar cells and gland acinar cells. Images of keratins in lung cells are shown in Figure 3. Detailed expression of keratins in normal and pathological lung has not been comprehensively documented as yet.

496 KERATIN

Figure 2 Keratin intermediate laments in vitro and in cells. (a) Negative stain electron micrograph illustrating spontaneous reconstitution of laments upon mixing keratin 5 and keratin 14 proteins in vitro. Reproduced from Coulombe PA and Omary MB (2002) Hard and soft principles dening the structure, function and regulation of keratin and intermediate laments. Current Opinion in Cell Biology 14: 110122, with permission from Elsevier. (b) Confocal light microscope image of human airway epithelial cells in culture. Keratin intermediate laments were detected with an anti-pan cytokeratin antibody (green), actin was detected with phalloidin (red), and the nucleus was stained with DAPI (blue).

Figure 3 Keratin expression in human lungs. Immunoperoxidase staining with different antikeratin antibodies as indicated. Magnication 400.

Transcriptional control of keratin gene expression in developing and mature epithelia has been extensively studied, using both in vitro cell models and transgenic mice. However, the complex mechanisms regulating tissue- and cell-type-specic expression of keratins are not fully understood. Furthermore, keratin expression is modulated by developmental stage

and physiologic or pathologic conditions. A dramatic example is development of squamous metaplasia in the airway epithelium as a function of chronic injury, carcinogen exposure, or vitamin A deciency. In this case, the keratin 4 and 13 pair, normally found in stratied oral, genital, and esophageal epithelia, is dramatically induced in the pathological airway.

KERATIN 497

Biological Function
Early investigators suggested that the cytoskeleton, including intermediate laments, functioned to maintain cellular shape and integrity, a theory subsequently proven by the phenotype of humans and mice with genetic abnormalities of keratin. Disruption of keratin 5 and 14 laments, found in basal cells, causes extreme mechanical fragility of the epidermis, resulting in blistering skin disorders collectively known as epidermolysis bullosa simplex. Nearly 20 human genetic disorders of keratin have been discovered. In addition, a large and growing panel of keratin mutants, knockouts, and double knockouts has been created in mice. In humans and mice, the pathologic consequences depend upon both, the expression pattern of the mutated keratin and the specic defect. Disruptive mutations may result in a more severe phenotype than complete absence, presumably due to functional redundancy in which an alternative keratin replaces a missing heteromeric partner. The spectrum of genetic alterations and their resulting phenotypes has provided key insights regarding keratin structure and function, rmly establishing the role of keratin intermediate laments in maintaining epithelial integrity. The diverse phenotypes of mice and humans with genetic alterations in keratins have suggested additional functions for keratin intermediate laments. Keratin 8 is important for placental function. Keratin 8 gene-deleted mice surviving after birth develop colorectal abnormalities including loose stools and colonocyte hyperplasia, as well as subtle liver pathology that becomes more evident upon liver stress and regeneration. Cells from keratin 8- and 18-decient animals are strikingly more sensitive to the pro-apoptotic effects of tumor necrosis factor (TNF). Keratin 8 and 18 intermediate laments bind to the TNF receptor 2, indicating a likely role in the modulation of TNF signaling. Physical association of laments with both receptors and signaling intermediates is likely a general mechanism for modulating diverse signal transduction pathways. The intestinal symptoms of keratin 8/18 deciency are linked to ion transport abnormalities and reect the importance of keratin intermediate laments to facilitate protein-targeting to the correct plasma membrane domain. Additionally, keratins are targets for, and participate in, the compartmentalization of caspases during apoptosis. Keratins may sequester growth-modulating proteins during physiologic regulation of the cell cycle, thereby participating in homeostatic cell turnover and regulation of proliferation during wound healing or in transformed cells. Although correlates of these functions have not been explicitly studied in lung cells, analogous roles are likely.

Interacting Molecules
There is a long and growing list of proteins that interact with keratins, falling into broad functional categories related to whether they are involved in intermediate lament synthesis and assembly (e.g., HSPs), cross-linking (e.g., laggrin), attachment to the plasma membrane, nucleus or other cell structures (e.g., desmoplakin), or in modulation of signaling pathways (e.g., TNFR2). The human type I keratin gene cluster is interrupted by a domain of multiple, structurally related genes encoding keratinassociated proteins, but these are mostly involved in disulde bridging of hair keratins.

Keratin in Respiratory Diseases

Genetic diseases due to abnormalities in keratin 5 and 14 (epidermolysis bullosa simplex) profoundly affect the skin, whereas alterations in keratin 8 and 18 primarily predispose to liver (cryptogenic cirrhosis) and bowel disease. In epidermolysis bullosa, upper airway pathology is not uncommon and manifests as hoarseness and inspiratory stridor, sometimes progressing to laryngotracheal stenosis and obstruction, which can be life threatening. To our knowledge, there are no reports of pulmonary manifestations of genetic diseases of keratins 8 and 18. Other known diseasecausing mutations affect keratins that are not highly expressed in lung, and thus are not expected to result in a pulmonary phenotype. In the gut, pathogenic salmonella strains may utilize keratins as scaffolds for cell-type-specic intracellular invasion and, in the lung, keratin 13 has been reported as a receptor for the cable pilus of Burkholderia cenocepacia, an epidemic pathogen of cystic brosis patients. There are reports that the presence of keratin fragments in bronchopulmonary lavage uid may be used to monitor disease intensity in chronic bronchitis, bronchiectasis, panbronchiolitis, idiopathic pulmonary brosis, alveolar proteinosis, and lung allograft rejection. Chronic airway and lung injury results in progressive epithelial changes, including modulation of keratin expression. As noted previously, a prominent example is induction of keratin 4 and 13 accompanying the development of squamous metaplasia. As metaplasia progresses to atypia and dysplasia during multistage carcinogenesis, changes in keratin expression are prominent; keratins have become important in the differential diagnosis and monitoring of thoracic neoplasms. Briey, all lung adenocarcinomas express keratins found in normal simple, cuboidal epithelial cells, namely keratins 7, 8, 18, and 19, and some express keratin 4. Squamous carcinomas show greater complexity in their keratin-expression pattern and may additionally

498 KERATINOCYTE GROWTH FACTOR

express keratins 5, 6, 10, 13, 14, 15, and 17. Small cell carcinomas of the lung and carcinoid tumors typically express low levels of keratin 8, 18, and 19, while cytokeratin 7 is strikingly absent in these tumors. Mesothelioma expresses keratins 5, 7, 8, 18, and 19, and some express keratins 4, 6, 14, and 17. Examination of keratin expression has proven useful to solve difcult diagnostic problems in lung cancer. Keratin 5/6 is amongst a panel of immunochemical markers discriminating between mesothelioma and adenocarcinoma. The combined use of keratin 7 (present) and 20 (absent) may help distinguish primary lung adenocarcinomas versus metastases from other sites, and in combination with other markers, may help to distinguish even highly related tumors such as pulmonary versus metastatic GI carcinoid or endocrine tumors. In addition, detection of keratin fragments in the serum of lung cancer patients may be of prognostic importance. Tumor growth is accompanied by increased apoptosis, and during apoptosis, keratins are cleaved in a highly specic manner, generating stable protein fragments detectable in serum. The CYFRA21.1 test detects keratin 19 fragments and appears to have value as a covariable for monitoring response to treatment and predicting outcome in non-small cell lung cancer.
See also: Mesothelioma, Malignant. Tumors, Malignant: Bronchogenic Carcinoma; Bronchial Gland and Carcinoid Tumor; Carcinoma, Lymph Node Involvement.

Further Reading
Broers JLV, de Leij L, Klein Rot M, et al. (1989) Expression of intermediate lament proteins in fetal and adult human lung tissues. Differentiation 40: 119128.

Broers JLV, Ramaekers FCS, Klein Rot M, et al. (1988) Cytokeratins in different types of human lung cancer as monitored by chain-specic monoclonal antibodies. Cancer Research 48: 32213229. Coulombe PA and Omary MB (2002) Hard and soft principles dening the structure, function and regulation of keratin and intermediate laments. Current Opinion in Cell Biology 14: 110122. Coulombe PA and Wong P (2004) Cytoplasmic intermediate laments revealed as dynamic and multipurpose scaffolds. Nature Cell Biology 6: 699706. Hesse M, Magin TM, and Weber K (2001) Genes for intermediate lament proteins and the draft sequences of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. Journal of Cell Science 114: 25692575. Ordonez NG (2003) The immunohistochemical diagnosis of mesothelioma: a comparative study of epithelioid mesothelioma and lung adenocarcinoma. American Journal of Surgery and Pathology 27: 10311051. Oshima RG (2002) Apoptosis and keratin intermediate laments. Cell Death and Differentiation 9: 486492. Owens DW and Lane EB (2003) The quest for the function of simple epithelial keratins. Bioessays 25: 748758. Porter RM and Lane EB (2003) Phenotypes, genotypes and their contribution to understanding keratin function. Trends in Genetics 19: 278285. Pujol JL, Molinier O, Ebert W, et al. (2004) CYFRA 21-1 is a prognostic determinant in non-small-cell lung cancer: results of a meta-analysis in 2063 patients. British Journal of Cancer 90: 20972105. Schaafsma HE and Ramaekers FCS (1994) Cytokeratin subtyping in normal and neoplastic epithelium: basic principles and diagnostic applications. Pathology Annual 29: 2162. Schutte B, Hening M, Koelgen W, et al. (2004) Keratin 8/18 breakdown and reorganization during apoptosis. Experimental Cell Research 297: 1126. Strelkov SV, Herrmann H, and Aebi U (2003) Molecular architecture of intermediate laments. Bioessays 25: 243251. Tot T (2002) Cytokeratins 20 and 7 as biomarkers: usefulness in discriminating primary from metastatic adenocarcinoma. European Journal of Cancer 38: 758763.

KERATINOCYTE GROWTH FACTOR

R J Mason and Y Chang, National Jewish Medical and Research Center, Denver, CO, USA
& 2006 Elsevier Ltd. All rights reserved. toxic chemotherapeutic agents used to prevent rejection in allogeneic bone marrow transplantation. In vitro KGF stimulates proliferation and differentiation of alveolar type II cells. KGF signals through a tyrosine kinase receptor (KGFR, FGFR2-IIIb) and activates several signal cascades, including ERK, Akt, and c-jun N-terminal kinase (JNK) as well as PAK4. The KGFR dominant negative mice have essentially no lungs, whereas the lungs of KGF knockout mice are relatively normal. These observations have been interpreted as indicating that FGF-10, not FGF-7, is required for fetal lung development and that FGF-10 can replace FGF-7 in the FGF-7 null animal. KGF has the potential as an important treatment for acute lung injury, and studies on the mechanism of how KGF increases differentiation in vitro may provide new therapeutic targets for the treatment of many lung diseases.

Abstract
Keratinocyte growth factor (KGF); also designated broblast growth factor-7 (FGF-7) and similar to FGF-10, is a mitogen produced primarily by broblasts and is a potent growth factor for alveolar and bronchial epithelial cells. The special significance of KGF is that it can prevent lung injury due to a variety of insults, if given before the injury. There have been no denitive reports of KGF effects given solely after lung injury. KGF is an approved growth factor for the treatment of mucositis due to

KERATINOCYTE GROWTH FACTOR 499

Introduction
Keratinocyte growth factor (KGF); also designated broblast growth factor-7 (FGF-7) is a potent relatively specic growth factor for epithelial cells produced primarily by broblasts. FGF-7 is very similar in its action to FGF-10, which is also referred to as KGF-2. Both use the same tyrosine kinase receptor (FGFR2-IIIb). Human recombinant KGF is available for the treatment of mucositis produced by toxic chemotherapeutic agents and is marked under the name of palifermin. The importance of KGF for lung biology is that it is a potent mitogen for normal alveolar type II cells as well as for bronchial epithelial cells and microvascular endothelial cells and has been used to prevent lung injury, if given before a variety of insults. It is not known if KGF can be developed to use effectively in the treatment of acute lung injury after the injury has occurred. KGF was cloned from fetal lung broblasts in the search for growth factors for epithelial cells. KGF was rst shown to be a potent mitogen for rat alveolar type II cells in vitro. These initial studies were performed to determine the mitogenic factors for type II cells in broblast-conditioned medium. The two growth factors identied were KGF and hepatocyte growth factor (HGF). Soon thereafter, recombinant human KGF was shown to induce proliferation of rat type II cells when it was instilled into the normal lung in vivo. KGF instillation causes a marked increase in BdrU labeling 2 and 3 days after instillation and increases the expression of sufactant protein A (SP-A), SP-B, SP-C, and SP-D. In contrast, KGF decreases the expression of the bronchiolar epithelial protein CC-10. KGF also increases the expression of the Na/K-ATPase, which is the pump for the transepithelial sodium transport and is important for transporting uid from the alveolar into the interstitial compartment, where it can be cleared by lymphatics. In other studies, KGF was also shown to stimulate the proliferation of bronchial epithelial cells and microvascular endothelial cells. Because KGF can cause the proliferative effect of both the epithelial and endothelial components of the alveolar wall, it is a potentially important therapeutic agent for the treatment of acute lung injury.

However, it is not known if gdT cells in the lung produce KGF. KGF (FGF-7) has significant homology to FGF-10 (KGF-2) (see Figure 1).

Biologic Function of KGF

Effects of KGF on the Lung In vivo

Structure and Regulation of Production of KGF

KGF is a small glycosylated heparin-binding 26 28 kDa protein. It is produced by broblasts, and secretion by broblasts can be stimulated by IL-1b and PDGF and inhibited by dexamethasone. KGF is also produced by gdT cells in the skin and intestine.

KGF can stimulate epithelial proliferation if administered intratracheally, intravenously, or subcutaneously, although the largest effect is seen after intratracheal administration. The effect is not species restricted, in that rats and mice also respond to human recombinant KGF. The response in the rat appears to be more marked than the effect in mice or with human type II cells in vitro. Marked type II cell hyperplasia occurs with an adenovirus overexpressing KGF or with conditional expression of KGF in transgenic mice (see Figure 2). KGF is also a major mitogen for keratinocytes, hair follicles, the epithelium lining the GI tract, and presumably any other epithelial tissue. The epithelial cells express KGFR2IIIb (the high-afnity receptor for KGF), but do not express KGF. The effect on the epithelium is a paracrine effect, whereby local mesenchymal cells produce KGF and regulate the local epithelial population. This occurs in the lung, the skin, the gastrointestinal track, and the prostate. Numerous studies have shown KGF to offer a preventative treatment for acute lung injury. Unfortunately, KGF administration has only been shown to be effective to protect against a given injurious agent if given before the insult, and postinjury treatment regimens have been ineffective. However, it is very effective as a pretreatment. KGF prevents lung injury due to oxygen, bleomycin, acid instillation, radiation, and a variety of cytotoxic agents. For example, instillation of bleomycin into rats reduces the total capacity by 25%; this effect is entirely blocked by pretreatment with KGF for 48 or 72 h but not by saline pretreatment. Bleomycin markedly reduces SP-C expression 1 or 7 days after instillation; this effect is completely inhibited by pretreatment with KGF for 48 h but not by saline pretreatment. KGF also prevents pulmonary complications of allogenic bone marrow transplantation. For injuries that are slow (oxygen toxicity), KGF can be given at the onset of injury; for injuries that take 12 days to develop (bleomycin injury), KGF needs to be given 48 h prior to the insult; and for injuries that cause immediate damage (HCl instillation), KGF needs to be given 3 days prior to the insult. It seems that the alveolar epithelium is maximally protected about 3 days after intratracheal instillation. Maximum proliferation also occurs 23 days after instillation. Time will tell

500 KERATINOCYTE GROWTH FACTOR

KGF receptor Heparan sulfate

KGF KGF

PIP3

PIP2

PIP3

RAS S p O grb2 Raf S

SHP2

FRS2

p
grb2 PDK-1 P85 P110

PLC

MEK1/2

MLK3

MEKK1/4

DAG P13K MKK3/6 MKK4/7 P70S6K ERK 1/2 p 38 p JNK ? SREBP1 ?

Figure 1

pH PKB/ AKT PKC

FAS SCD-1 E-FABP ? ? ?

GSK-3

C/EBP

Normal alveolar epithelium

KGF

Hyperplastic alveolar epithelium

Figure 2 KGF stimulates type II cell hyperplasia. The normal alveolar surface is covered primarily by type I cells, and there is a low rate of cell proliferation by the progenitor type II cells. However, after KGF treatment type II cells undergo rapid cell division and form an alveolar epithelium composed primarily of hyperplastic type II cells. This is readily seen in the rat lung, and there may be a less dramatic response in other species. After a week without additional stimulation by KGF the hyperplastic type II cells undergo apoptosis, and the lung appears normal.

if a treatment regimen can be developed for clinical human acute lung injury. Since the injury is usually ongoing, there appears to be a window of opportunity if the drug is given early. In addition, in bleomycin lung injury, KGF and HGF protein increase in lavage. In our studies, we were not able

to demonstrate a convincing increase in KGF mRNA after a variety of lung injuries. Importantly, KGF can also enhance postpneumonectomy lung growth. As contrasted with epidermal growth factor (EGF) and retinoic acid, KGF can induce formation of new alveoli. It should be noted that KGF also has the

KERATINOCYTE GROWTH FACTOR 501

capability of inducing autocrine growth factor signals in epithelial cells. In rat alveolar type II cells and keratinocytes, KGF increases the expression of EGF ligands such as transforming growth factor alpha (TGF-a), heparin-binding epidermal growth factor (HB-EGF), and amphiregulin.
Effects of KGF on Lung Cells In Vitro

KGF has marked effects on type II cells in vitro. It causes more type II cell proliferation than any other growth factor tested to date. The mitogenic effect of KGF can be blocked by TGF-b. KGF also induces type II cell differentiation in vitro. This is likely related to proliferation and an increase in the cuboidal cytoarchitecture of type II cells, which appears to be critical for type II cell differentiation, but the effect is also seen on matrigel where the cells are already cuboidal. KGF increases type II cell differentiation, as measured by secretion of SP-A and SP-D much more effectively than other growth factors such as HGF, FGF-10, and HB-EGF. KGF signals through Akt and c-jun N-terminal kinase (JNK) to increase a variety of transcription factors. Our laboratory has focused on the ability of KGF to stimulate lipogenesis in type II cells. KGF increases SREBP-1c and C/EBP a and d, which in turn increases the expression of lipogenic enzymes specifically acetyl CoA carboxylase, ATP-citrate lyase, fatty acid synthase, stearoyl CoA desaturase, and glycerol3-P acyltransferase. KGF also increases SP-A, SP-B, SP-C, and SP-D expression through other pathways.

through the KGFR (FGFR2-IIIb). For rat type II cells, the concentration of FGF-10 required for maximal thymidine incorporation is about 500 ng ml 1, whereas the concentration for maximal activity of KGF is 510 ng ml 1. FGF-10 binds heparin and presumably heparin sulfate proteoglycans more avidly than KGF. Heparin can stimulate mitogenic activity of FGF-10, whereas it can inhibit the mitogenic activity of KGF. FGF-10 binds to cells and extracellular matrix avidly and is not released easily into culture medium. FGF-10 likely has a very local effect, whereas some KGF may circulate. In the fetal lung, FGF-10 also causes more migration but less proliferation than KGF. FGF-10 is also being investigated as a therapeutic agent under the name repifermin.

Role of KGF in Respiratory Disease

KGF has the potential to be very important in the treatment of acute lung injury. However, its potential role has not been validated in studies on humans. KGF protects rodent lungs from a variety of acute lung injuries, if it is administered before the injury. However, postinjury treatment regimens have not been reported. The apparent cytoprotection probably requires a time of 23 days and the stimulation of type II cell proliferation. KGF increases rapidly and dramatically in the dermis after cutaneous wound healing. However, the increase in KGF mRNA or protein levels in the rodent lung after injury has been difcult to demonstrate. We know relatively little about KGF levels in human lung diseases. It is also not clear if KGF-decient animals are more susceptible to lung injury. There is much more to learn about the role of KGF and FGF-10 in human lung diseases.
See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Fibroblast Growth Factors. Stem Cells.

Receptors
KGF binds to the KGFR in association with heparan sulfate proteoglycans, such as syndecan-1. The KGFR contains an extracellular domain composed of three immunoglobulin-like loops, a transmembrane segment, and an intracellular domain with tyrosine kinase activity. Its third IgG loop is encoded by two exons, IIIa and IIIb, producing a receptor with afnity for KGF, FGF-10, FGF-1, and FGF-3. The KGFR signals through several pathways, including ERK, JNK, Akt, and PAK 4. However, the precise signaling pathways for the divergent functions of KGF are not dened. Since the biologic effects of KGF take 2428 h, numerous signaling pathways and a host of transcription factors and coactivators are likely involved. SU 5402 is an FGF receptor kinase inhibitor that can block the effect of KGF.

Further Reading
(2004) Palifermin: AMJ 9701, KGF-Amgen, recombinant human keratinocyte growth factor, rHu-KGF. Drugs in R&D 5: 351 354. auf demKeller U, Krampert M, Kumin A, Braun S, and Werner S (2004) Keratinocyte growth factor: effects on keratinocytes and mechanisms of action. European Journal of Cell Biology 83: 607612. Bao S, Wang Y, Sweeney P, et al. (2005) Keratinocyte growth factor induces akt kinase activity and inhibits fas-mediated apoptosis in A549 lung epithelial cells. American Journal of Physiology: Lung Cellular and Molecular Physiology 288: L36L42. Danilenko DM (1999) Preclinical and early clinical development of keratinocyte growth factor, an epithelial-specic tissue growth factor. Toxicologic Pathology 27: 6471.

Properties of FGF-10
FGF-10 (KGF-2) is similar to KGF (FGF-7) but differs from it in some important respects. It also signals

502 KININS AND NEUROPEPTIDES / Bradykinin

Deterding RR, Havill AM, Yano T, et al. (1997) Keratinocyte growth factor prevents bleomycin lung injury in rats. Proceedings of the Association of American Physician 109: 254268. Finch PW and Rubin JS (2004) Keratinocyte growth factor/broblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Advances in Cancer Research 91: 69136. Kaza AK, Kron IL, Leuwerke SM, Tribble CG, and Laubach VE (2002) Keratinocyte growth factor enhances post-pneumonectomy lung growth by alveolar proliferation. Circulation 106: I120I124. Mason RJ, Pan T, Edeen KE, et al. (2003) Keratinocyte growth factor and the transcription factors C/EBP{alpha}, C/EBP{delta}, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells. Journal of Clinical Investigation 112: 244255. Morikawa O, Walker TA, Nielsen LD, et al. (2000) Effect of adenovector-mediated gene transfer of keratinocyte growth factor on the proliferation of alveolar type II cells in vitro and in vivo. American Journal of Respiratory Cell and Molecular Biology 23: 626635. Panos RJ, Bak PM, Simonet WS, Rubin JS, and Smith LJ (1995) Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats. Journal of Clinical Investigation 96: 20262033. Panos RJ, Rubin JS, Aaronson SA, and Mason RJ (1993) Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in broblast-conditioned medium. Journal of Clinical Investigation 92: 969977. Ulich TR, Yi ES, Longmuir K, et al. (1994) Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. Journal of Clinical Investigation 93: 12981306. Ware LB and Matthay MA (2002) Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inammation, and repair. American Journal of Physiology: Lung Cellular and Molecular Physiology 282: L924L940. Werner S (1998) Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Reviews 9: 153165. Yano T, Deterding RR, Simonet WS, Shannon JM, and Mason RJ (1996) Keratinocyte growth factor reduces lung damage due to acid instillation in rats. American Journal of Respiratory Cell and Molecular Biology 15: 433442. Yano T, Mason RJ, Pan T, et al. (2000) KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in the adult rat lung. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L1146L1158. Yi ES, Williams ST, Lee H, et al. (1996) Keratinocyte growth factor ameliorates radiation- and bleomycin-induced lung injury and mortality. American Journal of Pathology 149: 19631970. Zhang F, Nielsen LD, Lucas JJ, and Mason RJ (2004) Transforming growth factor-beta antagonizes alveolar type II cell proliferation induced by keratinocyte growth factor. American Journal of Respiratory Cell and Molecular Biology 31: 679686.

KININS AND NEUROPEPTIDES

Contents

Bradykinin Neuropeptides and Neurotransmission Tachykinins Vasoactive Intestinal Peptide Other Important Neuropeptides

Bradykinin
J Eddleston, S C Christiansen, and B L Zuraw, University of California San Diego, La Jolla, CA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Bradykinin (BK) and Lys-BK are vasoactive peptides generated via the cleavage of kininogen by the action of kallikrein proteases. Both have a half-life of only seconds within the circulation, being rapidly hydrolyzed to biologically active des-Arg kinin derivatives. BK and Lys-BK cause the classical signs of inammation redness, local heat, swelling, and pain whereas their des-Arg derivatives induce increased collagen synthesis, broblast proliferation, and cytokine release from macrophages. The effects of these peptides are mediated by two distinct G-coupled receptors. The kinin system has been implicated in

the pathophysiology of human airway inammation. Inhalation of BK by asthmatics results in chest tightness, cough, and bronchoconstriction. Furthermore, increased levels of BK and Lys-BK, their derivatives, and tissue kallikrein are observed within the human airway following allergen provocation, with levels of BK correlating with symptom severity. Respiratory viral infections are also associated with increase levels of BK and Lys-BK within the airway. Research has demonstrated that BK is intricately involved in the pathogenesis of allergic airway inammation and respiratory viral infections; as such, therapeutic methods for intervening in the activation of the kinin system may alleviate airway inammation and symptoms of respiratory infections.

Introduction
A substance in urine that was capable of causing prolonged hypotension when injected into animals

KININS AND NEUROPEPTIDES / Bradykinin 503

was identied in the early 1900s and named kallidin (Lys-bradykinin). The enzyme required for its generation was called kallikrein (named after the Greek word for pancreas). In 1940, Rocha e Silva et al. discovered an enzyme in snake venom that generated a smooth muscle contracting factor in plasma that they labeled bradykinin (BK). The primary structure of BK, however, was not unraveled until the 1960s. A series of elegant pharmacologic studies dened two distinct kinin receptors whose agonists were BK and its desArg product (des-Arg-BK), respectively. Molecular analysis of the kallikreinkinin system has conrmed the general organization described previously, whereas the diverse biological effects of kinins have been claried by functional and genetic studies. Kinins have a range of benecial actions, including effects on insulin sensitivity, skeletal muscle function, uid balance, vascular tone, and cardioprotection. As is the case for other potent bioactive substances, however, kinins can mediate adverse effects on the host when present either inappropriately or in excessive concentrations. The control of kinin generation, degradation, and receptor interactions is therefore central to understanding the role of kinins in respiratory disease.

Structure, Production, and Regulation

Kinins are positively charged vasoactive peptides generated by proteolytic cleavage of the b2-globulin kininogen. Two human kininogens, high-molecularweight kininogen (HMWK) and low-molecular-weight kininogen (LMWK), are synthesized via alternative splicing from a single gene and circulate as single-chain glycoproteins. The primary sequences of HMWK (115 kDa) and LMWK (68 kDa) are identical from the N terminus through the BK moiety (heavy chain)
Exon 5 Kininogen NH2 Kallikrein 380 1

but differ at the C terminus (light chain). The common heavy chain mediates cysteine protease inhibitory activity, whereas the light chain of HMWK (but not LMWK) mediates procoagulant activity. Kinin is generated following cleavage of kininogen by one of several serine proteases, termed kallikreins (Figure 1). Plasma kallikrein circulates in plasma as the zymogen prekallikrein primarily bound to the light chain of HMWK. During contact system activation, prekallikrein is cleaved into active plasma kallikrein, which then cleaves HMWK at two sites releasing BK. Plasma kallikrein is efciently inhibited by C1 inhibitor and, to a lesser extent, by a2-macroglobulin. A separate and distinct family of tissue kallikreins is widely distributed in mammalian tissues. At least one of these tissue kallikreins (hK1) has kininogenase activity and releases Lys-BK (kallidin) from LMWK or HMWK. hK1 is synthesized as a preprokallikrein protein that is sequentially cleaved to generate active tissue kallikrein. Once activated, tissue kallikrein is relatively resistant to inhibition. BK and Lys-BK are rapidly hydrolyzed by peptidases called kininases. In blood, the half-life of kinin is estimated to be only a few seconds, and BK is essentially completely cleaved during one passage through the pulmonary circulation. As such, BK is active only close to the site of formation in a paracrine manner, acting as a local hormone (autocoid). The major kininases in the lung are carboxypeptidase N (kininase I), angiotensin-converting enzyme (kininase II), neutral endopeptidase (enkephalinase), and aminopeptidases. Of particular importance is the fact that carboxypeptidase processes BK and Lys-BK via the removal of the N-terminal arginine to produce the biologically active kinins des-Arg9-BK and Lysdes-Arg9-BK, respectively.
10 11 3 COOH 389

2 3 4 5 6 7 8 9

Lysyl-Bradykinin (tissue kallikrein + LMWK ) Kinins 1 Bradykinin (plasma kallikrein + HMWK ) 2 3 4 5 6 7 8 9

Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Kininases

Aminopeptidase M

Carboxypeptidase N Neutral endopeptidase (kininase I ) (enkephalinase) Angiotensin I-converting enzyme (kininase II )

Figure 1 Generation and metabolism of kinins. The kininogen gene is alternatively spliced to yield HMWK (exons 110) or LMWK (exons 19, 50 end of exon 10, and exon 11). Kininogen is cleaved by kallikrein yielding kinins, which are then metabolized by kininases.

504 KININS AND NEUROPEPTIDES / Bradykinin

Biological Function
In the 1960s, Elloit and colleagues demonstrated that synthetic BK injected into human or animal tissue reproduced the four classical signs of inammation: redness, local heat, swelling, and pain. The redness and local heat can be attributed to BK-induced local endothelium-dependent vasodilation, whereas the swelling is due to increased vascular permeability. BK also causes pain and triggers the autonomic nervous system as a result of activation of afferent neurons. In addition, BK causes the contraction of several types of smooth muscle, including human small bronchi and strips of human colon and urinary bladder. Furthermore, stimulation with BK can result in increased intracellular levels of arachidonic acid, eicosanoids, diacylglycerol (DAG), inositol-1,4,5triphosphate (IP3), cAMP, and Ca2 . BK has been shown to activate multiple phospholipases (PLs), including PLA2 (leading to arachidonic acid release and eicosanoid generation), PLC (leading to generation of DAG and IP3), and PLD. DAG can subsequently activate protein kinase C (PKC), whereas IP3 can release intracellular Ca2 stores. BK stimulation is associated with activation and membrane translocation of multiple PKC isoforms, including the Ca2 -independent PKCe and the atypical PKCz isoforms. It has also become clear that BK can contribute to inammation by activating transcription factors such as nuclear factor kappa B and activation protein 1. BK and Lys-BK in general share the same spectrum of biologic activity. The des-Arg-derivatives of BK and Lys-BK, however, do not induce signs of acute inammation or smooth muscle contraction. They do activate many of the same intracellular signaling pathways as BK, share some of the vascular effects of BK, and have also been found to mediate increased collagen synthesis, broblast proliferation, and cytokine release from macrophages (Figure 2).

SMC contraction, mucus secretion, neuronal activation, vascular effects, inflammation

B2 BK receptor

Plasma Kallikrein

BK

Lys-BK kininase I Lys-des-Arg-BK

Tissue Kallikrein

des-Arg-BK

B1 BK receptor

Vascular effects, inflammation, cell proliferation

Figure 2 Biological effect of kinins. BK and Lys-des-Arg-BK act through the B2R and transduce the classic kinin effects. Their des-Arg metabolites act through the B1R and transduce vascular effects as well as effects on inammation and cell growth.

Bradykinin Receptors
Kinins exert their biological effects by stimulating seven-transmembrane domain G-protein-coupled receptors (GPCRs). Two distinct kinin receptors, termed B1 and B2 receptors (B1R and B2R, respectively), were characterized by Regoli and co-workers in the 1970s. Tissues that responded to des-Arg9-BK or Lys-des-Arg9-BK better than BK or Lys-BK were considered to have B1R, whereas tissues that responding to BK or Lys-BK better than the des-Arg derivatives were considered to have B2R. These predictions were subsequently conrmed by cloning of two distinct genes for the B1R and the B2R that map

in close proximity to each other on human chromosome 14 but are only distantly related, with their amino acid sequence sharing 36% identity. The B1R gene has a single exon, whereas the B2R gene is composed of three exons separated by two introns, with the third exon encoding the receptor. In the noncoding region, several SNPs have been found that affect the transcriptional rate of the receptors. Coupling of B2R and B1R to hererotrimeric G proteins and the downstream signaling pathways that are activated appear to be tissue and cell specic. BK has also been shown to bind to the orphan GPCR, GPR100, although the significance of this is uncertain. B2R appear to be constitutively expressed and mediate most of the classic inammatory action of BK, whereas B1R are generally absent from normal tissue but are rapidly induced following certain types of injury. In vitro, B1R numbers are induced in many tissues by a number of factors, the most potent of which is interleukin (IL)-1b. Upregulation of B1R expression has also been shown to occur in vivo in inamed tissues isolated from human subjects. Following ligand stimulation, B2R show specic and rapid internalization and desensitization. B1R, however, display high basal activity even in the absence of ligand and do not show appreciable internalization or desensitization. Thus, the function of B1R during inammatory insult may be to amplify the tissue effects of kinins.

Bradykinin in Respiratory Disease

The pluripotent activities of kinin make it an attractive candidate for participating in the pathogenesis of respiratory tract disease. BK is a potent

KININS AND NEUROPEPTIDES / Bradykinin 505

bronchoconstrictor ex vivo, causing a biphasic response consisting of a brief contraction followed by sustained relaxation. Indeed, symptomatic and physiologic changes following BK challenge have suggested a key role for the kinin system in asthma and allergic rhinitis. Nasal insufation of BK causes acute symptoms of nasal congestion, drainage, and throat irritation, and inhalation of BK in asthmatic but not normal subjects results in cough, chest tightness, and measurable decreases in ow rates indicative of bronchoconstriction. These effects appear to be mediated in large part by neural mechanisms with involvement of both vagal cholinergic and sensory C bers. Other cardinal features of asthma induced by BK include mucus hypersection and airway edema. In subjects with perennial asthma, airway responsiveness to BK was found to correlate with the intensity of eosinophilia in bronchial biopsies, an accepted marker of asthma severity. The significance of these effects is supported by direct evidence of kinin generation in the airway during allergic inammation and other respiratory diseases. Proud and colleagues rst reported that BK and Lys-BK were generated in nasal secretions following allergen provocation, and they showed that the kinin levels correlated with reported symptoms and release of mast cell mediators. Elevated levels of both tissue kallikrein activity and kinins were found by Christiansen et al. in the bronchoalveolar lavage uid of patients with active asthma or chronic bronchitis. Furthermore, local segmental challenge with relevant allergen in allergic asthmatics increased tissue kallikrein and kinin levels in conjunction with histamine release at both immediate and late-phase time points. Kinins are also generated during naturally occurring rhinitis. Autoradiographic and immunohistochemical studies have conrmed the expression of B2R on a variety of airway structural cells, including epithelial cells, broblasts, and smooth muscle cells. Although airway challenge with B1R agonists results in a conspicuous lack of acute effects, B1R have been shown to acquire an increasingly primary role during chronic inammation in a variety of animal models. Indeed, allergic rhinitis subjects, but not normal subjects, were found to express B1R in nasal tissue, and allergen challenge further increased nasal expression of B1R mRNA. Nasal challenge with the B1R agonist Lys-des-Arg9-BK in subjects with mild allergic rhinitis activated the ERK MAPK in allergic rhinitis but not in normal subjects, suggesting that the B1R expressed in nasal tissue is functional and may participate in human airway inammation. Subsequent animal studies have supported these human results. Murine tracheal explants display enhanced

contractile responses to des-Arg9-BK and BK following long-term culture with IL-4, a signature cytokine for allergic inammation. This effect was mediated by upregulation of B1R. Furthermore, allergen-induced bronchial hyperresponsiveness was shown to parallel B1R upregulation in Brown Norway rats and was partially attenuated by the B1R antagonist des-Arg10-Hoe-140. Several lines of evidence support a role for kinins in the propagation of airway inammation beyond their direct effects on permeability, mucus production, and smooth muscle contraction. Kinins activate inammatory cells, thereby amplifying the release or production of other mediators of asthma, notably histamine, lipid-derived mediators, and neuropeptides. Treatment of moderate asthmatics for 4 weeks with an inhaled B2R antagonist (HOE-140) resulted in a gradual dose-dependent increase in FEV1, suggesting an anti-inammatory effect resulting from the BR blockade. HOE-140 has also been shown to abrogate the allergen-induced increases in nasal histamine hyperresponsiveness and the appearance of eosinophils, ECP, kinin, and IL-8 in lavage uid in allergic rhinitis patients. Conversely, nasal BK challenge selectively upregulated in vivo expression of the chemokine receptors CXCR1 and CXCR2 in allergic rhinitis subjects. Kinins may also exert effects on cell growth, and treatment with B2R antagonists has been shown to result in the killing of small cell lung carcinoma cells in vitro. The kinin system may also have an important role in infectious diseases affecting the respiratory tract. Objective signs of respiratory disease in ferrets inoculated with inuenza virus corresponded with the appearance of kinins in the nasal lavages, and airway hyperresponsiveness following parainuenza3 infection in guinea pig correlated with generation of BK. Furthermore, experimental rhinovirus infections in human subjects resulted in the generation of both Lys-BK and BK that directly correlated with symptom severity. Other studies have suggested a relationship between kinin generation and activation of the innate immune response. Exposure of mouse trachea to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide or the TLR-3 agonist poly I:C increased B1R expression and enhanced sensitivity of the tissue to both B1R and B2R ligands, suggesting that bacterial and viral infections of the airway could lead to increased sensitization of the airways to kinins. Molecular definition of the kinin system has led to the development of animals with targeted deletions or overexpression of kinin system components as well as improved tools to assess the role of the kinin system in human respiratory diseases. The availability of potent and specic BK receptor antagonists

506 KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission

provides an opportunity to translate these advances into potential therapeutic interventions.

See also: Capsaicin. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide; Other Important Neuropeptides. Proteinase Inhibitors: Cystatins.

Turner P, Dear J, Scadding G, and Foreman JC (2001) Role of kinins in seasonal allergic rhinitis: icatibant, a bradykinin B2 receptor antagonist, abolishes the hyperresponsiveness and nasal eosinophilia induced by antigen. Journal of Allergy and Clinical Immunology 107: 105113.

Further Reading
Bryborn M, Adner M, and Cardell LO (2004) Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogenactivated protein kinase pathways. Clinical and Experimental Allergy 34: 12911298. Casalino-Matsuda SM, Monzon ME, Conner GE, Salathe M, and Forteza RM (2004) Role of hyaluronan and reactive oxygen species in tissue kallikrein-mediated epidermal growth factor receptor activation in human airways. Journal of Biological Chemistry 279: 2160621616. Christiansen SC, Eddleston J, Woessner KM, et al. (2002) Upregulation of functional kinin B1 receptors in allergic airway inammation. Journal of Immunology 169: 20542060. Christiansen SC, Proud D, and Cochrane CG (1987) Detection of tissue kallikrein in the bronchoalveolar lavage uid of asthmatic subjects. Journal of Clinical Investigation 79: 188197. Eddleston J, Christiansen SC, Jenkins GR, Koziol JA, and Zuraw BL (2003) Bradykinin increases the in vivo expression of the CXC chemokine receptors CXCR1 and CXCR2 in patients with allergic rhinitis. Journal of Allergy and Clinical Immunology 111: 106112. Fuller RW, Dixon CM, Cuss FM, and Barnes PJ (1987) Bradykinin-induced bronchoconstriction in humans. Mode of action. American Review of Respiratory Disease 135: 176180. Huang TJ, Haddad EB, Fox AJ, et al. (1999) Contribution of bradykinin B(1) and B(2) receptors in allergen-induced bronchial hyperresponsiveness. American Journal of Respiratory and Critical Care Medicine 160: 17171723. Leeb-Lundberg LM (2004) Bradykinin specicity and signaling at GPR100 and B2 kinin receptors. British Journal of Pharmacology 143: 931932. Leeb-Lundberg LM, Marceau F, Mu ller-Esterl W, Pettibone DJ, and Zuraw BL (2005) Classication of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacologic Reviews 57: 2777. Marceau F and Regoli D (2004) Bradykinin receptor ligands: therapeutic perspectives. Nature Reviews: Drug Discovery 10: 845852. Naclerio RM, Proud D, Lichtenstein LM, et al. (1988) Kinins are generated during experimental rhinovirus colds. Journal of Infectious Disease 157: 133142. Pan ZK, Zuraw BL, Lung CC, et al. (1996) Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human broblasts. Journal of Clinical Investigation 98: 20422049. Proud D, Togias A, Naclerio RM, et al. (1983) Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. Journal of Clinical Investigation 72: 16781685. Roisman GL, Lacronique JG, Desmazes-Dufeu N, et al. (1996) Airway responsiveness to bradykinin is related to eosinophilic inammation in asthma. American Journal of Respiratory and Critical Care Medicine 153: 381390.

Neuropeptides and Neurotransmission

N R Prabhakar and G K Kumar, Case Western Reserve University, Cleveland, OH, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Neurotransmitters inuence breathing by acting either on central neurons that regulate breathing or on the peripheral nerves that innervate lungs, airways, and O2-sensing chemoreceptors. In addition to amino acids and biogenic amines, neuropeptides emerged as another class of molecules that profoundly inuence neuronal activities. Substance P (SP) is one such neuropeptide that profoundly impacts breathing via its actions on central and peripheral nervous systems. SP belongs to a group of structurally related peptides called tachykinins. It participates in the generation of respiratory rhythm by acting on pre-Botzinger neurons in the medulla oblongata and phrenic motoneurons in cervical cord that innervate the diaphragm. Selective destruction of neurons expressing SP-receptors leads to apneas, irregular breathing, and hypoxemia. SP also contributes to the integration of sensory inputs from lungs, airways, and peripheral chemoreceptors by acting on the neurons in the nucleus of the tractus solitarious. In the peripheral nervous system, SP acting as a sensory transmitter participates in the hypoxic sensing at the carotid body. In the lungs and airways, SP stimulates irritant receptors innervated by the vagus nerves and participates in the generation of cough reex. Also, SP is linked to asthma and hyperreactivity. In response to allergens, SP-like peptides are released from the nerve bers innervating airways, and SP is a potent constrictor of central and peripheral airways. It also stimulates airway secretions. SP-receptor antagonists are emerging as a new class of therapeutic interventions to alleviate asthma and chronic cough.

Introduction
Breathing is a rhythmic motor act that requires generation of respiratory rhythm by brainstem neurons, which is continuously under the control of information from lungs, airways, and chemoreceptors that senses changes in arterial blood gases via their respective sensory receptors. Neurotransmitters profoundly inuence breathing by acting either centrally or peripherally on sensory receptors and/or airways. For instance, amino acid transmitters (e.g., glutamate and g-amino butyric acid) modulate breathing by acting on central neurons. Biogenic amines such as acetylcholine (ACh), on the other hand, exert their inuence on breathing by acting peripherally on the

KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission 507

Tachykinins SP

Central Effectors Nucleus of the tractus solitarious

Peripheral

Pre-Botzinger neurons

Carotid body

Irritant receptors

Airways

Generation of respiratory rhythm

Integration of afferent inputs (lungs, airways, chemoreceptors)

Hypoxic sensing

Cough

Airway hyperreactivity

Figure 1 Schematic representation of the effects of substance P (SP), a tachykinin peptide, on breathing. The gure depicts the central and peripheral targets of SP and effect on breathing.

Effects

airways more so than their actions on the central nervous system. In recent years, neuropeptides have emerged as another class of molecules that profoundly inuence neuronal activities. Substance P (SP) is one such neuropeptide that modulates breathing by acting centrally as well as peripherally (Figure 1). SP belongs to a group of structurally related peptides called tachykinins. Other members of the tachykinin family include neurokinin A, neuropeptide K , neuropeptide g, and neurokinin B. Amongst the different tachykinins, SP is the most extensively studied with regard to its action on breathing.

the pre-Botzinger neurons. In adult animals, in addition to its effects on pre-Botzinger neurons, SP modulates respiratory rhythm by directly stimulating phrenic motoneurons that innervate the diaphragm.
SP and Integration of Respiratory Afferent Information at Brainstem Neurons

Central Effects of SP
SP and Generation of Respiratory Rhythm

Neural circuitry that is critical for generating the respiratory rhythm (at least in neonates) has been localized to the pre-Botzinger complex, a region in the medulla oblongata located ventrolateral to the nucleus ambiguous. Microinjections of SP into the pre-Botzinger area stimulate breathing primarily by increasing respiratory frequency in newborn rats. Biological actions of tachykinins are mediated by G-protein-coupled receptors that are classied as neurokinin-1, -2, and -3 receptors (NK-1, -2, and -3). Many of the neurons in this region express NK-1 receptors. More importantly, selective destruction of these neurons leads to irregular breathing, frequent occurrence of apneas, and hypoxemia in awake rats. However, in adult animals, generation of respiratory rhythm is more complex and involves other areas of the brain that make extensive synaptic contacts with

Respiratory responses to activation of pulmonary afferents innervated by vagal sensory bers and peripheral chemoreceptors require integration of afferent information by brainstem neurons, especially those localized in the nucleus of the tractus solitarius (nTS). Neurons in the nTS translate afferent information into appropriate respiratory motor behavior. Information from lungs (lung volume, intrapulmonary pressure) during each inspiration is conveyed to nTS neurons via sensory stretch receptors innervated by vagus nerves (pulmonary stretch receptors, PSR). After reaching a certain threshold, pulmonary stretch receptors turn off inspiration reexly involving brainstem neurons and expiration ensues passively. This phenomenon, known as inspiratory off-switch is critical for determining the magnitude of the tidal volume and duration of inspiration and, as a consequence, respiratory rate. SP alters the threshold of the inspiratory off-switch reex primarily by affecting nTS neurons. Local application of SP produces longlasting activation of nTS neurons, and stimulates breathing by increasing both tidal volume and respiratory rate. SP antagonists prevent these responses. In addition to its effects on inspiratory off-switch threshold, SP is also involved in integration of sensory information from peripheral chemoreceptors

508 KININS AND NEUROPEPTIDES / Neuropeptides and Neurotransmission

that sense hypoxia in arterial blood. Hypoxia facilitates SP release in the nTS region, and denervation of peripheral chemoreceptors prevents this effect.

Peripheral Effects of SP
SP and Peripheral Chemoreceptors: Implications for Hypoxic Ventilatory Response

Compensatory hyperventilation that ensues during hypoxemia is reex in nature and mediated entirely by activation of peripheral chemoreceptors, especially the carotid bodies. Carotid bodies are composed of two cell types: type I and type II. Type I cells (also called glomus cells) are of neural crest origin and express a variety of neurotransmitters. Type II cells resemble glial cells and are supporting cells. Type I cells are critical for sensing hypoxia. During hypoxia type I cells release neurotransmitter(s), which by acting on the nearby afferent nerve-ending, produces an increase in sensory activity that is eventually conveyed to brainstem neurons to evoke stimulation of breathing. SP satises several of the criteria for its role as a transmitter in hypoxia-induced sensory activation of the carotid body. SP occurs at femtomole levels in the carotid body, and is localized to type I cells of the carotid body in several species including humans, cats, and rabbits. Carboxypeptidase E, an enzyme essential for SP biosynthesis, is expressed in the carotid body. Hypoxia releases SP from the carotid bodies and requires activation of voltage-dependent calcium channels. SP augments carotid body sensory activity in several species both in vivo and ex vivo. Studies on ex vivo preparations suggest that the stimulatory effects of SP are due to its direct action on the carotid body rather than secondary to its systemic effects on the cardiovascular system. The stimulatory effects of SP on carotid body sensory activity requires the C-terminus region of the peptide to be intact, whereas the N-terminal fragment of the peptide was without any effect. Other tachykinin peptides including NKA, which has a similar C-terminal sequence to SP, also augment the sensory response of the carotid body. Autoradiographic analysis revealed SP-binding sites in the cat carotid body, and blockade of NK-1 receptors prevented SP-induced carotid body stimulation. In addition to its receptor-mediated actions, SP may directly inuence mitochondrial metabolism by acting as a protonophore as shown in mitochondrial preparations in vitro. Systemic administration of phosphoramidon, a selective and potent inhibitor of neutral endopeptidase, an enzyme that

terminates the actions of SP, potentiates sensory response of the carotid body to hypoxia. Blockade of NK-1 receptors prevents this effect. There is considerable evidence that SP profoundly inuences hypoxic ventilatory responses in humans and experimental animals. In conscious human subjects, systemic administration of SP potentiates hypoxic but not hypercapnic ventilatory response. In experimental animals (rats), NK-1 receptor antagonist attenuates hypoxic ventilatory response. In rats, depletion of SP, by neonatal exposure to capsaicin, markedly reduces the magnitude of the hypoxic ventilatory response, by attenuating the respiratory frequency. Furthermore, hypoxic but not the hypercapnic ventilatory responses are selectively blunted in genetically engineered mice with targeted deletion of the genes encoding pre-pro-tachykinin-1, the precursor for SP.

SP and Pulmonary and Airway Sensory Receptors: Implications in Generation of Cough

Cough is critical for maintaining the defense of airways and in clearing mucus and other foreign particles. It is a complex reex and involves (1) sensory receptors, (2) the afferent pathway, (3) the cough center, (4) the efferent pathway, and (5) the effectors. Irritant receptors (also called rapidly-adapting type of mechanosensory receptors) in airways innervated by vagus nerves constitute the sensory afferents and the afferent pathway. The cough center is located in the brainstem, and efferent signals are transmitted to glottis, diaphragm, intercostals, and abdominal muscles via cranial and spinal motor pathways. It has been proposed that chemical stimuli that induce cough do so by directly stimulating irritant receptors, and also secondarily by stimulating C bers (slowly-conducting mechanosensory receptors of pulmonary origin), which release tachykinins. Once released, tachykinins act on irritant receptors to promote cough further (positive feedback). SP is present in vagal sensory bers innervating the bronchopulmonary system. SP stimulates irritant receptors in rabbits and cats and SP-antagonists abolish these effects. Further support for the involvement of tachykinins in generation of cough has come from a number of studies demonstrating that neurokinin receptor antagonists prevent cough in several species including humans. Furthermore, cough can be elicited by exogenous administration of SP or by increasing the endogenous level of SP by preventing its degradation by neutral endopeptidase (NEP). Thus, interventions with selective neurokinin receptors might be of therapeutic value in alleviating cough.

KININS AND NEUROPEPTIDES / Tachykinins 509 SP and Airway Hyperreactivity: Implications in Asthma Symptoms of Respiratory Disease: Cough and Other Symptoms.

Asthma is a chronic airway inammatory disorder and is characterized by increased airway responsiveness to various physiologic and environmental stimuli. Airway hyperreactivity has been attributed in part to the transmitters released from the nerve bers innervating airways. There is also considerable evidence for the role of tachykinins, especially SP in airway hyperreactivity. SP-like peptides are present in the nerve bers innervating airways. Antigens, ozone, and inammatory mediators release SP from airway sensory nerves. Challenging guinea pigs with antigen markedly potentiates action-potential-driven tachykinin release from afferent bers in the airways. Anatomical and electrophysiological studies indicate that tachykinin-containing sensory bers directly innervate the local parasympathetic ganglion neurons in the airways. SP is a potent bronchoconstrictor and affects both the central and peripheral airway tone, via NK-1 receptors. Atropine and hexamethonium reduce SP-induced bronchoconstriction. Allergic reaction increases vagal sensory and parasympathetic activity that involves cross-talk between tachykinins and the cholinergic system. Thus, tachykinins control airway reactivity by directly affecting airway smooth muscle via NK-1 receptors as well as via their interaction with cholinergic neurons innervating airways. Eosinophils, macrophages, lymphocytes, and dendritic cells (inammatory cells) that are resident to airways also produce SP. Immune stimuli boost the production and secretion of SP. Apart from its action on bronchomotor tone, SP also promotes airway secretions and bronchial circulation (vasodilatation and microvascular leakage). Bronchomotor actions of SP are terminated by the enzymes NEP and angiotensin-converting enzyme (ACE). Lung contains large amounts of ACE. Therapeutic interventions with ACE are commonly associated with increased incidence of cough, secondary to increased airway sensitivity. The side effects of ACE inhibitors on breathing may in part be due to elevated levels of endogenous SP in lungs and airways. In view of their potent effects on the airways, tachykinins have been put forward as possible mediators of asthma, and neurokinin receptor antagonists are emerging as a potential class of anti-asthmatic medications.

Further Reading
Barnes PJ (1987) Neuropeptides in human airways: function and clinical implications. American Review of Respiratory Diseases 136: S77S83. Fox AJ (1996) Modulation of cough and airway sensory bers. Pulmonary Pharmacology 9: 335342. Hokfelt T, Pernow B, and Wahren J (2001) Substance P: a pioneer amongst neuropeptides. Journal of Internal Medicine 249: 2740. Joos GF, Germonpre PR, and Paulwels RA (2000) Role of tachykinins in asthma. Allergy 55: 321337. Kumar GK and Prabhakar NR (2003) Tachykinins in the control of breathing by hypoxia. Pre- and Post-genomic era. Respiration Physiology and Neurobiology 135: 145154. Prabhakar NR and Chearniack NS (1989) Importance of tachykinin peptides in hypoxic ventilatory drive. In: Lahiri S, Forster RE, Davies RO, and Pack AI (eds.) Chemoreceptors and Reexes in Breathing: Cellular and Molecular Aspects, pp. 99112. New York: Oxford University Press. Widdicombe JG (1995) Neurophysiology of the cough reex. European Respiratory Journal 8: 11931202.

Tachykinins
G F Joos and K De Swert, Ghent University Hospital, Ghent, Belgium
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The four major mammalian tachykinins are substance P, neurokinin A, neurokinin B, and hemokinin 1. They are derived from three preprotachykinin genes, termed TAC1, TAC3, and TAC4. Tachykinins are present in human airways, both in sensory nerves and in non-neuronal cells, including airway epithelium, airway smooth muscle, and immune cells such as eosinophils, monocytes, macrophages, lymphocytes, and dendritic cells. Tachykinins can be recovered from the airways after inhalation of ozone, cigarette smoke, or allergen. They interact in the airways with tachykinin NK1, NK2, and NK3 receptors to cause bronchoconstriction, plasma protein extravasation, and mucus secretion and to attract and activate immune cells. In preclinical studies substance P and neurokinin A have been implicated in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD), including airway inammation and bronchial hyperresponsiveness induced by allergen or cigarette smoke. Tachykinin receptor antagonists are potential new drugs for the treatment of asthma and/or COPD.

Acknowledgment
This work was supported by grants from National Institutes of Health, HL-25830.
See also: Asthma: Overview. Hypoxia and Hypoxemia. Kinins and Neuropeptides: Tachykinins.

Introduction
The tachykinin peptide family represents one of the largest peptide families described in the animal organism. They have been isolated from invertebrate, protochordate, and vertebrate tissues. The tachykinins substance P and neurokinins A and B have

510 KININS AND NEUROPEPTIDES / Tachykinins

previously been considered as a group of neuropeptides because of their widespread distribution in the central and the peripheral nervous system. Tachykinins are released from sensory nerves by a variety of stimuli such as capsaicin, electrical nerve stimulation, low pH, ether, formalin, toluene diisocyanate, histamine, bradykinin, methacholine, prostaglandins, leukotrienes, and cigarette smoke. In addition, tachykinins are present in a variety of non-neuronal cells, including immune cells.

Structure
Tachykinins, dened as peptides having the characteristic C-terminal pentapeptide F-X-G-L-M-NH2, are identied as aromatic when X is an aromatic amino acid residue (F or Y) or aliphatic when X is an aliphatic amino acid residue (V or I). All tachykinins are amidated at their C-terminus and this is crucial for their biological activity. The four major mammalian tachykinins are substance P, neurokinin A, neurokinin B, and hemokinin 1. Neurokinin A has also two N-terminally extended forms, neuropeptide g and neuropeptide k. Mammalian tachykinin peptide sequences are species invariant except for hemokinin 1. Human hemokinin 1 has also two elongated forms, endokinin A (47 amino acids) and endokinin B (41 amino acids) (Table 1).

Synthesis, Localization, and Degradation

Synthesis

Mammalian tachykinins are derived from three preprotachykinin genes which according to the Human Genome Organization (HUGO) Gene Nomenclature Committee are termed TAC1, TAC3, and TAC4 (Figure 1). The TAC1 gene (previously known as the

PPT-I or PPT-A gene) is coding for substance P, neurokinin A, and its extended forms. The TAC3 gene (also known as PPT-II or PPT-B) encodes the sequence for neurokinin B. Hemokinin 1 is coded by the TAC4 gene (or PPT-C). The precursor RNA of the TAC1 gene is alternatively spliced to yield four different mRNAs, a-, b-, g-, and d-TAC1 mRNA. All four mRNAs can generate substance P. Posttranslational processing of the b-TAC1 mRNA product can yield neurokinin A or neuropeptide k, while the g-form can generate neurokinin A and neuropeptide g. Usage of two differential promoters and alternative splicing of the TAC3 DNA transcript yields two mRNAs coding for neurokinin B (a- and b-TAC3). In contrast to the mouse and rat TAC4 gene which predicts one homologous transcript, the human ortholog encodes a different undecapeptide (Table 1) and has also the potential to generate a truncated form of the hemokinin 1 (hemokinin 1(4-11)). It should be noted that endokinin A and B are elongated forms of the human hemokinin 1. Translation of the mature mRNA yields a large polypeptide. The preprotachykinin consists of a signal peptide, one or several copies of a neuropeptide, and one or more spacer parts. The signal peptide is cleaved off after polypeptide synthesis leading to the propeptide. Tachykinins are liberated from their propeptides through proteolytic cleavage at specic sites of single basic residues (R, L) or at two adjacent basic residues, carried out by six groups of enzymes called convertases. The C-terminal methionine residues of the preprotachykinin precursors are all followed by glycine that donates its amine group thereby generating the C-terminal amidation.
Localization

Table 1 Amino acid sequences of mammalian tachykinins Peptide Substance P Neurokinin A Neuropeptide g Neuropeptide k Amino acid sequence R-P-K-P-Q-Q-F-F-G-L-M-NH2 H-K-T-D-S-F-V-G-L-M-NH2 D-A-G-H-G-Q-I-S-H-L-R-L-DS-F-V-G-L-M-NH2 D-A-D-S-S-I-E-L-Q-V-A-L-L-KA-L-Y-G-H-G-Q-I-S-H-K-RH-G-Q-I-S-H-L-R-L-D-S-FV-G-L-M-NH2 D-M-H-D-F-F-V-G-L-M-NH2 (R)-S-R-T-R-Q-F-Y-G-L-MNH2 T-G-K-A-S-Q-F-F-G-L-M-NH2 R-H-R-T-P-M-F-Y-G-L-M-NH2

Neurokinin B Hemokinin 1 m (mouse) Hemokinin 1 h (human) Chromosome 14 tachykininlike peptide 1 (C14TKL-1)

Tachykinins (substance P, neurokinin A, and neurokinin B) have previously been considered as a group of neuropeptides because of their widespread distribution in the central and the peripheral nervous system (capsaicin-sensitive primary afferent neurons and capsaicin-insensitive intrinsic neurons). This terminology is no longer held since their presence in a variety of non-neuronal structures has been proved repeatedly. Furthermore, mRNA expression studies suggest that hemokinin 1 has a unique distribution outside neuronal tissues. To date, this interpretation is altered to a more abundant expression outside neuronal tissue as low levels are detected in brain tissue. The alternative splicing of the TAC1 gene (Figure 1) affects the regional distribution of substance P and neurokinin A. Substance P can be expressed alone (translation of the a- and the d-form) while neurokinin A expression is always

TAC1 gene 5 1 2 SP 4 5 NKA 7 3 5 1 1 1

TAC3 gene 2 3 4 5 6 7 3 5 1

TAC4 gene 2 3 4 5 3

Transcription and RNA splicing

2 SP 4

7 AAA

2 SP

NKA

7 AAA

7 AAA

5 AAA

5 AAA

-TAC1 mRNA

-TAC1 mRNA

-TAC3 mRNA

-TAC4 mRNA

-TAC4 mRNA

2 SP 4 NKA -TAC1 mRNA

7 AAA

2 SP 5 -TAC1 mRNA

7 AAA

1 1

7 AAA

5 AAA

5 AAA

-TAC3 mRNA

-TAC4 mRNA

-TAC4 mRNA

Translation and posttranslational processing

Substance P

Substance P Neurokinin A Neuropeptide

Substance P Neurokinin A Neuropeptide

Substance P

Neurokinin B

Neurokinin B

hHemokinin 1 Endokinin A (Endokinin C)

hHemokinin 1 Endokinin B

hHemokinin 1 Endokinin B

hHemokinin 1 Endokinin B (Endokinin D)

Figure 1 Tachykinin peptides, mRNAs, and genes. Mammalian tachykinins are encoded by three different genes (TAC1, TAC3, and TAC4). TAC1 yields four different mRNA splice variants, which generate substance P, neurokinin A, and the elongated forms of neurokinin A. The TAC3 gene encodes for neurokinin B. The TAC4 gene is not species invariant. In humans, the gene yields also four splice variants which are nally translated into endokinin A, B, C, and D.

512 KININS AND NEUROPEPTIDES / Tachykinins

Table 2 Tissue distribution of mammalian tachykinins Peptide Substance P MRNA a-, b-, g-, d-TAC1 Main sources CNS, primary afferent neurons, enteric neurons, immunocytes, inammatory cells, endothelium, epithelium, Leydig cells, enterochromafn cells, broblasts, smooth muscle, female reproductive tracta CNS, primary afferent neurons, enteric neurons, immunocytes, inammatory cells, female reproductive tracta CNS, spinal chord, syncytiotrophoblasts of human placenta, lunga Lymphoid B and myeloid lineage cells of bone marrow,a T cell precursors of the thymus, uterus, blastocyst-stage embryos, ovary, testis, spleen, stomach, skin, lactating breast, heart, kidney, liver, lung, adrenal tissue, brain Heart,a skeletal muscle, skin, thyroid, kidney, liver, lung, stomach, testis, placenta, prostate Adrenal gland,a fetal liver, spleen Adrenal gland,a placenta, heart, liver, bone marrow, prostate, testis Liver,a kidney, prostate, bronchus, CNS

Neurokinin A

b-, g-TAC1

Neurokinin B

TAC3

muscle layer. In guinea pigs, airway sensory nerves containing tachykinins are easily demonstrated, but in human airways, tachykinergic innervation is sparse. In the bronchial tree, tachykinergic sensory innervation originates from the (right) vagus nerve, but in the lung, the bers are both from vagal parasympathetic and thoracic spinal origin. Non-neuronal sources of tachykinins in the airways are: airway epithelium, airway smooth muscle cells and immune cells such as eosinophils, monocytes and macrophages, lymphocytes, and dendritic cells. Immunoreactivity for neurokinin B has not been found in the airways yet. Transcripts of the mouse and the human TAC4 gene in lung tissue have been reported and these may result in the formation of the hemokinin 1.
Degradation

Hemokinin 1 m

mTAC4

Hemokinin 1 h

hTAC4

Endokinin A Endokinin B

a-hTAC4 b-, g-, d-hTAC4 ?

C14TKL-1

a Only mRNA expression evaluated. CNS, central nervous system.

accompanied by substance P expression (translation of the b- and the g-form). The most abundant mRNAs are the b- and the g-form. This means that in many occasions, substance P and neurokinin A are co-synthesized and co-released. The tissue distribution of mammalian tachykinins is reported in Table 2. In the airways, a distinct subpopulation of primary afferent nerves that are characterized by their sensitivity to capsaicin are considered as the principal source of substance P and neurokinin A. Although they can also be expressed in capsaicin-resistant neurons, capsaicin pretreatment of experimental animals causes an almost total depletion of substance P and neurokinin A immunoreactivity. Radioimmunoassay and immunohistochemistry demonstrated substance P and neurokinin A positive nerves beneath and within the epithelium, around blood vessels and submucosal glands, and within the bronchial smooth

The physiological actions of neuropeptides are normally terminated by extracellular metabolism. As far as the metabolism of substance P and neurokinin A is concerned, much attention has been paid to the role of the neutral endopeptidase 24.11 (also called NEP, enkephalinase, CALLA, CD10, or E.C.3.4.24.11) and to the role of peptidyl dipeptidase-15.1, better known as angiotensin-converting enzyme (ACE, kininase II, or E.C.3.4.15.1). A series of potent and relative selective inhibitors of NEP are available, such as the thiol inhibitors (e.g., thiorphan), and phosphorus-containing inhibitors (e.g., phosphoramidon). In guinea pigs, both NEP and ACE participate in the metabolism of substance P when administered intravascularly, while aerosolized tachykinins are degraded by NEP only. NEP mRNA and NEP-immunoreactive material has been identied in human airways. NEP is present in the basal cells of the epithelium, alveolar cells type II, submucosal glands, neutrophils, airway smooth muscle, postcapillary venules, and nerves.

Biological Functions
Tachykinins elicit a broad spectrum of biological actions in the cardiovascular, gastrointestinal, urogenital, immune, and nervous system. These may vary in different species and even in the various strains of single species. This suggests the concept of a general, important functional significance of these peptides. However, mice with a disrupted preprotachykinin I gene (TAC1 gene, encoding for substance P, neurokinin A, and its extended forms) or a disrupted tachykinin NK1 or tachykinin NK3 receptor gene (the TACR1 and TACR3 gene) are in good health conditions and fertile, which demonstrates that the

KININS AND NEUROPEPTIDES / Tachykinins 513

tachykinins substance P and neurokinin A and the tachykinin NK1 and NK3 receptor are not essential for life and health, at least in mice. The adequate stimuli for tachykinin release from the sensory nerves in the airways are of chemical nature, especially those chemicals that are produced during inammation and tissue damage.
Airway Smooth Muscle Tone and Airway Responsiveness

The bronchoconstrictor effect of exogenous substance P was rst reported in 1977, in guinea pigs and cats. Since then, substance P was shown to cause a dosedependent bronchoconstriction in various animal species, in vitro as well in vivo. Exogenously administered tachykinins also contract human airways, in vitro and in vivo. In vitro, substance P contracts human bronchi and bronchioli, but is less potent than histamine or acetylcholine. Neurokinin A however is a more potent constrictor, being, on a molar base, two to three orders of magnitude more potent than histamine or acetylcholine. Neurokinin B does not exert a contractile activity on human airways. The role of tachykinins in the regulation of airway function has been under even more intense investigation, since the demonstration that the atropine-resistant contraction (about 60% of the contraction induced by electrical eld stimulation) in guinea pig airways was ascribable to tachykinins released from sensory nerves. Such a noncholinergic bronchoconstriction has, however, not been consistently demonstrated in human airways.
Cough

(and pathological) regulators of secretion in the upper and lower airways. A local release of tachykinins in the nasal mucosa plays a role in the defensive response to irritants. Intranasally administered capsaicin induces sneezing, pain, and nasal secretion in both experimental animals and humans. Similarly, local tachykinin administration evokes secretion. In the lower tracheobronchial tree, seromucous glands and goblet cells produce mucus. Both sources are under neural control. Depending upon species and airway level, innervation comprises parasympathetic, sympathetic and sensory-efferent pathways. The transmitters of the latter pathway are substance P and neurokinin A, which act via tachykinin NK1 receptors. An involvement of tachykinin NK3 receptors has been suggested. Mucus secretion is closely coupled to liquid secretion to ensure optimal ciliary transport and presence of adequate concentrations of antibacterial substances on the airway surface. Substance P has also been shown to be involved in this process.
Alveolar Epithelial and Microvascular Permeability

An afferent (sensory) and an efferent arm that synapses in the brainstem areas, including the nucleus tractus solitarius, make up the reex arch that mediates the cough reex. Mediators and mechanisms of the sensory arm of the reex pathway are not completely understood. Pharmacological evidence exists for an involvement of substance P. The role of tachykinins in the regulation of the cough response appears to be complex since antagonists for the three tachykinin receptors have antitussive activity in preclinical models. In guinea pigs, substance P locally applied into the airways has been shown to produce or potentiate cough produced by other stimuli. These ndings support a role for substance P at a peripheral site of action. Central sites of action have also been reported.
Secretion of Mucus, Water, and Electrolytes

Relatively strong evidence exists that tachykinins released from sensory nerve bers are physiological

The involvement of sensory nerves in producing increased vascular permeability and plasma extravasation was rst described in skin and conjunctiva and termed neurogenic inammation. Neurogenic inammation has also been described in the airways. Substance P, released from the sensory nerves, mediates the neurogenic inammation in the airways. Plasma extravasation, induced by cold air, hypertonic saline, or isocapnic hyperpnea, depends on stimulation of sensory bers and is inhibited by tachykinin receptor antagonists. Once plasma extravasation occurs, leukocytes initiate a process that results in slowing down their velocity, rolling on and adhering to the venular endothelium. The involvement of the tachykinin NK1 receptor in all these phenomena has been demonstrated by the use of specic antagonists. Tachykinin NK2 receptors also mediate part of the neurogenic plasma extravasation in the secondary bronchi and intraparenchymal airways of the guinea pig. Direct evidence for neurogenic plasma extravasation in human airways could not be demonstrated for long time due to difculties in the assessment. However, microvascular leakage can now be measured in human airways with a dual induction model. First, substance P is inhaled and then sputum is induced by inhalation of hypertonic saline solution. With this method, it was demonstrated that inhalation of substance P induced significant increases in the levels of a2-macroglobulin, ceruloplasmin, and albumin in induced sputum.

514 KININS AND NEUROPEPTIDES / Tachykinins Cell Growth and Proliferation of Mesenchymal Cells

Tachykinins stimulate the proliferation of a number of cell types, including broblasts, smooth muscle cells, epithelial cells, and endothelial cells. The substance P-induced proliferation of human broblasts is mediated by interaction with tachykinin NK1 receptors. The proliferation of rabbit airway smooth muscle involves also tachykinin NK1 receptors. The proliferation of smooth muscle cells in response to tachykinins is coupled to inositoltriphosphate formation and DNA synthesis.
Immunomodulation

Tachykinins are known to have a wide variety of modulatory effects on inammatory and immune cells. Substance P degranulates mast cells, leading to the release of histamine and 5-hydroxytryptamine (5HT, serotonin), and the production of tumor necrosis factor alpha (TNF-a). Tachykinins cause adherence and chemotaxis of human neutrophils. Substance P induces chemotaxis of human eosinophils. Several in vitro and in vivo studies have shown that substance P is able to modulate the chemotaxis, proliferation, and activation of lymphocytes. Tachykinins also have a number of stimulatory effects on monocytes and macrophages: substance P has been shown to evoke superoxide anion production, enhance phagocytosis, and cause production of interleukin-1 and interleukin-12 by macrophages. Tachykinins are also able to activate mesenchymal cells in the airways.

Receptors
The biological activity of tachykinins depends on their interaction with three specic receptors: the
ATG 1 130 2 195 3 245

tachykinin NK1, NK2, and NK3 receptors. These receptors belong to the rhodopsin-like G-proteincoupled receptor family. This group of proteins share the same structural motif: a bundle of seven hydrophobic transmembrane domains with three extracellular loops, three intracellular loops, an extracellular N-terminus, and a cytoplasmic C-terminus. The human tachykinin NK1 and NK2 receptors are proteins of 407 and 398 amino acids respectively. The human tachykinin NK3 receptor is N-terminally extended and has 465 residues. The genes encoding the three mammalian tachykinin receptors have a similar structural organization. They all contain ve exons, with introns interrupting the protein-coding sequence in identical positions (Figure 2). Within the family of the G-protein-coupled receptors, the tachykinin receptor family is one of the few that retains introns. The splice sites of the exons occur at the border of the sequences encoding the putative transmembrane domains. The tachykinin receptor displaying the highest afnity for substance P was termed the tachykinin NK1 receptor. The receptor showing the highest afnity for neurokinin A was termed the tachykinin NK2 receptor and the receptor with the highest afnity for neurokinin B was called the tachykinin NK3 receptor. Hemokinin 1 and its elongated forms act as tachykinin NK1 receptor preferring agonists. It should be emphasized that all tachykinins can act as full agonists on the three different receptors but with lower afnities than on the preferred receptor. The extracellular loops of the receptors are involved in the selection of a specic ligand, whereas the interaction of the ligand with the transmembrane domains is responsible for receptor activation. Upon ligand binding, receptor activation and subsequent interaction with a G-protein occurs. The
311 4 5 TAG (407) TACR1

ATG

131

196

247

313

TGA (398) TACR2

ATG

183

246

296

362

TAA (465) TACR3

Figure 2 Schematic structure of the genes encoding for the tachykinin NK1 receptor (TACR1), the NK2 receptor (TACR2), and the NK3 receptor (TACR3). The coding regions of the genes are interrupted by four introns. The dark blue boxes indicate the transmembrane segments. Amino acid positions at splicing sites are indicated.

KININS AND NEUROPEPTIDES / Tachykinins 515

main second messenger system coupled to the receptors is the stimulation of phospholipase C (PLC), leading to phosphoinositide breakdown, inositol triphosphate (IP3) formation which leads to elevation of intracellular calcium and formation of diacylglycerol (DAG) which activates protein kinase C (PKC). Receptor activation leads also to activation of adenylyl cyclase (AC) and subsequent increases in cAMP levels but this requires about one order of magnitude higher concentrations of the tachykinin peptides (Figure 3). In Chinese hamster ovary (CHO) cells, transfected with the human tachykinin NK1 receptor, activation of the receptor results not only in IP3 and DAG accumulation and increase of cAMP levels but also in the mobilization of arachidonic

acid. In these cells, receptor activation also leads to stimulation of the phospholipase D (PLD), with formation of phosphatidic acid (PA) and sustained activation of protein kinase C. Also Rho family GTPases are involved in NK1 receptor signaling. Native tachykinin NK1 receptors, expressed in CaCo-2 (colon adenocarcinoma cell line), tracheal smooth muscle, U-373 MG (astrocytoma), and peritoneal mast cells, are coupled downstream to the activation of mitogen-activated kinases (MAPKs). Downstream involvement of NF-kB has been demonstrated in murine macrophages and dendritic cells, U-373 MG cells, and lymphocytes. An overwhelming amount of functional data demonstrates indirectly the broad expression of

HK SP NKA

PLD

Arachidonic acid
RhoA PLC G q GTP Prostaglandins Leukotrienes Lipoxins PLA2 PKC [cAMP] G s GTP

Cytoplasm

G i GTP

AC

[PA] [IP3] PI3K NO [DAG]

eNOS

AKT PKA

[Ca2+] MAPK
NF- B I B

[Ca2+]

[Ca2+] I B

[Ca2+]

ER

MAPK Nucleus
NF- B

Transcription

CREB

Figure 3 Intracellular signaling pathways. The tachykinin NK1 receptor is known to interact with several G proteins (Gq, Gs, Gi), which in their turn interact with effector molecules as adenylyl cyclase (AC) and phospholipase C (PLC). Protein kinases are depicted in red, GTPases in blue, and nuclear factors in gray. CREB, cAMP response element-binding protein; DAG, diacylglycerol; eNOS, endothelial nitric oxide synthase; HK, hemokinin; lkB, inhibitor kappa B; NKA, neurokinin A; NO, nitric oxide; P13K, phosphatidylinositol-3-kinase; PKA, protein kinase A; RhoA, a small GTP-binding protein; SP, substance P.

516 KININS AND NEUROPEPTIDES / Tachykinins

Epithelium Secretion Submucosal gland Inflammatory cell Stimulation Sensory nerve

Vasodilatation Blood vessel Plasma leakage Contraction Smooth muscle

Facilitation

Figure 4 Nerve bers containing substance P and neurokinin A are present from the pharynx down to the bronchioli, occasionally extending into the alveoli. They are located beneath and within the respiratory epithelium, among bundles of smooth muscle, around blood vessels, submucosal glands, and local tracheobronchial ganglion cells; and in close association with lymphoid aggregates and 5-hydroxytryptamine (5-HT)-positive cells. The sensory nerves can be activated by mechanical and chemical stimuli, generating antidromic impulses and a local axon reex, which lead to noncholinergic bronchoconstriction and neurogenic inammation.

tachykinin NK1 and NK2 receptors in the airways. Also functional evidence for the presence of tachykinin NK3 receptors exists. TACR1 and TACR3 mRNA are found in human pulmonary veins, arteries, and bronchi. TACR2 mRNA is abundantly expressed in human bronchi. A low expression of this receptor is found in human pulmonary veins and arteries. On surgical specimens, using specic antibodies, tachykinin NK1 and NK2 receptor protein can be detected in human bronchial glands, bronchial vessels, and bronchial smooth muscle. Tachykinin NK1 receptors are occasionally found in nerves and tachykinin NK2 receptors in inammatory cells, such as T lymphocytes, macrophages, and mast cells. On endobronchial biopsies, immunoreactivity for the tachykinin NK1 receptor is found in the epithelium and the submucosa. Goblet cells appear to be the cells with the strongest staining. In the submucosa, staining is localized on the endothelial cells of the blood vessels, the surfaces of inammatory cells, and some smooth muscle cells. Immunohistochemistry on human pulmonary vessels from tissue obtained at lobectomy reveals positive staining for tachykinin NK1 receptors on the endothelium of both veins and arteries. Inammatory cells such as macrophages, lymphocytes, neutrophils, dendritic cells, and mast cells, whose presence becomes important in inammatory states, also express functional tachykinin NK1 receptors. In guinea pig airways, tachykinin NK2 receptors and to a lesser extent tachykinin NK1 receptors are involved in the bronchoconstrictor response to exogenous tachykinins, capsaicin, and electrical eld

stimulation. In humans, it has long been thought that only tachykinin NK2 receptors are involved in the direct contraction of isolated bronchi. However, in small- and medium-sized bronchi, tachykinins also cause contraction via tachykinin NK1 receptor stimulation.

Role of Tachykinins in Respiratory Diseases

The tachykinins substance P and neurokinin A are present in human airways, sensory nerves, and immune cells. Tachykinins can be recovered from the airways after inhalation of ozone, cigarette smoke, or allergen. They interact in the airways with tachykinin NK1, NK2, and NK3 receptors to cause bronchoconstriction, plasma protein extravasation, and mucus secretion and to attract and activate immune cells (Figure 4). In preclinical studies they have been implicated in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD), including airway inammation and bronchial hyperresponsiveness induced by allergens and cigarette smoke. Clinical studies with potent tachykinin receptor antagonists (such as dual NK1/NK2 or triple NK1/NK2/NK3 tachykinin receptor antagonists) can bring a definite judgment on their importance in obstructive airway diseases.
See also: Capsaicin. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Other Important Neuropeptides. Symptoms of Respiratory Disease: Cough and Other Symptoms.

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517

Further Reading
Advenier C, Joos G, Molimard M, Lagente V, and Pauwels R (1999) Role of tachykinins as contractile agonists of human airways in asthma. Clinical and Experimental Allergy 29: 579584. Advenier C, Lagente V, and Boichot E (1997) The role of tachykinin receptor antagonists in the prevention of bronchial hyperresponsiveness, airway inammation and cough. European Respiratory Journal 10: 18921906. Barnes PJ (2001) Neurogenic inammation in the airways. Respiratory Physiology 125: 145154. Barnes PJ, Baraniuk JN, and Belvisi MG (1991) Neuropeptides in the respiratory tract. Part I. American Review of Respiratory Disease 144: 11871198. Canning BJ and Fischer A (2001) Neural regulation of airway smooth muscle tone. Respiratory Physiology 125: 113127. Carr MJ and Undem BJ (2003) Pharmacology of vagal afferent nerve activity in guinea pig airways. Pulmonary Pharmacology & Therapeutics 16: 4552. Groneberg DA, Quarcoo D, Frossard N, and Fischer A (2004) Neurogenic mechanisms in bronchial inammatory diseases. Allergy 59: 11391152. Joos GF, De Swert KO, and Pauwels RA (2001) Airway inammation and tachykinins: prospects for the development of tachykinin receptor antagonists. European Journal of Pharmacology 429: 239250. Joos GF, Germonpre PR, Kips JC, Peleman RA, and Pauwels RA (1994) Sensory neuropeptides and the human lower airways: present state and future directions. European Respiratory Journal 7: 11611171. Joos GF, Germonpre PR, and Pauwels RA (2000) Role of tachykinins in asthma. Allergy 55: 321337. Maggi CA (1997) The effects of tachykinins on inammatory and immune cells. Regulatory Peptides 70: 7590. Pennefather JN, Lecci A, Candenas ML, et al. (2004) Tachykinins and tachykinin receptors: a growing family. Life Sciences 74: 14451463. Rogers DF (2001) Motor control of airway goblet cells and glands. Respiratory Physiology 125: 129144. Tai CF and Baraniuk JN (2002) Upper airway neurogenic mechanisms. Current Opinion in Allergy and Clinical Immunology 2: 1119.

airway and vascular smooth muscle, inhibition of the proliferation of both smooth muscle types, modulation of inammatory and immune responses, and protection of the lung against acute injury and apoptotic cell death. These actions suggest potential therapeutic potential in bronchial asthma, pulmonary hypertension and acute lung injury.

Introduction
Although vasoactive intestinal polypeptide (VIP) was rst isolated from porcine small intestine, its principal biological activity, as a vasodilator, had previously been discovered in lung extracts. VIP was later rediscovered as a neuropeptide with neurotransmitter/neuromodulator properties, and is now known to occur widely in the central and peripheral nervous systems and to have a wide spectrum of biological actions.

Structure
VIP is a 28 amino acid residue peptide (Figure 1). It belongs to a family of structurally related peptides that include a peptide with N-terminal histidine and C-terminal isoleucine amide (PHI) and its counterpart in human tissues, a peptide with N-terminal histidine and C-terminal methionine amide (PHM), secretin, glucagon, gastric inhibitory peptide or glucose-dependent insulinotropic peptide (GIP), corticotropinreleasing hormone (CRH), growth hormone-releasing hormone (GHRH), sauvagine, urotensin I, helodermin, and pituitary adenylate cyclase-activating peptide (PACAP).

Distribution and Localization

The distribution of VIP in the central and peripheral nervous systems has been dened by immunouorescence, radioimmunoassay, and, more recently, by measurement of VIP mRNA. In the brain the peptide is found in highest concentrations in the cerebral cortex, suprachiasmatic nucleus, amygdala, and striatum, but it is also present in the midbrain periaqueductal gray and the sacral spinal cord. In the peripheral nervous system, VIP is localized in the sympathetic ganglia, the vagus nerve, predominantly
HOOC His Ser Asp Ala Val Phe Thr Asp Asn Tyr Ala Val Lys Lys Tyr Leu Asn Ser lle Leu Asn NH2 Met Gln Lys Arg Leu Arg Thr

Relevant Website
http://www.gene.ucl.ac.uk The Centre for Human Genetics.

Vasoactive Intestinal Peptide

S I Said, State University of New York, Stony Brook, NY, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Vasoactive intestinal peptide (VIP) is a neuropeptide that is widely distributed in the central and peripheral nervous systems. Both VIP and its receptors are expressed in various pulmonary structures, including airway epithelial cells, airway and pulmonary vascular smooth muscle, secretory glands, and immune and inammatory cells. Pulmonary effects include relaxation of

Figure 1 Amino acid sequence of human VIP. The sequence is well conserved in mammalian and other species.

518 KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

motor nerves such as the sciatic nerve, and nerves supplying exocrine glands, blood vessels, and nonvascular smooth muscle. In the lungs, VIP-containing nerve bers and nerve terminals are principally localized in the smooth muscle layer of the airways from the trachea through the small bronchioles, around submucosal mucous and serous glands, and in the walls of both pulmonary and bronchial arteries. Immunoreactive VIP is also present in neuronal cell bodies forming microganglia that provide a source of intrinsic innervation of pulmonary structures. In addition to its neuronal localization, VIP is present in abundance in the immune system, including inammatory cells such as mast cells, eosinophils, and B and T lymphocytes.
Colocalization with Other Peptides and Neurotransmitters

exocrine glands brings together the vasodilator effect of VIP and the secretory effect of acetylcholine. Bronchial and pulmonary vessels VIP dilates the vessels supplying the nose, upper airways, trachea, and bronchi, as well as pulmonary vessels. As a pulmonary vasodilator, VIP is 50 times as potent as prostacyclin, and its action is independent of endothelium. Immunologic release of mediators VIP has a moderate inhibitory effect on antigen-induced release of histamine from guinea pig lung. Since the peptide is normally present in mast cells, it may thus act as a natural modulator of mast cell function. Other immunomodulator activities of VIP are outlined below.

Strong evidence suggests that VIP coexists with acetylcholine in many postganglionic cholinergic neurons supplying exocrine glands and blood vessels. VIP may also be colocalized with other peptides: It coexists with PHI (or PHM, its human counterpart), both peptides being synthesized from the same precursor molecule, and may be colocalized with substance P. Other neurotransmitters that coexist with VIP in some of the neurons of bronchial ganglia include nitric oxide (nitric oxide synthase) and glutamate.

Biological Actions Pertinent to the Lung

Multiple biological actions of VIP have been identied. Those relevant to lung function and disease are summarized in Table 1.

Receptors
Specic, high-afnity receptors for VIP have been identied in membrane preparations of normal lungs and human lung tumor cells. These receptors, localized immunocytochemically and by the increased cAMP levels resulting from activation by VIP, exist ;in a variety of pulmonary cells. Molecular and pharmacologic studies have led to the identication, characterization, and cloning of at least three receptors
Table 1 Actions of VIP in the lung

Actions on Lung Structures

Airways

VIP has potent effects on all major structural components of the airways. Airway smooth muscle VIP relaxes airway smooth muscle both in vitro and in vivo, as demonstrated in airways of guinea pigs, rabbits, dogs, and humans. VIP also prevents or attenuates airway smooth muscle contraction by a variety of agents, including histamine, prostaglandin F2a, kallikrein, leukotriene D4, neurokinin A, serotonin, and endothelin. VIP-induced airway relaxation is long lasting and is unaffected by blockade of adrenergic or cholinergic receptors or of cyclooxygenase activity. However, in human asthmatics VIP has been found to be generally less effective in relieving bronchoconstriction, probably because of its rapid inactivation by proteases in airway secretions. Tracheobronchial secretion VIP stimulates water and ion transport in canine tracheal epithelium and may also promote the secretion of sulfated macromolecules. The coexistence of VIP and acetylcholine in cholinergic neurons supplying bronchial and other

Site Airways

Action Relaxation of smooth muscle, inhibition of smooth muscle proliferation, suppression of airway inammation and airway hyperreactivity Relaxation of smooth muscle, inhibition of smooth muscle proliferation Modulation of multiple aspects of the immune and inammatory responses: (a) inhibition of proinammatory cytokines (TNF-a, IL-6, IL-12, IL-18), chemokines (RANTES, MCP-1, 1a, 1b, 2), i NOS, proteases (MMP-2 gelatinase), (b) upregulation of antiinammatory cytokine IL-10 Inhibition of apoptotic cell death, promotion of cell survival, suppression of certain neoplastic cell proliferation, e.g., small cell cancer

Pulmonary vessels Immune and inammatory responses

Cell death, cell survival, or proliferation

IL, interleukin; i NOS, inducible NOS; MMP, matrix metalloprotease; MCP, monocyte chemotactic protein.

KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

519

for VIP and the related peptide PACAP. Now called VPAC receptors, these receptors belong to a distinct family of seven transmembrane-domain receptors coupled to G proteins. The three VPAC receptors are VPAC1 and 2, which respond to VIP and PACAP with comparable afnity, and PAC1, which binds PACAP with high afnity and VIP with considerably lower afnity.

together to bring about the relaxation and to inhibit mitogenesis and smooth muscle proliferation.

Physiological Roles
Evidence, largely collected from experimental studies, suggests that VIP probably plays a number of important physiological roles in relationship to the lung, including: 1. mediation of neurogenic (noncholinergic, nonadrenergic) airway smooth muscle relaxation; 2. mediation of pulmonary vascular relaxation; 3. modulation of inammation (see Table 1); 4. modulation of airway and pulmonary smooth muscle proliferation; 5. modulation of the pulmonary immune response; and 6. protection against apoptotic cell death and promotion of cell survival (Figure 2).

Signal Transduction Pathways

The actions of VIP are mediated principally by adenylyl cyclase stimulation and cAMP production. VIP biosynthesis itself is promoted by higher cAMP levels, and thus the peptide is potentially capable of stimulating its own generation. In some VIP actions, additional second messengers and signal transduction pathways may be involved, for example, intracellular mobilization of Ca2 in the case of the PACAP-preferring PAC1 receptor. The actions of VIP on some tissues and organs, including airway and vascular smooth muscle, may be mediated in part by the release of nitric oxide (NO) from neurons or smooth muscle cells. Because of its activation of a constitutive NO synthase (NOS) in neural elements and smooth muscle, and the subsequent activation of cytosolic guanylyl cyclase, VIP may stimulate intracellular cyclic GMP formation. The role of neuronal NOS in mediating the effects of VIP in the airways was recently assessed in neuronal NOS-knockout mice, and estimated to account for a significant proportion of VIP-induced tracheal relaxation. It is now clear that NO, along with carbon monoxide (CO), mediates VIP-induced relaxation of airway, gastrointestinal, and other smooth muscle. The cyclic AMP and cyclic GMP pathways work

Antiapoptotic Actions of VIP

VIP exerts its antiapoptotic activity by three complementary mechanisms: inhibition of the activation of caspases, key effectors of apoptosis; upregulation of antiapoptotic bcl2; and suppression of cytoplasmic translocation of cytochrome c, a critical step in mitochondria-mediated apoptosis.

VIP in Pulmonary Disease

Bronchial Asthma

The airways of several severely asthmatic subjects who had died in accidents were found to be lacking

VIP

Mediation of neurogenic airway smooth muscle relaxation

Mediation of pulmonary vascular relaxation

Modulation of airway & pulmonary vascular smooth muscle proliferation

Modulation of inflammation

Modulation of immune responses

Protection against apoptotic cell death, promotion of cell survival

Figure 2 Physiological roles of VIP relevant to the lung.

520 KININS AND NEUROPEPTIDES / Vasoactive Intestinal Peptide

VIP

Airways

Pulmonary circulation

Parenchymal lung cells

Small cell lung cancer cells

Relaxation of smooth muscle Inhibition of smooth muscle proliferation Suppression of inflammation & hyperreactivity

Relaxation of smooth muscle Inhibition of smooth muscle proliferation

Inhibition of apoptotic cell death; promotion of cell survival Modulation of inflammatory/immune damage

Suppression of proliferation in vitro & in vivo

Potentially beneficial in bronchial asthma (VIP-immunoreactive nerves may be lacking in asthmatic airways)

Potentially beneficial in primary pulmonary hypertension (VIP-containing nerves are lacking in pulmonary arteries in PPH)

Protection against acute lung injury

Potentially beneficial in small cell lung cancer

Figure 3 Key effects of VIP on various structural components of the lungs, and their potential therapeutic implications. PPH, primary pulmonary hypertension.

in VIP-immunoreactive nerves, raising the possibility of a causative link between VIP deciency and the pathogenesis of asthma. This observation still awaits conrmation, but further evidence suggesting such a link comes from knockout mice. Mice with targeted deletion of the VIP gene exhibited airway hyperreactivity and, upon sensitization and challenge with ovalbumin, showed pathologic features of airway inammation (see Figure 3).
Cystic Fibrosis

Therapeutic Potential
Strong preclinical data and early clinical trials support a therapeutic role for VIP, or more appropriate agonists, in a variety of lung diseases (Table 2).
Bronchial Asthma

VIP-containing nerves were absent around the sweat glands of a group of children with cystic brosis (see Figure 3). How this deciency may relate to the pathophysiology of cystic brosis has not been established.
Primary Pulmonary Hypertension (PPH)

The airway-relaxant, anti-inammatory, and antiproliferative effects of VIP make it an attractive candidate as an antiasthma medication. As mentioned above, early clinical trials of VIP as an aerosol in asthmatics showed modest benet, probably because of its degradation by airway proteases. On the other hand, VIP-like peptides that are more resistant to degradation, such as heloderma and PACAP, have not been adequately tested.
Acute Lung Injury/Acute Respiratory Distress Syndrome

In PPH patients, serum VIP levels are low, and VIPcontaining nerves are lacking in pulmonary arterial walls. Conversely, VIP receptors are upregulated in pulmonary arterial smooth muscle cells, and VIP inhibits the proliferation of these smooth muscle cells in vitro. These ndings suggest that a deciency of VIP may be a factor in the genesis of PPH (see Figure 3).

VIP prevents or delays acute lung injury (ALI) in 10 experimental models, in isolated lungs and in vivo. This protection is attributable to a combination of anti-inammatory, antioxidant, and antiapoptotic properties. Recently, male VIP knockout mice were reported to be more susceptible than wild-type mice to endotoxemia. Clinical trials of VIP in acute respiratory distress syndrome (ARDS) are in progress,

KININS AND NEUROPEPTIDES / Other Important Neuropeptides

Table 2 Possible therapeutic applications of VIP Disorder Airways Bronchial asthma Potential benet from VIP Suppresses bronchoconstriction, airway hyperreactivity, airway inammation, airway remodeling

521

Pulmonary circulation Pulmonary hypertension Other ALI/ARDS Small cell lung cancer

Reduces pulmonary hypertension

Attenuates or prevents ALI Inhibits growth and proliferation of cancer cells

ALI, acute lung injury; ARDS, acute respiratory distress syndrome.

with the peptide being delivered by IV infusion or as an aerosol.

Pulmonary Hypertension

Maruno K, Absood A, and Said SI (1998) Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proceedings of the National Academy of Sciences, USA 95: 1437314378. Petkov V, Mosgoeller W, Ziesche R, et al. (2003) Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. Journal of Clinical Investigation 111: 13391346. Said SI (1988) Vasoactive intestinal peptide in the lung. Annals of the New York Academy of Sciences 527: 450464. Said SI (1989) Vasoactive intestinal polypeptide and asthma (Editorial). New England Journal of Medicine 320: 12711273. Said SI (1996) Molecules that protect: the defense of neurons and other cells (editorial). Journal of Clinical Investigation 97: 21632164. Said SI (2004) Vasoactive intestinal polypeptide. In: Adelman G and Smith BH (eds.) Encyclopedia of Neurosciences, 3rd edn. (CD-ROM). Amsterdam: Elsevier. Said SI and Rattan S (2004) The multiple mediators of neurogenic smooth muscle relaxation. Trends in Endocrinology and Metabolism 15: 189191.

VIP, given as an aerosol over 1224 weeks, markedly improved hemodynamics and exercise tolerance in patients with PPH.
Lung Cancer

Other Important Neuropeptides

M G Belvisi and D J Hele, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Because VIP suppresses the growth of small cell lung cancer cells in culture and in athymic nude mice in vivo, it may offer a less toxic alternative to conventional chemotherapy in the management of this highly malignant form of cancer.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Cystic Fibrosis: Overview. Vascular Disease.

Abstract
Neuropeptides have been implicated in the physiology and pathophysiology of chronic inammatory diseases of the airways such as asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Detailed distribution studies have provided data on the putative role of neuropeptides in the control of normal respiratory functions, and studies point to the involvement of regulatory neuropeptides in diseases of the lung. Neuropeptides such as bradykinin, the tachykinins, and vasoactive intestinal peptide have been described as having a role in inammatory diseases of the airways but a number of other neuropeptides, which include calcitonin gene-related peptide, secretoneurin, bombesin, galanin, somatostatin, cholecystokinin, neuropeptide-Y, enkephalins, neurotensin, and pituitary adenylate cyclase-activating polypeptide, have been implicated in the etiology or pathophysiology of inammatory diseases of the airways.

Further Reading
Delgado M (2003) VIP: A very important peptide in T helper differentiation. Trends in Immunology 24: 221224. Delgado M, Abad C, Martinez C, Leceta J, and Gomariz RP (2001) Vasoactive intestinal peptide prevents experimental arthritis by down-regulating both autoimmune and inammatory components of the disease. Nature Medicine 7: 563568. Fahrenkrug J (1989) Transmitter role of vasoactive intestinal peptide. Pharmacology and Toxicology 72: 354363. Goetzl EJ, Voice JK, Shen S, et al. (2001) Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide. Proceedings of the National Academy of Sciences, USA 98: 1385413859. Gozes I and Brenneman DE (1989) VIP: Molecular biology and neurobiological function. Molecular Biology 3: 201236. Harmar AJ, Arimura A, Gozes I, et al. (1998) International Union of Pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Pharmacological Reviews 50: 265270. Laburthe M, Couvineau A, and Marie JC (2002) VPAC receptors for VIP and PACAP. Receptors and Channels 8: 137153.

Introduction
Neuropeptides have been implicated in the pathophysiology of chronic inammatory diseases of the airways such as asthma, allergic rhinitis, and chronic obstructive pulmonary disease (COPD). Many factors stimulate primary sensory neurons including cigarette smoke, airborne pollutants, and allergens, all of which may participate in the generation of airway inammation. Pathological activation of sensory neurons and the resulting inammatory events are known as neurogenic inammation and appear to be

522 KININS AND NEUROPEPTIDES / Other Important Neuropeptides

important in many organ systems, including the lung. Neuropeptides form part of that inammatory response and neurogenic inammation may participate in the development and progression of chronic airway inammatory diseases such as asthma or COPD. The molecular mechanisms underlying neurogenic inammation are orchestrated by a large number of neuropeptides. Neuropeptides in the lung originate either from local neurons (e.g., vasoactive intestinal peptide (VIP) and galanin), extrinsic neurons localized in sensory ganglia (e.g., substance P and calcitonin generelated peptide (CGRP)), or the sympathetic chain (e.g., neuropeptide Y (NPY)). Detailed distribution studies have provided data on the putative role of the peptides in the control of normal respiratory functions and further studies point to the involvement of regulatory peptides in diseases of the lung. Their widespread distribution and physiological effects suggest they may have a role to play and in fact plasma levels of neuropeptides such as CGRP, substance P, and NPY have been shown to be raised during exacerbations in asthma patients. Neuropeptides such as bradykinin, the tachykinins, and VIP have been described elsewhere (see Kinins and Neuropeptides: Bradykinin; Tachykinins; Vasoactive Intestinal Peptide). This article will discuss the neuropeptides CGRP, secretoneurin, bombesin, galanin, somatostatin, cholecystokinin, NPY, enkephalins, neurotensin, and pituitary adenylate cyclase-activating polypeptide (PACAP), all of which have been implicated in the etiology or pathophysiology of airway inammatory disease.

Calcitonin Gene-Related Peptide

CGRP is a 37 amino acid peptide that is constitutively expressed in normal lungs where it localizes to a specialized subset of epithelial cells (neuroendocrine cells) and sensory C bers distributed in the pulmonary airways, blood vessels, and lymphoid tissue. Two CGRP isoforms generated from different genes (a- and b-CGRP) have been identied in rats and humans. Comparison of the peptide sequences of a- and b-CGRP reveals a striking degree of sequence homology (human a-CGRP differs from b-CGRP in 3 of 37 amino acids, and rat a-CGRP differs from b-CGRP in 1 of 37 amino acids), and the two isoforms have been considered to be almost equivalent in function. Two CGRP receptors have been identied pharmacologically on the basis of their differential afnities for the competitive peptide antagonist CGRP837. A candidate receptor was initially cloned from the sequence of the calcitonin receptor by polymerase chain reaction (PCR) in rat lung and was named calcitonin receptor-like receptor (CRLR).

Human CRLR has subsequently been cloned and proposed as the CGRP1 receptor. It has recently been determined that when coexpressed with receptor activity-modifying proteins (RAMPs), in particular RAMP1, CRLR functions as a CGRP1 receptor in a variety of cells. In addition, an intracellular accessory protein termed CGRP-receptor component protein (RCP) has recently been identied and shown to be required for signal transduction through CGRP receptors. A recent study identied a 66 kDa protein as a new receptor for CGRP, which is distinct from CRLR and RAMP1, and proposed a new classication for CGRP1 and CGRP2 receptors as CGRP-A and CGRP-B, respectively. Binding of CGRP to its receptor is known to activate adenylyl cyclase and increase cAMP production, a pathway that involves Gas protein. CGRP has long been suggested to participate in airway physiology and pathophysiology and may play a key role in the pathogenesis of a number of respiratory diseases such as asthma, COPD, and chronic cough (Figure 1). A study has shown CGRP to be a constrictor of human bronchus airway smooth muscle in vitro, which is surprising since it stimulates intracellular cAMP generation; an effect normally associated with bronchodilation. There are two possible mechanisms that may account for the constrictor effect: either CGRP activates phospholipase C-b1 and leads via the inositol 1,4,5-trisphosphate-related pathways to constriction or CGRP does not have a direct effect on the airway smooth muscle itself but liberates other constrictor mediators. In this regard, a few binding sites for CGRP were found in the airway smooth muscle layer of human lung, suggesting an indirect mode of CGRPmediated bronchoconstriction in human airways. In the guinea pig respiratory tract, the effects of CGRP on airway smooth muscle are controversial and no consistent bronchoconstrictor or bronchodilator effect has been demonstrated. In addition, CGRP also causes other pulmonary effects including the induction of eosinophil migration and the stimulation of T cell adhesion to bronectin at sites of inammation. Furthermore, an increase in CGRP content in the lungs of rats and guinea pigs by allergic inammation has been reported. Interestingly, a recent study evaluated the role of CGRP in the development of airway hyperresponsiveness (AHR) in a-CGRP knockout mice and described increases in CGRP expression in the lungs of ovalbumin-sensitized and challenged wild-type mice, and an attenuated AHR in sensitized and challenged a-CGRP knockout mice. It was concluded that CGRP is a mediator of AHR, but not eosinophilia, in this model.

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Inflammatory cells Eosinophil Migration Structural cells Vessels

523

T lymphocyte Vasodilation Adhesion CGRP Airway smooth muscle Macrophage

Secretion T-cell activation

Figure 1 Cellular effects of CGRP.

Bronchospasm?

In contrast to the effects in allergic animal models described above, another study has shown that CGRP was significantly depleted in pulmonary neuroepithelial bodies (NEBs) and submucosal nerve bers following airway allergen challenge of sensitized mice and that these mice developed a robust allergic airway inammation and AHR. In addition, the CGRP1 receptor antagonist CGRP837 did not inhibit the development of AHR or airway inammation in these animals but exogenously administered CGRP (systemically or by inhalation) did inhibit the development of AHR. Similar observations of CGRP depletion have been reported in the guinea pig following sensitization and airway challenge with toluene-diisocyanate. Furthermore, CGRP has been also shown to inhibit macrophage secretion and the capacity of macrophages to activate T cells, indicating a potential anti-inammatory effect. A role for CGRP in tissue repair is also suggested based on its capacity to induce migration and proliferation of broblasts. The ability of CGRP to exert multiple effects such as vasoregulation, anti-inammatory actions and tissue repair suggests a role in airway homeostasis. However, in order to clearly dene the functional effects of CGRP it is necessary to develop potent and highly selective pharmacological agents, which can be used as tools to dissect the role of this peptide in airway physiology and pathophysiology.

Secretoneurin
Chromogranins belong to an evolutionarily conserved family of proteins that serve as neuropeptide proproteins and the secretogranin-II-derived peptide secretoneurin is a 33 amino acid polypeptide that exerts its

effects by binding to specic receptors. Secretoneurin is derived by proteolytic processing from its precursor secretogranin II (chromogranin C). Expression data suggests the presence of secretoneurin in rat tracheal tissue and in human bronchial neuroendocrine cells. Studies in rats have shown that afferent nerve bers may contain secretoneurin. Secretoneurin appears to have a similar distribution to the sensory neuropeptides substance P and CGRP in the nervous system of the rat. Furthermore, studies in rats have demonstrated that levels of secretoneurin are reduced in peripheral organs, including the trachea, after neonatal capsaicin treatment, although the reduction may be less pronounced than that of substance P and CGRP. Interestingly, it has been shown that this neuropeptide may have effects on various immune cells. For example, secretoneurin has been demonstrated to attract immature, and arrest mature, blood-derived dendritic cells. Moreover, secretoneurin may trigger migration of human monocytes in vitro and in vivo, and combinations of secretoneurin and sensory neuropeptides (substance P, somatostatin) synergistically stimulate this migration. Intriguingly, secretoneurin may also act as a potent eosinophil chemoattractant. Taken together, these previous studies indicate that secretoneurin may play a role in inammatory conditions. In fact, recent data have demonstrated that secretoneurin is widely distributed in the nasal mucosal nerves of patients with seasonal allergic rhinitis and ongoing allergen exposure is characterized by increased levels of secretoneurin in nasal lavage uid. Therefore, secretoneurin may have a role in the local trafc of immune cells in human airway mucosa and may play an important role in the early stages of allergic inammation.

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Bombesin
Bombesin is a 14 amino acid bioactive peptide that was rst isolated from frog skin; bombesin-like peptides (BLPs) are mammalian homologs of bombesin. BLPs comprise a family of related peptides, several of which could account for the BLP immunoreactivity localized to pulmonary neuroendocrine cells. One major pulmonary BLP has been identied as gastrinreleasing peptide (GRP). GRP and bombesin are highly similar both structurally and functionally, and neuromedin B (NMB) is also closely related. BLPs are growth factors for broblasts and normal airway epithelial cells. They have neuroregulatory functions in the brain and gut; and are potent bronchoconstrictors. GRP and bombesin are potent stimulators of airway mucus secretion in animals and humans. Multiple receptor subtypes mediate the effects of bombesin-related peptides. The bombesin receptor family is currently comprised of four receptor subtypes. These subtypes are classied as the NMB subtype (BB1), the GRP subtype (BB2), the orphan receptor subtype (BB3), and the bombesin (BBN) receptor subtype (BB4). BLPs promote fetal lung development, including lung-branching morphogenesis, epithelial and mesenchymal cell proliferation, and type II cell differentiation. A possible role has been suggested for BLPs in promoting cellular responses that may contribute to inammatory processes, including macrophage activation and phagocytosis, chemotaxis of macrophages and broblasts, smooth muscle constriction, and proliferation of broblasts, lymphocytes, and endothelial cells. BLPs have also been shown to be elevated in newborns who later develop bronchopulmonary dysplasia. Bombesin stimulates superoxide anion production and interleukin (IL)-8 release by peripheral monocytes. Increased lung levels of BLPs have been described in smokers and patients with COPD. Bombesin significantly enhanced the phagocytosis of monocytes and alveolar macrophages from normal and COPD patients and restored decient phagocytosis in a group of COPD patients. Increased numbers of BLP-positive neuroendocrine cells have been associated with other chronic inammatory lung diseases, including cystic brosis, lung cancer, eosinophilic granuloma, and pulmonary hypertension. Evidence suggests that BLPs are involved in immunoregulatory activities in the lung.

actions via three subtypes of G-protein-coupled receptor (denoted GALR1, GALR2, and GALR3), and has modulatory effects on sensory nerve bers. Galanin potently modulates the mechanosensitivity of gastroesophageal vagal afferents with either facilitatory or inhibitory actions on individual afferent bers. Both intrinsic and extrinsic vagal neurons contain galanin and are therefore potential sources of endogenous galanin. Galanin immunoreactive bers have been detected in the nerve cell bodies of the airways and in the trachea, bronchus, and major intrapulmonary airways, predominantly in smooth muscle, around seromucous glands, and in the adventitia of blood vessels but rarely in lung parenchyma. Galanin-containing nerve bers in the airways originate from nerve cell bodies of intrinsic airway ganglia, and galanin and VIP are frequently colocalized in these neurons. The distribution of galanin suggests that it may have some inuence on airway, vascular, and secretory functions in the mammalian respiratory tract although its role appears to be in nociception rather than as an inammatory mediator. There is evidence to suggest that secretion of mucus into airways is regulated by a complex network of peptidergic stimulators and inhibitors, which includes galanin, and it has been demonstrated that galanin inhibits secretion of mucus.

Somatostatin
The neuropeptide somatostatin (or somatotroph release inhibiting factor (SRIF)-14) is a cyclic decatetrapeptide initially isolated from hypothalamus extracts but has been found widely distributed in the body. Somatostatin is the predominant isoform of the SRIF family (the other being SRIF-28). The actions of these peptides are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors containing ve distinct subtypes (termed SSTR15) that are encoded by separate genes segregated on different chromosomes. The ve receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar afnity. The ve receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated proteinkinase (MAPK) through G-protein-dependent mechanisms. Some of the subtypes are also coupled to inward rectifying K channels (SSTR2, 3, 4, 5), to voltage-dependent Ca2 channels (SSTR1, 2), a Na / H exchanger (SSTR1), AMPA/kainate glutamate channels (SSTR1, 2), phospholipase C (SSTR2, 5), and phospholipase A(2) (SSTR4). Five different G-protein-coupled receptors for somatostatin have been cloned and at least three

Galanin
Galanin is a 29 (30 in human) amino acid neuropeptide found in central and peripheral nervous systems. It appears to have excitatory and inhibitory

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subtypes have been found in the lung. One of these receptors, sst4, is elevated in disease states in the lung. Furthermore, the expression of sst2A and sst3 receptor mRNAs has been reported in cells of the immune system such as activated macrophages and T and B lymphocytes. Significant quantities of somatostatin have been found in the lung and there is evidence to suggest that it may have anti-inammatory properties and may play a regulatory role in secretion of mucus. Somatostatin has been shown to have potent immunomodulatory actions on secretion by immune cells such as immunoglobulin (Ig) production by activated B cells and cytokine production by activated T cells and macrophages. It has also been shown to be involved in the regulation of the Th1/Th2 pattern of cytokine secretion. Somatostatin also inhibits IgE-stimulated histamine from basophils and tumor necrosis factor alpha (TNF-a), IL-1b, and IL-6 from monocytes. Somatostatin has been shown to be involved in the inhibition of neutrophil chemotaxis, natural killer cell activity, and the phagocytic activity of monocytes/macrophages. The proliferative response to antigens and mitogens is modulated by somatostatin; this can be inhibitory or stimulatory depending on the concentration of somatostatin present. Somatostatin has been shown to inhibit secretion of mucus and somatostatin analogs have been shown to inhibit the bronchoconstriction that occurs in patients with carcinoid syndrome. The evidence suggests that somatostatin may be of importance in the pathophysiology of a variety of lung diseases and may have a benecial anti-inammatory role in inammatory diseases of the airways.

and alveolar epithelial cells where they mediate the regulatory actions of CCK-8 on these cells. Although CCK-8 is not closely associated with nerves in the airways, its constrictor effect on airway smooth muscle and its presence in many cell types may suggest a role in airway disease.

Neuropeptide Y
NPY is a 36 amino acid neuropeptide that participates via at least ve G-protein-coupled receptors in the regulation of a large number of physiological and pathophysiological processes in the respiratory system, immune system, nervous system, and endocrine system. Subtypes Y1, Y2, Y4, and Y5 are expressed in humans. Their physiologic ligands are the neurotransmitter NPY and the two hormones peptide YY (PYY) and pancreatic polypeptide (PP). NPY is distributed in the region of sympathetic nerves found in the nasal mucosa, bronchial vessels, and seromucous glands and is often colocalized with catecholamines. NPY has long been proposed to play a role in the pathogenesis of inammatory diseases and serum levels of NPY are increased during asthma exacerbations. The number of NPY-immunoreactive nerves in the airways may remain constant in patients with airway inammatory disease although studies have shown NPY-immunoreactive nerves to be signicantly decreased in the smooth muscle of patients with asthma and chronic bronchitis. NPY exerts a major inuence on humoral and cellular immune functions and is known to modulate immune cell distribution, T-helper cell differentiation, mediator release, and natural killer cell activation. In addition to these direct effects, NPY also acts as an immunomodulator by inuencing the effects of a variety of other neurotransmitters. NPY has no direct effect on airway smooth muscle or on mucus secretion but may cause bronchoconstriction via the release of prostaglandins and has been shown to enhance cholinergic and adrenergic stimulation of mucus secretion. Conversely, local treatment of the nasal mucosa with NPY reduced nasal obstruction and mucus secretion evoked by allergen challenge in humans. This evidence suggests a possible role for NPY in the development of pulmonary inammatory disorders and studies with selective antagonists and agonists may help to elucidate that role.

Cholecystokinin
The neuropeptide cholecystokinin octapeptide (CCK-8) has been found in significant quantities in the lungs of various mammalian species but the portion colocalized with nerve bers is found to be very low. CCK-8 has been shown to protect the lung against injury by endotoxin, reducing the inammatory response and protecting against the development of pulmonary hypertension. Conversely, CCK-8 potently constricts airways in guinea pigs and humans and this effect is potentiated by prior sensitization to allergen. This direct constrictor effect on airway smooth muscle can be reversed by pretreatment with a specic CCK antagonist. There are two known receptors, CCK-A and CCK-B, and the constrictor effect is thought to be mediated via the CCK-A receptor, which is more widely distributed in the peripheral tissues. The CCK-A and CCK-B receptor genes have been found in the lung in vascular endothelial cells, macrophages, bronchial epithelial cells,

Opioids
Endogenous opioids are formed by proteolysis of larger precursor molecules. There are three such precursor molecules: proenkephalin A, which is processed to form the family of enkephalins; prodynorphin,

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which yields dynorphin A and B; and a- and b-neoendomorphins and propriomelanocortin, which give rise to b-endorphin. Endogenous opioids endomorphin-1 and -2 have also been described that act at the MOP (m mu for morphine) receptor in a naloxonesensitive fashion. Relatively recently another endogenous opioid-like peptide, nociceptin/orphanin FQ, was isolated which has some homology with dynorphin but lacks the N-terminal tyrosine residue that denes the classical opioids and is required for receptor activation. Both the endomorphins and nociceptin differ structurally from one another and from the classical opioids and are thought to derive from different precursor molecules. Endogenous opioids are synthesized by nerves innervating the airways. Immunoreactivity for [Met]enkephalin-Arg6-Gly7Leu8 and Met and Leu-enkephalin have been demonstrated in nerve bers innervating the trachea and major bronchi in the rat and guinea pig. These bers were present in smooth muscle bundles, around airway glands, in the lamina propria, and in the walls of pulmonary vessels. Pulmonary neuroepithelial endocrine cells have been shown to contain a number of regulatory neuropeptides, including enkephalins. Enkephalins have been found in normal lung tissues and possess both immunostimulant and immunosuppressive properties. The cells of the immune system such as T and B cells can synthesize and secrete significant amounts of enkephalins. Leu-enkephalin has been localized to neuroendocrine cells in mammalian airways and met-enkephalin immunoreactive nerves have also been found. To date, four opioid receptors have been cloned: the MOP, the KOP (k kappa for ketocyclazocine), the DOP (d delta for deferens because it was rst identied in mouse vas deferens), and the NOP-R (initially called LC132, ORL-1, or nociceptin/orphanin FQ receptor). Met-enkephalin and enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes and play an important regulatory role in the immune response. If the production of met-enkephalin and enkephalin-containing intermediary peptides is interrupted, it results in an enhancement of the proliferative T cell response and an inhibition of monocyte IL-6 secretion. The immunomodulatory effects of met-enkephalin are blocked by the opioid antagonist naloxone, suggesting these effects are mediated by opioid receptors on the cells involved. Opioids have also been shown to inhibit neurogenic mucus secretion and vagal nerve mediated plasma leakage in the airways by presynaptic inhibition of the release of neuropeptides from sensory nerve endings. Furthermore, an inhibitory action was

also revealed on plasma exudation induced by cigarette smoke in guinea pig bronchi. Similarly, opioids have also been reported to inhibit nonadrenergic, noncholinergic, sensory nerve-mediated bronchoconstriction in guinea pig airways and cholinergic neurotransmission in both guinea pig and human airways in vitro. The opioid receptor mediating these inhibitory actions on airway nerves appears to be the MOP subtype. The mechanism of the inhibition is unknown but may be via the opening of large conductance calcium-activated potassium channels. Opioids such as codeine, morphine, and dihydrocodone have also been demonstrated to be antitussives due to their action at central and peripheral MOP opioid receptors located on sensory nerve endings. Recently, both DOP receptor antagonists and agonists have been found to be antitussive in animal models. Additionally, antitussive KOP receptor agonists have been reported. Therefore the evidence suggests that endogenous opioids may act as regulators of the neural and inammatory response and may have a role to play in inammation in the airways and in the suppression of reex events such as cough.

Nociceptin
Nociceptin/orphanin FQ (N/OFQ) is an opioid-like peptide and is the endogenous ligand for the NOP receptor. NOP receptors are distributed widely in the CNS and on airway nerves. Nociceptin has been found to inhibit the release of sensory neuropeptides following depolarization of C bers and to inhibit bronchospasm in the guinea pig. Recently, it has been demonstrated that N/OFQ inhibited cough induced by mechanical stimuli or capsaicin in guinea pigs and cats. This suggests that NOP receptors are involved in the modulation of the cough reex and that selective NOP receptor agonists might therefore have potential as novel peripherally acting antitussives, although to date there have been no studies with such drugs in humans.

Neurotensin
Neurotensin (NT) is a 13 amino acid tridecapeptide localized to epithelial cells and nerves that fulls a dual function: as a neurotransmitter/neuromodulator in the nervous system, and a paracrine and circulating hormone in the periphery. Three NT receptors, NTS1, NTS2, and NTS3, have been cloned to date. NTS1, and NTS2 belong to the family of seven transmembrane G-protein-coupled receptors, whereas NTS3 is a single transmembrane domain protein (also known as sortilin) that belongs to a recently identied family of sorting receptors. Most of the known peripheral

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and central effects of NT are mediated through NTS1. NT is a potent bronchoconstrictor, an effect that is modulated by the enzyme neutral endopeptidase. Neutral endopeptidase levels are reduced in airway inammatory disease states and this leads to enhanced neurogenic inammation, which may involve NT. Further evidence for the proinammatory nature of NT is that it promotes the adhesion of neutrophils to bronchial epithelial cells and causes the degranulation of certain types of mast cell. There are no reported studies as yet on the effects of NT in human airways.

investigation to examine their potential as putative treatments for asthma.

See also: Asthma: Overview. Capsaicin. Chronic Obstructive Pulmonary Disease: Overview. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins; Vasoactive Intestinal Peptide. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells; Pulmonary Macrophages. Mucus. Neurophysiology: Neuroendocrine Cells. Smooth Muscle Cells: Airway.

Pituitary Adenylate Cyclase-Activating Polypeptide

PACAP is a 38 amino acid neuropeptide widely distributed in the central and peripheral nervous systems. PACAP-containing nerve bers have been found in association with bronchial and vascular smooth muscle and appear to be more abundant than VIP-containing nerves in human bronchi. PACAP receptors have been found at all levels in the respiratory system. There are three distinct receptor subtypes: the PACAP-specic PAC1 receptor, which is coupled to several transduction systems; and the two PACAP/VIP-indifferent VPAC1 and VPAC2 receptors, which are primarily coupled to adenylyl cyclase. PAC1 receptors are particularly abundant in the brain and pituitary and adrenal glands whereas VPAC receptors are expressed mainly in the lung, liver, and testis. PACAP exists in two endogenous forms, PACAP-27 and PACAP-38, and these display several activities that may be relevant to the understanding of airway diseases such as asthma and COPD. They modulate inammatory cell activity, mediating effects on lymphocyte mobility. PACAP-38 is present in human lung and is a more potent dilator of human bronchi than VIP, suggesting a role for PACAP-38 in the endogenous regulation of airway tone. This effect on smooth muscle tone is more sustained than that seen with PACAP-27. PACAP-38 has also been shown to enhance phagocytosis in macrophages. PACAP-38 inhibits chemotaxis and the release of the proinammatory cytokines TNFa, IL-2, IL-6, and IL-12, and promotes the release of the anti-inammatory cytokine IL-10, suggesting an overall anti-inammatory action. PACAP-38 has also been shown to be a potent stimulator of airway mucus secretion. Owing to the bronchodilator properties of PACAP, synthetic analogs are currently under

Further Reading
Barnes PJ, Baraniuk JN, and Belvisi MG (1991) State of the art: neuropeptides in the respiratory tract. Part 1 (review). American Review of Respiratory Diseases 144(5): 11871198. Barnes PJ, Baraniuk JN, and Belvisi MG (1991) State of the art: neuropeptides in the respiratory tract. Part 2 (review). American Review of Respiratory Diseases 144(6): 13911399. Bartfai T, Hokfelt T, and Langel U (1993) Galanin a neuroendocrine peptide (review). Critical Review of Neurobiology 7(34): 229274. Crawley JN and Corwin RL (1994) Biological actions of cholecystokinin (review). Peptides 15(4): 731755. Cutz E (1982) Neuroendocrine cells of the lung. An overview of morphologic characteristics and development (review). Experimental Lung Research 3(34): 185208. Groneberg DA, Folkerts G, Peiser C, Chung KF, and Fischer A (2004) Neuropeptide Y (NPY) (review). Pulmonary Pharmacology & Therapeutics. 17(4): 173180. Groneberg DA, Quarcoo D, Frossard N, and Fischer A (2004) Neurogenic mechanisms in bronchial inammatory diseases. Allergy 59: 11391152. Krantic S (2000) Peptides as regulators of the immune system: emphasis on somatostatin (review). Peptides 21(12): 1941 1964. Scheuermann DW, Adriansen D, Timmermanns JP, and De Groodt-Lasseel MH (1992) Comparative histological overview of the chemical coding of the pulmonary neuroepithelial endocrine system in health and disease (review). European Journal of Morphology 30(2): 101112. Springer J, Geppetti P, Fischer A, and Groneberg DA (2003) Calcitonin gene-related peptide as inammatory mediator (review). Pulmonary Pharmacology and Therapeutics 16(3): 121130. Sunday ME (1997) Neuropeptides and lung development. In: McDonald JA (ed.) Lung Growth and Development, pp. 401 494. New York: Dekker. Uddman R, Hakanson R, Luts A, and Sundler F (1997) Distribution of neuropeptides in airways. In: Barnes PJ (ed.) Autonomic Innervation of the Respiratory Tract, pp. 2137. London: Harwood Academic. Vaudry D, Gonzalez BJ, Basille M, Fournier A, and Vaudry H (2000) Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions (review). Pharmacological Reviews 52(2): 269324. Wiedermann CJ (2000) Secretoneurin: a functional neuropeptide in health and disease (review). Peptides 21(8): 12891298.

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LARYNGITIS AND PHARYNGITIS
H Coates, The University of Western Australia, Perth, WA, Australia
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Pharyngitis and laryngitis are inammatory conditions affecting the upper airway. The majority of these inammatory conditions are viral in origin and self-limiting. In adults, cigarette smoking and gastroesophageal reux are major causes of chronic laryngitis, whilst in infants and children gastroesophageal reux and viral and bacterial infections play the major role in etiology. Symptoms that occur secondarily to the pathologic features of mucosal inammation and submucosal edema include hoarseness, sore throat, cough, pain, or difculty in swallowing or, occasionally, airway obstruction. Diagnostic tests, depending on the presumptive diagnosis, may include bacterial culture and serology, histopathologic biopsy, barium radiography, or pH studies. Management may include avoidance or treatment of the underlying cause with viral conditions managed symptomatically, some bacterial infections treated with antibiotics and for more serious conditions such as epiglottitis (supraglottitis) and diphtheria occasionally requiring endotracheal intubation or tracheotomy.

or rheumatic fever, glomerulonephritis, or bacteremia from group A beta hemolytic streptococcus (GABHS) infection. Historically, laryngitis and pharyngitis have been known since the earliest medical writings but it is only in the last two centuries that visualization of the larynx has been achieved. The incidence of epiglottitis (supraglottitis) and diphtheria with severe airway obstruction and the need for urgent tracheotomy has declined precipitously since the introduction of vaccinations for both of these conditions.

Epidemiology
Pharyngitis and laryngitis are relatively common conditions, especially those spread by viruses, and are more common in late winter and spring as is laryngotracheobronchitis (croup). Three to ve per cent of children have at least one episode of croup and it reoccurs in about 5% of these cases. The majority of episodes occur between the ages of 6 months and 4 years, and boys are affected twice as commonly as girls. It has been estimated that prior to the introduction of the Haemophilus inuenzae (Hib) vaccine, there were between 3.2 and 8.6 cases of epiglottitis (supraglottitis) per 10 000 pediatric hospital admissions.

Introduction
Laryngitis is an inammation of the larynx, which frequently results in hoarseness or loss of the voice. Laryngitis may be acute or chronic and the most common form associated with a viral upper respiratory tract infection is self-limiting although bacterial infection may also cause laryngitis in association with bronchitis or pneumonia. Chronic laryngitis is frequently seen in smokers, and also in adults with gastroesophageal reux disease (GERD). In children acute laryngotracheobronchitis (croup) and epiglottitis (supraglottitis) can lead to severe or even fatal respiratory obstruction. Pharyngitis is an inammatory condition of the pharynx accompanied by a sore throat and occasionally difculty in swallowing. It is usually viral but may be caused by bacterial or fungal infection. GERD or particularly extraesophageal reux (EER) can also cause an acid pharyngitis in adults and children. Serious complications of pharyngitis may include peritonsillar abscess or retropharyngeal abscess, airway obstruction with infectious mononucleosis

Etiology
The major etiologic causes of laryngitis are shown in Table 1 and include infective, allergic, and traumatic causes. The great majority of cases of laryngitis, both acute and chronic, are caused by infective agents and GERD in children and adults, as well as cigarette smoking in adults. Vocal cord abuse in both age groups is another common cause of laryngitis. Viral croup covers a group of infections caused by viruses, including laryngotracheitis, laryngotracheobronchitis, and spasmodic croup. The organisms involved include adenoviruses, inuenza viruses, parainuenza virus, rhinovirus, and respiratory syncytial viruses. In addition, there are other factors that predispose a child to croup, including cold temperatures, low humidity, pollution, and passive smoking, as well as

530 LARYNGITIS AND PHARYNGITIS

Table 1 Etiology of laryngitis Infective Viral Common cold virus Adenovirus Inuenza virus Parainuenza virus Rhinovirus Respiratory syncytial virus Herpes simplex viruses Bacteria Haemophilus inuenzae Diphtheria Syphilis Tuberculosis Fungal Candida albicans Histoplasmosis Allergic Angioedema Trauma GERD/EER Cigarettes Alcohol Burns Corrosives Vocal cord abuse Table 2 Etiology of pharyngitis Infective Viral Common cold virus Adenovirus Inuenza virus Infective mononucleosis (EpsteinBarr virus) Human immunodeciency virus Bacteria Group A streptococcus Corynebacterium diphtheriae Neisseria gonorrhoea Haemophilus inuenzae Borrelia vincentii Ludwigs cellulitis (Anaerobic fusobacteria and spirochetes) Chlamydia Syphilis Tuberculosis Fungal Candida albicans Trauma GERD/EER Cigarette abuse Alcohol abuse Other Kawasaki disease

EER. In pediatric patients, epiglottitis (supraglottitis) is almost always caused by H. inuenzae infection although Streptococcus pneumoniae, Streptococcus group A, B, and C, Staphylococcus aureus, Pseudomonas aeruginosa, Candida, and Herpes simplex viruses have been implicated in less than 1% of cases in this age group and usually in immunocompromised patients. Diphtheria remains endemic in many developing countries although it has virtually disappeared from most developed countries with the introduction of diphtheria toxoid and immunization programs. The causative organism is Corynebacterium diphtheriae. GERD and, in particular, EER affect the larynx giving a high incidence of hoarseness, excessive throat clearing, chronic cough, and other otolaryngologic complaints with little in the way of esophagitis. The larynx and pharynx do not have protective mechanisms to prevent mucosal injury, while the esophagus demonstrates a mucosal barrier, bicarbonate production, and peristaltic clearance. Pharyngitis includes inammation of the structures of the nasopharynx, oropharynx, and laryngopharynx (see Table 2). Nasopharyngitis is common during winter months with adenovirus, inuenza virus, parainuenza viruses, and enterovirus being the most common etiologic factors. Ninety per cent of pharyngotonsillitis is related to GABHS, adenoviruses,

inuenza viruses, parainuenza viruses, enteroviruses, EpsteinBarr virus, and mycoplasma infection. GABHS is the most common and serious bacterium causing pediatric pharyngitis. There is a small risk of rheumatic fever (0.3%) and acute glomerulonephritis (1015% of those infected with nephritogenic strains). Infectious mononucleosis is caused by EpsteinBarr virus, a member of the herpes virus family, and is a major cause of pharyngitis in adolescents and young adults. EER may cause an acid pharyngitis and has been implicated in sinus and middle ear disease.

Pathology
Viral croup occurs when the causative virus spreads from the nose and pharyngeal airway to the larynx and trachea. Local cellular defenses are impaired and the ciliary function is inhibited resulting in edema and inltration of inammatory cells producing narrowing of the subglottis. Secondary bacterial infection may occur causing purulent secretions, which may compromise the airway. Epiglottitis (supraglottitis) is caused by H. inuenzae B bacterium, which is a mesomorphic Gramnegative organism capable of both anaerobic and aerobic growth. The type B strain is unique in that it possesses an antigenic capsule and is capable of invading mucosal tissue. The organism may be spread

LARYNGITIS AND PHARYNGITIS 531

hematogenously and blood cultures may be positive. Invasion of the supraglottic structures results in an intense inammatory and infective reaction and occasionally microabscess formation in the region that causes the epiglottis to swell and compromise the airway. Diphtheria caused by C. diphtheriae Gram-positive bacillus affects the respiratory passages with an exotoxin released after 24 days of incubation causing tissue necrosis and exudate. This exudate eventually can develop into an adherent gray membrane of brinous nature, which may cause edema and airway obstruction. EER causes laryngeal and pharyngeal erythema with edema of the vocal cords and false vocal cords, larynx, and pharynx. Because of the lack of protective factors mentioned earlier, less acid or pepsin exposure is needed to cause mucosal damage to the pharynx and larynx. Tonsillitis may be viral or bacterial, particularly with GABHS. Chronic tonsillitis where there is cryptic debris secondary to bacterial interaction with undigested food may also show bacterial biolm formation within the crypts. Peritonsillar and retroesophageal abscesses occur when bacterial organisms proliferate in between the tonsillar capsule and tonsillar bed and the retropharyngeal tissues and the prevertebral fascia, respectively. Chronic laryngitis in adults is often related to cigarette abuse and may be aggravated by alcohol consumption and EER. Pathologically, there is edema of the submucosa and epithelial changes that may undergo malignant transformation.

throat, difculty in swallowing, and occasionally cough or wheezing. Signs Signs of pharyngitis include erythematous pharynx including the tonsils and soft palate on occasion with a coating or membrane. There may be accompanying cervical lymph node enlargement and tenderness. Classically, streptococcal pharyngitis shows a red pharynx with exudate on the tonsils and petechiae on the soft palate. It may be accompanied by scarlet fever with a red rash on the trunk primarily with a strawberry tongue and pallor around the mouth. The main sign of gastroesophageal reux in infants and children is diffuse erythema of the oropharynx, unaccompanied by exudate or fever. Investigations If a diagnosis of strep pharyngitis is suspected then throat culture or an antigen detection test should be performed. The throat culture is 9095% sensitive while the rapid strep test is less sensitive but as specific as the throat culture. Culture must be taken prior to starting antibiotic treatment. There is a 10% false-positive rate in patients who are carriers of GABHS without clinical symptoms. Testing for infectious mononucleosis is indicated if there is generalized lymphadenopathy together with a membrane over the pharynx. If there is suspicion of other infections such as gonorrhea or HIV, then these should be tested for using the appropriate swab or serologic testing. Gastroesophageal reux may be tested for using a barium swallow examination or esophageal pH monitoring. Intraluminal impedance testing is a new modality for assessing GERD. An endoscopy may be necessary to demonstrate ulceration or inammation of the esophageal walls or obtain biopsies of the mucosa. In adults, esophageal manometry is used to demonstrate abnormal sphincter pressure.
Tonsillitis

Clinical Features
Pharyngitis

General features Major symptoms are a sore throat, usually of sudden onset, accompanied by pain and difculty in swallowing, and fever. In cases of viral pharyngitis there will be rhinitis, conjunctivitis, diarrhea, or cough, which will be absent in strep throat, caused by GABHS. Fever, headache, and swollen cervical lymph nodes may accompany strep throat. Gastroesophageal reux in infants may be normal and in small quantities but persistent reux may be accompanied by excessive vomiting during the rst few weeks of life, forceful vomiting, chronic cough, wheezing, apnea or brief breath holding spells, and excessive crying. In more severe cases, there may be weight loss or slow weight gain. In adults, GERD symptoms may include heartburn, particularly at night, belching, nausea and vomiting, and regurgitation of food with some hoarseness, sore

General features Acute tonsillitis presents with fever, sore throat, and odynophagia and usually in the absence of acute coryzal symptoms. Chronic tonsillitis is less well dened but may present with chronic sore throat, halitosis, malaise, and coughing up of cryptic debris. Signs The tonsils are usually enlarged, erythematous, and with an exudate on the medial surface, accompanied by tender cervical lymph nodes. The chronically infected tonsil may show dilated surface vessels and it may be possible to express cryptic debris with compression of the tonsil.

532 LARYNGITIS AND PHARYNGITIS

Investigations Throat cultures may show GABHS on testing surface swabs while full blood counts, monospot, or EpsteinBarr virus serology will differentiate infectious mononucleosis from other conditions.
Laryngitis

General features Laryngitis may be accompanied by an upper respiratory tract infection or have a history of a recent infection. There is often hoarseness. In the acute form, there will be fever and a possible cervical lymphadenopathy. Chronic laryngitis will have a history of months to years of hoarseness, possible cough or accompanying heartburn and there may be a history of cigarette or alcohol abuse. Laryngotracheobronchitis (croup) often is associated with a mild upper respiratory tract infection with a seal bark type cough. This may progress to inspiratory stridor and labored breathing. There may be signs of chest retraction and on auscultation of the chest there may be decreased breath sounds, wheezing, or prolonged inspiration or expiration. Epiglottitis (supraglottitis) presents with a sore throat, difculty in swallowing, drooling, and difculty in breathing with a crowing inspiratory stridor. There may be hoarseness, fever, chills, and occasionally cyanosis (Figures 1 and 2). Signs Apart from an accompanying upper respiratory tract infection, the larynx may show a diffusely erythematous larynx with swelling of the vocal cord. It is critical in adults with a long smoking history for a careful mirror or ber-optic examination of the larynx to rule out carcinoma of the larynx. With epiglottitis (supraglottitis), it is contraindicated to examine the larynx with a tongue blade as this may cause an acute laryngospasm and obstruction.

Investigations In adult smokers with persistent hoarseness, a microlaryngoscopy examination under anesthesia may be necessary for diagnosis and biopsy purposes to rule out carcinoma of the larynx. Radiography of the neck may reveal a classical rat-tail appearance of the subglottis in croup. Epiglottitis (supraglottitis) with airway obstruction is an emergency situation and requires careful assessment and investigation under controlled conditions. Neck X-rays, which might show an enlarged epiglottis, may well be academic and pose a danger of obstruction while the patient is in the radiology suite (Figure 3). A blood or throat culture may show H. inuenzae or other bacteria and a full blood count may indicate elevated white blood cell levels.

Figure 2 Classical appearance of child with epiglottitis, sitting up, mouth open, head forward, drooling.

Figure 1 Transoral view of classical epiglottitis.

Figure 3 Radiograph showing lateral view with swollen epiglottis.

LARYNGITIS AND PHARYNGITIS 533

Pathogenesis
Pediatric esophageal reux and EER is increasingly being diagnosed with a range of symptoms from postprandial vomiting through to failure to thrive and airway manifestations. GERD in children is generally dened using four categories: 1. Physiologic reux. This is usually largely asymptomatic with infrequent emesis in children. 2. Functional reux. This is silent or asymptomatic and can be conrmed by pH monitoring. 3. Pathologic gastroesophageal reux. This can interfere with growth and cause respiratory complications. 4. Secondary GERD. This is related to a secondary disorder such as neurologic or anatomic abnormality of the esophagus. Respiratory complications of reux include recurrent bronchitis, pneumonia, croup, and chronic asthma and it may complicate or possibly cause laryngeal contact granulomas and ulcers.

Infrequently, intubation is necessary. Epiglottitis (supraglottitis) requires hospitalization, usually in the intensive care unit, and endotracheal intubation may be required and, occasionally, emergency tracheotomy. Intravenous uids, systemic steroids, and antibiotics are often required. Family members should be treated in view of the contagious nature of the infections.
GABHS Pharyngitis

Management and Current Therapy

Gastroesophageal Reux in Children and Adults

There are three phases for treatment of EER in children, which include lifestyle modications, pharmacologic treatment, and antireux surgery; the severity of the reux determines the level of treatment. Conservative measures including elevating the head when in bed, frequent small feeds, thickening of milk, and fasting before bedtime are useful. Adjuncts for conservative management of EER may include H2 receptor blockers, prokinetic agents, or protein pump inhibitors. Conservative measures, antacids, and H2 antagonists are the treatment of choice for mild reux and in the more severe cases prokinetic agents or protein pump inhibitors are suggested. Surgical intervention includes Nissen fundoplication, which has a 90% success rate in symptom control and a low mortality rate. Management of adults with gastroesophageal reux is similar to that in children but particular attention should be paid to weight reduction.
Laryngitis

Viral pharyngitis is treated symptomatically but GABHS pharyngitis is managed with penicillin V for 10 days. There are concerns about the length of the regimen and bacteriologic treatment failures occur in up to 35% of young patients. Cephalosporins are an alternative treatment to penicillin therapy. The new generation macrolides such as azithromycin and chlorithromycin are effective in penicillin-resistant patients although there appears to be increasing GABHS resistance to this group of antibiotics. Clindamycin may be used in cases of GABHS carrier states or failure with other antibiotics. Bacterial biolm in tonsillar crypts may mitigate against effective antibiotic therapy for the chronic tonsillitis states. Bacterial replacement therapy utilizing probiotic bacteria, particularly Streptococcus salivarius, may be applied to prevention of streptococcal pharyngitis.
Tonsillitis

Tonsillectomy is indicated for severe recurrent tonsillitis where there are three or more tonsillar infections per year despite adequate medical therapy that are accompanied by fever, dysphagia, cervical adenopathy, or positive GABHS culture and tonsillar exudates. Other indications for tonsillectomy include quinsy (peritonsillar abscess) as well as hypertrophy causing obstructive sleep disorder. If chronic tonsillitis does not respond to a 36 week therapeutic trial of clindamycin or amoxicillin clavulanate, then tonsillectomy may need to be considered.

Facets of Treatment
Although vaccination for diphtheria and Hib has significantly decreased the presentation of these diseases, a high index of suspicion should still be exercised. There may be compliance problems with a 10 day penicillin treatment for GABHS pharyngitis but a 5-day course of azithromycin or macrolides, in view of the high rate of failure with these agents, must be regarded with caution. Recurrent croup may require further investigation to rule out coexisting anatomical abnormalities such as subglottic stenosis. Adults with chronic laryngitis who persist in smoking require regular follow-up to detect malignant change in the larynx.

The majority of cases of viral laryngitis correspond to conservative management with voice rest, humidication, and anesthetic lozenges. Croup of mild degree can be managed at home with a cool air nebulizer and symptomatic therapy. Occasionally, oral or inhaled steroids are necessary. Serious illness requires hospitalization with aerosolized racemic epinephrine, oxygen, humidication, and consideration of antibiotic therapy for bacterial infections.

534 LEUKOCYTES / Mast Cells and Basophils See also: Aerosols. Allergy: Overview; Allergic Reactions. Asthma: Extrinsic/Intrinsic. Bronchoscopy, General and Interventional. Chronic Obstructive Pulmonary Disease: Smoking Cessation. Corticosteroids: Glucocorticoid Receptors. Croup. Endotoxins. Environmental Pollutants: Overview; Particulate Matter, Ultrane Particles; Particulate and Dust Pollution, Inorganic and Organic Compounds. Gastroesophageal Reux. Human Immunodeciency Virus. Mucus. Occupational Diseases: Inhalation Injury, Chemical. Pediatric Aspiration Syndromes. Pediatric Pulmonary Diseases. Tracheostomy. Tumors, Malignant: Overview. Upper Airway Obstruction. Upper Respiratory Tract Infection. Vaccinations: Bacterial, for Pneumonia; Viral.
De Souza C, Stankiewicz J, and Pellitteri PK (1999) Textbook of Pediatric Otorhinolaryngology: Head and Neck Surgery, vol. 11. San Diego: Singular Publishing Group. Del Mar CB, Glasziou PP, and Spinks AB (2000) Antibiotics for sore throat. Cochrane Database of Systematic Reviews 4: CD000023. Discolo CM, Darrow DH, and Koltai PJ (2003) Infectious indications for tonsillectomy. Pediatric Clinics of North America 50: 445458. Jacobs RF (2000) Judicious use of antibiotics for common pediatric respiratory infections. Pediatric Infectious Diseases Journal 19(9): 938943. McGuirt WF (2003) Gastroesophageal reux and the upper airway. Pediatric Clinics of North America 50: 487502. Pichichero ME (2000) Short course antibiotic therapy for respiratory infections: a review of the evidence. Pediatric Infectious Diseases Journal 19(9): 929937. Wetmore RF, Muntz HR, McGill TJ, et al. (2000) Pediatric Otolaryngology: Principles and Practice Pathways. New York: Thieme. Woodson GE (2001) Ear, Nose and Throat Disorders in Primary Care. Philadelphia: Saunders.

Further Reading
Bisno AL (2001) Acute pharyngitis. New England Journal of Medicine 344(3): 205211.

LEUKOCYTES
Contents

Mast Cells and Basophils Eosinophils Neutrophils Monocytes T cells Pulmonary Macrophages

Mast Cells and Basophils

F Hollins, P Bradding, and C E Brightling, University of Leicester, Leicester, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Mast cells and basophils develop from pluripotential stem cells in the bone marrow. Mast cells migrate into tissue, where they mature into resident tissue mast cells. In contrast, basophils are resident in the circulation and migrate into tissue as part of an allergic response. Following activation, both cell types can degranulate, releasing preformed mediators from their granules and synthesizing lipid mediators and cytokines. Mast cell and basophil degranulation is classically mediated by cross-linking of IgE bound to the high-afnity IgE receptor, but there is increasing recognition that several mechanisms can result in degranulation of these cells. Mast cells have an important role in the immune response to infections, and both mast cells and basophils play a pivotal role in the allergic response and are implicated in the pathogenesis of a number of respiratory diseases, particularly asthma and allergic rhinitis.

In the late 1870s, Paul Ehrlich rst identied the mast cell and basophil. For many years, mast cells and basophils were thought to be the same cell or one a progenitor of the other. It has only been in recent years that the emergence of a differing developmental pathway from a common precursor has come to light and is gradually becoming accepted. Mast cells develop from pluripotential hematopoietic stem cells within the bone marrow. They enter the circulation as nongranular immature mast cell precursors and express CD34 , CD13 , and CD117 on their surface. They migrate to tissue, and under the inuence of local mediators they differentiate into mature tissue mast cells. Mature human mast cells are classied based on their serine protease content into cells that contain chymase and tryptase (MCTC) or cells that contain tryptase alone (MCT). Basophils are the rarest blood leukocyte, accounting for less than 1% of the total cell population. Like mast cells, basophils develop from pluripotential

LEUKOCYTES / Mast Cells and Basophils 535

Bone marrow Circulation Tissue

Fc RI+ CD34+ Basophil precursor Mature basophil bsp-1 CD117high

CD117low Pluripotent hematopoietic stem cell Mast cell precursor

+ Immature CD13 mast cell

Fc RI+

+ Mature CD13 mast cell

Figure 1 Comparison of the ontogeny of the mast cell and basophil. Although they both develop from a common pluripotent hematopoietic stem cell precursor, they have separate development and maturation pathways. Much of the maturation process is yet to be investigated.

respiratory diseases. This article summarizes mast cell and basophil biology and describes their role in health and disease.

Mast Cell and Basophil Biology

Cell Trafcking to the Lung

Figure 2 Electron micrograph of a mast cell within the lung tissue showing the rounded nucleus and granules of varying density. Note the dissolution of some of the granules.

hematopoietic stem cells within the bone marrow, where the cell differentiates into a basophil precursor. The basophil matures upon entering the circulation, expressing basophil-specific antibody-1 (Bsp-1). The ontogeny of mast cells and basophils is illustrated in Figure 1, whereas the structure of the mature mast cell is shown in Figure 2. Mast cells have an important role in both the innate and the adaptive immune response to infections. Mast cells and basophils play a central role in the allergic inammatory response, and both cells are implicated in the pathogenesis of a number of

Immature mast cell precursors are drawn into the circulation from the bone marrow by chemotactic factors and chemokines. Chemokines are a superfamily of low-molecular-weight heparin-binding molecules that serve as potent chemoattractants for cells of the immune system. The chemokine family is divided into four groups, designated CXC, CC, C, and CX3C, depending on the spacing of conserved cysteines (X represents an amino acid). The CC and CXC chemokines are the major chemokine families, consisting of more than 50 members. The C and CX3C chemokines are much smaller families, with each consisting of only 1 or a few members. Chemokines exert their effects through binding to heterotrimeric, seven-transmembrane-spanning, G-protein-coupled receptors. CCR1, -3, -4, -5, and -7 and CXCR1, -2, -3, -4, and -6 receptors have been identied on mast cells, which can bind a multitude of ligands. Some chemokines found to be important in the recruitment of mast cells include interleukin (IL)-8 (CXCL8 binding to CXCR1), SDF-1a (CXCL12 binding to CXCR4), IP-10 (CXCL10 binding to CXCR3), RANTES (CCL5 binding to CCR1, -3, and -5), and eotaxin (CCL11 binding to CCR3).

536 LEUKOCYTES / Mast Cells and Basophils

Bone marrow Circulation Smooth muscle Bronchus Epithelium Airway lumen

SCF TGFCXCL10 CCR3 CCR1 CCR4 CD117 CXCR4 CXCR1 CXCR3 CD117 CXCR4 CCR3

Rolling adhesion and migration

Mast cell survival and differentiation

CXCL8 CXCL10 CXCL12 CCL11 TGFNGF

Precursor recruitment SCF CCL11 CXCL12 Submucosal gland

Figure 3 Mast cell trafcking from the bone marrow into the circulation and then into the tissue in asthma. The important receptors expressed on the mast cell at each point and the cytokines and chemokines that are critical in controlling the mast cell movement are shown.

These chemokines, in conjunction with other chemotaxins such as stem cell factor (SCF), transforming growth factor beta (TGF-b), and nerve growth factor (NGF), are involved in the migration of mast cell precursors into the circulation and the movement of mast cells into tissue. Like mast cells, basophils express several chemokine receptors, including CCR1, -2, -3, and -5 and CXCR1, -2, and -4. The chemokine CCL11, acting via CCR3, induces the most potent basophil migration. In addition, basophils express the prostaglandin-D2 receptor DP2/CRTh2, and ligand activation of this receptor results in basophil migration. Mature mast cells localize to specific structures in the lung, namely nerves, blood vessels, lymphatics, airway epithelium, glands, and airway smooth muscle. This microlocalization of mast cells is likely to be important in the pathogenesis of many lung diseases. The mechanisms involved in mast cell trafcking are summarized in Figure 3.
Mast Cell Differentiation and Survival

IL-3, IL-4, IL-9, IL-10, and NGF. Basophil growth and survival are mediated by granulocyte macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5.
Mechanisms Mediating Degranulation

Once within the tissues, mast cells can survive for a relatively long period of time, possibly days to weeks, in comparison to neutrophils, which survive only for hours. Mast cell survival is thought to occur through the action of cytokines, particularly SCF, which also plays an important role throughout the development and migration of mast cells. Several other growth factors and cytokines are also known to affect the growth and differentiation of mast cells, including

The extracellular release of both preformed and newly synthesized mediators by mast cells and basophils, a process termed degranulation, can be mediated by both IgE-dependent and IgE-independent mechanisms. IgE molecules produced by B lymphocytes bind to high-afnity IgE receptors (FceRI) on the surface of mast cells and basophils. IgE can also bind to FcgRII, FcgRIII, and galectin-3, which are also found on the surface of mast cells. However, IgE elicits its functions only through binding to FceRI. FceRI is a heterotetrameric receptor composed of an IgE-binding a subunit, a four-transmembranespanning b subunit, and two disulde-bonded g subunits (abg2). The g subunits are signal-transducing molecules, producing a signal that can then be amplied by the b subunits. Cross-linking of the FceRI with IgE and antigen induces activation of protein tyrosine kinases, including those of the Src, Syk, and Tec families. Pathways that involve the activation of these protein tyrosine kinases ultimately lead to degranulation. In addition, several IgE-independent mechanisms can cause degranulation: (1) physical factors (i.e., high temperature, ionizing irradiation, and mechanical trauma); (2) chemical substances (i.e., proteases,

LEUKOCYTES / Mast Cells and Basophils 537

toxins, and venoms); (3) endogenous mediators (i.e., tissue proteases and cationic proteins derived from eosinophils and neutrophils); (4) osmotic effects; (5) adenosine, via activation of the adenosine A2B receptor; and (6) anaphylatoxins C3a, C4a, and C5a, formed during activation of complement can trigger degranulation through activation of the C5a receptors on the surface of mast cells and basophils. However, it is important to note that human lung mast cells are devoid of the C5a receptor; hence, it is unlikely that this mechanism of mast cell degranulation is important in the lung. Following degranulation of mast cells and basophils, there is an immediate release of granule products. Subsequently, there is synthesis and release of lipid mediators over several minutes and the production and secretion of cytokines, chemokines, and growth factors over several hours.
Preformed Mediators

Histamine Two major sources of histamine within the body are mast cells and basophils, containing approximately 3 and 1 pg per cell, respectively. Histamine plays a pivotal role in allergic inammation. Following mast cell degranulation, there is a local and systemic increase in histamine concentration. Basophils are then attracted into the tissue by cytokines and chemokines, at which point they too degranulate, releasing histamine, and contribute to the delayed late-phase reaction. The histamine receptor family is a group of seventransmembrane-spanning units that couple ligandbinding with intracellular reactions through guanosine triphosphate (GTP)-binding heterotrimeric proteins. The histamine receptors are subclassied into four groups: H1H4. The different receptor subtypes also have differing intracellular secondary messengers; H1 and H4 receptors mobilize intracellular calcium, whereas H2 receptor utilizes cyclic adenosine monophosphate and the H3 receptor can signal through both of these pathways. There is evidence of H1, H2, and H4 expression on both human mast cells and basophils. These receptors act to induce or modulate cytokine synthesis in allergic inammation, having varying effects on different cell types. Proteases The mast cell granules contain neutral proteases. Four such proteases have been identied: tryptase, chymase, carboxypeptidase, and cathepsin G. Tryptase Mast cell tryptase is a tetrameric neutral serine protease with a molecular weight of 134 kDa, and it composes 20% of the total cellular mast cell protein. Tryptase is found in negligible quantities in

other cells, making it a good mast cell marker for identication of both the cells and their causative properties. The enzyme is made up of four noncovalently bound subunits, with each subunit having one enzymatically active site. There are two main types of mast cell tryptase, a-tryptase and b-tryptase, with approximately 90% sequence homology between them. a-Tryptases are classied into aI- and aII-tryptases, and b-tryptases are classied into bI-, bII-, and bIII-tryptases. bII-Tryptase is stored in the secretory granules of mast cells. In contrast, a-protryptase is secreted constitutively from mast cells as an inactive proenzyme. The activation of b-II-pro-tryptase involves two proteolytic steps. The rst is an autocatalytic intermolecular cleavage, which occurs optimally at an acidic pH and in the presence of heparin or dextran sulfate. The resulting product is a monomer, which is approximately 50 times less active than the nal tetramer. The second step involves the removal of the remaining precursor dipeptide by dipeptidyl peptidase I, thus allowing the mature peptide to spontaneously form the active tetramer. This process also requires the presence of heparin or dextran sulfate. Tryptase can activate a number of peptides by cleaving certain extracellular substrates, such as vasoactive intestinal peptide, calcitonin gene-related peptide, bronectin, kininogens, and the protease activated receptor (PAR)-2. Airway epithelial and smooth muscle cells, endothelial and vascular smooth muscle cells, terminal bronchial epithelium, type II pneumocytes, and mast cells within the respiratory tract express PARs. Tryptase is a potent mitogen for epithelial cells, airway smooth muscle cells, and broblasts. During inammation, tryptase can stimulate the release of granulocyte chemoattractant IL-8 and upregulate expression of ICAM-1 on epithelial cells. It induces the expression of IL-1b mRNA, which may be important for the recruitment of inammatory cells to the sites of mast cell activation. The release of tryptase also promotes the degranulation of neighboring mast cells via activation of PAR-2, thereby initiating a positive feedback mechanism. Chymase As outlined previously, there are two types of mast cells: MCT and MCTC. MCT mast cells are the predominant mast cell subtype in the lung, comprising 9095% of the lung mast cell population. The MCTC mast cells are found in increased numbers in the airway smooth muscle, epithelium, and glands, and their numbers increase in disease. Mast cell chymase is a glycosylated chymotryptic serine protease with a molecular weight of 30 kDa. It is stored in the same secretory granules as those that contain tryptase, but it is released from mast cells in

538 LEUKOCYTES / Mast Cells and Basophils

a macromolecular complex with carboxypeptidase and proteoglycans, which is distinct from tryptase. Like tryptase, chymase is present in the granules as a catalytically active form, but due to the acidic condition within the granules, this activity is suppressed. Chymase has a net charge of 13, allowing it to bind strongly with negatively charged granule proteoglycans and to cells and basement membranes. Angiotensin I is readily hydrolyzed by chymase to form angiotensin II, a reaction that is catalyzed more efciently than with the angiotensin enzyme. Circulating a-antichymotrypsin and a1-antitrypsin and the respiratory tract inhibitors bronchial secretion inhibitor and antileukoprotease inhibit chymase. Chymase can modulate inammatory processes. It can convert cytokines such as IL-1b from their inactive to active form and it can degrade others, such as IL-4. In addition, chymase has a role in tissue destruction and matrix remodeling because chymase can activate stromelysin and interstitial collagenase, and it can convert procollagen I to collagen-sized fragments and degrade type IV collagen. Interestingly, chymase, like tryptase, can activate cells via PARs. Chymase acts on PAR-1, which is expressed on several inammatory and mesenchymal cells and can lead to cell activation and proliferation. Carboxypeptidase Human mast cell carboxypeptidase is a zinc metalloexopeptidase, 34.5-kDa monomer only found in mast cells of the tryptase/ chymase phenotype. It cleaves the carboxy terminal of the His-Leu bond of angiotensin I, generating desLeu10 angiotensin I, an inhibitor of angiotensinconverting enzyme. Cathepsin G Various physiological effects are attributed to cathepsin G, including antimicrobial activity, degradation of extracellular matrix, vasoregulation, activation of neutrophil elastase, processing of IL-8, and mast cell degranulation.
Synthesized Mediators

of inammatory cells at the site of inammation, aided by its effect on increasing the microvascular permeability. There are two types of receptor for PGD2: DP1 and DP2 (CRTH2). Both of these receptors are G-protein-coupled rhodopsin-type receptors with seven transmembrane domains. DP1 receptors are prostanoid receptors found on various types of hematological and nonhematological cells, including platelets, neutrophils, and smooth muscle cells. DP2 (CRTH2) receptors are a member of the chemokine receptor family and are found exclusively on the hematological cells: basophils, eosinophils, and Th2 cells. Leukotrienes are produced upon the activation of mucosal mast cells and basophils. They bind to receptors on smooth muscle and cause prolonged bronchoconstriction (101000 times more potent than histamine), induce prolonged wheal-and-are response, enhance venular permeability, promote mucus secretion, and act as chemotactic agents for neutrophils and eosinophils. The other type of lipid mediator produced by mast cells and basophils is platelet-activating factor (PAF). PAF has a diverse range of physiological actions, varying from aggregation and degranulation of platelets and neutrophils to a variety of cellular effects, including stimulation of chemotaxis and chemokinesis. It can also result in bronchoconstriction and vasodilatation, and it may cause retraction of endothelial cells. Cytokines/Chemokines Mast cells and basophils produce many different cytokines and chemokines, at differing amounts, that may contribute toward an inammatory response. The cytokines and chemokines produced are shown in Table 1. Mast cell and basophil activation results in the de novo synthesis of these cytokines; however, TNF and IL-4 may already be stored preformed in granules and rapidly released upon FceRI cross-linking.

Lipid-Derived Mediators Arachidonic acid is released from the phospholipid membrane following activation, the metabolites of which are cyclooxygenase and lipooxygenase. The cyclooxygenase pathway results in the synthesis of prostaglandin D2 (PGD2), whereas the lipooxygenase pathway liberates leukotrienes, particularly leukotriene C4 (LTC4). PGD2, which is synthesized by mast cells but not basophils, can bind to receptors on smooth muscle and can act as a vasodilator and a bronchoconstrictor. It can also cause histamine release from basophils, and it causes chemotaxis and accumulation

Mast Cell and Basophil Functions in the Normal Lung

Mast cells are found within tissues that interface with the local environment, such as the skin, airways, and intestine, and are therefore well placed to initiate and enhance early responses to a variety of challenges. In response to pathogens, mast cells produce cytokines, proteases, and lipid-associated mediators. This leads to immune vascular permeability and recruitment of other inammatory cells, such as neutrophils and eosinophils. The acquired immune response is enhanced through inuencing the function and maturation of dendritic cells and increasing recruitment

LEUKOCYTES / Mast Cells and Basophils 539

Table 1 Mast cell basophil contents Mast cell Resting Histamine Proteases Tryptase Chymase Carboxypeptidase Cathepsin G Lipid-derived mediators Prostaglandin D2 Leukotrienes Activated Basophil Activated Resting Vascular permeability Function

MCTC only MCTC only MCTC only

MCTC only MCTC only MCTC only

Tissue remodeling

Platelet-activating factor

Recruitment of effector cells, immune response regulation, and angiogenesis, bronchoconstriction, and edema promotion Effector cell activation and chemoattractant

Cytokines IL-1a and -b IL-6 IFN-a and -b GM-CSF TNF-a IL-3 IL-4 IL-5 IL-13 IL-12 IFN-g IL-10 TGF-b Chemokines CCL1 (TCA3/I309) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CCL5 (RANTES) CCL20 (LARC) CXCL8 (IL-8) CXCL10 (IP-10)

Inammation induction

Th2 cytokines

Th1 cytokines Inammation and angiogenesis induction

Recruitment of effector cells

Recruitment of effector cells and immune response regulation

, presence; , absence; blank, unknown.

of T cells in the lymph nodes through the actions of TNF-a, which optimizes the environment for effective antigen presentation. Both mast cells and basophils are able to detect and respond to pathogens through the use of direct or indirect receptors. Direct cell activation is essential for initiation of an early innate immune response as well as the generation of appropriate acquired immunity. For this direct interaction to occur, these

cells have cell surface receptors that can interact directly with pathogens. Toll-like receptors (TLRs) are an example of such a receptor type. TLRs recognize products from pathogens such as gram-positive and gram-negative bacteria. Binding of these pathogen products to TLRs can result in their activation, which could ultimately lead to a proinammatory response. In mammals, 11 TLRs have been identied, each having a distinct function with regard to its

540 LEUKOCYTES / Mast Cells and Basophils

immune recognition properties. Mast cells have been found to express TLR1, -2, -3, -4, -6, and -9, each capable of activating mast cells upon binding of the appropriate ligand. Indirect cell activation is important in the event of secondary or subsequent infections and can occur through several mechanisms, including Fc receptormediated activation.
Wound Repair

Mast cells are resident in tissues, particularly in association with endothelial and epithelial cell basement membranes, and are increased at sites of inammation, injury, and brosis. Although mast cells are known to both release and generate proinammatory mediators in response to inammatory stimuli, the cells express large amounts of mRNA for collagen IV, laminin, and heparin sulfate proteoglycan, suggesting that these cells may contribute to normal tissue repair.

Mast Cells and Basophils in Respiratory Disease

Asthma

Asthma is an important cause of morbidity and mortality worldwide, and its prevalence is increasing. More than ve million people in the United Kingdom are currently taking treatment for asthma. It is characterized by the presence of variable airow obstruction and airway hyperresponsiveness (AHR), which is the exaggerated bronchoconstrictor response of the airways to a wide range of specific and non-specific stimuli. In asthma, mast cells are present in an activated secretory state. Mast cell mediators have the capacity to contribute to the development and maintenance of the airway inammation and associated disordered airway physiology, which characterizes asthma. Secretion of the autacoid mediators histamine, PGD2, and LTC4 induces bronchoconstriction, mucus secretion, and mucosal edema and thus contributes to the symptoms of asthma. Basophils are increased in number in the bronchial submucosa of asthmatics and play an important role in the late asthmatic reaction in response to allergens. The mast cell has been implicated as playing an important role in the development of AHR. Strong correlations have been observed between the severity of AHR and mast cell numbers, histamine concentrations, and spontaneous histamine release in bronchoalveolar lavage (BAL). In biopsy and postmortem studies, mast cell numbers were found to be increased in the airway epithelium, glands, and airway smooth

muscle. The strongest evidence supporting the role of the mast cell in the development of the asthmatic phenotype is derived from comparisons of the immunopathology of asthma and nonasthmatic eosinophilic bronchitis. Eosinophilic bronchitis accounts for 15% of the cases of cough referred to respiratory specialists. It is characterized by corticosteroid responsive cough and the presence of a sputum eosinophilia occurring in the absence of variable airow obstruction or AHR. Thus, any differences in the immunopathology of asthma compared to eosinophilic bronchitis may yield specific clues as to the essential components of asthma. Both conditions share many immunopathological features, including eosinophilic airway inammation, Th2 cytokine expression, and subbasement membrane collagen deposition. The only immunopathological feature that is different is that there are significantly increased numbers of mast cells within the airway smooth muscle bundles in bronchial biopsies in patients with asthma but virtually none in patients with eosinophilic bronchitis or in normal subjects. The majority of these mast cells are of the MCTC phenotype containing both tryptase and chymase, with increased IL-4 and IL-13, but not IL-5, expression. In contrast, there are almost no T cells or eosinophils in the smooth muscle of any of these groups. The number of airway smooth muscle mast cells in asthma is negatively correlated with the degree of AHR. Taken together, this body of evidence suggests that the microlocalization of mast cells is a critical event in the development of the asthmatic phenotype.

Allergic Rhinitis

Allergic rhinitis is a very common condition, affecting 1025% of the worlds population. Allergic rhinitis is an inammatory disorder of the nasal mucosa typied by the symptoms of nasal itch, sneeze, anterior nasal secretions, and nasal blockage. These symptoms arise from the interaction between mediators and neural, vascular, and glandular structures within the nose. Nasal itch, sneezes, and rhinorrhea are predominantly neural in origin, whereas nasal obstruction is predominantly vascular. Nasal biopsy studies show accumulation of eosinophils, mast cells, and basophils in an activated state within the lamina propria and epithelium in both seasonal and perennial allergic rhinitis. The role of the mast cell and basophil in allergic rhinitis is supported by the observation that histamine and leukotriene nasal insufation induces the symptoms of rhinitis, and that these symptoms are improved by nasal corticosteroids and H1 receptor antagonists.

LEUKOCYTES / Mast Cells and Basophils 541 Anaphylaxis

Systemic anaphylaxis arises when mast cells, possibly along with other cell types, are provoked to secrete mediators that evoke a systemic response. Increased tryptase levels in the circulation provide a precise indicator of mast cell involvement. Although it is recognized that the mast cell is the predominant cell mediating anaphylaxis, in some circumstances there are elevations of histamine but not tryptase, suggesting that basophil-dependent anaphylaxis may occur.
Chronic Obstructive Pulmonary Disease

lung, but evidence suggests that blood-derived broblast precursors, brocytes, may play a critical role in the deposition of matrix proteins in the development of lung brosis. How mast cells and brocytes interact in terms of brocyte activation, differentiation, and survival is unknown.
Lung Cancer

Chronic obstructive pulmonary disease (COPD) is a common condition predominantly caused by smoking. COPD is the major cause of respiratory failure and a common cause of chronic disability. In contrast to asthma, COPD is characterized by irreversible airow obstruction. The physiological abnormalities observed in COPD are due to a combination of emphysema and obliteration of the small airways. In some, but not all, reports, histamine and tryptase sputum concentrations are increased in COPD and chronic bronchitis. However, mast cell numbers are not increased in the airway wall. Thus, the role of mast cells and basophils in COPD is unclear.
Lung Fibrosis/Interstitial Lung Disease

Lung brosis is characterized by inappropriate tissue remodeling and enhanced vascular remodeling, broproliferation, and deposition of extracellular matrix. Without resolution, continued brosis leads to loss of lung function and ultimately death. In chronic brotic lesions, a correlation exists between the number of mast cells and the severity of brosis. Mast cells and broblasts are usually found in close proximity to one another within the alveolar septae in both the normal and the brotic lung. These mast cells often appear to contain fewer numbers of granules, indicating that there is partial ongoing degranulation. In support of this, elevated histamine levels have also been described in the BAL of patients with lung brosis. Basic broblast growth factor (bFGF) is also produced by mast cells and is a potent brogenic cytokine. Mast cells are thought to be the predominant cells within the lung interstitium expressing bFGF. Indeed, the distribution of bFGF mast cells correlates with the deposition of extracellular matrix and, accordingly, the extent of brosis. This therefore suggests that mast cells do contribute to the brotic process. Fibroblasts have been held to be the predominant cells responsible for repair and remodeling within the

Mast cell numbers are increased in several malignant tumors, including non-small cell carcinoma (NSCLC). The functional significance of the accumulation of mast cells within lung tumors is a subject of controversy because of contradictory experimental and clinical data. Mast cells are a rich source of TNF-a, cytotoxic for some tumors, and mast cell-derived cytokine, IL-4, inhibits growth and induces apoptosis in human carcinoma cells. In patients with NSCLC, especially lung adenocarcinoma, the high mast cell count group has a significantly worse prognosis than the low mast cell count group. In vitro, mast cell conditioned medium stimulates capillary endothelial cell migration. Mast cell granules localize within endothelial cells and stimulate their proliferation. Mast cell products also degrade connective tissue matrix to provide space for neovascular sprouts to form. Mast cell histamine induces angiogenesis and heparin promotes new vessel formation in vivo. Tryptase derived from mast cells stimulates vascular tube formation and functions as a mitogen for microvascular endothelial cells. Chymase promotes angiogenesis through the local chymase-dependent angiotensin II-generating system in pathophysiological angiogenesis. Mast cells produce angiogenic factors vascular endothelial growth factor (VEGF) and bFGF; VEGF expression is documented in intratumoral stromal mast cells in NSCLC. Mast cells synthesize and store large amounts of matrix metalloproteinases-2 and -9, which can degrade collagen types IV, V, VII, and X and bronectin, the major components of the interstitial stroma and subendothelial basement membrane. This may contribute to the progression from in situ to invasive and metastatic solid tumors.

Summary and Therapeutic Considerations

Mast cells are present in the respiratory mucosal surface and are localized with epithelial and mesenchymal cells. They have an important role in the innate immune response and wound repair, and they play a role in the development of lung brosis and angiogenesis in lung cancer. Both mast cells and basophils have an established role in allergic

542 LEUKOCYTES / Eosinophils

disease. Mast cells are the key in the development and maintenance of chronic airway inammation in asthma, and their microlocalization within the airway smooth muscle bundles is critical in the development of disordered airway physiology in the disease. Furthering our understanding of mast cell and basophil migration to the lung and their survival, maturation, and activation in tissue may provide novel targets for the effective treatment of lung disease. In particular, treatments directed at modifying interactions between mast cells and airway smooth muscle may provide a novel approach to the effective treatment of asthma.
See also: Adenosine and Adenine Nucleotides. Adhesion, CellCell: Epithelial. Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Angiogenesis, Angiogenic Growth Factors and Development Factors. Apoptosis. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchiolitis. Bronchodilators: Beta Agonists. Chemokines. Chronic Obstructive Pulmonary Disease: Overview; Acute Exacerbations; Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General. Chymase and Tryptase. Complement. Defense Systems. Eotaxins. Fibroblast Growth Factors. Fibroblasts. Histamine. Interstitial Lung Disease: Overview; Cryptogenic Organizing Pneumonia; Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Lipid Mediators: Overview; Leukotrienes; Platelet-Activating Factors; Prostanoids. Pulmonary Fibrosis.

Eosinophils
D S Robinson and H H Kariyawasam, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Eosinophils are predominantly tissue resident cells with a bilobed nucleus and granular cytoplasm and are derived from bone marrow CD34 hemopoietic progenitor cells. Lineage selection and maturation is critically dependent on interleukin-5 (IL-5), and release from marrow occurs in response to further IL5 and eotaxin. In disease states, selective accumulation into tissue occurs in response to a series of interactions between eosinophils and endothelial cells with migration along specific chemokine gradients. Lung eosinophilia is implicated in a variety of allergic and nonallergic diseases. Mature eosinophils are an important source of basic proteins, lipid-derived mediators, growth factors, and proinammatory cytokines that can result in a spectrum of lung diseases that range from allergic airway inammation to parenchymal and vasculitic diseases. As a result, there is a substantial effort to target therapy at critical events involved in the eosinophil life cycle and functional pathways.

Further Reading
Bradding P and Holgate ST (1999) Immunopathology and human mast cell cytokines. Critical Reviews in Oncology/Hematology 31: 119133. Brightling CE and Bradding P (2005) The re-emergence of the mast cell as a pivotal cell in asthma pathogenesis. Current Allergy and Asthma Report 5(2): 130135. Galli SJ, Nakae S, and Tsai M (2005) Mast cells in the development of adaptive immune responses. Nature Immunology 6(2): 135142. Gurish MF and Austen KF (2001) The diverse roles of mast cells. Journal of Experimental Medicine 194(1): F1F5. Kawakami T and Galli SJ (2002) Regulation of mast cell and basophil function and survival by IgE. Nature Reviews: Immunology 2: 773786. Marone G, Triggiani M, and de Paulis A (2005) Mast cells and basophils: friends as well as foes in bronchial asthma? Trends in Immunology 26(1): 2531. Marshall JS (2004) Mast cell responses to pathogens. Nature Reviews: Immunology 4: 787799. Nagata K and Hirai H (2003) The second PGD2 receptor CRTH2: structure, properties and functions in leukocytes. Prostaglandins, Leukotrienes and Essential Fatty Acids 69: 169177.

Originally described by Paul Ehrlich in 1879, the eosinophil was named with regard to the cells avidity for the negatively charged uorescein compound eosin that confers on the cell a characteristic pink color. In health, eosinophils comprise only 13% of the circulating leukocyte population and are essentially tissue resident cells with connement predominantly to the gastrointestinal (GI) tract. In disease states, however, the cells can be rapidly mobilized from the bone marrow into the peripheral circulation and selectively accumulate at sites of tissue inammation with an estimated half-life of 13 days. Eosinophils are approximately 8 mm in diameter (this can vary with the state of activation) and are morphologically distinct with a bilobed nucleus and ovoid granules that confer the characteristic granular cytoplasm to the cell. Electron micrographs reveal four distinct populations of cytoplasmic granules that house specific cellular contents. Primary granules are the main site of CharcotLeyden crystals that display intrinsic lysophospholipase activity. Secondary granules are composed of a prominent crystalloid core containing major basic protein (MBP) and a noncrystalloid matrix that houses other highly cationic proteins, such as eosinophilic-cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPO), together with a number of cytokines and proteins with enzymatic activity. Lipid bodies are the site of major lipidderived mediator synthesis. Finally, there are small granules seen in mature tissue eosinophils that store proteins such as arylsulfatase B and acid phosphatase (Figure 1). The role of the small-granule proteins in eosinophil biology is not known.

LEUKOCYTES / Eosinophils 543

E P L E E

Figure 1 Electron micrograph of a bronchial biopsy from a mild stable asthmatic showing three eosinophils (E) (one degranulating) in close association with a lymphocyte (L) and plasma cell (P). Scale 2 mm. Kindly provided by P K Jeffery and Dr A Rogers, National Heart & Lung Institute, Imperial College London.

Life Cycle
Eosinophils are derived from CD34 bone marrow (BM) hemopoietic progenitor cells that differentiate into precursors with both basophil and eosinophil properties in response to T helper type 2 (Th2)secreted cytokines interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF). Ensuing cell surface expression of the IL-5Ra chain receptor confers IL-5 responsiveness. Cells can then undergo early lineage selection, proliferation, and terminal differentiation in direct response to IL-5. Although the marrow is the predominant site of eosinophilopoiesis, progenitors (CD34 IL-5Ra) are detectable in both the bronchial tissue and the sputum of atopic asthmatics, conrming that progenitors can trafc to tissue and thus eosinophilopoiesis is not marrow restricted. There is thus a pool of immature and mature eosinophils that can be rapidly mobilized into the circulation during inammatory responses. This mobilization is critically dependent on IL-5 and the eosinophil-specific chemokine eotaxin. Chemokines are chemoattractant cytokines selectively produced at sites of inammation and are subdivided into families based on the position of cysteine residues, such as CC, CXC, and CX3C, where X is any amino acid. Eotaxin (a CC chemokine) and the homologs eotaxin-2 and -3 display very high selectivity for eosinophils exclusively through the chemokine receptor CCR3. Recently differentiated eosinophils constitutively express CCR3, but in response to priming signals generated by the local tissue cytokine milieu, eosinophils can upregulate the expression of several other chemokine receptors, including CCR1. The chemokines RANTES (regulated

upon activation, normal T cell expressed and secreted) and MCP-3 and -4 (monocyte chemoattractant protein) can regulate eosinophils through CCR3, and MIP-1a (macrophage inammatory protein) can regulate them through CCR1. However, such chemokines are not eosinophil specific, with effects on both T cells and basophils. The selective trafcking of eosinophils from the circulation into tissue sites is a highly specific and regulated stepwise process between the eosinophil and endothelium that requires cellendothelial adhesion and then extravasation. Cell rolling is facilitated by enodothelial expression of P-selectin that specically binds eosinophil-expressed P-selectin glycoprotein-1 (PSGL-1). As eosinophil activation proceeds in response to the local inammatory and chemokine microenvironment, such as platelet-activating factor (PAF) and particularly eotaxin, conformational change of the b2 integrins Mac-1 and lymphocyte function-associated antigen (LFA)-1 on the eosinophil surface allows binding to endothelial intercellular adhesion molecule (ICAM)-1, thus allowing the cell to halt. Expression of very late activation antigen (VLA)-4 integrin (a4b1) is specific to eosinophils. VLA-4 attaches to the vascular cell adhesion molecule (VCAM)-1 on endothelium, allowing selective eosinophil transmigration across the vessel wall into tissues. Both IL-4 and IL-13, key inammatory cytokines, markedly upregulate the expression of VCAM-1. Also, eosinophil a4b7 can bind the mucosal addressin cell adhesion molecule 1 (MadCam-1), enabling further adhesion. This interaction is particularly important for eosinophil recruitment into the GI tract. High chemokine concentrations induce chemokine receptor internalization, thus preventing eosinophils from egressing from the tissue. IL-5 plays a key role in priming eosinophils for selective chemotaxis into tissues and local IL-5, and GM-CSF prolongs eosinophil survival by delaying apoptosis (Figure 2).

Eosinophil Functions in the Normal Lung

Granule Proteins

Eosinophils are ideally equipped to provide the primary defense against parasitic infection. The immune response to such invading organisms is characterized by IgE production, marked blood eosinophilia, and eosinophil accumulation in infected tissues and tissueresident mast cells. Thus, allergic responses may be considered as an aberration of this defense mechanism whereby environmental agents elicit a Th2 polarized adaptive immune response with production of the key cytokines IL-4 and IL-5. Eosinophil contents exert direct toxicity against parasites. MBP, which

544 LEUKOCYTES / Eosinophils

Eotaxin CCR3

Marrow

Eosinophil

Structural cells Tissue infiltration

Vessel
Lineage selection Maturation Release

Chemotaxis Eotaxin MCP - 4, RANTES CCR3 Migration Adhesion VCAM - 1 Endothelial cell

IL- 5

VLA- 4

PAF CCR3

Activation mediator release

IL - 3 GM - CSF

IL- 4 IL- 13

Th2

Survival IL - 3, IL - 5, GM - CSF

Figure 2 The key stages involved in the recruitment of eosinophils from the bone marrow to the tissue are illustrated. Eosinophilopoiesis is dependent on IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and particularly IL-5. On entering the circulation in response to IL-5 and eotaxin, the cells are recruited specifically to sites of tissue inammation through interaction of various adhesion molecules, including VLA-4/VCAM-1. Several cytokines can upregulate such adhesion molecules. Eosinophils undergo conformational change to transmigrate into tissues along chemokine gradients before interacting with the surrounding inammatory microenvironment to undergo activation. VLA, very late antigen; VCAM, vascular cell adhesion molecule; PAF, platelet-activating factor; MCP-4, monocyte chemoattractant protein; RANTES, regulated upon activation normal T cell expressed and secreted.

comprises 50% of the granule proteins, and ECP are both helminthotoxic and cytotoxic. Their highly basic property is conferred by the high arginine content and allows surface charge interactions with target cells that disrupt membrane permeability with consequent cell damage. In addition, both possess bactericidal activity. MBP can degranulate basophils and ECP mast cells. Both ECP and EDN have ribonuclease activity, but ECP is 100 times less potent than EDN in this respect. EDN displays markedly reduced toxicity compared to ECP due to lack of basicity. In addition, eosinophils can directly generate nitrite compounds, superoxide, and hydroxyl radicals through the action of EPO and NADPH oxidase in response to cell activation and priming.
Lipid Mediators and Cytokines

Primed eosinophils have the capacity to rapidly produce an array of lipid mediators and cytokines. The eicosanoids are a family of bioactive lipids derived from the cell membrane phospholipid arachidonic acid (AA). AA is cleaved by activated phospholipase A2 (PLA2), with the cleavage product entering the principal pathways of eicosanoid production in

eosinophilia. The cyclooxygenase (COX) pathway is responsible for the generation of prostanoids that include prostaglandins, prostacyclin (PGI2), and thromboxane A2 (TxA2). The 5- and 15-lipooxygenase (LO) pathway generates leukotrienes (LTs). Eosinophils are a predominant source of cysteinyl leukotrienes, particularly LTC4, which can induce bronchoconstriction, mucous hypersecretion, airway hyperresponsiveness (AHR), and increased microvascular permeability. Eosinophils have the capacity to produce and store cytokines that include IL-16, -10, -16, and GM-CSF. They are an important source of transforming growth factor (TGF)-b1, a potent stimulus for broblast activation and extracellular matrix (ECM) production. TGF acts as a chemotactic factor for mast cell recruitment, and mast cell-derived tumor necrosis factor (TNF) can induce further TGF release from eosinophils. ECP has been shown to inhibit the degradation of proteoglycans, and eosinophils store and release matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and -2. This suggests an important novel role for eosinophils in the process of lung repair and remodeling. Several brotic diseases are associated with eosinophilic inltration.

LEUKOCYTES / Eosinophils 545

Cytotoxic compounds Hydrogen peroxide Superoxides Halide acids Cytokines / Growth factors IL-1,IL-2,IL - 3,IL - 4,IL - 5,IL - 6,IL - 8,IL - 12,IL - 16, IL- 18 GM - CSF,TNF - ,IFN TGF - / ,VEGF SCF Lipid mediators LTC4 LTD4 LTE4 PAF Prostanoids

Chemokines Eotaxin RANTES MCP - 3,4 MIP - 1 Granule proteins MBP ECP EPO EDN

Mucous hypersecretion

Smooth muscle constriction AHR

Cellular toxicity

Vascular leakage / airway edema Remodeling

Inflammation

Figure 3 Important eosinophil secretory products and the resultant effector functions of the cell. AHR, airway hyperresponsiveness; IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; TNF, tumor necrosis factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; SCF, stem cell factor; PAF, platelet-activating factor; MCP-4, monocyte chemoattractant protein; RANTES, regulated upon activation normal T cell expressed and secreted; MIP, macrophage inammatory protein; LT, leukotriene; MBP, major basic protein; ECP, eosinophil cationic protein; EPO, eosinophil peroxidase; EDN, eosinophilderived neurotoxin.

Eosinophilia of tissues is often associated with mast cell recruitment. Eosinophils secrete mast cell stem cell factor (SCF), which is essential for mast cell growth, activation, chemotaxis, and degranulation. Mast cells in turn regulate eosinophils both directly and indirectly, thus creating a regulatory dependence between cells. Eosinophil maturation and migration is in turn effected by mast cell secreted cytokines (including IL-35 and GM-CSF) and chemokines, including eotaxin, RANTES, and MCP-1. Mast cell-derived TNF-a is a potent stimulus for EPO release. The possibility that eosinophils may act as antigen-presenting cells to T cells has been raised by the in vitro demonstration of the expression of molecules involved in antigen presentation on the eosinophil surface. The emerging theme is that eosinophils and other key effector inammatory cells crossregulate each other (Figure 3).

Eosinophil Function in Respiratory Diseases

Asthma

Airway eosinophilia is a hallmark of asthma. The degree of tissue eosinophilia and the level of eosinophil activation correlate with the severity of asthma. The

characteristic late phase response following allergen exposure in asthma is associated with airway eosinophilic inltration. Many of the products released by eosinophils, such as LTC4 and MBP, can promote the pathophysiological hallmarks of asthma, such as airway hyperresponsiveness. MBP can also enhance vagally mediated bronchoconstriction by acting as an antagonist at the neuronal M2 muscarinic receptor. Collectively, these ndings suggest that eosinophils are the major effector cells in the pathogenesis of asthma (i.e., the eosinophil hypothesis in asthma). However, this view has been challenged by the results of human studies in asthma that targeted tissue eosinophil depletion with an anti-IL-5 monoclonal antibody. Eosinophil depletion failed to show much effect on the asthma phenotype in man, such as airway hyperresponsiveness. This may be a reection of the fact that only a 55% reduction in bronchial wall eosinophil numbers was achieved, and further studies are needed before definite conclusions can be made. Importantly, however, eosinophil reduction is associated with decreased airway remodeling, conrming that eosinophil-derived growth factors such as TGF-b1 are pivotal to this process. This has important therapeutic implications given that structural changes in asthmatic airways are correlated with the severity of disease and progressive decline in lung function.

546 LEUKOCYTES / Eosinophils Eosinophilic Bronchitis

The degree of airway eosinophilia and other inammatory cell inltrates in EB is comparable to that of asthma, and both EB and asthma display the characteristic basement membrane thickening. However, EB syndrome is characterized by cough, normal lung function, and, importantly, a complete absence of AHR. Glucocorticoid eosinophil suppression leads to rapid improvement. The significance of eosinophilic inltration in this condition is unknown.
Eosinophilic Parenchymal Diseases

predominantly conned to the GI tract, with respiratory symptoms comprising mild cough and wheeze. Loefers syndrome is considered a response to the migration of parasitic larvae, usually Ascaris, through the lung. A more severe clinical presentation is seen in tropical pulmonary eosinophilia caused by the pulmonary presence of the larial worms Wuchereria bancrofti and Brugia malayi. The corresponding eosinophil recruitment and activation in the lung is a host defense response.
Pulmonary Vasculitis

Pulmonary eosinophilia describes the association of a raised blood eosinophil count with areas of pulmonary consolidation, sometimes itting in nature. Pulmonary eosinophilia can occur either as an association with another underlying disease process or as an idiopathic disease entity. The common causes are listed in Table 1.
Allergic Bronchopulmonary Aspergillosis

ChurgStrauss syndrome is characterized by systemic vasculitis in association with hypereosinophilia and asthma. A background of rhinosinusitis with atopy is common, and asthma may precede the syndrome by up to 30 years.
Eosinophilic Pneumonia

This is a syndrome of pulmonary eosinophilia and proximal bronchiectasis that can sometimes complicate asthma and cystic brosis. Airway inammation and subsequent damage is driven by eosinophil recruitment to the airway in response to fungal allergen-driven Th2-secreted IL-4 and IL-5.
Drug Reactions

This is a disorder of unknown etiology that mimics pneumonia-like illness, with both respiratory and systemic symptoms. There is an association with atopy and recent-onset asthma.
Therapeutic Manipulation of Eosinophils in Respiratory Disease

A number of drugs associated with pulmonary eosinophilia have been documented, and drug reactions are probably the most common cause of eosinophilia in the United Kingdom. The symptoms are generally mild, sometimes with cough and systemic symptoms, and usually improve on stopping the medication. In more severe or persistent cases, treatment with corticosteroids is required.
Parasitic Infections

A number of parasitic infections can be associated with pulmonary eosinophilia. Symptoms are
Table 1 Causes of pulmonary eosinophilia Drugs Sulfonamides Nitrofurantoin Minocycline Penicillins Tetracycline Clarithromicin Leukotriene inhibitors Sulfasalazine Aspirin Parasites Ascaris species Toxocara species Ancyclostoma species Wuchereria bancrofti Brugia malayi Schistosoma species Strongyloides species

Corticosteroids are considered the most effective treatment for eosinophilic disorders by virtue of their ability to inhibit eosinophil recruitment and activation by suppression of a variety of inammatory genes, including IL-5. Corticosteroids induce eosinophil apoptosis by the release of proapoptogenic factors and also by stimulating intracellular apoptotic events. The non-specific effects of corticosteroids and a failure of some patients with asthma to respond to steroids have driven the search to nd more specific therapies targeted at eosinophils alone, directed at different stages of the eosinophil life cycle. Anti-IL-5 therapy failed to affect lung function and symptoms in asthma in clinical trials. Only a 55% reduction in the degree of tissue eosinophilia was achieved. Such therapy may be ineffective because the pathways that

Systemic disease Hodgkins lymphoma Bronchogenic carcinoma Ulcerative colitis Mycobacterial infection Vasculitis

Idiopathic Acute eosinophilic pneumonia Chronic eosinophilic pneumonia

LEUKOCYTES / Neutrophils 547

recruit and activate eosinophils remain active. Eotaxin and CCR3 receptor blockade offer obvious therapeutic potential. Antagonists against these molecules are expected to enter clinical trials in the near future.
See also: Allergy: Overview. Asthma: Overview. Chemokines. Eotaxins. Interleukins: IL-5. Lipid Mediators: Overview; Leukotrienes; Prostanoids.

regulation of their function, it is unsurprising that neutrophilic inammatory diseases are prevalent; these include asthma, chronic obstructive pulmonary disease, the acute respiratory distress syndrome, and several vasculitides.

Introduction
The existence of white blood corpuscles was described by Hewson in the eighteenth century. In the mid-nineteenth century, Ehrlich was able to demonstrate the existence of several subtypes of these cells using a triacid stain; he identied acidophils (eosinophils), neutrophils, and basophils. The ability of white blood cells to migrate through blood vessel walls and comprise the chief cellular constituent of pus was established at around this time by Addison and Cornheim, but it was Metchnikov who, in the late nineteenth century, realized that these cells ingested and destroyed microbes, coining the term phagocyte from the Greek phagein (to eat). This crucial view of the benets of inammation was in contradiction to beliefs held since the time of the early Greek physicians in and before the rst century, in which inammation was viewed as an entirely negative and destructive process. Furthermore, it dened the principal role of neutrophils: to mobilize to sites of infection by emigration from the circulation and to destroy invading microbes. Since that date, the goals of neutrophil research have changed little: to understand how recruitment and antimicrobial responses are regulated. The neutrophil is our most numerous phagocyte (numbering 2.57.5 109 cells l 1 blood), whose function is primarily to defend against bacterial infections, but it may also play a role in antiviral responses and particle clearance. A striking feature of this cell type is its short life span, enhancement of which is one of the most important ways of regulating its antibacterial capabilities. Neutrophils only survive for about 68 h in the circulation. To maintain their numbers in spite of a short life span, the bone marrow manufactures 1 1011 neutrophils per day in health, with the capacity to markedly upregulate this rate of production when required. Development from CD34 stem cells to mature neutrophils typically takes about 14 days, and is dependent upon cytokines such as granulocytemacrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), the latter cytokine being a useful therapeutic agent to enhance neutrophil production in the neutropenic patient (e.g., during chemotherapy). The bone marrow contains a large pool of mature or near-mature neutrophils available for rapid mobilization, and specific pathways regulate the release of mature cells,

Further Reading
Alberts WM (2004) Eosinophilic interstitial lung disease. Current Opinion in Pulmonary Medicine 10(5): 419424. Bochner BS (2004) Verdict in the case of therapies versus eosinophils: the jury is still out. Journal of Allergy and Clinical Immunology 113(1): 310. Flood-Page PT, Menzies-Gow AN, Kay AB, and Robinson DS (2003) Eosinophils role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. American Journal of Respiratory and Critical Care Medicine 167(2): 199204. Gleich GJ (2000) Mechanisms of eosinophil-associated inammation. Journal of Allergy and Clinical Immunology 105(4): 651663. Kay AB, Phipps S, and Robinson DS (2004) A role for eosinophils in airway remodelling in asthma. Trends in Immunology 25(9): 477482. Klion AD and Nutman TB (2004) The role of eosinophils in host defense against helminth parasites. Journal of Allergy and Clinical Immunology 113(1): 3037. Menzies-Gow A and Robinson DS (2001) Eosinophil chemokines and chemokine receptors: their role in eosinophil accumulation and activation in asthma and potential as therapeutic targets. Journal of Asthma 38(8): 605613. Robinson DS, Kay AB, and Wardlaw AJ (2002) Eosinophils. Clinical Allergy & Immunology 16: 4375. Rothenberg ME (2004) Eosinophilic gastrointestinal disorders (EGID). Journal of Allergy and Clinical Immunology 113(1): 1129.

Neutrophils
I Sabroe, University of Shefeld, Shefeld, UK E R Chilvers, Addenbrookes Hospital, Cambridge, UK M K B Whyte, University of Shefeld, Shefeld, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
The neutrophil is a phagocytic leukocyte, the most abundant in the human body and the most short-lived of all myeloid cells. This polymorphonuclear cell is packed with granules containing potent antimicrobial compounds, and is absolutely essential for day-to-day successful defense against bacterial and fungal infections. Its recruitment and activation is carefully regulated, and control of its life span coupled with mechanisms allowing for a noninammatory death pathway (apoptosis) reduce risks of tissue damage and disease arising from unwanted neutrophil activation. Given their destructive potential, and despite intensive

548 LEUKOCYTES / Neutrophils

and the return of senescent cells, from and to the marrow (see below). Physiological removal of circulating cells also takes place in the reticuloendothelial systems of the liver and spleen. Within the circulation, a substantial proportion of neutrophils marginate into microvascular capillary beds such as the lung. Their large size (1012 mm diameter, i.e., larger than red cells) delays their transit through these beds, and it is possible that in these environments, neutrophils play important roles ltering out bacteria and other particles from the circulation. Certainly, sudden systemic exposure to bacteria or chemoattractants such as activated complement results in neutrophil activation in the circulation and a dramatic increase in margination, with a consequent transient neutropenia. Neutrophils contain large numbers of primary and secondary granules. Primary (azurophil) granules contain hydrolases, matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and cationic proteins; secondary granules contain lactoferrin, histaminase, and collagenase. Delivery of these proteins to phagosomes containing ingested microorganisms results in their destruction (though some bacteria have evolved strategies to protect themselves from these microbicidal systems). These act in concert with the NADPH oxidase system, which generates reactive oxygen species, and inuences phagosome alkalinization and the activation of proteases within the phagosome. Release of granule contents and superoxides into the extracellular milieu can be extremely destructive (see below), and excessive or inappropriate neutrophil activation is a major potential cause of tissue damage. (The oft-repeated and over-used phrase describes this as the double-edged sword of inammation, but we should bear in mind that the daily survival of every individual depends on neutrophil function, and whilst diseases involving unwanted neutrophil activation are prevalent in the developed world, the relative number of individuals killed by their own neutrophils remains small.) The histotoxic potential of neutrophils dictates the need for efcient physiological mechanisms to safeguard against inadvertent activation. The key mechanism for this is neutrophil priming, whereby degranulation and activation of the NADPH oxidase occurs only in cells pre-exposed to agents such as lipopolysaccharides (LPS) or GM-CSF. As well as controlling secretory responses, priming also affects neutrophil shape (creating a cell with a highly irregular polarized cell contour), deformability, integrin expression and longevity, and, as a consequence, has a profound impact on the rheological, adhesive, and survival properties of these cells and their injurious potential.

Neutrophil Function in the Normal Lung

The distal airways and acini are generally held to be sterile, yet small numbers of neutrophils may often be found on cellular analysis of normal bronchoalveolar lavage uid, or in normal endobronchial biopsies. Constitutive trafcking of granulocytes to normal mucosa has been seen in other organs (e.g., eosinophil trafcking to the gut); thus, whether neutrophil recruitment to the lung is the result of chronic low-grade inammatory insults (infectious organisms, inhaled endotoxin, pollution), or a surveillance and defense pathway that would act in the absence of any measurable stimulus, is unknown (and, since our only sterile environment is antenatal, broadly irrelevant). Large numbers of neutrophils reside temporarily in the pulmonary microvascular bed, where they may help lter the blood of pathogens and particles. In either setting, the principal role of the cell remains host defense.

The Neutrophil in Respiratory Diseases

The neutrophil is principally active against bacterial infections such as those that cause community- and hospital-acquired pneumonias, though there is also some evidence that neutrophil activation may contribute to the early response to tuberculosis (TB) and successful granuloma formation. Neutrophils also provide important responses to fungal infections and contribute to antiviral responses (e.g., to inuenza A).
Trafcking and Recruitment

Recruitment to inamed sites is regulated at multiple levels. Neutrophils are actively released from the marrow in processes regulated by adhesive interactions (blockade of CD11b/CD18 enhances release, whilst blockade of CD49d (VLA-4) inhibits release) and chemokine signaling. Neutrophils express the chemokine receptors CXCR1 and CXCR2, whose ligands, in particular IL-8/CXCL8, mobilize neutrophils from the marrow. Surprisingly, recent work has also shown that immature neutrophils express CXCR4, and SDF-1a/CXCL12 signals to retain neutrophils within the marrow. Furthermore, expression of CXCR4 increases with age and provides a mechanism that causes senescent neutrophils to trafc back to the marrow, presumably for removal by bone marrow macrophages. Recruitment into tissues from the microcirculation is driven by a series of adhesive interactions between the cell and the vessel wall, dependent chiefly on L-selectin and CD11b/ CD18, and directed by a large panel of neutrophil chemoattractants. Again, the chemokines have received considerable attention, as ligands of CXCR1

LEUKOCYTES / Neutrophils 549

and CXCR2 provide a degree of selectivity for neutrophils (though these receptors may be expressed by monocytic cells and lymphocytes to some degree). Activated neutrophils are themselves a source of CXCL8, potentially mobilizing more phagocytes to sites of inammation. However, many other molecules, such as activated complement fragments (C5a) and bacterially derived peptides, are potent chemoattractants; in contrast to chemokines these are also excellent stimulators of neutrophil degranulation and respiratory burst, and are important regulators of neutrophil function. In pneumonia, there is evidence from animal models that neutrophil recruitment, activation, and apoptosis proceeds in relatively discrete waves over a period of days, rather than continuous neutrophil recruitment occurring whilst inammation persists.
Detection of Pathogens

Neutrophils detect pathogens through a broad range of pathogen-recognition receptors, including integrins, Toll-like receptors (TLRs; see Toll-Like Receptors), and recently identied molecules such as dectin-1. Their signaling is typically primed by cytokines such as GM-CSF and lipid mediators such as platelet-activating factor (PAF), through signaling pathways including activation of MAPKs and PI-3K. The same pathways are involved in antimicrobial defenses activated at sites of infection, including generation of reactive oxygen species (ROS), cationic proteins (defensins), and release of granule contents into phagolysosomes or the extracellular milieu. Neutrophilic responses are regulated at every level by other lung cells and leukocytes. Neutrophil-recruiting chemokines are manufactured by many cell types, including epithelia, airway smooth muscle, and endothelia. Monocytic cells serve important roles in enhancing responses of lung tissue cells to infection, synergistically increasing inammatory responses. Furthermore, monocytic and other cells are also responsible in large measure for the increase in neutrophil survival at sites of infection, generating factors that activate nuclear factor kappa B (NF-kB), probably one of the most important signaling pathways in granulocyte survival.
Disruption of Function and Disease

failure of granule fusion with the phagosomes also results in vulnerability to staphylococcal infection. Patients with deciencies in CD11b/CD18 also show multiple defects in aspects of neutrophil recruitment and activation, and are subject to multiple bacterial and fungal infections. The latter abnormality may affect other leukocytes; similarly, defects in signaling of interleukin-1 (IL-1) and TLRs (particularly TLR4) resulting from abnormalities in the intracellular protein IRAK-4 affect both neutrophils and monocytes, and dramatically increase susceptibility to bacterial, though not viral, infections. Mutations significantly affecting neutrophil function are extremely rare, presumably as the odds of the affected individuals making it to reproductive age are dramatically reduced. In the war between host and pathogen, the neutrophil retains the upper hand, though some pathogens have also developed strategies to evade neutrophil function, for example, by expression of capsules that inhibit phagocytosis (some streptococci) or production of toxins that cause neutrophil death (Pseudomonas aeruginosa).
Resolution of Inammation

The most extraordinary feature of neutrophilic inammation is its ability to resolve. Lobar consolidation dissipates to leave normal lung architecture, an amazing feat given the delicacy of alveoli and lung soft tissue. Neutrophils typically die by apoptosis, a programmed cell death that retains granules intact inside the dying cell, and prevents unwanted local release of cellular contents. These apoptotic neutrophils are taken up locally by macrophages, without activating the macrophage inammatory responses that typically follow phagocytosis. Where cell death proceeds more rapidly than macrophages can handle, progression to necrosis results in neutrophil lysis, release of granule contents, and tissue damage.
Neutrophilic Inammation and Disease

When normal neutrophil functions are disrupted by disease, severe infection can result. Chronic granulomatous disease is an X-linked recessive condition in which neutrophils are unable to make hydrogen peroxide: in these patients, severe infections with organisms such as Staphylococcus aureus affect multiple organs. In the Che diakHigashi syndrome,

Unwanted neutrophil inammation has substantial potential to cause disease. Classical neutrophilic inammatory diseases include the acute respiratory distress syndrome (ARDS), where a predominantly neutrophilic inltrate causes massive lung tissue damage and is associated with a high mortality. Animal models using ischemic reperfusion injury (in the lung and other organs) recapitulate acute insults, and demonstrate the dependence of tissue injury on neutrophil recruitment and activation. In the human, levels of CXCL8 in bronchoalveolar lavage of patients at risk of ARDS are predictive of subsequent disease development. The inability of corticosteroids to suppress neutrophil activation or induce neutrophil

550 LEUKOCYTES / Neutrophils

apoptosis may, in part, explain their lack of effectiveness in this disease. Activation of the neutrophil in the vasculature in diseases such as Wegeners granulomatosis (WG) is associated with a severe multisystem vasculitis that has its greatest impact in the lung and kidney. In WG, patients typically have autoantibodies that target neutrophils, eliciting a cytoplasmic binding pattern (cytoplasmic antineutrophil cytoplasmic antibodies (cANCAs) that bind proteinase 3

(PR3)), as opposed to the perinuclear binding pattern (pANCAs) associated with antibodies to myeloperoxide. Increasing evidence shows these antibodies to be pathological. Priming of the neutrophil by cytokines such as tumor necrosis factor alpha (TNF-a) moves PR3 to the cell surface; engagement by cANCAs delivers an activating signal that is reinforced by interaction of the Fc portion of the cANCA with IgG receptors on the same neutrophil. Generation of ROS

Normal function

CXCRs 1, 2, 4 C5aR
rece ptor s Che moa ttrac tant

Pathogen clearance fMLPR

PAFR External regulation (e.g., by monocytes) = Cytokine production

Asthma

Bronchiectasis/CF

Vasculitis

COPD External regulation (e.g., by monocytes)

ARDS

tors

Nonrespiratory diseases CD11b/ CD18 CD14 TLR2 In disease

Path

oge

n re

cog

nitio

Resolution by apoptosis

n re

Figure 1 Neutrophil function in health and disease. Neutrophils function to maintain health by removal of pathogens. Their recruitment into tissues is regulated by many chemoattractants, principal amongst which are chemokines acting on CXCR1 and CXCR2 (such as CXCL8), and the activated complement fragment, C5a. Bacterial peptides may also activate the fMLP receptor, though this receptor may also respond to as yet unidentied endogenous molecules. Recruitment into tissues and to the site of infection is followed by neutrophil activation, as the cells detect pathogens through a range of pathogen-recognition receptors, which probably act in concert to switch on bacterial killing systems such as ROS generation and the release of toxic proteases. Simultaneously, neutrophils generate more CXCL8, facilitating further cell recruitment. It is likely that other cell types, e.g., monocytes and macrophages, oversee the neutrophil response, providing survival cytokines that control the duration and amplitude of the inammatory response. After removal of pathogens, neutrophils die by apoptosis and are engulfed by monocytic cells, which remove them from the local environment, preventing tissue damage. However, where inappropriate or excessive neutrophil activation occurs, there is considerable potential for tissue damage. Neutrophil-mediated tissue damage has been linked with the pathology of a range of respiratory and nonrespiratory diseases, and to date remains extremely difcult to target therapeutically.

cep

TLR4

LEUKOCYTES / Neutrophils 551

results, and this neutrophil activation appears central to disease pathogenesis. CXCL8 generation and some measure of neutrophil recruitment is a common feature of many other respiratory diseases, but it can be surprisingly hard to discern the unique contribution of neutrophils to the pathology. Such contributions have occasionally been obscured further when roles for neutrophils ran counter to dogma, even when pointers to their importance were apparent. There is now clear evidence that subgroups of asthmatics show marked airway neutrophilia (alone or with eosinophilia), which may be amplied by treatment with corticosteroids, since they induce eosinophil apoptosis but prolong neutrophil survival. Acute severe asthma is relatively hard to study, but neutrophils often dominate the airway inammation in these settings. Treatments for neutrophilic asthma variants may offer new ways of intervening in asthma that has responded poorly to conventional management strategies. Likewise in COPD, airway neutrophilia correlates with severity of airway obstruction, and overexpression of IL-1b in the lung results in airway inammation with neutrophil and monocyte recruitment, airway remodeling, and emphysema. In COPD, neutrophil proteases and ROS may contribute to tissue damage and remodeling, though the exact contribution of the neutrophil versus the monocyte/macrophage is difcult to determine in such a chronic setting. Likewise, arguments over the role of the neutrophil in some variants of brotic lung disease persist, and they may be involved in tissue damage where chronic infection is present, for example, bronchiectasis and cystic brosis.

Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deciency; Emphysema, General. Complement. Defense Systems. Defensins. Endotoxins. Granulomatosis: Wegeners Disease. Interstitial Lung Disease: Overview. Lipid Mediators: Platelet-Activating Factors. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens; Fungal (Including Pathogens). Toll-Like Receptors. Transcription Factors: NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading
Burdon PC, Martin C, and Rankin SM (2005) The CXC chemokine MIP-2 stimulates neutrophil mobilization from the rat bone marrow in a CD49d-dependent manner. Blood 105: 25432548. Cowburn AS, Cadwallader KA, Reed BJ, Farahi N, and Chilvers ER (2002) Role of PI3-kinase-dependent bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 100: 26072616. Donnelly SC and Haslett C (1992) Cellular mechanisms of acute lung injury: implications for future treatment in the adult respiratory distress syndrome. Thorax 47: 260263. Haslett C, Savill JS, Whyte MK, Stern M, Dranseld I, and Meagher LC (1994) Granulocyte apoptosis and the control of inammation. Philosophical Transactions of the Royal Society of London 345: 327333. Hopken UE, Lu B, Gerard NP, and Gerard C (1996) The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 383: 8689. Lamblin C, Gosset P, Tillie-Leblond I, et al. (1998) Bronchial neutrophilia in patients with noninfectious status asthmaticus. American Journal of Respiratory and Critical Care Medicine 157: 394402. Martin C, Burdon PC, Bridger G, et al. (2003) Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 19: 583593. Medvedev AE, Lentschat A, Kuhns DB, et al. (2003) Distinct mutations in IRAK-4 confer hyporesponsiveness to lipopolysaccharide and interleukin-1 in a patient with recurrent bacterial infections. Journal of Experimental Medicine 198: 521531. Parker LC, Whyte MK, Dower SK, and Sabroe I. The expression and roles of Toll-like receptors in the biology of the human neutrophil. Journal of Leukocyte Biology (in press). Peters AM (1998) Just how big is the pulmonary granulocyte pool? Clinical Science (London) 94: 719. Segal AW (2005) How neutrophils kill microbes? Annual Review of Immunology 23: 197223. Tonnel AB, Gosset P, and Tillie-Leblond I (2001) Characteristics of the inammatory response in bronchial lavage uids from patients with status asthmaticus. International Archives of Allergy and Immunology 124: 267271. Ward C, Chilvers ER, Lawson MF, et al. (1999) NF-kappa B activation is a critical regulator of human granulocyte apoptosis in vitro. Journal of Biological Chemistry 274: 43094318. Wenzel SE, Schwartz LB, Langmack EL, et al. (1999) Evidence that severe asthma can be divided pathologically into two inammatory subtypes with distinct physiologic and clinical characteristics. American Journal of Respiratory and Critical Care Medicine 160: 10011008. Williams JM, Kamesh L, and Savage CO (2005) Translating basic science into patient therapy for anca-associated small vessel vasculitis. Clinical Science (London) 108: 101112.

Summary
In summary, the neutrophil is central to host defense, particularly against bacterial and fungal infections. Although diseases with a basis in neutrophilic inammation are prevalent, given the extraordinary efcacy of these cells in causing microbial and tissue destruction, it is impressive how well controlled neutrophil function is on a day-to-day basis in the majority of the population. In the future, targeting of neutrophils may provide effective treatments for inammatory diseases where currently treatments are absent or rely on heavy immunosuppression (Figure 1).
See also: Adhesion, CellCell: Vascular; Epithelial. Adhesion, CellMatrix: Focal Contacts and Signaling; Integrins. Asthma: Overview; Acute Exacerbations; Extrinsic/Intrinsic. Autoantibodies. Bronchiectasis. Bronchiolitis. Bronchoalveolar Lavage. CD11/18. CD14. Chemokines. Chemokines, CXC: CXCL12 (SDF-1); IL-8; CXCL1 (GRO1)CXCL3 (GRO3). Chronic

552 LEUKOCYTES / Monocytes

Monocytes
P A Stumbles and C von Garnier, The University of Western Australia, Perth, WA, Australia
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Monocytes are bone marrow-derived phagocytic cells that circulate through the blood and into tissues, playing a major role in respiratory defense. Through their phagocytic and lysosomal activity, monocytes play a major primary role in innate immunity and pathogen clearance. Furthermore, through their capacity for cytokine-driven differentiation into macrophages (Mf) and dendritic cells, monocytes play an important role in respiratory homeostasis by acting as a resident precursor pool for these two major classes of immune cells. Dendritic cells (DCs) are the major antigen-presenting cell type of the mucosal surfaces and interstitial tissues of the respiratory tract and play an important role in the regulation of adaptive T-cell immunity to inhaled allergens and pathogens. Alveolar, interstitial, and mucosal Mf are the major phagocytes within the respiratory tract that, in addition to their important innate immune activity, also play a crucial role in regulating the antigen-presenting activity of DCs and the prevention of unchecked lymphoproliferation. In addition to their homeostatic activity, products of monocytes, such as chemokines, and the recruitment and deregulated differentiation of these cells are relevant in a wide variety of important pulmonary diseases such as tuberculosis and allergic asthma. Strategies aimed at modifying the recruitment and/or differentiation of these cells, as well as regulating their production of proinammatory mediators, may provide new therapeutic directions for the treatment of a wide variety of respiratory inammatory disorders.

Introduction
Monocytes are phagocytic cells that contribute to innate immunity through their phagolysosomal activity and adaptive immunity through their ability to differentiate into antigen-presenting cells. Cells of the mononuclear phagocyte system originate from stem

cells in the bone marrow (BM) that exit into the blood as circulating monocyte precursors prior to migration into tissues and differentiation into macrophages (Mf) and dendritic cells (DCs) (Figure 1). Pulmonary alveolar Mf can be obtained by lung lavage and are responsible for the clearance of inhaled particles and lung surfactant. They contribute to innate immunity through their capacity to phagocytose and are able to modulate the adaptive immune response of the dendritic cellT cell axis. In contrast, respiratory tract DCs (RTDCs) are pivotal in the initiation of T and B cell-mediated immunity due to their potent antigen uptake and presenting capacity. Ultrastructurally, monocytes are large (10 18 mm) cells with a characteristic lobulated nucleus and prominent intracytoplasmic lysosomes (Figure 2(a)). Based on surface expression of CD11c, major histocompatibility complex (MHC) class II, and other markers, cells that phenotypically and ultrastructurally resemble tissue monocyte precursor cells can be isolated from peripheral lung tissue of mice that, under appropriate stimulatory conditions, can differentiate into Mf and RTDCs (Figure 2(a)). In situ, these can be identied as Mf located at the alveolar interseptal junction in parenchymal lung tissue in association with type I alveolar epithelial cells. Monocytic precursor cells can also give rise to RTDCs, which lie within the underlying interstitium in close proximity to Mf (Figure 2(b)). Mf populations have also been identied in the pleural lining and peribronchial and perivascular connective tissue of peripheral lung. Within the mucosal tissue of the conducting airways, RTDCs are found both within the epithelium and in the proximal (luminal) regions of the underlying lamina propria, overlaying the cartilage rings. In contrast, airway mucosal Mf are located distally in a band overlaying smooth muscle and underlaying the cartilage rings, and are also

Bone marrow Stem cell Common myeloid precursor

Blood Circulating monocyte precursor

Respiratory tract Resident monocyte tissue precursor Macrophage F4/80+ CD11c+ CD11b Gr-1

DC Sca1+ cKit+ Thy1low CD34low/+ F4/80+ CD11b+ Gr-1+ Sca1+ cKit F4/80+ CD11clow CD11b+ Gr-1+

F4/80 CD11c+ CD11blow Gr-1

Figure 1 Monocyte origins and ontogeny. Monocytes develop from the bone marrow stem cells and enter the blood as circulating monocyte precursors, which enter tissues as resident monocyte precursors. Local factors produced within the respiratory tract will then promote differentiation into either Mf or dendritic cells.

LEUKOCYTES / Monocytes 553

RTDC MHC class II

M
Mono

CD11c (a)

Alveolar interseptal junction

M
Cap

Cap Type I alveolar epithelial cell

Type I epithelial cell membrane

RTDC (b) Ep DC

Airway lumen

Ep bm C C

Lp DC

C M
sm

(c)
Figure 2 Morphology and distribution of monocytes, Mf, and RTDCs. (a) Transmission electron micrograph (TEM) of a monocytic precursor cell puried from mouse lung tissue (left) and a schematic representation of the differentiation pathway of these cells into Mf or RTDCs following in vitro differentiation as dened by expression of CD11c and MHC class II. (b) Schematic representation of a crosssection of lung tissue showing the location of Mf at the alveolar interseptal junction and in close proximity to RTDCs and capillaries (Cap). (c) Schematic representation of a longitudinal section of conducting airway showing the distribution of RTDCs within the epithelium (Ep DC), underlying the basement membrane (bm) and within the lamina propria (Lp DC), and the location of Mf below the cartilage rings (c) and in juxtaposition with smooth muscle cells (sm).

554 LEUKOCYTES / Monocytes

present in mucous layers covering the epithelial cells of small and large airways (Figure 2(c)).

Table 1 Monocyte-derived or -acting chemokines in respiratory disease Systematic name Common name MCP-1 Receptor CCR2 Respiratory disease Allergic asthma Bacterial pneumonia Mycoplasmainduced BPD Interstitial pneumonia Allergic asthma Allergic asthma COPD COPD

Monocyte Function in the Normal Lung

The primary function of cells of the monocyte/macrophage lineage in peripheral lung is phagocytosis and killing of inhaled microorganisms. However, these cells also play an equally important role in the maintenance of tissue-specific homeostatic processes. Inuxing blood monocytes are thought to be the principal cell source for renewal of resident Mf and DCs in lung and airway tissue, although the processes governing these differentiation pathways in vivo are poorly understood. In vitro, macrophage colonystimulating factor (M-CSF) has been identied as a potent Mf differentiation factor in humans and rodents. Expression of the M-CSF receptor (CD115) on monocytes is regulated by a number of cytokines including IL-6, IL-10, and interferon gamma (IFN-g). Exposure of monocytes to these factors shifts monocyte differentiation towards Mf. In vitro studies of human peripheral blood monocytes also identied the capacity of these cells to differentiate into DCs following exposure to granulocyte-macrophage colonystimulating factor (GM-CSF) together with IL-4. Similar differentiation pathways exist in vitro for mouse monocytes and have also been demonstrated to occur in vivo in this species. GM-CSF receptor expression is also regulated by cytokines, with GMCSF itself acting dominantly to suppress M-CSF-driven Mf differentiation. While monocyte differentiation in the respiratory tract is poorly understood, this process is likely to be controlled by products of epithelial cells and other stromal cells in the steady state acting to replenish populations of local tissue RTDCs and Mf. Given that the half-life of RTDC at mucosal surfaces is estimated to be in the order of 1224 h, and that rapid recruitment of these cells occurs following inammatory challenge within a similar time frame, the differentiation of monocytes into RTDCs is likely to represent a dominant role for monocytes in the maintenance of respiratory tract homeostasis. Signaling through surface receptors for bacterial lipopolysaccharides (LPS) such as CD14 and Toll-like receptor 4 (TLR4) and through TLRs specific for other conserved microbial sequences will also accelerate the differentiation of respiratory monocytes into Mf and RTDCs during infection-induced inammation.
Chemokines

CCL2

CCL7 CCL13 CCL22 CXCL1 CXCL7

MCP-3 MCP-4 MDC GROa NAP-2

CCR1,2,3 CCR2,3 CCR4 CXCR2 CXCR2

COPD, chronic obstructive pulmonary disease.

cell migration (chemotaxis), and their abnormal production has been implicated in a large range of disease states. While monocytes produce and respond to a wide range of chemokines, only two families of chemokines have been described to act in a relatively monocyte-specific manner: activity of the monocyte chemoattractant protein (MCP) chemokines MCP-1 (CCL2), MCP-3 (CCL7), and MCP-4 (CCL13) is restricted to recruitment of monocytes and basophils and has been associated with monocyte recruitment in a variety of respiratory diseases; it is most often associated with allergic airways disease (Table 1). In contrast monocyte-derived chemokine (MDC, CCL22) is a potent attractor and activator of Th2type T cells and has been implicated in the pathogenesis of allergic asthma (Table 1). Enhanced chemotactic responses of monocytes towards other chemokines such as growth-related oncogene alpha and neutrophil-activating peptide (GROa/NAP-2, CXCL1, CXCL7) have also been identied in diseases such as cigarette smoke-induced chronic obstructive pulmonary disease.
Immunoregulation

Chemokines are produced by, and act on, virtually all cells of the immune system to direct homeostatic

An important homeostatic process within lung tissue is the regulation of local immune reactivity in order to avoid local immunopathology and damage of the blood-to-air interface. In this respect, a major function of cells of the monocyte/macrophage lineage in normal lung tissue is their capacity to downmodulate local T cell responsiveness through cytostatic signals delivered to both T cells and antigen-presenting DCs. Initial studies in rodents showed that in vivo depletion of alveolar Mf enhanced immune priming to aerosolized antigens. Subsequent studies showed that this effect could operate on both RTDCs through inhibition of the maturation of these cells into competent antigen presenters, and also on the T cell

LEUKOCYTES / Monocytes 555

CD4+ T cell NO GM-CSF TNF IL-4 TGFM
RTDC M-CSF GM-CSF IL-4 RTDC GM-CSF IL-4

CD4+ T cell

NO

(a)

Monocyte precursor

(b)

Monocyte precursor

Figure 3 Immunoregulatory functions of monocytes and Mf. (a) In normal tissue, monocytes differentiate into RTDCs and Mf, the latter acting to inhibit CD4 T cell activation by RTDCs through production of NO. (b) Under inammatory conditions, proinammatory cytokines (GM-CSF, TNF-a, IL-4, TGF-b) act to overcome the immunosuppressive activity of Mf while at the same time acting to promote the differentiation of monocytes into immunostimulatory RTDCs.

through inhibition of lymphoproliferative activity. The principal mediator of the lymphostatic effect was shown to be Mf-derived nitric oxide (NO), which inhibits IL-2 receptor signaling in T cells through disruption of the Jak3/STAT5 intracellular signaling pathway. Similarly, NO was also demonstrated to be the principal mediator of alveolar Mf-mediated inhibition of RTDC maturation. The inhibitory effects of alveolar Mf can be abrogated by exposure to the cytokine GM-CSF and to a lesser extent transforming growth factor beta (TGF-b), IL-4, and tumor necrosis factor alpha (TNF-a), the latter acting in a synergistic fashion with GM-CSF to inhibit NO production by Mf and also promote maturation of RTDCs. Thus, monocyte-derived alveolar Mf and RTDCs participate in a tricellular interaction with T cells that in normal lung tissue inhibits T cell activation and priming to nonpathogenic inhaled antigens through a suppressive mechanism mediated by Mf-derived NO (Figure 3(a)). Under inammatory conditions, production of cytokines such as GM-CSF together with other proinammatory cytokines such as TNF-a and IL-4 will act to promote RTDCT cell interactions and T cell priming through inhibition of Mf-mediated T cell suppression as well as upregulating the antigenpresenting capacity of Mf and the differentiation of monocytes towards mature antigen-presenting RTDCs (Figure 3(b)).

clinical course of this disease can range from severe respiratory failure to spontaneous resolution, but an important feature is susceptibility to pulmonary infections with opportunistic infections. Pulmonary alveolar proteinosis may present in three clinically different forms: congenital, secondary, and acquired. The congenital form is caused by mutations in the genes encoding surfactant protein B or C, or the bc chain of the GM-CSF receptor. Certain infections, inhalation of toxic fumes or inorganic dust, hematological malignancies, and pharmacological immunosuppression cause the secondary form, which results in functionally impaired or reduced numbers of alveolar Mf. In acquired (or idiopathic) pulmonary alveolar proteinosis, a neutralizing autoantibody against GM-CSF causes a functional defect in alveolar Mf, including impaired surfactant lipoprotein catabolism and homeostasis. Though mechanisms for cytokine changes are unknown, both MCP-1 and M-CSF are raised in acquired disease. Treatment with GM-CSF improves acquired pulmonary alveolar proteinosis in some patients, though the mechanism of this effect remains to be elucidated.
Tuberculosis

Monocytes in Respiratory Diseases

Pulmonary Alveolar Proteinosis

Accumulation of surfactant protein leads to a rare disorder termed pulmonary alveolar proteinosis. The

In primary pulmonary tuberculosis (TB), Mycobacterium tuberculosis organisms reaching the alveolar space are phagocytosed by alveolar Mf and are either killed or survive. In the latter instance, bacteria multiply and kill their host Mf; this is followed by the release of mycobacteria and subsequent infection of other host cells. Chemotactic factors attract circulating monocytes, lymphocytes, and neutrophils, none of which kills bacteria efciently, leading to the formation of granuloma. These granulomas display a

556 LEUKOCYTES / Monocytes

high rate of monocyte and lymphocyte turnover, indicative of the toxicity of M. tuberculosis bacilli and requiring continuous replacement of host cells. For protective immunity, a strong nonantigen-specific granulomatous reaction within days is essential, followed by an antigen-specific T cell response within weeks, which is embodied by a delayed type hypersensitivity (DTH) reaction. The DTH response leads to the killing of infected Mf as the periphery of the granuloma becomes brotic and caseated. Necrotic areas are surrounded by a layer of epithelioid histiocytes and multinucleated giant cells, which in turn are enclosed by broblasts, lymphocytes, and bloodderived monocytes. If a sufcient cell-mediated immunity is generated, infection is permanently terminated at this stage. If this response is insufcient, Mf containing viable M. tuberculosis escape from the granuloma, or tubercle, rapidly leading to lymphatic dissemination of organisms to regional lymph nodes. In an attempt to control infection, the host DTH response leads to further pulmonary parenchymal destruction and eventually causes liquefaction of the caseous granuloma center, providing a rich and oxygenated environment for enhanced mycobacterial proliferation. A repeat infectious episode, or postprimary TB can be caused either by reactivation of a dormant lesion, or by inhalation of additional mycobacteria, leading to an infection that proceeds in spite of existing immunity. Tissue necrosis and caseation then occur due to a DTH response. Several cell surface molecules are involved in the binding and internalization of M. tuberculosis: complement receptors, macrophage mannose receptors (MMR), and CD14. Following binding and phagocytosis of mycobacteria, intracellular organisms are able to proliferate only if they avoid eradication by lysosomal enzymes, reactive oxygen intermediates (H2O2 and O2 ), and reactive nitrogen intermediates (NO and NO2 ). One of the mechanisms by which intracellular M. tuberculosis might evade destruction is through decreased fusion of mycobacteriumcontaining phagosomes with lysosomes. The presence of live mycobacteria may lead to a reduction in antigen processing, loading, and transport of MHCII to the cell surface. Following infection by M. tuberculosis, activated Mf may efciently kill mycobacteria, while immature monocytes recruited from the periphery following infection are less effective and serve as the preferred host. Leukocyte-derived local production of IFN-g and TNF is central to activation and differentiation of recruited monocytes. Activated Mf present mycobacterial antigen to T cells in association with MHC class I, MHC class II, or nonpolymorphic MHC-like proteins such as CD1. Moreover, Mf have been shown to transfer

mycobacterial antigen to dendritic cells, thereby enhancing activation of naive T cells.

Allergic Asthma

Alveolar Mf must display the capacity to both enhance and suppress an inammatory response. Suppressive actions of Mf are mediated by soluble molecules and mechanisms requiring cell-to-cell interaction. NO has been suggested to promote Th2 responses, but may also suppress other inammatory responses including leukotriene synthesis, cytokine production, and smooth muscle contraction. Regulatory cytokines TGF-b and IL-10 elaborated by Mf have generalized suppressive effects on inammatory and immune responses. The gaseous molecule CO possesses generalized anti-inammatory effects. Its administration following allergen sensitization/challenge reduced eosinophilia and levels of inammatory cytokines in bronchoalveolar lavage. The enzymatic source of CO, heme oxygenase I, is increased in alveolar Mf from individuals with uncontrolled asthma. Prostaglandin E2 (PGE2) has been reported to possess contradictory effects on eosinophilic inammation. PGE2 causes bronchodilatation, inhibits proinammatory leukocyte function, and suppresses functions of broblasts as well as smooth muscle cells. While PGE2 promotes lymphocyte-dependent Th2 cytokine secretion in vitro, it suppresses Th2 cytokine secretion and allergic asthmatic responses. The PGD2 degradation product, 15-deoxy-PGJ2, exerts an in vitro anti-asthmatic effect by inhibition of T cell-dependent IL-5 generation and epithelial cell-derived IL-8 production. Both 15-deoxy-PGJ2 and the arachidonate 15-lipoxygenase product 15 hydroxyeicosatetraenoic acid (15-HETE) bind to the nuclear hormone receptor, peroxisome proliferatoractivated receptor-g (PPAR-g), which inhibits activation of the transcription factor nuclear factor-kB. Lipoxin A4, a dual 5- and 15-lipoxygenation product from arachidonic acid, exerts in vivo and in vitro antiinammatory actions. Apoptotic leukocytes generated during any inammatory process are phagocytosed by alveolar Mf, a process that triggers production of anti-inammatory molecules such as PGE2 and TGF-b. In contrast, phagocytosis of pathogens via opsonin receptors such as the Fcg receptor for IgG leads to proinammatory responses. Alveolar Mf from human asthmatics have been shown to have impaired Fcg receptor-mediated phagocytosis due to decreased surface expression of Fcg receptor I.
See also: Asthma: Overview. Chemokines. Chemokines, CC: C10 (CCL6); MCP-1 (CCL2)MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17); TECK (CCL25).

LEUKOCYTES / T Cells 557 Chemokines, CXC: CXCL12 (SDF-1); IL-8; CXCL9 (MIG); CXCL10 (IP-10); CXCL1 (GRO1)CXCL3 (GRO3). Dendritic Cells. Interstitial Lung Disease: Alveolar Proteinosis. Leukocytes: Pulmonary Macrophages.
role in innate, nonantigen-dependent immunity. Substantial research efforts are being directed at identifying mechanisms by which T or NK cells may contribute to disease pathogenesis or as targets for preventative strategies.

Further Reading
Bilyk N and Holt PG (1993) Inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor. Journal of Experimental Medicine 177: 17731777. Bingisser RM, Tilbrook PA, Holt PG, et al. (1998) Macrophagederived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. Journal of Immunology 160: 57295734. Blusse van oud Alblas AB and van Furth R (1979) Origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state. Journal of Experimental Medicine 149: 15041518. Holt PG, Oliver J, Bilyk N, et al. (1993) Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. Journal of Experimental Medicine 177: 397407. Imhof BA and Aurand-Lions M (2004) Adhesion mechanisms regulating the migration of monocytes. Nature Reviews: Immunology 4: 433444. Trapnell BC, Whitsett JA, and Nakata K (2003) Pulmonary alveolar proteinosis. New England Journal of Medicine 349: 25272539. Zlotnik A and Yoshie O (2000) Chemokines: a new classication system and their role in immunity. Immunity 12: 121127.

Introduction
Leukocytes, erythrocytes, and platelets are all derived from common progenitor cells, known as hematopoietic stem cells, which are located in the bone marrow. The myeloid progenitor is the precursor of granulocytes, macrophages, dendritic cells, and mast cells, whereas the lymphoid progenitor is the precursor of lymphocytes, which are the main topic of this article. The interaction of T cells with mononuclear cells including antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B lymphocytes is crucial for the onset of specific immune responses. T cells are derived from hematopoietic stem cells in the bone marrow or fetal liver (Figure 1). Following stimulation by stem cell factor (SCF), they differentiate into lymphoid stem cells, a common precursor for both T (thymic-derived) and B (bonemarrow derived) cells as well as for natural killer (NK) and some dendritic cells (DCs). Like SCF, interleukins such as IL-7 contribute to induce maturation of lymphoid stem cells into T- or B-cell precursor cells. Mature lymphocytes emerging from the thymus or bone marrow are in a quiescent or resting state. In a healthy state, T cells in the blood are present at a concentration of approximately 2500 cells mm 3. Most naive lymphocytes, unless activated, are programmed to die within a few days after leaving the bone marrow or thymus. Once dispersed into the bloodstream, the lymphocytes migrate efciently to various secondary (peripheral) lymphoid organs, such as the white pulp of the spleen, lymph nodes, tonsils, or mucosal regions of the respiratory or digestive tract, to carry out the different immune functions described below. The mucosal immune system of the lung includes antigen-dependent organized lymphoid tissues consisting of mucosal follicles of the respiratory tract, i.e., bronchus-associated lymphoid tissue (BALT) and diffuse lymphoid tissue consisting of widely distributed cells in the mucosal lamina propria.

T Cells
P W Finn, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA B Schaub, University Childrens Hospital, Munich, Germany
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Leukocytes emanate from hematopoietic stem cells in the bone marrow. T cells, derived from lymphoid progenitor cells, and natural killer (NK) cells mature and emerge from the bone marrow and thymus. Following antigen-presentation and costimulation, T cells proliferate and elicit their effector functions, including cytokine secretion. Communication between T and antigen-presenting cells is crucial in normal immune function, and key in immune dysfunction. T cells, whose main function is host defense, are classically thought to be important in allergic pulmonary diseases, including asthma. Recently, it has been suggested that T cells also play a prominent role in other pulmonary diseases including tuberculosis, sarcoidosis, Wegeners granulomatosis, and cystic brosis. The main function of NK cells is to kill lymphoid tumor cells by producing effector proteins that penetrate the cell membrane and induce programmed cell death. NK cells are recognized as being important in asthma and tuberculosis, among other pulmonary disorders. In addition to the role of T cells and NK cells in adaptive, antigen-dependent immunity, there is also great interest in understanding their

Activation of T Cells and NK Cells

This article will focus on antigen-dependent adaptive immune responses, which involve T and B cells, and are characterized by recognition of antigen via receptors such as the T-cell receptor (TCR) or B-cell receptor (BCR). In contrast, innate immune responses are antigen-independent, involving multiple cell

558 LEUKOCYTES / T Cells

Bone marrow Thymus

SCF Hematopoietic stem cell Lymphoid stem cell

IL - 7 SCF

IL - 7 SCF Secondary Iymphoid organ

T- and B-cell precursors Naive T and B cells

NK cells
Figure 1 Lymphopoiesis: schematic overview of lymphocyte development. SCF, stem cell factor; IL, interleukin; NK cells, natural killer cells.

Antigen presentation co-stimulation

Proliferation amplification

Differentiation cytokine production IL - 10 IL - 13 IL - 4 IL - 5 IL - 6

Th 2 cell Activated APC

CD86 / CD80 CD28 / CTLA4

MHC II TCR

T reg cell

TGF IL - 10

Activated T cell Th 1 cell IFN TGF IL - 15

Figure 2 Antigen presentation and T-cell activation. Induction of different T-cell populations and cytokine secretion. APC, antigenpresenting cell; CTLA4, cytotoxic T lymphocyte-associated antigen 4; MHC, major histocompatibility complex; TCR, T-cell receptor; T reg cell, T regulatory cell.

types and receptors that recognize pathogen-associated molecular patterns (PAMPs) such as the Toll-like receptors (TLRs), which in turn recognize endotoxins and bacteria, among other ligands. The interaction of adaptive and innate immunity in pulmonary diseases such as asthma is a topic of current interest. The rst step in adaptive immunity is the activation of naive T cells, requiring the presentation of specific antigen via major histocompatibility complex (MHC) II on APC to the TCR (Figure 2). This recognition of antigen occurs in the lymphoid organs and tissues, such as the lung, through which T cells are constantly crossing. TCRs recognize peptide fragments of intracellular pathogens transported to the cell surface by glycoproteins of the MHC. Two classes of MHC molecule (MHC I, II) transport peptides from different intracellular compartments to present them to distinct types of effector T cells: cytotoxic T cells (CD8 ), which kill cells infected with viruses; and

T-helper (Th) cells (CD4 ), which differentiate into Th1 and Th2 cells producing Th1 (e.g., interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), IL-15), Th2 cytokines (e.g., IL-4, IL-5, IL-6, IL-10, IL-13), and chemokines, activating other cells such as B cells and macrophages. The balance of Th1/Th2 cytokines, named the Th1/Th2 paradigm, has been a key immunological concept, and is a model that will be repeatedly referred to in this article. In addition, we will also consider the recent discovery of new Tcell subsets that may be as important or more important than the Th1/Th2 balance in the control of immune responses. T cells are crucial for both humoral and cellmediated responses of adaptive immunity. In addition to APC recognition and presentation of antigen to TCRs, additional co-stimulatory pathways mediate full T-cell activation (Figure 2). The best-characterized co-stimulatory molecules on T cells and APCs

LEUKOCYTES / T Cells 559

are the CD80 (B7.1) and CD86 (B7.2) molecules on APC, which interact with CD28 on T cells. CD28 is a glycoprotein homologous with cytotoxic Tlymphocyte-associated antigen 4 (CTLA4). Both CD28 and CTLA4 bind CD80 and CD86, but are positive and negative signals, respectively, for T-cell activation. In addition to the negative signal CTLA4, other negative signals suppress T-cell activation including programmed death-1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) molecules. PD-1 and BTLA also interact with members of the B7 family, providing negative regulation of immune function. Proliferation and differentiation of T cells is driven by interleukin-2 (IL-2), which is itself produced by activated T cells. In the case of specific antigen encounter, there is induction of IL-2 synthesis together with the a-chain of the IL-2 receptor (CD25). The b- and g-chains of the IL-2 receptor are expressed in resting T cells. Following activation, the association of the a-chain with the b- and g-chains induces a high afnity for IL-2, permitting the T cells to respond to very low IL-2 concentrations. These activated T cells are characteristically CD25 , a marker expressed in the maturation of thymocytes following expression of CD44 and c-kit. After rearrangement of the b-chain locus, expression of T a: b on the cell surface is associated with small amounts of CD3, followed by loss of CD25. This triggers progression through the cell cycle from the initial G1 phase. For complete T cell activation, signal 1 (antigen-presentation) and signal 2 (co-stimulation) are required. In the absence of co-stimulation, anergy occurs, characterized by the inability to produce IL-2, resulting in a lack of proliferation and differentiation even when antigen is presented. Anergy ensures the tolerance of T cells to self-tissue antigens. Besides macrophages and B cells, DCs are the most potent APCs, eliciting distinct functions; these are discussed in a separate article (see Dendritic Cells). NK cells develop in the bone marrow from the common lymphoid progenitor cells and circulate in the blood. Larger than T cells, they have distinct granules in the cytoplasm. NK cells, which lack expression of the TCR/CD3 complex, kill lymphoid tumor cell lines, releasing granules onto the surface of the bound target cell. The effector proteins penetrate the cell membrane, inducing programmed cell death. Activation of NK cells is induced by interferons or macrophage-derived cytokines. Unlike NK cells, NK T cells display rearranged TCR chains in association with the CD3 complex and express constitutive markers and receptors of both NK cells and T cells, supporting a potential relationship between NK T cells and NK cells.

T-Cell Function in the Normal Lung

The main functions of T cells in the lung are host defense at mucosal surfaces and prevention of the entry of mucosal antigen. T cells protect the systemic immune system from inappropriate antigenic exposure, and the induction of helper or cytotoxic activities of T cells in the diffuse lamina propria. Follicular T cells are scattered throughout the dome area, including the germinal centers (densest in the interfollicular area), expressing activation markers such as CD25. CD4 T cells are widely distributed throughout the mucosal follicle, and CD8 T cells are found exclusively in the interfollicular area. T cells produce a variety of cytokines characterized as Th1 or Th2, but overlapping expression of these cytokines is increasingly being recognized. Rates of antigen exposure and use of adjuvants (substances that boost immunity through undened mechanisms, likely involving innate immunity) are important factors in determining immune responses. For example, in murine models, Th1 responses follow oral protein antigen administration (without adjuvant). Th2 responses predominate following administration with certain adjuvants such as aluminum hydroxide (alum) and cholera toxins. In the diffuse mucosal lymphoid tissue, lymphocytes cluster in the intraepithelial lymphocyte (IEL) compartment (predominantly CD8 T cells) and the lamina propria lymphocyte (LPL) compartment (predominantly CD4 T cells), expressing the ab TCR and the human memory cell marker CD45 RO (495%). Specific to the lung, alveolar T cells are a primed memory cell phenotype that does not usually proliferate, nor does it normally undergo apoptosis. The percent of B and NK cells found in bronchoalveolar lavage (BAL) is very low; B cells are localized in the interstitial space around the pulmonary alveoli, while NK cells do not seem to play an important role in the local pulmonary defense. The evaluation of the CD4 /CD8 ratio in BAL uid can be used as an auxiliary tool in the diagnosis of lung diseases. For example, the BAL CD4 /CD8 amounts in interstitial lung disease (ILD) may be decreased, reecting a local imbalance between T helper primed memory cells and cytotoxic T cells.

T Cells in Respiratory Disease

In the following sections, we detail pulmonary diseases involving immune cells, primarily T cells, and NK cells, as relevant. The mucosal surface of the lungs poses tremendous problems for an immune system charged with maintaining a sterile pulmonary environment. Despite challenges posed by different

560 LEUKOCYTES / T Cells

diseases, the immune system is quite effective in controlling most pulmonary dangers. To date, most data regarding pulmonary immunity derive from animal models or BAL in humans. The results of BAL-cell differentials indicating lymphocytic, neutrophilic, eosinophilic, or a mixed cellular pattern have been associated with diseases such as sarcoidosis, occupational exposure, or eosinophilic pneumonia, respectively. In some cases, BAL may aid in the diagnosis of immune-mediated pulmonary disease, when accompanied by disease history and clinical and radiological ndings. Evaluation of the lymphocyte surface antigen phenotype may suggest sarcoidosis (increased proportion of CD4 lymphocytes) or extrinsic allergic alveolitis (increased proportion of CD8 lymphocytes). The BAL characterization of idiopathic pulmonary brosis (IPF) has been associated primarily with Th2 cytokines, while sarcoidosis or hypersensitivity pneumonitis are associated with a Th1 prole. High neutrophil counts in the BAL are characteristic of brosing processes or of occupational diseases caused by inhalation of inorganic dust.

T Cells in Asthma
T cells play a critical role in the pathogenesis of atopic asthma, a disease characterized by mucus hypersecretion, bronchoconstriction, edema, and a Th2-prominent immune response. CD4 T-helper cells respond to inhaled antigens from a variety of nonmicrobial sources. The cytokines secreted by CD4 T cells recruit a variety of other cell populations, either directly via cytokine receptors on the secondary effector cells, or indirectly, via IgE antibody. The variety of recruited cells includes B cells, neutrophils, mast cells, macrophages, platelets, and eosinophils. Recently, the neonatal period has been recognized as playing an important role in the determination of immune responses and the later onset of diseases like asthma. Focusing on the major population of T cells, those that express ab-chains of the TCR, neonatal populations typically contain CD4 /CD8 -positive cells. In neonatal immunity, 90% of CD4 T cells express CD45RA, representing the naive precursors of CD45RO cells. This proportion of CD45RO cells, loosely termed memory, steadily increases during childhood with exposure to environmental antigens. Some data suggest a predominance of specific cytokine patterns according to age. At birth, a Th2 cytokine pattern appears to dominate. During healthy childhood, Th1 cytokines increase. Since 1986, the Th1/Th2 paradigm, originally described in murine cells as an increase in Th2

(e.g., IL-4, IL-5, IL-13) and a decrease in Th1 (e.g., IFN-g) cytokine secretion, has dominated our understanding of asthma and allergic diseases. In asthmatic patients, this Th2 predominance may be modulated by factors not yet identied. An opposing paradigm, termed the hygiene hypothesis, suggests that exposure to a less hygienic environment is associated with a lower prevalence of allergic diseases. Exposure to different microbial substances, family size, older siblings, environment, and infections may account for differences in the development of allergen disease or severity. To date, the immunological mechanisms that protect against allergic responses are not well understood, but should take into account cell types and distinct pathways in addition to the Th1/Th2 paradigm. Thus, a spectrum of T cells including Th1 cells, CD4 T cells, different subsets of T regulatory cells (T regs) (e.g., Th3, Tr1 cells), and NK T cells may play a critical role in regulating allergy and asthma. CD4 CD25 cells (T regs), a minor fraction of CD4 T cells (10%), express the IL-2 Ra chain and are produced by the thymus as a functionally mature T-cell subpopulation. The CD4 CD25 T regs are characterized by markers such as the transcription factor Foxp3, glucocorticoid-induced TNF-receptor (GITR), lymphocyte activation gene (LAG)-3, and constitutive levels of CTLA4. T regs in the circulation, and possibly at the site of injury, may play an important role in the determination of disease or a healthy state by providing a balance between Th1 and Th2 cells, inducing tolerance, or other as yet undescribed mechanisms. Th3 cells produce transforming growth factor beta (TGF-b) as well as IL-4 and IL-10, and suppress the development of experimental autoimmune encephalomyelitis. Th3 suppressive effects in autoimmune models suggest that they may inhibit the effects of pathogenic autoantigenspecific T cells. Tr1 cells, induced with IL-10, can regulate antigen-specific immune responses, inhibiting the function of pathological autoantigen-specific T cells in vitro. NK T cells recognize self-glycolipid antigens in the respiratory tract after exogenous allergens enter the lung, followed by production of large quantities of cytokines. NK T cells produce IL-4 and IL-13, cytokines presumed to regulate the development of airway hyperresponsiveness (AHR) in allergic asthma. Whether NK T cells potentiate the development of asthma via cytokine production of IL-4 and IL-13 has yet to be determined. Therapies enhancing the development of regulatory T cells and inhibiting the function of NK T cells in patients with allergic disease offer exciting opportunities in the prevention or treatment of these disorders.

LEUKOCYTES / T Cells 561

T Cells in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is dened as chronic irreversible airow limitation, characterized by slowly progressive and irreversible deterioration in lung function, often induced by heavy smoking. T cells are increased in patients with COPD, showing a correlation between the number of T cells mm 3 of lung and the extent of emphysema. While both CD4 and CD8 T cells are increased in the airways and lung parenchyma, CD8 T cells predominate in studies to date. In addition to macrophages, CD8 T cells are the prevalent cell population in COPD. Increased CD8 T cells are also seen in healthy smokers. In contrast, there is a predominance of CD4 T cells in asthma. In exacerbations of COPD, bronchial biopsies demonstrated increased T-cell inltration; in addition, increased inltration of T cells is noted in the alveolar walls, small airways, and by the presence of lymphoid follicles that contain a core of B lymphocytes surrounded by T cells. The CD4 and CD8 T cells in COPD seem to be fully activated, showing a predominant Th2/cytotoxic T1 (Tc) pattern with increased IFN-g and Th1 chemokines. These data suggest that specific T-cell populations focused toward T-cell activation in COPD may be of importance.

T Cells in Tuberculosis, Sarcoidosis, and Wegeners Granulomatosis

In tuberculosis, the mycobacterium enters mainly by the respiratory route, and the alveolar macrophage is the important cell type combating the pathogen. However, in tuberculosis, within 1 week of infection, the number of activated CD4 and CD8 T cells in the lung-draining lymph nodes increases, followed by migration to the lung and to site of infection. In murine experiments, lack of CD4 cells results in delayed distribution of activated CD8 T cells from draining lymph nodes to distant organs and, consequently, delayed acquisition of immune protection. In humans, the tuberculosis granuloma contains both CD4 and CD8 T cells. Both CD4 and CD8 cells sustain infection within the granuloma and prevent reactivation. CD4 T cells are most important in the protective response to control the infection, with the primary effector function being IFN-g production and, possibly, other cytokines such as TNF-a, which are sufcient to activate macrophages. IFN-g is presumed to activate macrophages to combat intracellular infection via acidication of the phagosomal compartment housing the infection and production of nitric

oxide (NO), which is thought to have bactericidal effects. Also, apoptosis or lysis of infected cells by CD4 T cells may play a role in controlling infection. Whether Th1 or Th2 cytokines predominate in tuberculosis is not yet fully understood, but appears to depend on the site of location (peripheral blood or lung) and different stage or severity of disease. Primarily Th1 responses have been observed at the site of infection, and the strength of the Th1 type response is known to relate directly to the clinical manifestation of the disease. While the mechanism by which mycobacterial proteins gain access to the MHC I molecules is not fully understood, CD8 T cells may play a role in the balance of pulmonary Th1/Th2 cells. NK cells, as effector cells of innate immunity, may directly lyze the pathogens or infected monocytes. Decreased NK cell activity during tuberculosis infection may be the effect, rather than the cause, of the disease. In addition, apoptosis is a likely mechanism of NK cytotoxicity. In sarcoidosis, the salient pathological feature is the formation of noncaseating granulomas in more than one tissue system. Cytokines and chemokines produced by CD4 T cells, and mononuclear phagocytes guide the development and function of granuloma formation in the lung. A Th1 polarized immune response is demonstrated by increase in IFN-g and IL-2, as well as IL-12 and IL-18 (monocyte/macrophage-derived), providing a positive feedback loop, consistent with enhanced Th1 responses. IL-15, secreted by mononuclear cells among others, is involved in T-cell activation; TNFa and chemokine expression contribute to trafcking of immune cells to the inammatory site. Sarcoidosis patients with persistent inammation can develop pulmonary brosis or brosis can develop de novo, for example, idiopathic pulmonary brosis (IPF). As IPF is rare, human data are limited regarding cytokine proles to assess the contribution of Th1/Th2 distribution. From experimental models, Th2 cytokines are probrotic, enhancing broblast production of collagen. In contrast, the Th1 cytokine, IFN-g acts against brosis by downregulating broblast production of collagen and TGF-b expression, but can also be proinammatory. Indeed, a phase III trial administering IFN-g to IPF patients is ongoing in the US. Alternatively, IFN-g may play a probrotic role by enhancing tissue injury and subsequent repair processes, depending on timing of expression, and the presence of other proinammatory cytokines. Whether patients who develop pulmonary brosis convert to a more probrotic Th2 phenotype later in the disease or initially present with a Th2 dominant response is unknown. Another possibility

562 LEUKOCYTES / T Cells

is that brotic changes occur in a Th1 environment, or that other probrotic mediators such as TGF-b, insulin-like growth factor-1, and platelet-derived growth factor may contribute to a brotic outcome regardless of Th1/Th2 cytokine milieu. In Wegeners granulomatosis, an autoimmune disease of unknown etiology, a clinical triad consists of necrotizing granulomatous inammation of the upper/ lower respiratory tract, necrotizing glomerulonephritis, and an autoimmune systemic vasculitis affecting predominantly small vessels. Recent data suggest that TNF-a-producing Th1 type CD4 CD28 T-cell effector memory T cells may be involved in the pathogenesis of Wegeners granulomatosis. Their presence as a Th1-type cytokine population within granulomas provided the rationale for using TNF-a blocking agents in Wegeners disease refractory to standard induction therapy with cyclophosphamide and corticosteroids. The discovery of complex immunoregulatory networks to downregulate inammation, for example, CD4 CD25 T regs, suggests novel potential immune pathways that may be useful in the modulation of granulomatous inammation.

T Cells in Hypersensitivity Pneumonitis, Cystic Fibrosis, and Allergic Bronchopulmonary Aspergillosis

In other diseases with pulmonary brosis such as hypersensitivity pneumonitis, pneumoconiosis, or chronic beryllium disease, Th1 cytokines may also play a role. In hypersensitivity pneumonitis, both type III and type IV hypersensitivity reactions are mediated by immune complexes and Th1-type cells. Proinammatory cytokines and chemokines activate alveolar macrophages, which secrete IL-12, promoting the differentiation of CD4 Th0 cells into T cells of the Th1 phenotype. IFN-g production by Th1 cells further stimulates alveolar macrophages to produce greater amounts of IL-1 and TNF-a, resulting in a positive feedback loop with additional IFN-g production. Also, alveolar macrophages induce an inux of CD8 and, to a lesser degree, CD4 T cells into the lung, facilitating granuloma formation, and promoting the development of pulmonary brosis. In cystic brosis (CF), chronic pulmonary infection and destructive inammation are still the major causes of morbidity and mortality. A dysregulation of immune responses in CF includes antibody production by B cells, and perpetuation of the inammatory response by neutrophils. Overall, T-cell function may be of crucial importance for inammatory control. For example, IL-10, a major regulatory cytokine, produced not only by macrophages and NK cells but

also by Th1, Th2, and T regs is increased in CF. Furthermore, an imbalance of cytokines, for example, high IL-2 (proliferation) and low IL-8 production (activation of granulocytes, chemotaxis), may contribute to the immune dysregulation in CF. Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inammatory response to Aspergillus fumigatus antigens that affects approximately 10% of patients with CF. ABPA is characterized by markedly elevated Aspergillus-specific and total IgE levels and eosinophilia, and manifested by wheezing, pulmonary inltrates, bronchiectasis, and brosis, which afict asthmatic and CF patients. The pathogenesis of ABPA seems to be a prolonged asthmatic late-phase reaction orchestrated by CD4 Th2-like cells in response to persistent pulmonary A. fumigatus allergen exposure. Thus, polyclonal and A. fumigatus-specific IgE antibodies (and IgA and IgG) and blood pulmonary eosinophilia are stimulated by Th2-derived cytokines such as IL-4 and IL-5. In addition, IL-4 would also promote pulmonary transendothelial migration of eosinophils, basophils, and lymphocytes via induction of cell adhesion molecules and their ligands. In addition, A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial cell layer by damaging the epithelial integrity and by interacting directly with epithelial cell-surface receptors, producing proinammatory cytokines and corresponding inammatory responses.

Consideration of Present and Proposed Therapeutic Options

Corticosteroids, by general dampening of the immune function directed at T cells as well as other immune cells such as eosinophils, monocytes, and neutrophils, have been used as the mainstay of therapy for a number of lung diseases, including asthma, sarcoidosis, IPF, hypersensitivity pneumonitis, and even pleural tuberculosis. Corticosteroids exert their effects through binding to cytoplasmic glucocorticoid receptors, and modulate the transcription rate of inammatory factors such as transcription factors. They act as inhibitors of proinammatory cytokine production, also suppressing broblast proliferation and collagen deposition. However, the side effects of generalized immunosuppressive therapy can result in devastating side effects in addition to those of the disease itself, for example, osteoporosis, increased infection, diabetes, and cataracts. Strategies of directed therapy in asthma have included efforts at inhibiting events downstream of antigen presentation to T cells. Administration of anti-IL-5 to block

LEUKOCYTES / Pulmonary Macrophages 563

eosinophils has yielded only marginal effects, while anti-IgE treatment has been shown to be efcacious in some severe allergic asthmatics. Recently, suppression of TNF-a in severe asthma was shown to be efcacious in a small open study, but further examination is necessary. Use of a leukotriene receptor antagonist, which blocks the leukotriene pathway and has weak anti-inammatory activity, has had variable success in decreasing asthma symptoms or reducing the need for corticosteroids. These data and others suggest that the success of therapeutic interventions may depend not only on the selection of one specific target molecule, but potentially on a combination of several molecules and importantly also on the genetic background of the patient as well as environmental exposures, supporting the expanding eld of pharmacogenetics and epigenetics. Potential new therapeutic approaches include target cell populations such as T regs or NK cells. T regs may perform essential functions in modulating immune responses, including regulation of Th1/Th2 cytokine secretion. In murine models of autoimmunity, the adoptive transfer of T regs cures ongoing disease. Manipulating T regs or NK cells may be important not only in common diseases of unknown etiology such as asthma, but also for less common pulmonary entities such as sarcoidosis or granulomatosis. Prior to any strategies regarding therapeutic intervention, it is crucial to evaluate the multifaceted functions of both cell types, and, importantly, as they interact with each other and other cells, including APCs. Substantial research efforts are being directed at identifying mechanisms by which T cells and/or NK cells may contribute to disease pathogenesis or may become targets for preventive strategies.

Further Reading
Akbari O, Stock P, DeKruyff A, and Umetsu DT (2003) Role of regulatory T cells in allergy and asthma. Current Opinion in Immunology 15: 627633. Barnes PJ and Cosino M (2004) Characterization of T lymphocytes in chronic obstructive pulmonary disease. Public Library of Science Medicine 1: 1. Costabel U and Guzman J (2001) Bronchoalveolar lavage in interstitial lung disease. Current Opinion in Pulmonary Medicine 7: 255261. Di Stefano A (2004) Cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview. Clinical and Experimental Allergy 34: 11561167. Finkelstein R, Fraser RS, Ghezzo H, and Cosio MG (1995) Alveolar inammation and its relation to emphysema in smokers. American Journal of Respiratory and Critical Care Medicine 152(5 Pt 1): 16661672. Korsgren M (2002) NK cells and asthma. Current Pharmaceutical Design 8: 18711876. Lamprecht P and Gross WL (2004) Wegeners granulomatosis. Herz 29: 1. Mohr LC (2004) Hypersensitivity pneumonitis. Current Opinion in Pulmonary Medicine 10: 401411. Moller DR (2002) Pulmonary brosis of sarcoidosis. New approaches, old ideas. American Journal of Respiratory Cell and Molecular Biology 29: S37S40. Mosmann TR and Coffman RL (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology 7: 145173. Raja A (2004) Immunology of tuberculosis. Indian Journal of Medical Research 120: 213232. Sharma OP (2002) Sarcoidosis and other autoimmune disorders. Current Opinion in Pulmonary Medicine 8: 452456. Umetsu DT, et al. (2003) Regulatory T cells control the development of allergic disease and asthma. Journal of Allergy and Clinical Immunology 112(3): 480487. Woodland D (2003) Cell-mediated immunity to respiratory virus infections. Current Opinion in Immunology 15: 430435.

Pulmonary Macrophages
Conclusion
Recent discoveries in molecular immunology have advanced our understanding of the role of immune cells in the pathogenesis of pulmonary diseases. Immune cells such as T cells, and T cell subsets such as T regs and NK cells, play important roles in the activation of regulatory pathways in several lung diseases and, importantly, in host defense. T cells, as crucial regulators of the interplay between innate and adaptive immune responses, could provide promising targets for future therapeutic options in pulmonary diseases.
See also: Asthma: Overview. Dendritic Cells. Interleukins: IL-7. Leukocytes: Monocytes; Pulmonary Macrophages. Toll-Like Receptors. G S Davis and M E Poynter, University of Vermont, Burlington, VT, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Macrophages serve as the rst cellular line of defense against inhaled particulates and infectious agents. They are key participants in both innate non-antigen-specic immune responses and adaptive antigen-specic immune responses in the lung. They may play central roles in the transition from lung injury and inammation to brosis in both chronic airway and diffuse interstitial lung diseases. The macrophages of the lung arise from hematopoietic stem cells in the bone marrow. Several populations of macrophages reside in the normal healthy lung: alveolar, interstitial, airway and intravascular macrophages, dendritic cells, and monocytes. The pulmonary macrophage plays a central role in the homeostasis of the lung. Under normal

564 LEUKOCYTES / Pulmonary Macrophages

resting conditions it protects against the daily burden of inhaled particulates, microbes, and cellular debris without triggering an inammatory or immune response that could injure the lung. When challenged by virulent infectious agents, the macrophage is a rst line of defense, a sentinel that calls forth additional immune inammatory resources, and when activated it is an effective participant in microbial killing. Its potent degradative enzymes, inammatory cytokines, and mediators that signal broblast growth and collagen production appear to be key elements in a wide variety of environmental and idiopathic lung diseases.

Introduction
Macrophages serve as the rst cellular line of defense against inhaled particulates and infectious agents. They are key participants in both innate non-antigen-specic immune responses and adaptive antigen-specic immune responses in the lung. They may play central roles in the transition from lung injury and inammation to brosis in both chronic airway and diffuse interstitial lung diseases.

Macrophage Subpopulations in the Lung

Several populations of macrophages reside in the normal healthy lung (Table 1). The macrophages of the lung arise from hematopoietic stem cells in the bone marrow. Resident alveolar macrophages are large mobile phagocytes, loosely adherent to the epithelial surface. They appear to move about in the epithelial lining uid in order to encounter, engulf, sequester, and dispose off the particulates, microbes, and debris that are inhaled as part of normal life. It is postulated that the primary job of these cells is sanitation: cleansing the alveolus promptly to prevent injury to structural surfaces and limit extracellular replication of microbial agents without triggering an immune or inammatory response. In

many instances the phagocytosed material can be killed or degraded within the alveolar macrophage. Even if the material cannot be eliminated effectively, it can be isolated and sequestered while the cell and its contents are removed from the lung by the mucociliary escalator or while immune inammatory responses are mobilized. Macrophages also exist embedded in the airway wall and parenchymal tissue of normal subjects, albeit at lower numbers than in the alveolar spaces. These interstitial macrophages are thought to represent a self-renewing population of monocytes, some of which may go on to populate the alveolar compartment as alveolar macrophages, as well as to serve functions as innate sentinels. Within the lungs of large animals (possibly including humans) but not of rodents, a population of macrophages, termed pulmonary intravascular macrophages (PIMs), exist adhered to the endothelial cells comprising the pulmonary capillaries. The PIMs are phagocytic, capable of ingesting bacteria and producing inammatory mediators. Dendritic cells (DCs) share a number of qualities with monocytes and macrophages, and are interspersed throughout the lung tissues. DCs, in their immature form, are especially procient at engulfing antigenic materials and processing antigenic proteins into smaller peptide fragments. Ingestion of bacterial products and/or exogenous cytokines stimulates activation, mobilization, and maturation of DC. Activated DC upregulates CCR7, a chemotactic receptor that induces the DC to travel to lymphoid aggregates or regional lymph nodes where large numbers of T cells and B cells are located. Upon maturation, the DC may present the antigenic peptide on the cell surface in the context of major histocompatibility complex (MHC) class II molecules. DCs are considered to be professional antigen-presenting cells

Table 1 Populations of macrophages in the lung Name Location in the lung Features Antimicrobial activity Excellent Good (better when activated) Good (better when activated) Good (better when activated) Poor Good (better when activated) Antigen presenting function Very poor Probably poor Probably poor Probably poor Excellent, primary role Poor Inammatory recruiting function Excellent recruitment Activation of broblasts Probably good Probably good Poor Good

Alveolar macrophage Interstitial macrophage Vascular macrophage Airway macrophage Dendritic cell Monocyte

Alveolar surface Alveolar interstitium Capillaries and venules Airway wall, submucosa Interstitium, lymphoid tissues Recruited from blood stream

Large complex cell, many inclusions Small cell, some inclusions Small cell, some inclusions Small cell, some inclusions Large cell, long processes Small cell, few inclusions

LEUKOCYTES / Pulmonary Macrophages 565

(APCs). With activation, the DC also upregulates cell-surface molecules that act as co-receptors in Tcell activation and greatly enhance the ability to activate T cells during antigen presentation. DCs are named for their characteristic appearance in tissues, wherein they extend cellular processes away from the main cell body perhaps to sample antigenic material at more remote sites. Several subclasses of DC have been described, including plasmacytoid and myeloid dendritic cells. In general, the plasmacytoid DCs (PDCs) elicit suppressive effects, while the myeloid DC (MDC) subset elicits immunostimulatory effects. The MDC subset shares many characteristics with the monocyte/macrophage population, and is likely to be derived from them. Monocytes are large mononuclear phagocytes of the peripheral blood, ranging in size from 10 to 30 mm in diameter with band- or kidney-shaped nuclei. Blood monocytes possess limited capabilities as phagocytes or as secretors of enzymes and cytokines. Instead, their main function may be to act as precursors for tissue macrophages. Blood monocytes form an important component of the lung macrophage population, both as precursors to mature macrophages and as recruited inammatory leukocytes. Monocytes from the blood enter tissues and increase in size, phagocytic activity, and lysosomal enzyme content and become macrophages. In addition to circulating blood monocytes, there appear to be local replicating populations of mononuclear cells within the parenchyma and lymphoid aggregates of the lung that may serve as precursors for the resident populations of pulmonary macrophages. Blood monocytes are recruited into the lung rapidly and abundantly by a variety of inammatory or defense-related signals. The number of macrophage/monocyte cells is greatly increased above resident levels in virtually all infectious or inammatory lung diseases, as evidenced by recovery from enzyme digestates or bronchoalveolar lavage (BAL) samples. Thus, lung inammatory cell populations contain a mixture of mature, activated macrophages and monocytes in transition.

Figure 1 Human alveolar macrophage cell surface features. Scanning electron micrograph of a human alveolar macrophage recovered by bronchoalveloar lavage and placed in cell culture. The cell is adherent and spreading on the plastic surface by extending thin cytoplasmic psuedopods. The complex folded and rufed surface of the cell is prominent. Courtesy of A R Brody, PhD, Department of Pathology, Tulane University, New Orleans, LA, USA.

Structure and Function

The most detailed information about macrophages in the lung pertains to the alveolar macrophage because this cell can be obtained in large numbers and relatively high purity by BAL from normal human subjects, from patients with lung diseases, and from laboratory animals. The alveolar macrophage is a large pleomorphic cell, spherical in suspension but spread into a fried egg shape on the alveolar surface or in cell culture (Figure 1). A highly rufed cell membrane with numerous folds, pseudopods, and

projections offers a very large surface area for the cell volume, and provides maximum contact with materials in its environment. The alveolar macrophage is mobile and can move across surfaces and through the pores of experimental membranes, although it moves more slowly and less readily than a blood monocyte. The shape of the folded surface is conferred by the extent of polymerization of cytoplasmic actin laments, and changes substantially with activation. The intracellular anatomy of the alveolar macrophage is also complex (Figure 2). The nucleus is typically crescent- or kidney-shaped and is placed eccentrically within the cell. Nuclear chromatin is clumped at its periphery. There is an abundant Golgi apparatus and plentiful mitochondria and endoplasmic reticulum, perhaps reecting the active role of the alveolar macrophage in producing enzymes, cytokines, and oxidants. There are numerous lysosomal granules that contain cathepsins, proteolytic enzymes, collagenases, and other enzymes. These chemicals may be excreted into the environment near the cell by exocytosis, or utilized for intracellular action by the merger of lysosomes with phagosomes containing engulfed materials. The alveolar macrophage cytoplasm usually features large and small phagolysosomes that represent engulfed debris in various stages of digestion or detoxication. Living or dead bacteria, inhaled particles and bers, and

566 LEUKOCYTES / Pulmonary Macrophages

N PL

Figure 2 Human alveolar macrophage intracellular structure. Transmission electron micrograph of a human alveolar macrophage recovered by bronchoalveolar lavage shows a complex ultrastructure. The eccentrically placed kidney-shaped nucleus (N) with peripheral chromatin, numerous cell surface folds, and abundant intracellular inclusions are typical of this cell type. Lysosomes (L), mitochondria (M), and phago-lysosomes (PL) containing ingested debris can be identied. Courtesy of A R Brody, PhD, Department of Pathology, Tulane University, New Orleans, LA, USA.

debris from nearby dead cells can be identied. Most alveolar macrophages also contain distinctive whorled inclusions (lamellar bodies) that represent phagocytosed surfactant lipids and proteins. The alveolar macrophage is believed to be an important mechanism for disposing of (or possibly for recycling) pulmonary surfactant. The inclusion of the alveolar macrophage is a marker of the history of the alveolus. Human subjects or experimental animals exposed to environmental dusts and bers carry these materials within the alveolar macrophage for months or years after exposure. Bacteria within alveolar macrophages in sputum samples are taken as proof of pneumonia, an active infection in the alveolar spaces. Hemorrhage is reected as erythrocytes in alveolar macrophages within minutes after alveolar bleeding, and hemosiderin pigment from hemoglobin digestion appears in alveolar macrophages about 48 h after bleeding and may last for several weeks. Retired granite workers show numerous silica particles in their alveolar macrophages recovered by BAL more than 20 years after leaving the work place.
Metabolic Functions

Enzymes Macrophages produce many enzymes that appear designed primarily to act within the phagolysosome to create an acidic environment, inhibit microbial growth, kill susceptible microorganisms, and degrade organic debris. Most of these hydrolytic enzymes are upregulated by the act of phagocytosis. If they leak into the extracellular milieu they may be irritating and destructive. The matrix metalloproteinases can degrade structural proteins such as elastin and collagen. While normal tissue remodeling depends on their action, lung disease can result from excess degradation (e.g., emphysema) or inhibited activity (e.g., pulmonary brosis). Reactive oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide appear to be essential agents for killing microbes within the phagolysosome. Accordingly, an oxidative burst of ROS generation is a central feature of macrophage activation following ingestion of microbes. Humans and experimental animals that lack the enzymes responsible for generating these oxidant materials have impaired pulmonary host defenses and develop pyogenic bacterial infections. If these products are released extracellularly they may have the potential to kill target cells or damage proteins. Lipid mediators Macrophages produce lipid mediators derived from cell membrane arachidonic acid. The lipid mediator linked most securely to pulmonary macrophages is leukotriene B4, an important mechanism for attracting and activating neutrophils. The cysteinyl leukotriene C4 (and its metabolites D4 and E4) is an important mediator in asthma and bronchoconstriction, acting through the CysLT1 receptor. Mast cells and eosinophils appear to be the major source, but pulmonary macrophages may contribute as well. New attention focuses on probrotic activities mediated through the CysLT2 receptor, and could be another role for macrophages in chronic interstitial lung diseases. Cytokines Pulmonary macrophages are a very important source for the cytokines that drive acute and chronic lung diseases. The synthesis and secretion of cytokines by macrophages probably does not occur substantially under basal conditions, although BAL alveolar macrophages from humans and animals do produce small quantities of many cytokines when placed in resting or unstimulated cell culture. Activation of macrophages by mediators or direct cell contact triggers an intense, selective, and specic upregulation of families of cytokines with dened functions. Although gene expression of many of these proteins is mediated through nuclear factor kappa B (NF-kB), the panoply of surface receptors carried by

The metabolic functions and products of the macrophage reect its many roles in lung homeostasis and disease. Most of these materials have many different activities, targets, and interactions with other mediators depending on the exact location and circumstances. Table 2 provides an overview, not a comprehensive summary.

LEUKOCYTES / Pulmonary Macrophages 567

Table 2 Macrophage products and their functions Macrophage product Enzymes Cathepsin B Lysozyme Acid hydrolases Angiotensin convertase Matrix metalloproteinases (MMPs) Collagenase Elastase Peroxidase Superoxide dismutase Tissue inhibitors of metalloproteinases (TIMPs) Oxidant products Superoxide (Od 2 ) Hydrogen peroxide (H2O2) Nitric oxide (NO) Hydroxyl radical (dOH) Peroxynitrite (ONOO ) Lipid mediators Leukotriene B4 (LTB4) Leukotriene C4 (LTB4) Platelet activating factor (PAF) Prostaglandin E2 (PGE2) Cytokines Tumor necrosis factor alpha (TNF-a) Interleukin-1-beta (IL-1b) Interleukin-6 (IL-6) Interleukin-12 (IL-12) Interleukin-15 (IL-15) Interleukin-18 Fibroblast growth factor Transforming growth factor beta (TGF-b1) Chemokines Interleukin-8 (IL-8; CXCL8) Macrophage chemotactic protein 1 (MCP-1; CCL2) Interferon gamma inducible protein 10 (IP-10; CXCL10) RANTES (CCL5, MCP-2) Macrophage inammatory protein 1 (MIP-1a; CCL3) Function Antimicrobial (intracellular) Antimicrobial (intracellular) Degradation of debris (intracellular) Activate angiotensin Degrade connective matrix proteins (extracellular) Degrade collagen (extracellular) Degrade elastin (extracellular) Generate hydrogen peroxide Convert superoxide to H2O2 and H2O Inhibit matrix metalloproteinases Antimicrobial Antimicrobial Antimicrobial Antimicrobial Antimicrobial (intracellular; (intracellular; (intracellular; (intracellular; (intracellular; ? ? ? ? ? extracellular extracellular extracellular extracellular extracellular injury?) injury?) injury?) injury?) injury?)

Attract/activate neutrophils Bronchoconstriction Vascular tone, bronchoconstriction Anti-inammatory

Lymphocyte activation, cachexia, death Lymphocyte activation Proinammatory reactant TH1 T-cell activation TH1 T-cell and natural killer (NK) cell activation TH1 T-cell activation Promote broblast growth Fibroblast collagen synthesis, inhibitor of macrophages Attract/activate neutrophils Attract/activate macrophages, TH1 T cells, NK cells Attract/activate TH1 T cells, NK cells Attract monocytes Attract monocytes, macrophages

the macrophage (see below) allows focused responses to many distinct ligands. Acute reactants such as tumor necrosis factor alpha (TNF-a), interleukin-1beta (IL-1b), and interleukin-6 (IL-6) are generated by many stimuli including bacteria (endotoxin), silica, or asbestos. The elaboration of IL-12 and IL-15 by macrophages appears to be essential for effective activation of T cells and natural killer (NK) cells in the innate immune response, and an essential step in converting to an antigen-specic adaptive immune response. Mice that lack either of these cytokines are highly susceptible to tuberculosis and other intracellular pathogens. Macrophages are believed to be a primary source for the cytokines that promote broblast proliferation and increased collagen production in pulmonary brosis. Unlike macrophages in other

tissues, alveolar macrophages play an important role in the modulation of inammatory and immune responses through the constitutive secretion of antiinammatory cytokines, especially IL-10 and transforming growth factor beta (TGF-b). These cytokines function to retain the lung in a relatively silent state under normal conditions, which allows the resident phagocytes to deal inconspicuously with foreign inhaled material without the need to elicit inammatory or adaptive immune responses. Chemokines When needed, attracting and activating other leukocytes for pulmonary inammation and immune responses is a vital function performed by macrophages through the production of chemokines. These chemoattractant proteins are structurally

568 LEUKOCYTES / Pulmonary Macrophages

related and signal through a family of G-protein-coupled membrane receptors. The most common chemokines are divided into CC and CXC families based on amino acid structure. There is substantial redundancy in the secretion, activities, and receptor expression related to these polypeptides, but chemokines and their cognate receptors do exhibit substantial specicity that allows selective recruitment of neutrophils, eosinophils, TH-1 or TH-2 T cells, or NK cells.
Cell Surface Receptors and Ligands

The cell surface molecules displayed by the pulmonary macrophage are its interface with the particles, microbes, signal peptides, and other cell types that surround it. Some of the more important surface molecules are listed in Table 3. Macrophages recognize material destined for phagocytosis through a myriad of cell surface receptors. The scavenger receptors are not specic for individual ligands, but can bind dust particles, bers, and debris. Mannosyl
Table 3 Cell surface molecules on pulmonary macrophages Cell surface receptors and ligands Phagocytic receptors Scavenger receptors Toll-like receptors Mannosyl receptors Immunoglobulin Fc receptors (IgG, IgM, IgA) Complement receptors (C3, C5) Cytokine receptors Tumor necrosis factor R1 and R2 Interferon gamma R1 and R2 (CDw119) Interleukin-1 receptor (IL-IR CD121b) Interleukin-4 receptor (IL-4R) Chemokine receptors CXCR1 (CD128a) CXCR2 (CD128b) CXCR3 (CD183) CXCR4 (CD184) CCR5 (CD195)

receptors bind glycoconjugates common on fungal and bacterial cell walls. Toll-like receptors (TLRs) facilitate interactions with microbes, including Gram-positive and Gram-negative bacteria, fungi, parasites, and viruses. Signaling through TLRs (particularly TLR4) is believed to be an important route for macrophage activation. Receptors for the Fc portion of immunoglobulins mediate phagocytosis of microbes (or other particulates) that have been coated with antibody, a more rapid and efcient ingestion than through other receptors. An important macrophage phagocytic function is clearance of apoptotic cells from the lungs. This occurs under normal conditions of homeostasis, as well as during resolution of inammation. The ingestion of apoptotic bodies, necrotic cells, and their debris does not elicit an inammatory response on the part of the macrophages. Receptors for cytokines and chemokines allow the macrophage to respond to endocrine, paracrine, and autocrine cell signals, to become activated, to migrate to sites of inammation, and to participate in

Function Bind Bind Bind Bind Bind particles, bers, debris bacteria, organic materials fungal cell wall elements antibody-coated particulates activated complement components

Bind TNF-a/TNF-b IFNGR1 binds IFN-g, activates macrophage Low levels, autocrine effects Binds IL-4, may downregulate macrophage function Binds Binds Binds Binds Binds IL-8 (CXCL8), chemoattractant signal IL-8 (CXCL8), chemoattractant signal IP-10 (CXCL10), ITAC CXCL4, chemoattractant signal MIP1a (CCL3), MIP-1b, RANTES (CCL5)

Adhesion molecules Intercellular adhesion molecule-1, 2, 3 (ICAM-1, 2, 3 CD54, 102, 50) Sialoadhesin receptor (CD169) Integrin aM (Mac-1a CD11b) PECAM-1 (CD31) L-Selectin (CD62L) Immune regulatory molecules MHC Class II MHC Class I CD4 Other epitopes Lipopolysaccharide receptor (endotoxin; CD14) Macrosialin (CD68) Fas (CD95; APO-1)

Intercellular adhesion Migration, neutrophil interactions Intercellular adhesion Intercellular adhesion Intercellular adhesion Present antigen peptides to CD4 T cells, B cells Confer cell type recognition, present antigen to CD8 cells Antigen presentation costimulatory molecule Signaling results in broad activation Binds oxidized lipoproteins (surfactants) Signaling may trigger apoptosis

LEUKOCYTES / Pulmonary Macrophages 569

immune responses. The surface repertoire of the monocyte evolves to that of a mature and/or activated tissue macrophage after recruitment into the lung. Adhesion molecules, sialoadhesin receptors, and integrins attach the macrophage to lung surfaces and facilitate close contact with other cells for signaling. A group of immune regulatory molecules allow macrophages, particularly DCs, to present processed antigen peptides to T lymphocytes and B cells in an appropriate context so as to trigger clonal expansion or antibody production. Endotoxin, a cell wall lipopolysaccharide (LPS) from Gram-negative bacteria, is one of the most potent and well-studied signals that trigger a stepchange in macrophage function. LPS in serum (or possibly in alveolar epithelial lining uid) is joined to a specic binding protein. This LPS-binding protein complex is recognized by the macrophage surface receptor CD14. Signal transduction via TLR4 results in prompt, strong cell activation and the new or increased production of TNF-a, IL-1b, and other cytokines. Macrophages carry abundant Fas (CD95) molecules, making them susceptible to death signaling for apoptosis upon encountering cells bearing Fas ligand.
Life History of the Pulmonary Macrophage

experiments utilizing colored latex particles show that alveolar macrophages from all parts of the lung migrate to mediastinal lymph nodes. Inhaled silica particles follow a similar path in both humans and experimental animals. This appears to be an important route for antigen processing and adaptive immunity, as well as a mechanism for sequestering durable particulates. The kinetics of macrophage turnover in the lung probably varies greatly between normal health and disease. Alveolar macrophages appear to be longlived cells under normal circumstances. Early observations used BAL to sample airspace cells from patients who had received bone marrow transplantation from a donor of the opposite sex, and to track the appearance or disappearance of the Y chromosome in their alveolar macrophages. At least 6 months was required for near-complete conversion from host to donor phenotype. With active infection, an intense immune response, or brisk inammation there may be large numbers of monocytes recruited to the lung and many macrophages that die or leave. Thus, some macrophages may serve a brief lifespan of several days, while others may live for months or perhaps longer.

Macrophages in the lung arise ultimately from hematopoietic stem cell precursors in the bone marrow. Monoblasts become promonocytes and then monocytes, enter the blood stream, circulate for up to 40 h, and then adhere to pulmonary vascular endothelium, migrate into the interstitium, and nally take up residence in the alveolus or at other sites as listed above. During this process of differentiation and maturation their metabolic functions and surface molecule repertoire evolve from monocyte to tissue macrophage. It is likely that there are also local replicating populations of precursors to macrophages and DCs within the lung. The alveolar macrophage serves its life primarily on the epithelial surface. The end of life may occur by death or by exit from the alveolus. Death may be by necrosis from virulent microbial replication or ingestion of toxic particles, or by apoptosis from Fas signaling or other means. Macrophages may leave the alveolus via the airway mucociliary escalator, carrying away potentially injurious materials before they can harm more delicate tissues, and viable macrophages are found in expectorated sputum. Macrophages may also leave the alveolus by traversing the epithelium, entering the lymphatics, and migrating to bronchial-associated lymphoid tissue (BALT) aggregates and to regional lymph nodes. Animal

The Role of the Macrophage in Pulmonary Infection

The respiratory system is protected from inhaled infection by a complex network of physical barriers such as ltration of air by inertial impaction and removal of particulates by the mucociliary stream. It is also protected by molecular mechanisms such as surfactant, defensins, complement proteins, and immunoglobulins in the epithelial lining uid. When microbes evade these mechanisms, the resident alveolar macrophages are the next line of defense. When these resident defenses are insufcient and microbes multiply then other non-specic innate immune defenses are called forth. If these are still not sufcient, and the host survives, the most powerful specic adaptive immune responses appear in the lung. Airborne and oropharyngeal bacteria are inhaled frequently. The alveolar macrophages can effectively recognize and adhere to bacteria utilizing scavenger, Toll-like, and mannose receptors, or immunoglobulin Fc receptors if the bacterium has been coated with antibody. Binding of these receptors usually triggers prompt phagocytosis. The alveolar macrophages are capable of killing most inhaled bacteria, including virulent strains of staphylococci and Gram-negative organisms. When the bacterial load is high or macrophage function is impaired, bacteria can grow

570 LEUKOCYTES / Pulmonary Macrophages

extracellularly in the epithelial lining uid and may replicate faster than they can be removed. While small numbers of bacteria or nonvirulent bacteria may be eliminated without stimulating an inammatory response, larger numbers of organisms, bacteria that produce endotoxin, or virulent multiplying bacteria will trigger inammation. Resident alveolar macrophages are stimulated to secrete chemokines that attract and activate other leukocytes, particularly neutrophils (see Table 2). Cytokines, lipid mediators, and ROS cause increased permeability, alveolar edema, and functional changes in epithelial cells. Disruption of macrophages by growing bacteria releases lysosomal enzymes that can further damage the alveolar wall. Pneumonia is the result of this expanding alveolar infection, injury, edema, and inammation. Some pathogens, such as Legionella species, corrupt the activities of alveolar macrophages, creating within the cells a niche for bacterial replication. These types of organisms are considered obligatory or facultative intracellular pathogens. Having entered the alveolar macrophage by coiling phagocytosis mediated through the complement receptor, Legionella pneumophila induces a blockade of phagosome fusion with the lysosome. The bacteria replicate within the phagosome and then the cytoplasm, reaching a burden of up to 10 000 bacteria per cell within 48 h. Ultimately, death and lysis of alveolar macrophages release these bacteria to infect other nearby cells. When the adaptive cellular immune responses come into play, interferon gamma (IFN-g) produced by TH1-like lymphocytes activates the alveolar macrophages and decreases the rate of intracellular bacterial replication. Humoral immune responses provide IgM and then IgG to assist phagocytosis, activate complement, and assist with killing. Mycobacterium tuberculosis (TB) enters the alveolar macrophages via mannose and complement receptors. It survives in the phagocytes by interfering with fusion of the phagosome with the acidic lysosome. Infected macrophages selectively undergo apoptosis (which engenders less inammation than necrosis) and the infected apoptotic bodies are subsequently ingested and by other macrophages. The effective clearance of TB requires the generation of a potent cell-mediated adaptive immune response in order to activate macrophages adequately to kill these bacteria. Most healthy human hosts are able to mount a successful initial immune response and kill or at least sequester TB bacteria without growth. Small numbers of these hardy organisms may survive for decades within macrophages in apparently healed lesions, only to break out again and cause active infection when the defenses of the host become impaired. Similar processes, albeit without long-term

sequestration, also occur during infection with most fungi and protozoan parasites.

Tobacco Smoke and the Macrophage

Tobacco smokers have a dramatic increase in the number of alveolar macrophages in their lungs, and show accompanying changes in the morphology and function of these cells. A 240 ml BAL sample from a normal nonsmoker yields approximately 20 million alveolar macrophages, while 80100 million are recovered from normal current smokers. Smokers have a significantly increased number of alveolar macrophages per unit lung volume and per unit surface area, though the relative increase is less than from BAL. The BAL uid from smokers contains more chemotactic activity for monocytes, apparently in keeping with enhanced recruitment to the airspaces. The smokers alveolar macrophages are generally larger, show a golden-brown color, and contain more cytoplasmic inclusions, some of which are small kaolinite mineral particles derived from eld dust or from clay added to tobacco in processing. Smokers alveolar macrophages exhibit a slightly higher basal oxidant production, but less capacity to increase superoxide production in response to endotoxin LPS. The smokers cells may also have a reduced ability to phagocytose particles and kill bacteria in vitro, implying an explanation for increased susceptibility to respiratory infection. Smokers macrophages stimulated with LPS secrete less IL-1 and other cytokines than nonsmokers cells. Smokers alveolar macrophages produce more elastin-degrading capacity and more matrix metalloproteinase activity than nonsmokers alveolar macrophages. When combined with the greatly increased cell number, the enhanced production of matrix-degrading enzymes on a per cell basis implies a very large increase in an elastolytic burden in the lungs of smokers. One hallmark of chronic obstructive pulmonary disease (COPD) is the accumulation of macrophages and neutrophils around small airways (respiratory bronchiolitis) and the subsequent development of centrilobular emphysema at these sites. Thus the pulmonary macrophage, recruited and modied by the effects of tobacco smoke, appears to play a central role in the pathogenesis of COPD and emphysema.

The Macrophage in Fibrotic and Inammatory Lung Diseases

Many chronic lung diseases feature accumulations of inammatory cells, proliferating broblasts, epithelial

LEUKOCYTES / Pulmonary Macrophages 571

cells, and connective tissue matrix in distinctive pathological patterns. Macrophages are part of this mixture in most conditions, and in many are seen as essential elements orchestrating inammation and brosis. Macrophage function has been studied extensively in sarcoidosis, idiopathic pulmonary brosis, asbestosis, silicosis, and other diseases. Sarcoidosis will serve as an example.
Sarcoidosis

The cardinal feature of sarcoidosis in all organs is the noncaseating granuloma, a spherical collection of lymphocytes, monocytes, and macrophages surrounding occasional large multinucleated giant cells. These lesions appear in lymph nodes with tremendous enlargement, and in the lung and other organs with resulting dysfunction. Although the inciting agent or antigen is not known, sarcoidosis appears to be a TH1-like chronic cell-mediated immune response. CD4 T cells predominate in sarcoid tissues and are greatly increased in BAL samples, both in number and proportion. Local and systemic levels of TNF-a and IFN-g are high, and are believed to be pathogenic in driving the inammatory response. BAL macrophages from patients with sarcoidosis have served as a source for many studies, and upregulation of a variety of functions has been observed. In most instances the changes were compartmentalized to the lung, and peripheral blood monocytes did not show similarly altered activities. Cytokines and other mediators are produced by macrophages in sarcoidosis. Alveolar macrophages from sarcoidosis patients show greatly enhanced mRNA expression, basal secretion, and stimulated production of TNF-a, believed to be a primary cytokine driving the inammatory process and granuloma formation in this disease. Levels of potential antagonists and soluble TNF receptors 1 and 2 are also increased. The production of other cytokines such as IL-1b, IL-6, platelet-derived growth factor beta (PDGF-b), and granulocyte-macrophage colonystimulating factor (GM-CSF) is also increased. ROS (superoxide, hydrogen peroxide) are generated in elevated amounts by alveolar macrophages from patients with active sarcoidosis compared to normal subjects or patients with inactive disease. Sarcoidosis macrophages attract lymphocytes and monocytes to evolving granulomas. Elevated levels of macrophage inammatory protein 1a (MlP-1a), a CD4 lymphocyte chemoattractant, and IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells and NK cells, appear in the BAL uid of patients with pulmonary sarcoidosis, and production of these

mediators is localized to macrophages. Stimulated alveolar macrophages from patients with active sarcoidosis release higher levels of the chemoattractant leukotriene B4 than those from normal subjects. Sarcoidosis macrophages appear to activate T cells. Alveolar macrophages from patients with active sarcoidosis produce IL-15 (while those from healthy subjects and patients with inactive sarcoidosis do not), and tissue staining localizes IL-15 mRNA and protein to alveolar macrophages. Thus alveolar macrophages may trigger the growth of sarcoid T cells through IL-2 receptor signaling, and could deliver proliferative signals for the development of the T-cell alveolitis. Alveolar macrophages that produce IL-18, an accessory cytokine with IL-12 and IL-15 that drives T cells toward a TH1-like phenotype, can be found within the boundary zone of sarcoid granulomas. Although normal alveolar macrophages do not bear costimulatory molecules, those from sarcoid patients carry CD80 and CD86 (members of the B7 family) and the CD5 coligand CD72, and thus may have enhanced lymphocyte activation and/or enhanced antigen presentation abilities. Sarcoidosis macrophages also exhibit increased abundance of cell adhesion molecules. The percentages of macrophages expressing the aM integrin CD11b (Mac1, CR3) and the adhesion molecule CD54 (ICAM-1) are significantly increased in patients with active sarcoidosis compared with patients with inactive disease or control subjects. These changes may, in part, reect the inux of less mature and more monocyte-like cells. Pulmonary macrophages in active sarcoidosis demonstrate an activated inammatory phenotype that accompanies, and may drive, a TH1-like activated T-cell response.

Conclusions
The pulmonary macrophage plays a central role in the homeostasis of the lung. Under normal resting conditions it protects against the daily burden of inhaled particulates, microbes, and cellular debris without triggering an inammatory or immune response that could injure the lung. When challenged by virulent infectious agents, the macrophage is a rst line of defense, a sentinel that calls forth additional immune-inammatory resources, and when activated an effective participant in microbial killing. Its potent degradative enzymes, inammatory cytokines, and mediators that signal broblast growth and collagen production appear to be key elements in a wide variety of environmental and idiopathic lung diseases. A better understanding of this key cell, and more selective ways to modify its functions, will be important directions for lung research.

572 LIPID MEDIATORS / Overview See also: Bronchoalveolar Lavage. Dendritic Cells. Interleukins: IL-6; IL-10; IL-12; IL-15. Leukocytes: Monocytes. Systemic Disease: Sarcoidosis. Toll-Like Receptors. Transcription Factors: NF-kB and Ikb.
Lohmann-Matthes ML, Steinmuller C, and Franke-Ullmann G (1994) Pulmonary macrophages. European Respiratory Journal 7: 16781689. Reynolds HY (2005) Lung inammation and brosis: an alveolar macrophage-centered perspective from the 1970s to 1980s. American Journal of Respiratory and Critical Care Medicine 171(2): 98102. Schneeberger EE and Suda T (1997) Ontogeny and heterogeneity of lung dendritic cells. In: Lipscom MF and Russell SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 403469. New York: Dekker. Warner AE (1996) Pulmonary intravascular macrophages: role in acute lung injury. Clinics in Chest Medicine 17: 125135. Wesselius LJ, Murphy WJ, Morrison DC, and Russell SW (1997) Macrophage activation: concepts and principles applicable to the lung. In: Lipscom MF and Russel SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 3386. New York: Dekker. Zhang P, Summmer WR, Bagby GJ, and Nelson S (2000) Innate immunity and pulmonary host defense. Immunological Reviews 173: 3951.

Further Reading
Barnes PJ (2003) New concepts in chronic obstructive pulmonary disease. Annual Review of Medicine 4: 113129. Delclaux C and Azoulay E (2003) Inammatory response to infectious pulmonary injury. European Respiratory Journal 42(supplement): 10s14s. Gal AA and Koss MN (2002) The pathology of scarcoidosis. Current Opinion in Pulmonary Medicine 8(5): 445451. Gordon SB and Hughes D (1997) Macrophages and their origins: heterogeneity in relation to tissue microenvironment. In: Lipscomb MF and Russell SW (eds.) Lung Macrophages and Dendritic Cells in Health and Disease, 1st edn., pp. 331. New York: Dekker. Gordon SB and Read RC (2002) Macrophage defences against respiratory tract infections. British Medical Bulletin 61: 4561.

LIPID MEDIATORS
Contents

Overview Leukotrienes Prostanoids Platelet-Activating Factors Lipoxins

Overview
M Peters-Golden, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

overproduction of pathogenic lipids as well as the underproduction of protective lipids. A variety of pharmacologic agents that block either the synthesis of, or the receptors for, lipid mediators are currently employed in the clinic, and additional applications for such therapy are on the horizon.

Abstract
Lipid mediators comprise a family of enzymatically generated phospholipid hydrolysis products that act via receptors to elicit a wide spectrum of biological responses. The best known of these are platelet-activating factor and the eicosanoids, oxygenated metabolites that include prostanoids, leukotrienes, and lipoxins. These molecules are synthesized and secreted within minutes of agonist stimulation by virtually all cell types of bone marrow and lung origin, and exert autocrine and paracrine effects that inuence almost every conceivable category of biological response. Lipid mediator synthesis is modulated by, and mediates many of the actions of, polypeptides such as hormones and cytokines. By virtue of their diverse actions, lipid mediators are implicated in a variety of lung diseases, including asthma, interstitial lung disease, pulmonary vascular disease, and acute lung injury. They are also increasingly recognized to contribute to homeostatic or protective responses, such as innate immunity. Precedent exists for disease states to reect both the relative

Introduction
The term lipid mediators generally refers to enzymatically generated derivatives of membrane phospholipids that are secreted from the source cell and act at nanomolar concentrations on target cells, most classically via G-protein-coupled receptors (GPCRs). These substances include platelet-activating factor (PAF) as well as oxygenated metabolites of the 20-carbon fatty acid arachidonic acid (AA), which are collectively known as eicosanoids (eicosa Greek for twenty). The best-studied eicosanoids include prostanoids (prostaglandins (PGs) and thromboxane), leukotrienes (LTs), and lipoxins. Although originally recognized for their capacity to elicit biological responses such as smooth muscle

LIPID MEDIATORS / Overview 573

contraction, edema, and platelet aggregation, lipid mediators are now known to inuence processes ranging from inammation and immune responses to chronic tissue remodeling and cancer. Indeed, there is scarcely an area of biology or medicine in which these molecules have not been implicated. Not surprisingly, the lung is an important source of and target for lipid mediators. A variety of pharmacotherapeutic strategies based on lipid mediators are in current use and others are on the horizon. The generation and lung biology of PGs, LTs, lipoxins, and PAF are specifically addressed elsewhere in this encyclopedia. This article provides an historical perspective on lipid mediators and an overview of their synthesis, receptors, signaling, actions, and roles in respiratory diseases. As lipid mediators are likely to be less familiar to many readers than peptide mediators such as cytokines, it is particularly instructive to compare and contrast these two classes of mediators. Finally, the interplay between lipid and peptide mediators is discussed.

PAF. During the last 20 years, the tools of modern cell and molecular biology, biochemistry, genetics, transgenic animal modeling, and clinical medicine have provided new insights into the generation and actions of these lipid mediators.

Generation of Lipid Mediators

Biosynthetic Pathways

Brief History of Lipid Mediators

Although not recognized as such at the time, research into what we now term lipid mediators dates back to two disparate lines of investigation that commenced in the 1930s. First, Von Euler described a smooth muscle contractile bioactivity found in seminal vesicle and prostate, which he termed prostaglandin. A few years later, Feldberg and Kellaway described a bioactivity released by cobra venom-challenged guinea pig lung that caused a very potent and protracted contraction of the smooth muscle, and which was termed slow reacting substance (SRS). Over the next 40 years, SRS was identied as a mixture of LTs, and both PGs and LTs were recognized as families of enzymatically generated oxygenation products of AA. Synthesis of PGs was initiated by the cyclooxygenase (COX) enzyme, and that of LTs by the 5-lipoxygenase (5-LO) enzyme. In 1982, the Nobel Prize in Physiology or Medicine was awarded to three investigators Sune Bergstro m, John Vane, and Bengt Samuelsson who pioneered studies of the chemical structures, biosynthetic pathways, pharmacologic modulation, and biologic actions of eicosanoids during this era. Subsequently, many other families of eicosanoids were identied, including products of other LO enzymes, products formed from the interaction of two different LO enzymes (lipoxins), and cytochrome P-450 metabolites of AA. Free AA, the substrate for eicosanoid synthesis, is the product of phospholipase A2 (PLA2)-mediated hydrolysis of membrane phospholipids. Lysophospholipid is the other product of PLA2 action, and is the precursor for

Synthesis of both PAF and eicosanoids (Figure 1) is initiated by a single enzymatic step, cleavage of AA (or other unsaturated fatty acids) at the sn-2 position of membrane phospholipids by the enzyme PLA2. This process is stimulated by cellular agonists, acting via the generation of second messengers. More than 15 different mammalian PLA2 enzymes have been identied, which differ in their intracellular localization and biochemical characteristics. The enzyme that appears to be the most universally important in mediating the stimulated synthesis of lipid mediators is called cytosolic PLA2 (cPLA2). This is an AA-preferring enzyme whose activation involves its calcium-dependent translocation from the cytosol to intracellular membranes. Its catalytic activity is enhanced following phosphorylation by mitogen-activated protein kinases. Besides PLA2 action, another factor that determines the availability of AA for conversion to eicosanoids is the relative proportion of AA among the unsaturated fatty acids esteried at the sn-2 position of phospholipids. This can vary among different cell types. It is also inuenced by dietary intake, as increased consumption of foods rich in n-3 fatty acids (e.g., sh oils), such as eicosapentaenoic and docosahexaenoic acids, results in decreased AA content within phospholipids and a corresponding decrease in synthesis of bioactive eicosanoids. PAF formation requires that the fatty acid at the sn-1 position of the parent phospholipid molecule be linked to the glycerol backbone by an ether rather than an acyl bond. Since ether-linked phospholipids tend to be enriched in AA, PAF synthesis and AA release are thereby linked. The sn-1 ether-linked lysophospholipid (lyso-PAF) generated by PLA2 is then acetylated by the enzyme acetyltransferase to generate PAF. Free AA is potentially available for bioconversion to eicosanoids via a variety of metabolic pathways, each generally referred to by the name of its initial oxygenase enzyme. The two pathways for which the most information exists are the PGH synthase or COX pathway and the 5-LO pathway. In both of these, the oxygenase catalyzes the synthesis of an unstable intermediate, which is further metabolized by downstream terminal enzymes.

574 LIPID MEDIATORS / Overview

Phospholipid PLA2 2 lyso-PAF AA P-450 Epoxides HETEs 5-HPETE LTA4 Agonist

AT PAF

5-LO COX-1 COX-2 PGH2 12-HETE 15-HETE Lx PGD2 PGF2 PGE2 PGI2 TxA2 12-LO 12-HPETE 15-LO 15-HPETE FLAP

5-HETE

LTB4

LTC4

LTD4

LTE4

15-d-PGJ2
Figure 1 Pathways of lipid mediator formation. Enzymes are indicated by enclosure in rectangles and terminal lipid mediators are indicated in italics. AA, arachidonic acid; PAF, platelet-activating factor; PG, prostaglandin; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; LT, leukotriene; Lx, lipoxins; 15-d-PGJ2, 15-deoxy-D12,14-PGJ2; PLA2, phospholipase A2; AT, acetyltransferase; COX, cyclooxygenase; LO, lipoxygenase; FLAP, 5-LO activating protein; P-450, cytochrome P-450.

COX is an integral membrane enzyme that acts on AA to form the unstable endoperoxide PGH2, which can then be converted by a variety of synthases to the individual bioactive prostanoids: PGE2, PGF2a, PGD2, thromboxane (Tx) A2, and PGI2 (prostacyclin). COX exists in two isoforms, COX-1 and COX-2, which differ in their tissue distribution and regulation of expression. Although the conventional notion holds that COX-1 is constitutive and COX-2 is inducible, many exceptions to this dogma are now recognized. COX is the target for the actions of nonselective nonsteroidal anti-inammatory drugs, such as indomethacin and aspirin, and for selective COX-2 inhibitors (coxibs). 5-LO is a calcium-dependent and ATP-activated enzyme that acts on AA to form the unstable epoxide LTA4. A helper protein for 5-LO, known as 5-LO activating protein (FLAP), serves to bind AA liberated by PLA2 and present it to 5-LO, thereby facilitating its enzymatic oxygenation. Agonist activation causes a calcium-dependent translocation of soluble 5-LO to intracellular membranes, where FLAP is located. LTA4 is either hydrolyzed by LTA4 hydrolase to LTB4, or is conjugated with a glutathione molecule by LTC4 synthase to form LTC4. Sequential removal of the amino acids of glutathione can be mediated extracellularly by: (1) g-glutamyl transpeptidases, including g-glutamyl leukotrienase, to yield LTD4; and then (2) a dipeptidase to yield LTE4. LTs C4, D4, and E4 are collectively known as the cysteinyl LTs

(cysLTs), and these account for the bioactivity originally described as SRS.
Regulation of Lipid Mediator Generation

Consistent with the biological importance of lipid mediators, their synthesis is carefully regulated (Table 1). The most obvious mechanism for regulation of lipid mediator synthesis is via changes in expression of their biosynthetic enzymes. Expression of virtually all of the proteins involved in lipid mediator synthesis is subject to regulation, either up or down. Changes in expression most often involve regulation at the transcriptional level. Factors modulating expression of eicosanoid-forming proteins include cytokines, hormones, vitamins, and microbial products. In addition to such exogenous modiers, genetic factors can also inuence the expression of many of these proteins, as polymorphisms in the promoter or coding elements of their corresponding genes have been identied. The catalytic efciency of eicosanoid-forming enzymes is also subject to regulation. This can occur through a variety of mechanisms. First, levels of essential cofactors for these enzymes can vary. For example, ATP is required for optimal 5-LO action, and reduced glutathione is required for optimal action of PGE synthase and as a cosubstrate for synthesis of LTC4; depletion of ATP or glutathione, as might occur during metabolic or oxidative stress, can thereby

LIPID MEDIATORS / Overview 575

Table 1 Mechanisms for the regulation of lipid mediator biosynthesis and secretion Alterations in expression levels of biosynthetic proteins Genetic Acquired (modulation by cytokines, hormones, lipopolysaccharide, etc.) Alterations in function of biosynthetic proteins Levels of essential cofactors (e.g., calcium, ATP, glutathione) for enzymes Posttranslational modications that inuence catalytic activity of proteins (e.g., phosphorylation, nitration) Intracellular localization of proteins Alterations in expression or function of transport proteins required for export Table 2 Representative complete agonists that activate lipid mediator biosynthesis Soluble model agonists Calcium ionophores Phorbol esters Receptor-mediated agonists Thrombin Bradykinin Interleukin-8 Endothelin Immune complexes Leukotrienes PAF Microorganisms Viruses Bacteria Fungi Protozoa Silica Phagocytic particles Zymosan Asbestos Silica Oxidants Hyperoxia Ozone Nitrogen dioxide

inuence the prole of eicosanoids generated. Second, posttranslational modication of enzymes can inuence their catalytic efciency; examples include phosphorylation by protein kinases and nitration by nitric oxide. In this way, mitogen-activated protein kinase can enhance the actions of cPLA2, protein kinase A can reduce the actions of 5-LO, and nitric oxide can directly inhibit 5-LO. Third, intracellular compartmentalization of eicosanoid-forming proteins has emerged as an important means of regulating their functions. As lipid mediators are predominantly secreted into the extracellular space, it was long assumed that their synthesis depended on phospholipid hydrolysis and metabolism at the plasma membrane. Unexpectedly, many of the enzymes involved in their generation have instead been localized to the nuclear membrane and perinuclear endoplasmic reticulum. It may be inferred that topographic colocalization of a group of enzymes that must function in series serves to optimize their coupling and the efciency for lipid mediator generation. As colocalization of enzymes may depend on specic molecular motifs or phosphorylation events, it too may be subject to genetic or acquired variation. Finally, the fact that lipid mediators are synthesized within the cellular interior mandates that mechanisms for their export must exist. Indeed, evidence suggests the presence of distinct cellular transporters for prostanoids, LTB4, and LTC4. Although much remains to be claried about these export mechanisms, they represent an additional target for variation and regulation.
Agonists for Lipid Mediator Synthesis

often because they increase intracellular calcium either directly or via receptor-dependent signal transduction. Examples include the model agonist calcium ionophore as well as physiologically relevant substances such as hormones, microbes, antigens, and oxidants. (2) Certain agonists are only weak triggers by themselves, but can potentiate the subsequent response to a more complete agonist in a process referred to as priming. A priming effect may reect activation of protein kinases that amplify the catalytic activity or translocation of eicosanoid-forming enzymes. Lipopolysaccharide and various cytokines act in this manner. (3) Finally, a number of substances can potentiate lipid mediator synthesis by upregulating expression of eicosanoid-forming enzymes, as noted above; cytokines exemplify such actions. The effects of a given agonist on lipid mediator generation may vary depending on the cell type.
Cellular Participation in Lipid Mediator Synthesis

A myriad of substances can stimulate or potentiate the synthesis of lipid mediators (see Table 2). Three types of stimuli are recognized. (1) Complete agonists are capable of rapidly triggering AA release and its subsequent metabolism to an array of eicosanoids,

Although virtually all cell types participate in lipid mediator synthesis, the specic pathways expressed and therefore the spectrum of products synthesized depend on the cell type. As a general rule, structural or parenchymal cell types (such as epithelial cells, endothelial cells, broblasts, and smooth muscle cells) metabolize AA primarily via the COX pathway, often with some modest capacity for 12- or 15-LO metabolism. By contrast, only bone marrow-derived cells

576 LIPID MEDIATORS / Overview

Table 3 Lipid mediator synthetic proles of various cell types Cell type Platelet Neutrophil Alveolar macrophage Mast cell Endothelial cell Epithelial cell Smooth muscle cell Fibroblast TxA2 7 7 7 PGI2 PGD2 PGE2 7 cysLT LTB4 PAF

tend to synthesize large amounts of 5-LO products, and some also generate substantial amounts of COX, 12-LO, or 15-LO metabolites. PAF is synthesized by endothelial cells, platelets, and various leukocyte populations. The specic prole of eicosanoids synthesized by a given cell type is dictated by its complement of distal enzymes (see Table 3). However, it is instructive to note that both tissue and species specicity in eicosanoid synthetic proles can also be manifest. For example, alveolar macrophages tend to synthesize substantially more LTs and less prostanoids than do macrophages from other sites. Furthermore, mouse alveolar macrophages differ from human or rat cells in that LTC4, rather than LTB4, is their predominant 5-LO product. In view of the differences in biological actions between these two LTs (see below), this species discrepancy must be kept in mind when seeking to interpret the human significance of studies employing transgenic mouse models in which macrophages are a key source of these mediators. In vivo, lipid mediator synthesis reects interactions among neighboring cells. In a process known as transcellular eicosanoid synthesis, one cell type releases an intermediate in the AA metabolic pathway, which is subsequently taken up and processed to an eicosanoid by a second cell type. Thus, studies in a variety of co-culture systems have documented transcellular metabolism of intermediates including AA itself, PGH2, and LTA4 to classical eicosanoids such as prostanoids and LTs. Furthermore, transcellular exchange of LO intermediates can result in dual oxygenation of AA by both 5-LO and either 12- or 15LO to yield lipoxygenase interaction products, or lipoxins. Such transcellular interactions can involve virtually any combination of bone marrow-derived and structural cell types, and are clearly important determinants of tissue output of lipid mediators.
Degradation of Lipid Mediators

degradation or subsequent metabolism. Such conversion can proceed either spontaneously or enzymatically. Importantly, it can result in either inactivation or activation of bioactivity. Given their inherent reactivity, some eicosanoids, but not all, undergo almost instantaneous spontaneous conversion in an aqueous milieu; examples include the conversion of PGI2 to 6-keto-PGF1a and of TxA2 to TxB2 that dictate that these bioactive eicosanoids be measured as their more stable but biologically inactive metabolites. Similarly, PGD2 can spontaneously break down to PGJ2 and then to 15-deoxy-D12,14-PGJ2, which has been implicated as a possible ligand for intranuclear receptors (see below). Likewise, certain eicosanoids are susceptible to oxidative attack and subsequent bioinactivation, and this has been described for LTs. Finally, lipid mediators can be enzymatically metabolized with divergent consequences for biological activity. The conversion of LTC4 to LTD4 by g-glutamyl transpeptidases generally results in enhanced biological activity (see below). By contrast, PGs can be inactivated in the lung by the enzyme 15-hydroxy PG dehydrogenase, PAF can be inactivated by the enzyme PAF acetylhydrolase, and LTs can be metabolized and inactivated by cytochrome P-450 enzyme pathways.

Measuring Lipid Mediators and Their Synthesis

Lipid mediators or their stable metabolites or breakdown products can often be successfully measured in biological samples. This can be accomplished using immunoassays; greater specicity may be obtained by an initial lipid extraction step or by utilizing chromatographic separation techniques. These approaches have been successfully applied to specimens including cell culture supernatants, plasma, urine, lung lavage uid, lung tissue homogenates, induced sputum, and even exhaled breath condensate. Knowledge of the pitfalls, sensitivity, and specicity of each of these approaches is essential to their successful implementation.

One further determinant of the levels of bioactive lipid mediators in tissues is the capacity for their

LIPID MEDIATORS / Overview 577

A variety of indirect or surrogate approaches can also provide information about lipid mediator synthesis. The enzymatic activity or the mRNA or protein expression of specic eicosanoid-forming enzymes can be assayed in cell or tissue lysates. Such expression can also be assessed in situ using microscopic techniques. The activation of enzymes such as cPLA2 or 5-LO that translocate to membranes during cell stimulation can also be inferred by microscopic detection of changes in their intracellular localization. Combining direct mediator measurements with assessments of specic enzymes provides the most comprehensive information about the processes underlying lipid mediator generation.

Lipid Mediator Receptors and Signaling

Lipid mediators can exert biological actions on the source cell (autocrine actions) or on neighboring cells (paracrine actions). In either case, their actions are generally presumed to be mediated by their binding to specic receptors on target cells. Ligation of these receptors results in the generation of intracellular signals that, in turn, transduce the biological actions. Receptors for most of the major lipid mediators have been characterized using classic pharmacologic techniques, and many have been cloned. These are GPCRs, distributed mainly on the plasma membrane, that are coupled to signal transduction events including alterations in intracellular calcium and cyclic AMP levels. These, in turn, inuence the activation of diverse protein kinases such as protein kinases A and C and mitogen-activated protein kinases. Certain lipid mediators and free fatty acids can also bind to an alternative type of receptor known as the peroxisome proliferator-activated receptor (PPAR). These are intranuclear receptors that also act as transcription factors, regulating the expression of proteins involved in lipid metabolism, inammation,
Table 4 Major biological actions of lipid mediators

and cellular proliferation. It is attractive to speculate that interaction with nuclear receptors may mediate some of the intracellular (intracrine) effects of lipids formed locally at the nucleus, without the need for their prior export. It is now recognized that, as is true for certain synthetic enzymes, receptors for a given eicosanoid can be encoded by multiple genes. This is best exemplied by the receptors for PGE2 (termed the E prostanoid, or EP, receptors), as four distinct EP receptors are known. Multiple receptors also bind PGD2, LTB4, and cysLTs. As these distinct receptors differ in their tissue distributions, ligand specicities or afnities, and downstream signaling pathways, it is likely that such a complex receptor variety provides a further means for functional and cellular specicity in the actions of lipid mediators. Studies employing selective receptor agonists and antagonists as well as transgenic mouse lines are beginning to clarify the role of specic receptors in the biological actions of lipid mediators. Although relatively little is currently known about the regulation of receptor expression, it is apparent that genetic and acquired variation in such expression will also be of pathophysiologic importance.

Actions of Lipid Mediators in the Lung

As noted in the introductory comments, lipid mediators are now recognized to inuence the entire spectrum of biological responses, ranging from macro-level processes such as smooth muscle contraction to more subtle intracellular processes such as transcription and growth regulation. Although they will be considered in greater detail elsewhere, some of their key actions most relevant to lung biology are summarized in Table 4. Certain central paradigms about lipid mediator actions are apparent. First, many of these mediators

Lipid Platelet Airway or Bronchial Vascular Leukocyte Release of Fibroblast Immune mediator aggregation vascular responsiveness permeability recruitment mediators proliferation function tone and collagen synthesis PGE2 PGI2 PGF2a TxA2 PGD2 LTB4 5-HETE LTC4/D4/E4 PAF Lx k m k k m m m m m k k k m m m m k m m m m m k k m m m m m k k k m m m m m k k k k k

m m m m

m m m m k

578 LIPID MEDIATORS / Overview

demonstrate redundancy in their actions. Thus, PGD2, TxA2, cysLTs, and PAF all cause bronchoconstriction, and PGD2, PAF, LTB4, and cysLTs all promote leukocyte recruitment. Second, the actions of particular lipid mediators demonstrate context dependency. For example, PGE2 can either promote or inhibit the generation of proinammatory cytokines as well as the egress of leukocytes from the vasculature; this may reect, at least in part, the roles of distinct EP receptors in different cells or circumstances. Third, certain of these molecules oppose or antagonize the actions of others. Such a yin-yang relationship is exemplied by LTs versus PGE2. For example, LTs generally promote leukocyte activation while PGE2 generally inhibits this process; cysLTs are bronchoconstrictive whereas PGE2 is bronchodilatory; and LTs promote while PGE2 opposes brogenesis.

Lipid Mediators in Respiratory Disease and Homeostasis

The determination that a given mediator participates in the pathogenesis of a disease process is based on the fulllment of three criteria: (1) it can be identied in appropriate body uids or tissues (or it can be synthesized by relevant cells); (2) its administration to experimental subjects (humans, animals, organs, or cells) reproduces the pathophysiologic features of the disease process; and (3) its antagonism or inhibition by pharmacologic, immunologic, or genetic strategies ameliorates the manifestations of the disease. By these criteria, lipid mediators have been implicated in a variety of respiratory disease states; a partial list of such conditions is presented in Table 5. The link between LTs and asthma is the most rmly established, but a growing body of evidence also suggests roles for LTs in chronic obstructive pulmonary disease (COPD), acute lung injury, and interstitial lung diseases. Likewise, potential roles have

emerged for PGD2 in asthma and for both LTs and PGE2 in lung cancer. More recently, the homeostatic role of these molecules has been recognized. Examples of homeostatic functions include the protective role of PGI2 in the pulmonary vasculature, of PGE2 and lipoxins in asthma, of PGE2 in brotic lung diseases, and of LTs in antimicrobial defense. Two important paradigms are noteworthy in this context. The rst is that disease states can be characterized not only by overproduction of injurious/ contractile/proinammatory lipid mediators (e.g., LTs, PGD2, and PAF), but also by underproduction of protective/relaxant/anti-inammatory mediators (e.g., PGE2, PGI2, and lipoxins). Although states of relative deciency of homeostatic lipid mediators are much less well recognized, relevant examples include decient synthesis of PGI2 in primary pulmonary hypertension, of lipoxins in asthma, and of PGE2 in idiopathic pulmonary brosis. The second is that value judgments regarding the actions of lipid mediators are of necessity context dependent. Thus, the proinammatory actions of LTs can be considered protective, rather than deleterious, in the context of antimicrobial defense; moreover, increased susceptibility to infection may result when capacity for LT biosynthesis is relatively impaired, as occurs in HIV infection and in malnutrition. Likewise, the antiinammatory effects of PGE2 may be deleterious in the context of antimicrobial defense, and increased susceptibility to infection may also result from states of PGE2 overproduction, as occurs in cancer and advanced age.

Therapeutic Strategies to Alter the Synthesis or Actions of Lipid Mediators

Given the pathophysiologic importance of lipid mediators, therapeutic opportunities exist for modulating their tissue concentrations or tissue responsiveness to them. The generic strategies that can be considered are: (1) administration of a potentially benecial substance (or overexpression of the gene(s) encoding for its synthesis); (2) inhibition of the synthesis of a potentially deleterious substance; and (3) inhibition of the actions of a potentially deleterious substance. Each of these strategies has been successfully employed. Examples include: (1) systemic or inhalational administration of PGI2, PGE2, or analogs thereof for the treatment of pulmonary vascular disease; (2) inhibition of COX for the treatment of pain, fever, and cancer, and of 5-LO for the treatment of asthma; and (3) cysLT receptor blockade for the treatment of asthma. It is easy to envision many other circumstances in which these basic approaches

Table 5 Lung diseases in which abnormalities of lipid mediator production are implicated Airway diseases Asthma Aspirin-sensitive asthma Chronic bronchitis Cystic brosis Acute lung injury ARDS Oxygen toxicity Vascular disorders Pulmonary hypertension Pulmonary thromboembolism Immune complex vasculitis

Lung cancer

Interstitial lung diseases Impaired antimicrobial defense Idiopathic pulmonary brosis HIV infection Asbestosis Malnutrition Scleroderma Smoking

LIPID MEDIATORS / Overview 579

Lipid mediator receptor

Lipid mediator

Cytokine

Cytokine receptor

Figure 2 Bidirectional interactions between lipid mediators and cytokines. Lipid mediators can modulate both the generation of other mediators such as cytokines as well as the expression level of their receptors. In this way, cytokines mediate some of the actions of lipid mediators. In an analogous manner, cytokines can also modulate both the production of lipid mediators as well as the expression of lipid mediator receptors. Thereby, lipid mediators mediate some of the actions of cytokines.

can be applied to further exploit the actions of lipid mediators in lung disease or homeostasis, and these are considered elsewhere. Apart from issues unique to a particular candidate therapeutic agent, the relative merit of each of the above therapeutic approaches ultimately depends on its specicity and the clinical context in which it is applied. In general terms, specicity of actions increases as one moves down the biosynthetic pathways and to specic receptors. For example, an inhibitor of 5-LO will have far greater specicity than will an inhibitor of PLA2, but far less specicity than will an antagonist of the cysLT1 receptor. Achievement of the maximal level of specicity, however, depends on the disease being treated and the relative roles of different mediators. If asthma is the disease target and the only mediators of relevance are cysLTs acting through cysLT1, a cysLT1 antagonist should prove optimal. By contrast, if cysLT2 also mediates actions of cysLTs relevant to asthma, a 5-LO inhibitor would be expected to yield a more desirable therapeutic outcome. With further advances in our knowledge of the actions of lipid mediators and the receptors through which they act, the versatility of our repertoire for therapeutic targeting of lipid mediators will continue to grow.

Lipid Mediators and Peptide Mediators: Similarities, Differences, and Interactions

It is interesting to note that the actions of lipid mediators bear striking similarities to those of other mediator classes, and to cytokines in particular. Like cytokines, lipid mediators can elicit intracellular signal transduction events, which ultimately inuence every aspect of cellular biology. Like cytokines, different lipid mediators exhibit functional redundancy. They also resemble cytokines in manifesting context dependence in their actions. Finally, pairs of lipid mediators exhibit functional opposition, or yin-yang relationships, in their actions. However, important differences from cytokines also exist. Thus, while lipid mediators can match virtually all of the actions of cytokines, they can

exert actions that cytokines generally cannot. These include the ability to modulate such processes as smooth muscle contraction, vascular permeability, and platelet aggregation. Another noteworthy difference relates to the temporal aspects of lipid mediator synthesis. Since cytokine generation usually requires transcriptional and translational events, it tends to require a lag period of at least several hours. By contrast, since the enzymes necessary for biosynthesis of lipid mediators are constitutively expressed, these can generally be elaborated within 115 min following agonist exposure. For this reason, lipids are far more important than cytokines in mediating immediate and early responses to various perturbations. Moreover, because enzymes involved in their biosynthesis can be further upregulated by induction or phosphorylation in response to stimuli or other mediators, subsequent waves of lipid mediator generation can also occur, permitting these substances to also be elaborated in the later phases of a biological response. Finally, a comprehensive understanding of the biological roles of both lipid mediators and peptide mediators, such as cytokines, growth factors, proteases, and histamine, mandates an appreciation of the interactions between them (Figure 2). In fact, lipid mediators can modulate the expression of peptide mediators, as well as of their receptors. Likewise, peptide mediators modulate the expression of eicosanoid-forming enzymes and of eicosanoid receptors. By virtue of such interactive networks, cytokines actually mediate many of the actions of lipid mediators and vice versa. Recognizing these interactions has important pathophysiologic as well as therapeutic implications.
See also: Asthma: Overview. G-Protein-Coupled Receptors. Lipid Mediators: Leukotrienes; Prostanoids; Platelet-Activating Factors. Peroxisome ProliferatorActivated Receptors.

Further Reading
Calder PC (2003) N-3 polyunsaturated fatty acids and inammation: from molecular biology to the clinic. Lipids 38: 343352.

580 LIPID MEDIATORS / Leukotrienes

Charbeneau RP and Peters-Golden M (2005) Eicosanoids: mediators and therapeutic targets in brotic lung disease. Clinical Science (London) 108: 479491. Christie PE and Henderson WR Jr (2002) Lipid inammatory mediators: leukotrienes, prostaglandins, platelet-activating factor. Clinical Allergy and Immunology 16: 233254. Diaz BL and Arm JP (2003) Phospholipase A2. Prostaglandins Leukotrienes, and Essential Fatty Acids 69: 8797. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 18711875. Laskin JJ and Sandler AB (2003) The importance of the eicosanoid pathway in lung cancer. Lung Cancer 41(supplement 1): S73S79. Leslie CC (2004) Regulation of arachidonic acid availability for eicosanoid production. Biochemistry and Cell Biology 82: 117. McMahon B and Godson C (2004) Lipoxins: endogenous regulators of inammation. American Journal of Physiology. Renal Physiology 286: F189F201. Peters-Golden M (2003) Eicosanoids as mediators and therapeutic targets in airway inammation. In: Eissa NT and Huston DP (eds.) Airway Inammation, pp. 627656. New York: Dekker. Peters-Golden M, Canetti C, Mancuso P, and Coffey MJ (2005) Leukotrienes: underappreciated mediators of innate immune responses. Journal of Immunology 174: 589594. Poff CD and Balazy M (2004) Drugs that target lipoxygenases and leukotrienes as emerging therapies for asthma and cancer. Current Drug Targets in Inammation and Allergy 3: 1933. Prescott SM, Zimmerman GA, Stafforini DM, and McIntyre TM (2000) Platelet-activating factor and related lipid mediators. Annual Review of Biochemistry 69: 419445.

Introduction
Leukotrienes (LTs) are endogenously produced inammatory lipid molecules that affect lung function and disease pathogenesis via receptor-mediated effects on a variety of cell types. The slow-reacting substance of anaphylaxis, rst described in the 1930s, was later shown to be comprised of the cysteinyl LTs (cysLTs). Since then, interest in the biochemical and physiological effects of LTs has stemmed primarily from their deduced and predicted roles in asthma pathogenesis. Efforts to diminish the proinammatory and bronchoconstrictive effects of LTs via inhibition of their production and/or blockade of their receptors have resulted in improved therapeutic modalities for this disease. Additionally, clinical data and experimental studies have identied LTs as having potentially significant roles in other lung diseases including chronic obstructive pulmonary disease, brosis, and cancer.

Structure
LTs are so called because they are generated by white blood cells (hence the term leuko) and contain a conjugated triene system of double bonds. They are metabolites of arachidonic acid that are comprised of a 20-carbon backbone, and differ from one another in subtle but important structural ways as a result of progressive enzymatic processing (Figure 1). Oxygenation of arachidonic acid leads to formation of LTA4; subsequent enzymatic hydroxylation of LTA4 at the 12 position or conjugation with reduced glutathione at the 6 position generates LTB4 or LTC4, respectively. LTD4 and LTE4 are formed by the enzymatic removal of L-glutamate and L-glycine, respectively, from the glutathione moiety of LTC4.

Leukotrienes
D C Zeldin and J W Card, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
Published by Elsevier Ltd.

Abstract
Leukotrienes (LTs) are potent bioactive lipid mediators whose synthesis is increased in response to inammatory stimuli. LTB4 and the cysteinyl LTs (LTC4, LTD4, and LTE4) are products of the 5-lipoxygenase pathway of arachidonic acid metabolism and exert their biological effects through specic receptors located on the surface of target cells. In addition to promoting the recruitment and retention of inammatory cells in the lung, LTs increase mucus secretion, enhance vascular permeability, and cause airway constriction. Genetic and pharmacologic manipulation of LT biosynthetic and signaling pathways in experimental animals has uncovered a variety of functions of LTs in models of respiratory disease including allergen-induced airway inammation and hyperresponsiveness, pulmonary brosis, and cancer. Clinical data, including increased bronchoalveolar lavage uid levels of LTs in numerous disease states, also indicate a prominent role for LTs in respiratory disease and dysfunction. While the effects of LT modiers in many lung diseases have not been thoroughly investigated, the use of LT biosynthesis inhibitors and cysteinyl LT receptor antagonists has proven valuable for the treatment of asthma. Continued investigation into the roles of LTs in the lung will undoubtedly yield significant advancements in the treatment of a variety of respiratory diseases.

Regulation of Production and Activity

The production of LTs is mediated by the coordinated activity of several enzymes (Figure 1) and is dependent on the availability of free arachidonic acid within the cell. Importantly, it is inuenced by a variety of endogenous (e.g., cytokines, chemokines, growth factors) and exogenous (e.g., dietary factors, toxins) factors. The primary cells that generate LTs are eosinophils, mast cells, polymorphonuclear leukocytes (neutrophils), and monocytes/macrophages, whereas structural cells (e.g., epithelial and endothelial cells) exhibit low LT synthetic capacity. LTs are not stored in cells, but are generated de novo following cellular activation by bacterial peptides, immune complexes, cytokines, growth factors, and other stimuli. Such stimuli lead to translocation of

LIPID MEDIATORS / Leukotrienes 581

Cell activation

cPLA2 5-LO FLAP

5-L
Arachidonic acid

COOH

FLAP LTC4S 5-LO

Nucleus

COOH

LTA4

LTA4H
OH OH COOH S-Cys-Gly Glu

OH COOH S-Cys-Gly Glu

LTB4

Receptor BLT1 (high affinity)

Cell/tissue expression Leukocytes Lung, spleen, thymus

Effect Chemotaxis, adhesion, activation Uncharacterized

LTC4 MRP1

LTB4 transporter
OH COOH S-Cys-Gly Glu OH COOH S-Cys-Gly OH COOH S-Cys

BLT2 (low affinity)

Most tissues

Uncharacterized

LTC4

cysLT1 (high affinity)

Leukocytes Airway smooth muscle Endothelium Mast cells, macrophages Endothelium

Activation, cytokine production Contraction Increased vascular permeability Activation, cytokine production Activation

LTD4 LTE4

cysLT2 (low affinity)

Figure 1 Synthesis and actions of LTs. Cell activation leads to liberation of arachidonic acid from cellular membranes by cPLA2. 5-LO, in association with FLAP on the nuclear membrane, then converts free arachidonic acid to LTA4. Formation of LTB4 occurs via the action of LTA4 hydrolase on LTA4, whereas LTC4 is formed via the action of LTC4 synthase on LTA4. Both LTB4 and LTC4 are actively transported out of the cell, LTB4 by an uncloned transporter and LTC4 by multidrug resistance protein-1 (MRP1). Extracellular metabolism of LTC4 leads to generation of LTD4 and LTE4; these three molecules are collectively referred to as the cysLTs. LTB4 and the cysLTs act on specic receptors on target cells to elicit a variety of biological effects.

cytosolic phospholipase A2 (cPLA2) to intracellular membranes, which results in the liberation of free arachidonic acid. 5-lipoxygenase (5-LO), which translocates from the nucleus and/or the cytoplasm to the nuclear envelope, converts free arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5HPETE) and then to LTA4. The presence of the cofactor 5-LO activating protein (FLAP) is required for 5-LO activity and hence for LTA4 generation. Indeed, cells lacking FLAP or that are treated with a FLAP inhibitor, and mice genetically decient in FLAP, are unable to generate LTs. LTA4 is the precursor for the intracellular generation of LTB4, formed via LTA4 hydrolase activity, and of LTC4, formed via LTC4 synthase activity. Following their generation, LTB4 and LTC4 are actively transported out of the cell, although LTB4 may also act intracellularly to regulate gene expression via nuclear receptor interaction. Extracellular metabolism of the glutathione moiety of LTC4 results in the formation of LTD4 and LTE4; collectively, these three molecules are referred to as the cysLTs or suldopeptide LTs. The cell-specic expression of LT biosynthetic enzymes contributes significantly to the regulation of

LT effects in the lung. Neutrophils and dendritic cells primarily synthesize LTB4, mast cells and eosinophils primarily synthesize cysLTs, and macrophages produce a balance of both. As the cells that generate LTs are primarily inammatory in nature, they are usually only present in the lung in substantial numbers following recruitment by inammatory mediators secreted by injured or stressed pulmonary epithelial and/or endothelial cells and resident macrophages. In addition to recruiting inammatory cells that generate LTs, inammatory mediators can upregulate the expression of LT biosynthetic enzymes and receptors within inammatory and other cell types, including airway smooth muscle and epithelial cells. The net result is an environment in which LT synthesis contributes to decrements in lung function and to the recruitment and retention of more inammatory cells via direct chemotactic effects and through the continued generation of proinammatory cytokines and chemokines. Inammatory mediators that amplify LT biosynthesis include interleukin-4, interleukin-5, transforming growth factor beta, and others. It is of interest that the hormone leptin, which is secreted by adipocytes in correlation with total body fat mass,

582 LIPID MEDIATORS / Leukotrienes

has also been shown to augment LT production, suggesting a possible mechanistic link between obesity and asthma. Regulation of LT production also appears to exist at the genetic level, as polymorphisms in the promoter regions of the 5-LO and LTC4 synthase genes have been identied and may contribute to respiratory diseases and inuence therapeutic responses to anti-LT agents. The biological actions of LTs are mediated by the regulated expression, distribution, and activation of their receptors. Specic cell surface receptors for LTB4 and for the cysLTs are found on a variety of lung and inammatory cells. Expression levels of these receptors can be altered by an assortment of factors including cytokines, chemokines, LTs, and other mediators whose levels themselves vary with disease state and severity.

effects of cysLTs indicate an additional significant role in asthma pathogenesis.

Receptors
The biological actions of LTs result from their interaction with the specic 7 transmembrane domain, G-protein-coupled receptors on the surface of target cells (Figure 1). Two receptors have been identied for LTB4, namely BLT1 and BLT2, which are highand low-afnity LTB4 receptors, respectively. BLT1 is not strongly expressed in lung tissue, but is present on a variety of leukocytes including neutrophils, eosinophils, and effector CD4 and CD8 T cells. BLT2 is widely expressed in most human tissues and is also found on leukocytes. Two receptors have also been identied for the cysLTs, namely cysLT1 and cysLT2, which are high- and low-afnity cysLT receptors, respectively. Both cysLT1 and cysLT2 are expressed in a variety of tissues and cell types. The expression of cysLT1 on airway smooth muscles is primarily responsible for asthmatic bronchoconstriction resulting from antigen-induced generation of the cysLTs, and targeted antagonism of this receptor has provided an effective adjuvant in the treatment of this disease. Expression of LTB4 and cysLT receptors can be modied by cytokines and other factors present in the microenvironment of injured or diseased lung tissue. For example, interferon gamma stimulates BLT1 expression in macrophages, whereas interleukin-4 or interleukin-13, proinammatory cytokines believed to play crucial roles in asthma pathogenesis, upregulate cysLT1 expression. As such, the effects of LTs in respiratory diseases are probably mediated by a complex interplay between LTB4 and cysLT receptors and the regulated/dysregulated production of LTB4 and the cysLTs. As LTB4 and LTC4 are synthesized deep within cells, the existence of novel intracellular receptors that mediate autocrine effects of LTB4 and the cysLTs has long been suspected. The nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-a) was the rst molecule identied as a receptor for LTB4 and may be involved in transcriptional regulation of genes involved in LTB4 inactivation and clearance. An intracellular cysLT receptor has been suggested based on the ability of cysLTs to liberate interleukin-4 from membrane-permeabilized but not from nonpermeabilized eosinophils treated with cysLT1/2 inhibitors.

Biological Functions
LTs exert several important effects in the lung including promotion of inammatory cell recruitment and adhesion, mucus secretion, airway smooth muscle contractility, and vascular permeability. The primary role of LTB4 is as a potent chemoattractant and activator of myeloid leukocytes including neutrophils, monocytes, and eosinophils. As an example, direct intratracheal administration of LTB4 to humans induces neutrophil recruitment into the airways without altering protein permeability. LTB4 is a recognized autocrine stimulator of neutrophils, and exerts anti-apoptotic effects to prolong the survival of neutrophils and eosinophils. It is also involved in the recruitment and trafcking of T cells in asthma, acting in cooperation with various cytokines and chemokines to direct T cell migration to sites of inammation. cysLTs are potent mediators of bronchoconstriction resulting from exposure to allergic and other stimuli, and are more potent than histamines in eliciting a decrease in airow in humans. Blockade of the cysLT1 receptor attenuates the bronchoconstrictive response to various stimuli including allergen, cold air, exercise, and adenosine inhalation, conrming the central role of cysLTs in airow obstruction. In addition to their bronchoconstrictive effects, cysLTs contribute significantly to the recruitment and retention of inammatory cells in the lung, promote the induction of cytokine generation by eosinophils and mast cells, and increase vascular permeability. cysLTs also exert numerous effects on dendritic cells including inuencing their development, trafcking, and immunomodulatory functions. Given the importance of dendritic cells as antigen-presenting cells, the

Leukotrienes in Respiratory Diseases

A wealth of clinical and experimental evidence supports the involvement of LTs and their receptors in several respiratory diseases (Table 1), the most

LIPID MEDIATORS / Leukotrienes 583

Table 1 Murine models of respiratory disease in which genetic and pharmacological approaches have identied detrimental roles for LTs Disease model Allergen-induced inammation Approach 5-LO gene deletion cysLT1 gene deletion cysLT1 antagonism cPLA2 gene deletion 5-LO gene deletion LTC4 synthase gene deletion cysLT2 gene deletion cPLA2 gene deletion 5-LO inhibition FLAP inhibition cysLT1 antagonism

Bleomycin-induced brosis

Chemical-induced carcinogenesis

thoroughly studied of which is asthma. Bronchoalveolar lavage (BAL) uid LT levels are increased in asthmatics compared with normal subjects, and animal models of allergen-induced airway inammation reveal similar ndings. Inhibition of LT biosynthesis or abrogation of cysLT signaling via cysLT1 blockade reduces allergen-induced airway inammation and hyperresponsiveness in mice, and clinically efcacious cysLT1 antagonists and inhibitors of cysLT synthesis have been developed and employed in the treatment of asthma. Recent recognition that LTs inuence dendritic cell maturation, trafcking, and immunomodulatory function may lead to the development of further benecial therapies for this disease. In addition to asthma, elevated levels of LTs have been reported in BAL uid or exhaled breath condensate from patients with pneumonia, cystic brosis, lung cancer, chronic obstructive pulmonary disease, and idiopathic pulmonary brosis. While little data is available regarding LTB4, cysLTs appear to be intimately involved in the pathogenesis of pulmonary brosis. Constitutive activation of 5-LO is observed in lung tissue of humans with idiopathic pulmonary brosis. In addition, bleomycin-induced pulmonary brosis in mice is characterized by increased lung cysLT production and is attenuated by disruption of the 5-LO gene. Among other mechanisms, cysLTs appear to promote a probrotic phenotype via inammatory cell recruitment and induction of broblast proliferation and collagen production. The phenomenon of transcellular metabolism may contribute to the increased LT biosynthesis observed in respiratory diseases such as asthma and brosis. Under normal conditions, pulmonary epithelial cells metabolize arachidonic acid primarily via

the cyclooxygenase pathway, leading to generation of the anti-inammatory, antibrotic, and bronchodilatory prostaglandin E2 (PGE2). In conditions of disease, epithelial cell injury or dysfunction results in impaired cyclooxygenase activity and decreased PGE2 production. Consequently, there is an increased availability of free arachidonic acid that can be metabolized by leukocytes located in close proximity to the epithelial cells. This transcellular metabolism of arachidonic acid results in increased LT biosynthesis by leukocytes and may contribute to the inammatory and bronchoconstrictive phenotype of various respiratory diseases. Interestingly, LTs have been shown to exert antimicrobial activity and to be involved in protecting the lung from injury in a mouse model of pneumonia, indicating that not all actions of these mediators in the lung are deleterious. While the observed involvement of LTs in an array of respiratory diseases underscores their central importance in the diseased lung, recent advances indicate that much remains to be understood with regard to the varied roles of these lipid mediators. The evolving recognition and elucidation of the diverse roles of LTs in the lung suggests a favorable future for the development of therapeutic approaches for the treatment of respiratory diseases based on LT biology.
See also: Asthma: Overview. Lipid Overview; Lipoxins. Pulmonary Fibrosis. Mediators:

Further Reading
Coffey M and Peters-Golden M (2003) Extending the understanding of leukotrienes in asthma. Current Opinions in Allergy and Clinical Immunology 3: 5763. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 18711875. Holgate ST, Peters-Golden M, Panettieri RA, and Henderson WR (2003) Roles of cysteinyl leukotrienes in airway inammation, smooth muscle function, and remodeling. Journal of Allergy and Clinical Immunology 111: S18S36. Kanaoka Y and Boyce JA (2004) Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inammatory responses. Journal of Immunology 173: 15031510. Luster AD and Tager AM (2004) T-cell trafcking in asthma: lipid mediators grease the way. Nature Reviews: Immunology 4: 711724. McMillan RM (2001) Leukotrienes in respiratory disease. Pediatric Respiratory Reviews 2: 238244. Peters-Golden M and Brock TG (2001) Intracellular compartmentalization of leukotriene synthesis: unexpected nuclear secrets. FEBS Letters 487: 323326. Peters-Golden M, Canetti C, Mancuso P, and Coffey MJ (2004) Leukotrienes: underappreciated mediators of innate immune responses. Journal of Immunology 173: 589594. Sampson AP, Pizzichini E, and Bisgaard H (2003) Effects of cysteinyl leukotrienes and leukotriene receptor antagonists on markers of inammation. Journal of Allergy and Clinical Immunology 111: S49S61.

584 LIPID MEDIATORS / Prostanoids

Soberman RJ and Christmas P (2003) The organization and consequences of eicosanoid signaling. Journal of Clinical Investigation 111: 11071113. Tager AM and Luster AD (2003) BLT1 and BLT2: the leukotriene B4 receptors. Prostaglandins, Leukotrienes and Essential Fatty Acids 69: 123134. Yokomizo T, Izumi T, and Shimizu T (2001) Leukotriene B4: metabolism and signal transduction. Archives of Biochemistry and Biophysics 385: 231241.

roles of prostanoids in pathologic and homeostatic processes. This article reviews the current state of knowledge of the synthesis, actions, and pathophysiologic roles of prostanoids in the respiratory tract, as well as the therapeutic potential of their modulation.

Pulmonary Prostanoid Synthesis and Metabolism

Prostanoids
M Peters-Golden, University of Michigan, Ann Arbor, MI, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Prostanoids are a family of lipid mediators that include prostaglandins, prostacyclin, and thromboxane. Their synthesis from arachidonic acid is initiated by the cyclooxygenase enzyme, which is the molecular target of the widely employed class of nonsteroidal anti-inammatory drugs. Terminal synthase enzymes, expressed in cell-specic fashion, complete the synthesis of bioactive prostanoids. Via ligation of G-protein-coupled receptors, they exert autocrine and paracrine effects including modulation of smooth muscle tone, microvascular permeability, tissue repair and remodeling, inammation, and immune responses. Either overproduction or underproduction of prostanoids has been implicated in a variety of lung diseases, including asthma, interstitial lung disease, pulmonary vascular disease, cancer, and infections. Current therapies employ both blockade and administration of prostanoids, and additional applications of increasing selectivity can be envisioned in the future.

Introduction
Prostanoids comprise a family of lipid mediators formed from arachidonic acid (AA) via the prostaglandin (PG) H synthase or cyclooxygenase (COX) pathway. They include PGE2, PGD2, and PGF2a, as well as prostacyclin (PGI2) and thromboxane A2 (TxA2). Prostanoids have long been known to modulate such biological processes as smooth muscle tone, vascular permeability, pain, fever, and platelet aggregation. More recently, roles for these molecules in cellular proliferation and survival, immune responses, and even sleep have been identied. The clinical importance of prostanoids is emphasized by the fact that their biosynthesis is the target for nonsteroidal anti-inammatory drugs (NSAIDs), one of the most widely used classes of pharmacotherapeutic agents. The last decade has witnessed remarkable advances in our understanding of both prostanoid synthetic mechanisms as well as the receptors that mediate prostanoid action. In addition, transgenic animal models have shed a great deal of light on the

Arachidonic acid liberated from membrane phospholipids by the actions of phospholipase A2 (PLA2) is transformed to the unstable endoperoxide PGH2 by the COX enzyme (see Figure 1). Since the early 1990s it has been recognized that COX exists in two isoforms, generally designated COX-1 and COX-2. Most cell types are capable of expressing both COX isoforms, and both can initiate prostanoid synthesis from AA. The initial paradigm held that COX-1 was constitutively expressed in most tissues and was responsible for basal synthesis of prostanoids that mediated housekeeping functions, while COX-2 expression required induction (by cytokines, hormones, growth factors, lipopolysaccharide), which was glucocorticoid inhibitable, and was responsible for augmented synthesis of prostanoids that mediated pathobiologic effects. From this came the notion that inhibition of COX-2 accounted for the desirable therapeutic effects of nonselective NSAIDs (e.g., aspirin, indomethacin, and ibuprofen) that inhibited both isoforms, while inhibition of COX-1 accounted for their unwanted side effects (e.g., gastrointestinal irritation and impaired renal blood ow). This paradigm promptly ascended to the level of dogma, and helped drive the swift development of highly selective COX-2 inhibitors (coxibs). In fact, only some of the predictions of the original dogma have been proven to be accurate. The prediction that synthesis of pathologic prostanoids most often derives from COX-2 has generally been borne out. Not anticipated, however, was the fact that COX-2 was expressed constitutively in certain tissues (e.g., the vascular endothelium and the respiratory epithelium) and was consequently responsible for elaboration of prostanoids serving certain homeostatic functions. The COX-derived intermediate PGH2 is converted to bioactive prostanoids by terminal synthase enzymes, whose expression proles exhibit cell type specicity. Although macrophages synthesize a broad range of prostanoids, most cell types elaborate a single predominant COX product, as follows: platelets, TxA2; mast cells, PGD2; endothelial cells, PGI2; and epithelial cells, broblasts, and smooth muscle cells, PGE2. PGF2a, PGI2, and TxA2 synthases appear to

LIPID MEDIATORS / Prostanoids 585

COOH

Arachidonic acid + GCs LPS TNF-

COX-1 NSAIDs
O

COX-2 Coxibs
COOH

PGH2

O OH

Prostaglandin synthases PGDS

OH COOH O O OH HO COOH O OH OH HO OH COO Na
+

PGIS

PGES
O

PGFS

TxS
OH COOH OH

COOH

TxA2

PGD2

COOH

HO OH

PGF2

PGI2 PGJ2
O COOH

PGE2

15-deoxy-12,14 PGJ2
Figure 1 Pathway of prostanoid production. Free AA is oxygenated by either COX-1 or COX-2 to form the unstable endoperoxide intermediate prostaglandin (PG) H2. Traditional NSAIDs such as aspirin, indomethacin, and ibuprofen nonselectively inhibit COX-1 and -2, while coxibs selectively inhibit COX-2. The COX-2 enzyme can be induced by inammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-a), a process that can be prevented by glucocorticoids (GCs). PGH2 can be converted by terminal PG synthase enzymes to the stable prostanoids, PGD2, prostacyclin (PGI2), PGE2, PGF2a, and TxA2, each of which ligates specic G-protein-coupled prostanoid receptors. PGD2 can be nonenzymatically dehydrated to PGJ2 and then to 15-deoxy-D12,14-PGJ2; the latter compound is a putative ligand for the intranuclear receptor peroxisome proliferator-activated receptor-g (PPAR-g).

represent single enzyme isoforms. Lipocalin and hematopoietic PGD synthases represent two distinct gene products present in brain and mast cells/macrophages, respectively. At least three PGE synthases exist, all of which require reduced glutathione as a cofactor. Cytosolic PGE synthase and membrane-associated PGE synthase-2 are both primarily constitutively expressed enzymes. Membrane-associated PGE synthase-1, by contrast, resembles COX-2 in its induction by inammatory stimuli, which can be inhibited by glucocorticoids. Incremental prostanoid synthesis requires either an increase in free AA or increased expression of COX or terminal synthase enzymes capable of metabolizing the relatively small amount of AA liberated during constitutive phospholipid remodeling. Substances such as bradykinin, thrombin, antigen, leukotrienes, oxidants, silica, and microbes can activate PLA2 to trigger AA release, while lipopolysaccharide, growth factors, and cytokines can enhance expression of downstream enzymes. Prostanoids produced in the lung can be measured directly in lung tissue, bronchoalveolar lavage uid, induced sputum, exhaled breath

condensate, or plasma, or indirectly as urinary metabolites. In addition to a substantial capacity to elaborate COX products, the lung plays an important role in their catabolism. During a single pass through the pulmonary vascular bed, the majority of prostanoids are inactivated. This occurs via transporter-mediated uptake into endothelial cells followed by metabolism via the enzyme 15-hydroxy PG dehydrogenase. Mice, in which this enzyme has been deleted, demonstrate enhanced levels of PGE2 and biological responses mediated by PGE2.

Prostanoid Receptors
The autocrine or paracrine actions of prostanoids are mediated via ligation of receptors. The best characterized receptors for prostanoids are high-afnity (EC50 values in the nanomolar range) G-proteincoupled receptors (GPCRs). Binding to these receptors results in the generation of second messengers and subsequent activation of signal transduction pathways. Figure 2 depicts the GPCRs for various

586 LIPID MEDIATORS / Prostanoids

PGI2 PGD2 IP DP1 EP2 EP4 PGE2 EP3 TxA2 TP DP2 Gi cAMP Gs cAMP
Table 1 Actions of prostanoids other than PGE2 in the respiratory tract PGD2 * sleep * airow obstruction * Th2 immune responses TxA2 * bronchoconstriction * vasoconstriction * platelet aggregation PGI2 * vasodilation * inhibition of platelet aggregation * inhibition of vascular remodeling PGF2a * bronchoconstriction * vasoconstriction

EP1 PGF2 FP Gq

Ca2 +

Figure 2 GPCRs for prostanoids. Prostanoids and the GPCRs, major G proteins, and second messengers through which they act are depicted. The dashed line represents the capacity of PGD2 to ligate the TP receptor with a relatively low afnity.

prostanoids, and the G proteins and second messengers through which they generally signal. Based on their expected effects on smooth muscle tone, those receptors that signal through Gs-mediated increases in intracellular cyclic AMP can be considered relaxant receptors, while those that signal through Gqmediated increases in intracellular calcium can be considered contractile in nature. The prole of prostanoid receptors varies with the cell type. In addition, the expression of prostanoid receptors is under genetic control and can also be modulated by exogenous factors, such as lipopolysaccharide, cytokines, and tissue injury. Certain prostanoids can bind to and act via multiple GPCRs that are encoded by distinct genes. PGE2 represents the most striking example of this phenomenon, as it acts via four distinct E prostanoid (EP) receptors; such receptor diversity probably contributes to the pleiotropic actions of this most ubiquitous of prostanoids. Certain prostanoids can bind to another class of receptor, the peroxisome proliferator-activated receptors (PPARs). PPARs comprise a family of intranuclear receptors and transcription factors. PPAR-g regulates the expression of genes involved in lipid metabolism, inammation, and cellular proliferation. While it is the target for the glitazone class of antidiabetic drugs, the nature of the endogenous ligand(s) for this receptor has remained elusive. Substantial attention in recent years has surrounded the possibility that 15-deoxy-D12,14-PGJ2, a nonenzymatic dehydration product of PGD2, may fulll this role and thereby serve as a break on inammatory and proliferative events. However, the EC50 of PPAR-g for

exogenous 15-deoxy-D12,14-PGJ2 is in the 10 5 M range, and sophisticated mass spectrometric analyses have cast serious doubts on whether such levels of 15deoxy-D12,14-PGJ2 are generated endogenously. For this and other reasons, the role of this prostanoid as an endogenous activator of PPAR-g remains to be established.

Prostanoids in Respiratory Disease and Homeostasis

Prostanoids have been implicated in virtually every type of pathologic or homeostatic process conceivable. The best-known actions of prostanoids pertinent to the respiratory tract are shown in Table 1 and Figure 3, and their key roles in clinically relevant pathophysiologic states are summarized below. It is essential to point out that respiratory disease states can be linked to both the overproduction as well as the underproduction of various prostanoids.
Asthma and Aspirin-Sensitive Asthma

Prostanoids have the potential to inuence every aspect of the pathobiology of asthma. The recognition of both contractile and relaxant groups of prostanoids has been previously noted, and the effects of individual prostanoids on bronchial tone are depicted in Table 1. The greatest interest currently surrounds the possible proasthmatic role of PGD2 and the antiasthmatic role of PGE2. PGD2 can reproduce many of the airway abnormalities observed in asthma and its levels are increased in asthmatics. The fact that DP1 null mice are protected in a model of antigen-induced asthma is somewhat surprising, since DP1 signals as a relaxant receptor. Therefore, the proasthmatic actions of

LIPID MEDIATORS / Prostanoids 587

Airway responses
Bronchodilation Protection against bronchoconstriction Bronchial hyperresponsiveness

Vascular responses
Vasodilation Microvascular permeability

Neoplasia
Tumor cell proliferation Tumor cell apoptosis Tumor cell adhesion & migration Angiogenesis

Epithelial cells
Wound closure by airway epithelium

PGE2

Surfactant secretion by alveolar type cells Apoptosis

Mesenchymal cell (fibroblast & smooth muscle cell) functions

Migration Proliferation Collagen synthesis and collagen degradation Fibroblast transition myofibroblasts Fibroblast production of hepatocyte growth factor, a survival factor for epithelial cells

Inflammatory responses
Myelopoiesis Leukocyte chemotaxis Adhesion molecule expression Generation of inflammatory mediators Generation of proinflammatory IL- 6

Immune responses
Maturation of immature dendritic cells

Generation of anti-inflammatory IL-10

Antigen presentation by mature dendritic cells T and B lymphocyte proliferation and functions Context-dependent effects on Th1 vs. Th2 polarization Phagocyte antimicrobial functions

Figure 3 Pleiotropic actions of PGE2 in the respiratory tract.

PGD2 may instead involve DP1-mediated increases in airway edema (or other unknown actions), contractile effects mediated by its ability to also ligate the TP receptor, and chemoattractant and proinammatory effects mediated via a chemokine family receptor previously termed chemoattractant receptor expressed on Th2 cells or CRTH2 and now designated as DP2. Antagonists for both DP1 and DP2 are currently under development. Inhalation of PGE2 or its analogs in humans causes bronchodilation, protects against bronchoprovocation, and attenuates induced airway inammation. Such actions reect not only its well-known relaxant properties, but also its diverse anti-inammatory and immunosuppressive actions (Figure 3). Most of these salutary effects are mediated by EP2- or EP4-mediated increases in cyclic AMP. Recent studies have shown a worsening in models of experimental asthma when COX inhibitors or COX null mice are employed. As PGE2 is the predominant prostanoid found in lung lavage uid, these data are most consistent with the conclusion that endogenously produced PGE2 serves a bronchoprotective role in vivo. Limited data from humans and animal models suggest that, in some instances, asthma may be associated with a relative deciency of endogenous PGE2. In most asthmatics, administration of NSAIDs has negligible effect, probably reecting the global inhibition of prostanoids that exert opposing actions on the

airways. In B510% of asthmatics, however, nonselective COX inhibitors dramatically worsen their airway function; this is termed aspirin-sensitive or aspirin-intolerant asthma. Interestingly, these patients can safely tolerate COX-2-selective inhibitors. These observations imply that in this subset of patients, COX-1-dependent synthesis of a prostanoid (most likely PGE2) serves a critical bronchoprotective role. Inhibitors of COX-1 remove this brake, and also allow the accumulation of unmetabolized AA, which can be metabolized to proasthmatic leukotrienes. A relative deciency of COX-2-derived PGE2 in aspirinsensitive patients may help to explain both their ability to tolerate COX-2-selective inhibitors as well as the overall severity of their asthma at baseline even when they avoid NSAIDs. As for other types of bronchoprovocation, inhalation of PGE2 can also protect against aspirin-induced bronchospasm. However, such treatment frequently provokes cough. If the EP receptors that mediate cough are distinct from those that exert bronchoprotective actions, inhalation of EP-selective PGE2 agonists may represent an attractive therapeutic approach in both aspirin-sensitive and aspirin-tolerant asthma.
Fibrotic Lung Disease

By virtue of its capacity to inhibit inammation as well as broblast migration, proliferation, differentiation

588 LIPID MEDIATORS / Prostanoids

into myobroblasts, and collagen synthesis, PGE2 attenuates brotic responses to lung injury (Figure 3). These actions are largely mediated via increases in cyclic AMP. In animal models of pulmonary brosis, pharmacologic or genetic inhibition of COX enzymes exaggerates the degree of brosis. Interestingly, patients with idiopathic pulmonary brosis have been shown to have a relative deciency of PGE2 in their lung lavage uid, and impaired PGE2 synthetic capacity has been documented in both alveolar macrophages and lung broblasts from such patients. PGE2 deciency would tend to promote an activated phenotype in broblasts. Activation of broblasts and smooth muscle cells also contributes to airway remodeling in asthma, and defective PGE2 synthesis has also been described in these cell types from asthmatics. Taken together, these data strongly suggest that a relative deciency of PGE2 synthesis characterizes and may contribute to remodeling of both parenchyma and airways in chronic lung disease. Again, administration of PGE2 or of PGE2 analogs that selectively activate cyclic AMP-coupled EP receptors is an appealing therapeutic strategy in these conditions.
Pulmonary Hypertension

(Figure 3). Trials of COX inhibitors in humans with lung cancer are currently underway.
Immune Function

PGE2 modulates immune responses in a complex and sometimes conicting manner (Figure 3). For example, it can either stimulate or inhibit antigen-presenting dendritic cells, depending on their degree of maturation. Its effects on Th1 versus Th2 T lymphocyte phenotypes have also been controversial. In general, however, PGE2 suppresses most aspects of lymphocyte, macrophage, and neutrophil functions. Therefore, it might be expected to interfere with immune responses to vaccines, immune surveillance of cancer cells, and antimicrobial defense against infections. It is interesting to speculate that overproduction of PGE2 as has been described in cancer, bone marrow transplantation, the elderly, and in infections themselves might increase susceptibility to infections and compromise innate immune responses to them. In such instances, inhibition of PGE2 synthesis or blockade of relevant EP receptors might have a therapeutically desirable immunostimulatory effect.
See also: Asthma: Overview; Aspirin-Intolerant. Corticosteroids: Glucocorticoid Receptors; Therapy. Epithelial Cells: Type I Cells; Type II Cells. Fibroblasts. G-ProteinCoupled Receptors. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Mast Cells and Basophils; Pulmonary Macrophages. Myobroblasts. Peroxisome Proliferator-Activated Receptors. Platelets. Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Signal Transduction. Smooth Muscle Cells: Airway. Transgenic Models. Tumors, Malignant: Bronchogenic Carcinoma. Vascular Disease.

It is well known that PGI2 is the major prostanoid product of vascular endothelial cells and that it inhibits platelet aggregation as well as contraction and proliferation of vascular smooth muscle cells. TxA2 is produced by platelets, and has opposing effects. Patients with primary pulmonary hypertension have been identied as having both increased TxA2 production and impaired PGI2 generation, with the former probably reecting platelet aggregation and the latter reecting a deciency of PGI2 synthase. In view of their deciency of this important endogenous vasodilator, it is not surprising that exogenous administration of PGI2, PGE2, or analogs thereof by the intravenous or inhaled route has become established as standard therapy for pulmonary hypertensive disorders. These prostanoids have proven not only to cause vasodilation, but also to ameliorate vascular wall remodeling.
Lung Cancer

Further Reading
Badesch DB, McLaughlin VV, Delcroix M, et al. (2004) Prostanoid therapy for pulmonary arterial hypertension. Journal of American College of Cardiology 43(12 supplement S): 56S61S. Breyer RM, Bagdassarian CK, Myers SK, and Breyer MD (2001) Prostanoid receptors: subtypes and signaling. Annual Review of Pharmacology and Toxicology 41: 661690. Carey MA, Germolec DR, Langenbach R, and Zeldin DC (2003) Cyclooxygenase enzymes in allergic inammation and asthma. Prostaglandins Leukotrienes and Essential Fatty Acids 69: 157162. FitzGerald GA and Patrono C (2001) The coxibs, selective inhibitors of cyclooxygenase-2. New England Journal of Medicine 345: 433442. Harris SG, Padilla J, Koumas L, Ray D, and Phipps RP (2002) Prostaglandins as modulators of immunity. Trends in Immunology 23: 144150. Hayaishi O and Urade Y (2002) Prostaglandin D2 in sleep-wake regulation: recent progress and perspectives. Neuroscientist 2002. 8: 1215.

Many cancers, including lung cancers, overexpress COX-2 and elaborate high levels of PGE2. Pharmacologic and genetic interruption of PGE2 synthesis or EP receptors reduces tumor growth in vitro and in vivo. Such strategies target the ability of PGE2 to promote tumor cell migration, survival, and proliferation, to stimulate tumor angiogenesis, and to reduce immune surveillance against malignant cells

LIPID MEDIATORS / Platelet-Activating Factors

Helliwell RJ, Adams LF, and Mitchell MD (2004) Prostaglandin synthases: recent developments and a novel hypothesis. Prostaglandins, Leukotrienes and Essential Fatty Acids 70: 101113. Ide T, Egan K, Bell-Parikh LC, and FitzGerald GA (2003) Activation of nuclear receptors by prostaglandins. Thrombosis Research 110: 311315. Narumiya S, Sugimoto Y, and Ushikubi F (1999) Prostanoid receptors: structures, properties, and functions. Physiological Reviews 79: 11931226. Sandler AB and Dubinett SM (2004) COX-2 inhibition and lung cancer. Seminars in Oncology 31(2 supplement 7): 4552. Szczeklik A, Sanak M, Nizankowska-Mogilnicka E, and Kielbasa B (2004) Aspirin intolerance and the cyclooxygenaseleukotriene pathway. Current Opinion in Pulmonary Medicine 10: 5156. Vancheri C, Mastruzzo C, Sortino MA, and Crimi N (2004) The lung as a privileged site for the benecial actions of PGE2. Trends in Immunology 25: 4046. Warner TD and Mitchell JA (2004) Cyclooxygenases: new forms, new inhibitors, and lessons from the clinic. FASEB Journal 18: 790804. Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, and De Marzo AM (2004) Cyclooxygenases in cancer: progress and perspective. Cancer Letters 215: 120.

589

Metabolism of PAF

Platelet-Activating Factors
K F Chung, Imperial College London, London, UK
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Platelet-activating factor (PAF) is a biologically active phospholipid mediator belonging to a family of alkylphosphoglycerides with potent actions as a mediator of inammation. It acts through a G-protein-coupled receptor linked to phospholipase A2 and C. PAF is metabolized by PAF acetylhydrolases which reduce its activity. Low plasma PAF acetylhydrolase activity has been associated with inammatory disorders such as asthma and rheumatoid arthritis. PAF is produced by endothelial cells, macrophages, neutrophils, eosinophils, monocytes, and mast cells, and activates many inammatory cells by intercellular and intracellular mechanisms. PAF may be an important inammatory mediator in various lung diseases such as asthma, acute respiratory distress syndrome, and pulmonary vascular diseases. Although there is some support for a role of PAF in animal models, the effects of inhibiting the actions of PAF in human asthma in particular have been negligible. PAF may play an important role in normal physiological homeostasis and in the primary response to inammation and injury. This role remains to be dened.

PAF is synthesized in many inammatory cells such as monocytes, macrophages, neutrophils, eosinophils, mast cells, and endothelial cells by the remodeling pathway (Figure 1), which involves the activation of phospholipase A2 to hydrolyze a longchain acyl group from 1-alkyl-2-acyl-sn-glycero-3 phosphocholine in membrane phospholipids to form 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (lysoPAF), followed by acetylation by an acetyltransferase enzyme. Acetyltransferase activity can be induced by the calcium ionophore A23187, thrombin, bradykinin, elastase, cathepsin G, leukotrienes C4 and D4, interleukin (IL)-8, and tumor necrosis factor (TNF) and IL-1a. Activation of the remodeling pathway results in the biosynthesis of excessive amounts of PAF. By contrast, the de novo pathway involves the direct generation of PAF from etherlinked phospholipids, such as alkylacetylglycerols, under the action of a highly specic cholinephosphotransferase present in several tissues including rat lung and human neutrophils. This production of PAF relates to basal physiological levels. PAF is rapidly inactivated by the removal of the acetate moiety to form lyso-PAF, which is biologically inactive, by acetylhydrolases, of which the most important one is PAF-specic acetylhydrolases (PAFAH) which is present in cells and in plasma. Cellular acetylhydrolase activity may be released into the supernatant after stimulation of platelets with various agonists, including PAF. Low levels of plasma PAF-AH have been associated with several inammatory diseases such as asthma, rheumatoid arthritis, and Crohns disease. PAF-AH deciency was found to be more frequent in Japanese children with severe asthma, and was accounted for by a single point mutation at position 279 (V279F) on the PAFAH. The significance of plasma PAF-AH deciency is unclear since those decient in plasma PAF-AH bronchoconstrict equally to inhaled PAF as those with normal PAF-AH levels.

PAFs
Although most studies have concentrated on two molecular species of PAF (i.e., C16-PAF and C18-PAF), stimulated inammatory cells also synthesize and release a variety of PAF homologs and analogs, including saturated and unsaturated alkyl-chain homologs and 1-O-acyl-PAF analogs and acetylated glycerylphosphoethanolamine. Some human cell types (such as neutrophils, eosinophils, and macrophages) produce almost exclusively PAF on stimulation. Other cell types (mast cells, basophils, and endothelial cells)

Introduction
Platelet-activating factor (PAF) or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine was rst discovered as an active moiety released from rabbit basophils after stimulation with immunoglobulin E (IgE). PAF has turned out since to be a pleiotropic lipid mediator of physiologic and pathophysiologic events occurring during inammation.

590 LIPID MEDIATORS / Platelet-Activating Factors

Remodeling De novo Ether-linked phospholipid l-Alkyl-2-acetylsn-glycerol

Membrane phospholipid l-Alkyl-2-acyl-sn-GPC

Phospholipase A2

Acyltransferase

Arachidonic acid Acetyltransferase

l-Alkyl-2-lyso-sn-GPC (lyso-PAF)

Cholinephosphotransferase Acetylhydrolase

Eicosanoids O PGs LTs CH3 C O

l-Alkyl-2-acetyl-sn-GPC (PAF) (CH2)n O O P O O

CH2 CH CH2

CH3 n = 15,17 Choline

Figure 1 Synthesis and catabolism of PAF. GPC, glycerol-3-phosphocholine; PGs, prostaglandins; LTs, leukotrienes. Reproduced with permission from Chung KF (1992) Platelet-activating factors in inammation and pulmonary disorders. Clinical Science 83: 127138, & the Biochemical Society and the Medical Research Society.

produce predominantly 1-alkyl-2-acyl-sn-glycero-3phosphocholine. This prole of PAF generation also depends on the stimulus; for example, human basophils synthesize predominantly 1-alkyl-2-acyl-snglycero-3-phosphocholine in response to the calcium ionophore A23187. Lung tissue can also be stimulated via cell-surface IgE to produce a large proportion of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine relative to PAF. The biological activities of these PAF analogs and homologs can differ. Alkyl-chain PAF homologs are also active in stimulating human neutrophil in vitro when compared with C16-PAF. There are differences in rank orders of potency of alkyl and acyl PAF molecules in vitro on human neutrophils and in vivo thrombocytopenic and leukopenic effects when injected into rabbits. 1-alkyl-2-acyl-sn-glycero-3phosphocholine is a relatively weak stimulus of human neutrophils and platelets, and may actually inhibit some of the stimulating effects of PAF on the human neutrophil.

Cellular Sources
A wide range of cell types produce PAF in vitro in response to several stimuli. Within a few minutes of

activation of neutrophils by opsonized zymosan or the calcium ionophore A23187, PAF is synthesized, although only 34% is released. Leukotriene B4 also stimulates PAF biosynthesis and 5-hydroxyeicosatetraenoic acid synergistically increases PAF-stimulated PAF synthesis, and PAF itself could stimulate PAF acetyltransferase in neutrophils. The cytokine granulocytemacrophage colony stimulating factor (GM-CSF) can augment the production of PAF and increase the activity of acetyltransferase in human neutrophils. Eosinophils from patients with eosinophilia show high activity of acetyltransferase and release PAF after stimulation with various chemotactic factors. Human eosinophils contain the most abundant PAF precursor, 1-alkyl-2-acyl-sn-glycero-3-phosphocholine. Puried human hypodense eosinophils release PAF after IgE (but not IgG) mediated activation. Human alveolar macrophages obtained by bronchoalveolar lavage of allergic asthmatic patients also release small amount of PAF after stimulation in vitro. Human alveolar macrophages also produce and release a large amount of lyso-PAF after stimulation with the calcium ionophore A23187 or after IgEmediated mechanisms. Cultured human endothelial cells release a small percentage of synthesized PAF

LIPID MEDIATORS / Platelet-Activating Factors

591

after activation by a calcium ionophore, bradykinin, thrombin, and IL-1. Puried human lung mast cells also generate PAF when stimulated with anti-IgE, but most of the PAF is also retained intracellularly. Human lymphocytes appear to lack acetyltransferase activity. PAF biosynthesis can be stimulated in human monocytes in response to the cytokines IL-1b, TNF, and interferon alpha, in a biphasic process. During the rst 2 h of stimulation, most of the PAF is retained, whereas during the later phase of activation, PAF is largely secreted.

This type of action of PAF may lead to the priming and activation of leukocytes at surfaces of endothelial cells that have been activated. One advantage of this mechanism to PAF is that there is less chance of PAF being rapidly degraded by acetylhydrolases and ensures that only low concentrations of PAF are needed. Thus, PAF may be considered as a juxtacrine signaling molecule.

PAF Receptors
PAF activates specic membrane receptors (Figure 2). Intracellular endosomal receptors have also been described but their function is unknown. The PAF receptor is a serpentine G-protein-coupled receptor containing seven a-helical membrane-spanning domains and is linked to phosphoinositide metabolism by a G-protein coupled to phospholipase C and A2. Stimulation of PAF receptors causes transient production of diacyglycerol, which activates protein kinase C, and of inositol triphosphate which leads to the release of internal calcium stores. PAF stimulates tyrosine phosphorylation of many proteins in neutrophils, macrophages, and platelets, induces the stimulation of nuclear factor kappa B (NF-kB) and transcription of c-fos and c-jun in inammatory cells. PAF can activate a mitogen-activated protein kinase (MAPK) kinase-3, a known activator of p38 MAPK,

Role for Intracellular PAF

Many cell types such as neutrophils, endothelial cells, and macrophages can produce large amounts of PAF but retain it within the cell. This has led to the possibility that PAF may have an intracellular role as a second messenger. The ability of endotoxin to prime neutrophils with an enhanced secretion of superoxide anions may be related to the primed increase in intracellular PAF. Intracellular PAF in endothelial cells stimulated by agents such as thrombin or leukotrienes may mediate neutrophil adherence. Thus, PAF generated intracellularly in endothelial cells can be translocated by mechanisms that are not yet clear to the outer part of the surface membrane, where it can bind to the PAF receptor on target neutrophils (or eosinophils).
PAF Acetyl Choline NH2 P O

Extracellular space PAF receptor

n2 P Base

n1 G-protein PLA2 GTP GTP G-protein PLC

n1

n2

n1 n2 DAG OH DAG PKC Ca2+

PtdInaP2 O P O

Arachidonic acid

COOH Eicosanoids Endoplasmic reticulum InsP3

Inositol-P2

Protein phosphorylation Cytosol

InsP3

Ca2+

Figure 2 Schematic representation of the PAF receptor and PAF signaling pathways. PLA2, phospholipase A2; PLC, phospholipase C; PtdinsP2, phosphatidyl 4,5-bisphosphate; Inositol-P2, inositol 4,5-bisphosphate; InsP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C. Reproduced from Montuccio G, Alloatti G, and Camussi G (2000) Role of platelet-activating factor in cardiovascular pathophysiology. Physiological Reviews 80: 16691699, used with permission from The American Physiological Society.

592 LIPID MEDIATORS / Platelet-Activating Factors

and also the Jak/STAT pathway. Following ligand activation, the PAF receptor is degraded via both the proteasome and lysosomal pathways. Potent selective PAF antagonists have been developed from natural products (e.g., ginkgolides and kadsurenone) or by synthesis (e.g., phospholipid analogs, tetrahydrofuran derivatives, and triazolobenzodiazepine derivatives). Another approach to inhibiting the effects of PAF has been to use recombinant PAF-AH.

5-lipoxygenase activity. GM-CSF also enhances PAF-induced eosinophil accumulation in the lungs of guinea pigs.

Actions of PAF in the Lung

Airways

Effects of PAF on Cell Activation and Lipid Metabolism

PAF, at picomolar concentrations, causes aggregation of washed rabbit and human platelets, with release of 5-hyroxytryptamine (serotonin). PAF causes the release of lysosomal enzymes and superoxide anions, the generation of LTB4, and chemotaxis from neutrophils. PAF causes the release of the granule-associated enzyme eosinophil peroxidase from human and guinea pig eosinophils. Eosinophils activated by PAF induce tracheal epithelial shedding in vitro, associated with a slowing of ciliary beat frequency. It induces a dose-dependent enhancement of eosinophil cytotoxicity. PAF is a potent chemotactic and chemokinetic mediator for eosinophils in vitro and promotes the adhesion of neutrophils and eosinophils to vascular endothelial cell. PAF inhibits human lymphocyte proliferation when stimulated with mitogens such as phytohemagglutinin or concanavalin A and also suppresses IL-2 production. At higher concentrations of PAF, CD4 T-cell proliferation is inhibited. PAF induce suppressor cells, accompanied by an increase in CD8 T cells and a small decrease in CD4 T cells. PAF interacts with cytokines and arachidonic acid products. Cleavage of the preformed precursor of PAF liberates two important precursor molecules, lyso-PAF and arachidonic acid, which are then metabolized to PAF and lipoxygenase and/or cyclooxygenase products. LTB4 and PAF production from neutrophils stimulated by the ionophore A23187 are tightly coupled while PAF itself can stimulate LTB4 synthesis in the neutrophil and the eosinophil. IL-1b, TNF, and interferon alpha can induce the synthesis of PAF from human monocytes and GM-CSF can augment the production of PAF from human neutrophils. PAF can also increase the release of cytokines such as TNF and IL-1 from alveolar macrophages and monocytes, respectively. Preincubation of blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and

PAF induces contraction of human bronchial smooth muscle in vitro. When inhaled by humans, PAF is a potent bronchoconstrictor but there is a rapid development of tachyphylaxis with repeated doses; the bronchoconstrictor effect is inhibited by Cyst-LT1 receptor antagonists. PAF induces a rapid increase in microvascular leakage throughout the respiratory tract, an effect independent of circulating platelets and of eicosanoid generation. Inhalation of PAF in normal subjects caused an increase in alveolararterial oxygen partial pressure gradient and a fall in the arterial oxygen partial pressure, together with ventilation/perfusion mismatching. PAF stimulates the secretion of mucus from tracheal explants, secondary to lipoxygenase activation. PAF increases the protein content of tracheal uid by inducing plasma extravasation. PAF also induces small changes in ion transport and epithelial conductance. Inhaled PAF impairs tracheobronchial mucociliary clearance in normal subjects. PAF causes an increase in bronchial responsiveness to metacholine in normal subjects and in a variety of animal species, including guinea pig, dog, and sheep; however, asthmatic patients do not demonstrate an increase in bronchial responsiveness after PAF.

Circulating and Bronchovascular Cells

PAF causes a profound neutropenia immediately after inhalation, followed by a rebound neutrophilia. Circulating neutrophils become hypodense within 15 min of inhalation as a result of an increase in cell volume. The falls in circulating blood cell levels could be due to a combination of hemodynamic factors, such as a slowing of pulmonary blood ow and increased adhesiveness of cells to the pulmonary vascular endothelium. Radiolabeled neutrophils transiently accumulate within the pulmonary vasculature after inhalation of PAF. PAF inhalation causes neutrophil inux into the airways of normal volunteers probably through increasing the expression of the b2-integrins, Mac-1 (CD11a/CD18) and LFA-1 (CD11b/CD18) on neutrophils. Intradermal injection of PAF in allergic subjects causes the selective accumulation of eosinophils. PAF induces eosinophilic inltration of the airways of guinea pigs.

LIPID MEDIATORS / Platelet-Activating Factors Pulmonary Vasculature and Microvascular Permeability

593

Infusion of PAF in sheep induces an acute transient rise in pulmonary arterial pressure, together with an increase in the lung lymph ow and the lymph-toplasma protein concentration ratio, indicating an increase in pulmonary microvascular permeability. PAF infusion causes pulmonary edema in many lung preparations, an effect that may be dependent on leukotrienes. PAF may mediate pulmonary edema through acid sphingomyelinase-dependent production of ceramide, and activation of the cyclooxygenase pathway particularly through the production of prostaglandin E2. In the rat, low doses of PAF reduced pulmonary hypertension induced by hypoxia, prostaglandin F2a, or norepinephrine. On the other hand, chronic infusion of PAF into rabbits over a 4-week period resulted in pulmonary hypertension, an enlargement of the right ventricle, and internal thickening and a decreased number of small pulmonary arteries.

PAF in Pulmonary Diseases

Because of its wide range of effects on cellular function and on the priming of the inammatory response, PAF has been considered as a mediator of inammation in many pulmonary diseases, including asthma and allergy, acute respiratory distress syndrome (ARDS), and pulmonary hypertension.
Asthma and Allergic Diseases

lacking PAF receptor do not develop bronchial hyperresponsiveness following chronic allergen exposure, but have persistent lung inammation. Recombinant PAF-acetylhydrolase (r-PAF-AH), which converts PAF into inactive lyso-PAF, inhibited allergen-induced bronchial hyperresponsiveness, mucus hypersecretion, and airway eosinophilia in a sensitized murine model. However, no such effects were found in asthmatic subjects undergoing allergen challenge when pretreated with r-PAF-AH. PAF receptor antagonists have been used to probe the role of PAF. They inhibit eosinophil inltration in the airways and bronchial hyperresponsiveness induced by ovalbumin in sensitized guinea pigs, and to reduce allergen-induced late phase response in allergic sheep and rabbits. However, in allergic asthmatic patients, the PAFWEB 2086 and MK-287 had no significant effects on the late phase response. Overall, while PAF may have a role in animal models of allergic inammation, it appears to contribute insignificantly in human asthma. This is consistent with a lack of clinical effect of a PAF antagonist in the treatment of mild-to-moderate asthma or of acute exacerbations of asthma.
Acute Respiratory Distress Syndrome

Because PAF can mimic many of the features of asthma such as bronchoconstriction, bronchial hyperresponsiveness, airway edema, and eosinophil chemotaxis and activation, it has been implicated in this disease. PAF has been measured in plasma and bronchoalveolar lavage uid from asthmatic patients. An increase in PAF activity derived from acetylation of lyso-PAF and detected using a platelet-aggregating assay has been observed in the plasma of asthmatic patients during the late asthmatic response, but not in those with a single immediate response after antigen challenge. Plasma PAF levels increased immediately after allergen challenge of mild seasonal asthmatic subjects, but not after methacholine challenge. High levels of lyso-PAF have been detected in bronchoalveolar lavage uid from allergic subjects after allergen challenge, but no significant increase in PAF levels was seen. Studies in mice support a role for PAF in allergic inammation. Transgenic mice overexpressing PAF receptors demonstrate bronchial hyperresponsiveness to inhaled methacholine. In addition, mice

Acute respiratory distress syndrome (ARDS) is a complex clinical syndrome characterized by refractory hypoxemia and high-permeability pulmonary edema in the presence of a normal pulmonary wedge pressure. PAF can cause alveolar-capillary damage, high-permeability pulmonary edema, pulmonary vasoconstriction, and bronchoconstriction. Lungs taken from PAF-treated animals reveal frank damage of endothelial cells. A PAF antagonist inhibited hypoxemia, pulmonary edema, and the increased permeability of the alveolar capillary membrane after endotoxin-induced lung injury in pigs. In overexpressing PAF receptor mice, acid-induced lung injury, lung edema, and hypoxemia were increased, while in mice with PAF receptor gene disruption, these effects were reduced. In a trial of patients with severe sepsis but without established ARDS, r-PAF-AH showed a trend towards prevention of ARDS and death.
Pulmonary Vascular Disease

The role of PAF in the regulation of the normal pulmonary circulation remains unclear. Lung homogenates obtained from normal rats contain measurable levels of PAF and PAF is released into bronchoalveolar lavage uids in rats exposed to hypoxia. High plasma levels of PAF have been detected in persistent pulmonary hypertension in the newborn. In some experiments, hypoxic pulmonary vasoconstriction

594 LIPID MEDIATORS / Lipoxins

can be reversed by exogenous PAF while it can be reduced by PAF antagonist WEB 2086. These conicting observations are difcult to reconcile.

Conclusion
The biosynthesis, metabolism, cellular response, and control of PAF is complex. Its role in normal homeostasis and inammation remains to be dened. By acting both as an intercellular and an intracellular messenger, it may participate as a cell-to-cell communicator and in so doing allow cooperation of cells in recruitment and activation of leukocytes to inammatory sites both under physiological or pathophysiological conditions. This would also be a mechanism for priming of inammation. The fact that no specic benecial effects in several pulmonary diseases has been observed in humans with inhibition of PAFs effects does not exclude these important roles for PAF as a mediator of inammation. A more localized targeted approach on PAF may be required.
See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Endothelial Cells and Endothelium. Leukocytes: Eosinophils; Neutrophils; Pulmonary Macrophages. Vascular Disease.

Montuccio G, Alloatti G, and Camussi G (2000) Role of Plateletactivating factor in cardiovascular pathophysiology. Physiological Reviews 80: 16691699. Nagase T, Ishii S, Shindou H, Ouchi Y, and Shimizu T (2002) Airway hyperresponsiveness in transgenic mice overexpressing platelet activating factor receptor is mediated by an atropinesensitive pathway. American Journal of Respiratory and Critical Care Medicine 165: 200205. Prescott SM, Zimmerman GA, Stafforini DM, and McIntyre TM (2000) Platelet-activating factor and related lipid mediators. Annual Review of Biochemistry 69: 419445. Rodriguez-Rosin R, Felez MA, Chung KF, et al. (1994) Plateletactivating factor causes ventilation-perfusion mismatch in humans. Journal of Clinical Investigation 93: 188194. Tjoelker LW and Stafforini DM (2000) Platelet-activating factor acetylhydrolases in health and disease. Biochimica et Biophysica Acta 1488: 102123. Tokuyama K, Lo tvall JO, Barnes PJ, and Chung KF (1991) Mechanism of airway narrowing caused by inhaled platelet-activating factor. Role of airway microvascular leakage. American Reiview of Respiratory Disease 143: 13451349.

Lipoxins
C N Serhan and B D Levy, Brigham and Womens Hospital at Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Lipoxins (LXs) are lipoxygenase interaction products of arachidonic acid metabolism that are distinct from leukotrienes and prostaglandins in structure and function. This interesting class of eicosanoids is generated via bidirectional transcellular biosynthesis during multicellular host responses to inammation, infection, or injury. Similar to other autacoids, LXs are rapidly formed, act locally, and then are rapidly inactivated by specic metabolic enzymes. Unlike the proinammatory prostaglandins and leukotrienes, LXs display counterregulatory actions. A broad (and growing) array of cell-type specic biological actions has been uncovered for LXs that together serve to promote resolution of acute inammatory responses. When administered to human cells in vitro or murine systems in vivo, at least two classes of receptors, CysLT1 receptors and LXA4 receptors (designated ALX), can interact with LXs to mediate their actions. LXs are generated during several thoracic inammatory conditions, including airway and parenchymal lung diseases and exudative pleural effusions. Of interest, respiratory diseases of chronic inammation, such as severe asthma and cystic brosis, display defective generation of these protective lipid signals. Together, these ndings indicate a pivotal role for LXs in mediating respiratory homeostasis.

Further Reading
Benveniste J, Henson PM, and Cochrane CG (1972) Leukocytedependent histamine release from rabbit platelets: the role of IgE, basophils, and a platelet-activating factor. Journal of Experimental Medicine 136: 13561377. Chung KF (1992) Platelet-activating factor in inammation and pulmonary disorders. Clinical Science 83: 127138. Goggel R, Winoto-Morbach S, Vielhaber G, et al. (2004) PAFmediated pulmonary edema: a new role for acid sphingomyelinase and ceramide. Nature Medicine 10: 155160. Gomez FP, Marrades RM, Iglesia R, et al. (1999) Gas exchange response to a PAF receptor antagonist, SR 27417A, in acute asthma: a pilot study. European Respiratory Journal 14: 622626. Henig NR, Aitken ML, Liu MC, Yu AS, and Henderson WR Jr. (2000) Effect of recombinant human platelet-activating factoracetylhydrolase on allergen-induced asthmatic responses. American Journal of Respiratory and Critical Care Medicine 162: 523527. Ishii S, Nagase T, and Shimizu T (2002) Platelet-activating factor receptor. Prostaglandins and Other Lipid Mediators 6869: 599609. Ishii S, Nagase T, Shindou H, et al. (2004) Platelet-activating factor receptor develops airway hyperresponsiveness independently of airway inammation in a murine asthma model. Journal of Immunology 172: 70957102. Kuitert LM, Hui KP, Uthayarkumar S, et al. (1993) Effect of a platelet activating factor (PAF) antagonist UK-74, 505 on allergen-induced early and late response. American Review of Respiratory Disease 147: 8286.

Introduction
Enzymatic oxygenation of arachidonic acid occurs in a wide range of human cell types in the lung and results in the formation of several classes of biologically active products, termed eicosanoids because they contain 20 carbons. These compounds are involved in the regulation of inammatory reactions,

LIPID MEDIATORS / Lipoxins 595

smooth muscle tone, hemodynamic events, allergy, and asthma, as well as many other important physiological and pathophysiological processes. Lipoxygenase (LO)-derived eicosanoids display diverse and potent actions on leukocytes, endothelia, and epithelia. While studying the transformation of [1-14C]arachidonic acid in suspensions of mixed human leukocytes, Charles Serhan and colleagues in 1984 reported that labeled arachidonate was transformed into polar compounds with physical properties distinct from prostaglandins (PGs), thromboxanes, and leukotrienes. Of particular interest was the initial 15-lipoxygenation of arachidonate by activated leukocytes because this enzyme is abundant in the lung and active during airway inammation. 15-lipoxygenase (15-LO)-derived intermediates were determined to be substrates for further conversion by leukocyte-derived 5-LO to novel derivatives, which were named lipoxins (lipoxygenase interaction products). It is now appreciated that lipoxin (LX) formation occurs in vivo and is conserved across species, from sh to humans. Further investigation has uncovered additional biosynthetic pathways, intriguing biological actions, and routes for metabolic inactivation.

Structure
LXs are trihydroxytetraene-containing eicosanoids. 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid is also called lipoxin A4 (LXA4) and its positional isomer, 5S,14R,15S-trihydroxy-6,10,12trans-8-cis-eicosatetraenoic acid, is denoted lipoxin B4 (LXB4) (Figure 1). The structural features of LXs are ideally suited for spectroscopic methods in their detection. The conjugated tetraene imparts a UV absorption spectrum on both LXA4 and LXB4, with three intense absorbance bands at lMeOH 278, 300, max and 315 nm. A fourth, weaker absorbance band at 270 nm is also present. The most intense absorbance band at 300 nm, with a molar extinction coefcient of 50 000 M 1 cm 1, is typically used for UV quantication. In addition, LXs tetraene structure provides uorescent properties for quantitation with an emission maximum at 410 nm with an excitation maximum at 320 nm at room temperature. Several different extraction and chromatographic methods have been employed for LX isolation from complex sample matrices. The charge state of the LX carboxylic acid can be easily changed to alter the afnity of the compound for chromatography

COOH Airway

Arachidonic acid EPI ALOX15 WBC ALOX5 O

COOH

COOH

OH 15(S ) - HETE

LTA4

HO

OH COOH

OH COOH

OH LXA4

OH HO LXB4

Figure 1 LX generation in airway inammation. As circulating leukocytes (WBC) trafc into the interstitium and airspaces of the lung, their 5-lipoxygenase activity (WBC ALOX5) can collaborate with airway epithelial 15-lipoxygenase activity (EPI ALOX15) to generate LXA4 and LXB4 during cellcell interactions. Biosynthetic intermediates, such as 15(S)-hydroxy-eicosatetraenoic acid (15(S)-HETE) from ALOX15 and LTA4 from ALOX5, can serve as substrate for conversion to LXs by the complementary enzyme during transcellular biosynthesis.

596 LIPID MEDIATORS / Lipoxins

stationary phases. For isolation by solid phase extraction, samples are acidied to protonate LXs prior to loading onto a C18 (i.e., octadecyl) cartridge. Following loading, the cartridge is rst washed with water to remove more polar moieties, such as salts, and then with hexane to remove more nonpolar materials. LXs are then eluted with a solvent of intermediate polarity, such as methyl formate. Because the tetraene geometry and conformation are fragile, careful handling of LXs is required to prevent cis- to trans-double bond isomerization. In this regard, LXs are thermally labile and sensitive to light. Therefore, they should be kept cold in an organic solvent, such as methanol, and in an environment enriched with nitrogen to prevent non-specic oxygenation. By taking advantage of LXA4s unique overall three-dimensional conformation, enzyme-linked immunosorbent assays (ELISAs) have been developed for rapid detection of LXA4 as well as stereoisomerspecic detection of 15-epi-LXA4 (vide infra). These ELISAs are commercially available, show no crossreactivity for 5S-HETE, 12S-HETE, 15S-HETE, LTB4, LTC4, LTD4 or arachidonic acid, and have detection limits of approximately 100 fmol ml 1.

Regulation of Production
At sites of injury or inammation, LXs are generated via biosynthetic routes engaged during cellcell interactions. 15-LO is present in airway epithelial cells, eosinophils, and other leukocytes, and is capable of both initiating LX biosynthesis as well as converting the leukocyte 5-LO-derived intermediate LTA4 to LX. In the lung, mobilization of an LX biosynthetic circuit occurs, for example, when inltrating polymorphonuclear leukocytes (PMN) (WBC ALOX5, Genbank accession #NP 000689 interact with airway epithelia (EPI ALOX15, Genbank accession #NP 001131) in inamed target organs (Figure 1). In addition to this 15-LO pathway, LX formation also occurs in the vasculature during plateletleukocyte interactions by platelet 12-LO-catalyzed conversion of leukocyte LTA4. While human platelets are unable to generate LXs on their own, they can be a major source of LXA4 and LXB4 when adherent to activated neutrophils (PMNs) at sites of vascular injury or inammation. At both anti-inammatory and low-dose, aspirin therapy can promote LX biosynthesis. Aspirin-acetylated cyclooxygenase-2 (COX-2) no longer produces PGs, but remains catalytically active in cells to generate the endogenous LX mimetics, 15-epi-lipoxins (15-epi-LXs). These pathways illustrate anti-inammatory mechanisms that can account for some of aspirins benecial actions. Because LXs and the aspirin-triggered 15-epi-LXs

display leukocyte-selective actions, they are the main candidates for functional roles as endogenous mediators of resolution for acute inammation. Like other autacoids, LXs are rapidly formed to act locally and rapidly inactivated by metabolic enzymes via pathways shared with other eicosanoids. LXs are primarily inactivated via dehydrogenation converting LXA4 to 15-oxo-LXA4, and LXB4 to 5-oxo-LXB4, followed by specic reduction of the double bond adjacent to the ketone. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of LXA4 and LXB4 to their oxo-metabolites. This compound is biologically inactive and, in the case of 15-oxo-LXA4, further converted to 13,14-dihydro15-oxo-LXA4 by the action of eicosanoid oxidoreductase (EOR) (also known as LXA4/PGE 13,14-reductase/LTB4 12-hydroxydehydrogenase). Reduction of the 15-oxo-group by 15-PGDH yields 13,14-dihydro-LXA4, which reveals an additional catalytic activity for this enzyme. LX dehydrogenation is highly stereospecific, as compared with native LXA4, 15-epi-LXA4 is converted less effectively to the 15-oxo-metabolite. When 15-epi-LXs are generated, their biologic half-life is increased by about twofold greater than LXA4, thereby enhancing their ability to evoke bioactions. LXB4 shares a common route of inactivation with LXA4. LX structural analogs have been constructed with specic modications of the native structures of LXA4 and LXB4 to block metabolic inactivation by 15-PGDH. Modications have included synthesis of an LXA4 analog that has a phenoxy group bonded to carbon-16 to replace the o-end of the molecule to protect from dehydrogenation in vivo and addition of halide to the para-position of the phenoxy ring to hinder degradation further. A 15-epi, 16-para-uorophenoxy-lipoxin A4 analog is active and, like LXA4 and 15-epi-LXA4, signals via competition for endogenous lipoxin A4 receptors (Figure 2). Thus, these modications to native LXA4 serve to prolong in vivo half-life in the circulation and enhance both the pharmacokinetic and biological properties of the compound. In response to inammatory stimuli, the in vivo generation of eicosanoid classes is temporally staged with increases in LXA4 levels in experimental exudates that occur later than maximal LTB4 and PGE2, coincident with decreases in exudate PMN during resolution of acute inammation. The expression and activity of the LX biosynthetic enzymes 5-LO and 15-LO are regulated by inammatory mediators, including select cytokines and early response lipids (e.g., PGE2) to increase LX biosynthetic capacity at sites of inammation. During an inammatory program, early administration of a metabolically stable

LIPID MEDIATORS / Lipoxins 597

HO OH

COOH X LXA4 stable analog HO OH COOH

O HO OH OH COOH

OH

LXA4

OH 15-epi-LXA4

ALX

G LSP-1

Cell-type specific responses: Neutrophils stops adhesion, chemotaxis, transmigration, degranulation Eosinophils stops chemotaxis, allergen-induced trafficking T-lymphocytes stops cytokine release, NK cell cytotoxicity Monocytes stimulates adhesion, chemotaxis, phagocytosis of apoptotic PMNs Epithelial cells stops cytokine release and promotes bacterial killing
Figure 2 ALX signals for cell-type specic responses. LXA4, 15-epi-LXA4, and bioactive LXA4 stable analogs can interact with ALX to regulate leukocyte and nonmyeloid cell functional responses in a cell-type specic manner. ALX signals LXs anti-inammatory effects in PMN, in part, by blocking phosphorylation of leukocyte-specic protein-1 (LSP-1) and activation of the p38-MAPK cascade.

LXA4 analog inhibits PMN recruitment, providing evidence for a functional link between LXA4 and cessation of PMN inltration. Increased LX metabolism by local instillation of recombinant EOR (pivotal to LX endogenous inactivation) dramatically exacerbates PMN inltration at sites of inammation. Together, these ndings indicate that the onset of LX formation is delayed from PGE2 and LTB4 in acute inammation and that LXA4 is a regulator of PMN trafcking during the progression of acute inammatory responses.

Biological Function
LXA4, 15-epi-LXA4 and bioactive structural analogs display leukocyte selective actions (Figure 2). This is exemplied by LXA4, which, on the one hand, is a potent stop signal for neutrophil chemotaxis, adhesion, transmigration, azurophilic granule degranulation, and superoxide anion generation, and on the other hand, stimulates monocyte locomotion, adherence, and monocyte-derived macrophage phagocytosis of apoptotic PMNs. Like PMNs, eosinophil chemotaxis and tissue accumulation, dendritic cell mobilization and IL-12 release, T-lymphocyte cytokine release, and natural killer (NK) cell cytotoxicity

are all inhibited by LXA4. In sum, LXs regulate both innate and acquired immune effector cells to promote resolution of acute inammation. Many nonmyeloid cell functional responses are also regulated by LXs. LXA4 is a potent regulator of proinammatory cytokine and chemokine release and gene expression via nuclear factor kappa B (NFkB) in broblasts and epithelial cells (Figure 2). In addition, LXA4 blocks endothelial P-selectin expression, renal mesangial cell and adenocarcinoma-transformed airway epithelial cell proliferation, and hepatocyte PPARa (peroxisome proliferator-activated receptor a) signaling. Distinct from an immunosuppressive compound, LX signaling in epithelial cells promotes expression of bacterial permeability-inducing protein to enhance mucosal bacterial killing. Thus, in addition to anti-inammation, LXA4 signals for host protection. These cellular effects have now been investigated in a wide range of experimental animal models through the use of LXA4 stable analogs and generation of transgenic animals that either overexpress 15-LO or utilize a component of the CD11b promoter to target myeloid expression of the human lipoxin A4 receptor (ALX). Outside the lung, LXA4 signaling increases glomerular ltration rate, renal

598 LIPID MEDIATORS / Lipoxins

plasma ow rate, and protects renal tissues from ischemic acute renal failure and glomerulonephritis. In the gastrointestinal tract, LXA4 decreases leukocyte rolling and can promote detachment to lessen mucosal injury from gastric and colonic toxins. LXA4 signaling blocks the development of allergy in a murine model of asthma and promotes resolution of allergic pleural edema. Dermal, ocular, peritoneal, and periodontal inammation is also potently regulated by LXA4. Increased 15-LO activity also increases LX formation in vivo and in 15-LO transgenic rabbits protects against the development of atherosclerosis and periodontitis. Human ALX transgenic mice display markedly decreased inammatory responses to zymosan-induced peritonitis and experimental asthma. LX signaling also potently regulates pulmonary responses, including relaxation of precontracted pulmonary arteries and bronchi, prevention of PMN-mediated second organ injury in the lung from hind limb ischemia, and inhibition of both airway hyperresponsiveness and allergic inammation in experimental asthma (vide infra). These and other ndings with LX generation and impact in vivo in the lung are reviewed in Table 1.
Table 1 Action of LXs and 15-epi-LXs in the lung animal models and human diseases Action/impact Animal model Asthma Inhibit airway hyperresponsiveness and allergic inammation

LXs have been identied in human subjects with a wide range of illnesses involving leukocyte activation. Of interest, a lower biosynthetic capacity of these potentially protective signals (i.e., LX or 15epi-LX) is linked to more severe or chronic forms of disease, including chronic myelogenous leukemia, chronic liver disease, aspirin-intolerant and severe asthma, and cystic brosis. Because of the rapid inactivation of LXs, there is only limited information on their actions in humans in vivo. In one clinical trial, LXA4 was administered to individuals with asthma, leading to protection from LTC4-induced bronchoconstriction. The recent development of new LX stable analogs (Figure 2) that are topically and orally active should enable further investigation on LX regulation of human illness. Together, ndings on the biological functions of LXs indicate that altered LX or 15-epi-LX production can regulate both inammation and organ-specic functions and is mechanistically related to the pathogenesis of human disease.

Receptors
The actions of LXs can be attributed to interactions at one or more specic receptors with three potential mechanisms: (1) acting at their own specic receptor; (2) interacting with a subclass of LTD4 receptors (i.e., CysLT1); or (3) acting at intracellular recognition sites after transport and uptake (or within its cell of origin). Specic LXA4 binding sites were rst identied on human PMNs. ALX (also known as formyl peptide receptor like-1 and formyl peptide receptor-2) is also expressed in several other mammalian cells and tissues, including human monocytes, enterocytes, synovial broblasts, murine spleen and lung, and rat leukocytes. ALX expression is dramatically induced in vitro by cytokines in airway epithelial cells and enterocytes, most notably by interleukin-13 and interferon-g. This cytokine regulation is also evident in vivo during disease states, as ALX is upregulated in airway epithelial and inammatory cells during allergic airway inammation in a murine model of asthma that has high levels of IL-13 in the respiratory tract. These functional receptors are 7-transmembrane spanning, G-protein-coupled proteins that bind LXA4 with high afnity (KD 1.7 nM) (Figure 2). Human, mouse, and rat ALX amino acid sequences share 74% or greater homology with the highest homology found in their second intracellular loop (100% identical) and sixth transmembrane segments (93% identical). As a class, ALX belongs to the cluster of chemoattractant peptide receptors. Of interest for asthma therapy, annexin 1 is induced by corticosteroids and enzymatically cleaved to peptides that,

Microbial infection Pseudomonas aeruginosa infection Angiostrongylus costaricensis infection Human lung diseases

A novel secreted microbial lipoxygenase

Shorten the duration of pleural exudation LX and 15-epi-LX stimulate host antimicrobial activity LXA4 detected in bronchoalveolar lavage uids from patients who have pulmonary disease and asthma LXA4 detected in human exudative pleural uids Production of LX by nasal polyps and bronchial tissue LXA4 inhalation shifts and reduces LTC4-induced contraction in patients who have asthma ASA-intolerant asthmatics display a lower biosynthetic capacity than ASA-tolerant patients LXA4 inhibits IL-8 release by monocytes in patients who have asthma LX generation decreased in severe asthma LX generation defective in cystic brosis

LIPID MEDIATORS / Lipoxins 599

similar to the lipids LXA4 and 15-epi-LXA4, can interact with ALX to initiate anti-inammatory circuits. In PMNs, ALX signals for the anti-inammatory effects of LXs, in part, by polyisoprenyl phosphate remodeling and by inhibition of leukocytespecic protein-1 (LSP-1) phosphorylation, a downstream regulator of the p38-mitogen-activated protein-kinase (MAPK) cascade. ALX was the rst receptor to be identied that binds both lipid and peptide ligands. In addition to low-afnity interactions with annexin-1 peptide and fMLP (KDB5 mM), ALX binds several other peptides in the micromolar range, including naturally produced, cleaved forms of the urokinase-type plasminogen activator. The functional consequences of lipid and peptide ligands for ALX differ, probably secondary to distinct intracellular signaling that depends on the cell type and system.

See also: Allergy: Allergic Reactions. Asthma: Overview; Aspirin-Intolerant. CD11/18. Cystic Fibrosis: Overview. Dendritic Cells. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. NADPH and NADPH Oxidase. Pleural Effusions. Pleural Fluid, Transudate and Exudate.

Further Reading
Badr KF, DeBoer DK, Schwartzberg M, and Serhan CN (1989) Lipoxin A4 antagonizes cellular and in vivo actions of leukotriene D4 in rat glomerular mesangial cells: evidence for competition at a common receptor. Proceedings of the National Academy of Sciences, USA 86: 34383442. Fiorucci S, Wallace JL, Mencarelli A, et al. (2004) A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inammation and immune dysfunction. Proceedings of the National Academy of Sciences, USA 101: 1573615741. Gilroy DW, Lawrence T, Perretti M, and Rossi AG (2004) Inammatory resolution: new opportunities for drug discovery. Nature Reviews: Drug Discovery 3: 401416. Godson C, Mitchell S, Harvey K, et al. (2000) Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. Journal of Immunology 164: 16631667. Karp CL, Flick LM, Park KW, et al. (2004) Defective lipoxinmediated anti-inammatory activity in the cystic brosis airway. Nature Immunology 5: 388392. Levy BD, Bertram S, Tai HH, et al. (1993) Agonist-induced lipoxin A4 generation: detection by a novel lipoxin A4-ELISA. Lipids 28: 10471053. Levy BD, Clish CB, Schmidt B, Gronert K, and Serhan CN (2001) Lipid mediator class switching during acute inammation: signals in resolution. Nature Immunology 2: 612619. Levy BD, De Sanctis GT, Devchand PR, et al. (2002) Multipronged inhibition of airway hyper-responsiveness and inammation by lipoxin A(4). Nature Medicine 8: 10181023. Ohira T, Bannenberg G, Arita M, et al. (2004) A stable aspirintriggered lipoxin A4 analog blocks phosphorylation of leukocyte-specic protein 1 in human neutrophils. Journal of Immunology 173: 20912098. Perretti M, Chiang N, La M, et al. (2002) Endogenous lipid- and peptide-derived anti-inammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor. Nature Medicine 8: 12961302. Samuelsson B, Dahlen SE, Lindgren JA, Rouzer CA, and Serhan CN (1987) Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science 237: 11711176. Serhan CN and Chiang N (2004) Novel endogenous small molecules as the checkpoint controllers in inammation and resolution: entree for resoleomics. Rheumatic Diseases Clinics of North America 30: 6995. Serhan CN, Fiore S, Brezinski DA, and Lynch S (1993) Lipoxin A4 metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products. Biochemistry 32: 63136319. Serhan CN, Jain A, Marleau S, et al. (2003) Reduced inammation and tissue damage in transgenic rabbits overexpressing 15lipoxygenase and endogenous anti-inammatory lipid mediators. Journal of Immunology 171: 68566865. Serhan CN, Maddox JF, Petasis NA, et al. (1995) Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils. Biochemistry 34: 1460914615.

LXs in Respiratory Diseases

Both LXs and 15-epi-LXs are generated during respiratory inammation. LXA4 (0.42.8 ng ml 1) has been detected in bronchoalveolar lavage and pleural uid from individuals with inammatory lung disease (Table 1). In an experimental model of asthma, LX formation at peak airway inammation is similar to LTB4, yet one to two log orders less than cysteinyl LTs and PGE2. An LX stable analog, designed using the 15-epi-LXA4 structure as a template, markedly inhibits (with as little as 1 mg; B0.05 mg kg 1) murine allergen-driven airway hyperresponsiveness and inammation. Targeted expression of human ALX to murine leukocytes in transgenic mice also dramatically inhibits allergen sensitization, eosinophil trafcking, and airway inammation. With an ED50 of less than 0.05 mg kg 1 in this murine model of asthma, the LX analog compares favorably with both the CysLT1 receptor antagonist montelukast (0.03 mg kg 1 in rats) and the synthetic glucocorticoid dexamethasone (0.53 mg kg 1 in mice). Thus, ALX activation by endogenous ligands or an LX stable analog evokes potent stop signals for airway responses. Diminished formation of these counterregulatory mediators has been identied in severe forms of human respiratory illness, including aspirinintolerant asthma, severe steroid-dependent asthma, and cystic brosis. Decreased production of these counterregulatory substances may predispose the host to more severe inammatory responses in the lung. Elucidation of endogenous regulators of LX pathways will provide further insight into the role of LX in inammatory lung disease and its potential as a new therapeutic strategy that emphasizes natural counterregulatory pathways.

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LUNG ABSCESS
H Koziel, Harvard Medical School, Boston, MA, USA
& 2006 Elsevier Ltd. All rights reserved.

Abstract
Lung abscess is a clinical condition characterized by focal suppurative necrosis of lung parenchyma. The incidence of lung abscess is unknown. Current concepts suggest that most lung abscesses occur as a complication of aspiration pneumonia and are often attributed to anaerobic organisms from the gingival crevices of the oral cavity. Patients typically present with indolent onset of fever, malaise, night sweats, weight loss, cough, pleurisy, and purulent sputum production, of several weeks duration and often with an antecedent history of loss of consciousness or aspiration. The chest radiograph typically demonstrates parenchymal radiolucency with surrounding thick wall, or a radiolucency with an airuid level, and may require chest CT to conrm the diagnosis. Anaerobic bacteria frequently account for the majority of community-acquired lung abscess cases (often polymicrobial), whereas anaerobes may account for most cases of hospital-acquired lung abscess (often a single pathogen). Evaluation of sputum is controversial and blood cultures are rarely positive, so isolating the etiological agent often requires invasive techniques. Treatment includes prolonged antimicrobial therapy, and in general, the prognosis is favorable. Surgical resection is reserved for patients with poor clinical responses. Techniques of percutaneous or endobronchial drainage may be effective alternatives for select patients with poor clinical responses who are considered high surgical risk.

Introduction
Lung abscess is a suppurative process often considered in the spectrum of anaerobic pleuropulmonary diseases that include aspiration pneumonia, necrotizing pneumonia, lung abscess and empyema. As abscesses can be of varied sizes, the distinction between necrotizing pneumonia (or pulmonary gangrene) and lung abscess is somewhat arbitrary as these entities represent a progression along a continuum of lung infections. Generally, lung abscess is often dened as X2 cm in diameter whereas abscesses o2 cm are considered necrotizing pneumonia. Lung abscess has been recognized since the ancient Greek times of Hippocrates. Anaerobic bacteria were rst identied in empyema uid in 1899, and subsequently determined in 1927 to be a critical component of lung abscess formation based on experimental animal data. The role of aspiration was appreciated in 1934 with the association of high rate of lung abscess complicating head and neck surgery performed in the upright sitting position. The benet of drainage of lung abscesses was realized in 1945,

and the introduction of penicillin was associated with an apparent reduction in the incidence (especially in persons o50 years of age) and mortality. The major role of anaerobes in lung abscess formation was conrmed during the 1970s through investigations using newly developed laboratory culturing techniques that allowed for improved isolation and culturing of anaerobic bacteria. The incidence of lung abscess is not known, although lung abscesses likely develop relatively infrequently and the incidence appears to have decreased since the introduction of antibiotics (especially penicillin). From 1943 to 1956, the Massachusetts General Hospital reported 1011 cases of lung abscess per 10 000 hospital admissions in the pre-antibiotic era compared to 12 cases per 10 000 admissions in the post-antibiotic era. The Beth Israel Deaconess Medical Centers experience during 198496 showed that lung abscesses represented approximately 0.2% of all cases of pneumonia requiring hospitalization. The decrease in cases of lung abscess is primarily attributed to early and broad use of effective antimicrobials, improved management of hospitalized unconscious patients, and improved management of patients undergoing anesthesia. Patients with lung abscess typically present with indolent onset of fever, malaise, night sweats, weight loss, cough, pleurisy, and purulent sputum production, of several weeks duration and often with an antecedent history of loss of consciousness or aspiration. The chest radiograph typically demonstrates parenchymal radiolucency with surrounding thick wall, or a radiolucency with an airuid level, and may require chest CT to conrm the diagnosis (Figure 1). Mortality for untreated lung abscess may approach 40%.

Etiology
Anaerobic bacteria are considered the most common pathogens in community-acquired lung abscess, reecting both pathogenic potential and representing the predominant component of normal ora of the upper airways (although any pathogen can create a lung abscess under the appropriate circumstances). Prior studies using transtracheal aspirates or transthoracic needle aspirates reported recovery rates of anaerobic bacteria from 85% to 93% of lung abscess cases. Anaerobes were the only isolates in up to 46% of cases, whereas 43% of cases had a mixture of aerobes and anaerobes (Table 1). Although anaerobic abscesses generally contain multiple anaerobic

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Table 1 Reported microbiology of lung abscess Anaerobic organisms (common)a Fusobacterium nucleatum Peptopstreptococcus species Prevotella melaninogenica Bacteroides species Clostridium species Eubacterium species Lactobacillus Propionibacteria Aeorobic organisms (common) Staphylococcus aureus Streptococcus pyogenes Klebsiella pneumoniae Pseudomonas aeruginosa Aeorobic organisms (uncommon) Escherichia coli Haemophilus inuenzae type B Other pathogens (rare) Nocardia asteroids Paragonimus westermani Legionella species Burkholdaria pseudomallei Burkholdaria mallei (glanders) Mycobacterium tuberculosis Mucoraceae species Aspergillus species Entameoba histolytica Organisms associated with lung abscess in immunocompromised hosts (including HIV) Pseudomonas aeruginosa Streptococcus pneumonia Pneumocystis jiroveci Klebsiella pneumoniae Staphylococcus aureus Haemophilus inuenzae Stenotrophomonoas maltophilia Legionella species (nonpneumophilia) Enterobacter species Streptococcus milleri Proteus mirabilis Cryptococcus neoformans Aspergillus species Mycobacteria (nontuberculous) Nocardia Rhodococcus Zygomycetes
a

Figure 1 Radiological demonstration of lung abscess development in a patient with necrotizing Staphylococcus aureus pneumonia. A 47-year-old HIV male admitted to the medical intensive care unit following an acute drug overdose and loss of consciousness. (a) Admission anteroposterior chest radiograph demonstrated a dense RUL inltrate, and bronchoscopy with quantitative BAL revealed Staphylococcus aureus; (b) chest CT scan on day 14 of hospitalization conrmed the interval development of a cavitary lesion in the posterior segment of the RUL despite appropriate antimicrobials.

isolates, occasionally lung abscesses may result from a single anaerobic organism recognized as highly virulent, such as Fusobacterium nucleatum or Peptopstreptococcus species. Anaerobes may also be in combination with aerobic or facultative anaerobic species such as microaerophilic streptococci. In contrast to community-acquired lung abscess, aerobic bacteria may be a more important etiological agent in hospital-acquired lung abscess. Nosocomial aspiration is often associated with Gram-negative bacteria and Staphylococcus aureus, including organisms with hospital-acquired antibiotic resistance patterns. Furthermore, the spectrum of microbiological agents responsible for lung abscess in

Mouth ora.

immunocompromised hosts may be quite different and distinct from that of immunocompetent patients. This is evident in the study of the Beth Israel Deaconess Medical Centers experience during 198496 in 34 cases of lung abscesses in adults. Aerobic bacteria were the most common isolates in both immunocompromised and non-immunocompromised patients. However, immunocompromised patients more often had multiple pathogens, and certain

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pathogens were exclusively isolated from the lung abscess of immunocompromised patients, including Pseudomonas aeruginosa, and Hemophilus spp. (non-inuenzae), Stenotrophomonas maltophilia, Haemophilus inuenza, Enterobacter spp., Klebsiella oxytoca, nonpneumophilia Legionella spp., Mycobacterium avium complex and Candida spp. Anaerobes were isolated in only 20% of the cases of non-immunocompromised patients, and not isolated in any specimens from immunocompromised patients. Other reports of lung abscess in HIV patients demonstrate bacteria in 65% of isolates, followed by fungi (9%), and mixed infections (16%), although despite effective treatment, high relapse (36%) and mortality (19%) rates were experienced. More recent data suggest that the microbiology of lung abscess may be evolving, perhaps in part in response to external pressures induced by the indiscriminate use of broad-spectrum antimicrobials, development of bacterial resistance, presence of multiple or complex medical comorbidities in patients, and the expanding population of immuosuppressed individuals. As the microbiology of communityacquired pneumonia changes (e.g., the expanding role of methicillin-resistant Staphylococcus aureus), the agents responsible for community-acquired lung abscess are likely to change. Although certain genetic diseases that result in chronic lung disease (e.g., cystic brosis, Kartangeners syndrome) or chronic infection (e.g., Mycobacterium tuberculosis) may predispose to lung abscess formation, there are no known specic genetic markers that identify persons at risk for the development of lung abscess.

Table 2 Predisposing conditions or risk factors to development of lung abscessa Primary lung abscessb Alcoholism Seizure disorder General anesthesia Drug abuse Esophogeal lesions Neurological deficits (e.g., CVA, bulbar disease) Insulin-treated diabetes mellitus Secondary lung abscessc Pulmonary embolism with infarction Septic emboli with pulmonary infarction Endobronchial obstruction by neoplasm (benign or malignant) Obstruction by foreign body Bronchiectasis Cryptogenic lung abscess Absence of identiable risk factor 1015% of patients with lung abscess may not have obvious risk factor. b Symptoms often indolent over weeks. c Symptoms often abrupt over days.
a

Pathology
Lung abscess is dened as a pus-containing necrotic lesion of the lung parenchyma. The cardinal histologic change in all abscesses is suppurative destruction of the lung parenchyma within the central area of cavitation. Lung abscess can be of any size, from a few millimeters to several centimeters, often manifest as a single lesion (e.g., as a complication of aspiration pneumonia), but can develop multiple foci (e.g., as a complication of right-sided endocarditis). Lung abscesses are often infected and generally develop after inammation and produce tissue necrosis with cavitation. In the absence of communication with an airway, the abscess cavity may be lled with suppurative debris. However, when the abscess cavity communicates with an airway, the contents may be partially drained to create an air-containing cavity. In cases of pre-existing cavitary lung disease (such as that associated with emphysema or prior

Mycobacterium tuberculosis infection), infection of the cavity may proceed without overt necrosis. The abscess cavity may become lined with regenerated lung epithelium, and in cases of chronic lung abscess, broblastic proliferation may result in a brinous wall. Early autopsy observations that bacteria in the walls of the lung abscess at autopsy were similar to the bacteria found in the oral gingival crevice (between the tooth and gum tissue) prompted investigators to hypothesize that aspiration was the major mechanism in disease pathogenesis. Acute lung abscess is arbitrarily dened as o4 weeks duration, whereas chronic lung abscess is 44 weeks duration, although the clinical implications of this distinction are not clearly established. Abscess formation as a consequence of aspiration and pneumonia in a relatively normal host are dened as primary lung abscess (Table 2). In contrast, abscess formation as a consequence of central airway obstruction, bronchiectasis, immunocompromised state, or due to hematogenous spread from a distant site is dened as secondary lung abscess. Cases with no recognized or identiable risk factors are termed cryptogenic lung abscesses. Approximately 80% of cases of lung abscesses are primary lung abscess.

Clinical Features
Lung abscess is suspected from the history and physical exam, but requires radiographic conrmation. Clinical suspicion for lung abscess should be heightened in persons with a recognized pre-existing

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condition that increases risk for development of lung abscess (Table 2). In primary lung abscess, symptoms of lung abscess are similar to other anaerobic lung infections, and include indolent (13 weeks) development of fever, cough, pleurisy, dyspnea, sweats, weight loss, and sputum production (often foulsmelling). Sputum production may be absent in the absence of a communication of the abscess cavity and the airways. Most patients present with fever (median T 39.11C) and leukocytosis (median peripheral WBC 15 000 cells mm 3). The production of putrid sputum occurs in approximately 49% of the cases, and is generally considered diagnostic of anaerobic infection (as aerobic bacteria are generally not capable of producing this characteristic odor). In patients with chronic sputum production (such as bronchiectasis or chronic bronchitis), a change in the character, quality, or quantity of sputum should raise suspicion of a new lung infection or development of lung abscess. In secondary lung abscess, the development of symptoms may be more rapid, evolving over days, and may include localized symptoms that provide a clue to the underlying condition predisposing to rapid lung abscess formation (such as endobronchial tumor or endocarditis with multiple septic emboli). Radiographic appearances include a radiolucency surrounded by a visible irregular thick wall, or an airuid level within an area of pneumonia. Chest radiographs generally reveal cavity formation distributed in dependent lung segments. In recumbent patients, these include the superior segments of the lower lobes and posterior segments of the upper lobes. In patients who aspirate in the upright position, the basilar segments of the lower lobes are favored. For all patients, the right lung is more often involved in aspiration events due to the more direct vertical takeoff of the right mainstem bronchus compared to the angulated takeoff of the left mainstem bronchus. A solitary lung cavity is generally associated with primary lung abscess, whereas multiple small cavities suggest secondary lung abscess associated with hematogenous spread of infection. Peripheral parenchymal lung abscess near the chest wall may require chest CT to distinguish an abscess from a loculated empyema with bronchopleural stula. Chest CT scan is more sensitive than a routine chest radiograph, and may detect small cavities, demonstrate obstructing endobronchial lesions, or distinguish lung abscess from airuid levels in the pleural space. Cavitary lung lesions may represent lung abscess, but also represent noninfectious causes of cavitary lung lesions including pulmonary infarction, neoplasm, septic emboli, vasculitis, bullae or cystic

bronchiectasis, pulmonary sequestration, or sarcoidosis. These conditions represent conditions that may become secondarily infected with the subsequent development of a lung abscess. Complications of lung abscess include extension into the pleural space, hemorrhage, and septic emboli (brain abscess or meningitis). In an aspiration-prone patient with gingival disease presenting with a subacute illness, foul-smelling sputum, and a cavitary lesion on chest radiograph, a putative diagnosis of anaerobic lung abscess can be made without further microbiological studies. In other cases, the major challenges to establishing the bacterial diagnosis of lung abscess requires sampling the cavity without contamination by other oropharyngeal microbes (to distinguish pathogenic from colonizing bacteria) and sampling prior to the administration of antimicrobials (which may reduce the ability to cultivate bacteria from lung specimens). Generally, most patients receive antecedent antimicrobials as treatment for a pneumonitis, prior to the development of lung abscess. Techniques used to obtain relatively uncontaminated specimens include transtracheal aspirates (rarely used in current times) or quantitative cultures following bronchoalveolar lavage or protected brush specimens using beroptic bronchoscopy. Quantitative culture of lower respiratory specimens using bronchoscopy may improve the diagnostic yield for aerobes, whereas the detection of anaerobic bacteria by these methods is less well studied. In addition to sampling techniques, proper specimen storage, rapid transportation, and appropriate processing by the clinical laboratory are essential to optimizing isolation of etiological agents, especially anaerobes. As laboratory isolation of pathogens and in vitro sensitivity testing may require several days, empirical antimicrobial selection is necessary for initial treatment in patients with lung abscess. Gram-stain of expectorated sputum may provide initial guidance to the selection of antimicrobials, based on characteristic ndings of polymicrobial ora or the unique morphological features of certain Gram-negative anaerobic organisms and may provide clues as to the presence of anaerobic infection. Furthermore, selection of empirical antimicrobials should also consider the environmental exposures and immune status of the host, and whether the abscess is primary or secondary.

Pathogenesis
Aspiration of oropharyngeal contents is considered the main mechanism for the development of lung abscess (Figure 2). The association of peridontal

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Pathogenesis of lung abscess Aspiration of oral flora (especially anaerobes)* Large inoculum Altered host defense Aspiration pneumonia Pathogen virulence factors Anatomical abnormalities Necrotizing pneumonia

Lung abscess * Increased risk in persons with peridontal disease and predisposing factor for aspiration
Figure 2 Predominant mechanism for the development of lung abscess.

disease with lung abscess formation underscores the importance of anaerobic pathogens and aspiration. The bacteria implicated in anaerobic lung infections represent the normal ora of the oral cavity, and concentrations of anaerobic bacteria in the gingival crevice may approach 1012 organisms per gram, especially in the context of gingivitis. Lung abscess is rare in edentulous persons. Although aspiration of oropharyngeal materials may occur in 45% of healthy individuals during sleep, these occult aspirations may be of relatively small volume and normal host defense and clearance mechanisms allow spontaneous resolution preventing the development of clinical disease. However, clinically significant aspiration of these organisms occurs in persons with compromised consciousness or dysphagia. Predisposing conditions require compromised host defense mechanisms that protect the lower airways (such as cough, glottic closure, or mucocilliary clearance), and an infectious inoculum sufcient to induce disease (such as a large inoculum that overwhelms defenses, induction of a host inammatory response, or direct toxicity to the host) or a combination of these factors. The decisive factor for the development of lung complications following aspiration may depend on the frequency of aspiration events, volume of the aspirate, and character of the aspirated inoculum. Conditions that promote stasis or necrosis of tissue also predispose to anaerobic lung abscess including pulmonary infarction, obstruction due to endobronchial carcinoma, obstruction due to foreign body, and bronchiectasis. Aspiration of substances such as gastric acid pHo 2.5 (Mendelsons syndrome), milk and mineral oil, and volatile hydrocarbons are directly toxic to the lower respiratory tract. The inammatory process

induced by these substances (especially acid) may predispose to bacterial superinfections and result in abscess formation. Based on serial radiographic studies in patients following a dened period of aspiration, lung cavity formation was observed in 714 days. Anaerobic virulence factors promote abscess formation. The propensity for tissue necrosis by anaerobic bacteria may relate in part to a specic family of capsular polysaccharide of anaerobic Gram-negative bacilli. Specic repeating oligosaccharides containing sugars with positively charged free amino acid groups and negatively charged carboxyl or phosphate groups mediate abscess formation in experimental animal models. In addition, metabolic production of short-chain fatty acids by anaerobic bacteria (which may be responsible for the putrid odor of anaerobic infections) may inhibit phagocytosis and bacterial killing by host cell at low pH levels. The pathogenesis of lung abscess is in part dependent on the nature of the specic organism. For example, amoebic lung abscess formation generally involves the right lower lobe by direct extension of the liver abscess through the diaphragm.

Animal Models
Development of a reliable animal model for lung abscess has been elusive. Validation of the hypothesis that aspiration of oral bacteria was the major mechanism for the development of lung abscess was demonstrated in the pre-antibiotic era by Smith in 1927. Inoculating the trachea of rabbits with human gingival crevice material resulted in pneumonitis followed by the development of lung abscess formation in 710 days. The bacteriologic studies of the gingival crevice inoculum revealed predominantly anaerobic bacteria, including Fusobacterium nuclatum, Prevotella melaninogenica, Peptostreptococcus, and an anaerobic spirochete. Interestingly, lung abscess formation required a combination of anaerobes, as lung abscess formation did not occur when animals were inoculated individually with these anaerobic bacteria. In 1929, Varneys inoculation of human abscess material into experimental dogs produced lung abscess, although the microbiology of the inoculum was not fully dened. More recent experiments using rabbits inoculated with well-characterized laboratory anaerobic bacterial isolates suggested an important role for Bacteroides fragilis, although the nature of the stored bacteria may not be clinically relevant. Murine models of lung abscess using Porphyromonas gingivalis and Treponema denticola are associated with a high mortality and thus of limited value.

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Management and Current Therapy

Antibiotics represent the main component of treatment for lung abscess (Table 3). However, most published trials of antimicrobials in the treament of lung abscess have been relatively small. Penicillin represented the main agent during 195075 as