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Essential Clinical Immunology

The ways in which we can better understand cancer, HIV, and other
autoimmune diseases through clinical immunology are of great interest
to all scientists, from students to post-graduate practitioners. Designed
as an introduction to science students (MD, MDPhD, PhD) as well as to
more advanced PhDs and medical practitioners, this book focuses on the
importance of immunological concepts in disease states. Essential Clinical
Immunology begins with the basic concepts and then details the immuno-
logical aspects of various disease states involving major organs of the body.
The book explores how we can better understand disease and its treatment
through clinical immunology. Looking forward, each chapter concludes with
patterns for future research.

John B. Zabriskie (M.D., Columbia College of Physicians and Surgeons) is

Professor Emeritus and former head of the Laboratory of Clinical Micro-
biology and Immunology at The Rockefeller University, New York, New
York. He has written numerous articles on immunology, microbiology, and
Essential Clinical
Edited by

John B. Zabriskie
The Rockefeller University
Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, So Paulo

Cambridge University Press

The Edinburgh Building, Cambridge CB2 8RU, UK
Published in the United States of America by Cambridge University Press, New York

Information on this title: www.cambridge.org/9780521516815
Cambridge University Press 2009

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List of Contributors vii

1 Basic Components of the Immune System 1

John B. Zabriskie, M.D.

2 Immunological Techniques 21
John B. Zabriskie, M.D.

3 Immune Regulation 33
Nina Bhardwaj, M.D., Ph.D., David ONeill, M.D., and Thomas Waldmann, M.D.

4 Immunological Aspects of Infection 45

Kumar Visvanathan, M.D., Ph.D., Christopher M. MacIsaac, M.D., Ph.D.,
William W. Hall, M.D., Ph.D., and Vincent A. Fischetti, Ph.D.

5 Immunological Aspects of Immunodeficiency Diseases 61

Dinakantha S. Kumararatne, M.D.

6 Autoimmunity 91
Haoyang Zhuang, Ph.D., Matthew Kosboth, M.D., Jennifer A. Sipos, M.D.,
Minoru Satoh, M.D., Ph.D., Lijun Yang, M.D., and Westley H. Reeves, M.D.

7 Chronic Lymphocytic Leukemia 119

Nicholas Chiorazzi, M.D., and Manlio Ferrarini, M.D.

8 Immunology of HIV Infections 131

Anders G. Vahlne, M.D., Ph.D.

9 Immunological Aspects of Allergy and Anaphylaxis 145

Paul M. Ehrlich, M.D., and Jonathan D. Field, M.D., F.A.A.A.I.

10 Immunological Aspects of Skin Diseases 163

James G. Krueger, M.D., Ph.D., and Lisa Zaba, M.D., Ph.D.

11 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 175
Dalit Ashany, M.D., and Mary K. Crow, M.D.

12 Immunological Aspects of Cardiac Disease 199

John B. Zabriskie, M.D., Allan Gibofsky, M.D., J.D., Wesley C. Van Voorhis,
M.D., Ph.D., Frederick S. Buckner, M.D., and Noel R. Rose, M.D., Ph.D.

13 Immunological Aspects of Chest Diseases: The Case of Tuberculosis 231

Ernesto Muoz-Elas, Ph.D., and Robert J. Wilkinson, Ph.D., FRCP

14 Immunological Aspects of Gastrointestinal and Liver Disease 251

Christine Moung, M.D., and Lloyd Mayer, M.D.

15 Immunological Aspects of Endocrine Disease 277

Jean-Franois Bach, M.D.

16 Immune-Mediated Neurological Syndromes 293

Jacqueline Friedman, M.D.

17 Immunological Aspects of Renal Disease 313

Gil Cu, M.D., and John B. Zabriskie, M.D.

18 Immunological Aspects of Transplantation 331

Jeffrey M. Venstrom, M.D., and James W. Young, M.D.

Index 349

vi Contents

Dalit Ashany, M.D. Department of Rheumatology

Assistant Attending Physician, Hospital for Special Surgery
Hospital for Special Surgery New York, NY
Assistant Professor of Medicine,
Weill Medical College of Cornell Gil Cu, M.D.
University Assistant Professor and Medical
Hospital for Special Surgery Director
New York, NY Department of Medicine, Division of
Nephrology and Hypertension
Jean-Franois Bach, M.D.
University of Florida, Jacksonville
Professor of Immunology,
Jacksonville, FL
Necker Hospital, Paris
Secrtaire perptuel
Paul M. Ehrlich, M.D.
Acadmie des sciences
Clinical Assistant Professor
Paris, France
Department of Pediatrics
Nina Bhardwaj, M.D., Ph.D. New York University School of Medicine
Director of Tumor Vaccine Center New York, NY
New York Cancer Institute
New York, NY Manlio Ferrarini, M.D.
Istituto Nazionale per La Ricerca sul
Frederick S. Buckner, M.D. Cancro, IST
Associate Professor Genova, Italy
Department of Medicine, Division of
Infectious Diseases Jonathan D. Field, M.D., F.A.A.A.I.
University of Washington Director, Pediatric Allergy and
Seattle, WA Immunology Clinic
Nicholas Chiorazzi, M.D. New York University/Bellevue
Investigator and Director, Laboratory Medical Center
of Experimental Immunology New York, NY
Feinstein Institute for Medical
Vincent A. Fischetti, Ph.D.
Head, Laboratory of Bacterial
Manhasset, NY
Mary K. Crow, M.D. Rockefeller University
Senior Scientist New York, NY

Jacqueline Friedman, M.D. Lloyd Mayer, M.D.
Clinical Professor Professor and Chairman,
Department of Neurology Immunobiology Center
New York Harbor Dorothy and David Merksamer
VA Medical Center Professor of Medicine
New York, NY Chief, Division of Clinical
Allan Gibofsky, M.D. Chief, Division of Gastroenterology
Professor Mount Sinai Medical Center
Department of Medicine and Public New York, NY
Weill Medical College of Cornell Christine Moung, M.D.
University Department of Pathology
New York, NY Mount Sinai Medical Center
New York, NY
William W. Hall, M.D., Ph.D.
Professor Ernesto Muoz-Elas, Ph.D.
Department of Medical Postdoctoral Fellow
Microbiology Department of Microbiology
University College Dublin Tufts University
Dublin, Ireland Boston, MA

Matthew Kosboth, M.D. David ONeill, M.D.

Rheumatology Fellow Assistant Professor of Pathology
Division of Rheumatology and Clinical Director, NYUCI Vaccine and Cell
Immunology Therapy Core Facility
University of Florida New York University School of
Gainesville, FL Medicine
New York, NY
James G. Krueger, M.D., Ph.D.
Professor/Medical Director Westley H. Reeves, M.D.
Rockefeller University Marcia Whitney Schott Professor of
New York, NY Medicine
Division of Rheumatology and Clinical
Dinakantha S. Kumararatne, M.D. Immunology
Consultant in Immunology University of Florida
Department of Clinical Biochemistry Gainesville, FL
and Immunology
Addenbrookes Hospital Noel R. Rose, M.D., Ph.D.
Cambridge, UK Professor of Pathology
Professor of Molecular Microbiology
Christopher M. MacIsaac, M.D., Ph.D. and Immunology
Candidate Director, Johns Hopkins Center for
Centre for Inflammatory Diseases Autoimmune Disease Research
Monash Institute of Medical Research Bloomberg School of Public Health
Clayton, Victoria, Australia Baltimore, MD

viii Contributors
Minoru Satoh, M.D., Ph.D. Adjunct Professor of Pathobiology
Associate Professor University of Washington
Division of Rheumatology and Clinical Seattle, WA
University of Florida Thomas Waldmann, M.D.
Gainesville, FL Head, Cytokine Immunology and
Immunotherapy Section
Jennifer A. Sipos, M.D. Metabolism Branch Chief
Assistant Professor Center for Cancer Research, National
Division of Endocrinology Cancer Institute
University of Florida National Institutes of Health
Gainesville, FL Washington, DC

Anders G. Vahlne, M.D., Ph.D. Robert J. Wilkinson, Ph.D., FRCP

Professor of Clinical Virology Institute of Infectious Diseases and
Department of Immunology, Molecular Medicine
Microbiology, Pathology, and University of Cape Town
Infectious Diseases Cape Town, South Africa
Karolinska University Hospital,
Huddinge Lijun Yang, M.D.
Stockholm, Sweden Associate Professor
Department of Pathology,
Jeffrey M. Venstrom, M.D. Immunology and Laboratory
Fellow, Hematology-Medical Medicine
Oncology University of Florida
Department of Medicine Gainesville, FL
Memorial Sloan-Kettering Cancer
Center James W. Young, M.D.
New York, NY Chief (Interim), Adult Allogeneic Bone
Marrow Transplantation
Kumar Visvanathan, M.D., Ph.D. Attending Physician and Member
Director, Innate Immunity Professor of Medicine, Weill Medical
Laboratory and Infectious College of Cornell University
Diseases Physician Division of Hematologic Oncology
Centre for Inflammatory Diseases Department of Medicine
Department of Medicine (Monash Memorial Sloan-Kettering Cancer
Medical Centre) Center
Monash University New York, NY
Clayton, Victoria, Australia
Lisa Zaba, M.D., Ph.D.
Wesley C. Van Voorhis, M.D., Ph.D. Biomedical Fellow
Training Program Director, Infectious Rockefeller University
Diseases Krueger Laboratory
Professor of Medicine New York, NY

Contributors ix
John B. Zabriskie, M.D. Haoyang Zhuang, Ph.D.
Professor Emeritus, Laboratory Graduate Assistant
of Clinical Microbiology and Division of Rheumatology and Clinical
Immunology Immunology
Rockefeller University University of Florida
New York, NY Gainesville, FL

x Contributors
1. Basic Components of the Immune System

John B. Zabriskie, M.D.

INTRODUCTION response to a given antigen, and these anti-

bodies are proteins, have similar structures,
This chapter is not a comprehensive review and can be divided into various classes of
of immunology but rather a condensed immunoglobulins. Cellular responses are
version of those aspects of immunology established by cells and can only be trans-
that have particular relevance to clinical ferred by cells. (See the Bibliography for
immunology. Refer to the Bibliography for the extraordinary beginnings of the con-
a more extensive discussion of the role of cept of a cellular arm of the immune sys-
each component. tem.) Up to the 1940s the general dogma
It is generally believed that the immune held that only antibodies were involved in
system evolved as the hosts defense against the immune response. Dr. Merrill Chase,
infectious agents, and it is well known that who began his experiments in a labora-
patients with deficiencies in the immune tory devoted primarily to the humoral
system generally succumb to these infec- response, clearly showed in a series of ele-
tious diseases. However, as we shall see, it gant experiments that immunity was not
may well play a larger role in the elimina- just humoral but that a cellular response
tion of other foreign substances, including by the lymphocytes could also produce
tumor antigens or cells and antibodies that immunity. Some of the best examples of
attack self. the power of cellular immunity may be
An immune response may be conve- found in the many experiments in which
niently divided into two parts: (1) a specific transfer of cells can induce autoimmune
response to a given antigen and (2) a more disease in animals and humans as well as
nonspecific augmentation to that response. rejection of an organ graft in both animals
An important feature of the specific and humans by cells.
response is that there is a quicker response The separation of human and cellular
to the antigen during a second exposure to immunity was further advanced by the
that antigen. It is the memory of the initial study of immunodeficient humans and
response that provides the booster effect. animals. For example, thymectomized or
For convenience, the specific immune congenitally athymic animals as well as
response may be divided into two parts: humans cannot carry out graft rejection,
(1) the humoral response and (2) the cellu- yet they are capable of producing some
lar response to a given antigen. As we shall antibody responses. The reverse is also
see, however, both responses are medi- true. Children (and animals) who have an
ated through the lymphocyte. Humoral immune deficit in the humoral response
responses are antibodies produced in do not make antibodies but can reject

grafts and appear to handle viral, fungal, Several types of molecules play a vital
and some bacterial infections quite well. role in the immune response, and we will
An extraordinary finding by Good and deal with each in detail. Antigens, both
colleagues in studying the cloacal lym- foreign and self, are substances that may
phoid organ in chickens revealed that, or may not provoke an immune response.
with removal of the bursa Fabricius, these Both T cells and B cells have receptors that
animals lost their ability to produce anti- recognize these antigens. In the case of B
bodies and yet retained the ability to reject cells, antibodies on the surface are a major
grafts. source (but not the only one) of antigen rec-
Out of these and many other contribu- ognition, and once activated, they differen-
tions, a clearer picture of the division of tiate into plasma cells that produce large
efforts by lymphocytes begins to emerge. quantities of antibodies that are secreted
Since cellular immune responses require an into blood and body fluids to block the
intact thymus, cellular immune responses harmful effects of the antigen.
are mediated through the T lymphocytes T cells have similar receptors known
(thymus), while antibody-producing cells, as T-cell receptors (TCR), and in the con-
which are dependent on the bone mar- text of the major histocompatibility com-
row (the bursa equivalent), are known as plex (MHC) molecules provide a means of
B (bursa) cells. The pathways of both cell self-recognition and T-lymphocyte effector
types are depicted in Figure 1.1. functions. Often these effector functions

Lymphocyte Development Peripheral Effector Cells

Tc TH2
Self reactive
cells deleted
T memory

Plasma cell

Pre-B B
stem cell
B memory

Myeloid cell

Premonocyte Monocyte

CLP Cell Lymphocyte Progenitor

CMP Cell Myeloid Progenitor

Dendritic cell

Figure 1.1 Development and differentiation of lymphocytes from pluripotential stem cells.

2 Basic Components of the Immune System

are carried out by messages transmitted IgM and IgG2 antibodies and do not stimu-
between these cells. These soluble messen- late long-lasting memory cells. Most bac-
gers are called interleukins or cytokines. terial polysaccharides (found in bacterial
cell walls) fall into this category. Certain
polysaccharides, such as LPS (lipopoly-
ANTIGENS saccharide), not only induce specific B-cell
activation but also can act as a polyclonal
Antigens are any substances that are B-cell stimulant.
capable, under appropriate conditions, of
inducing the formation of antibodies and
reacting specifically with the antibodies ANTIBODY
so produced. They react with both T-cell
recognition receptors and with antibodies. The basic structure of the antibody mol-
These antigenic molecules may have sev- ecule is depicted in Figures 1.2A and B. It
eral antigenic determinants, called epitopes, consists of a four-chain structure divided
and each epitope can bind with a specific into two identical heavy (H) chains with
antibody. Thus, a single antigen can bind a molecular weight of 25 kDa. Each chain
to many different antibodies with different is composed of domains of 110 amino acids
binding sites. and is connected in a loop by a disulfide
Some low-molecular-weight mol- bond between two cysteine residues in the
ecules called haptens are unable to evoke chain.
an immune response but can react with The amino acid N-terminal domains of
existing antibodies. These molecules need the heavy and light chains include the anti-
to be coupled to a carrier molecule to be gen-binding site. The amino acids of these
antigenic. variable domains vary between different
For some molecules such as drugs, the antibody molecules and are thus known as
molecule needs to be conjugated to a car- the variable (V) regions. Most of these dif-
rier. The carrier may be a host protein. The ferences reside in the hypervariable areas of
tertiary structure of the molecule as well the molecule and are usually only six to
as the amino acid sequence is important ten amino acid residues in length. When
in determining antigenicity. Certain struc- the hypervariable regions in each chain
tures such as lipids and DNA are generally come together along with the counterparts
poor antigens. on the other pair of H and L chains, they
Most antigens are either thymus- form the antigen-binding site. This part
dependent or thymus-independent anti- of the molecule is unique to the molecule
gens. Thymus-dependent antigens require and is known as the idiotype determinant.
T-cell participation: Most proteins and In any individual, 106 to 107 different anti-
foreign red cells are examples of these body molecules can be composed from
molecules. Thymus-independent antigens 103 different heavy and light chains of the
do not require T-cell participation for anti- variable regions. The part of the molecule
body production. Instead, they directly next to the V region is called the constant
stimulate specific B lymphocytes by cross- (C) region made up of one domain in the
linking antigen receptors on the surface of light chain (C1) and three or four in a heavy
B cells. These molecules produce primarily chain (CH). A Cl chain may consist of either

Basic Components of the Immune System 3




Light chain Heavy chain

Figure 1.2A Heavy and light chains of an IgG antibody. An IgM

antibody would be a pentameric structure of an IgG molecule.

two kappa () or two lambda () chains but tion. This activation permits the segment
never one of each. Of all the human anti- removal of antigenantibody complement
body molecules, approximately 60%, are complexes via complement receptors on
chains and 40% contain chains. Although phagocytic cells or complement-mediated
there are no known differences in the func- lysis of the organism. However, in contrast
tional properties of and chains, there are to the IgG molecule, it has relatively low
several different types of the CH domain. affinity binding to the antigen in question.
These differences are reflected in determin- Second, because of its size, it does not usu-
ing the class (isotype) of the antibody and ally penetrate into tissues.
thereby the physiological function of a par- In contrast, IgG is a smaller molecule
ticular antibody molecule. that penetrates easily into tissues. There
The IgM molecule is the oldest class of are four major classes of IgG: IgG1 and
immunoglobulins, and it is a large mol- IgG3 activate complement efficiently and
ecule consisting of five basic units held clear most protein antigens, including the
together by a J chain. The major role IgM removal of microorganisms by phago-
plays is the intravascular neutralization of cytic cells. In contrast, IgG2 and IgG4 react
organisms, especially viruses. The reason mostly with carbohydrate antigens and are
for this important physiological role is that relatively poor opsonins. This is the only
it contains five complement-binding sites, molecule that crosses the placenta to pro-
resulting in excellent complement activa- vide immune protection to the neonate.

4 Basic Components of the Immune System

binding antigen

Fab domain

Fc domain

Figure 1.2B Antigen-binding sites and antigen binding in an IgG antibody.

Hinge region allows for rotational and lateral movements of the two antigen-
binding sites.

The major mucosal immunoglobulin, important role in allergic reactions and

IgA, consists of two basic units joined by a J expelling intestinal parasites, which is
chain. The addition of a secretion molecule accomplished by increasing vascular per-
prevents its digestion by enzymes present meability and inducing chemotactive fac-
in mucosal and intestinal secretions. Thus, tors following mast cell degranulation.
IgA2 is the major IgA molecule in secretions Given this extraordinary ability to gen-
and is quite effective in neutralizing anti- erate large numbers of antibody molecules,
gens that enter via these mucosal routes. how does the immune system recognize
IgA1, the main IgA molecule in serum, is, all pathogens, including past, present,
however, susceptible to inactivation by and future? This diversity is achieved by
serum proteases and is thus less active for the way in which the genetics of antibody
defense. Its function is unclear at present. production is arranged (see Figure 1.3).
Two other classes are worthy of note. The light and heavy chains are carried on
IgD is synthesized by antigen-sensitive B different chromosomes. The heavy chain
cells and is involved in the activation of genes are carried on chromosome 14. These
these cells by antigen. IgE is produced by genes are broken up into coding systems
plasma cells and binds to specific IgE recep- called exons with intervening segments of
tors on most cells and basophiles. This mol- silent segments called entrons. The exons
ecule (see Chapter 9) plays an extremely represent the central region of the heavy

Basic Components of the Immune System 5

Chromosome Silent area = Intron

(V)n D J C C C1 C1 C1 C2 C4 C C2
14 H

VHC, = an lgM heavy chain with

a particular variable region
VDJC Final
(V)n J C lgM
2 or

(V)n J C


Figure 1.3 The genetics of antibody production.

chain and a large number of V regions. in sequence while using the same variable
Between the V and D genes are two small regions (see Figure 1.4). The heavy chain
sets of exons called the D and J. With each gene recombinations are controlled by two
single B cell, one V gene is joined to one D recombination activity genes called RAG1
and J in the chromosome. The product, the and RAG2. If these genes are eliminated by
VH domain, is then joined at the level of knock-out techniques in mice, profound
RNA processing to Cu and the B cell makes immunodeficiency status occurs in these
an IgM molecule. By omitting the Cu gene animals, characterized by absent mature B
and joining VHDJ to a C an IgG molecule is and T cells.
produced. This enormous versatility allows Thus, the diversity of antigen bind-
the cell to make IgM, IgD, IgG, IgA, or IgE ing is achieved by the large number of V

VDJ Region Constant Region
Mature B-cell 3 1 2b 2a
Primary response
Functional gene
Producing lgM

Class Switching

Mature B-cell 1 2b 2a
Secondary response
Functional gene
Producing lgG1

Figure 1.4 Recombination events necessary for generation of class and subclass switching.

6 Basic Components of the Immune System

genes available and their combination with the cell-surface complex or at the level of
different D and L genes to provide differ- the kinases may result in various forms of
ent antibodies. Furthermore, the inherited immunodeficiency. Two other important
set of genes may be increased by somatic antigens present on TCR2 cells recognize
mutation during multiple divisions of lym- histocompatibility antigens and will be
phoid cells, thereby increasing the number discussed later. The genes for TCR chains
of antibody specificities to 1014, which far are on different chromosomes with the
exceeds the number of B cells (1010) in the and molecules on chromosome 7, while
body. the and are on chromosome 14. As seen
Once a given B cell is preselected to pro- in Figure 1.5, the four chains are made up
duce a particular VH and VL domain, all the of a variable region and a constant region
ensuing progeny of that B cell will produce similar to those observed with the immu-
the same VH or VL domain. The sequence noglobulins. The variable regions are also
of events is as follows: initially, the B cell numerous and joined at D and J regions
produces intracellular antigen-specific by RAG1 and RAG2. This permits a diver-
IgM, which becomes bound to the cell sur- sity of antigen recognition similar to that
face. The B cell is now antigen responsive observed with immunoglobulin, but addi-
with exposure to a given antigen. The com- tional somatic mutation is not involved in
mitted B cell begins producing a certain T cells. These similarities have led to the
isotype or class of immunoglobulins and concept that genes for antigen-specific T
begins dividing, and all the progeny will cells evolved in the same manner as immu-
produce the identical immunoglobulin mol- noglobulin from a parent gene, and both
ecules. These B cells will later mature into are members of a superantigen family.
either plasma cells or long-term memory The TCR complex recognizes small
B cells. peptides presented to it by the MHC class
I and II and depends on the type of T cell.


Each T cell is also committed to a given or chain or chain

antigen and recognizes it by one of two
TCRs. They may have TCR2s composed of
gamma () and delta () chains or TCR2s Variable region
composed of another heterodimer of alpha
() and beta () chains. These TCR2s are
associated with a group of transmem-
Constant region
brance proteins on the CD3 molecule,
which takes the antigen recognition sig-
nal inside the cell. Signal transduction via
the CD3 complex is regulated by a series Plasma membrane

of kinases, which are associated with the

tails of the CD3TCR complex and regulate
phosphorylation. Deficiencies or blocks Figure 1.5 Diagram of the structure of a T-cell
in the T-cell signaling pathways either at receptor.

Basic Components of the Immune System 7

Helper T cells (CD4) recognize class II anti- Figure 1.6). This extensive polymorphism
gens while suppressor cytotoxic T cells is important when viewed in the context of
(CD8) recognize class I antigens. Because an immune system that needs to cope with
of the rather low affinity of the reactions, an ever-increasing range of pathogens.
recognition of processed antigen alone is These pathogens in turn are extremely
not sufficient to activate T cells. Soluble adept at evading the immune system.
interleukins are needed to complete the Thus, the battle between invading microbe
picture and are generated during the anti- and immune recognition is constant and
gen processing. ever changing. Recognition of antigen by
T cells is MHC restricted. Therefore, any
given individual is only able to recognize
MAJOR HISTOCOMPATIBILITY antigen as part of a complex of antigenic
COMPLEX peptide and self.
The importance of this concept is under-
Human histocompatibility antigens are scored by the experiments of Dougherty
also known as human leucocyte antigens and Zinkernagel. Using virus-specific
(HLA), a term that is synonymous with (virus 1) cytotoxic T cells, Figure 1.7 illus-
the MHC complex. These antigens are cell- trates their remarkable discovery. If anti-
surface glycoproteins classified as type I gen-presenting cells (APCs) of mouse
or type II. They can produce genetic poly- A are mixed with T cells of mouse A in
morphism with multiple alleles at each the context of virus 1 peptides, the T cell
site, thus permitting a great deal of genetic responds and kills the virus. If the MHC
variability between given individuals (see complex is from mouse B and the T cells

Peptide binding groove

1 2 1 1



2m s s s s
s s s s

3 2 2

Class I Class II

Figure 1.6 Diagrammatic representation of class I and II MHC antigens with

B2 microglobulins and CHO carbohydrate side chains.

8 Basic Components of the Immune System

type a type b type a
Ag1 Ag1 Ag2

T cell T cell T cell

Response No Response No Response

Figure 1.7 MHC restriction of antigen recognition by T cells. If APC and

T cell are of the same genetic lineage as virus I, the T cell responds and
kills the virus. If APC and T cell are of different lineage, no response occurs.
If APC and T cell are of same lineage but virus 2 is present, no response

are from mouse A, no killing occurs. groups of class II antigens: namely, HLA-
Finally, if MHC and T cells are both from DP, HLA-DQ, and HLA-DR.
mouse A but virus 2 is unrelated to virus Depending on the nature of the anti-
1, no response will occur. gen (endogenous or exogenous), the MHC
The MHC class I antigens are divided response is different. For example, endog-
into three groups (A, B, and C), and each enous antigens (including viral antigens)
group belongs on a different gene locus on are presented by MHC class I antigen cells
chromosome 6. The products of all three exclusively to CD8 cells. The endogenous
loci are similar and are made up of a heavy antigen is first broken down into small pep-
chain (45 kDa) and associated 2 micro- tides and transported by shuttle proteins
globulin molecule (12 kDa) gene, which called Tap I and Tap II to the endoplasmic
resides on chromosome 12. The MHC class reticulum. There they complex with MHC
I antigen differences are due to variations class I molecules and are delivered to the
in the chains, the 2 microglobulin being cell surface for further processing to the
constant. X-ray crystallography studies CD8 cells.
have shown that as few as nine amino In contrast, MHC class II molecules
acids can be tightly bound in the chain are held in the endoplasmic reticulum and
groove. are protected from binding to peptides in
MHC class II antigens also exhibit a the lumen (not human) by a protein called
similar structure with the groove being MHC class II associated invariant chain.
formed by the 1 and 1 chains. Unlike Finally, there are class III antigens, such
class I antigens present on most nucleated as complement components C4 and C2,
cells, the class II antigens are restricted to a plus certain inflammatory proteins, such
few types: macrophages, B cells, and acti- as tumor necrosis factor (TNF), which are
vated T cells. In humans, there are three encoded in adjacent areas.

Basic Components of the Immune System 9

ADHESION MOLECULES designated by three separate prefixes:
E (endothelial), P (platelet), and L (leu-
In spite of the known MHC complex cocyte). The letters denote the cells on
consisting of binding of a TCR to the pro- which they were first observed. These
cessed antigen, which in turn is bound to groups of selectins bind avidly to car-
the class II molecule of APCs, this is not bohydrate molecules on leucocytes and
enough for T-cell activation. One must endothelial cells.
have additional stimuli that are provided c. Immunoglobulin superfamily: The
by a series of adhesion molecules on the molecules in this family are so called
two cell surfaces. because they contain a common
These molecules are composed of a immunoglobulin-like structure. They
diverse set of cell-surface glycoproteins and strengthen the interaction between the
play a pivotal role in mediating cell-to-cell T cells and APCs. They include some
adhesion. Adhesion molecules are divided of the most powerful molecules in the
into four major groups, (a) integrins, (b) immune system, such as the CD4, CD8,
selectins, (c) immunoglobulin superfamily, CD2, lymphocyte function antigen
and (d) caherins. (LFA-3 or CD58), and the intercellular
adhesion molecules such as ICAM-1
a. Integrins are heterodimers: These through 3.
are divided into and subunits. d. Cadherins: These molecules are
Depending on the substructure of the calcium-dependent adhesion mole-
unit, there are five families, but for cules and are mainly important in
convenience 1 and 2 integrins are establishing molecular connections
involved in leucocyteendothelial inter- between epithelial cells. Their particular
actions. 1 integrins, also known as very importance is during embryonic devel-
late activation proteins, are so named opment.
because they appear on lymphocytes
several days after antigenic stimula-
tion and are composed of a common CYTOKINES
chain (CD29) paired with a different
chain. They mediate lymphocyte and This group of soluble molecules plays an
monocyte binding to the endothelium extremely important role in clinical immu-
receptors called vascular adhesion mol- nology. They are secreted by macrophages
ecule. 2 integrins also have a common and may act as stimulatory or inhibi-
chain (CD18), which pairs with dif- tory signals between cells. Cytokines that
ferent chains (CD11 a, b, c) to form initiate chemotaxis of leucocytes are called
a number of separate molecules. These chemokines.
two sets of integrins mediate strong Among the group of cytokines, there
binding of leucocytes to the endothe- are a few of particular interest because
lial cell while 35 are concerned with of their stimulatory activity. Interleu-
binding to extracellular matrix proteins kins 1 (IL-1) and 2 (IL-2) are of particu-
such as fibronectin and vitronectin. lar importance secondary to their role in
b. Selectins: These molecules are com- amplifying the immune response. IL-1
posed of three glycoproteins and are acts on a wide range of cells including T

10 Basic Components of the Immune System

Table 1.1 Lymphocytes Involved in Immune Response

Cell Type Function of Cell Product of Cell Function of Product

B Antibody production Antibody Neutralization

Cell lysis
TH2 cells B-cell antibody production Cytokines IL-3, -4, -5, Help B and Tc
Activated Tc -10, -13
TH1 Inflammation: Initiation IL-2, IFN-, TNF Inflammatory
Augmentation mediators
Ts B-cell antibody production Suppressor factor(s) Suppress TH and
Activated Tc indirectly B and Tc
Tc Lysis of antigenic target cells INF- Enhance MHC
Perforins expression,
activate NK cells,
disrupt target cell

Tc, cytotoxic T cell; TH1-2, helper T-cell types; Ts, suppressor T cell.

and B cells. In contrast, IL-2 primarily acts specific T cells. This interaction between
on lymphocytes, although it has similar APCs and T cells is strongly influenced by
trophic effects on IL-receptor B cells and a group of molecules called co-stimulators.
natural killer (NK) cells. (See Table 1.1 and For example, it is CD80 (B7-1) and CD86
functions.) (B7-2) on the APC cells with receptors
CD28 and CTLA-4 on the T cell that pro-
vides this interaction. The absence of these
INITIATION OF THE IMMUNE co-stimulators leads to T-cell unrespon-
RESPONSE siveness. The importance of this pathway
is emphasized by the fact that antagonists
The effector cells are really divided into to these co-stimulators do interrupt the
two types: B cells and T cells. B cells are immune response in both in vitro and in
primarily responsible for antibody produc- vivo experiments. For example, mice with
tion, whereas T cells act as effector cells and a severe form of lupus exhibit a milder dis-
may function as both helpers and suppres- ease following a CTLA-4 antagonist.
sors, depending on the stimulus provided As stated before, processed antigen is
by APCs. presented to the T cells in the context of
The first step in initiation of the immune the MHC complex present on the surface
response to an antigen must necessarily of APCs. In this regard, the most efficient
involve modification of the antigen, and APCs are the dendritic cells. These cells
these specialized cells are called APCs. have high concentrations of MHC class I
Without such processing, T cells cannot and II antigens, co-stimulatory molecules,
recognize antigen. Thus, it is the secretion and adhesion molecules on their surface.
of cytokines by APCs activated by antigen These cells may be divided into two
presentation that further activates antigen- major groups. The dendritic cells of the

Basic Components of the Immune System 11

skin are called the Langerhans cells and cells to switch from IgM molecules to other
play an important role in immune defenses isotypes. Deficiencies in either molecule
since they are present in the largest protec- lead to severe immunodeficiency states
tive organ of the body. Because they are with only IgM produced but no IgG or IgA
mobile, Langerhans cells can capture anti- antibodies. This syndrome is called the
gen in the periphery and migrate to sec- hyper-IgM syndrome, and in this case of
ondary lymph nodes where they become CD40L deficiency, it is an X-linked immu-
mature dendritic cells and interact with nodeficiency.
nave T cells. As mentioned before, each B cell is
In contrast, the follicular dendritic committed to the production of antibody
cells reside in the follicular germinal cen- expressing a unique VHVL combination,
ter (B-cell area) of a lymph node. These and the surface and excreted immuno-
cells have receptors for complement and globulin are the same. These observations
immunoglobulins and their function is to form the basis of Burnets clonal selection
trap immune complexes and feed them to theory in that each B cell expresses a sur-
B cells. This processed immune complex face immunoglobulin that acts as its anti-
containing antigen is closely associated gen-receptor site. Contact with the antigen
with MHC class II molecules on the APC and helper T-cell factors commit each B
surface and thus activates B cells. cell to divide and differentiate to pro-
duce more of the same VHVL antibody. A
number of these B cells become memory
ANTIBODY PRODUCTION cells so that a greater number of antigen-
specific B cells are available on a second-
To achieve antibody production, at least ary contact with the same antigen. This
four types of cells are required: APC, B phenomenon is known as clonal expan-
cells, and two types of regulating cells. sion and helps to account for the greater
secondary response.
Perhaps more important is that the sec-
B Cells
ondary response of antibodies has a higher
Antibodies are produced by nave B cells affinity binding for these antigens. These
and are called plasma cells. These cells latter antibodies will bind to antigen even
express immunoglobulins on their sur- when complexed to antibody and help
face. In the early stages, B cells first show clear the antigen more effectively from the
intracellular -chains and then surface circulation. It is important to remember,
IgM. Through the process described ear- however, that B cells alone do not respond
lier, these cells can later express IgG, IgA, to antigen directly, even in the presence of
or IgE, a phenomenon known as isotype APC cells. They must have a second signal,
switching. The final type of surface immu- normally provided by the T cells. This point
noglobulin determines the class of anti- was elegantly shown in a series of transfer
body secreted. experiments using irradiated recipient ani-
Isotype switching is mediated through mals. As seen in Figure 1.8, antigen alone
two important protein interactions: CD40 or antigen + B cells produced no antibody
on the B cell interacts with CD40L on acti- production in these animals. Similarly,
vated T cells (IL-4 induced) to stimulate B T cells alone were ineffective. However,

12 Basic Components of the Immune System

Irradiated mouse

Antigen + Antigen + B cells Antigen + T cells Antigen + B+T cells

Ab Ab Ab Ab+++

Figure 1.8 Adoptive cell transfer experiments in irradiated animals shows

both T and B cells important for antibody production; Ab, antibody.

when all three were transferred into irradi- T cells. These cells constitute an expanded
ated animals, this resulted in excellent anti- clone and the immune response is quicker
body production. and more vigorous.
This activation is also aided by two
other mechanisms. First, memory cells
T Cells
have increased numbers of adhesion mol-
One must first emphasize that helper T ecules (LFA-1, CD2, LFA-3, and ICAM-1)
cells can only respond to antigen presented on their surface as well as a higher popu-
by macrophage MHC class II antigens as a lation of affinity receptors. Thus, memory
complex on APC cells. In turn, they recog- cells produce high concentrations of IL-2
nize the same combination of antigen and to recruit more helper cells of both types
class II MHC antigens on the correspond- TH1 and TH2. The recognition of antigen
ing B cells. It is only then that the helper involves several receptors on the surface
T cell secretes its cytokines to activate the of the T cells. In contrast, B cells recognize
reaction. As seen in Figure 1.8, T cells rec- antigen by surface-bound immunoglobulin
ognize antigen in the context of their own and recognize the same epitopes somewhat
MHC configuration. They will not cooper- differently. T cells only recognize haptens
ate with B cells and macrophages express- (small molecules) when the haptens are
ing antigens of a different genetic back- coupled to a carrier protein while antibody
ground. can recognize free haptens easily.
When helper T cells meet an antigen for
the first time, only a limited number of cells
Differentiation of T Cells
are activated to provide help for the B cells.
However, when the animal is re-exposed, T cells have characteristic cell-surface
there is a marked increase of specific helper glycoproteins that serve as markers of

Basic Components of the Immune System 13

differentiation of these cells. These such as viruses, certain bacteria, and para-
markers are recognized by specific mono- sites inaccessible to antibodies.
clonal antibodies and divide them into two Cytotoxic T cells lyse cells infected with
particular subsets. viruses. This cytotoxicity is virus specific,
TH1 cells secrete TNF and INF- and and only cells expressing those proteins on
mediate cellular immunity. Conversely, the surface of the infected cell are killed. As
TH2 cells secrete IL-4, IL-5, IL-10, and IL-13 stated before, this destruction occurs only
and are needed for stimulating antibody in the presence of the same MHC class I
production by B cells. T cells secreting both molecules. This combination directly acti-
cytokine profiles are designated THO. vates CD8+ cells and is a potent killer of
What influences a nave T cell to select virally infected cells. The induction of the
which cytokine profile to secrete is not cytotoxic T cell requires precursor cells
known. However, experiments in which and IL-2 from helper cells and is subject to
cells are exposed to IL-1 and IL-6 promote regulation by other T cells.
TH2 cells while IL-12 and IFN- stimulate Cytotoxic T cells also play a role in graft
production of TH1 T cells. rejection. This was shown years ago in a
In humans, a TH1 cytokine profile mixed lymphocyte reaction in which the
is primarily directed toward protection lymphocytes from two genetically differ-
against intracellular pathogens while a ent individuals were placed in culture. In
TH2 profile interacts with diseases charac- this case, helper cells responded to a for-
terized by overproduction of antibodies eign MHC class II antigen, but cytotoxic
including IgE. T cells were able to lyse target cells carry-
An elegant example of these different ing the MHC class I molecules of the stim-
pathways of the TH1 and TH2 response is ulating (genetically different individual)
seen in the disease leprosy. Patients that cells. The in vivo reactions between indi-
develop a TH1 response develop only viduals undergoing transplantation will be
limited disease (tuberculoid leprosy). In discussed in more detail in a later chapter.
contrast, those patients mounting a TH2 In contrast, delayed-type hypersensi-
response develop debilitating and spread- tivity reactions are mediated by specific
ing lepromatous leprosy since the antibody T cells that produce TH1-type cytokines
response will not protect against an intra- upon exposure to antigen. An example of
cellular pathogen. this type of reaction is the PPD reaction, or
tuberculin test. When the antigen is injected
under the skin of an individual who was
Cellular Immunity
previously infected with Mycobacterium
Cell-mediated responses are implemented tuberculosis, a reaction in the skin evolves
by T lymphocytes. The major functions over 48 to 72 hours in which there is local
of T cells can be divided into two catego- swelling and induration >10 mm. If the site
ries: the first (cytotoxicity) is to lyse cells is biopsied, one finds a T-cell and macro-
expressing specific antigens; the second phage infiltration. Injection of the same
(delayed hypersensitivity) is to release material in a noninfected individual pro-
cytokines, thereby triggering an inflam- duces little or no induration, and the his-
matory response. These two types of cells tology is essentially negative. Whereas the
are used to combat intracellular pathogens cells in this case do not kill the organism,

14 Basic Components of the Immune System

most individuals infected surround the readily bound and phagocytosed through
organism in a caseous inflammatory IgG: Fc and C3b receptors. Immune com-
lesion, which does not allow the organism plexes are handled in a similar fashion,
to spread. The in vivo state of the lesions activating the classical pathway comple-
will be discussed in more detail in a later ments. Individuals who lack one of the
chapter. classical pathway components are prone to
immune complex disease.
The minor complement fragments con-
Nonspecific Effector Molecules
tribute to the immune response by activating
There are a number of nonspecific mol- the inflammatory response. For example,
ecules that affect the immune response, some increase vascular permeability (C3a);
especially antibody production. These others are chemotactins for neutrophils and
major factors are as follows: phagocytic macrophages (C5a) and not only promote
cells such as neutrophils and macrophages, leucocytosis in the bone marrow but attract
which remove antigens and bacteria, and these cells to the site of inflammation.
complement, which can either destroy The critical step in complement activa-
the organism or facilitate its destruction. tion is the cleavage of the C3 component
The role of many of these factors will be by complement-derived enzymes called
discussed in more detail in later chapters, C3 convertases. This results in the presence
but a brief outline of their functions is war- of C3b, which mediates a number of vital
ranted here. biological activities. The cleavage of C3b
can be initiated by three routes (classical,
COMPLEMENT alternative, and lectin), but each route is
The complement component system in response to different stimuli. Individu-
consists of a series of heat-liable proteins, als who are deficient in C3 are obviously
and they normally exist as inactive precur- predisposed to bacterial infections and
sors. However, once activated each compo- immune complex disease.
nent may act as an enzyme and cleaves the Each of these routes will be examined
next component in the sequence. in more detail (see Figure 1.9).
Each precursor is cleaved into two or
more components, and the major frag- 1. Classical pathway: As its name implies,
ment (designated b) has two biologically this is the usual pathway whereby anti-
active sites. One is for binding to cell mem- genantibody complexes in the presence
branes and the other is for enzymatic cleav- of complement destroy the invading
age of the next component. The control of organism. The antibody (either IgM or
the sequence relies on either spontaneous IgG) causes a conformational change in
decay or specific inactivation of these com- the Fc portion of the antibody to reveal
ponents. Minor fragments play a role in the a binding site for the first component
fluid phase, acting as chemotactins. of complement C1q. This component
The major function of the complement consists of six subunits and reacts with
system is to help in the opsonization of micro- the Fc via its globular heads. The acti-
organisms and immune complexes. These vation of this component requires the
components plus antibody are more read- binding of two globular heads for acti-
ily recognized by macrophages and more vation. Thus, one molecule of IgM with

Basic Components of the Immune System 15

Complement Pathways
Classical Alternative
Antigen-antibody MBL*
complexes Endotoxin, bacterial cell walls

C1q C3

C4 C2 Factor D & B

C4b2b (C3 convertase)



C3b4B2b (C5 convertase)




*Mannose binding lectin (MBL) bound to surface-binding carbohydrates

Figure 1.9 Pathways of complement activation by the classical, alternative,

or lectin activation; MBL, mannose binding protein.

its pentameric conformation can easily activation of C3. In contrast to the clas-
activate C1q, while the ability of IgG, sical pathway, however, this pathway
which has only two sites to activate bypasses antibody, C1, C4, and C2, and
C1q, is low. IgA, IgD, and IgE do not it is bacterial cell walls or endotoxin that
activate the classical pathway. C1q in activates C3. C3b here is unstable and,
turn activates C4 and C2, generating the if an appropriate receptor is not found,
complex C4b2b, which is the C3 con- it decays and the molecule becomes
vertase of the classical pathway. After inactive. However, if a receptor surface
the splitting of C3 into C3a and C3b is is present, then the C3b molecules bind
achieved, C3a possesses anaphylatoid and remain active. Then C3b can use
and chemotactic activity. However, factors D and B of the alternative path-
more important is C3b, which forms way to form the active enzyme C3bBb;
the complex C3b4b2b, which is the C5 this complex then becomes stabilized in
convertase and initiates the final lytic the presence of properdin.
attack complex. This molecule then can chose
2. The alternative pathway is phyloge- between two pathways. It can break
netically older than the classical but down more C2, providing more C3b.
was not generally accepted until the Or it becomes stabilized to form the C5
1960s. Again, the control reaction is the convertase of the alternative pathway.

16 Basic Components of the Immune System

3. Lectin pathway: The third pathway of pathways such as clotting, fibrinolytic, and
complement activation is created by kimin pathways.
the mannose-binding lectin MBL, a
circulating protein that binds to carbo-
hydrate on the surface of many micro- FUNCTIONAL COMPONENTS OF THE
organisms. MBL (structurally related IMMUNE SYSTEM
to C1q) activates complement through
a serine protease known as MBL- Each of the cells in the immune response
associated serine protease. Deficiencies has a particular role to play. While many
in circulating levels of MBL are asso- of these cells will be discussed in detail in
ciated with frequent infections in subsequent chapters, a brief review of the
childhood. functional capabilities is presented here.

Once these components are activated,

that is, C3b, 4b2b or C3bBb, and proper-
din, these molecules trigger sequentially These cells may be divided into two main
C5, C6, C7, C8, and C9, which leads to the groups: the dendritic cell and the mature
final lytic pathway and lysis of the target macrophage. The dendritic cells major
cell. The target can be a red cell, a virally function is to present antigen to the lym-
infected cell, or a bacterium. Electron phocyte, and it is the earliest cell to recog-
microscopy has shown that this complex nize foreign antigen.
binds to the cell membrane and actually There are two forms of dendritic cells:
punches a hole in the cell. Salts and water immature and mature. The induction of an
pass through the hole and the water fills adaptive immune response begins when a
the cell, eventually leading to swelling and pathogen is ingested by an immature den-
destruction of the cell. dritic cell. These cells reside in most tissues
The control of the complement activa- and are relatively long-lived. As seen in
tion is important since many of its compo- Figure 1.1, they are derived from the same
nents induce inflammation. This control is cell myeloid precursor as the macrophage.
executed in the following ways. First, many This immature cell carries receptors on its
of the activated components are unstable surface that recognize common features of
and will decay rapidly if the next sequence many pathogens such as cell wall carbo-
is not present. Second, there are specific hydrates of bacteria. Once the bacterium
inhibitors of each component, such as C1 is in contact with these receptors, the den-
esterase, which inhibits factors I and H. dritic cell is stimulated to engulf the patho-
Finally, the cells themselves contain pro- gen and degrade it intracellularly. These
teins that increase the rates of breakdown cells also continue engulfing extracellular
of these products. material (both viruses and bacteria) by a
In summary, all acute phase comple- receptor-independent mechanism of mac-
ment components are acute phase proteins ropinocytosis. Once accomplished, the main
and the rate of increase occurs shortly function of the activated dendritic cell is
after injury or infection. As will be seen to carry pathogenic antigens to the periph-
later, there is considerable interaction eral lymphoid organs to present them to T
between the complement system and other lymphocytes. Once arrived, the dendritic

Basic Components of the Immune System 17

cell matures into an APC, which now per- molecule receptors, permitting them to
mits it to activate pathogen-specific lym- adhere to and migrate from the blood
phocytes. Another function of activated vessels to the site of infection. They are
dendritic cells is to secrete cytokines that attracted to the site by IL-8, C3a, and C3b,
influence both the innate and adaptive cytokines released by TH1 cells, and finally
immune responses (see Chapter 4). factors produced by mast cells. These cells
The mature macrophage also derives are also phagocytic cells, and the process
from primitive stem cells in the bone mar- of phagocytosis is similar to that seen in
row, but unlike the lymphocyte, it matures macrophages. They are particularly effec-
in the tissues. Thus monocytes, the pre- tive when the invading organism becomes
cursors of mature macrophages, circu- coated with antigen-specific antibodies
late for only a few hours before entering (often called opsonins) along with acti-
the tissues where they live for months as vated complement components.
mature macrophages. There is great vari-
ety in the tissue macrophages; they are
Other Functional Cells
heterogeneous in appearance and metab-
olism. They include mobile alveolar and NK cells also can kill target cells in the
peritoneal macrophages. There are also absence of either antigen or antibody
fixed cells in the liver called Kupffer cells stimulation. Their lineage is not known,
and skin macrophages called Langerhans but they are probably in some manner
cells. related to T cells. Unlike other cells, they
The primary function of these mono- can be nonspecifically activated by mito-
nuclear cells is to phagocytose invading gens, interferon, and IL-12. These cells are
organisms, dead cells, immune complexes, particularly useful in the early response to
and antigens. To do this, these cells are viral infection. As in other cells, they have
equipped with powerful lyososomal gran- receptors on their surface that recognize
ules containing acid hydrolases and other particular ligands. For example, NKR-PI is
degrading enzymes. Macrophages need a lectin-like receptor that recognizes carbo-
activation to carry out these functions. hydrate moieties on target cells, which ini-
These include cytokines, which can bind to tiates killing. As in other cell systems, there
IgG: Fc receptors or most importantly (as is also an inhibiting receptor called KIR.
we shall see later) receptors for bacterial This molecule binds to ligands on MHC
polysaccharides. In addition, they can be class I ligands, and this prevents killing of
activated by soluble inflammatory prod- the target cell.
ucts such as C5a. In turn, the macrophages NK cells are not immune cells, and
can release monokines, such as TNF or they have a broad range of specificity and
IL-1, which increase the inflammation in no real memory. Studies of animals with
inflamed tissues. NK deficiencies indicate that they have a
greater incidence of viral infections and
malignancies. This suggests that they
have broad immunological surveillance
These circulating cells also play an impor- properties but the exact mechanisms
tant role in the bodys defense against whereby they exert those properties are
infection. These cells produce adhesin not known.

18 Basic Components of the Immune System

The use of an antibody-coated target to help and suppression, including route of
destroy foreign target cells is called anti- administration, physical nature of the sub-
body-dependent cell-mediated cytotoxic- stance, and the genetic background of either
ity, or ADCC. This killing is dependent on animals or humans. In the latter, there is a
the recognition by cells bearing Fc receptors family tendency to these reactions but exact
and includes monocytes, neutrophils, and genetic factors are still ill defined.
NK cells. These cells do not need simulta-
neous recognition by MHC molecules. The TYPE II: CELL BOUND
mechanisms of killing most likely involve These reactions are initiated by anti-
the release of cytoplasmic components of body reacting with antigen on the cell mem-
the target cells and perforin, but additional branes. IgM and IgG can be involved in
factors are also probably involved. these reactions. Clinical examples include
organ-specific autoimmune diseases and
immune hemolytic anemia. The role of
TISSUE DAMAGE PATHWAYS autosensitized T cells in some diseases such
as rheumatoid arthritis and multiple sclero-
Although the major function of the com- sis have been postulated, but the evidence
ponents of the immune system is to neu- for their involvement is far from clear. In
tralize or destroy the invading organisms Graves disease (hyperthyroidism), auto-
or antigen, these reactions often cause antibodies have a primary pathogenic role
bystander tissue damage as well. These but specific reactive T cells are also present.
are called hypersensitivity reactions, and Gell However, it is not clear whether the T cells
and Coombs conveniently divided them exert a primary role in stimulating anti-
into five types. body production or are really secondary to
the tissue damage.

Hypersensitivity Reactions
TYPE I: IMMEDIATE These reactions result from the pres-
These reactions are those that involve ence of either circulating immune com-
antigens that react with IgE bound to tissue plexes or immune complexes in the tissues.
mast cells or basophils. Activation of the Deposition of immune complexes depends
mast cell results in the release of large on their size, charge, local concentration
amounts of pharmacologically active sub- of complement, and perhaps most impor-
stances. These reactions are rapid (hence tant the nature of the antigen. An excel-
immediate) and if injected into the skin lent example of this type of reaction is the
a wheel and flare reaction can be seen arthritis reaction in which antigen is injected
within five to ten minutes. Most anti- into the skin of an animal previously sensi-
gens stimulating IgE are either inhaled or tized to the same antigen and has produced
ingested. A perfect example of the inhaled antibody to that antigen. The preformed
antigen is ragweed pollen. The IgE pro- antibody goes to the site of the injected
duction requires helper T cells and T-cell- antigen and forms a complex, thereby
derived cytokines. IL-4 and IL-13 stimulate inducing complement activation and neu-
IgE production while IFN- is inhibiting. trophil attraction. The result is intense local
Many factors regulate the balance between inflammation, hemorrhage, and necrosis.

Basic Components of the Immune System 19

There are numerous examples of this type Janeway CA, Travers P, Walport M,
of hypersensitivity reaction, including Schlomchik M, eds. Immunobiology: The
serum sickness, glomerulonephritis, and Immune System in Health and Disease.
systemic lupus erythematosus. Many of New York: Garland Publishing; 2004.
these conditions will be described in detail Paul WE. Fundamental Immunology. 2nd ed.
in later chapters. New York: Raven Press; 1994.


T cells drive this reaction when they Chase MW. The cellular transfer of cutane-
react with antigen and release TH1 cyto- ous hypersensitivity to tuberculin. Proc
kines. The cytokines in turn attract other Soc Exp Biol Med. 1942;59:134135.
cells, such as macrophages, which release Chase MW. Hypersensitivity to sim-
their lysosomal enzymes. Histologically, ple chemicals. Harvey Lect. 1967;61:
the lesions consist of lymphocytes, mac- 169203.
rophages, and occasionally eosinophilic Del Prete G. The concept of type 1 and
polymorphonuclear leucocytes, leading type 2 helper T cells and their cytokines
to a chronic lesion of necrosis fibrosis and in humans. Int Rev Immunol. 1998;16:
granulomatosus reaction. An excellent 427455.
example of this reactivity is seen when Papermaster BW, Dalmasso AP, Martinez
PPD is injected into the skin of a person C, Good RA. Suppression of antibody
who has been previously infected with the forming capacity with thymectomy
tuberculosis organism. in mouse. Proc Soc Exp Biol Med.
Zinkernagel RM, Doherty PC. H-2 com-
patibility requirement for T-cell medi-
ated lysis of target cells infected with
REVIEWS lymphocytic choriomeningitis virus:
Abbas AK, Lichtman AH, Pober JS, eds. different cytotoxic T-cell specificities
Cellular and Molecular Immunology. are associated with structures coded
2nd ed. Philadelphia, PA: W. B. for in H-2K or H-2D. J Exp Med.
Saunders Co.; 1994. 1975;141:14271436.

20 Basic Components of the Immune System

2. Immunological Techniques

John B. Zabriskie, M.D.

INTRODUCTION precise results. Many of these assays are

automated and can be related to interna-
This chapter is not designed to cover all tional standards. Qualitative assays are
the techniques and assays used in clinical less specific and will give answers such
immunology. Rather, it is an introduction as normalabnormal, or positivenegative
to various techniques commonly used in results. The problem is that interpretation
diagnosing human disease or, rather, assays of results may be subjective and require
to evaluate the competence or incompe- special expertise in carrying out the test.
tence of the immune system. Finally, it will Many research tests are in this category at
serve as an introduction to the many new first, and many become quantitative assays
techniques emerging in the past several when more fully developed.
years that have widened our knowledge of
the complex relationship of microbehost
interactions in human disease. ANTIBODY PRODUCTION
Laboratory tests vary widely in clinical
immunology. Some are essential for diag- Antibodies for various tests can be pro-
nosis while others are useful in subclassify- duced in a number of different ways, and
ing disorders. Finally, some are of research we will discuss the prototype of each in
interest only but may add to our immuno- turn.
logical armamentarium in the future. In
this regard, it is important to understand a. Polyclonal antibodies: Many mammals
that these tests do vary in their sensitivity have been used to produce antibodies,
and specificity. ranging from the horse, sheep, and goat
The sensitivity of a test is defined as the down to mice and guinea pigs. Often an
number of diseased individuals that are animal species is selected for antibody
positive for the test compared with those production because it will produce less-
who are negative. The specificity of a test cross-reactive antibodies to a given tis-
is the proportion of individuals without sue. Larger mammals, such as goats and
a given disease that are negative. Thus, a sheep, are used to obtain larger volumes
positive test is really restricted to the dis- of serum to be used therapeutically in
ease in question. humans. A recent fear has been that ani-
The various assays to be discussed mals such as sheep or cows may have
later in this chapter can be conveniently eaten animal foddage contaminated
divided into two main divisions. Some with prion disease. Thus, polyclonal
assays are quantitative in that they produce antibody production for therapeutic

uses has often been limited to countries remains the same as the original clone. As
like Australia or New Zealand where will be seen in many other chapters in this
there have been no recorded cases of book, the use of monoclonal antibodies has
prion disease in mammals. expanded enormously in the past ten to fif-
b. Monoclonal antibodies: Over the past teen years. They may be humanized by
two decades, the revolutionary experi- the introduction of human heavy and light
ments of Kohler and Milstein have chains so that they can be used as thera-
been a major advance in the production peutic agents in many human diseases,
of antigen-specific antibodies. In brief, ranging from rheumatoid arthritis to many
the key to this remarkable advance was forms of cancer.
the ability to obtain spleen cells from
mice that had been immunized with a
given antigen and fuse these cells to a
non-secreting myeloma cell line, which
Measurements of Immunoglobulins
then produces a single antibody clone
when fused with a given B cell pres- The introduction of automated machines
ent in the spleen cells. Antibody clones to measure immunoglobulins and other
are only produced when the mouse B proteins has proceeded rapidly in recent
cell fuses with the myeloma line. Non- decades. Most clinical immunology labo-
fused B cells are eliminated by a spe- ratories rely almost exclusively on these
cial factor in the medium. The beauty machines, and research labs are also intro-
of the hybridoma (fused) cell is that it ducing these automated techniques at a
produces only the antibody of a single rapid pace. Precise measurement of serum
mouse B cell and is therefore identical immunoglobulins is an essential corner-
throughout its variable and constant stone in this area and is important for
regions, and the antibody reacts only repeated and serious infections secondary
with a single determinant on a given to immunosuppressive agents, immunode-
antigen. Finally, it is immortal and will ficiencies, in lymphoproliferate disorders,
produce the same specificity of antibody and for detection of autoantibodies.
for generations. Large-scale culture The main principle behind this test is
of these antibodies can provide large related to the formation of immune com-
quantities of antibody that are precise in plexes between the antibody and a given
their reactivity. antigen. If the concentration of antigen
antibody complex is low, then the immune
However, a word of caution is war- complexes remain in suspension as fine
ranted. These hybridoma clones can some- particles, which can disperse a beam of
times partially lose their antibody produc- light. As the complexes increase with
tion so that they no longer secrete as much concentration of antibody, the complexes
antibody as before and may even stop will precipitate, and light scattering will
production altogether. Finally, they may decrease. This degree of dispersion can be
also lose their specificity so that a given measured on a nephelometer.
hybridoma line must be checked periodi- Using this method, a wide variety of
cally against the original antigen to deter- proteins in serum, amniotic fluid, cerebro-
mine whether production or specificity spinal fluid, saliva, and gastrointestinal

22 Immunological Techniques
juices can be determined. The method spectrophotometer using a 450-nm filter.
includes a wide range of immune reac- By keeping the known antigen constant
tants, acute phase proteins, and tumor and diluting the serum to be tested, one
markers. Standard preparations are used can produce a curve of decreasing optical
and have been calibrated against interna- density readings, thereby indicating the
tional World Health Organization stan- amount of antibody in a given serum when
dards. These tests primarily use polyclonal compared with a standard control.
antibodies for each antigen since monoclo- For detection of small amounts of a
nal antibodies do not form immune pre- given antigen or antibody in a test sample,
cipitates because there are too few relevant the capture assay is used (Figure 2.1 bot-
epitopes. tom). In this case, an unlabeled antibody to
a given molecule is laid down on the plate
to capture the small amount of antigen
Radioimmunoassay and Enzyme-
or antibody present in the test sample. The
Linked Immunosorbent Assays
second antibody to this antigen or anti-
body is labeled with the appropriate fluo-
The use of these highly sensitive assays in rochrome, and the rest of the tests proceed
human disease has virtually exploded in as in the direct assay described previously.
the past two decades. They can be used While the radioimmunoassay remains the
to detect the levels of a given antibody or gold standard for many clinical labora-
hormone in human serum, and they are tories, more research and clinical laborato-
extremely sensitive methods of detecting ries are turning to the ELISA since it does
low levels of autoantibodies. not present the problem of radioactivity
In the radioimmunoassay, one can hazards or, perhaps more important, the
radiolabel a particular antigen or antibody removal of radioactive wastes (mainly a
using either 125I or 14C tagged to the antigen problem of disposal sites).
or antibody. Once the serum or purified
antibody or antigen to be tested is placed in
the well, a second radiolabeled antihuman
IgG antibody is placed in the well. After This immunological technique has gained
appropriate binding and further washes, great favor with both basic immunolo-
the degree of activity of the antibody to gists and clinical immunologists over the
a given antigen can be determined in a past decade. Its beauty is its simplicity
counter (see Figure 2.1 top left). and the fact that one can compare differ-
The description of ELISAs; (Figure 2.1 ent proteins, toxins, and cellular products
top right) is similar to that described for all at the same time and reach conclusions
the radioimmunoassay, but in place of concerning their commonality or differ-
the radioactively labeled antibody or anti- ences or purity. The procedure is relatively
gen, various fluorochromes have been simple. The proteins to be studied are run
substituted in place of the radioactive on a standard SDS gel, the percentage of
label. In the presence of an appropriate which depends on the known or estimated
substrate, the fluorochrome-labeled anti- size of the protein: larger proteins are run
body is activated to produce a given color, in 10 percent gels, while smaller proteins
and the intensity of the color is read on a are run on 15 percent gels. The gel is then

Immunological Techniques 23
Radioactive Assay Direct ELISA
Radio active label Fluorochrome

Antibody in serum
Active substrate

Antigen Antigen



Second Antibody


Figure 2.1 Schematic depiction of the radioimmunoassay and the direct and
indirect ELISA. Both methods are extremely sensitive and will detect very
small amounts of antibody in a given serum. One can label either antigen or
antibody to determine amounts of antigen or antibody in a given serum or
preparation. The indirect or sometimes called the capture. ELISA is also

removed and the proteins in the gel are

Complement Assays
transferred by another electrical charge to
a cellulose membrane. The membrane is Perhaps the most useful assays for comple-
treated overnight with a blocking buffer, ment are the immunochemical assays of C3
washed, and then layered over the mem- and C4. As noted in Chapter 1, which out-
brane with the antibody designed to pick lines the alternative and classical modes
up the binding to the protein (S) in ques- of complement breakdown, a low C3 and
tion. This incubation usually lasts one C4 but normal factor B suggest that activa-
hour; following washes, the membrane is tion of the classical pathways has occurred.
treated with a species-specific second anti- Examples would be patients with systemic
body tagged to an enzyme and developed lupus erythematosus or vasculitis. In con-
with an enzyme substrate to form a colored trast, if C3, C4, and factor B are all low,
band (see Figure 2.2). the alternative pathway is also activated

24 Immunological Techniques
1 2 3 4 5 6 7
poststreptococcal glomerulonephritis, or
30- Antibodies to common microbial anti-
gens have been used to detect infection
20- with different microbes for years. However,
the presence of a single antibody serum
Figure 2.2 The photo is an example of an
specimen only tells you that the person
immunoblot in which the proteins in question
are electrophoresed in a 15 percent SDS-gel, was exposed to this microbe in the past.
then transferred to nitrocellulose paper and To diagnose an acute infection, one must
incubated overnight in blocking buffer. This have paired sera usually taken two weeks
is followed by incubation with first antibody
(1:1,000 dilution) followed by the species-
apart that demonstrate a significant rise
specific second antibody tagged to alkaline in antibody titers in the second specimen
phosphatase. The bands are developed using compared to the first. One can also do these
an alkaline phosphatase substrate. In this case, antibody tests in reverse. Normally, we are
a single antipeptide antibody covering two
sections of homology of the superantigens was exposed to many microbial antigens during
used as the immunogen. Wells 27 contain three growth and development either as a result
superantigens, each from the streptococcal and of exposure to a given microbe or after
staphylococcal family of superantigens.
immunization with a given antigen (i.e.,
tetanus toxoid, pneumococcus polysac-
via either feedback loops or simultaneous charide, measles, or mumps viral antigen).
activation. This would point to a gram- Antibodies to the microbial products are
negative bacteremia. Normal C4 levels usually found in normal individuals, but
with low C3 and factor B levels suggest the if they are not, one should suspect abnor-
alternative pathway alone. Elevation of all malities of antibody production like those
three components usually suggests acute seen in immunodeficiency states.
or chronic infection. Acute rheumatic fever
is such an example.
Assays for immune complexes are best
directed toward an analysis of the immune
Fluorescein-Activated Cell Sorter
complexes or their deposition in various
human disease tissues. In most cases, the With the renewed interest in the role of
best approach is to receive freshly biop- lymphocytes in disease states over the past
sied nonfrozen material that is then snap thirty years, a systematic study of the mark-
frozen and sections cut and stained to test ers present on B and T lymphocytes has
for the presence of appropriate antigen or been undertaken. The knowledge that many
antibody (see Figure 12.3 in Chapter 12). such markers exist on a given cell was made
In some cases, the antigen is still intact possible by the introduction of monoclonal
after formalin fixation and paraffin blocks antibodies specific for each marker. Thus,
are prepared. But in these specimens, one antibodies could quickly identify lympho-
always runs the risk of destroying the cytes as B (CD19) or T lymphocytes (CD3)
appropriate antigen or antibody during the and later into helper (CD4) or suppressor
fixation process. Examples of the diseases cells (CD8) and many other markers.
studied in this manner are renal immune The second major advance was the
complex disease such as seen in SLE, acute introduction of the flow cytometer, which

Immunological Techniques 25
measures the fluorescence of each labeled calculated. All of these values are obtained
antibody. The different cell populations are rapidly due to the speed with which cells
aspirated into the machine, which forces are counted with different labels, and the
the cells to flow through the chamber singly results are accurate because of the large
past a laser beam and light sensors. Light number of cells that are counted (in the
emitted by the excited fluorescent dye on thousands). It should be emphasized that
the cell surface is detected by the sensors values for lymphocyte subsets will change
and analyzed by computer software. Using with increasing age from children to adults,
this system, one can divide cells into dif- especially during the first year of life.
ferent populations, depending on their size Using a more sophisticated machine
and granularity (see Figure 2.3). Once the called a fluorescein-activated cell sorter
different cell populations are identified, a (FACS), individual subsets of lymphocytes
specific population can be gated for fur- can be separated with fluorescein-labeled
ther study, such as identification of helper antibodies into unique cell populations or
cells (CD4) and suppressor cells (CD8-PE), even single cells. This procedure is done
as well as many other lymphocyte subsets. under sterile conditions and is useful in
A variation of this technique is illus- isolating a single cell or cell population for
trated in Figure 2.4 in which the lympho- further cell culture, growth, and study.
cytes are identified with anti-CD3 labels.
Of this population, the number of B cells
Lymphocyte Proliferation Assays
in the population is identified by CD19-
PE labeled antibodies. Finally, the num- This test assumes greater importance in
ber of B cells expressing a novel antigen clinical immunology both at a research
is identified, in which the percentage level and in the clinical laboratories. These
of CD19+ exhibiting D8/17+ B cells is tests can be carried out in whole blood
Side scatter (granularity)


Red cells Monocytes

Foward scatter (size)

Figure 2.3 Schematic depiction of cell separation on a flow cytometry

machine or fluorescein-activated cell scan. The different cell populations are
separated both by size (forward scatter) and granularity (side scatter) and
a given population is then separated into B or T cells and within the T cells
into helper (CD4+) and suppressor (CD8+) cells. Isolation and purification of a
given subset of cells can be achieved by a fluorescein-activated cell sorter.

26 Immunological Techniques







100 101 102 103 104 100 101 102 103 104 B







100 101 102 103 104 100 101 102 103 104 D

Figure 2.4 FACS analysis of D8/17-positive lymphocytes. Whole venous blood

was obtained from a normal patient (panels A and B) and an obsessive-compulsive
disorder (OCD) patient (panels C and D). Panels A and C are controls in which the
D8/17 and CD19 antibodies were omitted. Panels B and D are dual-labeled cells
from a normal individual and a patient with OCD. The D8/17 fluorescence as a
percentage of total B cells was 0.5 percent in the normal and 33.4 percent in the
OCD patients in panels B and D. Each point on this plot represents fluorescence
recorded from an individual lymphocyte as it passes through the counter. Reprinted
with permission from Elsevier; Chapman F, Visvanathan K, Carreno-Manjarrez R,
Zabriskie JB. A flow cytometric assay for D8/17 B cell marker in patients with
Tourettes syndrome and obsessive compulsive disorder. J Immunol Methods.

that has been anticoagulated so as to per- trophils and red blood cells are centrifuged
mit the use of viable cells. The preferred to the bottom, while the mononuclear
method is the isolation of the lymphocytes cell population stays in the middle of the
from blood using a density gradient assay gradient. Finally, the serum components
method. The anticoagulated blood sample and platelets are mainly in areas above
is usually diluted 1:1 with PBS (pH 7.4) and below the mononuclear cells. Once
and slowly layered over the density gradi- the mononuclear cells are removed from
ent solution, which has been prepared so the gradient and washed several times, a
that the cell populations will separate into relatively pure population of mononuclear
different layers. As seen in Figure 2.5, neu- cells is obtained.

Immunological Techniques 27
of microbial agents, human genetics, and
analysis of tissue samples, to name but a
+ few of the uses of DNA technology today.
whole blood The analysis of blood or serum samples
also has far-reaching implications in our
legal system. The following is a short sum-
mary of these techniques and their impor-
Diluted blood tance in clinical immunology.
layered on top

Known unique segments of nucleic acid
Centrifugation sequences can be used as DNA probes to
determine the presence of complemen-
Platetet-rich, diluted plasma tary sequences of DNA in a sample from a
given patient. The probe, which is a single
Layer of lymphocytes and
monocytes platelets strand of a given DNA, is presented to the
Red cells and granulocytes target DNA, which is composed of thou-
Remove + wash + test sands of nucleotides. The complementary
strands from target and probe DNA will
Figure 2.5 Lymphocyte and monocyte anneal to each other, a process known as
separation using a ficoll gradient. As noted, DNA hybridization. The high affinity of the
red cells and granulocytes spin to the
bottom of the tube while a clear layer of probe for a complementary segment in the
lymphocytes and monocytes is separated target DNA is the most specific intermo-
by the gradient. These cells are counted lecular interaction between biological mac-
and adjusted to a given number of cells for
use in the proliferation assay.
This technique may be used not only on
fresh samples but also in tissues that have
When these cells are stimulated by a formalin-fixed and paraffin-embedded
specific antigen, a few of these resting cells tissues. In this technique of in situ hybrid-
undergo proliferation (activated cells) and ization, the probes can be applied directly
the transformation of activated cells can be to tissue sections on microscope slides.
measured by incorporation of radiolabeled However, this technique works only after
thymidine into the DNA of the cells and the deparaffinization and proteolytic diges-
number of counts per minute measured in tion are performed to expose intracellular
-counter. More recently, these same acti- nucleic acid targets. The probe is detected
vated cells can be measured by the num- in the sample either by radiolabeling the
ber of cells expressing cell surface antigens probe or radiolabeling an antibody to the
using markers such as CD69. probe DNA.
Another approach is the use of restric-
tion endonucleases (see Figure 2.6). These are
DNA Technology Assays
enzymes that cleave DNA at sites specifi-
The emergence of molecular biology and cally related to the nucleotide sequences.
DNA technology has ushered in a whole Using enzymes with different specificities,
new series of methods for the detection a DNA fragment containing a particular

28 Immunological Techniques
Restriction Denaturation Labeled
endonuclease cDNA
Identification of
Agarose gel Nitrocellulose labeled gene
electrophoresis blotting Hybridization fragment

gene of

dsDNA fragment containing gene of interest dsDNA fragment after denaturation

Figure 2.6 The use of restriction endonucleases to carry out gene mapping using the Southern
blotting technique. DNA hybridization is the most specific intermolecular interaction between
biological macromolecules.

gene can be cut out from the DNA mole- tion in detecting DNA material occurred
cule. In the Southern blot, these fragments with the introduction of the PCR assay.
of DNA are electophoresed on agarose gels This method is particularly valuable since
with the smaller fragments migrating fur- it can markedly amplify a small piece
ther than the larger ones. Alkaline dena- of DNA before cleavage with a restric-
turation of these fragments uncoils the tion enzyme. Complementary oligonu-
fragment so that a single stranded DNA cleotide primers from either end of the
will hybridize with the complementary target DNA are added to the denatured
DNA after transfer to a special nitrocellu- sample, along with a heat-resistant DNA
lose filter. Blotting the gel with the nitrocel- polymerase. If the target sequence is pres-
lulose filter fixes these DNA fragments ent, the primers anneal to it and provide
after the electrophoresis. Finally, a radio- a starting point for the polymerase to
labeled probe containing DNA known to begin the synthesis of second-strand DNA
be complementary to the DNA of interest (see Figure 2.7). The newly synthesized
hybridizes to it, and the fragment can be double-stranded DNA is then denatured
identified by autoradiography of the filter. by heating and exposed again to the poly-
The Northern blotting technique is very merase enzyme at a lower temperature.
similar to the DNA experiment but uses In this way, newly synthesized molecules
RNA instead of DNA in the system. and original DNA can reassociate with
the primer and act as templates for fur-
POLYMERASE CHAIN REACTION (PCR) ASSAY ther rounds of DNA synthesis. After com-
Although the DNA analysis systems pleting about thirty cycles (usually two
described earlier are still used in research to three hours in an automated machine),
and clinical laboratories, a major revolu- the specific target sequence is amplified

Immunological Techniques 29
Primer 1 DNA sequence
sequence of interest
5 3
3 5
Primer 2
sequence Primer 1 Primer 2
Cycle 1
Anneal (primer excess)

DNA synthesis

Cycle 2
Anneal (primer excess)

DNA synthesis


Figure 2.7 Schematic description of a PCR. To use this method, it is necessary to know the
sequence of a short region of DNA on the 3 and 5 of the sequence. It is a very powerful tool for
detecting DNA sequences in tissues, dried blood spots, and pathological specimens. The specimen
must be handled carefully to avoid introduction of other DNA material, that is, the DNA of the handler
of the specimen in question.

more than 1-million-fold. This power- 1960s identified a series of antigens present
ful and sensitive technique can detect a on mononuclear cells called the human leu-
specific DNA sequence from a single cell cocyte antigens (HLAs). Using a somewhat
(e.g., lymphocyte, sperm), fixed pathologi- crude agglutination assay of leucocytes,
cal specimens, and dried blood spots. The they noticed that these sera agglutinated
main disadvantage is that contamination leucocytes from unrelated donors and that
of the reaction mixture with traces of the patterns of such co-agglutination dem-
DNA from another source will lead to onstrated definitive antigens in human
false positive results. Thus extreme care in populations. After extensive research and
handling specimens to be tested as well as workshop meetings, it became apparent
in the test itself is obligatory. that these antigens were present on all tis-
sues of the body, but their high concen-
MAJOR HISTOCOMPATIBILITY (MHC) ASSAYS tration on peripheral blood lymphocytes
Using sera obtained first from multi- enabled investigators to perform immu-
transfused patients and later from multipa- nogenetic studies more easily on these
rous women (alloantigens), the early work cells. While most of the original testing
of Dausset and others in the 1950s and for the antigens on cells and for use in

30 Immunological Techniques
transplantation relied on serological tech- The method is relatively straightfor-
niques using purified antibodies to detect ward. One takes the tissues or cells to be
these antigens, the introduction of the PCR studied and prepares them as follows:
assay changed the rapidity with which
these antigens could be identified, and Tissue mRNA cDNA
both MHC class I and class II antigens are Biotinylated cRNA
now routinely identified by the method.
Generally, this technique is available The biotinylated material is layered on
only in centers that specialize in organ and glass slides on which DNA fragments
bone marrow transplantation. In addition of the human genome have been placed
to the transplantation, these techniques (12,000 probe sets/chips). The chips are
are used for familial genetic studies as then washed, stained with streptavidin-
well as for specific disease states (see phycoerythrin, and scanned with a probe
other chapters in this book). One example array scanner.
is the identification of HLA B27, which is As an example, Figure 2.8 represents
highly associated with anklyosing spon- the staining patterns seen in six psoriatic
dylitis, and as the research continues one patients before and six hours after treat-
can expect that other genetic MHC mark- ment with a T-cell suppression agent. Note
ers will be identified in particular disease the decreased expression of red intensity in
states. The converse is also true. It is now
known that certain MHC antigens confer
N 1 2 3 4 5 1 2 3 4 5 6 7 8
resistance to a given disease, while others
confer susceptibility. A word of caution
is warranted. These markers only sug-
gest a susceptibility to the disease. Most
disease states appear to be polygenic,
and factors such as the environment and
past exposure to a given microbe also
play a part in establishing the full disease

Over the past decade, this assay system
has become popular in many branches of
science, including immunology. In essence,
these assays have become the standard
tools for gene expression profiling as the
mRNA levels of a large number of genes
Figure 2.8 Photograph showing
can be measured in a single assay. Of par- genomic analysis of lesional versus
ticular interest, one can compare the lev- nonlesional skin in psoriatic skin. The
els of gene expressions across the human heat map describes 1,119 genes that
have significantly higher expression (red
genome in one subset of patients in a given
area) by 1.2-fold (P < 0.05, after BH
disease to another subset of patients with correction) in the lesional skin versus
the same disease. nonlesional skin (green area).

Immunological Techniques 31
the patterns following treatment, indicating Rose NR, de Macario EC, Folds JD, Lane
a decrease in gene regulation. HC, Nakamura RM, eds. Manual of Clin-
ical Laboratory Immunology. Washington,
DC: American Society for Microbiol-
von Muhlen CA, Tan EM. Autoantibod-
The use of monoclonal antibodies, espe- ies in the diagnosis of systemic rheu-
cially humanized ones, will play an matic diseases. Semin Arthritis Rheum.
increasingly important role in both basic 1995;24:323358.
immunology and clinical immunology.
Their use will be in both detection of small SUGGESTED READING
and early tumors and in the treatment of Alvarez-Barrientos A, Arroyo J, Canton R,
tumors. The success of monoclonal anti- Nombela C, Sanchez-Perez M. Appli-
bodies in metastatic prostate cancer is cations of flow cytometry to clini-
already well established. It is believed that cal microbiology. Clin Microbiol Rev.
other tumor therapies will follow in the 2000;13:167195.
future. Churchill GA. Fundamentals of experi-
The use of microarray assays has grown mental design for cDNA microarrays.
in the past decade and will continue to Nat Genet Suppl. 2007;32:490495.
grow. The use of these assays has a role in Craig FE. Flow cytometric evaluation of B
diseases associated with human genetics cell lymphoid neoplasms. Clin Lab Med.
and provides information on which cyto- 2007;27:487512.
kines or lymphokines are important in dis- Dausset J. The major histocompatibil-
ease states. ity complex in man. Science. 1981;213:
Davies MA, Marstone AJ, Viza DC, Colon-
BIBLIOGRAPHY bani J, Dausset J. Human transplan-
tation antigens: the HLA (Hu-1) sys-
tem and its homology with the mouse
Abbas AK, Lichtman AH, Pober JS, eds.
H-2 system. Transplantation. 1968;4:
Cellular and Molecular Immunology. 2nd
ed. Philadelphia, PA: W. B. Saunders
Dyer P, Middleton D. Histocompatibility
Co.; 1994.
Testing: A Practical Approach. Oxford:
Kohler G, Milstein C. Continuous cul-
IRL Press; 1993.
tures of fused cells secreting anti-
body of predefined specificity. Nature.

32 Immunological Techniques
3. Immune Regulation

Nina Bhardwaj, M.D., Ph.D., David ONeill, M.D.,

and Thomas Waldmann, M.D.

INTRODUCTION or reversal of graft rejection (see Chap-

ter 18). The other is in the treatment of
The immune system in general responds autoimmune and malignant diseases.
appropriately to the presence of foreign anti- Corticosteroids modulate inflammation
gens. However, there are certain diseases by suppressing cytokine- and chemokine-
that arise from either a defective or over- encoding genes, which inhibits the acti-
responsive immune system on the part of vation and recruitment of inflammatory
the host. Two major therapeutic approaches cells.
are possible: either immunosuppression or The side effects of steroids are numer-
immunopotentiation of the immune system. ous and often depend on both the dose used
The object of this chapter is to introduce the and duration of treatment. These include
reader to the various approaches that have an increased susceptibility to infection,
been used to either suppress or stimulate osteoporosis, and growth disturbances in
the immune response. children, as well as gastric ulcers, hyperten-
sion, acne, and hirsutism. By giving larger
doses for shorter periods, many of these
IMMUNOSUPPRESSION side effects are lessened.
The development of the thiopurines
Immunosuppressive Drugs
in the 1950s ushered in a new group
Several groups of drugs suppress the of immunosuppressive agents, the most
immune system (see Table 3.1). Among important of them being azathioprine. It is
the oldest of these drugs are the corticoste- inactive until it is metabolized in the liver
roids, which have long been known to alter and takes three to four weeks to be effec-
immune responses. When corticosteroids tive. The metabolites work by inhibiting
are given, the result is a transient lympho- DNA synthesis in dividing cells (such as
penia peaking at four hours and lasting activated lymphocytes). Like many other
up to twenty-four hours. Helper T cells drugs, it has side effects, mainly in bone
are predominantly affected, and at higher marrow toxicity, and long-term use even-
doses of steroids inhibition of interleukin-2 tually results in granulocytopenia and
(IL-2) production by helper T cells becomes thrombocytopenia.
increasing important. Another major effect Another group is the alkylating agents, of
in humans is on resting macrophages which cyclophosphamide is one of the best
(activated macrophages are not sensitive). examples. This drug also requires activa-
In humans, steroids are used for two tion by the liver. It inhibits cell division and
main purposes. One is the prevention can suppress antibody production, and it

Table 3.1 Monoclonal Antibodies Approved for Cancer Therapeutics

Agent Principal Mode of Actiona

Corticosteroids Inhibition of activation of cytokine and chemokine genes by nuclear

factor B
Mercaptopurine Inhibits nucleic acid synthesis in activated lymphocytes
Azathioprine Inhibits nucleic acid synthesis in activated lymphocytes
Myocphenolate mofetil Inhibits inosine monophosphate and lymphocyte proliferation
Methotrexate Inhibits dihydrofolate reductase; anti-inflammatory
Leflunomide Inhibits pyrimidine synthesis; anti-inflammatory; antiproliferative
Cyclophosphamide Cross-links DNA; blocks cell division
Cyclosporine Binds calcineurin, inhibits nuclear factor of activated T cells; early
events in T-cell activationb
Tacrolimus (FK506) Binds tacrolimus-binding protein; inhibits nuclear factor of activated
T cells; early events in T-cell activation
Sirolimus FTY720 Blocks T-cell proliferation
Analogue of sphingosine 1-phosphate; inhibits lymphocyte homing
Rapamycin Interferes with cyclins; blocks mitogen-activated signals and cell cycle

Nuclear factor of activated T cells is a transcription factor that regulates production of IL-2 and
other cytokines; tacrolimus-binding protein is a member of a family of at least eleven proteins,
some of which inhibit calcineurin.
Ligation of the T-cell receptor activates calcineurin, a serine-threonine phosphatase and a member
of the family of intracellular regulatory proteins termed cyclophilins. Some cyclophilins can inhibit
calcineurin, regulate intracellular calcium flux, and activate the NFAT gene. Cyclosporine binds to
the cyclophilin CyPA, thereby inhibiting the phosphatase activity of calcineurin.

decreases delayed-type hypersensitivity. as nephrotoxicity and hepatotoxicity and

Methotrexate, which inhibits cell division particularly lymphoma induction. Taco-
by disrupting folic acid metabolism, has limus is a newer-generation drug with a
similar immunomodulatory effects. similar mechanism of action.
Cyclosporin, a naturally occurring
fungal metabolite, also inhibits T-cell acti-
vation and cell-mediated immunity. The ANTIBODIES AND OTHER
drug becomes active only when complexed IMMUNOSUPPRESSIVE METHODS
to its intracellular receptor cyclophilin, and
it inhibits early calcium-dependent events, There are a number of instances in which
especially the activation of several cytokine antibodies have been used to suppress the
genes. Its major effect is the inhibition of immune response. Among the earliest mea-
IL-2 production and the CD4+ proliferation sures has been the use of anti-RHD anti-
responses. Cyclosporin has been extremely bodies to prevent hemolytic disease of the
useful in the control of transplant rejec- newborn due to incompatibility between
tion and is also used in several autoim- the mother (RHD) and an RHD+ fetus.
mune diseases such as psoriasis and severe The disease is prevented by the adminis-
rheumatoid arthritis. However, long-term tration of anti-D antibodies to the mother
use has demonstrated severe toxicity such immediately after delivery. This inhibits

34 Immune Regulation
the formation of anti-D antibodies in the infusions must be repeated frequently to
mother, thereby avoiding the development sustain results.
of serious disease in the infant. This thera- Monoclonal antibodies can also be used
peutic measure has virtually eliminated as antitumor agents (see review by Reichert).
the incidence of RH disease in developed Specific targeting and killing of tumor cells
countries. can be enhanced by linking tumor antigen-
More recently, monoclonal antibodies specific monoclonal antibodies to agents as
are being used to suppress the immune follows: (1) a cytotoxic drug such as metho-
system, and several antibodies are now trexate, (2) a radioisotope such as iodine-
approved for the treatment of autoimmune 131 or ytrium-90, or (3) a toxin such as ricin.
diseases. These antibodies are typically Monoclonal antibodies are now approved
humanized mouse monoclonals, created for the treatment of non-Hodgkins lym-
by transposing the mouse antigen-binding phoma, myeloid and lymphocytic leuke-
sites onto a human antibody framework mia, breast cancer, and colorectal cancer
(see Figure 3.1). This technique retains the (Table 3.2).
full range of effective properties of human Some new studies, still in clinical trials,
Fc while minimizing the immunogenicity are occurring in the treatment of prostate
of the mouse component. Antibodies that cancer, especially metastatic bone lesions,
target the immune system can target cell and it is hoped many more monoclonal
surface molecules on T or B cells or can antibodies will be forthcoming against var-
target soluble mediators of inflammation ious other tissues.
such as cytokines. Among the most effec- Other methods of immunosuppression
tive uses of monoclonal antibodies has are plasmapheresis or plasma exchange. In the
been in treating severe rheumatoid arthri- first method, improvement may be due to
tis, using monoclonal antibody directed removal of mediators of tissue damage,
against tumor necrosis factor (TNF-). whereas in plasma exchange, it may be due
The drawback to this therapy is that the to replacement of deficient factors or to

Variable region
Mouse CDR


Muromonab-CD3: Infliximab: mouse Trastuzumab mouse

all mouse anti-foreign variable region, CDRs, humanized
antibody chimeric antibody antibody

Figure 3.1 Three types of monoclonal antibodies now in clinical use. Reprinted with permission from
Schwartz RS. The Shattuck Lecture: diversity of the immune repertoire and immunoregulation. New
Engl J Med. 2003;348:10171026.

Immune Regulation 35
Table 3.2 Immunosuppressive Drugs in Clinical Use or Clinical Trials

Year Approved Brand Name Generic Name Type of mAb Target Indication

1997 Rituxan Rituximab Chimeric CD20 NHLa

1998 Herecptin Trastuzumab Humanized HER2 Her-2/nev
positive breast
2000 Mylotarg Gemtuzumab Humanized CD33 AML
2001 Campath-1 Aliemtuzumab Humanized CD52 B-cell CLLb
2002 Zevalin Ibritumomab Murine CD20 NHL
to 111In or 90Y
2003 Bexxar I-tositumomab Murine CD20 NHL
2004 AvastinTM Bevacizumab Humanized VEGF Colorectal
2004 Erbitux Cetuximab Chimeric EGFR Colorectal
(HER-1) cancer

EGFR, epidermal growth factor receptor; VEGF, vascular endothelial growth factor.
NHL: Non-Hodgkins lymphoma.
CLL: Chronic lymphocytic leukemia.

the immunomodulatory effects of human the host immune system, so potentiation

immunoglobulins. of the host immune system may prove
Total lymphoid irradiation produces long- useful in treating these cancers. There
term suppression of helper T cells and has are three principal ways to potentiate the
been used in some severe autoimmune immune response in humans: through
diseases like lupus or rheumatoid arthritis. cytokines, adoptive immunotherapy, or
The side effects of this treatment may be vaccination.
severe and sometimes fatal.

Cytokine Therapy
IMMUNOPOTENTIATION Interferons are antiviral glycoproteins,
which are secreted as a result of a viral
Chronic infections such as HIV and hepa- infection and have wide-ranging antitu-
titis C are characterized by an inability mor and immunomodulatory effects. They
of the host to control viral replication. have attracted much interest as immuno-
The ability to potentiate the host immune therapeutic agents. Interferons bind to cell
response to control chronic infections is an surface receptors and activate secondary
important goal and is under active investi- intracellular changes which inhibit viral
gation. It has also been shown that at least replication. They can be divided into three
some types of cancer can be controlled by groups: alpha (), beta (), and gamma

36 Immune Regulation
() interferons. All three interferons have pulmonary edema, and neuropsychiatric
been genetically engineered, and recom- symptoms.
binant IFN-, -, and - are available, but
IFN- is the best studied. IFN- is the
Adoptive Immunotherapy
treatment of choice for hepatitis B and C;
when given systemically, it produces sig- Adoptive immunotherapies involve
nificant clearing of hepatitis B in chronic the transfer of either cells or antibodies
carriers. IFN- has some side effects, into a host. These are also referred to as
mainly flu-like symptoms such as fever, passive therapies, since the host does not
malaise, and anorexia all symptoms that actively mount its own immune response.
can be tolerated. More severe effects are Examples include infusion of hepatitis B
reversible: bone marrow depression, liver immune globulin and the adoptive trans-
dysfunction, and cardiotoxicity. fer of antigen-specific T lymphocytes to
IFN- has been shown to be of benefit in treat a chronic viral infection or cancer (see
patients with relapsing-remitting multiple Gattinoni et al. 2006).
sclerosis, and IFN-1 appears to decrease
the rate of progression of disability. Despite
these results, the precise therapeutic role of
IFN- is still controversial. Prevention of infectious diseases depends
IFN- is a potential activator of mac- on many factors. Foremost is the presence
rophages and is most often used in con- of a clean water supply, development of
ditions in which defective macrophage sanitary facilities, good nutrition, and good
function occurs. Examples of these disor- personal hygiene. More recently, immuni-
ders are lepromatous leprosy, leishmani- zation against a particular agent has been
asis, and chronic granulomatous disease. the most effective measure in controlling
IFN- works by increasing phagocytic bac- infectious disease. Yet, with the emergence
tericidal activity, but only some patients of new infectious agents such as hepatitis C
show enhanced superoxide activity, imply- and HIV, novel approaches will be needed
ing that IFN- works by several different to generate new and effective vaccines.
IL-2 is produced by stimulated CD4 T IMMUNITY
cells and induces clonal expansion of IL-2+ The two ways to achieve immunity are
T and B cells. is used in immunodeficiency actively and passively. Active immunity is
states such as HIV infection where IL-2 achieved when exposure to a foreign stim-
production is defective. In patients with ulus triggers an immunological response
HIV infection and baseline counts of CD4 to the agent by the host. The best immunity
above 200 ml, intermittent IL-2 infusions to an agent is achieved by natural infection,
have been shown to produce substantial which evolves with a clinical or subclinical
and sustained increases in CD4 counts. IL-2 response to the agent by the host. Artificial
has side effects similar to IFN-, with most active immunization is the administration
of the effects being flu-like. The most seri- of an immunogen as a vaccine. Vaccines
ous side effect is IL-2 action on IL-1, IFN-, may be live organisms, killed organisms,
and TNF, all mediators of vascular perme- or modified toxins. Although no vaccine
ability, resulting in marked hypotension, is ideal and each has its problems, the

Immune Regulation 37
problems of live vaccines are generally humans is aluminum compounds, which
related to their safety, while the problems are generally safe for human use. Others
of killed vaccines are related mainly to include muramyl dipeptide, biodegradable
their effectiveness. polymers, and a glycoside adjuvant called
Live attenuated vaccines are useful Quil A from the bark of an Amazon oak
because they infect, replicate, and immu- tree. However, many others are being
nize in a manner similar to natural infection developed or will probably be given U.S.
but with milder clinical symptoms. Exam- Food and Drug Administration (FDA)
ples include many of the childhood infec- acceptance in the future. The key feature
tions such as measles, mumps, and rubella will be their immunogenic enhancement
(MMR vaccine), chicken pox (varicella) and their strength of safety for use in
and Bacille Calmette-Gurin (BCG) for humans.
tuberculosis. Although millions of doses Although we have mainly been dis-
have been administered with no complica- cussing various forms of vaccination to
tions, if given to an immunocompromised protect against the invading organism,
host (such as primary immunodeficiency one of the most interesting new vaccines
or secondary to HIV infection), these live has not been developed to eliminate the
vaccines may cause serious disease. infectious agent but rather to prevent the
Killed vaccines consist of suspen- development of another far more serious
sions of killed organisms such as typhoid, disease a complication of the initial infec-
cholera, and pertussis (although there is tion. This is the Gardisal vaccine manufac-
now an acellular vaccine) or one of the tured by Merck to protect against human
products or fractions of the organism. papilloma virus (HPV). HPV infection is
These include toxoids of diphtheria and usually sexually acquired, and it is esti-
tetanus and subunits of viruses such as mated that currently 20 million people are
surface hepatitis B antigen. Among the infected in the United States. The infection
most successful of these types of vaccines has no real signs or symptoms, and HPV
has been the use of polysaccharides in may lead to cervical cancer. It is estimated
the pneumococcal, meningococcal, and that ten of the thirty different serotypes
Haemophilus influenza vaccines. In general, of the virus can induce cervical cancer, so
the killed vaccines are not as effective as the vaccine has been directed at eliminat-
the live viruses because they do not give ing those serotypes. Thus, if 10,000 women
long-lasting immunity as a live infection are infected with one of the high-risk viral
does. For example, although the tetanus serotypes, approximately 3,900 of them
toxoid vaccine is effective, it requires a will die of cervical cancer. The new vac-
booster dose every ten years. cine, if given before active sexual activity
The immunological response to the in women, can prevent the viral infection
killed organism or product thereof has been and thereby markedly diminish the risk of
enhanced by the use of adjuvants. Although cervical cancer. Because of the possible suc-
the most common adjuvant for animal cess of this vaccine, it may be worthwhile
studies has been the complete Freunds to look at how to prevent the Epstein-
adjuvant, it cannot be used in humans Barr virus in at-risk children to prevent or
because it causes liver, skin, and spleen dys- diminish the risk of Burketts lymphoma
function. The most common adjuvant for in children infected with the virus.

38 Immune Regulation
CYTOKINE IMMUNOMODULATION and promote transcription of target genes
(see Figure 3.2).
Although many of the immunoregulation There are several distinct differences
techniques revolve around the humoral in the functions of IL-2 and IL-15. IL-2
arm of the immune response, there is is involved in checkpoints or brakes on
increasing interest in targeting T cells or the immune system. IL-2 is required to
the cytokine regulators to regulate the maintain the competitiveness of forkhead
elimination of autoreactive T cells, tumor box P3- (FoxP3-) expressing regulatory
cells, and the maintenance of a specific T cells, and plays a crucial role in activa-
memory response to these pathogens. Such tion-induced cell death (AICD), a process
immune responses are normally regulated that leads to the elimination of self-reactive
by cytokines, and the activities of the cyto- T cells. Thus, the unique feature of IL-2
kines have a high degree of redundancy. is to prevent T-cell immune response to
The common cytokine receptor chain is self that would lead to autoimmunity. In
used by IL-2, IL-4, IL-7, IL-9, IL-15, and contrast, IL-15 expression has no effect on
IL-21 (see Figure 3.2). The IL-2R and the regulatory T cells but is an anti-apoptotic
IL-15R have a second ( chain) subunit in factor in several systems. Furthermore,
common as well, but have unique sub- IL-15 promotes the maintenance of CD8+
units (see Figure 3.3). These two cytokine CD44 memory T cells. Thus, IL-15 has as its
receptors use a common signaling path- primary role the maintenance of a robust
way that involves JAK1, JAK3, and STAT5. memory response to invading pathogens.
The signaling involves the phosphoryla- These observations from ex vivo func-
tion of STAT5 that results in the dissocia- tional studies are supported by studies in
tion of STAT5 from the receptor and the mice deficient in cytokine or cytokine recep-
subsequent dimerization of STAT5. The tor genes. For example, IL-2-deficient and
dimers then translocate to the nucleus IL-2R-deficient mice develop a marked

IL 2R IL 15R

IL-2 IL-15 IL 4 IL 7 IL 9 IL 21

IL-2/15R IL-2/15R IL-4R IL-7R IL-9R IL-21R

Cell surface


Nucleus DNA

Copyright 2006 Nature Publishing Group

Nature Reviews | Immunology

Figure 3.2 The structure and signaling pathways of the common cytokine-receptor chain family
of receptors. Reprinted by permission from Macmillan Publishers: Waldmann T. The biology of
interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat Rev
Immunol. 2006;6:595.

Immune Regulation 39
Monocyte or dendritic cell IL-2 functions as a secreted cytokine, act-
ing on preformed heterotrimeric receptors
expressed on activated T and NK cells
(Figure 3.3). In contrast, IL-15 acts as part
of an immunological synapse. IL-15 and
IL-15R IL-15R expressed on the surface of anti-
gen-presenting cells are presented in trans
to CD8 T cells and NK cells that express
c IL-2/15R c
only the and chain of the IL-15 receptor
Cell IL-2/15R
(Figures 3.3 and 3.4).
The unique IL-2 and IL-2R system has
been a valuable target for immunotherapy
CD8+ T cell or NK cell because IL-2R is not expressed by any
resting cells with the exception of T regu-
Copyright 2006 Nature Publishing Group
Nature Reviews | Immunology latory cells. Yet, it is expressed by many
Figure 3.3 The mode of interaction of IL-2
malignant cells of various T- and B-cell
and IL-15 with the subunits of their receptors. leukemias, T cells that participate in organ-
Reprinted by permission from Macmillan allograft rejection, and finally by T cells
Publishers: Waldmann T. The biology of involved in autoimmune diseases. Thus,
interleukin-2 and interleukin-15: implications for
cancer therapy and vaccine design. Nat Rev antibodies directed against IL-2R (sev-
Immunol. 2006;6:595. eral such antibodies have been approved
by FDA for use in humans) specifically
destroy those cells that are IL-2 dependent.
enlargement of the peripheral lymphoid One of these antibodies (daclizumab by
organs and polyclonal expansion of T and Hoffmann La Roche) is in phase II trials
B cells, and this proliferation reflects the in patients with uveitis, in certain multiple
impairment of regulatory T cells and AICD. sclerosis patients, and in asthma patients.
These mice develop autoimmune diseases Finally, it was shown to be effective in a
such as hemolytic anemia and inflamma- subset of patients with adult T-cell leukemia
tory bowel disease. due to human T-cell lymphotropic virus I.
In contrast, mice that are deficient in IL- It is believed that IL-15 might contrib-
15, or its receptor IL-15R, do not develop ute to autoimmune diseases by inducing
enlarged lymphoid tissues, in creased the expression of TNF-, by inhibiting
serum immunoglobulin, or autoimmune self-tolerance mediated by IL-2-induced
disease. Rather they have a marked reduc- AICD, and by facilitating the maintenance
tion in the number of thymic and periph- of CD8+ memory T-cell survival, includ-
eral natural killer (NK) cells, NK T cells, ing that of self-reactive memory cells. For
and intestinal intraepithelial lympho- example, dysregulated IL-15 expression
cytes. Thus, IL-15R-deficient mice show has been reported in patients with a range
a marked reduction in CD8+CD44hi mem- of autoimmune diseases such as multiple
ory T cells. The differences in the func- sclerosis, inflammatory bowel disease, and
tions of IL-2 and IL-15 reflect the distinct psoriasis. In this context, cytokine-directed
modes of actions of these two cytokines. blockade of TNF with specific monoclonal

40 Immune Regulation


Endocytic vesicle


Activated c
monocyte or IL-2/15R
dendritic cell
of IL-15
CD8+ T cell
or NK cell

Copyright 2006 Nature Publishing Group

Nature Reviews | Immunology

Figure 3.4 Following their co-induction on antigen-presenting cells by addition of an interferon, the
interleukin receptor chain presents IL-15 to neighboring natural killer cells and memory phenotype
CD8 T cells that express the other IL-15 receptors beta and gamma. Reprinted by permission from
Macmillan Publishers: Waldmann T. The biology of interleukin-2 and interleukin-15: implications for
cancer therapy and vaccine design. Nat Rev Immunol. 2006;6:595.

antibodies or soluble TNF receptors has For example, in vivo, the IL-15 mutant
been an important target for many of markedly diminished antigen-specific
the diseases mentioned previously. Yet delayed-type hypersensitivity responses
TNF-directed therapies do not provide in Balb/c mice and increased survival of
effective therapy for all these patients, pancreatic islet cell allografts. The use of
and new therapeutic targets are needed. soluble high-affinity IL-15R inhibited
Furthermore, although TNF-directed the development of mouse collagen-
therapy has an anti-inflammatory effect, induced arthritis and inhibited allograft
it does not have an effect on self-reactive rejection. An antibody specific for IL-15
memory T cells that might play a role in has been efficacious in mouse models
the pathogenesis and maintenance of auto- of psoriasis. This antibody is now in a
immune diseases. Thus, it is hoped that by phase I/II clinical trial in patients with
targeting IL-15 it might be possible to both rheumatoid arthritis. Finally, a humanized
achieve the anti-inflammatory effects and antibody specific for IL-2/IL-15R when
reduce the number of CD8+ self-reactive administered as a single agent prolonged
memory T cells. cardiac-allograft survival in cynomolgus
Several agents that inhibit IL-15 activ- monkeys, and only minimal toxicity was
ity have been developed, including sol- noted when this antibody was given in a
uble IL-15R, mutant IL-15 molecules, phase I trial to patients with T-cell large
and antibodies specific for IL-2/IL-15R. granular lymphocytic leukemia.

Immune Regulation 41
CELLULAR VACCINES AND by various stimuli such as toll-like recep-
MODULATIONS THEREOF tor (TLR) ligands (LPS or poly I:C), inflam-
matory cytokines (IL-1, TNF-, IL-6, and
Dendritic Cell Vaccines
PGE2), or CD40L. The use of DCs that have
Recently, much attention has focused on the received a maturation stimulus is likely to
area of dendritic cell (DC) vaccines in the be important to induce strong immunity. It
treatment of cancers. The immunological has become clear that antigen delivered on
basis of current approaches to therapeu- immature or incompletely matured DCs
tic cancer vaccination (often called vacci- can even induce tolerance. However, the
treatment) has been established over the type and the duration of the maturation
past decade or longer. These new devel- stimulus remain to be determined and may
opments are mainly based on the lessons influence efficacy. At this time, monocyte-
learned from the clinical testing of these derived DCs are the most accessible and
approaches. In particular, three lessons are homogeneous populations of DCs.
worthy of note: First, recent randomized Monocyte-derived DCs were the first to
phase III trials suggest that vacci-treatment be used for treating melanoma patients, and
with autologous DCs expressing pros- several pilot studies have been published.
tatic acid phosphatase or with autologous Most used defined antigens in the form of
tumor-derived heat shock protein (HSP peptides, but in some studies, tumor lysates
gp96) peptide complexes are showing or autologous DC tumor hybrids were also
progress in cancer patient survivals. Sec- employed. The first trial, published in 1998
ond, immunological monitoring of many by Nestle and colleagues, aroused great
clinical trials has failed to identify a sur- interest given an overall response rate of
rogate marker for clinical outcomes. Third, 30 percent in stage IV patients (i.e., distant
many articles and reviews suggest that metastases), including complete responses.
protective immunity to human cancer is An important point in the first study by
elicited by the mutated antigenic repertoire Nestle and colleagues was that they use
unique to each cancer. fetal calf serum (FCS) during DC genera-
Focusing more closely on the type of tion, and this might have contributed to
DC needed to achieve its vaccine poten- the observed effects by providing nonspe-
tial, the subsets of DCs could prove criti- cific helper epitopes and by promoting the
cal. Much of the current research is being maturation of DCs.
carried out with monocyte-derived DCs, Jonuleit and colleagues directly com-
which are potent and homogeneous stimu- pared within each of eight patients the
lators of immunity. Monocyte-derived DCs immunogenicity of immature DCs (gen-
can be readily generated within a few days erated according to Nestle and colleagues
in large numbers (300 million500 million in FCS-containing media using GM-CSF
mature DCs per apheresis) from precursors and IL-4) to mature DCs generated in the
in the blood without the need for pretreat- absence of FCS and matured by a cocktail
ing patients with various cytokines such consisting of TNF-, IL-1, IL-6, and PGE2
GM-CSF or FLT 3-L. Rather, one obtains mimicking the composition of monocyte-
populations of immature DCs by exposing conditioned medium. These two differ-
monocytes to GM-CSF and IL-4, and then ent DC populations were administered
they are differentiated into mature DCs intranodally into opposite inguinal lymph

42 Immune Regulation
nodes. FCS-free mature DCs induced stron- at breaking tolerance than their wild-type
ger T-cell responses, both to the two recall counterparts. Finally, there is a multiplic-
antigens used (tetanus toxoid and PPD/ ity of choices of delivery agents for these
tuberculin) and to tumor peptides. Interest- antigens whole proteins, peptides, RNA,
ingly, however, both immature and mature DNA, viral vectors, DCs, and so on.
DCs showed an expansion of peptide-spe- The importance of innate immunity
cific T cells by tetramer staining; yet, only and the emergence of the toll-like receptors
mature DCs induced IFN--producing and have led to the recognition that immune
lytic CD8+ T cells. These findings suggest modifiers must be an essential compo-
the interesting possibility that the imma- nent of any cancer vaccine. The inclusion
ture DCs might have induced regulatory T of QS21, ISCOMS, Montanide, heat shock
cells rather than effector T cells, an obser- proteins, CpG, BCG, and granulocyte-
vation previously noted in studies with macrophage colony-stimulating factor
normal volunteers. with cancer vaccines reflects this. Of inter-
Cancer patients and healthy subjects est in this respect are the anecdotal success
often harbor a repertoire of self-reactive of Coleys toxin at the turn of the nineteenth
T cells and antibodies. This led to the idea century (a heat-killed mixture of strep-
that if one could break immunological tococcal cells and probably superantigen
tolerance to these self-antigens in a con- broths), which may now be reinterpreted
trollable manner one would find a thera- in the light of the newly identified immune
peutic window in which an autoimmune response modifiers.
response might damage cancers more than Finally, a better understanding of the
normal tissues. This approach has worked controls that act on T cells to stimulate or
reasonably well with chemotherapies, inhibit them has led to the use of reagents to
which, although not cancer specific, can enhance antitumor T-cell activity. For exam-
confer clinical benefit with acceptable mor- ple, blocking antibodies to the inhibitory
bidities. T-lymphocyte antigen CTLA-4, manipula-
The efforts to create cancer vaccines tion of regulatory T cells, antibodies to PDL
using allogeneic cell lines, differentiation or its ligand PDLI, and enhancement of co-
antigens (such as gp100 and MART1), stimulating molecules like B7 on antigen-
cancer testes (CT) antigens (such as presenting cells are a few examples of
MAGE, NY-ESO-1), or other common reagents that are being used in this manner
molecules (such as carcinoembryonic anti- in conjunction with vacci-treatments.
gen, mucins, prostate-specific antigen, and
prostatic acid phosphatase) represent this
approach. Within this approach lie several
subthemes. Thus, CT antigens, which are
not expressed on normal somatic tissues REVIEWS
but only on cancers or gonads, might be Barnes P. Molecular mechanisms and
a better target for breaking tolerance than cellular effects of glucosteroids. Immu-
are differentiation antigens expressed on nol Allergy Clin North Am. 2005;25:
somatic tissues. Another subtheme is the 451468.
idea that artificially mutated differentiation Berzofsky JA, Ahlers JD, Belyakov IM.
or CT antigens as vaccines might be better Strategies for designing and optimizing

Immune Regulation 43
new generation vaccines. Nat Rev Immu- Jonuleit H, Giesecke-Tuettenberg A, Tting
nol. 2001;1(3):209219. T, et al. A comparison of two types of den-
Fehniger TA, Caligiuri MA. Interleukin 15: dritic cell as adjuvants for the induction
biology and relevance to human dis- of melanoma-specific T-cell responses in
ease. Blood. 2001;97:1432. humans following intranodal injection.
Gattinoni L, Powell DJ Jr, Rosenberg SA, Int J Cancer. 2001(2):243251.
Restifo NP. Adoptive immunotherapy Nestle FO, Alijagic S, Gilliet M, et al. Vacci-
for cancer: building on success. Nat Rev nation of melanoma patients with pep-
Immunol. 2006;6(5):383393. tide or tumor lysate-pulsed dendritic
Schwartz RS. The Shattuck Lecture: diver- cells. Nat Med. 1998;4:328332.
sity of the immune repertoire and Steinman RM, Palucka K, Bancherceau J,
immunoregulation. New Engl J Med. Schuler-Thurner B, Schuler G. Active
2003;348:10171026. immunization with dendritic cells bear-
Strand V, Kimberly R, Isaacs JD. Biologic ing melanoma antigens. In: Levine
therapies in rheumatology: lessons MM, Kaper JB, Rappuoli R, Liu MA,
learned, future directions. Nat Rev Drug Good MF, eds. New Generation Vaccines.
Discov. 2007;6:7492. 3rd ed. New York: Marcel Dekker;
SUGGESTED READING Vincenti F, Kirkman R, Light S, et al.
Blachre NE, Darnell RB, Albert ML. Apop- Interleukin-2-receptor blockade with
totic cells deliver processed antigen to daclizumab to prevent acute rejection
dendritic cells for cross-presentation. in renal transplantation. N Engl J Med.
PloS Biol. 2005;3:e185. 1998;338:161165.
Dubois S, Mariner J, Waldmann TA, Waldmann TA. The biology of interleukin-
Tagaya Y. IL-15R alpha recycles and 2 and interleukin-15: implications for
presents IL-15 in trans to neighboring cancer therapy and vaccine design. Nat
cells. Immunity. 2002;17:537 547. Rev Immunol. 2006;6:595601.

44 Immune Regulation
4. Immunological Aspects of Infection

Kumar Visvanathan, M.D., Ph.D., Christopher M. MacIsaac, M.D., Ph.D.,

William W. Hall, M.D., Ph.D., and Vincent A. Fischetti, Ph.D.

INTRODUCTION rophages and polymorphonuclear leuko-

cytes that ingest invading microorganisms
From the moment of birth, the host is and kill them.
constantly exposed to a wide variety The polymorpholeukocytes are a large
of bacteria and viruses. In general, the pool of phagocytic cells that are both cir-
host manages to either eliminate or ward culatory and in the bone marrow. Invad-
off these invading organisms, and a ing organisms trigger an inflammatory
symbiosis is achieved between microbes cascade, which stimulates these cells to
and the host. How does this occur? There adhere to vascular epithelium and actively
are two major pathways to achieve this migrate toward the infection. Phagocytosis
resistance: nonspecific and adaptive. is promoted by opsonins (usually IgG anti-
body) and complement.
The macrophages reside in the sub-
NONSPECIFIC RESISTANCE epithelial tissues of the skin and intestine
and line the alveoli of the lungs. Microbes
Nonspecific or natural resistance refers to that penetrate an epithelial surface will
barriers, secretions, and normal flora that encounter local tissue macrophages called
make up our external defenses. Phago- histocytes. If the organism enters via blood
cytes and complement are also involved. or lymph, then defense is provided by
Mechanical barriers are highly effective, fixed macrophages called Kupffer cells,
and the skin (our largest organ) is highly which line the sinusoids of the liver. Simi-
suited to this protection (see Figure 4.1); larly fixed macrophages called Langerhans
loss of a major part of the skin (second- cells are also present in the epidermis of
ary to burns, acids, etc.) immediately the skin. Once engaged with the organ-
exposes the host to marked susceptibility ism, these macrophages release a number
to infection. The mucosal lining of mouth of macrophage-derived cytokines, which
and respiratory tract is another excellent nonspecifically amplify the immuno-
defense mechanism. Yet, a defect in the logical and inflammatory reactions to the
mucosal lining of the respiratory tract, invading microbe.
which occurs in cystic fibrosis, results in Most pathogenic microorganisms have
a heightened susceptibility to many infec- evolved methods of resisting phagocy-
tions. These are examples of a defect in tosis. For example, group A streptococci
the epithelium or epithelial lining. In gen- have cell surface structures called M pro-
eral, however, it is the mobilization of the teins of which there are now more than
phagocytic cells such as monocytes/mac- 120 antigenically distinct molecules that


Respiratory tract

Digestive system

Urogenital tract


Figure 4.1 Schematic drawing of the body showing the various organs and
systems that offer nonspecific resistance to infection.

inhibit direct phagocytosis, mainly by saccharides, lipoproteins or peptidogly-

preventing deposition of complement cans have profound effects on human
on the organism. Another example is the cells. Although the structures of many dif-
pneumococcal polysaccharide capsule ferent pathogenic microbial compounds
of which there are thirty to forty distinct have been extensively studied, the molec-
polysaccharides. Another approach (taken ular basis of their recognition by the cells
by both group A streptococci and staphy- of the innate immune system remained
lococci) is the release of potent extracel- elusive. Charles Janeway first devel-
lular toxins, which kill phagocytes with oped the concept of microbial structures
the formation of pus. An intriguing bac- forming pathogen-associated molecular
terium, Mycobacterium tuberculosis, can be patterns (PAMPs), which would be rec-
ingested by phagocytes but resists intra- ognized by pattern recognition receptors.
cellular killing, often persisting for years The discovery of a family of toll receptors
in the macrophage. (toll refers to the toll gene of Drosophila,
Over the past decade, our knowledge initially identified as an essential receptor
of how we sense the microbial world controlling dorsoventral polarization dur-
(innate or adaptive) has fundamentally ing embryonic development of the fly lar-
changed. It has been known for decades vae) in species as diverse as Drosophila fly
that microbial products such as lipopoly- and humans and the recognition of their

46 Immunological Aspects of Infection

role in distinguishing molecular patterns is expected that many more receptors will
that are common to microorganisms led be discovered in the future.
to a renewed appreciation of the innate
immune system.
From these humble beginnings, the ADAPTIVE IMMUNITY
field of mammalian toll-like receptors
(TLR) quickly evolved as a crucial sys- A specific immune response to invad-
tem for alerting the host to the presence ing microbes is conveniently divided
of numerous infectious agents. Infectious into humoral and cellular immunity. The
microbes display certain molecular pat- importance of each arm of the specific
terns that are necessary for microbial response varies from infection to infection.
virulence (see Table 4.1). Many of these Experimental animal models and naturally
molecular patterns such as lipopolysaccha- occurring immunodeficiency states clearly
ride in the outer membranes of gram-nega- demonstrate that certain components of
tive bacteria seem to be particularly potent the immune response are crucial for con-
activators of mammalian cells. The mam- trolling a particular infection.
malian receptors responsible for recogni- For example, individuals with anti-
tion of PAMPs are called pattern recognition body deficiencies are particularly prone to
receptors. The failure of the immune system repeated infections with pyrogenic bacte-
to recognize a pathogens PAMP could ria. Yet, replacement therapy with immu-
lead to a delay or blunting of the immune noglobulin greatly reduces the number of
response, resulting in unchecked invasion infections. Interestingly, these individu-
by the microbe. als can mount a normal response to most
Perhaps the best example of an incom- viruses (varicella, measles, mumps, etc.).
plete recognition by the TLR system is a Yet the absence of mucosal antibody does
gram-negative bacterial infection in the make them susceptible to some enterovi-
C3H/HeJ mouse. As few as two colony- ruses.
forming units of Salmonella typhimurium can Although it is clear that innate immu-
kill this mouse. Further exploration of this nity is the first line of defense against
extraordinary virulence revealed that the invading organisms, the TLRs are also
mouse harbored a point mutation (P712H) playing a role in adaptive immunity, and
in the TLR4, which results in defective sig- the dendritic cell (DC) appears to be play-
nal transduction in response to LPS and a ing a key role in linking the innate and
heightened susceptibility to gram-negative adaptive immune responses. As immature
infections. cells, they are present in the peripheral
The family of TLRs is a highly special- tissues. However, with the appearance of
ized system that can identify a number of invading organisms, DCs recognize these
microbial and endogenous ligands and acti- pathogens through their TLRs. Fortunately,
vate the immune system to respond. The they express the full repertoire of TLRs.
body must be able to respond differently to After activation of the TLRs, the DCs are
various challenges. Thus, the specificity in transformed into more mature cells with a
the immune response via TLRs is becoming high expression of major histocompatibil-
increasingly complex. Currently, probes of ity complex (MHC) and the co-stimulatory
the TLR family have been described, and it molecules CD80 and CD86. The DCs then

Immunological Aspects of Infection 47

Table 4.1 List of Known Toll-like Receptors, What Cells Express Them, and What the Ligand Is for Each

Receptors Cells Ligand

TLR1 Platelets, atherosclerotic plaques, monocytes, Lipoprotein from

neutrophils, germinal center B cells, natural killer cells, Mycobacterium
DCs, B cells tuberculosis
Soluble factors from
Neisseria meningitidis
TLR2 Atherosclerotic plaques, monocytes, neutrophils, Peptidoglycan
natural killer cells, B cells, spleen, lymph nodes Lipoteichoic acid
Triacyl lipoproteins
Diacyl lipoproteins
(with TLR6)
TLR3 DCs, natural killer cells dsRNA
TLR4 Atherosclerotic plaques, neutrophils, monocytes, B LPS
cells Fusion protein from
respiratory syncytial
TLR5 DCs, natural killer cells, monocytes, T cells Flagellin
TLR6 Platelets, resting B cells, germinal center B cells, Diacyl lipoproteins
natural killer cells, monocytes, DCs (with TLR2)
TLR7 Monocytes, resting B cells, germinal center B cells, ssRNA

TLR8 DCs, monocytes ssRNA

TLR9 Resting B cells, germinal center B cells, natural killer Bacterial CpG DNA
cells, monocytes, spleen

TLR10 Resting B cells, germinal center B cells, natural killer Unknown

cells, DCs

TLR11 ? not expressed in humans murine kidney and bladder Unknown

migrate to the lymph nodes to activate the incidence of allergic disease and atopy
antigen-specific nave T cells. The cytokine has markedly increased in the industrial-
milieu being expressed around the cells ized countries compared with developing
determines their fate; that is, the produc- countries over the past decades, and one
tion of IL-12 drives these cells to TH1 cells, hypothesis is that this increase is linked
which produce interferon-, whereas IL-4 to the reduction of bacterial infections,
drives them toward TH2 cells producing which occurs in a cleaner environment, a
IL-4, IL-5, Il-10, and IL-13. These latter hypothesis known as the hygiene hypoth-
cytokines are of interest as they are also esis (see Chapter 15). The discovery that
responsible for the development of allergic TLR signaling might be crucially involved
diseases such as asthma and account for in the establishment of TH1/TH2 path-
the regulation of antigen-specific IgE pro- ways opens up the field to look for new
duction, accumulation of eosinophils, and strategies against diseases such as asthma
activation of mast cells. In this connection, and atopy.

48 Immunological Aspects of Infection

In summary, the fields of both innate breast-fed infants who are less susceptible
and adaptive immunity have experienced to infection than non-breast-fed babies. It
a new and rapid growth in interest in the now appears that it is not polysaccharides
past eight to ten years. Concomitant with or antibodies that are responsible for this
this growth, the number of genomes that protection but rather a multimeric form of
have been sequenced is expanding, thus lactoalbumin (present in high concentra-
enabling researchers to identify and to tions in human breast milk (see Suggested
characterize the receptors and adaptors Reading). A broader approach to protec-
involved in the recognition. tion has been the production of pneumo-
Although much of the work over the coccal polysaccharide vaccines specially
past years has focused on defining ligands designed to induce antibody induction in
for the different TLRs, the molecular basis the young child.
for this recognition is not known for a sin- In the following discussion, strepto-
gle ligand. Furthermore, the characteriza- cocci, particularly S. pyogenes, are used as
tion of TLR-dependent signaling for the the example of a bacterial infection, but
instruction of adaptive immune responses many other organisms produce a similar
has just started to be explored. Because response. Streptococcal antigens include
TLRs play such a crucial role in innate and specific toxins such as streptolysins O and
adaptive immune responses to distinct vir- S that lyse blood and tissue cells and pyro-
ulence factors, the development of selec- genic exotoxins, which act as superanti-
tive inhibitors/activators may be a worth- gens to overstimulate the host responses.
while endeavor to help manage a number There are also specific enzymes such as
of infectious and immunologic diseases. hyaluronidase and streptokinase, which
help promote the spread of infecting strep-
tococcus. Perhaps most important is the M
BACTERIAL INFECTION protein (Figure 4.2), a cell surface antigen
of the group A streptococcus that allows
The immune system responds to bacterial the bacteria to evade immune defenses
infections in two major ways. First, it may (especially neutrophils and complement).
respond to soluble products of the cell such One way in which M protein functions is to
as toxins or released structural antigens like bind host factor H, which prevents comple-
LPS of a given gram-negative bacterial cell. ment C3 from depositing on the streptococ-
Most bacterial antigens are T-cell depen- cal surface. Since efficient phagocytosis by
dent and require helper T cells for initia- neutrophils requires interaction with its C3
tion of the immune response. Yet certain receptor, factor H prevents this interaction.
cell antigens, such as the pneumococcal Antibodies to streptococcal antigens
polysaccharides, are T-cell independent. other than M protein are slow to appear,
They are large-molecular-weight mol- and most likely do not play a role in lim-
ecules, and in children, antibody response iting the infection. However, antibodies
to these antigens may take four to six years. to streptolysin O and deoxyribonuclease
Thus, younger children are susceptible to B have become important clinical tools to
these infections. determine whether a given individual has
An interesting sidelight to protection had a recent streptococcal infection. This
against these infections can be seen in is partially true if a blood sample drawn

Immunological Aspects of Infection 49

r va
Hy Variable Conserved

N A1 A2 A3 A4 A5 B1 B2 B3 B4 B5 C1 C2 C3 Pro/Gly C

600 nm

Cell Wall
Figure 4.2 Coiled-coil structure of the M protein. The streptococcal M protein is a coiled-coil
molecule that extends about 600 nm from the bacterial cell surface. The C-terminal region is
embedded within the cell wall and the C-terminus is located in the cytoplasmic membrane in the
nascent molecule. The coding region for the M protein is distributed in repeat blocks designated
AC in which the C-repeat region is conserved among M-protein serotypes, and the A and B
repeats are variable among these serotypes. The N-terminus is the hypervariable, type-specific
region for the M proteins. Pro/Gly designates the region of the M protein that is rich in proline
and glycine.

at the onset and one drawn 10 to 14 days most antigens, these toxins bind to the
later show a marked rise in the titer. Con- lateral surface of both the T-cell receptor
trary to the dogma, both skin infection and and the MHC (see Figure 4.3). Thus, in
pharyngeal infection with group A strepto- contrast to a conventional peptide anti-
cocci can stimulate the production of both gen, which stimulates only one in 104 to
antibodies. 108 T cells, the superantigen may bind up
Some bacterial antigens such as endo- to approximately 1:50 T cells. The result is
toxins can be powerful stimulators of the a veritable explosion of cytokines result-
immune response and lead to polyclonal ing in high fever, hypotension, and mul-
activation of B lymphocytes. This rise in tiorgan shock. Death often occurs within
immunoglobulin levels is believed to be 24 hours of the release of these toxins.
nonspecific since only a small portion of The most widely studied superantigens
the total immunoglobulin level is directed are produced by Staphylococcus aureus
to the endotoxin. and group A streptococcus. These supe-
rantigens bind the T-cell receptor at its
region, and that exact V region is dif-
BACTERIAL SUPERANTIGENS ferent for each superantigen. Table 4.2
lists the known superantigens and their
Among the toxins secreted by bacteria V specificities.
is a special group from the streptococcal The role of SAg has also been explored
and staphylococcal family of toxins called in a number of disease states, including
superantigens (SAg). Instead of binding to atopic dermatitis, psoriasis, Kawasaki
the specific antigen receptor known for disease, rheumatic fever, and tuberculosis.

50 Immunological Aspects of Infection

Lymphocyte Lymphocyte



MHC Class II MHC Class II

Antigen Antigen
Presenting Presenting
Cell Cell

Figure 4.3 Binding of conventional (left) and superantigen (right) antigens to

the T-cell receptor and antigen-presenting cell. Note that the conventional
antigen binding is associated with the T-cell receptor and the major
histocompatibility complex (MHC) directly and only a small proportion of T
cells are activated. In contrast, the superantigen binds to the lateral sides
of the T-cell receptor and MHC molecule, thereby triggering a much larger
group of activated T cells (up to 25 percent of the cells).

BACTERIAL EVASION OF IMMUNE organism and mucoid secretions of these

DEFENSE polysaccharides can block the activation of
the alternate pathway of complement.
There are several ways in which bacteria Antigenic variation or drift is another
can survive in the host by evasion of the mechanism whereby bacteria evade the
immune defenses. These mechanisms will immune system. The M protein (the most
be briefly summarized here, but the list of important virulence factor of the group A
evasive mechanisms used by each bacterial streptococcus) has been shown to exhibit
species may be much longer in nature. antigen variation in the environment, and
Capsules play an important role for new M protein molecules appear regularly
long-term survival of pathogens. For in human isolates of group A streptococci.
example, group A streptococci have a Another example is the relapsing fever
hyaluronic acid capsule that is identical to by Borrelia. During the first episode, anti-
hyaluronic acid in humans. While antibod- bodies kill the bacteria and the fever sub-
ies can be obtained following immuniza- sides. However, some antigenic variants
tion with the streptococcal hyaluronic acid, of the bacteria persist, and after five to
they are nonprecipitating antibodies and seven days these new variants can cause
are not effective in eliminating the organ- a relapse in the patient with fever reap-
ism. Polysaccharide antigens of both the pearing. Other examples of evasion of the
pneumococcus and the meningococcus immune response are bacteria such as N.
capsules can inhibit phagocytosis of the gonorrhea, N. meningitidis, Haemophilus

Immunological Aspects of Infection 51

Table 4.2 List of All the Known Superantigens and Their V Specificities

Superantigen Human V specificity

Staphylococcal enterotoxins
SEA 1, 5.3, 6, 7, 9, 16, 22, 21.3
SEB 3, 9, 12, 14, 15, 17, 20
SEC1 3, 6, 12, 15
C2 12, 13.2, 14, 15, 17, 20
C3 3, 5, 8, 12, 13.2
SED 5, 12
SEE 5.1, 6, 8, 18
SEG 3, 7, 9, 12, 13.1, 13.2, 13.6, 14, 15
SEH Nil? V10
SEI 1.1, 3, 5.1, 5.3, 13, 23
SEK 5.1, 5.2, 6.7
SEL 5.1, 5.2, 6.7, 16, 22
SEQ 2.1, 5.1, 21.3
TSST-1 2
Streptococcal exotoxins
SPE-A 2, 12, 14, 15
SPE-C 1, 2, 5.1, 10
SPE-F 2, 4, 8, 15, 19
SPE-G 2.1, 4.1, 6.9, 9.1, 12.3
SPE-H 2.1, 7.3, 9.1, 23.1
SPE-I 6.9, 9.1, 18.1, 22
SPE-J 2.1
SPE-K/L 1.1, 5.1, 23.1
SPE-M 1.1
SMEZ1 2.1, 4.1, 7.3, 8.1
SMEZ2 4.1, 8.1

influenza, and others that secrete proteases One must also consider that some bac-
that hydrolyze IgA antibody. Some strains teria sequester themselves in nonphago-
of staphylococci secrete catalase, which cytic cells where they are not exposed to
prevents them from being killed inside the immune system. An excellent example
phagocytic cells. is M. tuberculosis, which can lie dormant

52 Immunological Aspects of Infection

for years inside a granulomatous, case-
Epstein-Barr Infection
ous lesion called the Ghon complex. Once
ground up, live M. tuberculosis organism Infectious mononucleosis is caused by the
can be extracted from this complex. Epstein-Barr virus (EBV), and in devel-
Finally, a number of organisms display oping countries, 99 percent of children
antigens on their surface that are cross- demonstrate antibodies to the virus by
reactive with human antigens. This could age 3. In developed countries, the age is
result in an enhanced immune response much later and clinical infection usually
to host tissue antigens or a diminished occurs in the age group of 15 to 25 years.
response secondary to similarities between The virus is excreted in the oropharyngeal
bacterial and self-antigens. fluids for some months after the infection
and is responsible for person-to-person
VIRAL INFECTIONS Hoagland observed that a female col-
lege student traveling from New York to
The clinical spectrum of viral disease is Boston met several different male students
wide, and because there is such variation, on the train and exchanged kisses with all
we will use the template of herpes viruses of them. All of these contacts came down
as an example. In general, viral infections with infectious mononucleosis, which is
are self-limited and usually produce long- often referred to as the kissing disease.
term immunity, and secondary attacks by The pattern of antibody response to
the same virus are uncommon. Of the large different EBV antigens helps distinguish
number of herpes viruses, only eight infect acute or subclinical infections from postin-
humans, and each will be discussed in some fections with the virus. IgM antibodies to
detail. However, two general features of viral capsid antigen (VCA) appear early
pathogens of these viruses are important. in the course of the infection, but at the
First, there must be close contact between time of clinical symptoms, IgG levels to
infected and noninfected individuals for VCA are quite high. Thus, paired sera at
transmission to occur and no intermediate the time of clinical infection do not help
host is involved. Second, after the primary diagnose the disease. In contrast, antibod-
infection, herpes virus will persist in the ies to EB nuclear antigen appear months
host for life. after the infection. Antibodies to early anti-
To eliminate virions from entering gen appear in about 70 percent of primary
noninfected cells and to eliminate virus- infections and are usually indicative of an
infected cells, two major pathways of acute infection.
the immune response are initiated. The The clinical signs of fever, such as
humoral response is primarily directed enlarged cervical lymph nodes, and red-
against virions, while the T-cell response dened pharynx with whitish exudates (also
is primarily directed against infected seen with other viruses) are often mistaken
cells. The humoral response may directly for group A streptococcal pharyngitis.
neutralize the virus, but complement- Thus, culture and antibody tests are used
dependent enhancement of viral phago- to distinguish between the two.
cytosis or complement lysis of virus also EBV has one unique feature compared
may occur. with other viruses in that it produces

Immunological Aspects of Infection 53

disease by infecting and transforming B chronic malaria of these children induces
lymphocytes via the CD21 molecule on EBV-infected lymphoproliferation, leading
the surface of B cells. These infected cells to chromosomal translocation in the long
multiply like tumor cells, and up to half of arms of chromosomes 8 and 14 and even-
the lymphoid cells from the tonsils of EBV- tually to the activation and translocation of
infected patients may be transformed. the c-myc oncogene to the active region of
The infection is stopped by a T-cell the cellular genome (see Figure 4.4).
response that kills virus-infected cells and Viruses have evolved many mecha-
a humoral response that neutralizes free nisms to evade the immune response, but
virions. The characteristic atypical lym- one of the key elements in these evasive
phocytes seen in this disease are CD8+ mechanisms has been their ability to induce
cytotoxic T cells that kill EBV-infected a latent status within the cells of the host.
B cells. Thus, all herpes viruses can remain latent,
Most individuals handle EBV infection and no viral antigen is expressed. When
quite well. However, rare individuals have there is a change in the host-virus equilib-
a specific defect that fails to handle EBV rium secondary to certain other infections,
infections and usually die of the disease. metabolic disturbances, immunosuppres-
More important is the failure to contain sion, or aging, only then does the virus
EBV infections secondary to immunosup- reactivate and cause disease. Examples
pressive therapy. For example, the x-linked are herpes simplex in the trigeminal gan-
lymphoproliferative syndrome that affects glia, which causes recurrent cold sores,
males from age 6 months to 20 years is in this or herpes zoster in a dorsal root ganglion,
category. These patients die of lymphomas, which causes shingles.
aplastic anemia, or immunodeficiency. A second example is antigenic variation
Immunosuppressive therapy may or drift in certain viruses. The best example
result in EBV reactivation, and about 1 to 10 is influenza A, an RNA virus surrounded
percent of certain transplants may be com- by a lipid envelope into which two impor-
plicated by EBV-induced lymphoprolifera- tant proteins (hemagglutinin and neuro-
tive disease. Similar results are also seen in minidase) are inserted. The virus evades
patients infected with the HIV virus. One the neutralizing antibodies to this virus by
of the most interesting effects of EBV infec- constantly modifying the structure of these
tion and transformation has been the occur- two proteins by either antigenic drift or
rence of Burkitts lymphoma. It is endemic antigenic shift. In the former, the change is
in certain African countries (especially in minor and accounts for minor epidemics of
regions of mosquito-borne disease), where flu during each winter. Antigenic shift is a
it represents 90 percent of childhood can- major change in the structure of those pro-
cers in contrast to 3 percent in developed teins, which can cause a major pandemic
countries. The link between EBV and of devastating proportions. Viewed in this
Burkitts lymphoma was established when light, the influenza vaccine currently in use
it was found that EBV antigens and genome is generally protective against common flu
were present in the tumor cells. Since the epidemics in the world but would not be
incidence of Burkitts lymphoma follows protective against a major antigenic shift.
the same belt as the chronic malaria belt of A third mechanism is viral persistence in
the region, it has been suggested that the which the virus is not cleared and a chronic

54 Immunological Aspects of Infection

1. Viral infection triggers 2. Translocation during
lymphocyte proliferation proliferation

3. If gene enhancer 4. Uninhibited proliferation

activating bcl-2 of monoclonal cells, i.e.,
gene to produce lymphoma
bcl-2 protein which
inhibits apoptosis

Figure 4.4 A schematic presentation of the steps involved in the transformation of Epstein-Barr virus
infected B cells into a B-cell lymphoma.

infection with persisting virus occurs. For to these antigens will not only recognize
example, HIV persists because it eventu- viral antigens, but some will bind to host
ally destroys the CD4 T cells needed to kill antigens as well, thereby possibly initiat-
the virus. Hepatitis C also may persist for ing an autoimmune response and disease.
years with continuous carriage of the virus An example of this type of reaction is the
in the liver. appearance of chronic autoimmune liver
While immunological reactions to the disease following chronic hepatitis B infec-
virus are generally beneficial to the host, tion or immune complex disease such as
they may also initiate or aggravate tis- vasculitis or glomerulonephritis.
sue damage in the host inadvertently. For Another example of viral bystander
example, EBV is a polyclonal B-cell activa- damage is dengue fever in which immune
tor, and the virus may combine with host enhancement plays an important part. Here,
antigens to form new antigens. Antibodies the dengue virus can infect macrophages

Immunological Aspects of Infection 55

via Fc receptors, and its capacity to enter which has a commensal relationship with
the target cell is enhanced if it is bound the lung in the normal host. However,
to IgG antibodies. Thus, the first infection the sudden onset of pneumonia with this
with a specific dengue virus type elicits organism secondary to HIV infection in a
antibodies to that specific virus. How- child or adult is often the first sign of an
ever, cross immunity to the other dengue underlying suppressed immune system
serotypes is fleeting and lasts only a few caused by HIV.
weeks. When the individual is exposed to In contrast to the superficial infections
a second dengue virus serotype, the pre- of fungi, systemic infections in the immuno-
existing antibody to the first type binds compromised host have a higher mortality
to it but does not neutralize the virus. The rate. This category includes Histoplasma cap-
antibody-enhanced second virus enters the sulatum (pulmonary infection), Coccidiodes
macrophage, setting off a veritable explo- immitis (acute pneumonitis), and Crypto-
sion of proteases, lymphokines comple- coccus neoformas (meningitis, lung lesions).
ment activation, coagulation cascade, and Such systemic infections may occur in
vascular permeability factors. The result is normal individuals, and the cell-mediated
a much more severe disease called dengue immune response is the more important fac-
hemorrhagic fever, whose mortality rate in tor in control or prevention of them. How-
areas with poor medical facilities and treat- ever, all of these fungal infections (superfi-
ment may be as high as 50 percent. cial and systemic) are more likely to occur in
immunosuppressed individuals, especially
those in which the cell-mediated immune
FUNGAL INFECTION response is compromised.
There are several plausible outcomes
Fungi cause many diseases, most of which to fungal infections. As stated before, an
are adequately handled by the immune active cellular immune response plus topi-
system of the normal host. However, the cal antifungal drugs usually handles most
increased use of immunosuppressive superficial infections. Systemic infections,
drugs the immunosuppression secondary especially in the immunocompromised
to HIV infection has raised our awareness host, carry a high mortality rate, and even
of these infections. For example, Candida the use of newer prophylactic and thera-
albicans can cause superficial infections in peutic agents only partially decreases the
the normal host and is found throughout mortality.
the intestinal tract and commonly in the As with viruses, there is a third possible
vagina. The organism is usually kept under outcome. If the fungal infection is not elim-
control by the bacterial flora, but changes inated or causes persistent infection, then
in these flora secondary to overuse of anti- the host response may trigger a hypersen-
biotics or changes in hormone balance will sitivity reaction. As an example, Aspergil-
favor a chronic superficial infection. All of lus fumigatus infections can persist as an
this is exacerbated in the immunocompro- aspergilloma in preexisting lung cavities
mised host. secondary to tuberculosis or bronchiecta-
Another organism that is receiving sis following childhood pertussis (whoop-
wide attention is the fungus Pneumocystis ing cough). Allergic bronchopulmonary
carinii (now called Pneumocystis jiroveci), aspergillosis may occur and is due to

56 Immunological Aspects of Infection

IgE-mediated hypersensitivity to the asper- Although most protozoa stimulate the
gillus antigens. Clinically, the condition production of IgG and IgM antibodies,
presents as recurrent episodes of increased these antibodies are probably not protec-
wheezing and coughing, fever, and pleu- tive, and thus vaccines have not yet been
ritic pain similar to that seen in asthmatics. successful in the control or prevention of
Another example is farmers lung in which malaria. Also in the case of malaria, pro-
antigen-antibody complexes of Micropoly- tozoa invade erythrocytes and hepatocytes
spora faeni cause a hypersensitivity reaction and thus are hidden from the immune
on the part of the host. The name farmers response. Clinically, many of the signs and
lung indicates that this particular fungus is symptoms of these patients are related to
found in moldy hay. the destruction of red blood cells and hepa-
tocytes; therefore, anemia, jaundice, sple-
nomegaly, hypostasis, hypotension, tender
PARASITIC INFECTION hepatomegaly, and biphasic fever are hall-
marks of the disease.
Protozoa are a diverse group of parasites, Interestingly, there have been several
but malaria, leishmaniasis, and trypanoso- mutations in the host that help provide
miasis globally account for most of the prob- resistance to malarial infection. Most strik-
lems encountered in parasitic diseases. The ing has been the appearance of the het-
balance between host and parasite is two- erozygous sickle cell trait (Hbas), which
fold. The parasite may be too virulent for confers a survival advantage in endemic
the host or may evade the immune surveil- disease. Second, the absence of the red cell
lance and thus kills the host. Conversely, Duffy antigen (receptor for plasmodium
the immune response may be vigorous and vivax) is quite protective. Finally, the pres-
kill the parasite, thereby jeopardizing its ence of HLA-B53 in individuals is associ-
survival. Thus, the survival of any parasite ated with resistance to the disease.
depends on a balance between induction of Evasion mechanisms of protozoa fall
immunity and escape from surveillance. into three major categories. The first is
entrance of the organism into the host cell,
where it avoids immune surveillance. One
example is malaria, as noted previously.
The worldwide incidence of malaria is esti- Others include toxoplasma, leishmania,
mated at 300 million500 million people, and Trypanosoma cruzi, which enter and can
and at least 1 million die each year of the grow inside macrophages. For example,
disease, mostly of cerebral malaria and leishmania binds C3 avidly and thus serves
usually young children. Cerebral malaria as a ligand for the CR3 receptor on macro-
is usually associated with infection with phages. If one uses monoclonal antibodies
Plasmodium falciparum and not Plasmodium to the CR3 receptor, this inhibits the uptake
vivax. Patients react to protozoal infec- of the parasite into macrophage. Another
tion with activation of macrophages and approach is used by toxoplasma in which
monocytes with the release of cytokines they prevent fusion of phagocytic vacuoles
TNF, IL-1, and IL-6. Clinically, they pro- containing the parasite with lysosomes
duce fever, leukocytosis, and acute phase and thus are not destroyed. Finally, try-
reactants. panosomes need activated macrophages for

Immunological Aspects of Infection 57

killing; thus they are resistant to intracellu- Fischetti VA. Streptococcal M protein. Sci
lar killing in nonactivated macrophages. Am. 1991;264(6):5865.
A second mechanism for evasion is anti- Heine H, Lien E. Toll-like receptors and
genic variation. Trypanosoma brucei is an their function in innate and adaptive
excellent example of this. In this scenario, immunity. Int Arch Allergy Immunol.
the trypanosomes are initially destroyed 2003;130:180192.
by host antibody. However, the organism Lavoie PM, Thibodeau J, Erard F, Sekaly
resurfaces in the body with a different set RP. Understanding the mechanism of
of antigens or glycoproteins. The process action of bacterial superantigens from
continues, and the parasite possesses a a decade of research. Immunol Rev.
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known as phenotypic variation and dif-
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press the immune response. Malaria and effect of Staphylococcus aureus-derived
leishmania organisms release soluble anti- exotoxins in atopic dermatitis. J Allergy
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al. Characterization of two novel pyro- tive LPS signaling in C3H/HeJ and
genic toxin superantigens made by an C57BL/10ScCr mice: mutations in Tlr4
acute rheumatic fever clone of Strep- gene. Science. 1988;282:20852088.
tococcus pyogenes associated with mul- Visvanathan K, Charles A, Bannan J,
tiple disease outbreaks. Infect Immun. Pugach P, Kashfi K, Zabriskie JB. Inhi-
2002;70(12):70957104. bition of bacterial superantigens by
Ohmen JD, Barnes PF, Grisso CL, Bloom peptides and antibodies. Infect Immun.
BR, Modlin RL. Evidence for a 2001;69:875884.

Immunological Aspects of Infection 59

5. Immunological Aspects of Immunodeficiency Diseases

Dinakantha S. Kumararatne, M.D.

INTRODUCTION characteristic pattern of clinical presenta-

tion (Table 5.3), which will be elaborated
The principal function of the immune on later.
system is to prevent microbial infection.
Therefore, disorders resulting in impaired
function of the immune system (immuno- DEFECTS IN ANATOMICAL
deficiency) result in increased susceptibil- OR PHYSIOLOGICAL BARRIERS
ity to infection. Immunodeficiency can arise TO INFECTION
from an intrinsic defect of a component of
the immune system (primary immunode- One of the commonest predisposing causes
ficiency, or PID). Alternatively, immuno- of infection is a defect in an anatomical or
deficiency may be secondary to another physiological barrier to infection. Intact
pathological condition, which adversely epithelial membranes, especially strati-
affects immune function (Table 5.1). Both fied squamous epithelial surfaces such as
primary and secondary immunodeficien- the skin, constitute an extremely effective
cies result in increased susceptibility to barrier to infection. Thus, integumentary
infection. The precise pattern of infection damage caused by burns, eczema, and
depends on the specific component of the trauma (including surgery), predisposes
immune system that is affected. Most PIDs to infection. Skull fractures, particularly
are caused by defects in single genes and damage of the cribriform plate, may result
are hence heritable. Others may represent in recurrent episodes of pyogenic menin-
the consequence of an interaction between gitis. The existence of sinus tracts between
the genetic phenotype and an environmen- deeper tissues and the skin surface or
tal influence, like viral infections. Primary alternatively, the presence of foreign bod-
immunodeficiencies are rare and based on ies or avascular areas (e.g., within bone)
information from national registers; these predisposes to infection. Obstruction to
diseases are estimated to occur between 1 the drainage of hollow tubes and viscera
in 2,000 to 1 in 10,000 live births. In con- also predisposes to infection, for example,
trast, secondary immunodeficiencies are obstruction of the biliary tract, urinary tract,
more commonly seen in clinical practice. or bronchi. Impaired vascular perfusion of
(See Table 5.1 for examples of secondary the tissues due to edema and angiopathy
immunodeficiency.) (including microvascular changes follow-
From a clinical perspective, immuno- ing diabetes mellitus) also predisposes to
deficiencies can be classified into eight infection. Alteration of the normal com-
categories (Table 5.2). Each category has a mensal flora by broad-spectrum antibiotic

Table 5.1 Secondary Immunodeficiency

Causes of Secondary
Immunodeficiency Defect

Defects in anatomical and Various

physical barriers to infection
(see text for explanation)
Malignancies of the B-cell Antibody
Non-Hodgkins lymphoma
Chronic lymphocytic leukemia
Therapeutic agents
Biological agents
Anti-B-cell antibodies: e.g., Antibody
Anti-TNF agents Innate immunity and CMI
Cytotoxic drugs: Alkylating Myelosupression and CMI
agents, cytotoxic antibiotics,
antimetabolites, Vinca
alkaloids, etc.
Immunosuppressive drugs: CMI
Corticosteroids, calcineurin
Inhibitors, antiproliferative
(azathioprine, mycophenelate)
Radiotherapy, metabolic/ CMI
nutritional deficiencies
Renal failure CMI and innate immunity
Liver failure CMI and innate immunity
Protein calorie malnutrition CMI
Increased loss of Antibody
Protein-losing enteropathy
Nephrotic syndrome
Virus infections CMI

CMI, cell-mediated immunity.

therapy predisposes to colonization by Finally, damaged tissues, for example,

antibiotic-resistant potential pathogens, damaged cardiac valves, provide a nidus
which may cause infectious or toxin- for the establishment of infection.
induced complications, for example, pseu- Infections that recur in the same ana-
domembranous colitis caused by Clos- tomical site are often due to defective
tridium difficile toxin, multidrug-resistant anatomical or physiological barriers and
Staphylococcus aureus infection. Surgical hence should induce a diligent search for
instruments, perfusion lines, and cath- such factors. Microorganisms that cause
eters may promote microbial invasion past infection in patients with this category of
the anatomical or physiological barriers. defects comprise pyogenic bacteria such

62 Immunological Aspects of Immunodeficiency Diseases

Table 5.2 Operational Classification of infections, for example, meningitis or
Imunodeficiency States bone and joint infection.
2. Structural lung damage (bronchiecta-
1. Immunodeficiency due to defective
anatomical or physiological barriers to sis, pulmonary fibrosis) can be a con-
infection sequence of recurrent respiratory tract
2. Deficiency of opsonins: (a) antibody
infections in inadequately treated, anti-
deficiency, (b) complement deficiency
3. T-cell deficiency body-deficient patients, and contributes
4. Combined T- and B-cell deficiency to morbidity and mortality.
5. Phagocyte deficiency
6. Defects in macrophage activation 3. Once respiratory tract damage is estab-
7. Defects in immunoregulation lished, patients are prone to sinopul-
8. Defects in homeostasis of inflammation
monary sepsis caused by nontypeable
Haemophilus influenzae strains.
4. Overgrowth of commensal bacteria in
as staphylococci and commensal organ- the small intestines or chronic infection
isms from the skin or intestinal tract. by intestine pathogens (Giardia, Salmo-
Fungi, especially Candida, may be another nella, Campylobacter) may give rise to
pathogen under these circumstances. diarrhea or malabsorption secondary to
villous atrophy.
5. In general, the course of uncomplicated
viral infection (chicken pox, measles,
DISORDERS CHARACTERIZED BY etc.) is not significantly different from
ANTIBODY DEFICIENCY those in normal individuals, indicat-
ing that antibody production is not
Antibody deficiency can be defined as a
essential for recovery from acute viral
condition characterized by a reduction in
infections. However, long-term immu-
serum immunoglobulin concentrations
nity, which depends on the ability to
below the fifth centile for age. Antibody
develop neutralizing antibodies does
deficiency may affect all classes of immu-
not develop and the infections can
noglobulins or may be confined to a single
6. Fungal and intracellular bacterial infec-
tions are not a feature of antibody
Clinical Manifestations of Antibody
7. About a fifth of patients with antibody
deficiency due to common variable
KEY CONCEPTS immune deficiency (which is described
1. Patients with antibody deficiency typi- in a later section) develop autoimmune
cally develop recurrent infection with disorders. These include autoimmune
encapsulated bacteria such as Strep- hematological disorders (hemolytic ane-
tococcus pneumoniae and Haemophilus mia, autoimmune thrombocytopenia,
influenzae type B. The common sites pernicious anemia), autoimmune endo-
affected are the upper and lower respi- crinopathies (e.g., thyroid disease) or
ratory tracts and the middle ear. From neurological diseases such as Guillain-
these sites, infection can spread via Barr syndrome, and, rarely, a lupus-
the bloodstream to produce metastatic like syndrome.

Immunological Aspects of Immunodeficiency Diseases 63

Table 5.3 Pattern of Microbial Infection in Immunodeficiency

Defect Microorganism Site

Antibody Encapsulated bacteria: Upper and lower

Streptococcus pneumoniae, Hib, respiratory tract
Mycoplasma, Giardia, Salmonella, Less commonly
Campylobacter systemic infection:
meningitis, bone and
joint infection
Localized or disseminated
Gastrointestinal tract
Complement Pyogenic bacteria, especially Septicemia, meningitis,
C3/Factor I Streptococcus pneumoniae pyoderma
C5, C6, C7, C8, Neisseria meningitidis Meningitis, septicemia
C2, C4 No infections or occasionally
infections with S. pneumoniae
Properdin, factor D Encapsulated bacterial sepsis
Phagocyte deficiency Staphylococci, enteric bacteria, fungi Systemic
Chronic granulomatous Staphylococcus aureus, Salmonella, Skin, visceral abscesses:
disease enteric bacteria, Burkholderia lymph nodes, lung, liver
cepacea, fungi: Aspergillus
Leucocyte adhesin Pyogenic bacteria Skin; any site, localized or
deficiency systemic
T-cell deficiency Viruses, fungi, protozoa, intracellular Any site, localized or
bacteria: Mycobacteria, Listeria, systemic; mucocutaneous
Salmonella candidiasis
Defects in macrophage Intracellular bacteria: Mycobacteria, Lymph node; bone;
activation: Type I Listeria, Salmonella disseminated
cytokine deficiency
Combined T- and B- As for antibody and T-cell deficiency As for antibody and T-cell
cell deficiency deficiency

Hib, Haemophilus influenzae type B

Major Categories of Antibody with V pre-), and the signal-transducing

Deficiency components Ig and Ig. The activities of
the protein BTK (Brutons tyrosine kinase)
These are summarized in Table 5.4.
and BLNK (B-cell linker protein) are also
essential for the transduction of signals
ANTIBODY DEFICIENCY ASSOCIATED WITH received via the B-cell (and pre-B-cell)
ABSENT B CELLS receptors. Therefore, it is not surprising that
B-cell maturation beyond the pre-B-cell mutations in each of these elements causes
stage found in the bone marrow, requires early-onset antibody deficiency associated
signals received through the pre-B-cell with lack of circulating B cells.
receptor complex. The pre-B-cell receptor Ninety percent of all such cases occur
is composed of the chains, surrogate light in boys due to mutation of the BTK
chains (heterodimers of constant region gene, which maps to the X chromosome

64 Immunological Aspects of Immunodeficiency Diseases

Table 5.4 Major Antibody and/or T-cell Deficiencies

Antibody Deficiency
Diseases Mutated Gene/Pathogenesis Associated Features

X-linked BTK Antibody deficiency and

agammaglobulinemia B lymphopenia
Autosomal recessive Mutations in genes for , Ig, Ig, Antibody deficiency and
agammaglobulinemia 5, or BLNK B lymphopenia
X-linked hyper IgM CD40 ligand Lack of CD40 ligand on
syndrome activated T cells. Failure
of Ig class-switching and
affinity maturation; low IgG/
IgA, raised or normal IgM;
may develop neutropenia,
autoimmune cytopenias,
opportunistic infections, and
gastrointestinal and liver
CD40 deficiency (a CD40 Lack of CD40 expression
type of autosomal on B cells. Other features
recessive hyper IgM similar to CD40L deficiency
Hyper-IgM syndrome UNG or AID Low IgG and IgA, raised
(autosomal recessive) IgM
Common variable Unknown in most; TACI in 10%, Antibody deficiency;
immunodeficiency rarely ICOS, CD19, or BAFFR may have autoimmunity,
systemic granulomata
Selective IgA deficiency Most unknown; few due to TACI Most remain healthy;
mutations increase in autoimmunity,
atopy, celiac disease
IgG subclass deficiency Unknown If associated with specific
antibody deficiency, (see
text) may have recurrent
sinopulmonary infections

T- and B-cell
Immunodeficiencies Mutated Gene/Pathogenesis Associated Features

Severe combined See Table 5.6 for details Lymphopenia, low serum
immunodeficiency Igs, failure to thrive, severe
(SCID) recurrent infections by
viruses, bacteria, and
parasites; fatal without bone
marrow transplant (BMT)
Omenns syndrome Hypomorphic mutation of RAG1, Variant of SCID; some T
RAG2, Artemis, or IL7Ra and B cells may develop
but are oligoclonal. Features
include erythroderma,
eosinophilia. Outcome poor
without BMT


Immunological Aspects of Immunodeficiency Diseases 65

Table 5.4 (continued)

Antibody Deficiency
Diseases Mutated Gene/Pathogenesis Associated Features

MHC class II deficiency MHC2TA, RFXANK, RFX5, RFXAP : Lack of MHC class II
SCID due to defective MHC class II expression resulting in CD4
transcription lymphopenia and severe
failure of T-cell and B-cell
MHC class I deficiency TAP1 or TAP2 Lack of MHC class I
expression on cells; CD8
Lymphopenia; present with
bronchiectasis or vasculitis

(Xp22). This condition is called X-linked antibody (this process is called affinity
agammaglobulinemia, which was the first maturation). Through these processes,
immunodeficiency to be described in 1952 memory B cells are generated within
by Colonel Ogden Bruton. Mutations in the germina centers.
genes, encoding , 5, Ig, Ig, and BLNK Defects in genes encoding molecules
cause rare autosomal recessive forms of required for the above processes, which
early-onset antibody deficiency with severe operate within germinal centers, result
B lymphopenia. in a form of antibody deficiency with
elevated (or normal) IgM levels but lack-
ANTIBODY DEFICIENCY DUE TO DEFECT IN ing IgG, IgA, and IgE. These conditions
During primary antibody responses, A key requirement for germinal center
B cells initially produce IgM and later on formation and function is the interaction
in the response switch to the production of of CD40 (belonging to the TNF-receptor
IgG, IgA, and IgE. superfamily) found on the surface of B
During the so-called immunoglobulin cells with an activation induced, CD40-
class-switching process, the heavy chain ligand (CD40L) protein expressed on the
constant region changes while antigen surface of CD4 lymphocytes. Mutations in
specificity is maintained. Immunoglobulin the CD40L gene or the CD40 gene result in
class switch takes place within germinal X-linked (relatively common) and autoso-
centers contained within B-cell follicles of mal recessive (rare) HIGM, respectively.
the secondary lymphoid organs. Another Patients with the CD40L deficiency suffer
process that occurs within germinal centers from recurrent bacterial infections typical
is somatic hypermutation, which results in of antibody deficiency. However, because
the sequential accumulation of point muta- CD40L function is required for optimal
tions in the Ig variable region gene. If the T-cell immunity, they also suffer from
point mutation(s) result in increased bind- opportunistic infections characteristic of
ing affinity to the inducing antigen, the B- T-cell deficiency. About a third of patients
cell blasts (centrocytes) survive, proliferate, with CD40L deficiency develop Pneumocys-
and eventually give rise to memory B cells tis pneumonia. Infections with cryptosporio-
and plasma cells that secrete high-affinity dosis, toxoplasmosis, and nontuberculous

66 Immunological Aspects of Immunodeficiency Diseases

mycobacteria also occur in this condition. with CVID develop autoimmune disor-
These opportunistic infections can be ex- ders (arthritis, cytopenias, endocrinopa-
plained on the basis that the interaction of thies). Some patients with CVID develop
CD40L on activated T cells with CD40 ex- noncaseating sarcoidlike granulomata
pressed on the surface of macrophages and infiltrating various organs (lungs, liver,
dendritic cells, which in turn undergo matu- spleen, skin). The mechanisms underlying
ration and activation, is required for the opti- autoimmunity and granulomatous disease
mum expression of antimicrobial immunity. in CVID are unknown.
A high proportion of CD40L-deficient The immunological phenotype of
patients develop progressive liver dam- CVID is heterogeneous with documented
age (sclerosing cholangitis), probably the defects in B-cell survival, generation of B
result of cryptosporidial infection of the memory cells, and in vitro B- and T-cell
bile ducts. activation. About 10 percent of cases of
Defects in the RNA-editing enzymes, CVID are familial, with a predominance of
activation-induced cytidine deaminase autosomal dominant or autosomal reces-
(AID), and uracil-DNA glycosylase (UNG) sive inheritance. CVID or selective IgA
result in two further types of HIGM syn- deficiency can affect different members of
dromes, which are defective class switch- the same kindred. Recently, a number of
ing and affinity maturation. gene defects have been identified in CVID
Signaling through CD40, which belongs patients, accounting for about 1015 per-
to the TNF-receptor superfamily, depends cent of the total patient pool.
on the activation of the inhibitor of kinase The commonest defect (in approxi-
(IKK) complex, resulting in the induction mately 10% of CVID patients) is a mutation
of NFB. Hypomorphic mutations of the of the gene TACI (transmembrane activator
gamma subunit of the IKK complex, which and calcium modulator and cyclophilin
is called NEMO (NFB essential modu- ligand interacter). TACI, which belongs to
lator), impairs NFB activation. Patients the TNF-receptor superfamily, is a ligand
with mutations in NEMO develop a com- for the cytokines, BAFF (B-cell-activat-
plex immunodeficiency, which includes ing factor of the TNF family) and APRIL
features of the HIGM syndrome. (a proliferation-induced ligand), which
induce immunoglobulin class-switch
Most patients with primary antibody Mice deficient in BAFF or its recep-
deficiency are collected under the heading tor have impaired B-cell development
common variable immune deficiency, and antibody deficiency. TACI/ mice
which is a condition characterized by low have reduced serum IgA and IgM levels,
serum IgG and IgA and a variable decrease reduced antibody responses to T-cell-
in IgM and the impaired production of independent antigens, and tend to develop
specific antibodies following natural autoimmunity and B lymphoprolifera-
microbial exposure or immunization. The tion. Mutations in TACI have been found
estimated prevalence of CVID is between 1 in CVID patients and their relatives,
in 10,000 and 1 in 50,000. Clinically, CVID with selective IgA deficiency, indicating
is dominated by the effects of antibody variable penetrance of this gene defect.
deficiency. About 20 percent of patients In the majority of currently documented

Immunological Aspects of Immunodeficiency Diseases 67

patients, TACI mutations affect only one IgA DEFICIENCY
allele, indicating a dominant negative effect IgA deficiency is characterized by
of the mutated gene. A possible explana- reduced (<0.07 g/L) or absent serum IgA
tion is that TACI, like other members of the levels. IgA deficiency is the commonest
TNF-receptor family, undergoes ligand- form of primary antibody deficiency and
independent preassociation and function affects about 1 in 700 Caucasians. IgA
as multimeric units. Thus, incorporation deficiency is rare in some racial groups
of a mutated TACI chain in this multimeric (Japanese, Africans). The majority of IgA-
complex may disrupt ligand binding or deficient individuals remain free of infec-
signal-transducing capacity. Recent stud- tion due to the ability of IgG and IgM to
ies have found that family members of compensate for the lack of IgA. Long-term
CVID patients possessing heterozygous studies have shown that a small propor-
TACI mutations may have completely nor- tion of IgA-deficient patients develop
mal serum immunoglobulin levels and in recurrent sinopulmonary or gastroin-
vitro B-cell function. This leads to the con- testinal infections. Most infection-prone
clusion that the development of antibody IgA-deficient patients have concomitant
deficiency in those carrying heterozygous IgG2 subclass deficiency and a selective
TACI mutations may depend on modifier inability to produce antibodies against
genes or environmental factors. bacterial capsular polysaccharides. IgA
ICOS (inducible co-stimulating recep- deficiency is associated with an increased
tor) is a co-stimulatory T-cell molecule incidence of atopy, celiac disease, and a
that induces cytokines required for sup- range of autoimmune diseases, including
porting class-switch recombination, Ig arthritis, lupus-like syndrome, autoim-
production and terminal B-cell differentia- mune endocrinopathies, and autoimmune
tion. ICOS/ and ICOS ligand/ mice cytopenias.
exhibit defective germinal center formation IgA-deficient patients with serum lev-
and antibody production. Mutations in the els below 0.07 g/L are at the risk of devel-
ICOS gene account for about 1 percent of oping anti-IgA antibodies after receiving
CVID patients. blood products. Such individuals are at
A single CVID patient was identified to risk of developing anaphylaxis on receiv-
have a mutation on the gene encoding the ing blood products.
BAFF receptor, a finding that could be pre- IgA deficiency and CVID can differ-
dicted from observations in BAFF/ mice. entially affect members of the same kin-
Finally, CD19 deficiency has been dred. Rarely, IgA deficiency can precede
described in two families with CVID. the development of CVID. Therefore, in
CD19 is a B-cell accessory molecule that is some instances, the molecular mechanisms
required for B-cell activation, proliferation, underlying CVID and IgA deficiency may
and hence B-cell development. CD19/ be identical. TACI deficiency can cause IgA
mice exhibit hypogammaglobulinemia deficiency in some family members while
and poor antibody responses to protein others develop CVID. However, the molec-
antigens. ular basis of IgA deficiency in most cases is
In the case of the large majority of largely unknown.
patients with CVID, the underlying molec- Genetic analysis of kindred with CVID
ular defect is unknown. and IgA deficiency have highlighted the

68 Immunological Aspects of Immunodeficiency Diseases

existence of susceptibility loci within the antibody-deficient individuals are likely
MHC region of chromosome 6. The stron- to have a normal life span. Replacement
gest linkage lies within the DR/DQ within immunoglobulin is prepared from plasma
the MHC class II region. pooled from large cohorts of normal
donors. Current practice of prescreening
IgG SUBCLASS DEFICIENCY donors for infection and multiple antivi-
Serum IgG comprises four subclasses ral steps employed during the manufac-
called IgG1, IgG2, IgG3, and IgG4, respec- ture of plasma have eliminated the risk of
tively reflecting the relative abundance HIV and hepatitis B and C transmission.
of these isotypes in the serum. IgG sub- Different immunoglobulin products are
class deficiency is diagnosed on finding licensed for administration via the intra-
a reduction in the serum concentration of venous or subcutaneous route. Detailed
IgG subclasses, of more than two standard management of a patients antibody defi-
deviations below the mean value for age, ciency is discussed in the references on
despite the total IgG level being normal. In the diagnosis and management of anti-
practice, IgG subclass assays are difficult body deficiency (see list of references at
to standardize due to the lack of an inter- end of chapter).
nationally accepted reference preparation.
Furthermore, genetic variations that influ-
ence serum IgG subclass levels exist among
different ethnic groups. Hence, age- and
population-related normal ranges are not
always available.
Some individuals with IgG subclass
These may be inherited (primary) condi-
deficiencies are asymptomatic. Others
tions, which are rare or secondary to other
with IgG subclass deficiencies are prone to
pathologies (Table 5.5). HIV infection,
recurrent sinopulmonary infections. Such
which is an important cause of secondarily
infection-prone patients exhibit reduced
impaired T-cell immunity is discussed in
antibody responses to bacterial capsular
Chapter 8.
polysaccharides. Defective anti-polysac-
charide antibody responses are most often
seen in individuals with IgG2 subclass
Manifestations of T-Cell Deficiency
deficiency with or without concomitant
IgA deficiency. The molecular mechanisms KEY CONCEPTS
underlying IgG subclass deficiencies are Patients with impaired T-cell function
unknown. may develop the following:

TREATMENT OF ANTIBODY DEFICIENCY 1. They show increased susceptibility to

Prospective clinical studies have infections with intracellular microbial
shown that optimal IgG replacement ther- pathogens (viruses, intracellular bacte-
apy reduces the incidence of sepsis and ria, and protozoa).
complications like structural lung dam- 2. Viral infections: Infections with exan-
age. If replacement therapy is introduced thematous viruses (measles, chicken
early before organ damage is established, pox) can be fatal in children with

Immunological Aspects of Immunodeficiency Diseases 69

Table 5.5 Causes of T-Cell Deficiency susceptible to infection with M.
tuberculosis or reactivation of latent
Primary See Table 5.6
(inherited) and text
tuberculosis. They are also suscep-
Secondary (a) Viral-induced
tible to disseminated infections with
HIV poorly pathogenic mycobacteria
Measles (transient) (e.g., nontuberculous mycobacteria,
(b) Iatrogenic
Irradiation BCG).
BMT until engraftment 5. Infants with T-cell deficiency are usually
is complete
lymphopenic and fail to thrive (i.e., fail
Chemotherapy for
malignancy to maintain normal rates of growth in
Immunosuppressive height and weight).
(c) Thymoma 6. Infants with SCID may develop der-
(d) Lymphoma matitis and hepatosplenomegaly due
(e) Severe renal or
to graft-versus-host disease caused
liver failure
by maternal lymphocytes that have
Idiopathic Idiopathic CD4 cell
deficiency (cause unknown) crossed the placenta.
7. Malignancies: T-cell-defi cient indi-
viduals are prone to develop a range
of malignancies where viral infection
T-cell deficiency. These viruses are acts as a co-factor (e.g., Epstein-Barr
not a problem in adults as they have virus [EBV-] induced non-Hodgkins
residual protective antibody responses lymphoma, Kaposis sarcoma where
generated by primary infection or human herpes virus 8 is a co-factor).
immunization. Adults with T-cell There is also an increase in cutaneous
deficiency are typically affected by the malignancies occurring in an individ-
reactivation of latent viruses (e.g., cyto- ual exposed to significant amounts of
megalovirus, Herpes simplex), which can ultraviolet light (basal-cell carcinoma
produce life-threatening disseminated and squamous carcinoma).
3. Fungal infections: T-cell-deficient pa-
Major Categories of Combined
tients are typically susceptible to fungal
a. Pneumocystis giroveci causes intersti- SEVERE COMBINED IMMUNODEFICIENCY
tial pneumonia, which is pathogno- SCID comprises a group of inherited
monic for T-cell deficiency; diseases characterized by a severe defi-
b. Mucocutaneous infection with cit in T-cell development and function
Candida; with variable defects in B-cell and natural
c. Systemic infections with filamentous killer or NK cell development. SCID leads
fungi (e.g., Aspergillus fumigatus); to death within the first two years of life,
d. Meningitis or systemic infection unless patients are rescued by hemato-
caused by Cryptococcus neoformans. poietic stem cell transplantation (HSCT).
4. Intracellular bacterial infection is a These are rare disorders with an estimated
particular problem in T-cell-deficient frequency between 1 in 50,000 and 1 in
patients. These patients are highly 100,000 live births.

70 Immunological Aspects of Immunodeficiency Diseases

Table 5.6 Classification of SCID

Type of SCID Frequency (%) Inheritance Cells Affected Defective Gene

Reticular <1 AR T, B, NK, ?

dysgenesis Leucocytes,
T B SCID 10 AR T, B RAG1/2
10 AR T, B Artemis
T B + SCID 50 XL T, NK IL2R6
10 AR T, NK JAK3
<1 AR T CD45
<1 AR T CD36,CD3E

AR, antosomal recessive; XL, X-linked

Classification of SCID cells following blood transfusion. GvHD

The classification of SCID, based on manifests as skin rashes or hepatospleno-
immunological and genetic criteria, is sum- megaly and lymphadenopathy.
marized in Table 5.6. The absence of tonsils or other lymphatic
tissue may be evident and radiographic
SCID: Clinical Features studies may reveal thymic-hypoplasia.
These patients typically present in the Lymphopenia (absolute lymphocyte count
first year of life with failure to thrive and < 3 109/L in the first year of life) is a char-
recurrent infections caused by bacterial, acteristic feature seen in over 80 percent
viral, and fungal pathogens. Infections of patients with SCID. (Therefore, SCID
typically affect the respiratory and gas- needs to be excluded in any infant with
trointestinal systems. The infections unexplained lymphopenia.)
may be caused by common pathogens
(adenovirus, respiratory syncytial virus, Immunological and Molecular Classification
parainfluenza virus), as well as by of SCID
opportunistic organisms of low-grade SCID can be classified in two groups
virulence (Candida, Pneumocystis carinii, based on the blood lymphocyte phenotype.
cytomegalovirus). Live vaccines such as
BCG can lead to disseminated life-threat- Patients lacking T cells with normal or
ening infections. The persistent infections increased B cells: TB+ SCID
developing in SCID patients rapidly lead Patients lacking T and B cells: TBSCID
to malnutrition, growth impairment,
and early death. Because of the patients Defects in one of four functionally
inability to reject allogeneic cells, graft- related genes causes TB + SCID. X-linked
versus-host disease (GvHD) can be SCID, which is the commonest form, is
caused by transplacentally acquired due to a mutation of the gene encod-
maternal lymphocytes or by allogeneic ing the IL-2 receptor chain, which is the

Immunological Aspects of Immunodeficiency Diseases 71

signal-transducing chain common to the Hypomorphic mutations of RAG1 or
receptors for six cytokines (IL-2, IL-4, IL- RAG2 result in a leaky form of SCID called
7, IL-9, IL-15, and IL-21). The absence of Omenns syndrome. In Omenns syn-
responses to these cytokines causes defects drome, a few T- and B-cell clones may be
in a broad range of T- and B-cell func- generated but the full T- and B-cell reper-
tions. IL-7 is required for early stages of toire fails to develop. The few T- and B-
T-cell development. Lack of response to cell clones that leak through may undergo
this cytokine results in T lymphopenia. secondary expansion. As a result, patients
IL-15 is required for NK-cell development with Omenns syndrome may not be mark-
and its lack results in the failure of NK-cell edly lymphopenic but the lymphocyte rep-
development. Signal transduction through ertoire is oligoclonal and severe immuno-
the aforementioned cytokine receptors deficiency is the outcome.
involves the interaction of the common T- and B-cell antigen receptors are
chain with the tyrosine kinase JAK3. This assembled from the recombination of
explains why mutations of the JAK3 gene variable region V(D)J and constant region
result in an autosomal recessive form of genes. A protein called ARTEMIS is
SCID, with a phenotype similar to X-linked required for DNA repair, including the
SCID. Mutations of the chain of IL-2 or repair of DNA breaks generated during
IL-7 receptors result in two rare forms of V(D)J recombination. Mutation of the gene
SCID. encoding ARTEMIS results in a rare form
T- and B-cell receptors consist of invari- of TBSCID. These patients also exhibit
ant signal-transducing elements combined increased sensitivity to ionizing radiation.
with elements that make up the variable About 15 percent of SCID cases are
regions, which contribute to the antigen- caused by deficiency of adenosine deami-
binding portion of the receptor. The gene nase (ADA), an enzyme required for the sal-
recombination required for generating vage of nucleotides within lymphoid cells.
these receptors requires the function of the The lack of ADA causes the accumulation
product of recombination activating genes of toxic metabolites of adenosine (deoxy-
1 and 2 and a number of proteins that are adenosine and deoxy-ATP) within lym-
required for DNA repair (DNA-PKcs, phoid cells, resulting in their demise. ADA
KU70, KU80, XRLC4, and DNA-IV). In deficiency results in profound lymphopenia
mice, mutations in any one of these genes affecting T cells, B cells, and NK cells. Rarely,
produces analogues of SCID. mutations of ADA causing milder forms of
In humans, TBSCID is most common enzyme deficiency lead to a milder form of
(50 percent of total) caused by mutations combined immunodeficiency presenting at
of the recombinase-activating genes, RAG1 a later stage in life. Purine nucleoside phos-
or RAG2. RAG1 and RAG2 are enzymes phorylase (PNP) is an enzyme required for
responsible for introducing double- purine salvage within lymphocytes, and
stranded DNA breaks, which initiate V(D)J PNP deficiency causes a milder phenotype
gene rearrangements, required for gener- of SCID than seen in ADA deficiency. SCID
ating T- and B-cell receptors for antigen. due to PNP deficiency not treated with
Without normal RAG1 and RAG2 func- HSCT is fatal in childhood.
tion, T- and B-cell development is arrested Mutations in proteins required for nor-
early in ontogeny, producing TBSCID. mal functioning and signal transduction

72 Immunological Aspects of Immunodeficiency Diseases

through the T-cell receptor (TCR) cause functions. Lack of thymic CD4+ CD8 cell
rare forms of SCID. Mutations of the tyro- selection for survival results in peripheral
sine phosphatase, CD45, which helps to CD4 lymphopenia. Because CD4 func-
initiate signaling by the TCR, results in tion is required for normal cell-mediated
TB+ SCID in humans. Mutation of com- immunity, as well as antibody production,
ponents of CD3-complex (CD3 , , and ) MHC class II deficiency results in a severe
result in a SCID phenotype. During signal form of SCID with a fatal outcome. MHC
transduction via TCRs, the protein tyrosine class II deficiency is due to a mutation in
kinases Lck and ZAP70 are required for one of four transcription factors (RFXAP,
phosphorylation of ITAMs on the intracy- CIITA, RFX5, RFXANK), which regulate
toplasmic segment of the TCR. Deficiency MHC class II expression.
of either of these kinases results in rare
TCRs of CD8 cells recognize antigenic Hematopoietic Stem Cell Transplantation
peptides that are complexed to MHC class Untreated SCID is invariably fatal in
I antigens, and TCR of CD4 cells recog- early infancy. Once a diagnosis of SCID is
nize antigen bound to MHC class II on the confirmed, irrespective of the molecular
surface of antigen-presenting cells. Cell- diagnosis, HSCT from a human leucocyte
surface expression of MHC class I mol- antigen (HLA-) identical or haplo-identical
ecules fails if either of the two transport- family donor is the treatment of choice.
ers of antigenic peptides (TAP1 or TAP2) Treatment of SCID with HSCT before 3.5
is lacking. TAP1 and TAP2 help to transfer months of age results in good immune
peptides from the cytosol into the endo- reconstitution and 95 percent survive long
plasmic reticulum, for subsequent loading term. Delay in treatment, or the occurrence
onto newly synthesized MHC class I mol- of infection, impairs outcome. Infection and
ecules. In the absence of peptide loading, GvHD are the main complications follow-
MHC class I molecules are degraded before ing HSCT. North American and European
reaching the cell surface. In the absence of data indicate that long-term survival after
MHC class I antigen expression, CD8 cell transplants from HLA-matched unrelated
function is deficient, and these cells are not donors was close to 60 percent. Review
generated within the thymus. The result- of European data between 1968 and 1999
ing immunodeficiency is milder than SCID indicates the progressive improvement of
and often presents in later life. Paradoxi- outcome, which is mainly because of better
cally, viral infections are not a problem in prevention of GvHD and the treatment
these patients. Some MHC class I deficient of infection. Analysis of the outcome of
patients develop progressive bronchiecta- European and U.S. bone marrow transplant
sis, while others develop vasculitis affect- programs for the treatment of SCID is
ing the face and upper respiratory tract. It ongoing and will be regularly reported.
has been postulated that vasculitis seen in
these patients may be due to self-destruc- Gene Therapy for SCID
tion of vascular endothelial cells by the Long-term immune reconstitution
unrestrained cytotoxicity of NK cells. has been achieved in patients with SCID
In contrast, MHC class II deficiency caused by the common -chain deficiency
results in a profound failure of CD4 cell or ADA deficiency, using gene therapy.

Immunological Aspects of Immunodeficiency Diseases 73

This was achieved by ex vivo gene transfer (<1 percent) exhibit profound T lympho-
to hematopoietic stem cells isolated from penia, associated with opportunistic infec-
the patients bone marrow. These gene- tions and a poor outlook unless rescued
reconstituted stem cells were retransfused with fetal thymic transplant.
into the patient. To date, gene therapy has The cardiac, velopharyngeal, thymic,
been restricted to patients without an HLA- and parathyroid abnormalities are due
matched family donor. to the defective development of third and
Several cases of leukemia have occurred fourth pharyngeal arches during ontogeny.
among -chain-deficient patients who The 22q. 11.2 region contains the TBX1
received gene therapy. In these cases, the gene, which belongs to the T-BOX fam-
retroviral vector had integrated close to ily of genes that incorporate proteins that
the LMO2 proto-oncogene in the leukemia regulate embryonic development. Patients
clone, leading to aberrant transcription with mutations in the TBX1 genes also
and expression of LMO2. Because of this develop the clinical features seen in 22q.
setback, clinical trials of gene therapy for 11.2 deletion syndrome, suggesting that
SCID are being carefully evaluated, and haplo-insufficiency of the TBX1 gene may
more experience is required before the be responsible for the clinical features seen
definitive role of gene therapy in SCID is in those with a deletion of the 22q. 11.2
established. region.

DiGeorges Syndrome (Thymic Aplasia)

DiGeorges syndrome (DGS) is second-
ary to a hemizygous deletion of the short Neutrophils are the principal circulating
arm of chromosome 22 (DEL 22q. 11.2). phagocyte. During inflammation, neutro-
This chromosomal defect causes a com- phils become activated and migrate into
plex inherited syndrome characterized by the tissues where they ingest, kill, and
cardiac malformations, thymic hypopla- digest invading bacteria and fungi. Neu-
sia, palatopharyngeal abnormalities with trophil function can be deficient because
associated velopharyngeal dysfunction, of a reduction in the number of circulating
hypoparathyroidism, and facial dysmor- neutrophils (neutropenia) or due to inher-
phism. The 22q deletion has an incidence ited defects in neutrophil function, which
of approximately 1 in 2,500 live births. The are rare disorders.
associated clinical phenotype is highly
variable. About 20 percent of individu-
als with 22q deletion have thymic aplasia,
resulting in T lymphopenia and impaired Neutropenia is defined as a decrease in the
CMI. In most such cases, the degree of T blood neutrophil count below 1.5 109/
lymphopenia is modest (partial DGS) and L. In some racial groups (e.g., Africans),
almost complete restitution of the T-cell the normal neutrophil count is somewhat
repertoire and function occurs by two years lower (up to 1.2 109/L). While mild neu-
of age. Therefore, infections characteristic tropnia may be asymptomatic, severe neu-
of T-cell deficiency are rare in these indi- tropenia (counts < 0.5 109/L) is invariably
viduals. A minority of infected individuals associated with the risk of life-threatening

74 Immunological Aspects of Immunodeficiency Diseases

Table 5.7 Causes of Neutropenia

Category of
Neutropenia Condition Defective Gene

Primary (due to rare Autosomal recessive congenital Homozygous mutations of

gene defects) neutropenia mitochondrial HS-1 associated protein
X1 (HAX1)
Autosomal dominant congenital In 60% heterozygous mutations of
neutropenia neutrophil elastase gene (ELA-2)
Cyclical neutropenia ELA-2
Shwachman Diamond syndrome Shwachman Bodian Diamond
syndrome gene (SBDS)

Category of
Neutropenia Mechanism of Neutropenia Cause

Secondary Reduced neutrophil production Secondary to viral infection

in bone marrow
Drugs (idiosyncratic) or immune
Bone marrow failure (leukemia,
myelodysplasia, aplasia)
Reduced neutrophil migration WHIM syndrome secondary to mutation
out of bone marrow of CXCR4 receptor
Increased peripheral destruction Autoimmune neutropenia
of neutrophils Hypersplenism

microbal sepsis caused by a broad range needs to interact with E selectin, which is
of endogenous Gram-positive bacteria (S. expressed on the luminal surface of the
aureus) and Gram-negative bacteria (E. coli, endothelial cells. Leucocytes, which are
Pseudomonas spp., Klebsiella spp.) as well transiently arrested by the previous inter-
as fungi (Candida). Neutrophils are particu- action, need to bind tightly to the endothe-
larly important for maintaining the integ- lial surface by a second set of interactions.
rity of mucous membranes. Hence, oral This is between the protein lymphocyte
ulceration and perianal inflammation can function-associated antigen-1(LFA-1) LFA-
be features of severe neutropenia. Causes of 1 expressed on the leucocyte surface and
neutropenia are summarized in Table 5.7. its ligand intercellular adhesion molecule-
1 expressed on the luminal surface of acti-
vated endothelial cells. Leucocyte emigra-
Defects in Leucocyte Migration
tion into the tissues follows these adhesion
To reach the sites of inflammation, events. LFA-1 is one of a set of three cell-
neutrophils need to migrate across the endo- surface heterodimers composed of a com-
thelium into sites of inflammation. For this mon chain (CD18) with three separate
process to be initiated, sialyl LewisX, which chains called CD11a, b, and c. CD18-CD11a
is expressed on the surface of leucocytes, heterodimers form LFA-1, CD18/CD11b

Immunological Aspects of Immunodeficiency Diseases 75

Table 5.8 Defects in Phagocyte Function

Phagocyte Deficiencies
(excluding congenital
neutropenias) Defective Gene/Pathogenesis Clinical Features

Chronic granulomatous Mutations in components of Pyogenic and fungal

disease the phagocyte oxidase (see infections; lymph node and
text for details) visceral abscesses; chronic
granulomata (see text for
Leucocyte adhesin deficiency Mutation of gene encoding Delayed umbilical cord
type 1 CD18, which is a component separating, omphalitis,
of leucocyte adhesins (see text pyoderma, periodontitis,
for details) leucocytosis
Leucocyte adhesin deficiency Mutation of gene encoding As above plus mental
type 2 GDP-fucose transporter (see retardation
text for details)
Rac 2 deficiency Mutation in RAC2 leading to Poor wound healing,
impaired actin polymerization/ leucocytosis
cytoskeletal function
Mendelian susceptibility to Defects in the production or Recurrent disseminated
mycobacterial infection response to IFN-g : Mutated infections with poorly
genes include IL12B, IL12RB pathogenic mycobacteria
IFNGR1, IFNGR2, STAT-1, (NTM or BCG) and systemic
TYK2. Intact response to infections with non-typhi
IFN- is essential for control Salmonella species
of intracellular bacterial
Some NEMO mutations cause
X-linked susceptibility to
mycobacterial infection.
See text for full explanation.
Hyper-IgE syndrome Mutations in STAT-3 leading to Complex phenotype
a functionally defective form of characterized by recurrent
STAT 3. Autosomal dominant abscesses, pneumonia with
inheritance. pneumatocoele formation,
elevated serum IgE,
dermatosis, dysmorphic
features, delayed shedding
of decidual teeth, bone
abnormalities, selective
impairment of anticapsular
antibody responses, and
impaired acute-phase
response following infection.
Spectrum of infecting
organisms include S. aureus,
H. infuenzae, S. pneumoniae,
and filamentous fungi.

heterodimers form complement receptor while CR3 and CR4 act as receptors for
3, and CD18 combined to CD11c forms activated complement, aiding ingestion of
complement receptor 4. LFA-1 is required opsonized microorganisms. Mutation of
for leucocyte adhesion to endothelial cells the gene encoding CD18 (resulting in the

76 Immunological Aspects of Immunodeficiency Diseases

lack of expression of LFA-1, CR3, and CR4) the proteolytic enzymes, cathepsin, and
results in an inherited primary immunode- elastase. The activity of the proteolytic
ficiency called leucocyte adhesin deficiency enzymes is bactericidal. The NADPH oxi-
type 1(LAD1). Mutations of the enzyme dase complex comprises two membrane-
GDP-fucosyl transferase prevents post- associated proteins, p91phox and p22phox
transcriptional fucosylation of proteins. (also called the and units of cytochrome
Such individuals cannot synthesize sialyl B558, respectively), complexed with three
LewisX. This condition is called leucocyte cytosolic cofactors, p47phox, p40phox, and
adhesion deficiency type 2. Leucocytes p67phox. Mutations have been identified
of the patient with LAD1 and 2 exhibit in four out of five components (p91phox,
impaired ability to adhere to endothelial p22phox, p47phox, p67phox), resulting
walls and therefore cannot migrate into in defective NADPH oxidase activity,
infected sites. leading to impairment of bacterial kill-
These patients typically present in ing by phagocytic cells. The result is a
early childhood with recurrent pyogenic clinical syndrome, called CGD, character-
infection of skin, respiratory, and gastro- ized by failure of bacterial degradation in
intestinal tracts as well as mucous mem- vivo, resulting in the persistence of tissue
branes. Poor wound healing and delayed inflammation with granuloma formation
umbilical cord separation are typical. in a variety of organs. CGD due to p91phox
Because of impaired neutrophil migration, deficiency is X-linked, while the other vari-
these patients develop a leucocytosis and ants of CGD are inherited in an autosomal
pus fails to form at sites of infection. These recessive manner.
inherited disorders are typically associated In CGD, the bacterial killing mecha-
with severe gingivitis and periodontal nisms that depend on the phagocyte
disease, again indicating the particular oxidase system are inoperative, but the
importance of normal neutrophil function nonoxidative killing mechanisms are
for maintenance of the health of the den- still intact. Therefore, patients with CGD
tal crevice. Outcome in both conditions is are not troubled by the broad range of
poor, with early death. BMT is curative in microbes that a neutropenic patient would
LAD1, and oral fucose supplementation is be susceptible to. Instead, CGD patients get
beneficial in LAD2. infections with a restricted range of micro-
organisms, which are only susceptible to
the bactericidal mechanisms initiated by
Defects in Bacterial Killing
NADPH oxidase activation. The spectrum
The best example of an immunodeficiency of microorganisms that causes infections
characterized by failure of phagocyte- in CGD include S. aureus, Gram-negative
mediated bacterial killing is chronic bacteria (Burkholderia cepacea, Salmonella,
granulomatous disease (CGD). In normal Serratia), and fungi (Aspergillus spp.).
neutrophils (and monocytes), bacterial Characteristic sites of infection include
phagocytosis results in the activation of the subcutaneous tissue, lymph nodes, lungs,
nicotinamide adenine dinucleotide phos- and liver. Oral and perioral ulceration are
phate (NADPH) oxidase complex, which common.
produces superoxide and generates a mi- The formation of chronic granulomata
lieu within the phagosome that activate in various tissues is a typical feature of

Immunological Aspects of Immunodeficiency Diseases 77

CGD. In critical locations, chronic granu- gamma (IFN-) production is critically
loma formation may cause pathology. important for immunity against intracel-
Granulomatous obstruction of the gastro- lular bacterial pathogens. This process is
intestinal or genitourinary tract may be a initiated by the stimulation of Toll recep-
consequence. Hepatosplenomegaly may tors on the surface of antigen-presenting
occur due to granulomatous infiltration cells by bacterial ligands such as mycobac-
of these organs. A granulomatous colitis, terial lipoarabinomannan. This results in
resembling Crohns disease, occurs in about the secretion of IL-12 and IL-23 and TNF-
15 percent of CGD patients, highlighting by the antigen-presenting cells. Binding of
the importance of normal phagocyte func- IL-12 and IL-23 to their respective recep-
tion for preventing harmful inflammation tors, expressed on the surface of activated
in the large intestine. T cells and NK cells, induces these cells
Diagnosis of CGD is based on the abil- to secrete a further cytokine IFN-. IFN-
ity of stimulated neutrophils from these acting in concert with TNF- activates
patients to oxidize dyes to give a colored macrophages, which are then capable of
product or a fluorescent product, detect- killing intracellular pathogens (e.g., Myco-
able by flow cytometry. Prophylactic treat- bacteria and Salmonella). Individuals with
ment with IFN- substantially reduces the impaired T-cell-mediated immunity (e.g.,
incidence of bacterial and fungal infections SCID or HIV infection) are highly suscep-
in CGD patients, for reasons that are not tible to mycobacterial infections, including
fully understood. infections caused by poorly pathogenic
The cytosolic GTPase Rac2 is required mycobacterial species (nontuberculous
for the normal actin polymerization and mycobacteria and BCG). However, dissem-
optimal function of the phagocyte oxidase inated life-threatening infections by these
system. Rac2 deficiency results in impaired organisms may also occur in the absence of
neutrophil mobility and poor superoxide a recognized primary or secondary immu-
responses to some stimuli. A single affected nodeficiency. Genetic analysis of affected
patient identified to date had a clinical kindred to date have identified mutations
presentation similar to that seen in LAD. in seven different genes that participate in
the production or response to IFN-. These
patients are classified under the heading
Defects in Killing of Intracellular
Mendelian susceptibility to mycobacte-
Bacteria by Activated Macrophages
rial disease (MSMD) (OMIM 209950). The
Some bacterial species (Mycobacteria, Lis- genetic lesions responsible for MSMD
teria, Salmonella) are resistant to the killing affect the integrity of the IL-12/23-depen-
mechanisms operating within phagocytic dent IFN- pathway.
cells and therefore can survive and multi- Thus, mutations have been identified in
ply within monocytes and macrophages.
Effective immunity against these organ- The P40 subunit shared by IL-12 and
isms depends on T-cell- (and NK-cell-) IL-23 (also called IL12B);
dependent macrophage activation. Studies The chain shared by the IL-12 and
in gene-disrupted mouse and human IL-23 receptors;
immunodeficiencies have identified that The TYK2 kinase required for signaling
IL-12- and IL-23-dependent interferon via the IL-12 receptor;

78 Immunological Aspects of Immunodeficiency Diseases

The and chains of the IFN- receptor As could be predicted, patients with
(IFN- R1 and IFN- 2); IL-12/23 defects or partial IFN-R defects
The STAT-1 signal transducing mol- respond to treatment with IFN- but those
ecule, which is required for signaling with complete IFN- defects are refractory
through the IFN- receptor; to this therapy.
The seventh defect responsible for
increased susceptibility to mycobac-
terial infection is that NFB essential COMPLEX IMMUNODEFICIENCIES
modulator gene, which is required for DUE TO MISCELLANEOUS DEFECTS
NFB activation, which is critical for
Wiskott-Aldrich Syndrome
signal transduction via the Toll, IL-1,
and TNF- receptors. Patients with Wiskott-Aldrich syndrome
typically develop eczema; purpura due to
The severity of the clinical phenotype thrombocytopenia with small-sized, defec-
of MSMD depends on the genotype. Com- tive platelets; and a variable immunode-
plete IFN-R1 or R2 deficiencies lead to ficiency. Antibody production to bacte-
the abrogation of responses to IFN-. Such rial capsular polysaccharides is deficient.
patients present in early childhood with Patients therefore commonly develop
disseminated NTM or BCG infections, recurrent sinopulmonary infections. T-
resulting in a high mortality, despite cell and NK-cell function is deficient and
chemotherapy. Mycobacterial lesions in progressive T lymphopenia develops with
such patients are multibacillary and asso- time. Hence, patients can develop opportu-
ciated with poor granuloma formation. nistic infections. Risk of malignancy (espe-
In contrast, partial IFN- R1 deficiency, cially leukemias or EBV-induced lympho-
complete IL-12B deficiency, and IL-12/23 mas) is increased in these patients.
receptor deficiency predispose to myco- The defective gene, which is on the X
bacterial infections, presenting at a later chromosome, encodes for the Wiskott-
age and with a favorable outcome, fol- Aldrich syndrome protein, which regu-
lowing chemotherapy. Lesions in these lates actin polymerization, and therefore
latter patients are paucibacillary and are cytoskeletal change is required for normal
associated with an intact granulomatous platelet and lymphocyte function.
response. Patients with partial or com-
plete STAT-1 deficiency, which impairs
DNA Repair Defects Associated with
the signaling via the IFN- R results in
susceptibility to mycobacteria of low-
grade virulence. Complete STAT-1 defi- ATAXIA TELANGIECTASIA
ciency also impairs signaling via type I Ataxia telangiectasia (AT) is a condition
interferon receptors, which predisposes characterized by cerebellar ataxia, oculo-
to life-threatening herpes viral infections cutaneous telangiectasia, growth retarda-
occurring in childhood. The commonest tion, and a variable immunodeficiency.
infections that affect patients with defects AT patients show increased sensitivity to
in the IL-12/23 are extraintestinal or sep- ionized radiation and radiomimetic drugs.
ticemic, relapsing infections with non- Increased susceptibility to leukemias
typhoidal Salmonella species. and lymphomas is also a feature of this

Immunological Aspects of Immunodeficiency Diseases 79

syndrome. Patients often fail to produce tion of apoptotic pathways. Defects in criti-
antibodies to bacterial capsular polysaccha- cal components of these pathways result
rides and therefore develop sinopulmonary in susceptibility to hemophagocytic lym-
infections. Chromosomal translocations, phohistiocytosis (HLH), which is usually
involving immunoglobulin heavy-chain triggered by an intercurrent viral infection
and TCR loci, are often detected in the T caused by viruses such as EBV or cytomeg-
cells of AT patients. alovirus. HLH is characterized by massive
The function of the protein encoded by infiltration of organs, such as the liver,
the affected gene (called ataxia telangiecta- spleen, bone marrow, and central nervous
sia mutated, or ATM) is to detect double- system, by activated CD8+ lymphocytes
strand breaks in DNA, initiating their repair. and macrophages, as well as a massive
Mutated ATM results in defective control of overproduction of IFN- and TNF-. Severe
the cell cycle. This explains the radiation sen- pancytopenia is typical of this syndrome
sitivity, abnormal immune cell development and is caused in part by phagocytosis of
and function, and the cytogenetic abnor- blood cells by activated macrophages and
malities seen in AT. in part is secondary to the infiltration of
bone marrow by activated macrophages
IMMUNODEFICIENCY A number of genetic defects that affect
Nijmegen breakage syndrome, which the efficiency of T-cell- and NK-cell-medi-
is phenotypically similar to AT, is due to ated cytolysis can predispose to the devel-
a mutation of the NBS1 gene that encodes opment of HLH. Cytolysis by T cells and
a protein that acts as a substrate for ATM. NK cells is initiated by the secretion of
DNA-ligase I defect, which also results contents of cytolytic granules at the immu-
in defective DNA repair, manifests with nological synapse between the T cells
growth retardation and immunodeficiency. and the target cells. The process involves
The product of the MRE11A gene is another the translocation of perforin-containing
component of the DNA damage sensing lytic granules onto the target cell inter-
machinery. Mutation of the MRE11A gene phase, followed by fusion of these gran-
results in a condition similar to AT. ules with the plasma membrane of the T
cell, with release of perforin onto the sur-
face of the target cell. Perforin punches
IMMUNODEFICIENCIES RESULTING holes in the target cell membrane, causing
THE IMMUNE SYSTEM Mutation of the perforin (PRF1) gene,
which encodes for perforin, is one of the
Defects in the Cytolytic Pathway genetic defects that predisposes to famil-
The homeostasis of immune responses ial HLH. Perforin-supported cytolysis
requires prevention of excessive lympho- (by CD-8 cells and NK cells) may damp
cyte activation (Table 5.9). The mecha- down immune responses triggered by
nism by which such regulation occurs viral infections by aiding the elimination of
includes activation-induced cell death of antigen-presenting cells or by promoting
T lymphocytes, which requires the activa- activation-induced death of T cells.

80 Immunological Aspects of Immunodeficiency Diseases

Table 5.9 Disorders of Homeostasis of Immune Function

Syndromes with
Autoimmunity Gene Defect/Pathogenesis Chief Clinical Findings

Autoimmune Defects of components in Lymphadenopathy,

lymphoproliferative the apoptosis pathway in hepatosplenomegaly,
syndrome (ALPS) lymphocytes: mutations in genes hypergammaglobulinemia, deficient
encoding CD95(TNFRSF6), CD95 lymphocyte apoptosis, autoimmune
ligand (TNFSF6), caspase 10, diseases, increased CD4, CD8,
caspase 8, or NRAS T cells
Autoimmune Mutation in autoimmune Multiple endocrine autoimmunity;
polyendocrinopathy, regulator gene (AIRE) encoding chronic mucocutaneous candidiasis
candidiasis, protein required for expression
ectodermal dysplasia of ectopic antigens in the thymic
syndrome epithelial cells. This is required
for induction of tolerance to
Immune Mutation of FOXP3 gene whose Childhood onset autoimmune
dysregulation, product is expressed by and endocrinopathy, enteropathy,
polyendocrinopathy, is required for function of eczema
enteropathy, X-linked T-regulatory cells; also mutations
(IPEX) in the IL2R gene can cause a
similar syndrome.
Defects in cytolytic pathways
Familial Defective T-cell- and NK-cell- Viral infection triggers
hemophagocytic mediated cytotoxicity due to hemophagocytosis
lymphohistiocytosis mutations in genes encoding
(HLH) perforin (PRF1), or MUNC protein
(MUNK13-4) needed for fusion of
intracellular vesicles.
X-linked SAP or XIAP (see text for Clinical manifestations precipitated
lymphoproliferative explanation of function) by EBV infection: hepatitis,
disorder (XLP) hemophagocytosis, aplastic
anemia, hypogammaglobulinemia,
Chediak-Higashi Mutation in LYST gene. Impaired Partial albinism, recurrent pyogenic
syndrome lysosomal function and defect infections, eventually 85% develop
in sorting cytolytic proteins into accelerated phase with syndrome
secretory granules; poor NK-cell- resembling HLH
and T-cell-mediated cytolysis.
Griscelli syndrome Deficiency of the RAB27A Partial albinism, recurrent pyogenic
(type II) GTPase required for secretory infections, eventually 85% develop
vesicle function. Exocytosis accelerated phase as in Chediak
of cytolytic granules deficient Higashi syndrome.
leading to poor NK-cell- and
T-cell-mediated cytolysis.
Disorders of homeostasis of inflammation (autoinflammatory syndromes)
Syndrome Gene defect/pathogenesis Chief clinical findings
Familial MEFV: leads to deficiency of Periodic fever, amyloidosis
Mediterranean fever pyrin


Immunological Aspects of Immunodeficiency Diseases 81

Table 5.9 (continued)

Syndromes with
Autoimmunity Gene Defect/Pathogenesis Chief Clinical Findings

Hyper-IgD syndrome Partial mevalonate kinase Periodic fever

deficiency; mechanism of
disease uncertain
TNF-receptor- TNFRSF1: results in decreased Periodic fever, amyloidosis
associated periodic availability of soluble TNF
fever receptor for mopping up TNF
Familial cold CIAS1: defect in cryopyrin Cold-induced urticaria, fever
autoinflammatory required for leucocyte apoptosis,
syndrome NFB signaling and IL-1
Neonatal-onset CIAS1: as above Neonatal-onset rash, fever, chronic
multisystem meningitis, arthropathy
inflammatory disease
Muckle-Wells CIAS1: as above Urticaria, deafness, amyloidosis

Studies of rare immunodeficiency syn- sorting of cytolytic proteins into secretory

dromes, all of which are characterized by granules. This is the underlying defect
an increased tendency to develop HLH in the condition called Chediak-Higashi
have identified a number of components syndrome, which is another condition
required for the normal expression of characterized by susceptibility to HLH.
cytolytic capacity by T cells and NK cells.
The intracellular migration and docking
Immunodeficiencies Characterized
of lytic granules requires the function of
by Increased Susceptibility to EBV
the small Rab GTPase, RAB27, which is
mutated in the Griscelli syndrome char-
acterized by partial albinism, immunode- XLP is a rare inherited immunodeficiency
ficiency, and susceptibility to developing characterized by life-threatening patho-
HLH. Defective cytolytic granule exocy- logical processes triggered by EBV infec-
tosis is also characteristic of patients with tions. The manifestations of this condition
mutations of the gene encoding the protein include fulminant infectious mononu-
MUNK13-4 (UNC13D). The tSNARE syn- cleosis, virally induced HLH, hypogam-
texin 11, which is present in the trans-Golgi maglobulinemia, and the propensity to
network, is also involved in intracellular develop malignant lymphomas. Mutation
vesicle trafficking. Mutation of these two in the gene SH2D1A encoding the signaling
genes is responsible for a further form of lymphocyte activation molecule (SLAM)
familial HLH. associated protein (SAP) are responsible
Lysosomal trafficking regulator is defi- for more than 60 percent of cases of XLP.
cient in individuals with a mutation in SH2D1A / mice lack NK T cells and this
the CHS1 gene. This causes a defect in defect could be corrected by reconstitution

82 Immunological Aspects of Immunodeficiency Diseases

of SH2D1A expression within bone mar- IMMUNE DYSREGULATION
row cells derived from SH2D1A / POLYENDOCRINOPATHY AND
mice. Patients with XLP also lack NK T ENTEROPATHY, X-LINKED SYNDROME
cells. Therefore, it is possible that NK T- The product of the FOXP3 gene (belong-
cell function is essential for controlling ing to the forkhead family) is required for
EBV infection as well as homeostasis of the generation and the function of CD4+
T-cell responses to this virus. Further CD25+ T-regulatory cells. Mice that are
support for this theory comes from the FOXP3 / lack these cells and develop a
recent observation that XLP can also be syndrome characterized by lymphoprolif-
caused by a mutation in the gene (BIRC4) eration and autoimmunity. Expression of
that encodes for the protein X-linked ectopic FOXP3 confers suppressive func-
inhibitor of apoptosis (XIAP). XIAP/ tion on CD4+ and CD25+ T cells. Human
patients also have reduced numbers of males with mutations of the FOXP3
NK T cells. gene (which is located on the X chromo-
The pathological manifestations of some) develop a syndrome characterized
the XLP cannot be completely attributed by neonatal endocrinopathies (including
to the reduced NK- and T-cell function type I diabetes) enteropathy, eczema,
seen in this condition. The SAP, which immune thrombocytopenia, and cachexia.
acts as a signaling adaptor molecule In these patients, pancreatic islet cell and
within lymphoid cells, and XIAP, which intestinal mucosal damage are secondary
regulates apoptosis, help in the homeo- to infiltration of the tissues with mononu-
stasis of immune responses in complex clear cells and plasma cells, signifying an
ways. Therefore, mechanisms underlying autoimmune pathogenesis. This condition
the pathogenesis of XLP are likely to be is called the immune dysregulation poly-
complicated. endocrinopathy and enteropathy, X-linked
(IPEX) syndrome. This condition provides
Immunodeficiencies Characterized evidence supporting current concepts on
by Increased Liability to Develop the role of T-regulatory cells in preventing
Autoimmunity autoimmunity.
Recently, mutations in the IL2R gene
Immune responses to self-antigens are pre-
encoding for the chain of the IL-2
vented by:
receptor have been described in patients
with the clinical phenotype of the IPEX
1. The elimination of self-reactive B and
syndrome. CD4 T cells of these patients
T cells in the thymus and bone marrow
fail to produce the immunosuppressive
(central tolerance).
cytokine IL-10.
2. Inhibition of self-reactive T and B cells
that escape central tolerance induc- AUTOIMMUNE LYMPHOPROLIFERATIVE
tion, by the action of regulatory T SYNDROME
cells found in the periphery. These T- During lymphocyte development,
regulatory cells, which are CD25+ and autoreactive cells are culled by undergo-
CD4+, are required for peripheral tol- ing apoptotic death. During the life span
erance and the active suppression of of mature lymphocytes, activation and
autoimmunity. effector function is followed by activation-

Immunological Aspects of Immunodeficiency Diseases 83

induced cell death by apoptosis. Apop-
Autoinflammatory Syndromes
tosis thus maintains homeostasis in the
immune system by minimizing autoim- The responses of acute inflammation and
mune reactions to self-antigens, as well fever are protective responses triggered by
as limiting the total size of the peripheral infection or tissue damage, acting through
lymphocyte pool. Antigen-mediated acti- the innate immune system. The study of
vation of T cells induces them to express a collection of disorders termed familial
a surface receptor FAS (CD95) as well periodic fever syndromes has contributed to
as its ligand, the FAS-ligand. FAS/FAS- our understanding of the physiological
ligand interaction activates a biochemi- processes underlying the homeostasis of
cal pathway culminating in cell death by inflammation and fever. These inherited
apoptosis. Mice that are FAS / (lpr) or disorders are characterized by recurrent
FAS-ligand / (gld) develop autoimmu- episodes of fever and systemic inflamma-
nity and develop expansion of their their tion. These conditions are not associated
lymphocyte pool. with autoantibody production or
ALPS is a rare disease caused by self-reactive T-cell responses. They are col-
defective cellular apoptosis. Failure lectively called autoinflammatory syndromes.
of lymphocytes to undergo apoptosis Unraveling the pathophysiology of these
results in impaired homeostasis of the disorders has given rise to the concept
lymphoid population. Patients develop that these conditions are due to an innate
lymphocytosis, hyperplasia of lymphoid immune system that is either oversensitive
organs (spleen, lymph nodes), hyper- and prone to activation by minor stimuli or
gammaglobulinemia, and autoimmunity is poorly regulated.
(autoimmune cytopenias, Guillain-Barr The cleavage of pro-IL-1b to its active
syndrome). Peripheral blood and periph- product IL-1 by the action of caspase-1
eral lymphoid tissues characteristically (interleukin converting-enzyme) is a key
contain an increased population of TCR event in the generation of an acute inflam-
positive CD4 and CD8 cells (dou- matory response. Activated caspase-1,
ble negative T cells). Mutations in the intern, is generated from pro-caspase-1 by
FAS-mediated pathway of apoptosis are the action of activated forms of two cyto-
responsible for most cases. These include solic proteins called pyrin and cryopyrin,
mutations of FAS, FAS-ligand, and cas- acting via adaptor proteins. The best-char-
pase 10. Caspase 8 defi ciency causes acterized adaptor protein is called ASC
impaired CD95-mediated apoptosis as (apoptosis-associated speclike protein
well as a defect in T-, B-, and NK-cell with a caspase recruitment domain). As
function, manifesting as an immunode- summarized in Table 5.9, mutations of
ficiency. Recently, an activating muta- components of this system (also called
tion in the NRAS gene encoding a GTP- an inflammasome) give rise to most auto-
binding protein with a broad spectrum inflammatory syndromes characterized
of signaling functions has been found in to date. The net effect of all these muta-
other patients with ALPS. Defective, IL-2 tions is to impair homoeostasis of the pro-
withdrawal induced, apoptosis of lym- inflammatory cytokine IL-1, NFB acti-
phocytes is a characteristic abnormality vation, and cellular apoptosis. Impaired
in these patients. cellular apoptosis leads to the persistence

84 Immunological Aspects of Immunodeficiency Diseases

of activated leucocytes that would other- deficiency may also be associated with the
wise undergo apoptosis, leading to the risk of pneumococcal sepsis.
resolution of inflammation. Collectively, Protection from Neisserial infection
these abnormalities permit the inappro- requires the ability to generate the MAC,
priate amplification and persistence of which lyses these bacteria. Patients with
inflammatory responses leading to the inherited homozygous deficiency of C5,
clinical features of the autoinflammatory C6, C7, C8, and C9 are susceptible to recur-
syndromes. rent meningococcal infections. In Japanese
populations, an inherited terminal comple-
ment component deficiency leads to about
INHERITED DEFICIENCY OF THE a 5,000-fold increase in the risk of Neisse-
COMPLEMENT SYSTEM rial sepsis. Primary or secondary CD3 defi-
ciency, which in turn reduces the ability
Activation of the complement system to generate MAC, also results in increased
underlies one of the main effector pathways susceptibility to meningococcal infection.
contributing to antibody-mediated immu- Complement deficiency is not a risk factor
nity. The normal function of the comple- in sporadic cases of meningococcal disease
ment system includes defending the body seen in the population at large.
against pyogenic bacterial infection and MBL is a serum collectin that can bind
helping to eliminate immune complexes to mannose residues on microbial cell
and damaged cells (Table 5.10). walls. Upon binding of MBL to pathogens,
The key event in complement activa- two serine proteases found in serum (MBL-
tion is the proteolytic cleavage of C3 to C3a lectin associated serine proteases 1 and 2)
and C3b. Three pathways can lead to C3 become activated. These serine proteases
cleavage, namely, classical, alternative, and in turn activate the classical pathway at C2
mannose-binding lectin (MBL) pathways. and C4. MBL deficiency may arise because
C3 cleavage leads on to the activation of of one of three point mutations in the gene
the terminal complement pathway, caus- encoding for this protein. Polymorphisms
ing the generation of the membrane attack in the promoter region of the gene also
complex (MAC), which assembles a lipo- influence serum levels of MBL. Low serum
philic complex capable of lysing plasma levels of MBL may result in an increased
membranes of susceptible cells. incidence of pyogenic infections in young
The inability to generate sufficient children below two years of age. MBL defi-
CD3b results in increased susceptibility ciency is also overrepresented in patients
to pyogenic sepsis, especially infections with pneumococcal or meningococcal
caused by encapsulated bacteria. C3 defi- sepsis. However, the clinical significance
ciency may be due to complement utiliza- of this finding has been disputed. This is
tion (e.g., in systemic lupus erythematosus because large, long-term prospective stud-
[SLE] or the presence of a C3 nephritic fac- ies of adults in Scandinavia have failed to
tor) or rarely autosomal recessive C3 defi- identify increased morbidity due to infec-
ciency. Factor I deficiency also leads to pro- tious diseases in MBL-deficient individuals.
found CD3 deficiency due to uncontrolled Therefore, except in young children who
progression of C3 cleavage. Hereditary C2 have not yet acquired a wide repertoire of
deficiency and less commonly inherited C4 protective antibodies, MBL deficiency on

Immunological Aspects of Immunodeficiency Diseases 85

Table 5.10 Complement Deficiency

Deficient Component Physiological Consequence Clinical Manifestations

C3 deficiency; factor I Inability to generate C3b and Recurrent pyogenic sepsis

deficiency; secondary C3 therefore failure to generate (especially by encapsulated
deficiency due to utilization membrane attack complex bacteria)
(e.g., SLE; C3 nephritic
Deficiency of C1q, C1r, Failure to generate classical SLE
C1s, C4, or C2 pathway C3-convertase;
defective scavenging of
apoptotic cells and immune
Deficiency of properdin or Failure to generate alternative Recurrent encapsulated
factor D pathway C3-convertase; failure bacterial sepsis
to opsonize encapsulated
bacteria when anticapsular
antibody is limiting
Deficiency of factor H or Failure to regulate C3 activation Hemolytic uremic syndrome,
factor I membrano proliferative
C1 inhibitor deficiency Failure to regulate C1 activation Angioedema
and kinin generation
CD59 Failure to protect cell surfaces Hemolysis
from endogenous C3b

SLE, systemic lupus erythematosus

its own does not appear to be a significant process described earlier for eliminating
risk factor for bacterial sepsis. immune complexes and safe disposal
of apoptotic cells. This would explain the
increased incidence of SLE-like disorders
Complement Deficiency
in patients with inherited C1q, C1r, C1s,
and Autoimmunity
C4, C2, C3, factor I, or factor H deficiency.
Under physiological conditions, activation
of the classical complement pathway helps
Factor H Deficiency
in the clearance of the circulating immune
complexes by the resident macrophages of Complete or partial factor H deficiency
the reticuloendothelial system. The surface is associated with the occurrence of the
of apoptotic cells activates the classical hemolytic-uremic syndrome, although
complement pathway, leading to their effi- the precise underlying pathogenic mecha-
cient clearance by phagocytic cells express- nisms are unknown.
ing complement receptors, preventing the
generation of autoimmune responses to
Cell-Surface-Based Inactivators
cellular components.
of Complement
Deficiency of components required
for generating the classical partly C3-con- CD59 and CD55 are cell-surface molecules
vertase may result in impairment of the anchored by glycosylphosphatidylinositol.

86 Immunological Aspects of Immunodeficiency Diseases

These two proteins inactivate any C3 con- mutation prevents transcription of the
vertase molecules deposited on cell sur- defective gene. In 15 percent of affected
faces. Somatic mutation of the enzyme individuals, the gene mutation abolishes
(PIGA: phosphatidylinositol glycan the activity of the secreted protein. Rarely,
class A), needed to generate phosphatidyl- autoantibodies to C1 inhibitor can lead to
inositol anchors for cell-surface proteins, acquired C1-inhibitor deficiency.
including CD55 and CD59, in erythroid
precursors, results in a condition called
paroxysmal nocturnal hemoglobinuria, DEFECTS IN INNATE IMMUNITY
which is due to the increased susceptibil-
ity of red cells to complement-mediated Defects in the complement pathway
hemolysis. Isolated CD59 deficiency also resulting in immunodeficiency are sum-
results in hemolytic anemia. marized in the previous section. Recent
work has identified defects in pathways
involved in the recognition and response
C1 Inhibitor Deficiency
to pathogen-associated molecular patterns.
C1 inhibitor is a serine-protease inhibitor Some of these defects are outlined next.
that inactivates the serine esterases gener- Defective NFB activation caused by
ated by complement activation (C1r and X-linked hypomorphic mutations of the
C1s), kallikrein of the kinin system, and essential modulator gene (NEMO) compro-
activated factors XI and XII of the clotting mises signaling mechanisms downstream
cascade. In the absence of C1 inhibitor, of Toll, IL-1, and TNF- receptors. These pa-
C1 activation results in the depletion of tients are susceptible to infections caused
the serum C4 level. (This is useful from by a range of microorganisms, including
a diagnostic point of view.) C1-inhibitor mycobacteria, Gram-positive and Gram-
deficiency also results in the inability to negative bacteria, fungi, and viruses.
inactivate bradykinin, resulting from the UNC93B is a protein of the endoplas-
unregulated activity of kallikrein. Produc- mic reticulum involved in Toll-receptor
tion of bradykinin in the tissues results in activation. Mutations in UNC93B impair
increased vascular permeability, manifest- the production of IFN- and IFN- in
ing as attacks of angioedema. Angioedema response to HSV and other viruses.
of the respiratory tract can lead to death Affected patients present with Herpes sim-
from asphyxia. Angioedema of the intes- plex viral encephalitis. Recently, heterozy-
tinal tissues results in recurrent episodes gous dominant-negative mutations in the
of severe abdominal pain due to partial gene encoding toll receptor 3 (TLR3) have
intestinal obstruction, which can mimic an been identified in patients with Herpes sim-
acute abdominal emergency. plex encephalitis. TLR3 is expressed in the
C1-inhibitor deficiency arises from a het- central nervous system where it helps to
erozygous mutation of C1INH gene, which initiate IFN- and IFN- responses to viral
acts in an autosomal dominant manner. duble-stranded DNA.
The single normal gene cannot maintain Interleukin-receptor-associated kinase-
the synthesis of physiologically sufficient 4 mediates signaling downstream of Toll
quantities of C1 inhibitor. In 85 percent receptors and members of the IL-1 receptor
of C1-inhibitor-deficient individuals, the superfamily. IRAK-4-deficient individuals

Immunological Aspects of Immunodeficiency Diseases 87

present during childhood with recurrent, features, delayed shedding of primary
severe, pyogenic sepsis. They are particu- dentition, osteopenia, and impaired acute-
larly susceptible to recurrent pneumococ- phase responses during infections. Most
cal infections. patients have an autosomal dominant
The signal-transducing molecule inheritance while others are sporadic cases.
STAT-1 is required for signaling via recep- Patients with classical HIES have heterozy-
tors to IFN- as well as IFN- and IFN- gous mutations in the gene encoding the
. IFN--receptor-mediated signaling signal-tranducing protein STAT-3. These
involves the dimerization of phosphory- mutations mainly involve the DNA-bind-
lated STAT-1 molecules. Signaling via ing domain or the SRC homology 2 domain
IFN- and IFN- receptors involves the of the protein and are permissive of protein
formation of a complex between STAT-1, expression. These mutant proteins severely
STAT-2, and a third protein called inter- impair the DNA binding of the phosphory-
feron-stimulated-gene factor 3-. Com- lated STAT-3 dimer in response to IFN-
plete (homozygous) defects of the signal- and responses to the cytokines IL-10 and
transducing molecules STAT-1 results in IL-6. Reduced response to IL-6 would
defective responses to IFN-, IFN-, and explain the defective acute-phase response,
IFN- eater leading to the susceptibility to and the defective response to IL-10 explains
disseminated mycobacterial infections as the overproduction of IgE. STAT-3 is essen-
well as fatal Herpes simplex viral infection. tial for the generation of TH17 cells and for
Partial STAT-1 deficiency, which interferes IL-12 signaling, which are required for the
with STAT-1 dimerization required for sig- secretion of the bactericidal peptides called
nal transduction via IFN- receptors, leads -defensins by epithelial cells of the skin
to increased susceptibility to mycobacte- and lungs. This may in part explain the
rial infections. In these patients, the cellu- increased incidence of severe sepsis.
lar responses to IFN- and INF- is intact,
thus preserving antiviral immunity.
The WHIM syndrome is a condition CONCLUSIONS
characterized by severe warts, hypogam-
maglobulinemia, and neutropenia. This is Studies in genetically manipulated animals
the first example of an immunodeficiency have helped in the development of mecha-
caused by aberrant chemokine-recep- nistic models of antimicrobial immunity.
tor function. WHIM syndrome is caused Gene knockout mice have been invaluable
by a mutation in the gene encoding the for the elucidation of the phenotypic conse-
CXCR 4 chemokine receptor. The mutant quences of single-gene defects. Collectively,
form of this receptor shows enhanced these animal studies have highlighted can-
responsiveness to its ligand. didate genes that should be investigated
The hyper-IgE syndrome (HIES) is a in patients with increased susceptibility
complex clinical entity characterized by to microbial infection. Comparison of the
recurrent bacterial (S. aureus, Gram-nega- phenotype of a mouse carrying a defined
tive bacteria) and fungal infections of skin, genetic defect with the clinical and immu-
lymph nodes, lungs, bones, and joints. These nological phenotype of patients has been
patients have elevated serum IgE levels, helpful in identifying candidate genes that
eosinophilia, dermatitis, facial dysmorphic might be affected in the human patient.

88 Immunological Aspects of Immunodeficiency Diseases

Studies in humans with PID have been deficiency), bone marrow transplantation
complementary. For example, human stud- (for SCID), and gene therapy (for the treat-
ies have highlighted that (1) defects in more ment of X-linked SCID and SCID due to
than one gene can give rise to a similar pri- ADA deficiency). In X-linked SCID and
mary immunodeficiency syndrome; (2) the in ADA-deficiency gene, corrected bone
same genetic mutation can give rise to a marrow cells have a selective advantage
variable phenotype, depending on the mod- over unmodified stem cells, contributing
ulating effect of genetic and environmental to successful engraftment by gene-recon-
influences; and (3) defects in components stituted cells. The correction of genetic
of innate and adaptive immunity that are defects in conditions where the expression
required for homeostasis of the immune of the normal molecule does not provide a
response may result in autoimmunity or selective survival advantage will be more
autoinflammatory syndromes rather than difficult and will require the development
increased susceptibility to infection, thus of more effective genetic vectors.The elu-
extending the clinical spectrum of primary cidation of molecular defects underlying
immunodeficiency diseases. PIDs helps in the development of better
In the past decade, we have realized methods of diagnosing these disorders and
that defects in some components of the genetic counseling of affected families. The
innate and adaptive immunity may result contribution of new genetic techniques for
in susceptibility to a narrow range of micro- elucidating molecular defects underly-
bial pathogens. This indicates that some ing PIDs have been described elsewhere
immune mechanisms have evolved to deal (see references list at end of chapter).
with specific pathogens but are redundant
for immunity against other microorgan-
isms. Such knowledge may lead to the
development of novel treatment strategies COMPREHENSIVE REVIEWS (PRIMARY
targeted against specific microbes. IMMUNODEFICIENCY DISEASES)
In this review, we have highlighted Primary immunodeficiency diseases.
how defects in individual components of Report of an IUIS Scientific Committee.
the immune system can lead to the sus- International Union of Immunological
ceptibility to different categories of patho- Societies. Clin Exp Immunol. 1999;118
gens. Such analyses help us to understand (suppl 1):128.
which components of immunity are non- Fischer A. Human primary immunode-
redundant for protection against different ficiency diseases: a perspective. Nat
microorganisms. This knowledge also helps Immunol. 2004;5(1):2330.
us to be guided by the pattern of micro- Geha, RS, Notarangelo, LD, et al. Pri-
bial infections in an individual patient in mary immunodeficiency diseases: an
devising a rational approach to the inves- update from the International Union
tigation of patients with suspected immu- of Immunological Societies Primary
nodeficiency. Immunodeficiency Diseases Classifica-
The past two decades have seen the tion Committee. J Allergy Clin Immunol.
development of novel treatment modali- 2007;120(4):776794.
ties for PIDs. These include the use of Ochs, HD, Smith, CIE, et al. Primary Immu-
Ig replacement therapy (for antibody nodeficiency Diseases: A Molecular and

Immunological Aspects of Immunodeficiency Diseases 89

Genetic Approach. New York: Oxford Novartis Found Symp. 2006;279:9298;
University Press; 2007. discussion 98100, 216219.
Smith TF, Johnston, RB Jr. Functions of
SELECTED REVIEWS (MOLECULAR the spleen in host defense against
MECHANISMS UNDERLYING PRIMARY infection. Am J Pediatr Hematol Oncol.
IMMUNODEFICIENCIES) 1979;1(4):355362.
Brydges S, Kastner DL. The systemic auto-
inflammatory diseases: inborn errors REFERENCES (DIAGNOSIS AND MANAGEMENT
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Microbiol Immunol. 2006;305:127160. Bonilla FA, Bernstein IL, et al. Practice
Doffinger R, Patel SY, Kumararatne, DS. parameter for the diagnosis and man-
Host genetic factors and mycobacte- agement of primary immunodeficiency.
rial infections: lessons from single Ann Allergy Asthma Immunol. 2005;94(5
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adaptive immunity. Microbes Infect. Cavazzana-Calvo M, Fischer, A. Gene
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A. Pathophysiology of B-cell intrinsic Invest. 2007;117(6):14561465.
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Fischer A, Le Deist F, Hacein-Bey-Abina designed for non-immunologists. Clin
S, et al. Severe combined immunode- Exp Immunol. 2006;145(2):204214.
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90 Immunological Aspects of Immunodeficiency Diseases

6. Autoimmunity

Haoyang Zhuang, Ph.D., Matthew Kosboth, M.D., Jennifer A. Sipos, M.D., Minoru
Satoh, M.D., Ph.D., Lijun Yang, M.D., and Westley H. Reeves, M.D.

DEFINITIONS AND TYPES specific T lymphocytes. Alternatively,

OF AUTOIMMUNITY autoimmune disease may be caused by
B cells that produce autoantibodies, as in
Autoimmunity versus Autoimmune the case of systemic lupus erythematosus
Disease (SLE). Autoantibodies bind to self-anti-
The classic studies of Paul Ehrlich in the gens (proteins, nucleic acids, or other mol-
early twentieth century laid the founda- ecules from ones own body, also known
tion for our current notions of the concept as autoantigens) and can damage cells
of autoimmunity. Ehrlich used the term either by binding directly to a cell surface
autoimmunity to signify an immune re- or extracellular matrix antigen or through
sponse against self and introduced the the formation of immune complexes
phrase horror autotoxicus, suggesting that (see the section Mechanisms of Auto-
there are mechanisms to protect against immune Tissue Injury and Examples).
autoimmunity. Over the years, autoim- Autoantibody-mediated auto immune
munity has been recognized as not uncom- diseases sometimes can be transmitted
mon and not necessarily detrimental. Thus, transplacentally, as in the case of neona-
an important distinction must be drawn tal Graves disease or congenital com-
between autoimmunity, which may be plete heart block and neonatal lupus. IgG
asymptomatic, and autoimmune disease, antibodies/autoantibodies can cross the
which occurs when autoimmunity leads to placenta, whereas IgM cannot. Thus, neo-
an inflammatory response, resulting in tis- natal autoimmune diseases are invariably
sue injury. An autoimmune response does caused by IgG, not IgM, autoantibodies.
not necessarily imply the existence of auto- In view of the half-life of IgG (twenty-
immune disease. one to twenty-eight days), nearly all
maternal IgG disappears from the circula-
tion of the baby by six to twelve months
postpartum. Thus, in most cases, neona-
T-Cell versus B-Cell-Mediated
tal autoimmune disease is transient. One
Autoimmune Disease
exception is congenital complete heart
Autoimmune disease may be mediated block, which is thought to be mediated
primarily by T cells, as in multiple sclero- by the transplacental passage of anti-Ro
sis or the animal model experimental auto- or anti-La autoantibodies that cross-react
immune encephalomyelitis (EAE). In that with cardiac antigens, causing permanent
case, disease can be transmitted from one inflammation-mediated damage to the
animal to another by transferring antigen- cardiac conduction system.

Examples include autoimmune hemolytic
Systemic versus Organ-Specific
anemia (AIHA), and autoimmune throm-
Autoimmune Disease
bocytopenia (Table 6.1). Some autoanti-
Autoimmune disease also can be classi- bodies bind to surface receptors, either
fied as systemic or organ specific. Systemic activating (e.g., anti-TSH receptor auto-
autoimmune diseases, such as SLE, involve antibodies in Graves disease) or inhibit-
multiple organs or tissues, whereas organ- ing (e.g., anti-acetylcholine antibodies in
specific autoimmune diseases involve a myasthenia gravis) their function.
single organ or tissue, such as the thy-
roid gland in autoimmune thyroiditis or
Type IIA Autoimmune Reaction:
the islets of Langerhans in type I diabetes
Autoimmune Hemolytic Anemia
(TID). Some of the more common systemic
and organ-specific autoimmune diseases AIHA is an example of type IIA autoim-
are listed in Table 6.1. munity. In this disorder, a self-antigen
on the surface of erythrocytes elicits an
autoantibody response, resulting in the
MECHANISMS OF AUTOIMMUNE binding of autoantibody to the erythro-
TISSUE INJURY AND EXAMPLES cyte surface followed by destruction of
the antibody-coated erythrocytes by the
Tissue damage in autoimmune diseases reticuloendothelial system of the spleen
can occur through several mechanisms, and liver. The mechanism of hemolysis
which are analogous to three of the classical depends on the type of autoantibodies.
types of hypersensitivity reactions: type II Autoimmune hemolysis is classified into
(caused by autoantibodies reactive with two groups on the basis of thermal reactiv-
cell surface or matrix antigens), type III ity of the autoantibodies. Warm autoanti-
(caused by immune complexes), and bodies react optimally at temperatures of
type IV (delayed-type hypersensitivity, 35C40C, whereas cold agglutinins and
mediated by T cells). other cold-reactive autoantibodies react
maximally at 4C. Warm autoantibod-
ies are typically polyclonal IgG but may
Type II Autoimmune Reactions
also be IgM or IgA. Most are IgG1 sub-
Type II hypersensitivity reactions are class antibodies reactive with Rh antigens.
caused by antibodies against altered self- These antibodies are detected by the direct
proteins, such as penicillinprotein con- antiglobulin (Coombs) test (Figure 6.1A).
jugates. In the case of autoimmunity, Erythrophagocytosis mediated by Fc
antibodies generated against cell surface receptors on Kupffer cells in the liver and
antigens/extracellular matrix proteins may macrophages in the splenic marginal zone
be cytotoxic (type IIA) or they may have is generally the major mechanism of eryth-
agonistic/antagonistic properties (type rocyte destruction in patients with warm
IIB). Autoantibodies to cell surface anti- autoantibodies.
gens may initiate cell destruction by com- In contrast, AIHA induced by cold
plement-mediated lysis (cell destruction), agglutinins is complement mediated.
phagocytosis, or antibody-dependent These autoantibodies are of the IgM class
cell-mediated cytotoxicity (ADCC). and cannot interact with Fc receptors

92 Autoimmunity
Table 6.1 Some Human Autoimmune Diseases

Organ(s) Prevalence Female:

Disease Involved per 100,000a Male Ratio Autoantibodies

Systemic autoimmune diseases

Systemic lupus Joints, skin, 24 9:1 anti dsDNAb
erythematosus nervous system, anti Sm b
kidneys, blood anti ribosomal P b
cells, heart, lungs anti RNA helicase*
Rheumatoid Joints, blood 860 3:1 anti citrullinated peptides b
arthritis vessels, lungs Rheumatoid factor
Sjgrens Exocrine glands 14 9:1 anti Ro60 (SS-A)
syndrome (salivary and anti Ro52
lacrimal glands), anti La (SS-B)
kidneys, nerves
Scleroderma Skin, blood 4 4:1 anti topoisomerase I b
vessels, GI tract, anti fibrillarin (U3 RNP) b
lungs, kidneys anti RNA polymerase I b
anti RNA polymerase III b
Polymyositis Muscles, lungs 5 2:1 tRNA synthetases
(histidyl, alanyl, threonyl,
glycyl, etc.) b
Signal recognition particle b
Organ-specific autoimmune diseases
Hashimotos Thyroid 982 9:1 Thyroid peroxidase
thyroiditis Thyroglobulin

Graves disease Thyroid 1152 9:1 Thyroid-stimulating

hormone receptor
Addisons disease Adrenal glands 5 9:1 2I-hydroxylase
Type I diabetes Pancreatic islet 192 1:1 Glutamic acid
cells dehydrogenase, insulin,
other islet cell antigens
Pemphigus Skin N/A N/A Desmoglein 3
Bullous Skin N/A N/A 230 kDa
pemphigoid hemidesmosomal antigen
Vitiligo Skin melanocytes 400 1:1 Unknown melanocyte
Goodpastures Kidneys, lungs 0.05 1:1 Type VII collagen
Myasthenia gravis Nervous system 5 2:1 Acetylcholine receptor
Multiple sclerosis Nervous system 58 2:1 Unknown myelin antigens
Pernicious anemia Gastric parietal 151 2:1 Parietal cell antigens,
cells intrinsic factor
Primary biliary Bile ducts N/A N/A Dihydrolipoamide
cirrhosis acyltransferase and other
antigens b
Autoimmune Liver 0.4 9:1 Smooth muscle antigens
hepatitis (F-actin)


Autoimmunity 93
Table 6.1 (continued)

Organ(s) Prevalence Female:

Disease Involved per 100,000a male ratio Autoantibodies

Thrombocytopenic Platelets N/A 3:1 Antiplatelet antibodies

purpura against GPIIbIIIa and/or the
GPIb complex
Autoimmune Erythrocytes N/A ~1.5:1 Rh, I, i, and other antigens
hemolytic anemia

N/A, not available.

a b
USA, 1996 estimate; disease-specific autoantibodies.

because there are no Fc receptors capable to recruitment of the terminal complement

of binding the heavy chain. Idiopathic components (C5C9, membrane attack
cold agglutinin disease generally is asso- complex) and intravascular hemolysis or
ciated with an IgM paraprotein against C3b receptor-mediated phagocytosis by
the I antigen, an erythrocyte surface reticuloendothelial cells.
protein. Unlike IgG, which must be cross-
linked, pentavalent IgM fixes complement Case 1. Autoimmune Hemolytic Anemia,
efficiently without cross-linking. After a Type II Autoimmune Reaction
binding to the erythrocytes surface at low A twenty-eight-year-old woman
temperature, IgM cold agglutinins acti- with a four-year history of SLE pre-
vate C1, C4, C2, and C3b. With rewarm- sented for a scheduled follow-up in
ing, the antibody can dissociate, but C3b clinic. Because she avoids the sun and
remains fixed irreversibly, which can lead started taking hydroxychloroquine
four years ago, her rash and arthritis
Patients red blood cells Patients serum had improved, but over the past six
Y months, she had become progressively
Y more fatigued and began to notice dark
Anti-lgG + urine. Review of medications, alcohol
antibodies ABO and Rh compatible RBCs
intake, recreational drug use, and sick
contacts was unrevealing. On physical
A Direct Coombs Test Indirect Coombs Test exam, she was mildly tachycardic at
105, with a two out of six systolic ejec-
tion murmur at the left sternal border,
dullness to percussion over Traubes
space (the normally resonant gastric
bubble), and a palpable spleen tip.
Her hemoglobin was 9.5 g/dl (nor-
B mal 1216 g/dl), mean cell volume
Figure 6.1 Autoimmune hemolytic anemia. (MCV, a measure of erythrocyte size)
A, Diagram showing the difference between the
was normal at 88 cu m, and platelets
direct and indirect Coombs test. B, Peripheral
blood smear illustrating microspherocytes were 75,000/l (normal 140400,000/
(arrows). l). Urinalysis revealed no blood

94 Autoimmunity
but was remarkable for urobilinogen her anemia, thrombocytopenia, and
of 8 mg/dl (normal <2 mg/dl). Hepatic some of her fatigue resolved. After
panel was notable for a total bilirubin of tapering the prednisone dose, she felt
2 mg/dl (normal <1.5 mg/dl) with indi- normal. Two years later, her symptoms
rect bilirubin of 1.5 mg/dl (normal <0.8 recurred and laboratory tests confirmed
mg/dl) and direct bilirubin 0.5 mg/dl evidence of active hemolytic anemia. A
(normal <0.7 mg/dl). Lactate dehy- liver-spleen scan indicated the presence
drogenase was elevated at 350 IU/L of an accessory spleen (present in 1030
(normal <250 IU/L), and corrected retic- percent of normal population), which
ulocyte count (immature erythrocytes) was removed. She is currently symp-
was 3 percent (normal <1 percent). tom free.
Direct Coombs test was positive
(Figure 6.1A). Haptoglobin (a scaven- COMMENT
ger of free hemoglobin) was reduced AIHA in patients with SLE is usu-
to <5 mmol/L (normal 1030 mmol/ ally due to the presence of warm-reac-
L). Parvovirus B19, thyroid-stimulat- tive autoantibodies against the Rh
ing hormone (TSH), vitamin B12 level, antigen. As the autoantibody-coated
folate level, iron profile, and ferritin erythrocytes pass through the spleen,
were unremarkable. A review of her phagocytes bearing Fc receptors
blood smear showed numerous sphe- remove some of the immunoglobulin
rocytes (spherical erythrocytes instead on the cell surface along with some of
of the usual biconcave disc shape, the the cell membrane, which subsequently
result of damage to the red cell mem- reseals, causing the erythrocyte to take
brane as it passes through the spleen; the form of a spherocyte. Eventually,
Figure 6.1B) and confirmed thrombocy- the erythrocyte is unable to be repaired
topenia (low numbers of platelets). An and is removed from the circulation. If
ultrasound of her abdomen revealed a this occurs faster than new erythrocytes
normal liver but an enlarged spleen. can be produced (normal life span of
On the basis of these clinical find- an erythrocyte is about 120 days) then
ings, the diagnoses of AIHA and anemia develops. The elevated indirect
thrombocytopenia were made. She was bilirubin (a measure of bilirubin before
treated with prednisone (a corticoste- the liver has a chance to process it) is
roid) at a dose of 60 mg/day. Initially, a result of the increased breakdown of
her platelet count improved to 120,000. hemoglobin.
However, after three months of treat- AIHA can occur in a variety of
ment, her anemia did not improve. She circumstances, including neoplas-
gained twenty pounds and noted easy tic diseases (most often lymphomas),
bruising, fatigue, and difficulty sleeping connective tissue diseases (such as
as well as feeling on edge all the time. SLE), and infections (viral, bacterial,
Since she had not improved and was or mycoplasma). Or it may be drug
experiencing side effects of prednisone, induced (classically penicillin). The
she was given a pneumococcal pneu- initial treatment is to diagnose and
monia vaccination before surgery to treat the underlying cause or remove
remove her spleen. After splenectomy, offending agents. If this is not possible,

Autoimmunity 95
corticosteroids such as prednisone are acetylcholine released from a nerve end-
often used. If patients do not respond, ing, transiently opening a calcium channel.
then consideration is given to the use The signal is terminated by acetylcholine
of cytotoxic drugs (e.g., azathioprine or esterase, an enzyme located in the basal
vincristine) or splenectomy. lamina between the nerve ending and the
postsynaptic membrane. As in mothers
with Graves disease, transplacental pas-
sage of IgG autoantibodies from mothers
Type IIB Hypersensitivity: Graves
with myasthenia gravis can cause transient
neonatal myasthenia gravis. Anti-AChR
Graves disease is an organ-specific auto- autoantibodies cause disease by down-
immune disease of the thyroid mediated regulating expression of the receptor and
by stimulatory (agonistic) autoantibod- by complement-mediated lysis of the cells
ies. Autoantibodies to the thyroid-stimu- bearing the AChR. Intermolecular cross-
lating hormone receptor (TSHR) cause linking of AChR by the autoantibodies
hyperthyroidism in patients with Graves may lead to antigenic modulation.
disease. The pathogenicity of anti-TSHR
autoantibodies is demonstrated by the
Type III Autoimmune Reactions
occurrence of neonatal Graves disease
(Immune Complex Disease)
after passive transplacental transfer of IgG
thyroid-stimulating autoantibodies from a Autoantibodies also cause disease by form-
mother with Graves disease to the fetus. ing networks of autoantibodies bound to
The anti-TSHR autoantibodies in Graves their antigens (immune complexes). The
disease inhibit binding of TSH to its recep- antigen-antibody complexes can deposit
tor by binding to a conformational epitope in tissues, causing inflammatory lesions.
(the part of the antigen recognized by an Studies of serum sickness led to the first
antibody) of the extracellular domain of description of an immune complex dis-
the TSHR. Although the autoantibodies ease. Serum sickness is manifested by
appear to interact with TSHR somewhat fever, glomerulonephritis, vasculitis, urti-
differently than the natural ligand, they caria, and arthritis, appearing seven to
nevertheless stimulate TSHR signaling, twenty-one days after primary immuniza-
causing increased production of thyroid tion or two to four days after secondary
hormone. immunization with a foreign protein. Two
consequences of immune complex forma-
tion are complement fixation and binding
Type IIB Hypersensitivity: Myasthenia
to Fc or complement receptors on phago-
cytes. Clearance is facilitated by the bind-
Myasthenia gravis is an autoimmune ing of immune complexes to C3b recep-
disease caused by inhibitory (antagonis- tors (CR1) on erythrocytes, which retain
tic) autoantibodies that bind and block the complexes in the circulation until their
the acetylcholine receptor (AChR), caus- removal by the reticuloendothelial cells of
ing muscular weakness and fatigue. The the spleen or liver.
AChR is found at postsynaptic membranes Immune complex formation is a nor-
of neuromuscular junctions and binds mal process that removes foreign antigens

96 Autoimmunity
from the circulation. Removal of immune be cytoplasmic. Immune complexes con-
complexes by phagocytes bearing Fc or taining these autoantibodies, especially
complement receptors prevents their anti-dsDNA antibodies, are selectively
deposition at other sites. The efficiency of enriched in the renal glomeruli (capillary
uptake of immune complexes by either tufts that produce urine as an ultrafiltrate
Fc receptors or CR1 is proportional to the of blood) of patients with lupus nephritis
number of IgG molecules associated with and are thought to play a critical role in
the complex. establishing the inflammatory response.
Immune complexes can activate either Immune complex deposition in the kidney
the classical or the alternative complement leads to proliferative glomerulonephritis
pathway. The classical pathway plays a and effacement of the normal glomerular
major role in maintaining immune com- architecture (Figure 6.2). As is the case in
plexes in a soluble form, preventing their serum sickness, active lupus nephritis is
deposition in tissues. C3b bound to the sol- frequently associated with hypocomple-
ubilized immune complexes promotes their mentemia (Figure 6.2). In addition to the
clearance by the erythrocyte complement kidneys (glomeruli), immunoglobulin
receptor CR1. If the rate of immune com- and complement deposits are found in the
plex formation exceeds the ability to clear blood vessels (vasculitis), skin (rashes),
these complexes via Fc receptors and CR1, nervous system, and other locations. Pre-
the immune complexes can deposit within formed immune complexes may become
tissues, leading to inflammation. This trapped in the glomerular filter, or immune
efficient immune complex transport and complexes may develop in situ because of
removal by Fc and complement receptors the interaction of cationic antigens (e.g.,
can be overwhelmed, however, leading to histones) with heparan sulfate glycos-
tissue deposition and immune complex aminoglycan in the glomerular basement
disease. This situation may result from membrane. The association of lupus with
overproduction of immune complexes, deficiencies of the early classical comple-
blockade of phagocytosis by the reticulo- ment components, especially C2 and C4,
endothelial system, or complement deple- is consistent with the role of complement
tion resulting in inefficient solubilization of pathways in solubilizing immune com-
immune complexes. plexes (see the section Pathogenesis of
SLE is the prototype of human immune Autoimmune Disease).
complex disease. Tissue damage in lupus
is mainly caused by immune complexes Case 2. Systemic Lupus Erythematosus,
containing autoantibodies to soluble anti- a Type III Autoimmune Reaction
gens. These autoantibodies include anti- A fifteen-year-old girl developed
bodies against RNA-protein complexes myalgias (muscle pain), painful and
(e.g., anti-Sm, RNP, Ro/SS-A, and La/ swollen joints, and low-grade fevers
SS-B antibodies) and DNA-protein com- and was found to have a positive anti-
plexes (e.g., anti-double-stranded DNA, nuclear antibodies (ANA) test. Kidney
antihistone, antichromatin antibodies). function was normal. She was given
The target antigens are found mainly in a diagnosis of SLE and treated with
the cell nucleus, although in some cases hydroxychloroquine (an antimalarial),
(e.g., antiribosomal antibodies), they may azathioprine (a nucleoside analog), and

Autoimmunity 97


MMF 3000 mg
Prednisone 40 mg
Prednisone 40 mg
200 Anti-DNA antibodies 100
Anti-dsDNA (1 titer)

C4 (mg/dL)

100 50

0 0
0 2 4
F Months

Figure 6.2 Systemic lupus erythematosus. A, Acute cutaneous lupus (malar or butterfly rash); B,
fluorescent antinuclear antibody test (HEp-2 cells were stained with patient serum followed by
fluorescein isothiocyanate-labeled goat antihuman IgG antibodies); C, Crithidia luciliae kinetoplast
staining assay for anti-double-stranded DNA autoantibodies (Crithidia organisms were stained with
patient serum followed by fluorescein isothiocyanate-labeled goat anti-human IgG antibodies).
Kinetoplasts are indicated by arrows; D, hematoxylin and eosin staining of a renal glomerulus
illustrating proliferative nephritis and crescent formation; E, direct immunofluorescence of a renal
glomerulus in lupus nephritis stained for IgG; F, classic inverse relationship between anti-dsDNA
antibody titer (broken line) and complement (C3, solid line) levels. Bars above the graph depict the
doses of prednisone and mofetil mycophenolate (MMF) over the same period.

10 mg/day of prednisone (a corticoste- nisone until she moved to another state

roid). Three years later, she developed and was unable to continue her health
alopecia (hair loss) and a red, ulcerat- insurance. Several months after stop-
ing rash of the legs. A skin biopsy was ping all of her medications, vasculitic
reported to be consistent with lupus. skin lesions recurred on the legs, and
The skin lesions resolved when the she developed a rash on the face (Fig-
dose of prednisone was increased. For ure 6.2A). Laboratory testing revealed
the next five years, her lupus remained that her creatinine (a measure of renal
well controlled with hydroxychloro- function) was now abnormally ele-
quine and intermittent low-dose pred- vated at 3.4 mg/dl (normal 1.0 mg/dl),

98 Autoimmunity
her albumin was low, and her urine stopping medications that keep the
tested positive for protein (protein- autoimmune response in check (pred-
uria, >300 mg/dl) and blood (hematu- nisone and hydroxychloroquine), lead-
ria). Microscopic examination revealed ing to the production of high levels of
seven erythrocytes per high-power anti-dsDNA antibodies that could be
field. Fluorescent antinuclear antibody detected by staining the kinetoplast
testing was positive at a titer of 1:640 (a circular DNA molecule) of Crithidia
homogeneous pattern and anti-dsDNA luciliae organisms. These autoantibodies
antibodies were detected at a titer of formed immune complexes, resulting
1:160 using the Crithidia luciliae kineto- in the consumption of classical comple-
plast staining assay (Figure 6.2B, 6.2C). ment components C3 and C4 (the classic
Complement components C3 and C4 inverse relationship between anti-DNA
were low (56 and 11 mg/dl, respec- and complement levels, as illustrated in
tively). She was treated with a high Figure 6.2F). Because the immune com-
dose of methylprednisolone (another plexes were inadequately cleared, they
corticosteroid) intravenously followed deposited in the renal glomeruli, result-
by prednisone. A renal biopsy was per- ing in the patients new onset of hema-
formed and showed proliferative lupus turia and proteinuria and the decline
nephritis (Figure 6.2D). Immunofluo- in her renal function (increased creati-
rescence showed staining of the glo- nine). With reinstitution of appropriate
merular basement membrane for IgG therapy, the anti-DNA levels declined,
(Figure 6.2E) as well as IgM and C3. C3 and C4 levels recovered, and renal
She was treated with mycophenolate immune complex deposition dimin-
mofetil (MMF, 1,500 mg twice a day), ished, resulting in an improvement of
and after four months, her proteinuria renal function.
and hematuria resolved, the creatinine
returned to near baseline (1.1 mg/dL),
Type IV Autoimmune Reactions
C3 increased to 85 mg/dl, and anti-
(T-Cell Mediated)
dsDNA antibodies decreased to 1:20
(Figure 6.2F). Type IV hypersensitivity reactions are
mediated by T cells that recognize pep-
COMMENT tides presented on the surface of antigen-
SLE is the prototype of human presenting cells in the context of class II
immune complex disease. For reasons major histocompatibility complex (MHC)
that are unclear, autoantibodies against molecules and that produce the cytokines
dsDNA are involved in the formation interferon (IFN-), interleukin 3 (IL-3),
of immune complexes that appear to be tumor necrosis factor (TNF) , TNF-, and
particularly prone to become trapped granulocyte-macrophage colony-stimulat-
in the renal glomeruli, where they ing factor (GM-CSF). These cells consti-
can cause inflammation (glomerulo- tute a subset of helper T cells termed TH1
nephritis). The levels of anti-dsDNA cells. Elaboration of TH1 cytokines leads
often are low during periods of dis- to macrophage recruitment and activa-
ease quiescence. In this case, a flare of tion, enhanced expression of adhesion
disease activity was precipitated by molecules, and increased production of

Autoimmunity 99
monocytes by the bone marrow. Delayed- Complete blood count was notable for
type hypersensitivity in response to the mild anemia (hemoglobin 11.3 g/dl).
intradermal injection of certain antigens, Her T4 level was low (1.9 g/dl), TSH
such as tuberculin (used for tuberculosis level was elevated at 25 mIU/L, and
skin testing), is a classic example of a type serum antithyroid peroxidase and anti-
IV hypersensitivity reaction. In the case thyroglobulin autoantibodies were
of autoimmunity, self-antigens (instead detected. Antithyroid-stimulating hor-
of foreign antigens) plus MHC molecules mone receptor antibody was negative.
are recognized by the antigen receptors of She was given a diagnosis of autoim-
the TH1 cells. Examples of type IV autoim- mune (Hashimotos) thyroiditis on the
mune reactions include insulin-dependent basis of the low T4 level, elevated TSH,
diabetes mellitus (pancreatic antigens, and the autoantibody profile and was
such as glutamic acid dehydrogenase, treated with thyroid replacement. Her
insulin, and other islet cell antigens are TSH levels normalized and the ane-
recognized), multiple sclerosis (unidenti- mia resolved and she noted a gradual
fied components of myelin are recognized), decrease in her fatigue. Her skin and
experimental antoimmune encephalomy- hair dryness improved.
elitis (an animal model of multiple sclero-
sis in which myelin basic protein (MBP) is COMMENT
recognized), and Hashimotos thyroiditis Pathologically Hashimotos thy-
(thyroid antigens such as thyroid peroxi- roiditis represents an infiltration of the
dase and thyroglobulin are recognized). thyroid gland with T and B lympho-
cytes, which often organize to form
Case 3. Hashimotos Thyroiditis: A Type IV germinal centers (Figure 6.3D). The
Autoimmune Disease lymphocytic infiltration may be visu-
A thirty-one-year-old woman was alized on positron emission tomog-
seen in the clinic because she had a raphy scanning as shown in Figure
sensation that something was stuck 6.3E. Patients with Hashimotos thy-
in her throat. Her older sister had a roiditis may exhibit a focal or diffusely
similar problem. She also noted feeling increased 2-[18F]fluoro-2-deoxy-d-glu-
tired and had gained weight since giv- cose (FDG) uptake, which correlates
ing birth to a child five years earlier. with the T-/B-cell infiltration. The
Her hair and skin seemed to be get- B cells make antibodies against thy-
ting drier. On examination, her thyroid roid antigens, as seen in this patient,
gland was mildly enlarged on palpa- whereas the T cells produce cytokines
tion (Figure 6.3A, 6.3B) and ultrasound that stimulate the B cells and induce
revealed multiple small nodules and a the thyroid cells to undergo apopto-
pseudonodule indicated by the arrow sis (programmed death). Eventually,
(Figure 6.3C). A needle biopsy of the the thyroid is destroyed and is unable
thyroid revealed a diffuse interstitial to secrete thyroid hormone, result-
lymphocytic infiltrate with formation ing in hypothyroidism. The diffusely
of lymphoid follicles (Figure 6.3D). micronodular appearance on ultra-
Residual thyroid follicles were small, sound (Figure 6.3C) is due to disrup-
and some contained inspissated colloid. tion of the normal microarchitecture of

100 Autoimmunity


Figure 6.3 Autoimmune (Hashimotos) thyroiditis. A, B, Appearance of goiter

(diffusely enlarged thyroid gland); C, ultrasound image showing a transverse
view of the right lobe of the thyroid. The gland is diffusely hypoechoic
with multiple small nodules and a pseudonodule. The arrow indicates a
pseudonodule (arrow) separated from the remainder of the gland by a fibrous
septum. D, hematoxylin and eosin staining of the thyroid biopsy illustrating
a diffuse lymphocytic infiltrate and the formation of well-organized ectopic
lymphoid follicles (arrows). E, positron emission tomography image showing
focally increased 2-[18F]fluoro-2-deoxy-D-glucose (FDG) uptake in the thyroid
gland, correlating with lymphocytic infiltration.

the thyroid gland. The small nodules of thyroid hormone (seen as increased
seen on ultrasound (pseudonodules) levels of T4), leading to hyperthyroid-
represent germinal centers and areas ism. Eventually, this too may cause
of focal infiltration in the gland, such destruction of the thyroid gland, result-
as shown in Figure 6.3D. ing in a hypothyroid state.
There may be overlap with Graves The cause of Hashimotos thy-
disease, which is manifested by agonis- roiditis is unknown. There are famil-
tic (activating) antibodies reactive with ial linkages (as seen in this patient).
the TSHR (see Type II Autoimmune Other conditions that may predispose
Reactions). Initially, this antibody may to Hashimotos are physical stress,
activate the thyroid into oversecretion radiation, viral infections, increased

Autoimmunity 101
iodine, medications (most notably 2.1 million Americans (1.5 million women
amiodarone, lithium, and interferon- and 600,000 men) at an annual cost of about
), other autoimmune diseases (most $6,000 per patient (direct medical costs and
notably Sjgrens syndrome), female indirect costs such as absence from work).
gender, and pregnancy. Lupus affects 500,000 Americans at an esti-
mated annual cost of $13,000 per patient, a
total $6.5 billion per year.

Epidemiology of Autoimmune Disease

There are nearly 100 different forms of auto- ANIMAL MODELS OF AUTOIMMUNE
immune disease, making these disorders DISEASE
a major cause of chronic illness, affect-
ing up to 3 percent of the general popula- The difficulty in carrying out randomized,
tion. Nearly any organ can be affected by well-controlled research in patients com-
either systemic or organ-specific autoim- plicates studies of the pathogenesis and
mune disease (Table 6.1). Women make up treatment of human autoimmune disease.
nearly 75 percent of all individuals afflicted Often the simplest course is to first study
by autoimmune disease, making these the disease in an appropriate animal model.
disorders one of the ten leading causes of However, because animal models of dis-
death in women less than sixty-five years ease rarely are identical to the human dis-
old. However, the female-to-male ratio var- order, the suitability of a particular model
ies widely among different diseases, being in any given situation must be considered
as high as 9:1 in SLE, Sjgrens syndrome, carefully before undertaking animal stud-
and autoimmune thyroiditis and as low as ies. The list of animal models of autoim-
1:1 in TID, Goodpastures syndrome, and mune disease is extensive, and only some
vitiligo (Table 6.1). The mean age of onset of the more commonly studied models can
also varies widely, with some disorders be reviewed here (see also Table 6.2).
typically occurring early in childhood (e.g.,
TID, juvenile rheumatoid arthritis), oth-
ers in the childbearing years (ages 1545,
e.g., SLE), and still others in later life (e.g.,
Sjgrens syndrome). There may be strik- Numerous mouse strains have been stud-
ing ethnic/racial predispositions to auto- ied over the years as animal models of SLE.
immune disease. For example, SLE is about Some strains develop lupus spontane-
three times more prevalent in individuals ously, such as NZB X NZW (F1) (NZB/W)
of African, Asian, or Latin ancestry than in hybrid mice, MRL mice, and BXSB male
individuals of European ancestry, whereas mice. Other models, such as tetramethyl-
Sjgrens syndrome and multiple sclerosis pentadecane (TMPD, pristane) induced
are more prevalent in those of European lupus, are inducible with chemicals. The
ancestry. The racial/ethnic differences are spontaneous models afford hope that if
likely to reflect differences in the frequen- the genetic defect(s) responsible for lupus-
cies of disease susceptibility genes. The like disease in these mice can be identified,
costs of these disorders to society are enor- similar defects will be found in human
mous. Rheumatoid arthritis (RA) affects lupus. However, the inducible TMPD

102 Autoimmunity
Table 6.2 Key Features of Selected Animal Models of Autoimmune Disease

Susceptible Similarities to Differences

Animal Model Disease Strains Human Disease from Disease

(NZB X SLE N/A Glomerulonephritis, Autoantibody

NZW)F1 ANA, anti-dsDNA, profile, no vasculitis
female > male
MRL lpr/lpr SLE (RA) N/A Lymphadenopathy, FAS deficiency
glomerulonephritis, causes mainly
erosive arthritis, hematological
vasculitis, female > autoimmunity
male in humans
BXSB male SLE N/A Glomerulonephritis Only male is
affected, limited
autoantibody profile
TMPD- SLE (RA) BALB/c, C57BL/6, Glomerulonephritis, Not genetically
induced SJL, DBA/1, erosive arthritis, mediated (but
lupus DBA/2, 129Sv, pulmonary vasculitis, influenced by the
and most others ANA, anti-dsDNA, genetic background)
anti-Sm/RNP, female
> male, increased
interferon /
Collagen- RA DBA/1 and Chronic, erosive Induced by
induced others inflammation of immunization.
arthritis peripheral joints In mice, males
have a greater
Primarily affect
ankles rather than
T TP RA 129Sv Erosive, polyarticular Patchy alopecia,
deficiency symmetrical arthritis dermatitis,
conjunctivitis, and
kangaroo hunched
K/BxN RA BALB/c and Erosive, polyarticular Induced by
others symmetrical arthritis autoantibodies;
no RF or anti-CCP
EAE MS SJL and Relapsing-remitting or Inducible instead
others chronic-progressive, of developing
highly variable spontaneously,
neurological disorder might require
NOD TID N/A Insulin-dependent Autoimmune
diabetes sialadenitis,
SLE-like disease,
and prostatitis

MS, multiple sclerosis; N/A, not applicable; RA, rheumatoid arthritis; SLE, systemic lupus
erythematosus; TID, type I diabetes; TMPD, tetramethylpentadecane; TTP, tristetrapolin; EAE,
experimental autoimmune encephalomyelitis.

Autoimmunity 103
model more closely mimics the abnormali- characteristic of SLE, including anti-Sm
ties in interferon (IFN) and production and anti-dsDNA as well as severe hyper-
seen in most lupus patients. gammaglobulinemia. These autoantibodies
develop earlier in the presence of the lpr
NZB/W F1 MODEL mutation, which generally accelerates the
The NZB/W model was the first onset of lupus-like disease in this strain. The
murine model of lupus nephritis. New abnormalities caused by the lpr mutation
Zealand Black (NZB) mice develop AIHA are due to an ETn retrotransposon inser-
and the female New Zealand White (NZW) tion into the Fas gene, which encodes an
mice develop mesangial glomerulonephri- important protein mediator of apoptosis.
tis late in life. In contrast, the F1 hybrid Defective apoptosis of lymphocytes leads
(NZB/W) develops early-onset severe to the accumulation of CD3+CD4CD8
(proliferative, immune complex-medi- (double negative) T cells, accounting for
ated) glomerulonephritis along with ANA, the massive lymphoproliferation seen in
antichromatin, and anti-dsDNA antibod- MRL lpr/lpr mice.
ies. However, these mice lack other classic
clinical and serological manifestations of BXSB MODEL
SLE, such as arthritis, inflammatory skin This strain was created by crossing
rashes, serositis, and anti-Sm autoanti- male SB/Le and female C57BL/6J mice.
bodies. Extensive genetic analysis of this Male, but not female, BXSB mice develop
strain has revealed three major susceptibil- severe glomerulonephritis, lymphade-
ity intervals on chromosomes 1, 4, and 7. nopathy, splenomegaly, AIHA, and anti-
Each of these intervals appears to contain dsDNA autoantibodies. Thus, the sex
multiple disease-susceptibility genes and predilection is an important difference
several candidate genes have been identi- from human lupus and most other murine
fied. NZB/W mice have been used widely lupus models. A mutant gene located on
for preclinical studies of various therapeu- the Y chromosome, designated Yaa (Y
tic interventions for lupus nephritis. chromosome-linked autoimmune accel-
eration), causes accelerated lupus-like dis-
MRL MODEL ease in male BXSB mice. A recent study
MRL mice, an inbred strain derived showed that the Yaa mutation results from
from several other strains, develop ANAs translocation of a 4-megabase portion of
and late-onset glomerulonephritis remi- the X chromosome to the Y chromosome,
niscent of SLE. A spontaneously occurring leading to increased expression of several
mutation led to impressive lymphoprolif- genes that are normally X linked, includ-
eration (lpr mutation), severe, early-onset ing TLR7.
nephritis closely resembling proliferative
lupus nephritis, the development of ero- TMPD-INDUCED LUPUS
sive arthritis (more characteristic of RA Intraperitoneal injection of pristane (2,
than SLE), salivary gland inflammation 6, 10, 14 tetramethylpentadecane, TMPD)
(reminiscent of Sjgrens syndrome), vas- can induce a lupus-like syndrome in non-
culitis, and skin disease resembling cuta- autoimmune-prone mice characterized
neous lupus. Both MRL and MRL lpr/lpr by proliferative glomerulonephritis,
mice develop a host of autoantibodies erosive arthritis, pulmonary vasculitis,

104 Autoimmunity
and a variety of lupus autoantibodies, the development of a severe polyarticular
including anti-dsDNA and anti-Sm. All arthritis resembling human RA. Although
or nearly all immunocompetent mouse induced by heterologous CII, immuniza-
strains are susceptible to lupus induced tion leads to a response against autologous
by this hydrocarbon oil. This inducible CII. CIA can be induced in susceptible
model of lupus is, at least so far, unique strains of mice, rats, and primates. Histo-
in reproducing the increased levels of logically, both RA and CIA are character-
IFN- and IFN- seen in the majority ized by an intense synovitis accompanied
of lupus patients. The disease is largely by erosion of cartilage and subchon-
abrogated in type I interferon receptor- dral bone by a pannuslike tissue. Unlike
deficient mice. human RA, CIA is monophasic. In addi-
tion, there are important serological differ-
ences. In general, rheumatoid factor is not
Rheumatoid Arthritis (Collagen-
produced in CIA and antibodies against
Induced Arthritis, TTP Deficiency,
CCP are absent.
K/BxN Model)
RA is a systemic autoimmune disease char- TTP DEFICIENCY
acterized by prominent joint involvement. Tristetraprolin (TTP) is a transcription
Arthritis is typically associated with ero- factor that can bind to and destabilize
sion of cartilage and subchondral bone, mRNAs encoding TNF- and granu-
formation of an inflammatory tissue, con- locyte-macrophage colony-stimulating
sisting of activated macrophages, T cells, factor (GM-CSF). Mice deficient in TTP
fibroblasts, and other immune cells (pan- develop a complex syndrome character-
nus). This can ultimately result in joint ized by cachexia, polyarticular arthritis,
destruction and significant joint deformi- dermatitis, autoimmunity, and myeloid
ties. In addition to the joints, RA can cause hyperplasia accompanied by extramedul-
vasculitis, splenomegaly, and leukopenia lary hematopoiesis (erythrocyte produc-
(Feltys syndrome), interstitial lung dis- tion outside of the bone marrow). TTP
ease, and other abnormalities. Rheuma- knockout mice exhibit exuberant inflam-
toid factor (an autoantibody against the matory pannus and bony erosions. These
Fc portion of immunoglobulin) and anti- mice also produce high titers of anti-DNA,
bodies against citrulline-modified proteins and ANAs; however, rheumatoid factors
or peptides (usually detected as antibod- are absent.
ies against an artificially produced cyclic
citrullinated peptide, or CCP) are typical K/B X N MODEL
serological findings in RA, although not Although RA has been considered pri-
all patients exhibit these abnormalities. marily a type IV autoimmune reaction for
Several animal models of RA exist, but many years, the finding that autoantibod-
they do not precisely reproduce the clinical ies against glucose-6-phosphate isomerase
and laboratory abnormalities. (GPI) can transfer RA-like joint disease to
normal mice has rekindled interest in the
COLLAGEN-INDUCED ARTHRITIS (CIA) possibility that antibody-mediated auto-
Immunization of susceptible rodent immune mechanisms (type II or type III)
strains with type II collagen (CII) leads to could play a role in the pathogenesis of

Autoimmunity 105
RA. K/BxN T-cell-receptor transgenic mice the lesions classically occur at different
express a transgenic T-cell receptor specific times and in different locations. Symp-
for a peptide of the ubiquitously expressed toms include sensory loss, paresthesias
self-protein GPI. Arthritis in this model is (numbness, tingling), visual changes due
initiated by antibodies against GPI. The to optic neuritis, tremor, ataxia, weakness,
resulting synovitis is chronic, erosive, and spasticity, and other neurological symp-
associated with pannus formation. Para- toms. Patients with MS can exhibit either
doxically, although the GPI antigen is ubiq- a relapsing-remitting or a progressive
uitous, autoimmunity is focused on the course.
joints. It appears that the GPI protein, not EAE is a model of MS induced in
a cross-reactive synovial antigen, is the tar- susceptible animals by immunization by
get of the pathogenic antibodies. Although intact myelin or components of myelin, the
the histological appearance of the affected sheath that surrounds certain neurons. Like
joints is reminiscent of RA, there is no evi- CIA for RA, EAE can be induced in several
dence that RA in humans can be caused by species, including mice, rats, guinea pigs,
antibodies against GPI. The classic sero- rabbits, and primates. Although induced
logical abnormalities, rheumatoid factor, by heterologous antigen(s), the disease is
and anti-CCP antibodies are not seen, and autoimmune. Several proteins have been
anti-TNF- antibodies have little effect in used to induce EAE, including MBP, pro-
this model. teolipid protein (PLP), and MOG. Different
antigens cause somewhat different clini-
cal manifestations. By administering the
Multiple Sclerosis (Experimental
antigen with complete Freunds adjuvant
Autoimmune Encephalomyelitis)
and pertussis toxin, the blood-brain bar-
Multiple sclerosis (MS) is a chronic rier is disrupted, permitting access by
autoimmune disease affecting the central immune cells. The resulting demyelinating
nervous system, including the brain and disease closely resembles human MS and
spinal cord. The disease affects about is thought to be mediated primarily by T
350,000 Americans and about 1.1 mil- cells because disease can be transferred to
lion worldwide. Age of onset is typically normal animals by T cells (type IV autoim-
twenty to forty years old, women are mune reaction). There is only limited evi-
affected more frequently than men (2:1 dence that an immune response to MBP,
ratio), and it is most prevalent in indi- PLP, or MOG is involved in human disease,
viduals of northern European ancestry. and it is hypothesized that other myelin
It is thought to be mediated primarily by antigens may be the targets of autoreactive
a T-cell-mediated attack on the myelin T cells in MS.
sheaths of certain nerve fibers, resulting
in inflammation, demyelination, and glio-
Type I Diabetes (Nonobese Diabetic
sis (scarring). In addition, autoantibod-
Mouse Model)
ies against components of myelin such
as myelin oligodendrocyte glycoprotein TID is an autoimmune disease in which
(MOG) may be seen and also may con- the insulin-producing cells in the pan-
tribute to disease pathogenesis by fixing creatic islets of Langerhans are gradually
complement. During the course of disease, destroyed by autoreactive T cells over

106 Autoimmunity
a period of months to years. After about Autoimmune Thyroiditis (Experimental
80 percent of the islet cells are destroyed, Autoimmune Thyroiditis)
insulin deficiency and a severe form of
Experimental autoimmune thyroiditis is
insulin-dependent diabetes marked by
induced in mice by immunization with
ketoacidosis develops. The disease usually
murine thyroglobulin plus complete
affects children and young adults but can
Freunds adjuvant. The mice develop
occur at any age. Males and females are
autoantibodies against thyroglobulin and
affected equally. The highest incidence is
histological changes consistent with those
in Scandinavians (35 per 100,000 per year).
seen in human autoimmune thyroiditis. It
Individuals with a genetic susceptibil-
is a useful model for studying the patho-
ity to the disease are thought to develop
genesis of human chronic (Hashimotos)
autoimmunity in response to an undefined
environmental trigger. Most patients with
TID produce anti-islet cell autoantibod-
ies reactive with insulin, glutamic acid
decarboxylase, ICA-512/IA-2, phogrin,
or other antigens. These autoantibodies
generally appear before the onset of clini-
Genetic Predisposition
cal diabetes and have been used for early
diagnosis of the condition. Genetic, environmental, and random (sto-
The nonobese diabetic (NOD) mouse chastic) factors all play a role in the patho-
is the most useful model of autoimmune genesis of autoimmune diseases. Family
TID. NOD mice spontaneously develop members of affected individuals are at
marked infiltration of T cells into the higher risk for developing autoimmune
pancreatic islets. The infiltrating T cells disease than the general population. The
selectively destroy the pancreatic cells. relative risk to siblings of affected individ-
In addition to diabetes, NOD mice spon- uals (probands) versus the risk in the gen-
taneously develop autoimmune responses eral population (s = disease prevalence in
involving other tissues, including sali- siblings of affected individuals disease
vary gland, lacrimal gland, thyroid gland, prevalence in the general population) is a
parathyroid gland, adrenal gland, testis, useful way to estimate the importance of
large bowel, and red blood cells. NOD genetic factors. The relative risk is between
mice also are susceptible to exogenously five- and fiftyfold higher in siblings of
induced autoimmune diseases, such as affected probands than in unrelated indi-
experimental autoimmune thyroiditis, viduals in most autoimmune diseases
colitis-like wasting disease, encephalomy- (Table 6.3). Part of this effect is accounted
elitis, and SLE. Defects related to several for by MHC-linked genes.
genes, including the MHC class II, CTLA- Twin studies illustrate the importance
4, and IL-2, have been associated with the of these genetic factors. If the concordance
susceptibility to diabetes. T cells play an rates in monozygotic and dizygotic twins
important role in the development and are about the same, the genetic effect is
progression of disease, whereas B cells are small. For most autoimmune diseases,
not required at the effector stage of TID in concordance rates are 1530 percent for
NOD mice. monozygotic twins versus 25 percent for

Autoimmunity 107
Table 6.3 Twin Studies IIlustrating the Importance of Genetic Factors in Autoimmune Disease

Concordance Rates Sibling Risk/Population Risk (s)

Disease Dizygotic Twins Monozygotic Twins Overall Attributable to MHC

Systemic autoimmune
Rheumatoid 4% 1215% 8 1.6
SLE 2% 24% 20 N/A
Organ-specific autoimmune
Type 1 diabetes 5% 33% 15 2.4
Multiple sclerosis 3.5% 2140% 20 2.4
Graves disease 0% 36% 15 N/A

Compiled from the following references: Vyse TJ, Todd JA. Genetic analysis of autoimmune
disease. Cell. 1996;85:311318; Jarvinen P, Aho K. Twin studies in rheumatic diseases. Semin
Arthritis Rheum. 1994;24(1):1928; Brix TH, Christensen K, Holm NV, Harvald B, Hegedus L. A
population-based study of Graves disease in Danish twins. Clin Endocrinol. 1998;48(4):
397400; Redondo MJ, Fain PR, Eisenbarth GS. Genetics of type 1A diabetes. Recent Prog
Horm Res. 2001;56:6989; Kahana E. Epidemiologic studies of multiple sclerosis: a review.
Biomed Pharmacother. 2000;54(2):100102.

dizygotic twins, consistent with a sizeable ning studies of a variety of systemic and
genetic effect (Table 6.3). Identification of organ-specific autoimmune diseases map
the actual mutations or genetic polymor- nonrandomly into eighteen chromosomal
phisms that confer susceptibility to auto- clusters, possibly explaining the occur-
immune diseases has been complicated by rence of several autoimmune diseases in
the fact that most autoimmune disorders a given individual or family. For example,
appear to involve multiple genes, each Hashimotos thyroiditis is associated with
with only a small effect. Moreover, many a variety of organ-specific (e.g., TID, per-
autoimmune diseases are actually syn- nicious anemia, autoimmune hepatitis,
dromes that may arise through a variety and Addisons disease) and systemic (e.g.,
of different pathogenic mechanisms and lupus, RA, and Sjgrens syndrome) auto-
genetic abnormalities. Even in the inbred immune diseases. Pedigrees with more
lupus-prone mouse strain NZB/W, ten than one systemic autoimmune disorder
or more susceptibility loci are thought to are not unusual. Of course, shared envi-
contribute to disease severity in an addi- ronmental influences could also explain
tive fashion (threshold liability model). familial clustering.
Human SLE and other autoimmune dis- Among candidate genes, the MHC
eases also are likely to be highly complex class II molecule is the most comprehen-
genetically. Interestingly, there may be sively studied. MHC polymorphisms are
some overlap genetically between different associated with development of RA, SLE,
forms of autoimmune disease, such as SLE MS, TID, and other autoimmune diseases
and type I diabetes. More than half of the (Table 6.3). RA is a striking example. A
linkages identified in genomewide scan- shared epitope, consisting of a 5-amino

108 Autoimmunity
acid sequence motif in the third allelic wavelengths most likely to induce lupus
hypervariable region of the HLA-DR1 fall in the UV range: UVC (200290 nm),
chain (QKRAA in the *0401 allele, QRRAA UVB (290320 nm), and UVA (320400
in the *0404 and *0101 alleles), is carried nm). UVB irradiation is mostly absorbed in
by 90 percent of patients with RA and is the upper layers of the epidermis, whereas
associated with disease severity. In addi- the longer wavelength UVA is able to reach
tion to MHC-linked genes, mutations or the dermis. UV exposure can induce apop-
genetic polymorphisms involving non- tosis and release of immune mediators and
MHC genes also are strong candidates for activation of resident dendritic cells and T
autoimmune disease susceptibility genes cells. Expression of certain self-antigens,
(Table 6.4). These include genetic polymor- such as Ro60/Ro52, on the surface of the
phisms or deficiency of molecules involved apoptotic cells may lead to antibody-medi-
in the response to or clearance of immune ated inflammatory responses that could
complexes (e.g., C1q, C4, FcRIIa, FcRIIb, play a role in the pathogenesis of skin
FcRIIIa, mannose-binding lectin), which rashes in lupus.
are associated with SLE, as well as genes The importance of environmental fac-
influencing T-cell activation (PTPN22, tors is further illustrated by the induc-
CTLA-4), cytokine responses (STAT-4, IRF- tion of a murine lupus syndrome by the
5, Tyk2), or programmed cell death (Fas, hydrocarbon pristane (see Animal Mod-
PDCD1; Table 6.4). els of Autoimmune Disease), which
Many, if not most, systemic and organ- appears to act, in part, through the induc-
specific autoimmune diseases are thought tion of type I interferon (IFN- and IFN-
to be multifactorial, involving multiple ) production. Many other chemicals and
genetic defects consistent with the thresh- drugs have been implicated as triggers
old liability model of multifactorial inheri- of autoimmunity or autoimmune disease.
tance. This model supposes a continuously Procainamide, hydralazine, chlorproma-
distributed genetically determined liability zine, methyldopa, quinidine, minocycline,
to the development of disease. Individuals and nitrofurantoin all have been associ-
who develop disease will bear multiple ated with the induction of ANAs and in
disease susceptibility genes. Because of some cases antineutrophil cytoplasmic
the normal distribution of genes, first- antibodies as well as in the pathogenesis
degree relatives will have much higher of drug-induced lupus, most frequently
risk of developing disease than the general manifested by serositis (inflammation of
population, second-degree relatives will the pleura or pericardium) and arthri-
have a moderate risk, and third-degree tis. Silica is recognized as a precipitating
relatives will have low risk. factor for scleroderma, cigarette smoke
may aggravate RA, and trichloroethyl-
ene is thought to promote lupus in ani-
Environmental Triggers of
mal models and possibly humans. Other
Autoimmune Disease
chemical agents implicated in the patho-
Environmental factors can trigger autoim- genesis of autoimmunity include heavy
mune disease in susceptible hosts, as illus- metals such as mercury, gold, and cad-
trated by the initiation of disease by sun mium, pesticides, herbicides, hydrazine,
exposure in a subset of lupus patients. The and certain dyes.

Autoimmunity 109
Table 6.4 Some Candidate Non-MHC Autoimmune Disease Susceptibility Genes

Gene Abnormality Disease Association Defect

C4 Deficiency SLE IC clearance

FcRIIa R131 SLE Inflammatory
response to ICs
FcRIIb T232 SLE Inflammatory
response to ICs
FcRIIIa F176 SLE Inflammatory
response to ICs
CTLA-4 +49G SLE, TID, Graves disease, Control of T-cell
Hashimotos thyroiditis, activation
Addisons disease, celiac disease
PTPN22 W620 SLE, RA, TID, RA, JRA, Graves Control of T-cell
disease, vitiligo activation
STAT4 SNP rs7574865 SLE, RA, TID, autoimmune Cytokine signaling
thyroiditis, myasthenia
IRF-5 SNP rs2004640 SLE Cytokine signaling
T allele
Tyk2 SNP rs2304256 SLE Cytokine signaling
PDCD1 SNP rs11568821 SLE Programmed cell
(PD-1.3) death

Fas Deficiency Autoimmune cytopenias Programmed cell

Foxp3 Deficiency Organ-specific autoimmune Control of
disease (TID, autoimmune T-cell activation
thyroiditis) (deficiency of Treg)

IC, immune complex; JRA, juvenile rheumatoid arthritis; RA, rheumatoid arthritis; SLE, systemic
lupus erythematosus; TID, type I diabetes.

Infections also are implicated in the (e.g., schistosomiasis, Chagas disease),

pathogenesis of autoimmune disease. bacterial (e.g., Helicobacter pylori, staphy-
The classic example is rheumatic fever, lococci, salmonella, Lyme borreliosis),
which is thought to be a consequence mycobacterial (e.g., tuberculosis, leprosy),
of cross-reactivity or molecular mim- and viral (e.g., cytomegalovirus, Epstein-
icry between antigens carried by certain Barr virus, hepatitis C, Coxsackie virus,
strains of streptococci and self-antigens parvovirus B19) infections can be compli-
of the heart. Mycoplasma pneumonia can cated by autoimmunity. Proposed mecha-
induce the production of cold agglutinins, nisms include molecular mimicry and the
polyclonal cold-reactive IgM autoantibod- chronic overproduction of cytokines, such
ies against the erythrocyte I, or i antigens as IFN-. Indeed, therapy with IFN- can
that can cause complement-mediated lead to the development of autoimmune
AIHA (see Type II Autoimmune diseases such as autoimmune thyroiditis
Reactions). A variety of other parasitic and SLE.

110 Autoimmunity
gen receptor leads to a state of anergy (the
Maintenance of Self-Tolerance
inability of the lymphocyte to respond to
Environmental triggers, such as sunlight, its specific antigen).
drugs/chemicals, and infectious agents, Many self-antigens are expressed at
act on a genetic background that regulates a very low level that is insufficient to
tolerance to self. The immune system has induce T-cell activation. In the case of T
evolved a remarkable ability to distin- cells, which recognize short peptides asso-
guish self from nonself. Immune tolerance ciated with MHC molecules, the induc-
is achieved by multiple mechanisms, tion of self-tolerance requires the genera-
operating both centrally and peripher- tion of a sufficient amount of self-peptide
ally. Central tolerance occurs during the in antigen-presenting cells to stimulate
development of T and B lymphocytes in T-cell deletion or anergy. Self-peptides
the thymus and bone marrow, respec- that are generated inefficiently by the anti-
tively. This mostly involves the deletion gen-presenting cells can neither stimulate
of autoreactive cells before they exit the immunity nor induce tolerance; that is,
primary lymphoid organs. In general, the immune system remains ignorant of
lymphocytes exhibiting strong reactiv- them. If these minor self-peptides are pro-
ity for ubiquitously expressed self-anti- duced in larger amounts and exposed to
gens are deleted in this manner, whereas the immune system in the presence of an
autoreactive cells of lower affinity for self inducer of co-stimulatory molecules (e.g.,
may escape central tolerance. These cells adjuvants), they have the capacity to stim-
are held in check by peripheral tolerance ulate an immune response. This has been
mechanisms. Peripheral tolerance is medi- shown experimentally with peptides gen-
ated by deletion, anergy, and suppression erated in vitro using proteolytic enzymes
as well as by neglect or ignorance, or using synthetic self-peptides.
acting on autoreactive lymphocytes after Regulatory T cells (Treg) also play an
they exit the primary organs. In general, important role in maintaining peripheral
lymphocyte activation requires two sig- tolerance. Several different subsets of Treg
nals, one delivered by the antigen receptor have been reported, but one of the most
(T-cell antigen receptor or surface immu- intensely studied is the CD4+CD25+Foxp3+
noglobulin) and second, a co-stimulatory subset, which represents about 10 percent
signal. For T-cell activation, this co-stimu- of total CD4+ cells. These cells regulate
latory signal is delivered by the interac- T-cell activation by a cellcell contact-
tion of molecules expressed on the surface dependent mechanism and through the
of professional antigen-presenting cells or secretion of inhibitory cytokines such as
B cells, such as CD80 and CD86, which IL-10 and transforming growth factor-beta
interact with CD28 (or other receptors) on (TGF). They suppress both nave and
the T-cell surface. In the case of B cells, memory T-cell responses and down-regu-
the co-stimulatory signal is delivered by late the expression of pro-inflammatory
CD40 ligand, a surface protein expressed cytokines and co-stimulatory molecules
by activated helper T cells that interacts on the antigen-presenting cells. These cells
with CD40 on the surface of B lympho- are induced in an antigen-specific man-
cytes. In the absence of a co-stimulatory ner, but the subsequent suppressive effects
signal, engagement of the T- or B-cell anti- are not antigen specific. Genetic defects in

Autoimmunity 111
Foxp3, a transcription factor that is the key immunosuppressive medications, as are
controller of Treg function, lead to organ- the majority of systemic autoimmune dis-
specific autoimmune or autoinflamma- eases. Immunosuppressive medications
tory diseases. The scurfy mouse has an can be categorized by mode of action.
X-linked defect of the Foxp3 gene that is
lethal in males, which exhibit hyperactiva-
Anti-inflammatory Agents
tion of CD4+ T cells and overproduction
of inflammatory cytokines. Foxp3 muta- Nonsteroidal anti-inflammatory drugs
tions in humans are the cause of the IPEX have been used since the 1800s when sali-
(immune dysregulation, polyendocrinopa- cin was extracted from willow bark (1828)
thy, enteropathy, X-linked syndrome) syn- and sodium salicylate (1875) and aspirin
drome. These individuals develop organ- (1899) were synthesized. A large number
specific autoimmune diseases, such as TID, of these drugs, which either selectively or
autoimmune thyroiditis, and inflammatory nonselectively inhibit the enzyme cyclo-
bowel disease. Interestingly, however, the oxygenase (a synthetic enzyme for pros-
development of systemic autoimmune dis- taglandins), are currently in use to treat
ease is not part of the syndrome in either inflammatory disease. Although most of
mice or humans. their anti-inflammatory properties derive
Finally, antigen-presenting cells play from the inhibition of prostaglandin syn-
an important role in the induction of tol- thesis, at high doses, there is inhibition of
erance. Dendritic cells can either initiate T- the transcription factor nuclear factor B
cell activation and proliferation or promote (NFB), a key mediator of inflammatory
peripheral tolerance through the deletion cytokine production. Corticosteroids have
of autoreactive T cells, depending on their a more potent effect on NFB and con-
maturation state. Tolerance is induced sequently a greater anti-inflammatory
when antigens are presented by immature effect.
dendritic cells and these cells also play a Philip S. Hench discovered the anti-
role in the generation and maintenance inflammatory properties of cortisone in
of Treg. 1949. Corticosteroids are a mainstay of
therapy for many systemic autoimmune
diseases, including SLE, RA, and inflam-
TREATMENT OF AUTOIMMUNE matory myopathies such as polymyositis.
DISEASE Corticosteroid therapy also is used for
the treatment of some of the more seri-
Treatment for autoimmune disease is ous organ-specific autoimmune diseases,
diverse, and in recent years, the options such as AIHA, autoimmune thrombocy-
have increased rapidly. Organ-specific topenia, multiple sclerosis, and Goodpas-
autoimmune diseases of endocrine func- tures syndrome. Corticosteroids reduce
tion, such as TID and autoimmune thy- inflammation by multiple mechanisms of
roiditis, may be treated with hormone action. One major action is enhanced tran-
replacement. In contrast, other forms of scription of an inhibitor of NFB called
organ-specific autoimmune disease such IB. IB dimerizes with NFB, inhibit-
as autoimmune thrombocytopenia, AIHA, ing the production of inflammatory cyto-
and multiple sclerosis are treated with kines mediated by this transcriptional

112 Autoimmunity
pathway. In addition, corticosteroids pro- monoclonal antibody), and adalimumab
mote the differentiation of a subset of anti- (a fully humanized monoclonal antibody
inflammatory macrophages that produce against TNF-) in RA demonstrated
the cytokine IL-10. that although multiple cytokines may
be involved in disease pathogenesis (in
RA, IL-1, and IL-6 in addition to TNF-),
Antimalarial Drugs
inhibitors of a single cytokine pathway
Antimalarial drugs have been used for may show therapeutic efficacy. In addi-
the treatment of SLE and RA since the tion to RA, TNF- inhibitors are used for
early 1900s. The precise mechanism of treating inflammatory bowel disease, pso-
action remains uncertain, but they have riasis, and psoriatic arthritis and are being
been shown to inhibit cytokine (IL-1 and tested in sarcoidosis, Wegeners granulo-
IL-6) production in vitro. The antimalari- matosis, pyoderma gangrenosum, SLE,
als pass freely through cell membranes at and Behcets syndrome.
neutral pH, but in acidic environments, Anti-TNF therapy is only the tip of the
such as endosomes, they become proton- biological iceberg. Recombinant IL-1
ated and can no longer diffuse freely. This receptor antagonist (anakinra) has been
leads to concentration of the drug within approved for the treatment of RA, and
endosomes and the collapse of endosomal numerous other cytokine antagonists are
pH gradients. It has been proposed that currently in clinical trials or under develop-
the inhibition of endosomal acidifica- ment.
tion interferes with antigen processing or,
alternatively, that there is an effect on the
interaction of microbial substances such
as unmethylated CpG DNA or uridine- Methotrexate is a folic acid analog used
rich RNA with endosomal toll-like recep- extensively for the treatment of RA. It
tors (TLR9 and TLR7/TLR8, respectively). appears that its ability to inhibit dihydrofo-
In addition to SLE and RA, antimalarials late reductase is not responsible for its effi-
are used in the treatment of juvenile rheu- cacy in RA, however. Instead, activity may
matoid arthritis, Sjgrens syndrome, and be related to effects on aminoimidazole-
inflammatory myopathies. carboxamide ribotide transformylase,
leading to the release of adenosine, a
potent anti-inflammatory molecule that
Anticytokine Agents
inhibits neutophil adherence to fibro-
The development of TNF- inhibitors in blasts and endothelial cells. Methotrexate
the 1990s ushered in a new era of therapy inhibits IL-1 and increases the expression
of autoimmune disease using biologi- of TH2 cytokines (e.g., IL-4), leading to
cals capable of interfering with the decreased production of TH1 cytokines
interactions between cytokines and their (e.g., IFN-).
receptors. The initial clinical use of TNF
inhibitors such as etanercept (a soluble
Anti-T-Lymphocyte Therapy
recombinant TNF receptor II linked to
the Fc portion of human IgG1), infliximab T cells play a key role in the pathogen-
(a chimeric human-mouse anti-TNF- esis of type IV autoimmune reactions

Autoimmunity 113
and also are critical for generating the NFAT. Although used most frequently to
T-cell-dependent autoantibodies mediat- prevent transplant rejection, these agents
ing type II and type III autoimmune dis- have been shown to have activity in the
eases. Consequently, considerable effort treatment of RA, SLE, and certain forms of
has gone into the development of thera- vasculitis.
peutic agents that selectively or nonselec- The CTLA4 (CD152) molecule is an
tively target T lymphocytes. Drugs that inhibitory receptor expressed by acti-
target primarily T cells include cyclo- vated T cells that block the co-stimulatory
phosphamide, azathioprine, cyclosporin interaction between CD80 or CD86 on the
A, tacrolimus, and the biological CTLA4- surface of antigen-presenting cells and
Ig. Cyclophosphamide is an alkylating CD28 on T cells. It acts by binding CD80/
agent that substitutes alkyl radicals into CD86 with greater affinity than CD28.
DNA and RNA. The drug is inactive by CTLA4 is expressed late in T-cell activation
itself but is converted to an active metabo- and serves to turn off the activated state.
lite responsible for its immunosuppres- CTLA4-Ig (abatacept) is a recombinant
sive effects. It is used for the treatment of chimera of CTLA4 and the Fc fragment of
lupus nephritis and other life-threatening IgG1. CTLA4-Ig/abatacept is used for the
complications of SLE and other systemic treatment of RA and is active in mouse
autoimmune diseases. models of lupus. Clinical trials in SLE
Azathioprine is a purine analog that patients are in progress.
inhibits the synthesis of adenosine and
guanine. Like cyclophosphamide, it is
Anti-B-Lymphocyte Therapy
converted to an active metabolite (6-mer-
captopurine), which inhibits the division Rituximab is a cytotoxic chimeric human-
of activated B and T cells. Azathioprine mouse monoclonal antibody with a high
is used in the treatment of RA, SLE, auto- affinity for CD20, a pan-B-cell surface anti-
immune hepatitis, inflammatory myopa- gen. It was developed originally for the
thy, vasculitis, and other autoimmune treatment of B-cell lymphomas. The kill-
disorders. ing of B cells by rituximab is thought to
Unlike cyclophosphamide and aza- depend on both the specific recognition of
thioprine, cyclosporine and tacrolimus B cells by this monoclonal antibody and
(FK506) have immunosuppressive prop- natural killer (NK) cell-mediated antibody-
erties that are highly selective for T cells. dependent cellular cytotoxicity (ADCC)
Both agents interfere with the phosphatase of those cells. There is considerable evi-
calcineurin, ultimately leading to an inhi- dence that the interaction of B-cell-bound
bition of the activation of the transcription monoclonal antibodies with NK cell CD16
factor NFAT (nuclear factor of activated T (FcRIIIA) is a critical event leading to
cells). Cyclosporine binds to the intracel- ADCC following treatment with rituximab.
lular protein cyclophilin and tacrolimus Rituximab appears to have activity in a
to a protein called FK binding protein. variety of autoimmune diseases associated
The cyclosporine-cyclophilin and tacroli- with autoantibody production, including
mus-FK binding protein complexes bind RA, SLE, polymyositis/dermatomyositis,
to calcineurin, preventing its activation by Sjgrens syndrome, and cryoglobulinemic
intracellular calcium, and the activation of vasculitis.

114 Autoimmunity
Intravenous Immunoglobulin T lymphocytes as well as antigen-present-
ing cells (monocytes, macrophages, and
Intravenous immunoglobulin (IVIG) is a
dendritic cells). The rationale for HSCT as
preparation of human immunoglobulin
a therapy for autoimmune disease is based
pooled from thousands of healthy indi-
on the concept that the peripheral expan-
viduals. It was originally developed for
sion of autoreactive T- and B-cell clones
replacement therapy in humoral immu-
is central to the pathogenesis of autoim-
nodeficiency syndromes but has more
munity. If these autoantigen-specific cells
recently become an important therapeutic
can be deleted and the immune system
modality in severe autoimmune disor-
regenerated with normal hematopoietic
ders, such as thrombocytopenic purpura,
stem cells, there is the potential to effect a
AIHA, neuroimmunological diseases such
cure of autoimmune disease. Therapy is
as Guillain-Barr syndrome, SLE, certain
based on the mobilization of hematopoi-
forms of vasculitis, and polymyositis/
etic stem cells using C-CSF or G-CSF plus
dermatomyositis. The mechanism of action
cyclophosphamide. There is the risk dur-
remains unclear, but IVIG may block the
ing mobilization of flares caused by G-CSF
function of Fc receptors expressed by
treatment. The stem cells are depleted of
phagocytes of the reticuloendothelial sys-
lymphocytes and enriched for CD34+ cells
tem and also induces FcRIIB (inhibitory
followed by expansion and reinfusion into
Fc receptor) expression on infiltrating mac-
the same donor after conditioning. The
rophages in the K/BxN model of RA. An
conditioning regimen involves cyclophos-
additional mode of action may involve the
phamide treatment or other immunosup-
presence of anti-idiotypic antibodies that
pressive treatments aimed at depleting
block the antigen combining sites of patho-
mature lymphocytes. Phase III clinical tri-
genic antibodies. The duration of action is
als of the efficacy of autologous HSCT in
limited by the metabolism of serum immu-
MS, SLE, RA, and scleroderma are ongoing
noglobulin, and generally, IVIG is regarded
or planned. Promising preliminary results
as a temporary measure that is followed by
have been obtained with all of these condi-
more definitive therapy.
tions, but further study is needed.

Autologous Hematopoietic Stem Cell

Transplantation (HSCT)
Therapy Directed at Inflammatory
The ability to adoptively transfer autoim-
mune diseases with bone marrow trans-
plantation in a variety of animal models The pace of changes in the field of auto-
provides strong evidence that these dis- immunity is rapid. Recent advances in
orders are mediated by cells derived from understanding the importance of key
hematopoietic cells. There is compelling inflammatory pathways involved in spe-
evidence that autoimmune disease results cific diseases, such as the TNF- path-
from a loss of B- or T-cell tolerance to cer- way in RA, inflammatory bowel disease,
tain self-antigens. Hematopoietic stem and other disorders, have been quickly
cells are the earliest progenitor cells of followed by new biological therapies
the immune system and give rise to B and designed to interfere with these pathways.

Autoimmunity 115
With recent data increasingly underscor- will be applicable to the therapy of autoim-
ing the importance of type I interferon mune disease remains to be determined.
pathways in the pathogenesis of lupus,
it seems reasonable to expect that thera-
Cell Therapy
pies directed at preventing the excessive
production of IFN-/- will be tested Another promising possibility involves
in the near future. As always, a major manipulating tolerance through the use
challenge in immune therapy will be to of suppressor T cells or immature den-
treat the key immunological defects selec- dritic cells. Decreasing the numbers of
tively, leaving the remainder of the immune CD25+CD4+Foxp3+ cells in mice can induce
system intact to deal with infections. As a variety of organ-specific autoimmune
the important immunological pathways conditions. Conversely, expansion of this
become better defined, it may be feasible to subset can be used to induce immune tol-
selectively target one part of the pathway erance in transplantation models. There is
while leaving others intact. For instance, considerable interest in the potential use
type I interferon is produced through sev- of Treg expansion either in vivo or in vitro
eral interrelated pathways. If only one of in the treatment for autoimmune disease.
them is found to be abnormal in SLE, it Treg are highly proliferative in vivo, and
may be feasible to selectively blockade that certain drugs, such as rapamycin, may
pathway, leaving the others intact to deal increase the ratio of Treg to T effector cells.
with viral and other types of infections. Alternatively, Treg can be expanded in vitro
in the presence of high doses of IL-2 and
self-antigen followed by reinfusion, an
Gene Therapy
approach that has been used successfully
Although considerable progress has been in the treatment of TID in NOD mice.
made in defining the genetics of autoim- Whereas mature dendritic cells are
mune disease, nearly all of the major sys- highly potent stimulatory antigen-
temic autoimmune diseases are highly presenting cells, immature dendritic cells
complex, multigene disorders, even in are tolerogenic. Dendritic cell therapy is
animal models. In comparison with single- being explored as a means of both promot-
gene diseases, such as cystic fibrosis or ing immunity (mature DCs) and inducing
alpha 1-antitrypsin deficiency, it may tolerance (immature DCs). Immune silenc-
prove considerably more difficult to correct ing may be made feasible by loading DCs
genetically complex autoimmune disorders ex vivo with self-antigens followed by
using standard gene therapy approaches. treatments that render them tolerogenic
However, there may be reason for cautious (TGF-, retinoic acid, or rapamycin, for
optimism because inhibition of a single instance). Reinfusion of these cells may
cytokine (TNF-) can have a significant be useful for inducing tolerance, though
beneficial effect in the treatment of a multi- numerous obstacles remain.
genic autoimmune disorder (RA). At least
in mice, retroviral transduction of Foxp3
Stem Cell Therapy
has been shown to convert nave T cells
into cells that phenotypically and function- Although the bulk of evidence suggests
ally resemble Treg. Whether this approach that autoimmune diseases arises primarily

116 Autoimmunity
due to defects in the immune system, ther- Interestingly, allogeneic bone marrow
apy directed at repairing the target organs derived MSCs or stromal cells suppress
also may be equally important. Thus, there in vitro T- and B-cell proliferation in a
is increasing interest in the possibility of non-MHC-dependent manner. The immu-
repairing damage at the level of the target nosuppressive activity of MSCs has been
organs with stem cell therapy. attributed to effects on the expansion of
the CD25+CD4+Foxp3+ Treg population.
MESENCHYMAL STEM CELLS Current tissue engineering strate-
Found in bone marrow, cord blood, gies tend to rely on the use of autologous
spleen, adipose tissue, and other tissues, sources of adult stem cells. However, data
mesenchymal stem cells (MSCs) are a well- demonstrating that the transplantation of
characterized population of adult stem cells allogeneic adult mesenchymal stem cells is
that can give rise to three main cell types, feasible have the potential to revolutionize
including adiopocytes, chondrocytes, and this field. With routine access to adult stem
osteoblasts. However, these cells may be cells at the point of care, physicians may
induced experimentally to undergo dif- be able to incorporate tissue-engineering
ferentiation into other cell types as well, approaches into the management of auto-
such as neural cells and myogenic cells. immune disease.
Isolation, amplification, and large-scale in
vitro culturing of MSCs has been mastered HUMAN EMBRYONIC STEM CELLS
to a degree appropriate for clinical appli- Very recent advances in stem cell
cations, making MSCs good candidates technology enable the generation of plu-
for use in tissue repair. These cells can be ripotent human stem cells from human
maintained and propagated in culture for somatic cells using a process known as
long periods, without losing their capac- reprogramming or dedifferentiation. The
ity to form the cell types discussed earlier. procedure exploits viral vector-mediated
Another advantage is that MSCs can take expression of only four genes (namely,
up and retain introduced genes, a prop- c-myc, oct3/4, sox2, and klf4) to reprogram
erty that can be exploited for the delivery mouse and human somatic cells (specifi-
of clinically beneficial proteins to targeted cally, skin fibroblasts) into embryonic-like
locations. MSCs are also amenable to cryo- induced pluripotent stem cells (iPS
preservation, allowing their future use in cells). These cells appear to be just as
off-the-shelf therapies. Animal studies plastic as embryonic stem cells, but one
seeking to reconstitute or repair damaged drawback to their clinical application in
cartilage, bone, muscle, heart muscle, and humans is the use of lentiviral or other
tendon using MSCs have shown great retroviral vectors to introduce the genes.
promise, raising the possibility that they Although still at an early stage, the use
might one day be used for repairing tis- of iPS cells could greatly advance the
sues damaged by autoimmune attack. practicality of regenerative medicine as
Indeed, cell therapy using MSCs can a therapeutic option. By creating patient-
prevent damage to the joints in collagen- specific, pluripotent human stem cells, cell
induced arthritis and also can ameliorate replacement therapies that avoid human
end-organ damage in EAE and murine embryo destruction may now be within
lupus. reach.

Autoimmunity 117
ACKNOWLEDGMENTS Bruhns P, Samuelsson A, Pollard JW,
Ravetch JV. Colony-stimulating
We thank Drs. Richard Lottenberg and factor-1-dependent macrophages are
Neil Harris (University of Florida Division responsible for IVIG protection in anti-
of Hematology-Oncology) for providing body-induced autoimmune disease.
the micrograph of spherocytes. Immunity. 2003;18:573581.
Clynes R, Dumitru C, Ravetch JV. Uncou-
pling of immune complex formation
BIBLIOGRAPHY and kidney damage in autoimmune
glomerulonephritis. Science. 1998;279:
REVIEWS 10521054.
Atkinson MA, Eisenbarth GS. Type 1 dia- Ehrchen J, Steinmuller L, Barczyk K, et al.
betes: new perspectives on disease Glucocorticoids induce differentia-
pathogenesis and treatment. Lancet. tion of a specifically activated, anti-
2001;358:221229. inflammatory subtype of human
Drayton DL, Liao S, Mounzer RH, Ruddle monocytes. Blood. 2007;109:12651274.
NH. Lymphoid organ development: Jacobson DL, Gange SJ, Rose NR, et al. Epi-
from ontogeny to neogenesis. Nat demiology and estimated population
Immunol. 2006;7:344353. burden of selected autoimmune dis-
Feldmann M, Brennan FM, Maini RN. Role eases in the United States. Clin Immunol
of cytokines in rheumatoid arthritis. Immunopathol. 1997;84:223243.
Annu Rev Immunol. 1996;14:397440. Kouskoff V, Korganow AS, Duchatelle V,
Goodnow CC, Cyster JG, Hartley SB, et Degott C, Benoist C, Mathis D. Organ-
al. Self-tolerance checkpoints in B lym- specific disease provoked by systemic
phocyte development. Adv Immunol. autoimmunity. Cell. 1996;87:811822.
1995;59:279368. Kremer JM, Westhovens R, Leon M, et al.
Miyara M, Sakaguchi S. Natural regulatory 2003. Treatment of rheumatoid arthritis
T cells: mechanisms of suppression. by selective inhibition of T-cell activa-
Trends Mol Med. 2007;13:108116. tion with fusion protein CTLA4Ig. N
Engl J Med. 349:19071915.
SUGGESTED READING Nacionales DC, Kelly-Scumpia KM, Lee
Anolik JH, Barnard J, Cappione A, et al. PY, et al. Deficiency of the type I inter-
Rituximab improves peripheral B feron receptor protects mice from
cell abnormalities in human systemic experimental lupus. Arthritis Rheum.
lupus erythematosus. Arthritis Rheum. 2007;56:37703783.
2004;50:35803590. Traynor AE, Schroeder J, Rosa RM, et al.
Boackle SA, Holers VM, Chen X, et al. Cr2, Treatment of severe systemic lupus
a candidate gene in the murine Sle1c erythematosus with high-dose chemo-
lupus susceptibility locus, encodes therapy and haemopoietic stem-cell
a dysfunctional protein. Immunity. transplantation: a phase I study. Lancet.
2001;15:775785. 2000;356:701707.

118 Autoimmunity
7. Chronic Lymphocytic Leukemia

Nicholas Chiorazzi, M.D., and Manlio Ferrarini, M.D.

INTRODUCTION B-cell development and the aberrant trans-

formation to leukemia.
Any cell of the immune system (see
Chapter 1, Figure 1.1) can undergo malig-
nant transformation giving rise to leukemia, B-CELL ACTIVATION AND
lymphoma, or myeloma. In this chapter, we MATURATION
focus on B-cell-type chronic lymphocytic
leukemia (B-CLL), the most common leu- The enormous diversity of the normal
kemia in the Western Hemisphere. This B-cell-antibody repertoire initiates in the
B-cell lymphoproliferative disorder arises bone marrow where B lymphocytes rear-
among the aging population, increasing in range their immunoglobulin (Ig) variable
incidence in a linear fashion after age 50. (V) region gene segments coding for the B
Therefore, its incidence is likely to increase cells receptor for antigen (BCR) (Figures
as the baby boomer generation enters the 7.1 and 7.2). The diversity in the repertoire
sixth decade. Because patients with the dis- continues to grow after binding antigen,
ease in general have an extended clinical when the B cell enters a lymphoid follicle
course (three to twenty-five years), B-CLL and, with the help of other cells and cyto-
is categorized as one of a group of indolent kines, creates a structure called the germinal
leukemias/lymphomas. However, despite center (GC) where the cell proliferates and
progress in therapeutic strategies, B-CLL accumulates somatic mutations in its BCR-
remains an incurable disease. encoding genes (Figure 7.3). These muta-
An abundance of new information has tions may produce amino acid changes in the
become available within the past decade, binding site of the BCR, which can improve
such that B-CLL is now divided into two or create new antigen-binding specificity.
related conditions, both originating from Enhanced affinity B cells survive, whereas
antigen-selected B lymphocytes but dif- those having BCRs that either do not bind
fering in clinical course. Some patients antigen or bind self-antigens die. The GC
live several decades, often without treat- reaction usually occurs in secondary lym-
ment, and others succumb to the disease phoid follicles with the help of T lympho-
in a few years. Here we shall discuss the cytes. This mutation and selection process
possible mechanisms causing expansion can take place in response to bacteria with-
and conversion of normal B lymphocytes out T-cell help in the marginal zones out-
to leukemic clones and determining the side of lymphoid follicles, although in this
differences between the two major B-CLL case, antigenic stimulation does not always
subtypes. First, we will review normal induce mutation of IgVH genes.

Fab CH1
-S - S-
Hinge CH2
Region Chromosome 14


Chromosome 2

Ig Molecule V Region Ig Genes

Figure 7.1 Ig molecules and their genes. Ig molecules can be divided into two
fragments, Fab and Fc, by enzymatic digestion; the former fragment engages antigen
and the latter fragment mediates effector functions. The two fragments differ in IgV gene
composition, with the Fab region, containing the antigen-binding site, being composed
predominantly of the Ig variable region gene segments (IGHV, IGHD, and IGHJ) and the
Fc region composed completely by an IGHC gene.

IGHV (46) IGHD (36) IGHJ (6) IGHC

3 1 2 1 2 4 1 2

5' 3'

Figure 7.2 IGHV gene rearrangements. To code for a complete Ig molecule

containing both Fab and Fc regions, several Ig genes need to be brought
together from distinct locations and joined. This is a multistep process
involving first the rearrangement of one of thirty-six IGHD gene segments
with one of six IGHJ segments. Subsequently this unit is rearranged and
combined with one of forty-six IGHV genes. In normal B-cell development,
the first rearrangement contains the IGHC gene.

CHARACTERIZING THE those whose B-CLL cells use unmutated

HETEROGENEITY OF B-CLL BY IgVH genes (U-CLL) and those whose B-CLL
MOLECULAR AND CLINICAL cells use mutated Ig VH genes (M-CLL). Ini-
SUBTYPES tially, it was believed that the two types of
B-CLL cells could be generated by virgin
Based on their mutation status, B-CLL and memory cells, respectively, although,
patients can be divided into two groups: as we shall discuss, there is now evidence

120 Chronic Lymphocytic Leukemia

the presence of monoclonal rearrange-
ments of VDJ genes in the leukemic cells.
Most B-CLL cells also express activation
markers and, like normal activated B cells,
GC MZ GC have low levels of surface IgM and IgD.
The expression of CD5, usually thought of
as a lineage marker, is characteristic of B-
CLL cells and should be considered part of
their activation status as it can with normal
B cells when stimulated through several
Activated B cell
receptors, including the BCR. The presence
of these markers and the degree of activa-
gene substitution
switch tion varies in different B-CLL cases.
B-CLL clones can be further subdivided
G.C. based on their activation marker expres-
VH D J Centroblast sion: in one subgroup, the majority of leu-
VK J kemic cells express two activation markers,
that is, CD38 and ZAP-70; and in the other,
the majority of the cells lack these markers.
Centrocyte The two molecules appear to be involved
in the regulation of signals delivered by the
BCR. The CD38+ ZAP-70+ B-CLL subset is
B primarily characterized by the presence
Figure 7.3 Germinal center and the germinal of more members of the leukemic clone-
center reaction. A, Photomicrograph of two bearing markers of activated B cells. These
adjacent germinal centers within a mesenteric activation marker-defined subgroups
lymph node, with germinal center (GC), follicular
mantle (FM), and marginal zone (MZ) areas. B, also differ in their expression of several
On close inspection of the DNA sequence of other molecules up-regulated by cellular
the rearranged IgVHDJH, one notices somatic activation.
changes that arise during the time the activated
In comparing these two subsetting
B cell resides in the GC. These changes involve
predominantly point mutations and isotype class schemes the first based on IgVH mutation
switching, although other less frequent events status and the second on activation
such as nucleotide deletion or insertion can markers CD38+ZAP-70+ B-CLL clones
occur. These changes occur at the centroblast
generally use unmutated IgVH genes,
stage of B-cell maturation; at the centrocyte
stage, rescue from apoptosis is achieved by whereas CD38ZAP-70 B-CLL clones
antigen binding through the BCR and T-cell mainly use mutated IgVH region genes.
interaction. Although the subdivision into CD38+ZAP-
70+ U-CLL and CD38ZAP-70 M-CLL is
that both B-CLL subtypes derive from true for the majority of patients, it is not
(auto)antigen-experienced B cells. universal since about 25 percent of cases
B-CLL cells are monoclonal B-cell ex- are discordant for the expression of the
pansions as documented by the expression three markers.
of monotypic (i.e., either or ) surface Ig It is interesting to note the correlation
and the CD19 antigen; this is in line with between the molecular characterizations

Chronic Lymphocytic Leukemia 121

of the patients clones with the patients between the number of unfavorable mark-
clinical course. Of major clinical and inves- ers and clinical outcome.
tigative importance are the observations
that IgVH gene mutation status and ZAP-
70 and CD38 expression have clinical DEVELOPMENT OF B-CLL FROM
relevance, since cases in whom the major- NORMAL B LYMPHOCYTES: SIGNALS
ity of the cells express CD38, ZAP-70, and AND MECHANISMS INITIATING THE
exhibit unmutated IgVH genes have a more GROWTH AND ACCUMULATION OF
aggressive clinical course (Figure 7.4). LEUKEMIC LYMPHOCYTES
Correspondingly, those patients whose
clones are in the main CD38ZAP-70 and The major events in tumorigenesis have
express mutated IgVH genes generally fare traditionally been grouped into inducing
better and have a good prognosis. In those and promoting factors. Inducing factors
individual cases that are discordant for cause transforming mutations, whereas
the expression of these markers, there is promoting factors sustain the proliferation
nevertheless generally a direct correlation and survival of cells undergoing or having
undergone transforming mutations. It is
2% mutation useful to apply such principles to B-CLL.
Despite active research, to date no
genetic aberration (inducing factor), shared
Percent Surviving

by all patients, has been found in B-CLL.

The monoclonality of B lymphocytes that
develop in this disease implies that such
< 2% mutation
40 a lesion existed in the cells initiating the
30 Unmutated p = 0.0001 clone. Although characteristic DNA abnor-
20 malities can occur later in the development
10 of B-CLL clones, these are rarely found in
0 the initial phases of the disease.
0 2 4 6 8 10 12 14 16 18 20
In contrast, several stimulatory signals
Figure 7.4 IGHV mutation status is delivered from the microenvironment may
a robust prognostic marker in B-cell-
type chronic lymphocytic leukemia (B-
represent important promoting factors
CLL). CLL cases can be divided into in the development and evolution of the
two categories based on the presence disease. One of these antigen stimula-
of somatic mutations in the expressed tion appears to play a major role in the
IGHV gene. Those patients whose CLL
cells have an IgVH gene that differs by pathogenesis of B-CLL; this conclusion
greater than 2 percent from its germ line is based on the existence of remarkable
counterpart have a much longer survival similarities in the structures of BCRs of
time than those patients whose cells use
unrelated patients. This similarity in BCR
an IgVH gene that differs by less than
2 percent. This distinction has major structure is especially striking for about
prognostic value. Reprinted by permission 25 percent of patients, with some patients
from Damle RN, Wasil T, Fais F, et al. clones using identical IgVH, D, and JH genes.
IgV gene mutation status and CD38
expression as novel prognostic indicators
Extraordinarily, in some of these cases,
in chronic lymphocytic leukemia. Blood. these rearranged Ig heavy-chain genes are
1999;94:18401847. paired with identical IgVL genes, yielding

122 Chronic Lymphocytic Leukemia

antigen-binding sites that are virtually vivo by a combination of nonprotein self-
identical at the amino acid level. Given and microbial antigens.
the enormous number of possible com- Similarly, such a mechanism could also
binations of IgV gene segments encoding promote the origin of M-CLL. As already
antibody-binding domains, one would not mentioned, IgVH gene mutations can occur
expect to find B-CLL patients having such without T-cell help in marginal zones
structurally similar stereotypic BCRs by outside of GCs. Because mutations can
chance until well over 1 million cases have sometimes favor autoreactivity, such auto-
been screened. Hence, their occurrence reactive B cells would become expanded.
is not likely random, making a plausible However, expansion would stop if IgV
argument for the importance of antigen gene mutations alter BCR structure in such
stimulation and drive in this disease. a way that antigen binding is no longer suf-
Foreign antigens or autoantigens could ficient to induce B-cell signaling.
prompt normal B lymphocytes to become With continued expansion leading (or
B-CLL cells by selecting B-cell clones with not) to accumulation of IgVH gene muta-
restricted stereotypic BCRs. The nature of tions as explained by the T-cell-dependent
the antigens that select for these highly versus T-cell-independent models men-
restricted BCRs (possibly unique to B-CLL) tioned earlier, it becomes increasingly likely
is largely unknown, although currently that a cell develops a genetic abnormality
under investigation. It may be they result as in an initial inducing lesion that would
from infection due to a specific microbe lead to relatively unrestrained expansion.
common among patients, which has been Such a cell is primed for leukemic transfor-
found for gastric lymphomas. Alternative mation.
possibilities are that both environmental A hypothesis such as this implies that
and autologous antigens may be involved. it might be possible to detect clonal expan-
Indeed, it appears that intermittent and sions in healthy subjects. In fact, small
interchangeable encounters with microbial numbers of apparently clonal B cells with
antigens and autoantigens, especially those B-CLL cell characteristics do exist in the
generated during cell death and oxidative blood of about 3.5 percent of disease-free
and other forms of stress, are key. individuals. An even higher proportion of
How would the transition from nor- such clones have been found in the blood
mal B cells to leukemic cells via antigen of first-degree relatives of patients with
stimulation occur? Normal B lymphocytes B-CLL (as often as 12 percent). Although
using unmutated IgV genes produce anti- such studies of the BCRs of B lymphocyte
bodies that are frequently polyreactive, expansions in normal disease-free individ-
binding carbohydrates, nucleic acids, and uals are limited, they further support this
phospholipids. Such antibodies provide hypothesis in that these expansions are not
the first line of defense against microor- only monoclonal but also use some of the
ganisms and promote the clearance of same genes commonly encoding the BCRs
autoantigens and their fragments. B-CLL of B-CLL clones.
cells frequently display polyreactive BCRs, In addition to antigen stimulation,
thereby making it possible that they derive B-CLL cells also receive receptor-medi-
from normal polyreactive B lymphocytes ated signals as well as soluble factors, such
that have been repeatedly stimulated in as cytokines and chemokines, from other

Chronic Lymphocytic Leukemia 123

lymphoid and nonlymphoid cells. In quently among patients exhibiting unfa-
particular, it is thought that in vivo B-CLL vorable prognostic markers. Thus, con-
cell interactions with stromal cells and tinuous (auto)antigenic stimulation would
nurselike cells can rescue normally likely represent a major factor for U-CLL
(ex vivo) apoptosis-prone B-CLL cells from cases and much less likely for M-CLL.
death. The natural ligand of CD38, CD31, Similarly, there is a rough correspon-
is displayed on stromal and nurselike cells dence between the clinical course of patients
as well as on endothelial cells and might be and the development of chromosomal
involved in setting up these rescue signals. abnormalities in their clones. Recurrent
Such contact-derived and soluble signals chromosomal lesions typically found in B-
can go on to up-regulate anti-apoptotic CLL patients include deletions at 13q14.3,
genes, such as Bcl-2, survivin, and Mcl-1, 11q2223, 17p13, and 6q21, and amplifica-
which could rescue B-CLL cells from apop- tions of all or portions of chromosome 12.
tosis and facilitate their growth. Deletion at 13q14.3 is found in greater than
half of B-CLL cases over time and is linked
to loss of two micro-RNAs that can regulate
CORRELATIONS BETWEEN THE Bcl-2 expression. However, this particular
CELLULAR AND MOLECULAR chromosomal abnormality is not especially
FEATURES OF THE DISEASE WITH dangerous because patients exhibiting this
THE CLINICAL COURSE OF B-CLL deletion on one allele and no other DNA
lesions in their clones have a clinical course
How might features of the repertoire of IgVH that is benign and comparable to normal
genes used by B cells in B-CLL, the muta- age-stratified individuals. In contrast, dele-
tion status of these genes, and expression of tions at 11q2223, 17p13, and 6q21 are gen-
molecules related to cellular activation and erally associated with more aggressive dis-
BCR signal transduction (CD38 and ZAP- ease, perhaps because these deletions may
70) be relevant to the clinical course of B- affect important genes such as p53 (17p13
CLL? The disease manifests differently in deletion), and ataxia telangiectasia mutated
different patients, depending on the utili- (ATM; 11q2223 deletion). Longitudinal
zation of mutated or unmutated IgVH genes studies, albeit on a relatively limited num-
and the expression of ZAP-70 and CD38 ber of patients, demonstrated that these
by the leukemic cells (Figure 7.5). One ominous cytogenetic abnormalities accu-
explanation is that activation via the BCR mulate progressively in the course of the
following recognition of (self-)antigens disease and more frequently in patients
activates the cells in vivo, accompanied by with U-CLL.
expression of CD38 and ZAP-70. Because These considerations are in line with
the majority of U-CLL clones contain a self- the results of in vivo labeling experiments
reacting BCR, while most M-CLL clones do that involve incorporation of nonradioac-
not, it is not unexpected that more activa- tive deuterium into newly synthesized
tion markers are found on U-CLL cells. In DNA of dividing cells. These studies have
addition, B-CLL clones from patients in shown B-CLL clones to be dynamic, hav-
different prognostic subsets differ in sig- ing measurable birth rates from about 0.1
naling capacity, with an intact BCR signal to >1.0 percent of the clone/day. Although
transduction pathway found most fre- only a minority of cells in a B-CLL clone can

124 Chronic Lymphocytic Leukemia

Unfavorable Favorable


Unmutated V genes Mutated V genes

ZAP-70+ ZAP-70.
CD38+ CD38-

Activation No activation

Potential survival signals:

IgD signaling
T lymphocytes
Natural killer cells
Stromal cells
Apoptosis Survival and proliferation

Figure 7.5 Differences in B cells receptor for antigen (BCR) signaling in the
two subgroups of B-celltype chronic lymphocytic leukemia (B-CLL). B-CLL
clones from patients with unmutated IgVH genes (U-CLL) frequently differ
from those from mutated IgVH (M-CLL) patients in the ability to transduce
an activation signal through the BCR. In the case of U-CLL, patients that are
more likely to have an unfavorable clinical course, BCR-mediated signals
deliver an apoptotic signal unless influenced by other survival signals. In the
case of M-CLL patients who are more likely to have a more favorable clinical
course, BCR-mediated signals are ineffective either because the BCR has
been altered and no longer binds available (auto)antigens or because after
antigen binding the BCR is incapable of signaling due to anergy. Reprinted
with permission from Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic
leukemia. N Engl J Med. 2005;352:804815.

be shown to divide using this approach, SUMMARY REMARKS ON THE

estimates of the leukemic cell burden of a DEVELOPMENT, GROWTH, AND
typical B-CLL patient are of the order of EVOLUTION OF B-CLL
about 1012 cells, and therefore about 109 to
1010 new leukemic cells would be gener- On the basis of the foregoing information,
ated daily. Such rates of cell division are we propose a model for the development
sufficient to permit more dangerous clonal of B-CLL (Figure 7.6). B-CLL cells are able
variants to emerge and to influence clinical to avoid apoptosis and even to prolifer-
course and outcome over time. ate by receiving growth and stimulatory

Chronic Lymphocytic Leukemia 125

A Initial inducing mutation B Response to self-antigen C Repetitive antigen stimu- D Conversion to leukemia E Development of subclones
or foreign antigen lation of polyreactive by new mutations

No V-gene mutations
Subclone B
Unmutated CLL

Antigens Antigens Antigens Antigens?

Clone A

Activated B cells
Clone A
Subclone C

Antigens? Clone D

V-gene mutations

Mutated CLL


Anergic B cells Clone D


Subclone E

Nonactivated B cells Clone F Clone F

Figure 7.6 Model to explain the development and evolution of B-CLL. See text for description.
Reprinted with permission from Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia.
N Engl J Med. 2005;352:804815.

signals from the environment delivered apoptotic pathways, our model posits the
through their BCR or other receptors; these absence of an intrinsic cell death defect in
signals likely involve autologous and for- the majority of the leukemic clones. We do
eign antigens, cytokines, and chemokines, not rule out the possibility that developing
as well as yet-to-be-defined ligands on genetic alterations in the evolving clone
accessory and stromal cells. The cells BCR can tip the balance between pro- and anti-
mediates major growth effects in cases for apoptotic molecules in such a way as to favor
which the receptor is polyreactive, binding B-CLL cell survival. However, the influence
autoantigens and foreign antigens, while of external signals appears to dominate
maintaining its capacity to transmit stimu- based on current knowledge. This is in line
latory signals to the cell nucleus. Both self- with the in vivo labeling studies indicating
reactivity and intact BCR signal-transduc- the dynamic nature of CLL clones.
ing capacity are more frequently found
in U-CLL; hence, the more active clonal
expansion and clinical aggressiveness of ANIMAL MODELS OF B-CLL
patients with such clones.
Because in vitro observations demon- While an enormous amount of new infor-
strate the absence of lesions in the major mation has been gleaned by directly

126 Chronic Lymphocytic Leukemia

studying human B-CLL cells, animal mod- eventually with massive splenomegaly
els are now contributing to our understand- and leukemia.
ing of the human disease. For example, In the third transgenic model, overex-
New Zealand Black (NZB) mice spontane- pression of APRIL ( a proliferation inducing
ously develop, with age, an expansion of ligand ) in murine T cells leads indirectly to
IgM+CD5+ B cells that resembles B-CLL. B-cell proliferation and survival because of
However, because frank leukemia occurs signaling through its receptors BCMA and
randomly in only a minor subset of animals, TACI. Unlike the previous two transgenic
this model has been used sparingly. animals, however, expansions of CD5+
In recent years, however, a variety of B cells occur in only 40 percent of animals,
transgenic mouse models have been devel- with these B cells locating predominantly
oped that lead to diseased phenotypes in the spleen and rarely passing into the
resembling human B-CLL more closely and blood. Nevertheless, as APRILs action
reproducibly. We focus on three models involves the TRAFs and leads to NFB
that have been especially helpful. activation, this model may prove helpful
Transgenic mice expressing the TCL1 in linking signals from soluble ligands and
gene in murine B cells develop a poly- surface receptors to the NF,B pathway, a
clonal expansion of B lymphocytes early pathway known to be constitutively active
in life that becomes progressively more in some B-CLL clones.
restricted until a monoclonal population Finally, transfer of human B-CLL cells
emerges after about one year in most ani- into immune-deficient mice may bypass
mals. The genetic and phenotypic features the ex vivo apoptosis tendency of B-CLL
of this murine leukemia resemble those of cells, enabling their survival and amplifi-
the aggressive, treatment-resistant cases of cation in vivo. Such an approach may also
human U-CLL. Although it is of interest make it possible to define and test new
that TCL1 is an activator of the PI3K-Akt therapeutics to treat this currently incur-
oncogenic signaling pathway, a pathway able disorder.
not infrequently active in human CLL,
the extent to which overexpression of this
gene leads to human B-CLL remain to be CLINICAL IMPLICATIONS AND
elucidated, as an overexpression of TCL1 in THE DEVELOPMENT OF NOVEL
human B-CLL patients is not uniform. THERAPEUTICS
Another mouse model that develops
features resembling human B-CLL in- Currently used clinical staging systems
volves the overexpression of two genes: defined by Rai and by Binet help clini-
BCL-2 and TRAF2 (TNF-receptor-associated cians to monitor disease progression and
factor 2). This double transgenic animal is decide when to treat patients. Because
especially intriguing because of the already of difficulties distinguishing patients
mentioned recent work showing that the with poor prognosis at the onset of dis-
deletion at 13q14, often seen in human ease using these staging systems, the
B-CLL, involves the loss of micro-RNAs generally used practice is to wait to
15a and 16-1, which affects expression of start therapy until the patients clini-
BCL2. As with the TCL1 transgenic mice, cal course becomes evident (wait and
these animals develop CD5+ B-cell clones, watch mode). However, the molecular

Chronic Lymphocytic Leukemia 127

and cellular markers that reflect intrin- markers have stereotypic antigen re-
sic properties of leukemic cells present ceptors, these common structures may be
at the disease onset can help to distin- feasible as vulnerable points of attack. As
guish patients that will follow worse the antigens that engage these receptors
clinical courses, regardless of their Rai become more precisely defined, it may be
and Binet risk categories at diagnosis. possible to use these to develop an arsenal of
Although determination of IgVH gene specific therapies.
mutation status is not yet routinely avail-
able, measurement of CD38 and ZAP-70
has become more widely available. Thus, BIBLIOGRAPHY
although the wait-and-watch mode is
still being followed in current clinical REVIEWS
practice, it may be substituted for by new Chiorazzi N, Ferrarini M. B cell chronic
more aggressive strategies in the future. lymphocytic leukemia: lessons learned
The new prognostic markers indicate from studies of the B cell antigen recep-
that 3050 percent of the patients have tor. Annu Rev Immunol. 2003;21:841894.
features portending a poor outcome, and Chiorazzi N, Rai KR, Ferrarini M. Chronic
therefore an early start of therapy may be lymphocyrtic leukemia. N Engl J Med.
justified in such poor-prognosis groups. 2005;352:804815.
This strategy is plausible, considering that Ferrarini M, Chiorazzi N. Recent advances
continued proliferation and expansion of in the molecular biology and immuno-
the neoplastic clone facilitates accumula- biology of chronic lymphocytic leuke-
tion of ominous cytogenetic abnormali- mia. Semin Hematol. 2004;41:207223.
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guidelines can be proposed, clinical trials lymphocytic leukemia: revelations
must test the use of early intervention in from the B-cell receptor. Blood. 2004;
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CLL can provide clues for novel therapeu- SUGGESTED READING
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must interact with the stroma in bone mar- Human chronic lymphocytic leukemia
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is worth being explored in clinical trials, tors and stromal cells in the homing
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128 Chronic Lymphocytic Leukemia

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Chronic Lymphocytic Leukemia 129

8. Immunology of HIV Infections

Anders G. Vahlne, M.D., Ph.D.


An estimated 33 million people globally
are infected with the AIDS virus (HIV-1). The pattern of disease progression has now
Most of the infected individuals are poor, been well documented. Following infec-
live in developing countries, and have little tion with the virus, the virus hones to and
access to health care. Although initiatives infects cells with CD4 receptors. During the
are under way to bring proper medications early phase, individuals may experience a
to these individuals via a large infusion flu-like illness with mild fever, cough, and
of money, this will benefit only approxi- occasional chills. The symptoms subside,
mately 2.5 million individuals and pre- and the individual may be asymptomatic
vent 12 million new cases, as pointed out for many years. In reality, the disease is
in a 2007 New York Times editorial. The progressing, and it is a long battle between
remainder of these infected individuals the immune response with production of
will continue to increase the number of new CD4+ cells and the dying (apoptotic)
newly infected individuals. In this context, HIV-infected CD4 cells. Eventually, the
the medications decrease the viral load in host immune system deteriorates, and the
treated individuals, but medications are individual succumbs to the complications
expensive and would not reach many of secondary to loss of the cellular immune
the infected individuals living in develop- system (see Figures 8.1 and 8.2). The pat-
ing countries. tern and complications are quite similar to
Thus, many researchers are focusing those seen in primary immunodeficiency
on studies of the immunological concepts diseases (see Chapter 5). However, it has
involved in the disease, not only to been a well-known observation that some
understand in greater detail why some individuals, especially sex workers with
individuals remain uninfected (they are repeated exposure to the virus, are rela-
not immune since some resistant women tively resistant to HIV acquisition.
later acquire human immunodeficiency One of the earliest markers in the
virus, or HIV), despite repeated exposure progression of HIV-1 infection was the
to infected individuals, but also to explore presence of NEF in the viral strain. NEF is
various vaccine candidates and concepts a 2734 D myrisolated protein unique to
that might prevent the disease before primate lente viruses. A functional NEF
exposure to the virus candidates that protein is important for the development
would be cheap to make and inexpensive of high viremia and AIDS in simian immu-
to deliver. nodeficiency virus (SIV) in infected rhesus

Crossing the barrier

Dendritic HIV virus Lipid
cell membrane


transcriptase Capsid

Microbreak in Cell becomes

epithelium infected, releases

Local expansion


Resting CD4+
Co-receptor T cells
CD4+ T cell

34 days

CD4+ T cell

Infected activated CD4+ T cell Infected resting CD4+ T cell

Dissemination to lymphatic tissue Lymph node


Local proliferation
12 Peak plasma virus levels,
weeks CD4+ memory cell loss
Partial immune control Class I MHC
Cytotoxic CD8+ Helper CD4+
T lymphocyte T lymphocyte Cytokine

Neutralizing Viral antigen Viral antigen

antibodies Class II MHC

Figure 8.1 Early events in the vaginal transmission of HIV, modeled after studies of simian
immunodeficiency virus infection in nonhuman primates. The binding of the HIV gp120 envelope
protein to CD4 T cells results in conformation change in the envelope, interaction with the co-receptor,
and fusion of the viral and cell membranes, thus giving the HIV genome access to the interior of the
cell. The infected cells are carried first to draining lymph nodes and then spread systemically. HIV-
specific immune responses, including increases in CD8+ T cells and eventually neutralizing antibodies,
only partially control infection. As a result, in the absence of effective vaccination or therapy, a slow
and continued depletion of CD4+ T cells ensues and there is a progression to AIDS. Reprinted by
permission from Macmillan Publishers: Haase AT. Perils at mucosal front lines for HIV and SIV and their
hosts. Nat Rev Immunol. 2005;5:783792.

132 Immunology of HIV Infections

A Unvaccinated
Primary infection Plateau-phase infection AIDS

Window of Vulnerability
107 Burst of viremia with dissemination
Plasma Viral Concentration (copies/ml) Early seeding and destruction of
gut-associated lymphoid tissue
Establishment of HIV reservoir with
a latent component

104 No



Weeks Years

B Vaccinated
infection Plateau-phase infection

107 Decrease in Window of Vulnerability

Smaller burst of viremia, with less
Plasma Viral Concentration (copies/ml)

106 Less seeding and destruction of gut-
associated lymphoid tissue
Smaller HIV reservoir established

104 vaccination

103 Vaccination with or without broadly

reactive neutralizing antibody

102 No
Weeks Years

Figure 8.2 Panel A shows the course of infection in unvaccinated

persons. The primary stage of HIV infection (yellow) starts with a burst of
viremia, dissemination of the virus, early seeding and destruction of gut-
associated lymphoid tissue, and establishment of a viral reservoir with a
latent component. HIV levels in plasma then decline to a set point that lasts
from months to years. Eventually, in the absence of effective therapy, the
virus escapes immune control and AIDS results (red). Panel B shows the
hypothetical course of infection in vaccinated persons. A T-cell vaccine
might decrease the burst of viremia and dissemination that occurs in primary
infection (yellow), preserving gut-associated lymphoid tissue, diminishing
the viral reservoir, decreasing virus levels at the set point, and increasing the
length of time that viral levels are controlled (blue).

Immunology of HIV Infections 133

macaques. An important observation first around DC and could enhance HIV-1
noted in the macaques and later in humans replication in CD4+ T cells.
was that animals infected with NEF-deleted In summary, the selective effects of the
virus were resistant to subsequent challenge NEF protein may markedly enhance viral
with pathogenic wild-type viruses. This pathogenesis and progression to disease.
was followed by the observation in humans It does this by hijacking DC functional
that some individuals with long-term non- activity and favoring HIV replication via
progressive HIV-1 infection (persons who bystander activation of CD4+ cells and
showed no clinical or immunological signs the escape of HIV-1 from immune surveil-
of immunodeficiency despite being HIV lance by blocking CD8+ cells functional
seropositive for over a decade) turned out competence. Thus, efforts directed toward
to be infected with viruses carrying deletion blocking NEF influence in viral replication
in their NEF gene. may have long-term beneficial effects on
More recently, a large body of evidence therapeutic management of the disease.
has appeared that shows that NEF has Approximately 510 percent of sex
many important effects on many cells of workers in Kenya remained HIV unin-
the immune system. Both exogenous and fected despite engaging in unprotected sex
endogenous NEF down-regulates HLA- with many male clients and the frequent
ABC molecules critical for the initiation of acquisition of other sexually transmitted
cytotoxic T lymphocyte (CTL) responses, infections. Because of the pioneering work
thus impairing antigen presentation to of Plummer and associates, the scientific
HIV-specific CD8+ lymphocytes. NEF and reasons for this extraordinary resistance to
gp120 also are able to down-regulate major the virus in these women are beginning to
histocompatibility complex class I (MHC-I) unfold.
in dendritic cells (DCs) and introduction Turning first to examination of CD4+
of exogenous NEF leads to up-regulation T-cell immune responses in seronegative
of MHC-II molecules, thereby favoring sex workers compared with HIV-positive
CD4+ T-cell activation. This step increases women, CD4+ T cells that produced inter-
the pool of lymphocytes permissive to feron gamma (IFN-) in response to HIV
infection. However, endogenous NEF does p24 were detected in exposed seronega-
not modulate MHC-II surface expression; tive sex worker (ESN) women, albeit at a
rather it induces a loss of co-stimulation. much lower level than in HIV-positive
These results underscore the pleiotrophic women. However, ESN women had a 4.5-
action of NEF. On one hand, exogenous fold stronger CD4+ proliferation response
NEF triggers ABC-mediated bystander to the p24 peptide compared with the HIV-
T-cell activation, ensuring viral spread, positive group. These data suggest that
while endogenous NEF induces a loss of CD4+ T cells in ESN women recognize HIV
co-stimulation, favoring immune evasion. and have an enhanced ability to proliferate
Another area of interest has been the to the p24 protein.
observation that NEF-pulsed DC produces Perhaps more important has been the
a wide variety of cytokines and chemokines examination of the CD8+ cells in these ESN
typical of mature DC. This up-regulation individuals. Because IFN- production cor-
of cytokine production might promote relates with cytotoxic functions, the CD8+
bystander stimulation of T cells that cluster T lymphocyte IFN- response to HIV p24

134 Immunology of HIV Infections

peptide in ESN women was compared with DC interaction with HIV is relevant to
HIV-positive individuals. Approximately the pathogenesis of AIDS because they are
40 percent of ESN women had a CD8+ IFN- present in the mucosa and skin of humans
-positive response, and this was five times and are believed to be the first HIV-1 tar-
lower in magnitude than that of HIV-posi- gets following sexual transmission of the
tive women. The breadth of the response virus. Both myeloid DC and plasmacytoid
was very narrow and focused primarily on DC possess the receptors for HIV entry; that
one peptide that is similar to KK10 protec- is, CD4, CXR4, and CCR5 can be infected
tive peptide when compared with the HIV- but with a lower efficacy than CD4+ cells
positive group. In HIV-positive women, or macrophages. The long-term result of
lower CD4+ counts influenced the num- this infection is that DC remains a reser-
ber of CD8+ cells producing IFN-, which voir for the production and persistence of
may undermine the ability to control HIV. the HIV-1 virus, and the virus induces sev-
These results indicate ESP women have eral functional impairments and variations
an HIV-1 p24 peptide-specific CD8+ IFN- in the DC population. Thus, the number of
response, providing evidence to the speci- both myeloid DC (MDC) and peripheral
ficity needed for an effective HIV vaccine. DC (PDC) are significantly decreased in
Further studies of these women cen- HIV-positive progressors, while remain-
tered around the known polymorphisms ing unaltered in HIV-positive long-term
of the IL-4 gene and resistance to infection. nonprogression. The destruction of these
Using microsatellite genotyping methods cells may be a consequence of direct lytic
coupled with the genomic sequencing, infection or as targets for specific CTL or
three polymorphisms in the interferon reg- through a block in DC development from
ulatory factor (IRF-1) located at 619a, 179, peripheral CD34+ stem cells. More recent
and 6516 of the gene, showed association evidence supports the notion that both
with resistance to HIV infection. Periph- midi and pad show impaired functional
eral blood mononuclear cells from these capacity in HIV-positive patients. Since
patients showed significantly lower-based both DC subsets participate in the initiation
IRF-1 expression and reduced respon- of innate and adaptive immune responses,
siveness to IFN- stimulation. This study infection, depletion, and dysfunction of
added IRF-1, a transcriptional immunoreg- DCs may contribute to the immunosup-
ulatory gene, to the list of genetic correlates pression seen in HIV disease. Therefore,
of altered susceptibility to HIV-1. DCs play a dual role in HIV infection: they
One of the key cells in the immune sur- trigger both innate and adaptic immune
veillance system is the native DC, which responses to control the infection, but they
is well equipped for activation of both the also represent a viral reservoir for infection
innate and adaptive immune response. of permissive CD4+ T cells.
It has been demonstrated that some DC- Although the story of the African
tropic viruses such as influenza virus leave sex workers who seem to be immune to
DC function intact while other viruses such HIV infection has captured the imagina-
as HIV and cytomegalovirus have evolved tion of many researchers, there is another
strategies to impair DC functions, thereby model of natural immunity that needs
enhancing the viruss ability to persist and examination in more detail. Most babies
escape immune surveillance. born to HIV-infected mothers also escape

Immunology of HIV Infections 135

infection even after potential intrauterine immature compared with later in life.
exposure and, more important, exposure However, their capacity to produce CC
to virus-contaminating blood and secre- cytokines is greater compared with their
tions during labor and delivery. Finally, mothers whether she is HIV infected or
breast-fed infants ingest hundreds of liters not (see Figure 8.3). This is consistent with
of virus-infected breast milk. Many factors the expected skewing toward a stronger
such as CD4 cell counts and viral load in the innate response rather than the adaptive
secretions must be considered. One should response.
add that, like the sex worker model, it is The initially unexpected finding of HIV-
a real-world situation of viral encounter specific CD4+ and CD8+ T-cell responses
opening a window to the in vivo infection among uninfected sex workers paved the
moment. Although interesting, there are, way to the fact that natural protective
however, several drawbacks to the model; immunity may exist. It has been difficult
notably, the newborn is immunologically to repeat these studies in infants because of

EU infants IP infants

P = 0.035 P = 0.003

CCL3 (ng/ml)



<4 4 <4 4

CCL3L1 copies per diploid genome

Figure 8.3 CCL3 production by cord blood cells. CCL3 concentrations in
mitogen-induced mononuclear cell cultures of umbilical cord blood from
exposed and uninfected (EU) and intrapartum-infected (IP) infants are
stratified on the basis of low numbers (less than 4) or high numbers (4 or
more) of CCL3L1 copies per diploid genome. Small, black horizontal lines,
median values; solid gray lines across low and high groups, median values
for each total group; gray dashed line, median value for uninfected controls.
Reprinted by permission from Macmillan Publishers: Tiemessen CT, Kuhn L.
CC chemokines and protective immunity: insights gained from mother-to-
child transmission of HIV. Nat Immunol. 2007;8:219222.

136 Immunology of HIV Infections

small blood volumes. Nevertheless, some encoded for one or both of the functional
studies, using IL-2 production as a marker genes (CCL3 or CCL3L1) is involved, sug-
following stimulation with envelope gesting that the abundance of this protein
peptides, have shown that none of the may be important in HIV-protective immu-
children with apparent HIV-specific T-cell nity. Functional capacity, not just gene cop-
response developed HIV infections despite ies (four or more in some cases) of these pro-
exposure to HIV through breast-feeding. teins, is important. This suggests that the
However, this observation does not susceptibility phenotype lives at the level
completely explain why some babies are of induction of gene expression (see Figure
infected with HIV and others are not. In a 8.3) of CCL3 and CCL3L1, resulting in quali-
landmark study, Gonzalez and colleagues tative differences in protein production.
(2005) showed that segmental duplication Turning to their use as vaccine candi-
occurred in the gene CCL3L1-like chemo- dates, Tiemessen and colleagues (2007)
kine. CCL3L1 as well as CCL3 are ligands suggest that, if CCL3 is a crucial molecule
for CCR5 and are associated with lower in protection against HIV by virtue of its
susceptibility to HIV. Moreover, a study adjuvant ability, it may be useful as a vac-
of maternal-infant HIV transmission has cine adjuvant. In this manner, one does not
shown that a phenotype of deficient mito- have to rely on the hosts ability to produce
gen-induced CCL3 production is associated it (production may be deficient in certain
with a greater risk of HIV infection during populations; see Figure 8.4).
labor and delivery. Thus the maternal-infant Innate immunity has been mostly
model has demonstrated that the protein neglected in favor of HIV-specific
Response magnitude

CCL3 adjuvant

Time after vaccination

WT production Deficient production Deficient production

+ CCL3 adjuvant

CCL3 production phenotype

CCL3 and CCL3L1 genotype

Figure 8.4 Possible influence of CCL3 and CCL3L1 genotype on the development of adaptive immunity to
HIV vaccines and restoration of loss of function in people with deficient production through the provision of
CCL3 as adjuvant. CCL3 and CCL3L1 genotypes determine the production phenotype as wild type (WT) or
deficient (red arrowheads). Left, wild-type production of CCL3 as the critical component of a rapid innate
immune response (red line) that instructs the effective development of subsequent adaptive immunity
(green line). Middle, deficient production of CCL3 translates into an ineffective adaptive immune
response. Right, provision of CCL3 as an adjuvant (blue line) compensates for deficient host production
and restores the development of adaptive immunity to wild-type capability (dashed green line). Reprinted
by permission from Macmillan Publishers: Tiemessen CT, Kuhn L. CC chemokines and protective
immunity: insights gained from mother-to-child transmission of HIV. Nat Immunol. 2007; 8:219222.

Immunology of HIV Infections 137

immunity in HIV vaccine studies. CCL3 In fact, a significant obstacle in HIV
production should be considered a cor- vaccine research has been the difficulty in
relate of protective immunity. A desired developing an appropriate animal model
response would be sufficient production with many of the features of human HIV
of CCL3 at the site of HIV and vaccine infection. The only animals susceptible
encounter. It is imperative to delineate the to experimental infections with HIV are
precise functions of CCL3 in the immune chimpanzees, Pantroglodytes, and pigtail
response to HIV, aside from its noncyto- macaques, Macaca nemestrina. These ani-
lytic inhibitory effect on HIV, as the abil- mals maintain low levels of persistent
ity of CCL3 to drive the development of virus but do not develop clinical manifesta-
adaptive immunity might be the crucial tions of AIDS. In contrast, Asian monkeys,
factor in overall protection. Many impor- especially rhesus monkeys from India,
tant questions arise, given the findings are highly susceptible to SIVmac infec-
outlined here. How should HIV vaccines tion and progressively develop an immu-
be designed? How can people who would nodeficiency syndrome that is similar to
be poor responders to vaccines (deficient human AIDS. Plasma viral levels during
CCL3 producers) be identified? How can acute and chronic SIVmac251 infection in
the genetically encoded loss of function these animals parallel those observed in
be overcome, and how can molecules be humans, with some animals containing
identified that can compensate for this? virus spontaneously and progressing to
Innate immunity must become a more disease slowly as in HIV-1 infected
integral component of studies of HIV vac- human long-term nonprogressors. In con-
cines, as understanding the interaction trast, others maintain high viral loads and
between innate and adaptive immunity behave like human rapid progresses. As
may hold the key to understanding what in HIV-1 infected humans, the cellular
constitutes protective immunity to HIV. immune response to SIVmac during acute
Studies of uninfected infants born to HIV- and chronic disease differs significantly,
infected mothers offer a unique human and evidence of immune escape has been
experimental model for extending the well documented. This is particularly true
understanding of such phenomena. of the mucosal immune system, especially
the many CD4+ CCR-5+ T cells in the gut-
associated lymphoid tissue, the major site
ANIMAL MODELS OF HIV INFECTION of viral replication and of CD4+ T-cell
depletion in SIV-infected macaques. This
Although many questions concerned with also mimics the human disease and thus
HIV biology were first learned in cul- the SIV macaque model is currently con-
tured cell lines infected with HIV or SIV, sidered the most appropriate animal
it became obvious that these cultured lines model for studies on potential protective
did not truly mimic the wild-type infec- immune responses against HIV.
tions seen in animal models. A number A second animal model of HIV vaccine
of small rodent models, such as the SCID research is based on the use of SIV/HIV
mouse, were developed and were helpful hybrid viruses that were engineered to
but somewhat artificial and did not really carry the env gene from HIV into the con-
mimic human disease. tent of the SIV genome. These viruses do

138 Immunology of HIV Infections

replicate in rhesus macaques and after genetic diversity of the virus and the unique
serial macaque passages do lead to the features of the HIV envelope protein. One
emergence of highly pathogenic SHIV thing we have learned from studies of HIV
variants that can wipe out circulating pathogenesis, however, has been that a vac-
CD4+ T cells within a few weeks and lead cine that induces a strong and specific T-cell
to a lethal immunodeficiency syndrome immune response in the absence of broadly
within a year of infection. neutralizing antibodies may blunt initial
However, the problem has been viremia, even if the infection is not com-
whether these variants, like SHIV 89.6P, pletely prevented. Moreover, they may pre-
are relevant to HIV infections. For exam- vent the mass destruction of CD4+ T cells,
ple, they exhibit an X4 cellular tropism thereby helping to control the infection and
and are therefore easier to contain by vac- prolong disease-free survival.
cine-induced immune response than R5 Before we go to the actual vaccine can-
viruses such as SIV. If we could obtain didates, some knowledge of the structure
SHIV strains with R5 tropism, this might of the HIV is warranted. HIV, together
be a better model to study. with the simian, feline, and bovine immu-
The real problem with the monkey nodeficiency viruses (SIV, FIV, and BIV)
models is that experiments to evaluate vac- all belong to the genus lentevirus in the
cine efficacy requires challenging the vac- family of Retroviridae. These viruses char-
cinated animals with high doses of virus acteristically produce slowly progressive
to get 100 percent infection, that is, 103 105 infections, and the replication depends on
TCID50 equivalent to 5 107 SIV RNA cop- an active reverse transcriptase enzyme to
ies/ml. This is in marked contrast to natu- transform the viral RNA genome into a
ral exposure to HIV in humans wherein the proviral DNA copy that integrates into the
doses of 103 HIV RNA copies/ml of semi- host cell chromosome. This step provides
nal plasma have been reported. However, the enormous genetic diversity of HIV iso-
these results are quite different in mucosal lates in the infected cell, since the reverse
challenge, wherein reported low doses transcriptase step is often fraught with
(1030 TCID50) with SIV results in the errors in replication.
same viral and immunological kinetics of Two types of HIV have been described:
infection as high-dose challenges. Thus, HIV-1 and HIV-2, the latter being less
this new type of mucosal challenge might virulent, less transmissible, and confined
change the results of preclinical vaccine mostly to West Africa. HIV-1 is phylo-
efficacy studies in the future. genetically close to SIVcpz commensural
virus in chimpanzees, which probably
arose as a specific transmission event from
VACCINES TO PREVENT HIV chimps to humans. However, SIVmac is
INFECTION the etiological agent of simian AIDS and
has many of the features of HIV AIDS.
Despite the many efforts to develop a safe HIV-1 is further divided into three major
and effective vaccine against HIV infection, groups: M (major), O (outlier), and N (non
the results to date have been negative or M or O). Most of the strains responsible
controversial at best. Among the reasons for for the AIDS epidemic belong to the M
this state of affairs have been the enormous group, and this group has been divided

Immunology of HIV Infections 139

into ten different subgroups known as host. The positive side is that although
clades (AK). the attenuated vaccine does not prevent
The genome of HIV is a single- infection with a wild-type SIV infection,
stranded positive small RNA molecule, it does prevent that infection from going
approximately 9.5 kbs in length and on to produce the disease AIDS. Thus, this
encodes the typical retrovirus proteins approach does not appear feasible now.
Gag further divided into M (matrix), C However, what is being explored is the
(capsid), and N (nucleocapsid); Pol, cleaved nature of the protective immune response
into protease, reverse transcriptase, and in these monkeys after infection with
integrase; and ENV 160 kD glycoprotein, the attenuated virus.
divided into an external gp120 and a trans- Several reports raise the question
membrane gp41 subunit that together whether these attenuated vaccines will give
form trimeric spikes on the surface of the the broad protection so eagerly sought. As
virion. Furthermore, the HIV genome Anne Piantadosi and colleagues (2007) have
encodes a variety of nonstructural proteins pointed out, HIV-1 superinfection (reinfec-
such as transactivation protein (Tat) splice tion) does not always protect against other
regulator protein (Rev) and accessory pro- strains. In their studies, they screened a
teins such as Nef, Vpr, and Vpn. cohort of high-risk Kenyan women by com-
paring partial gag and envelope sequences
over a five-year period, beginning with the
VACCINE CANDIDATES primary infection. Of thirty-seven women
screened, seven were found to have
There have been many attempts to con- superinfections, including cases in which
struct a vaccine that is both protective and both viruses were from the same HIV-1
has a low cost of production; yet, there is subtype A. In five cases, the superinfect-
still not a promising candidate. Part of the ing strain was detected in only one of the
problem is that to be effective the vaccine two genomes examined, suggesting that
has to be given early or before exposure to recombination frequently occurs following
the virus. Ideally a vaccine could prevent HIV-1 superinfection. They conclude that
the disease entirely or at least shut down superinfection commonly occurs after the
the viremie phase (see panel B of Figure immune response against the initial infec-
8.4). For the sake of convenience, we have tion has had time to develop and mature.
listed the vaccine approaches separately Supporting this claim was an earlier report
with their promises and caveats. by Altfeld et al. in Nature in which this
superinfection occurred despite broad
CD8+ T-cell responses (twenty-five distinct
Live Attenuated Vaccines
epitopes) to many HIV viral proteins. They
The observation that NEF strains could conclude that superinfection can occur in
offer protection against challenge with the setting of a strong and broadly directed
pathogenic SIV infections in rhesus virus-specific CD8+ T-cell response.
macaques served as the model for the use of Yet, other reports by Chakraborty and
this type of attenuated vaccine. However, colleagues indicate that superinfection may
this mutants drawback is that it produces not be so high. They studied fourteen HIV
a lifelong persistent viral infection in the seroconcordant couples (i.e., partners were

140 Immunology of HIV Infections

independently infected with different HIV- Subunit Vaccines
1 strains) with high risk of re-exposure to
Most of the research efforts in HIV vaccines
the virus. Phylogenetic analyses based on
have gone into the subunit vaccines involv-
pol and env global sequences obtained from
ing the gp120 envelope proteins, which also
more than a 100 longitudinal plasma samples
includes the gp41 domain. Both of these
over one to four years failed to detect HIV-1
proteins do elicit neutralizing antibodies
superinfection in this cohort of patients. They
to the homologous vaccine strain but not to
conclude that chronic HIV infection seems
heterologous primary isolates in the animal
to confer protection against superinfection
model. Despite these caveats, two large-
with a second HIV-1 strain. Obviously, more
scale phase III clinical trials of this type of
work needs to be done to determine what
vaccine have been carried out involving
factors are responsible for superinfection in
7,500 high-risk individuals. Neither of these
some individuals and protection in others,
trials showed a significant reduction in HIV
both at high risk of reinfection.
infection in the vaccinated individuals,
even with continuous booster shots dur-
Inactivated Viral Vaccines ing the three-year trial. A number of other
trials involving trimeric gp140, a DNA vac-
Although the initial results with inacti-
cine encoding a V2-deleted gp140 and other
vated vaccine were negative with high
combinations, are in various phases, but the
doses of formalin treatment (loss of anti-
results are not known at present.
genicity of the viral envelope proteins), it
was quickly discovered that by using low
doses of formalin, the antigenicity of the
Cellular Vaccines
envelope proteins was preserved. This
preparation was capable of inducing viral A strong and specific T-cell immune
neutralizing antibodies in both mice and response in the absence of broadly neu-
nonhuman primates. This area is presently tralizing antibodies may blunt the ini-
being actively pursued, but the problem tial viremia, even if the infection is not
will be overcoming the rapidly changing completely prevented. Thus, more recent
antigens of the wild-type virus. vaccine efforts have been directed toward
A second approach has been to inacti- stimulating the cellular immune response.
vate the domains of two nucleocapsid pro- Particular attention has been paid to those
tein zinc fingers. This has been achieved vaccines that induce an HIV-specific
by treating these complexes with mild CD8+ CTL response whose role in the con-
oxidation or alkylation procedures, which trol of virus load and evolution of disease
completely inactivates both HIV-1 and SIV has been well documented in the macaque
but keeps the envelope glycoprotein spikes model. Although the T-cell vaccines do not
intact and functional. Studies in the SIV prevent the HIV infection, they do help
macaque model revealed that monkeys vaccinees who get infected to control viral
vaccinated with the inactivated virus were replication and reduce viral loads, thus
not protected against infection with the resulting in less risk of transmission of the
wild-type virus, but the levels of SIV vire- disease to seronegative partners.
mia were low and there was no depletion Vaccines have been based on this strat-
of CD4+ T cells. egy with particular emphasis on the use of

Immunology of HIV Infections 141

naked DNA vaccines and live recombinant a limited number of infected individuals,
vectors, for example, naked DNA vaccines especially in developing countries where
expressing the HIV-1 gag gene and either most of the epidemic is occurring.
IL-12 or Il-15, which was developed by Thus, the need for a safe, effective, inex-
Wyeth and is presently in several phase pensive vaccine to prevent further spread
I trials in the United States, Brazil, and of the infection becomes an increasingly
Thailand. DNA vaccines have been found important goal. Unfortunately, most of the
to be most useful as priming vaccines in vaccine trials to date have not been success-
prime-boost strategies, using live recombi- ful in this regard, and many investigators
nant vaccines for booster immunization. have turned their attention to vaccines that,
While the original recombinant HIV if given early in the infection, may pro-
vaccines used vaccinia virus and were well tect individuals from developing AIDS.
tolerated, safety considerations in immu- A number of these vaccines show prom-
nodeficient hosts has led investigators to ise and are in various stages of clinical or
substitute the canarypox virus for the vac- nonhuman primate trials. As stated in the
cinia virus. However, the immunogenicity 2007 New England Journal of Medicine article
of the pox-virus-based vaccines in humans by Johnson and Fauci, There is optimism
has been relatively modest with less than that even a less than perfect vaccine could
35 percent of the vaccinees scoring positive benefit individual recipients and the at risk
for T-cell responses. In contrast, replication- community. However such a vaccine will
defective adenovirus type 5 (ad5) appears have to be delivered as part of a comprehen-
to be one of the most promising live virus sive multifaceted prevention program.
vectors for HIV vaccines. Merck has used These are, however, to be considered as
this complex and showed that 50 percent first-generation vaccines. The ultimate goal
of the volunteers had significantly long- will be to produce an effective preventive
lasting CD8+ T-cell responses to HIV-1 vaccine. This will require new thinking about
peptides. A trivalent recombinant ad5-gag/ vaccine approaches, innovative solutions, and
pol/nef complex has been engineered and persistence, but the total prevention of this
retested in human volunteers. A phase II devastating disease is a worthwhile goal.
trial of this candidate is being investigated
in 1,200 men and 400 women at high risk
of exposure to HIV infection who will be
followed for three years. The results of this REVIEWS
trial will appear in 2008. Duerr A, Wasserheit JN, Corey L. HIV vac-
cines: new frontiers in vaccine develop-
ment. Clin Infect Dis. 2006;43:500511.
RESEARCH review of vaccine research and devel-
opment: the human immunodeficiency
The use of antiretroviral combination virus (HIV). Vaccine. 2006;24:4062
regimes has made significant advances in 4081.
the treatment of HIV infections in indus- Johnson MJ, Fauci AS. Current concepts:
trialized countries. However, these treat- an HIV vaccine evolving concepts.
ments are expensive and would only reach New Engl J Med. 2007;356:20732081.

142 Immunology of HIV Infections

SUGGESTED READING Heeney JL. Primate models for AIDS vaccine
Altfeld M, Allen TM, Yu XG, et al. HIV-1 development. AIDS. 1996;10(suppl A):
superinfection despite broad CD8+ T cell S115S122.
responses containing replication of the Kaul R, Dong T, Plummer FA, et al. CD8+
primary virus. Nature. 2002;420:434439. lymphocytes respond to different
Chakraborty B, Valer L, De Mendoza C, HIV epitopes in seronegative and
Soriano V, Quiones-Mateu ME. Fail- infected subjects. J Clin Invest.
ure to detect human immunodefi- 2001;107(10):13031310.
ciency virus type 1 superinfection in 28 Kaul R, Plummer FA, Kimani J, et al.
HIV-seroconcordant individuals with HIV-1-specific mucosal CD8+ lym-
high risk of reexposure to the virus. phocyte responses in the cervix of
AIDS Res Hum Retroviruses. 2004;20: HIV-1-resistant prostitutes in Nairobi.
10261031. J Immunol. 2000;164:16021611.
Devito C, Broliden K, Kaul R, et al. Kaul R, Rowland-Jones SL, Kimani J, et al.
Mucosal and plasma IgA from HIV- Late seroconversion in HIV-resistant
1-exposed uninfected individuals Nairobi prostitutes despite pre-existing
inhibit HIV-1 transcytosis across HIV-specific CD8+ responses. J Clin
human epithelial cells. J Immunol. Invest. 2001;107:341349.
2000;165:51705176. Piantadosi A, Chohan B, Chohan V, et al.
Devito C, Hinkula J, Kaul R, et al. Muco- Chronic HIV-1 infection frequently fails
sal and plasma IgA from HIV-exposed to protect against superinfection. PLoS
seronegative individuals neutral- Pathog. 2007;3(11):e177.
ize a primary HIV-1 isolate. AIDS. Quaranta MC, Mattiol B, Giordoni L,
2000;14:19171920. Viora M. The immunoregulatory
Editorial. New York Times. June 1, 2007. effects of HIV-1 Nef on dendritic cells
Ellenberger D, Otten R, Li B, et al. Evidence and the pathogenesis of AIDS. FASEB J.
of protection after reported mucosal SHIV 2006;20:21982208.
challenges to HIV-1 vaccinated rhesus Rowland-Jones S, Pinheiro S, Kaul R.
types. AIDS Vaccine Meeting, Montreal New insights into host factors in
Quebec, Canada. Abs. 110; 2005. HIV-1 pathogenesis. Cell. 2001;104:
Gonzalez E, Kulkarni H, Mangamo A, 473476.
et al. The influence of CCL3L1 gene- Tiemessen CT, Kuhn L. CC chemokines and
containing segmented duplications M protective immunity: insights gained
HIV-1/AIDS susceptibility. Science. from mother-to-child transmission
2005;307:14341440. of HIV. Nat Immunol. 2007;8(3):219222.

Immunology of HIV Infections 143

9. Immunological Aspects of Allergy and Anaphylaxis

Paul M. Ehrlich, M.D., and Jonathan D. Field, M.D., F.A.A.A.I.

INTRODUCTION immediate hypersensitivity reaction. This

reaction will be described in the next
Adverse responses to otherwise innocuous section.
substances we are exposed to constitute
the crux of allergic reactions. Reaction to
exposure to these substances may vary ALLERGIC HYPERSENSITIVITY
from a slight rash, easily treated with an
antihistamine, leukotriene modifier, or In 1964, Gell and Coombs classified four
corticosteroid cream, to a multisystemic types of immunologically mediated hyper-
reaction, with catastrophic consequences sensitivity states. The majority of disease
or anaphylaxis. All that will be discussed states encountered in the clinical practice
in this chapter have, as a common factor, of allergy are related to type I, or immediate-
various aspects of the immune system with type hypersensitivity. In this model, an
inflammatory responses involving these allergen interacts with preformed IgE
seemingly innocuous substances. on the surface of a mast cell or basophil.
Allergic reactions may be found in up This interaction causes cross-linking of
to 20 percent of the general U.S. popula- the FceRI receptor and release of multiple
tion, but by the age 6, 40 percent of the mediators, including histamine, leukotri-
children in the United States have some enes, and various interleukins. Depending
sort of allergic problems. Although most on the relative localization of release, clini-
of these children have respiratory prob- cal states such as allergic asthma, allergic
lems, such as allergic rhinitis or bronchial rhinitis, or systemic anaphylaxis occur. Of
asthma, many of those with allergies may the remaining clinical allergy hypersensi-
also have atopic reactions to foods or medi- tivity states, type IV, or delayed-type hyper-
cations. As antigens are slowly introduced sensitivity, is most common. In this type,
into an infants environment or diet, the T-cell antigen receptors on TH1 or TH2
childs propensity to deal with these new lymphocytes bind to tissue antigens, caus-
substances may not be developed. In addi- ing clonal expansion of lymphocytes and
tion, childrens airways are small, their release of pro-inflammatory lymphokines.
gastrointestinal tracts are not developed, Distinct clinical entities such as contact der-
and their immune systems are not ready matitis (e.g., poison ivy) or tuberculin skin
to meet the challenges of these newly test sensitivity in pulmonary tuberculosis
introduced proteins called allergens. Most may occur relative to the site of the tissue
responses are Gell and Coombs type I or antigen.

Table 9.1 Gell and Coombs Classification of interleukin-4 (IL-4) and interleukin-13
Immune-mediated Allergic Responses (IL-13). These interleukins interact with
receptors on B lymphocyte cell surfaces,
Type Mechanism Responses
which promote class switching to the IgE
I IgE mediated Anaphylaxis, antibody subclass. The subsequent class
urticaria switch produces antigen-specific IgE anti-
II Complement- Cytopenias bodies with specificity toward common
cytotoxicity allergens such as pollen, animal dander,
III Immune Vasculitis/
food, or venom.
complex nephritis Genetic studies of atopic families have
deposition identified regions on chromosome 11q and
IV Delayed-type Dermatitis or 5q that affect IgE production. Chromosome
hypersensitivity hepatitis
5 contains multiple genes, including those
for IL-4, IL-5, and granulocyte-macrophage
colony-stimulating factor. Eosinophil sur-
ATOPY vival and mast cell proliferation are just a
few pro-allergic effects of these cytokines.
Clinical allergic diseases are predominately Chromosome 11 encodes the beta subunit
type I, or IgE mediated. Approximately of the high-affinity IgE receptor. Increased
40 percent of people in Western nations expression of this receptor on mast cells
are inclined toward an exaggerated IgE leads to a more vehement response to
response to multiple environmental aller- small numbers of antigens. This increased
gens such as pollen or animal dander. This expression explains how exposure to min-
allergic state, known as atopy, is the result ute amounts of allergen, such as venom
of multiple genetic and environmental from a stinging insect, can produce sys-
factors. temic anaphylaxis.
Our current understanding of the devel- Although atopy has a strong genetic
opment of an IgE response favors a TH2 component, environmental factors best
T-cell induction. When specific inhaled, explain the recent global trend toward
ingested, or absorbed proteins, or aller- increased prevalence of allergic disease.
gens, appropriately stimulate this subset Predictive factors include the following:
of the T-cell population, a series of cellular (1) decreased exposure to infectious dis-
reactions occurs that leads to IgE antibody ease during early childhood, (2) changes
production. in diet, (3) higher levels of allergen expo-
Inhalation of most proteins does not sure, and (4) increased environmental
cause IgE-mediated responses, whereas pollution. Of these factors, variances in
a limited number of small protein aller- exposure to infectious disease appear to
gens can elicit such reactions. Although have the greatest correlation with atopy.
the mechanism of allergic induction is Epidemiological studies point out a nega-
not completely clear, some general prin- tive association between atopic disease in
ciples have emerged. Allergens presented children and a history of measles or hepa-
transmucosally at very low doses induce titis A virus infection. It is hypothesized
IgE responses by TH2 cells. This subset that infections such as these tilt the pro-
of cells produces the primary cytokines, duction of cytokines toward interferon

146 Immunological Aspects of Allergy and Anaphylaxis

gamma (IFN-) and the TH1 cytokines, mediating anaphylaxis. Mast cells are
thereby decreasing production of TH2 found in large numbers beneath cutaneous
allergic cytokines such as IL-4. This the- and mucosal surfaces and are closely asso-
orys attractiveness may be its ability to ciated with blood vessels and peripheral
explain the global increase in atopy due nerves. Basophils, most closely related
to decreased infection rates in Western- to eosinophils, function similarly to mast
ized regions with aggressive vaccination cells. Basophils are present in the circula-
programs. Current research trials use this tion, while mast cells are present only in
theory with protein vaccines that promote tissue but in much greater numbers. When
TH1 responses to shift the immune sys- either of these cell types is triggered, a
tem away from this allergic phenotype. biphasic release of mediators occurs. The
In this chapter, we highlight the common pathophysiology of anaphylaxis is clini-
clinical manifestations of atopy and dem- cally defined by the physiological effects of
onstrate how immunological reactivity to the immediate-phase and late-phase medi-
key antigens underscores each condition. ators on the target organs. By definition,
Although the Gell and Coombs classifica- anaphylaxis involves the cardiovascular,
tion is not universally applicable, the fun- respiratory, gastrointestinal, or epidermal
damental immunological processes apply system; in most cases, multiple organs are
in most of the common clinical hypersen- involved.
sitivity states discussed next. In the immediate phase, preformed
enzymes and toxic mediators such as his-
tamine, tryptase, and heparin are the pre-
SYSTEMIC ANAPHYLAXIS AND dominant bioactive substances released.
ANAPHYLACTOID REACTIONS Acting on H1 and H2 receptors, histamine
promotes multiple effects such as increased
vascular permeability, vasodilatation, con-
Systemic anaphylaxis represents the clinical striction of bronchial smooth muscle, and
manifestation of type I hypersensitivity increased mucous secretion. Clinically,
that occurs when a specific antigen and a these reactions lead to
homocytotrophic antibody interact. The
reaction can be sudden and progress rap- 1. Airway and laryngeal edema and bron-
idly, often without a clear cause. Death chospasm with potential for complete
can occur because of vascular collapse or asphyxiation;
airway obstruction. The term anaphylaxis is 2. Gastrointestinal tract smooth muscle
derived from the Greek ana, meaning back- contraction, causing pain, vomiting,
ward, and phylaxis, meaning protection. and diarrhea;
Poiter and Richet coined the term in 1902 3. Blood vessel dilatation with potential
after sea anemone antigen injected into a for progression to circulatory collapse;
previously tolerant dog caused a fatal reac- 4. Cutaneous vascular permeability,
tion instead of the expected immunological resulting in flushing, urticaria, and
protection, or prophylaxis. angioedema.
Generally, antigen-specific IgE anti-
bodies on mast cells, or basophils, inter- Late-phase reactions are characterized
act with previously encountered antigens by induced production of mediators such

Immunological Aspects of Allergy and Anaphylaxis 147

as leukotrienes, chemokines, and cytokines, and IgG production. After fourteen days,
which are not preformed. Therefore, the they challenged the mice with antigen (goat
effects of these mediators are seen later into IgG) or rat antimouse IgE monoclonal anti-
the course, usually hours after the onset of body (mAb). The severity of anaphylaxis
an anaphylactic reaction. The late-phase was gauged by changes in body tempera-
effects include activation and recruitment ture, physical activity, and mortality. Find-
of TH2 inflammatory cells, including neu- ings in this experimental model included
trophils and eosinophils, as well as prom- similar anaphylactic responses regardless
ulgation of the smooth muscle contraction of anti-IgE, mAb-induced, or goat IgG
and vasodilatation initiated by the imme- antigen challenge. Anti-IgE, mAb-induced
diate-phase reactants. anaphylaxis was FcRI dependent and
Treatment of anaphylaxis is aimed at mediated predominantly by histamine. In
restoring blood pressure, decreasing tis- contrast, antigen-induced anaphylaxis was
sue edema, and reversing bronchospasm. Fc RIII and macrophase dependent with
Subcutaneous or intravenous epinephrine, PAF as the predominant mediator. This
oral or intravenous antihistamines, and mouse model provides evidence that ana-
systemic steroids are the mainstay of treat- phylaxis may have alternative pathways
ment. Patients should be monitored for at and mediators contributing to its pathogen-
least six to twelve hours because late-phase esis. Table 9.2 summarizes key substances
reactions are possible. and their role in anaphylactic reactions.

Experimental Models Representative Agents Causing

Animal models have been use to under-
stand various manifestations of anaphy- Multiple substances have been implicated
laxis. Circulatory collapse typically occurs as possible causes of anaphylaxis. The
in dogs; rabbits may suffer acute pulmo- most common substances include drugs,
nary hypertension; and guinea pigs may specifically low-molecular-weight com-
experience acute respiratory obstruction. pounds. In most cases, the parenteral mode
These reactions are classically mediated of administration is implicated, although
by the interaction of IgE; the high-affinity oral, inhaled, and even cutaneous exposure
IgE receptor found on mast cells, FcRI; and can induce anaphylaxis as well.
histamine. However, findings from several Penicillin, the prototypic beta-lactam
rodent studies suggest an alternative path- antibiotic, is the most frequent cause of
way involving the IgG receptor (Fc RIII), anaphylaxis in humans. The reaction
macrophages, platelets, and platelet- occurs when the compound is covalently
activating factor (PAF) may be more bound to tissue carrier proteins to form
important in anaphylaxis than previously drug-protein conjugates known as haptens.
realized. Strait and colleagues immunized Ninety-five percent of tissue-bound peni-
wild-type, IgE-deficient, FcRI-deficient, cillin is haptenized as benzylpenicilloyl,
and mast celldeficient mice using goat termed the major antigenic determinant. IgE
antimouse IgD antibody. This technique antibodies directed at this hapten appear
induced mastocytosis and a large response to be implicated in type I hypersensitivity
to goat antigen (IgG) with increased IgE reactions.

148 Immunological Aspects of Allergy and Anaphylaxis

Table 9.2 Mediators of Anaphylaxis

Mediator Action Signs/Symptoms

Histamine Vasodilatation Pruritis, edema, wheezing,

diarrhea, hypotension
Leukotrienes Bronchoconstriction Wheezing
Platelet-activating Bronchoconstriction, Wheezing, hypotension
factor vasodilatation
C3a, C5a Smooth muscle contraction, Wheezing, hypotension
Tryptase Proteolysis Edema, smooth muscle contraction

Skin testing using a commercially avail- risk. IgE antibodies to latex antigen Hev b1
able benzylpenicilloyl polylysine conjugate have been demonstrated by immunoassay
(Pre-Pen) may be used to predict the likeli- and are felt to play a pathogenic role in this
hood of an immediate-type reaction. Skin clinical entity.
testing cannot predict non-IgE-mediated Anaphylactic reactions involve an
reactions; therefore, a thorough history to immunological mechanism. However,
determine the necessity for skin testing is there are subsets of reactions clinically
important. indistinct from anaphylaxis, which occur
Insect venom, another common cause in a non-IgE-mediated fashion. In these
of anaphylaxis, accounts for approximately anaphylactoid reactions, certain substances
forty to fifty deaths each year in the United such as morphine and other agents such as
States. The Hymenoptera order, including radiocontrast media are common triggers.
hornets, wasps, honeybees, and yellow jack- In contrast to IgE-mediated anaphylaxis,
ets, contains the most common allergenic prior exposure is generally not required. In
species. Immunotherapy is 90 to 95 percent non-IgE anaphylaxis, some patients with
effective after five years of therapy. selective absence of IgA demonstrate ana-
Foods, including nuts and crustaceans phylactic symptoms following transfusion
(i.e., shrimp), are frequently the cause of IgA-containing plasma. Physiological
of anaphylactic reactions. With exercise- changes such as exercise, emotions, and
induced anaphylaxis, certain foods that are overheating can provoke symptoms in
normally tolerated such as celery, apples, patients with cholinergic anaphlyactoid
and shrimp can induce anaphylaxis when reaction. In this rare condition, an increase
ingestion is followed by exercise. in core body temperature causes a rise in
In recent years, natural rubber latex plasma histamine from mast cells. This
has been recognized as a source of ana- disease is an exaggerated form of urticaria,
phylaxis. Exposure can occur through con- described later in this chapter. In all cases,
tact or inhalation, and various scenarios no single pathogenic mechanism has been
of exposure include surgical procedures, defined, but it is likely that direct mast
dental exams, or sexual intercourse with cell activation accounts for most of these
latex condoms. Health care workers and disorders; complement activation has also
spina bifida patients are particularly at been reported.

Immunological Aspects of Allergy and Anaphylaxis 149

ALLERGIC CONJUNCTIVITIS number of cells provoking allergy symp-
toms, usually in spring and in fall. The
Overview aforementioned cell types, such as mast
The eye, one of the initial exposure inter- cells and eosinophils, interact and release
faces for allergens, is a common site for a variety of allergic mediators when
allergic disorders. Symptoms can range exposed to seasonal aeroallergens such as
from mild eye itching to chronic cataracts tree or grass pollen. Preformed mediators
and blindness. These disorders are differ- are released in an immediate phase, and
entiated by their clinical presentation as newly formed mediators appear approxi-
well as by the nature of the immunologi- mately eight to twenty-four hours after
cal changes occurring in the conjunctival exposure. These mediators have overlap-
surface. Ocular allergic inflammation is ping biological functions that contribute
typically associated with IgE-mediated to the typical ocular itching, redness, and
mast cell activation. Mast cellderived watery discharge associated with allergic
mediators (e.g., histamine, proteases, leu- eye disease. Inflammatory markers include
kotrienes, and cytokines) initiate a cascade up-regulation of intercellular adhesion
of events that culminate in the infiltration molecules such as ICAM-1. Increased levels
and migration of inflammatory leukocytes of helper T cell 2 subset (TH2) cytokines,
(neutrophils, eosinophils, and lympho- including IL-4, are found in the eye tissues
cytes) to the ocular tissue. This trafficking during allergy season. Clinically, the con-
of inflammatory cells requires attraction junctiva is red, with a clear discharge. The
of these cells initiated by chemokines, and eye is almost uniformly pruritic. Seasonal
directed migration of the inflammatory allergic conjunctivitis is commonly asso-
cells out of the bloodstream to the surface ciated with rhinitis, but it may be the
epithelium using adhesion molecules and predominant symptom of allergy.
their receptors. These immunological reac- Perennial allergic conjunctivitis, in
tions lead to the common allergic ocular contrast, is associated with a persistent
diseases described next. increase in the number of these allergic cell
types throughout the year as well as spe-
cific IgE (dust, mold, animal dander) found
Clinical Disease States
in the tears of affected individuals.
Allergic eye disease consists of four over- Vernal conjunctivitis is a chronic, bilat-
lapping conditions, including (1) seasonal eral conjunctival inflammatory disease
and perennial allergic conjunctivitis, (2) found primarily in young males with
vernal conjunctivitis, (3) giant papillary history of atopy during spring months.
conjunctivitis, and (4) atopic keratocon- Initial symptom onset is before puberty,
junctivitis. Distinct immunological changes and symptoms dissipate by the third
occurring at the conjunctival surface give decade of life. In severe cases, vernal con-
rise to the clinical spectrum seen in ocular junctivitis can lead to corneal scarring
allergy. and permanent vision loss. Histopatho-
Seasonal allergic conjunctivitis is logically, conjunctival infiltration with
the most common form of allergic ocu- basophils, eosinophils, plasma cells, lym-
lar disease. Changes in the conjunctiva phocytes, and macrophages characterizes
include a visible increase in the type and vernal conjunctivitis. With this cellular

150 Immunological Aspects of Allergy and Anaphylaxis

milieu, it appears that vernal conjunctivi- normal tissue. This infiltration is likely
tis is a combined immediate- and delayed- critical to the sight-threatening nature of
type hypersensitivity reaction. the disease. Table 9.3 summarizes the his-
Giant papillary conjunctivitis is a topathological and other laboratory mani-
chronic inflammatory process that leads festations of ocular disease.
to the production of giant mucosal ducts, Treatment is aimed at reducing the local
or papillae, on the conjunctival lining of inflammation. Ocular mast cell stabilizer
the upper eyelids. The immunopathologic drops such as olopatadine are the mainstay
mechanism is complex and is theorized to of treatment. In addition, treatment of nasal
be a mechanical trauma culminating in a symptoms with nasal anti-inflammatory
mast cellmediated delayed-type hyper- sprays promotes patency of the nasolac-
sensitivity. rimal duct. This combination treatment
The major signs and symptoms include allows the eye to drain excess allergens,
itching and a clear or white stringy dis- therefore diminishing allergic responses in
charge. A cobblestone appearance is the eye.
typical. It is commonly associated with
contact lens use. Foreign bodies and ocu-
Experimental Models
lar sutures or prosthetics may be causal as
well. Treatment typically involves avoid- Animal models of ocular allergies have
ance of the inciting process or use of ocular been developed in the past years to estab-
anti-inflammatory agents such as cromo- lish new therapeutic approaches and assess
lyn sodium or topical corticosteroids. immunological mechanisms. The current
Atopic keratoconjunctivitis (AK) is animal models are based on sensitization
observed in approximately 15 to 40 percent to and subsequent challenge with small
of patients with atopic dermatitis. Whereas numbers of allergens such as ovalbumin
allergic conjunctivitis is usually a self-lim- (Ova), ragweed pollen, or cat epithelium.
iting process, AK is chronic and can poten- The murine model of allergic conjunctivitis
tially cause loss of vision. Immunological represents the presently preferred species
and immunohistochemical studies reveal for investigating the immunological basis
mast cells, IgE antibody, eosinophils, and of the disease.
other inflammatory cells in similar quanti- Magone and colleagues applied such a
ties to those found in allergic conjunctivi- model to evaluate the role of various cyto-
tis. The finding of lymphocyte involvement kines, including IL-4, IFN-, and IL-12, in
explains the chronic nature of the disease the early and late phases of ocular allergy
as well as the threat to sight. Additional using knockout (KO) mice and neutral-
findings include antibodies to ICAM-1 and izing antibodies. In this model, multiple
HLA-DR throughout the ocular epithe- mice experimental groups, including
lium, suggesting increased antigen presen- (1) ragweed-sensitized wild type, (2) IL-
tation. Also, increased level of RANTES, 4KO type, (3) IL-12KO type, (4) IFN- KO
an eosinophil homeostasis chemokine, are type, (5) anti-IL-12/mAb-treated, and (6)
observed in immunohistochemical studies anti-IFN- mAb-treated mice were chal-
of the epithelium. Fibroblast numbers are lenged with ragweed allergen ten days
increased in the connective tissue with an after immunization. An additional group
increased level of collagen compared with received recombinant murine IL-12. They

Immunological Aspects of Allergy and Anaphylaxis 151

Table 9.3 Histopathological and Laboratory Findings in Allergic Eye Disease

Disease Histopathology Laboratory Manifestations

Allergic Mast cell/eosinophil infiltration in Increased tear-specific IgE

conjunctivitis epithelium and substantia propria; antibody; histamine, tryptase,
mast cell activation; up-regulation TNF-
of ICAM-1 on epithelial cells
Vernal Increased eosinophils, mast cells Increased specific IgE/IgG
keratoconjunctivitis in conjunctival epithelium and antibody in tears; reduced serum
substantial propria; eosinophil histaminase activity; increased
major basic protein deposition in serum levels of nerve growth
conjunctiva, CD4+ cells; increased factor and substance P
collagen; increased ICAM-1 on
corneal epithelium
Giant papillary Giant papillae; mast cells in Increased tryptase in tears;
conjunctivitis epithelium; conjunctival thickening no increased histamine in tears
Atopic Eosinophils in conjunctival Increased serum eosinophils and
keratoconjunctivitis epithelium and substantia propria; IgE; increased specific IgE in tears;
increased mast cells, increased decreased cell-mediated immunity;
collagen; increased CD4/CD8 ratio increased conjunctival eosinophils
in conjunctival epithelium

found the anti-IL-12 antibody-treated and and lungs, are disproportionately affected
IL-12KO type mice failed to show allergic by allergic disease. In clinical practice, the
cellular infiltration into the conjunctiva. common manifestations include seasonal
IFN- KO type mice, however, had a sig- or perennial allergic rhinitis, allergic sinus-
nificantly stronger immediate-type hyper- itis, and allergic asthma. The former two
sensitivity reactions and prolonged allergic are often described together as allergic rhi-
cellular infiltration after ragweed exposure. nosinusitis. Although clinically distinct,
Overall, their data provided evidence of IL- each condition involves similar underlying
12 as an inducer of the late phase of ocular immunologic processes that will be dis-
allergy. Additionally, the data suggest IFN- cussed next.
as a limiting factor of the late phase and
a potential therapeutic cytokine in the pre-
vention of chronic allergic disease. Further
Allergic Rhinitis
research is aimed at preventing initial sen- OVERVIEW
sitization to allergens. The nose has five major functions,
including (1) olfaction, (2) aiding speech,
(3) airflow to the lungs, (4) humidifying and
ALLERGIC DISEASE OF THE warming air, and (5) filtering potentially irri-
RESPIRATORY TRACT tating particles from the air. Allergic reac-
tions occurring in the nose can have severe
The nature of the respiratory process effects on all levels of function, usually
involves inhalation of airborne allergens. divided into primary and consequential,
As a result, the components of the respi- or secondary symptoms. Typical primary
ratory tract, including the nose, sinuses, symptoms include nasal congestion, runny

152 Immunological Aspects of Allergy and Anaphylaxis

nose or rhinorrhea, as well as itchy palate as histamine and tryptase in the early-
and ears. Secondary symptoms include phase response. Shortly afterward, de
involvement of the middle ears, eustachian novo generation of mediators, including
tubes, and sinuses, inducing symptoms cysteinyl-leukotrienes (LTC4, LTD5, and
such as headache, ear pain, and decreased LTE4) as well as prostaglandin D2 (PGD2)
hearing. Multiple central nervous system occurs. These substances cause a pro-
complaints may occur, including fatigue, nounced inflammatory response leading
irritability, anxiety, and even depression. to the typical symptoms of paroxysmal
As in other atopic diseases, the pathophys- sneezing, nasal pruritis and congestion,
iology of allergic rhinitis involves specific clear rhinorrhea, and palatal itching.
IgE production after exposure to airborne Over the next four to eight hours,
allergens. The clinical manifestations and the mediators released during the initial
underlying immunological mechanisms of response set off a sequence of events, with
the disorder will be discussed later. enhanced inflammatory responses known
as the late-phase response. In this phase,
CLINICAL DISEASE STATES cytokines and various mediators released
Allergic rhinitis is the most common earlier promote the influx of other immune
allergic disease, affecting 25 percent of the cells by enhancing expression of vascular
population. It consists of two forms, sea- cell adhesion molecules (VCAMs) that help
sonal and perennial. Seasonal allergic rhi- traffic circulating eosinophils, neutrophils,
nitis is commonly referred to as hay fever and lymphocytes to the nasal endothelium.
or rose fever and is triggered by pollens Although each of these cell types plays a
with a typically well-defined season of ger- role in the late response, eosinophils appear
mination. Perennial allergic rhinitis has to be the main effector cell in allergic rhi-
similar symptoms, yet involved substances nitis. Nasal obstruction and secretions can
are present year-round, including animal lead to secondary effects, including ear and
dander, dust mites, and mold. In both sinus infections, sleep apnea, and asthma
forms, allergens interact with mast cells or exacerbations. Furthermore, the inflamma-
basal cells in the nasal mucosa. They are tory cytokines may circulate to the central
then presented by antigen-presenting cells nervous system, eliciting malaise, irritabil-
such as dendritic cells and macrophages. ity, and impaired concentration.
CD4+ lymphocytes are stimulated by this Treatment of allergic rhinitis involves
presentation to release interleukins includ- environmental control for indoor aller-
ing IL-3, IL-4, IL-5, IL-13, and other cyto- gens, reduction of swelling and congestion
kines that promote local and systemic IgE by nasal corticosteroids or oral leukotriene
production by plasma cells. In addition, receptor antagonists, and relief of rhinor-
these cytokines lead to enhanced chemo- rhea and nasal pruritis by oral or nasal
taxis, inflammatory cell recruitment, prolif- antihistamines. Recalcitrant cases may
eration, activation, and prolonged immune require desensitization by immunotherapy
cell survival in the airway mucosa. (allergy shots).
Within minutes of allergen inhalation
in sensitized individuals, IgE antibodies EXPERIMENTAL MODELS
fixed to mast cells and basophils trigger Recent research into the underlying
release of acute preformed mediators such immunological mechanisms of rhinitis

Immunological Aspects of Allergy and Anaphylaxis 153

has borne new insights into the patho- Other models focus on the eosino-
physiology of allergic rhinitis. Prior under- philic inflammation that characterizes
standing favored initial IgE production in allergic rhinitis as well as modulation of
regional lymph nodes or bone marrow. the allergic response. Hussain and col-
Failure of prior experiments to co-localize leagues sensitized BALB/c mice using Ova
IgE protein within B cells to the local tis- intraperitoneally. Subsequent challenge
sue environment weighed heavily against with aerosolized Ova took place. Vari-
localized tissue IgE production. In the past ous outcomes, including nasal symptoms,
ten years, however, a growing body of nasal submucosal eosinophilia, and bone
research has challenged the former dogma marrow eosinophilia, were measured.
that IgE production occurred remotely Afterward, a subset of mice received CpG
from the allergentissue interface. Studies oligodeoxynucleotides (ODNs), potent
of local messenger RNA (mRNA) sup- inducers of a TH1 nonallergenic response.
port the hypothesis of local protein syn- Using enzyme-linked immunosorbent
thesis. Durham and colleagues, using a assay, cytokine levels were measured.
combination of in situ hybridization and Findings included elevated IL-4 and IL-5
immunohistochemistry, showed that cells and suppressed IFN- in Ova-sensitized
expressing epsilonheavy chain mRNA mice compared with ODN-treated mice.
(C) were present in the nasal mucosa of In addition, the administration of ODNs
allergic individuals, and marked increases abrogated nasal symptoms and upper air-
of these cells occurred on exposure to aller- way eosinophilia compared with controls.
gen. Furthermore, the increases seen in Collectively, these models elucidate the
IgE and C mRNA after allergen challenge complex immunological basis of allergic
were inhibited by topical corticosteroids, rhinitis and demonstrate how manipula-
favoring a localized process. In addition, tion of the immune response can be used
Kleinjan and colleagues used similar tech- clinically as a potential treatment.
niques to demonstrate finally IgE-produc-
ing B cells in the nasal mucosa. Biopsies
Allergic Asthma
were obtained from normal subjects and
from seasonal and perennial subjects dur- OVERVIEW
ing pollen season and during house dust The human lung provides the funda-
exposure. The study found no differences mental function of gas exchange. As the
in B-cell numbers, either CD19+ (B cells) terminal level of the respiratory tract after
or CD138+ (immunoglobulin-secreting the nasal cavity and pharynx, it is constantly
plasma cells) in allergic and normal exposed to airborne particulate matter.
patients. Allergic patients, however, exhib- Allergic asthma is the manifestation of a pul-
ited significantly greater numbers of IgE- monary immune response to various inhaled
positive B cells than normal patients, and substances. Clinically, the cardinal symptoms
allergen-positive cells were only found in include (1) generalized but reversible air-
allergics with almost all such cells staining way obstruction, (2) wheeze, (3) dyspnea,
positive for IgE or CD138. The combination and (4) cough. Symptoms can range from
of these factors suggests local IgE produc- mild to life threatening. Typical allergens
tion may take place in the mucosa during include house dust mite, pollen, cockroach
natural allergen exposure. epithelium, animal dander, and fungi.

154 Immunological Aspects of Allergy and Anaphylaxis

As in allergic rhinitis, recent advances in exposure should always be attempted.
understanding the immunology of asthma Inhaled corticosteroids (ICS) remain the
have important therapeutic value, and mainstay of medical treatment as they
immune manipulation will likely become down-regulate multiple inflammatory
an important modality in the treatment of reactions in the lungs. Other adjuncts such
asthma. as leukotriene receptor antagonists modify
significant mediators of allergic inflamma-
tion present in asthmatic airways. Newer
Immunology of Asthma
treatments include monoclonal antibod-
From a histopathological standpoint, the ies directed against IgE (anti-IgE therapy),
inflammation in allergic asthma involves which have shown some success in
the entire thickness of the airway. Findings decreasing asthma symptoms and the need
include generalized edema, denudation for oral or inhaled corticosteroids.
of the epithelium, subbasement mem-
brane thickening, and smooth muscle and
Experimental Models
mucous gland hypertrophy. This process
begins when dendritic cells, a subset of Although our understanding of the
antigen-presenting cells found in the lung pathogenesis of allergic asthma is incom-
tissue, process inhaled antigens and pres- plete, animal models have been of great
ent them to T lymphocytes through the utility in elucidating the mechanisms of this
interaction of the receptor molecule CD28 disease.
on T cells and its ligand CD80 (B7.1) on A major topic of current research has
dendritic cells. This interaction results revolved around airway changes in the
in T lymphocyte development down the chronic asthmatic. This process, known
TH2 pathway. TH2 lymphocytes are char- as remodeling, is believed to result in
acterized by release of a family of pro- irreversible changes in the lung. McMillan
inflammatory cytokines, including IL-3, and Lloyd induced acute pulmonary
IL-4, IL-5, IL-13, tumor necrosis factor- eosinophilia and bronchial hyperreactivity
(TNF-), and granulocyte-macrophage in mice using multiple allergen challenges.
colony-stimulating factor. These cytokines They subsequently induced a chronic
promote development, activation, and sur- phase in a subset of mice using Ova chal-
vival of eosinophils. In addition, IL-4, IL-5, lenge. Evaluation at one month after Ova
IL-13, and TNF- activate endothelial cell challenge showed significant changes
adhesion proteins, ICAM-1 and VCAM-1, in the Ova-challenged mice. Compared
which assist inflammatory cell movement with the acutely challenged mice, the Ova
from blood vessels into the airway. IL- group showed deposition of collagen as
4 and IL-13 are key stimuli of B cells for well as airway smooth muscle and goblet
antigen-specific IgE production, which cell hyperplasia. Cytokine profiles in the
initiates the allergic cascade. As a whole, chronic phase revealed increases of IL-4,
these complex immunological processes transforming growth factor beta 1 (TGF-
lead to the pathologic processes that char- 1), and IFN- . These findings strongly
acterize asthma. support the concept of airway remodeling
Treatment is multifactorial. Environ- and reveal a dual TH1 and TH2 cytokine
mental measures to eliminate allergen profile in the chronic phase of asthma.

Immunological Aspects of Allergy and Anaphylaxis 155

Another important focus of asthma
Diagnosis of Food Allergy and
research centers on so-called inner-city
asthma, an increasing epidemic in devel-
oped countries. Many epidemiological The gold standard of food allergy test-
studies have shown disproportional rates ing is the double-blinded, placebo-con-
of asthma in urban, lower socioeconomi- trolled food challenge, which will establish
cally stratified patients. Although many whether the patient is truly allergic to a
socioeconomic factors are thought to play specific food. Because this method of test-
a role, distinct allergic triggers specific to ing for food allergies is time consuming,
these environments appear to be impor- expensive, and potentially dangerous, in
tant. Using recombinant proteins, Sarpong, vivo testing is usually performed in food
Zhang, and Kleeberger (2004) evaluated allergy centers. Occasionally, straight food
two such allergens, cockroach (Bla g 2) challenges may be tried when the chances
and dust mite (Der f 1), in inbred mouse of reactions are minimal and the serum IgE
strain (A/J). Mice were immunized with (radioallergosorbent test, or RAST) specific
Bla g 2 and Der f 1 or a combination on for the allergen is low or negative.
days 0 and 7 and were inhalant challenged Approximately 20 percent of the U.S.
on day 14. Airway hyperreactivity and population may perceive food as causative
airway cellular content were subsequently reaction in food allergies, whereas the true
studied. Findings included dose-related prevalence of food allergies is approxi-
statistically significant increases in airway mately 1 percent. Food allergies are usually
reactivity and inflammatory and epithe- found in individuals with a strong per-
lial cell measurements. Compared with sonal and family history of allergies. Atopic
individual antigens, however, enhanced responses are associated with many foods.
inflammatory cell levels and epithelial cell In children under the age 2, 90 percent of the
numbers, but not airway reactivity, were incriminating foods are eggs, milk, legumes
noted in the combined group. This model, (such as peanuts, which are not nuts), and
which has been subsequently validated, soy. In adults, fish, shellfish, fruits, and tree
has practical implications for preventing nuts might be added to the list.
Case 9.1 Peanut Allergy
N.G. is a two-year-old female who
FOOD ALLERGY AND INTOLERANCE was well until the age of seventeen
months when she developed a watery,
Perhaps of all allergic reactions, the pub- runny nose that persisted throughout
lic, clinicians, and medical personnel mis- the spring but was gone by the sum-
understand food allergy most. As little as mer. She responded well to conventional
1 percent of the U.S. population has true antihistamines and did well during the
food allergies compared with 20 percent summer. In the middle of August, she
who are perceived to have them. True broke out in hives and wheezing soon
food allergy is a typical, and sometimes after eating a peanut butter sandwich,
catastrophic, type I, IgE-mediated reaction, but the symptoms were reversed with
which preferably must be proven by in the antihistamine. She had never had
vitro or in vivo testing. peanut butter before that event, but her

156 Immunological Aspects of Allergy and Anaphylaxis

mother was a self-described peanut but- that she described as maddening. She
ter addict and consumed large amounts assumed she had food allergies, so she
during her pregnancy with and breast- tried to eliminate various substances
feeding of N.G. N.G.s subsequent skin from her diet, including dairy products,
prick test with peanut revealed a 20 mm fish, and nuts without any positive
by 30 mm wheal and flair response with response. Eventually, she took antihis-
a markedly elevated RAST to the peanut tamines, with marginal improvement
protein. She has remained peanut free and sought medical attention after a
since that time. She is able to eat tree nuts three-month ordeal. An extensive his-
(e.g., almonds and cashews) without a tory was taken and allergy tests (all
problem because peanuts are not nuts. negative) and a variety of serum stud-
ies were performed. She was diagnosed
with a thyroid abnormality. She was
Food Intolerance
referred to an endocrinologist and did
True allergic reactions (IgE mediated) to well on therapy, which had nothing to
food must be differentiated from food intol- do with an allergic diathesis.
erance, which is rarely multisystemic, not
necessarily found among atopic patients,
Urticaria and Angioedema
and with no positive skin prick tests or in
vitro responses (RAST). A nonallergic reac- Angioedema and urticaria are two of the
tion that typifies food intolerance would more perplexing problems evaluated by
be an acute gastrointestinal response in a physicians, and cases of chronic urticaria
lactose-intolerant individual. This patient (hives of more than six weeks duration)
does not have the enzyme lactase and, may yield diagnoses in about 20 percent of
therefore, cannot break down sugar lactose those evaluated. Perhaps most important
in milk and other foods into glucose and in the evaluation is the medical history, fol-
galactose. Profound diarrhea or vomiting lowed by understanding whether physical
usually results without any other system issues such as pressure, cold, and scratch-
involved (respiratory or skin, for example). ing affect the skin. These issues must be
Food avoidance is the treatment of choice, carefully explored in the initial questioning
as it is with food allergy, and over time, of the patient.
food intolerance may resolve. Lactaid is a Certain types of urticaria may be
brand of milk in which the enzyme lactase is brought on by physical disruption of mast
added so that the milk may be consumed. cells by either cold, scratching, or pressure.
There is no IgE mediation of these reac-
tions, unlike those from foods, but hista-
SKIN DISEASES AND ALLERGY mine release from the cells is common to
all. Swelling of the skin due to damage to
Case 9.2 Skin Reactions small blood vessels causes burning and
A.A. is a fifty-six-year-old female pain of the areas involved and may last
who was well until she began to develop for several days. The burning and pain are
acute, episodic, or transient swelling of unrelated to allergies or physical mast cell
her skin (urticaria), with intense pru- disruption. These might typically be related
ritis (itching) and erythema (redness) to a systemic disorder, and precipitating

Immunological Aspects of Allergy and Anaphylaxis 157

Box 9.1 Types of Urticaria evaluated by an allergist. Prick skin
tests revealed a significant reaction to
Classification Diagnosis
dermatophagoides farinae or dust mite,
Plain urticaria Acute and chronic and subsequently, he covered the
Physical urticaria Aquagenic urticaria sheets, mattress, box spring, and pil-
Cholinergic urticaria
Cold-induced lows with sealable covers of less than 2
urticaria of mesh. He also used a mild cortico-
steroid cream; the scratching stopped,
Dermatographism and his skin cleared up.
Angioedema C1 esterase inhibitor
deficiency Case 9.4 Atopic Eczema
Atopic eczema is an inflammatory
skin disease characterized by severe
IgG autoantibodies against C1q may be pruritis with a chronic, relapsing
found along with other organ involvement. course. Acute eczema is associated with
Fevers, bone pain, and arthralgia may also marked erythema, superficial papulae,
occur along with the skin involvement. and vesiculae, which easily excoriate
Swelling of the skin without wheals, leading to crusting. It is the most com-
or angioedema, may be due to C1 esterase mon dermatologic problem for which
inhibitor deficiency, and this may be a a dermatologist is consulted. The IgE
hereditary or an acquired disease. The reac- level in most people with atopic eczema
tions may be painful subcutaneous or sub- is elevated and is specific to foods and
mucosal reactions initiated by trauma and environmental allergens.
may involve the gut, causing colic, or the Provocative factors of atopic eczema
larynx, causing difficulty breathing. This are varied and may include stress, skin
type of angioedema is rare but potentially irritants, food allergens, environmen-
life threatening. tal allergens, climate, season, and hor-
mones. Avoiding the suspected foods
Case 9.3 Atopic Dermatitis has a mixed response. Often food restric-
J.E. is a nineteen-year-old male with tion during a childs early life leaves the
a family history of allergies who devel- child open to detrimental psychological
oped severe pruritis, which is usually and growth issues during those impor-
worse at night. As a toddler, he had aller- tant developmental years. Fortunately,
gic reactions to milk and eggs, which many food allergies tend to resolve
resolved by age 4. In addition, he was a over time; however, in the beginning
mild asthmatic until puberty. In his mid- of avoidance techniques, careful evalu-
teens, he developed a pruritic, crusty ation is mandatory. In addition, exces-
rash, which was relieved by cortico- sive amounts of certain foods such as
steroid creams and antihistamines and citrus in children and alcohol in adults
resolved somewhat during the summer make eczema worse, and these reactions
months, where he lived near the ocean. may be unrelated to an IgE-mediated
After switching to cotton sheets problem. It seems that food allergy and
and eliminating his wool blanket, he the intolerance (non-IgE mediated) are
improved slightly and was finally important factors in atopic dermatitis.

158 Immunological Aspects of Allergy and Anaphylaxis

Box 9.2 Classification of Common Eczematous animals, or dust mites. Prevalence is com-
Skin Diseases mon, and the treatment usually involves
mast cell inhibitors and topical and sys-
Disease Feature
temic antihistamines. Topical ophthalmic
Allergic contact Provoked by local steroids are usually avoided.
dermatitis contact with allergen
Atopic keratoconjunctivitis is a rare,
Atopic dermatitis Extrinsic type (i.e.,
atopic dermatitis) lifelong problem, seen more commonly in
Irritant contact Provoked by local
adults with atopic disease such as atopic
dermatitis contact dermatitis. This facial eczema usually
involves the eyelids, and the lid margins
usually show chronic inflammation of the
Although those with atopic eczema lash follicles (blepharitis) and staphylococ-
often test positive to various foods and cal organisms. In addition, the lid margins
house dust mites, strict avoidance of these may thicken and keratonize, and the lids
allergens does not improve the problem. may turn out or turn in. Corneal plaques,
Although allergies are an important cataracts, and defects of the corneal epithe-
component in atopic eczema, an esti- lial may lead to loss of sight.
mated 90 percent of patients with moder- Topical mast cell stabilizers may be
ate to severe eczema have staphylococcal used for atopic keratoconjunctivitis, but
infection of the skin. This condition and often topical corticosteroids are required.
large insensible water loss make atopic Facial eczema must be controlled, and the
eczema a difficult medical problem. lid margins must be treated. Conventional
The effects of the weather, including treatment for seasonal or perennial allergic
temperature and humidity, on eczema conjunctivitis is not sufficient. Atopic kera-
add to these immunological and nonim- toconjunctivitis may be difficult to manage.
munological factors. A day at the beach
may have a positive effect on atopic Case 9.4 History Contact Dermatitis
dermatitis, while a dry, cold winter has A.A. is a fifty-six-year-old woman
the opposite effect. Wet wraps are ben- who decided, after many years of pro-
eficial because they replace the skins crastination, to have her ears pierced.
moisture loss. Corticosteroid creams, Within four to six weeks of the proce-
hydrotic creams and ointments, and dure, she developed protracted pruritis
occasional oral antibiotics are the main- (itchiness) at the site of the piercing, with
stays of treatment. Avoiding allergens, a crusty rash. She then found she could
local skin care, and treatment of the pru- no longer wear her wedding band or
ritis are the best approaches for treating bracelets because of similar reactions.
atopic eczema. Patch testing to various allergens,
including nickel, was performed. She
Atopic Keratoconjunctivitis
had a severe forty-eight-hour reaction
Conjunctivitis is an inflammation of the to nickel, a metal used in jewelry to
conjunctiva, the inner eyelid surfaces and harden the gold or silver. A.A. could
the mucous membrane lining the sclera. no longer wear her earrings, rings, or
When the cause is seasonal or perennial bracelets. She could only wear 24-karat
(very common), the etiology may be pollen, gold or .999 fine silver.

Immunological Aspects of Allergy and Anaphylaxis 159

Box 9.3 Where Contact Agents are Found

Contact Agents Where Contactant is Found

Chloroisothiazolinone Preservatives in creams

Chromate Leathers and bleaches
Formaldehyde Cosmetics, newsprint, fabric
softeners, wrinkle-resistant clothing
Mercaptobenzothiazole Rubber products like boots and
Thiuram Rubber products, clothing dye
Vegetation Rhus (poison ivy), tulips

Contact Dermatitis many as 90 percent of normal individuals,

which is why the patients history with
Classical contact dermatitis is a Gell and
suspected contact dermatitis is impor-
Coombs type IV reaction, mediated by
tant. Among those situations patients
previously sensitized lymphocytes, which
need to elucidate are occupation, cosmet-
is exhibited by raised, very pruritic rash
ics, topical or systemic drugs, recreational
at the sight of the contact. Unlike allergic
activities, effects of holidays, and time
reactions of a type I, IgE-mediated contact
dermatitis, as a type IV reaction, involving
Testing for contact dermatitis involves
low-molecular-weight allergens (less than
placing a patch of the suspected substance
1 kDa). These contact allergens are haptens
or substances on the back. The area must
and need to link with proteins in the skin
be kept clean and dry for forty-eight
to become allergenic. These haptens may
hours, after which the various patches
be readily absorbed into the skin, a reac-
are removed, and the individual areas are
tion that renders them antigenic. If the skin
evaluated for inflammatory responses. A
is exposed to humidity or warmth, the
positive response then characterizes a type
penetration of the hapten is greater, and the
IV reaction, and avoidance of the offending
chance of developing contact dermatitis is
allergen is the treatment of choice. Thirty
greater. As these haptens make their way
milligrams per day of systemic steroids
into extravascular spaces, they combine
may be required when large areas of the
with serum proteins or cell membranes
skin are involved (>25 percent of body
of antigen-presenting cells. The processed
surface). Otherwise, corticosteroid creams
antigens is presented by Langerhans cells
may be employed; however, avoidance of
to T cells leading to a cascade of events
the contactant is most crucial.
that result in an influx of mononuclear
cells into the dermis and epidermis, hence
Although most people exposed to
these presenting allergens do not develop GENERAL
contact dermatitis, certain substances such Adkinson NF Jr, Yunginger JW, Busse
as dinitrochlorobezene may sensitize as WW, et al., eds. Middletons Allergy:

160 Immunological Aspects of Allergy and Anaphylaxis

Principles and Practice. 6th ed. Philadel- oligodeoxynucleoides. Laryngoscope.
phia, PA: Mosby; 2003. 2002;112(10):18191826.
Janeway CA Jr, Travers P, Walport Magone MT, Whitcup SM, Fukushima
M, Sholmchik M. Immunobiology: A, Chan CC, Silver PB, Rizzo LV.
The Immune System in Health and Disease. The role of IL-12 in the induction of
6th ed. New York: Garland Science; late-phase cellular infiltration in a
2004. murine model of allergic conjunctivitis.
Patterson R, et al. eds. Allergic Diseases: J Clin Immunol. 2000;105:299308.
Diagnosis and Management. 6th ed. McMillan SJ, Lloyd CM. Prolonged aller-
Philadelphia, PA: Lippincott-Raven gen challenge in mice leads to persistent
Publishers; 2002. airway remodeling. Clin Exp Allergy.
ANIMAL MODELS Sarpong SB, Zhang LY, Kleeberger SR.
Gronenberg DA, Bielory L, Fischer A, A novel mouse model of experimen-
Bonini S, Wahn U. Animal models of tal asthma. Int Arch Allergy Immunol.
allergic and inflammatory conjunctivitis. 2004;132(4):346354.
Allergy. 2003;58(11):11011113. Strait RT, Morris SC, Yang M, Qu X-W,
Hussain I, Jain VV, Kitagaki K, Businga Finkelman FD. Pathways of anaphy-
TR, OShaughnessy P. Modulation of laxis in the mouse. J Clin Immunol.
murine allergic rhinosinusitis by CpG 2002;109(4):658668.

Immunological Aspects of Allergy and Anaphylaxis 161

10. Immunological Aspects of Skin Diseases

James G. Krueger, M.D., Ph.D., and Lisa Zaba, M.D., Ph.D.

INTRODUCTION proteins, chemokines, cytokines, and lipid-

derived molecules. Keratinocytes can also
The skin is the largest human organ, and activate acquired immunity through syn-
its surface (measuring about 2 square thesis of heat shock proteins that activate
meters) protects the body from invading dendritic cells (DCs) in the skin.
organisms, toxins, and viruses. Loss of Normal skin contains at least two resi-
the protective epidermal barrier (second- dent populations of antigen-presenting
ary to burns or disease states) results in an DCs Langerhans cells and dermal DCs
increased risk of infection from a variety of Langerhans cells are randomly distrib-
organisms. uted throughout the living cell layers of
Normal skin may be divided into the epidermis and constitute about 1 per-
three major parts: epidermis, dermis, and cent of all epidermal cells. Although this
hypodermis. The epidermis is a stratified, cell type originates from precursors in the
squamous epithelia composed mainly of bone marrow, continued proliferation of
keratinocytes that differentiate to a physi- Langerhans cells in the epidermis appears
cal barrier (the stratum corneum). The to sustain steady state levels. However, if
dermis is composed largely of collagen large numbers of Langerhans cells are lost
and elastin fibers synthesized by dermal from the epidermis (e.g., induced migra-
fibroblasts. The dermis also contains a tion after antigen exposure or epidermal
rich vascular supply, nerves, and various damage), bone marrow precursors can
appendages such as hair follicles, eccrine replete epidermal stores. Langerhans cells
glands, and apocrine glands. The hypoder- may be identified visually by their char-
mis contains subcutaneous adipose tissue acteristic tennis racketshaped organelles
with associated vascular and neural ele- known as Birbeck granules that are a sub-
ments and contributes the largest bulk of domain of the endosomal recycling com-
the cutaneous organ. partment. CD1a and langerin (CD207) are
The skin is also an important immuno- antigens used to identify Langerhans cells
logical organ and is capable of mediating or on a molecular level. CD1a is an MHC-like
initiating both innate and acquired immune protein that mediates the presentation of
responses. Keratinocytes synthesize a range nonpeptide antigens to T cells, and lan-
of proteins such as defensins that directly gerin (CD207) is an endocytic receptor that
kill bacteria. Activated keratinocytes can recognizes bacterial mannose residues and
also rapidly recruit neutrophils and other transports them to the Birbeck granules.
innate immune cells through release of a Langerhans cells are immature DCs that
large number of mediators such as S100 survey the epidermal environment for

foreign antigens. If these cells capture an cells accumulating in the skin may play
antigen, or are triggered by cytokines or an active role in disease pathogenesis.
other danger signals, maturation ensues Selective targeting of activated T cells
such that the cells up-regulate MHC and and immune-related cytokines was then
co-stimulatory molecules and migrate shown to reverse psoriasis in a large num-
through dermal lymphatics to skin-drain- ber of cases. It is now believed that the
ing lymph nodes. Activated, or mature, epidermal hyperplasia is a consequence
Langerhans cells, in turn, activate nave of the immune activation of the focal skin
T cells, inducing T-cell proliferation and lesions. We consider that psoriasis and
differentiation into effector T cells. These atopic dermatitis are the most prevalent
Langerhans cell-induced effector T cells T-cell-mediated inflammatory diseases in
home specifically to the skin because they humans.
express cutaneous lymphocyte-associated To understand the cellular features of
antigen (CLA) that binds to E-selectin on the psoriasis plaque, a clinical and patho-
endothelial cells. logical description of the lesion is war-
Presumably, dermal DCs (HLA-DR+ ranted. Clinically, the plaque is a red,
cells that possess co-stimulatory markers raised, and scaling (flaking) region of the
and the integrin CD11c, but lack Birbeck skin 1 cm in size. Usually, many indi-
granules, CD1a, and CD207) have a similar vidual lesions cover the skin separated by
potential to activate, mature, and migrate normal skin. In extreme cases, virtually all
to lymph nodes; however, there is less of the skin surface can be affected. Histo-
experimental evidence for this outcome. logical features include (1) marked thicken-
Potentially, activation of CLA+ T cells is ing of the epidermis with rete elongation,
not limited to lymph nodes via Langerhans keratinocyte hyperplasia, and incomplete
cell migration but may also occur directly terminal differentiation of keratinocytes
in the skin with activated dermal DCs. (parakeratosis); (2) infiltration of skin
This chapter will explore in detail some lesions by many types of leukocytes; and
skin diseases that have a major immuno- (3) increased vascular growth (angiogen-
logical component in disease pathogen- esis) and dilation of blood vessels (Figure
esis. Two other immune-mediated skin 10.1). Leukocyte alterations in psoriatic
diseases atopic eczema and contact der- skin are extensive and include marked
matitis are dealt with in other chapters. numbers of type I T cells (TH1) and DCs,
both activated and nonactivated subsets of
each cell type, and neutrophils are present
PSORIASIS in the stratum corneum in most cases. T
cells are mainly skin-homing CLA+ mem-
Psoriasis vulgaris is an inflammatory dis- ory cells, either CD4+ TH1 or CD8+ TC1
ease of the skin that affects 23 percent of cells. A subset of CD8+CD103+ T cells is
people across North America and Europe. further specialized for epithelial homing
Until recently, this disease was thought through surface expression of the E-cad-
to be an abnormality in skin cell differ- herin binding integrin e7. Few allergic
entiation, resulting in keratinocyte hyper- type II T cells (TH2) cells exist in psoriasis
proliferation. However, work begun in the skin lesions. Instead, strong TH1 differen-
late 1970s showed evidence that immune tiation is suggested from genetic profiling

164 Immunological Aspects of Skin Diseases

Normal Skin Psoriatic Plaque
with nucleus
Corneocytes differentiate
without nucleus (orthokeratosis)
(CDB+)TC1 cell
granular layer absent TC1

granular layer present CD11C+

Mature Keratinocytes
EPIDERMIS Langerhans

Langerhans Cell CD11C+
Langerhans cell
cell (CDB+)TC1


DC Flow CD11C+
DC plasmacytoid (CD123+)DC DERMIS

Figure 10.1 Schematic drawings of normal skin and psoriatic plaque with infiltrating lymphocytes,
Langerhans cells, and proliferating keratinocytes.

of psoriatic lesional skin T cells compared neutrophils, which is unusual for a pure
with normal, including increased expres- T-cell-mediated response in the skin.
sion of GATA-3 and STAT-1 transcription An alternative hypothesis is that, in the
factors and TH1-deviating inflammatory genetically programmed psoriatic patient,
cytokines. Furthermore, systemic immune initial T-cell activation is not necessarily a
deviation in psoriasis patients is evidenced reaction to a cutaneous antigen but rather
by the threefold increase in circulating to bacterial antigens known to be immu-
blood TH1 cells compared with normal nologically cross-reactive with keratino-
controls. cytes such as the group A streptococcus.
On the basis of finding clonal popula- Evidence for this pathway is suggested by
tions of T cells in psoriatic skin lesions, it an elevated immune response to strepto-
has been hypothesized that pathogenic coccal antigens in guttate psoriasis and the
T cells are reactive to as yet an unidentified presence of antigens similar to streptococ-
cutaneous antigen(s) and that the process cal M proteins in the psoriatic lesions.
of generating skin-homing effector T cells The model in Figure 10.1 considers
is initially the same as a normal cellular mainly the pathogenic activity of T cells in
response. The formation of psoriatic lesions disease pathogenesis, but one must con-
differs from lymphocytic infiltration in sider the influence of the innate immune
acute hypersensitivity reactions in that response on chronic cellular activation in
activation does not resolve spontaneously psoriasis plaques. Yet, the T-cell model
(as, for example, after the elimination of has been a working hypothesis on which
an infectious agent). Furthermore, chronic the therapeutic development of immune-
lesions contain a significant infiltration of targeted biological drugs has been based.

Immunological Aspects of Skin Diseases 165

The use of various animal models, but unaffected (nonlesional) skin or lesional
most especially transplanted human skin skin from a psoriasis patient on severe
on immunodeficient mice, has also been combined immunodeficiency (SCID) mice.
helpful in our understanding of the patho- Injection of cytokines into the uninvolved
genic mechanisms involved in the disease. tissue grafts induces some mild hyperpla-
sia, whereas injection of superantigen-acti-
vated mononuclear leucocytes obtained
Animal Models
from peripheral blood of the same patient
At the onset, there is no spontaneous model induces a full psoriasis phenotype (but
of psoriasis in animals. However, the use of without neutrophil infiltration), suggest-
genetically engineered mice or the implan- ing that the psoriasis phenotype can be
tation of psoriasis plaque xenografts in induced in genetically predisposed skin by
immunodeficient mice has aided greatly bacterial antigen-primed leucocytes. When
in understanding some of the basic mecha- psoriasis lesional skin is grafted, long-term
nisms of skin inflammation as they apply to grafts continue to show viable T cells and
psoriasis and other inflammatory diseases. other infiltrating mononuclear leucocytes.
Several types of transcription factors, Thus, it appears that viable T cells can con-
including STAT-1 and interferon (IFN) reg- tinue to expand in situ in skin lesions at a
ulatory factors (IRFs), convert IFN-induced rate that matches the rate of programmed
genes. IRF-1 is a transcriptional activator, cell death, and one does not need new
whereas IRF-2 suppresses IRF-1 activity. T cells from the peripheral circulation to
The IRF-2 null mouse chronically overex- perpetuate the lesion.
presses IRF-1. These mice spontaneously Perhaps of greater importance has been
develop inflammatory skin lesions similar the observation that when nonlesional
to psoriasis, including CD8+ T-cell infiltra- normal skin from psoriasis patients
tion in the epidermis, CD4+ T cells in the der- is grafted onto highly immunodeficient
mis, and marked epidermal hyperplasia. AGR129 mice, the bystander T cells in
IL-12 and IL-23 (related cytokines that the graft expand in situ and cause a full-
share a p40 subunit) stimulate T cells to blown psoriatic lesion. Unlike SCID mice,
differentiate and produce IFN-. Mice were AGR129 mice lack both natural killer (NK)
genetically engineered to express constitu- cells and IFN receptors, which may leave
tively p40 in the skin by joining the kera- them unable to reject graft T cells or create
tin-14 promoter with the p40 gene. These a cytokine environment more conducive to
animals developed inflammatory skin T-cell activation. Either a tumor necrosis
lesions with marked epidermal hyperpla- factor (TNF) antagonist or anti-CD3 anti-
sia and increased levels of DCs and T-cell- bodies can block the onset of lesions.
derived cytokines similar to those found
in psoriasis lesions. Thus, IL-23 appears to
Global View of Psoriasis Through
be an important upstream inflammatory
product leading to possible IFN- produc-
tion and synthesis of downstream genes Another informative approach to under-
controlled by IFN-, STAT-1, and IRFs. standing the underlying mechanisms
Another approach that has yielded of pathogenesis of the psoriatic plaque
useful information has been to transplant has been the study of differences in gene

166 Immunological Aspects of Skin Diseases

expression detected through transcrip- products and are synthesized in large part
tional profiling on 63,000-element gene by keratinocytes in plaques. These che-
arrays. Using this technique, it appears mokines direct CXCR3+ T cells to migrate
that 1,338 genes have altered expression to the epidermis, where they may trigger
in psoriasis. Having a global view of these epidermal hyperplasia as a result of physi-
differences in gene expression between cal damage (disruption of basement mem-
psoriatic plaques and normal skin back- brane and desmosomes) done to epider-
ground is important because it provides mal structure through T-cell trafficking or
an unbiased means to assess activation through secreted products.
pathways in psoriasis. Through genomic Interleukin-8 (also induced by IFN-) is
analysis, new inflammatory or regulatory a key regulatory chemokine for neutrophil
cytokine and chemokine products have trafficking into lesions. Inducible nitric
been identified as overexpressed in psori- oxide synthase (iNOS) transcription is also
atic lesions. Unexpectedly, there is strong regulated by IFN- and is highly overex-
expression of numerous lymphoid-orga- pressed in psoriasis lesions, so that its prod-
nizing chemokines, for example, CCL19, uct (NO, or nitric oxide) may be responsible
CCL21, and SDF-1 in psoriasis, as expres- for either cell damage or vascular dilation
sion is normally confined to lymph nodes in lesions. Hence, IFN- plays a key role in
or formal lymphoid tissues. Most likely, leukocyte migration, as well as in epider-
these chemokines orchestrate a striking mal and vascular alterations. The T cells
accumulation of immature dendritic cells in skin lesions do not become activated to
(iDCs) and mature dendritic cells (mDCs) release TNF, IFN-, and other cytokines
in skin lesions; T-cell activation in situ may unless triggered by antigen recognition,
be regulated through interaction with these specific cytokines, or both. Hence, both
DCs in the skin or through release of acti- the clonal nature of these T-cell infiltrates
vating cytokines (such as IL-23) produced in psoriasis and the ability of CTLA4-Ig to
by these infiltrating DCs. If one considers reverse disease activity argue for an ongo-
that the vascular changes in psoriasis bear ing activation of T cells via classical TCR
a striking resemblance to those in lymph engagement and co-stimulation. Two cyto-
nodes, then the picture of dense perivas- kines produced by activated DCs, IL-12
cular DC/T-cell aggregates and increased and IL-23, have been detected in elevated
expression of lymphoid chemokines sug- levels in psoriasis lesions. Both of these
gests that the dermal infiltrates are actually factors augment IFN- production from
a type of secondary lymphoid tissue. T cells and may stimulate excessive expan-
Genomic profiling permits us to iden- sion of TH1-biased clones. Therefore, in
tify which of the many cytokines detected this model, the presence of activated DCs
in psoriasis plaques are transcriptional in skin lesions may be as important in sus-
activators. Thus, expression of more than taining disease activity as are T-cell infil-
sixty-five genes with increased expres- trates. Long-term disease persistence in the
sion in psoriasis lesions can be linked to skin (individual plaques can last for years
IFN- and subsequent activation of STAT-1 without suppression by therapy) has been
signaling by this cytokine. For example, the hypothesized to be a consequence of orga-
chemokines CXCL9/MIG, CXCL10/IP-10, nized lymphoid tissue that forms in these
and CXCL11/I-Tac are STAT-1-regulated inflammatory skin lesions.

Immunological Aspects of Skin Diseases 167

a twelve-week treatment course. For
Insights into Psoriasis Pathogenesis
unknown reasons, responses to these tar-
from Treatments
geted agents are more variable than to
In 2003, on the basis of many observa- more general immunosuppressive treat-
tions that T cells play an important role in ments. Possible explanations include vari-
the pathogenesis of psoriasis, two T-cell- able expression of redundant T-cell activa-
targeting biologics, alefacept and efali- tion pathways, restrictions in the access of
zumab, were initially tested for activity in large molecules to relevant T-cell pools, or
psoriasis and are now approved by the U.S. more complex interactions between T cells
Food and Drug Administration (FDA). Ale- and other types of leukocytes that contrib-
facept is a fusion protein that contains the ute to disease pathogenesis in different
extracellular domain of LFA-3 fused with patients. Genetic/genomic heterogeneity
immunoglobulin constant region domains. of humans seems likely to underlie the vari-
This agent binds to CD2, which is expressed able response to molecule-specific antago-
at high levels on memory T cells. Although nists, but more consistent improvements in
this binding interaction with CD2 could disease are seen with less selective T-cell
inhibit normal LFA-3/CD2 signaling, suc- antagonists, for example, cyclosporine,
cessful therapeutic outcomes with this agent suggesting that psoriasis is fundamentally
are most closely associated with depletion caused by cellular immune system dis-
of T cells from psoriasis skin lesions and, to regulation. In general, suppression of T-
a lesser extent, depletion of memory T cells cell-mediated inflammation functionally
(CD8 > CD4) from the circulation. defined as genomic suppression of IFN-,
Efalizumab is a humanized monoclonal STAT-1, and downstream genes regulated
antibody that binds to the alpha subunit by STAT-1 in psoriasis skin lesions leads
(CD11a) of the integrin lymphocyte func- to objective improvement or clearing of
tional antigen (LFA-1). LFA-1 is expressed disease, whereas failure to suppress these
at high levels on T lymphocytes, while other inflammatory genes leads to continuing
integrins are specific for monocytes, macro- disease activity. A review of therapeutic
phages, neutrophils, and DCs. Thus, LFA- trials with many different agents indicates
1 blockade with efalizumab, specifically that there are no documented cases in
targets T cells, and alters T-cell function in which T cells and associated inflammatory
several ways: (1) prevents firm adhesion of genes were reduced without correspond-
T cells to inflamed (ICAM+) endothelium ing improvements in disease-defining epi-
and thus blocks entry of cells from the cir- dermal hyperplasia. Hence, no data argues
culation; (2) prevents binding of T cells to against the fundamental hypothesis that
ICAM+ keratinocytes, thus reducing traf- activation of the cellular immune system
ficking of cells into the epidermis; and (3) triggers psoriasis.
reduces T-cell activation, with attendant Another therapeutic approach in pso-
cytokine release, either directly or by block- riasis has been to antagonize inflamma-
ing DC/T-cell immune synapse formation. tory cytokines. Although an antibody to
Although alefacept and efalizumab IL-8 was administered to psoriasis patients
are potent immune therapies, only 2530 with moderate improvement in disease
percent of treated patients experience activity, the first major success of the anti-
maximal disease improvement after cytokine approach was antagonizing TNF

168 Immunological Aspects of Skin Diseases

with the chimeric antibody infliximab. sequence. PSORS2 is shown to encode a
Subsequently, etanercept, a TNF/lympho- mutated binding site for the transcrip-
toxin antagonist fusion protein, consist- tion factor RUNX1. Two adjacent genes,
ing of the extracellular domain of TNF- SCL9A3R1/EBP50 and RAPTOR, each
R2 and immunoglobulin constant region associated with activation-related signal
domains, was shown to improve psoriasis. transduction, may be affected in a way
This agent is now an FDA-approved drug that leads to T-cell activation or keratino-
for treating psoriasis and psoriatic arthri- cyte hyperplasia, but more work is needed
tis. The success of these trials suggests the to understand fully the functional conse-
need to consider psoriatic inflammation in quences of this mutation.
a broader context than just IFN- release In summary, psoriasis vulgaris can be
from activated T cells. Although TNF is viewed as a cell-mediated autoimmune dis-
often considered a key cytokine of innate ease characterized by interaction between
immune responses, activated type I T-cells DCs, T cells, and inflammatory cytokines.
co-synthesize IFN- and TNF. Many dif- In this reaction, there is transcriptional
ferent inflammatory genes, including IL-8 activation of a broad set of inflammation-
and iNOS, which are central mediators in producing gene products that change traf-
psoriasis, have composite promoters for ficking of leukocytes and growth patterns
both STAT-1 and NFB transcription fac- of resident skin cells. Although evidence
tors that are activated by IFN- and TNF, suggests that type I T cells are probably
respectively. Thus, the extent to which reacting with an autoantigen in diseased
many type I genes are transcribed may be skin, this is not yet proven. Alternatively,
determined by combined levels of TNF and the psoriasis could be a disease of an over-
IFN- in the lesions. A recent study shows active innate immune system or underac-
that etanercept induces strong suppression tive T regulatory pathways. The use of
of type I inflammatory genes in psoriasis animal models, the study of cell types in
lesions, consistent with the view that T- the lesions, the use of genomics to detect
cell-mediated pathways are being modu- transcriptional profiling, and clinical trials
lated by TNF inhibition. One should also have all been useful in understanding the
add that TNF stimulates differentiation and disease process; however, a good deal of
activation of DCs, cells that present antigen work still needs to be done to treat this seri-
to T cells. Thus, interference with DCs may ous disease of the skin.
break DC/T-cell immune synapses neces-
sary for psoriasis pathogenesis.
Pathogenic Insights Provided
Alopecia areata (AA) is most likely a T-cell-
by Genetics
mediated disease of a skin appendage, the
Several genetic susceptibility loci have hair follicle, and thus can be classified as an
been associated with an increased risk inflammatory skin disease. The condition
of developing psoriasis, but the identity is quite common because about 1.7 percent
of susceptibility genes has been elusive. of the population experience an episode of
Finally, a susceptibility region (PSORS2) AA during their lifetime. AA may be focal,
has been mapped to a discrete DNA affecting only one small region of the skin

Immunological Aspects of Skin Diseases 169

of focal hair loss. These pathognomonic
exclamation point hairs are broader at their
distal ends, hence the name.
Although the exact mechanism of path-
ological events is still unknown, there is a
growing body of evidence indicating that
it is a T-cell-mediated autoimmune disease
as follows:

1. The mononuclear infiltrate surrounding

the hair follicle is primarily composed
of CD4 and CD8+ T cells as well as mac-
2. Type I T cells produce IFN-, which is
associated with increased expression
of HLA-DR, HLA-ABC, and ICAM-1
in the follicular epithelium, resulting
in increased leukocyte trafficking from
blood into the hair follicle.
Figure 10.2 The patient has nonscarring alopecia
3. Hair regrowth is reproducibly induced
areata covering >90 percent of his scalp. White
tufts of hair near the temples are evidence of by immunosuppressive drug treat-
hair regrowth during active inflammation in the ment, including local corticosteroid
hair bulb, which inhibits pigment transfer from injections and the use of systemic
melanocytes to keratinocytes and hair.
4. Lesional scalp from AA patients grafted
(Figure 10.2), or more generalized total onto SCID mice regrows, coinciding
hair loss from the scalp (alopecia totalis) or with the loss of the infiltrating lympho-
scalp and body (alopecia universalis). cytes in the graft. Furthermore, hair
Normal hair growth cycle can be broken loss can be transferred to human scalp
down into three phases (1) anagen growth explants in SCID mice by injection of
phase lasting three or more years, (2) cata- lesional T cells.
gen transitional period lasting two to four
weeks, and (3) telogen phase when the hair Circulating autoantibodies to follicular
follicle advances from the inferior segment structures have been reported in biopsies
of the follicular sheath into the isthmus and of AA patients but have also been reported
is eventually shed, either by traction or from in normal controls. These autoantibodies
being pushed out by a new hair in anagen have also been seen in C3H/HeJ mice and
phase. Hairs affected by AA end the ana- DEBR rats but do not appear pathogenic
gen phase prematurely and enter into telo- in either mice or humans. Also one cannot
gen, resulting in precipitous hair shedding. transfer AA by injection of patient IgG into
Because AA does not result in destruction/ human skin explants.
scarring of the hair follicle, lost hairs may In contrast, C3H/HeJ mice develop
eventually grow back, first appearing as spontaneous hair loss with aging and dem-
exclamation point hairs along the border onstrate many features of AA, including

170 Immunological Aspects of Skin Diseases

inflammatory infiltrates and response to animal models. Using SCID mice with skin
intralesional steroids. More important, grafts obtained from AA patients, mela-
grafts from C3H/HeJ mice when implanted nocyte-associated peptides are capable of
into C3H/SmNCPrkd (SCID/J) mice do activating lesional T cells to induce hair
not result in hair loss, which emphasizes loss. However, melanocyte antigens may
the role of the host immune system. In not be the only autoantigens capable of
common with human AA, there is elevated stimulating these cells.
expression of MHC class I and II antigens Treatment at present is related to the
and ICAM-1 in the skin of these animals, observation that AA appears to be medi-
and they respond to topical immunother- ated by a TH1 response with production of
apy like humans. IFN-. Psoriasis also has a TH1 response.
As in many other autoimmune dis- IL-10, which inhibits TH1 responses, was
eases, there is a genetic susceptibility to found to be effective in psoriasis in a phase
the disease. AA has HLA associations II clinical trial. On the basis of these results
with DQB/*03 and possibly HLA-A. and a small trial in AA, the use of IL-10 in
These associations suggest a role for CD4+ intralesional AA might be warranted. The
T cells in AA but association with HLA, same might be said of alefacept and mono-
ABC was not examined in these studies, clonal antibodies that are directed against
thus excluding the possible role of CD8+ CD11a (LFA-1) or CD4 cells. All of these
T cells. Autoimmune diseases result from immunomodalities are presently available
many factors, and it is probable that AA is and could be considered as potential new
polygenic with multiple potential routes of therapeutics if an appropriate risk-to-bene-
genetic susceptibility. In addition to HLA fit equation is established in clinical trials.
genes, there is known genetic polymor-
phism in cytokine receptors and antigen-
processing molecules. Thus, genetics alone ANTIBODY-INDUCED BULLOUS
is unlikely to supply a complete explana- SKIN LESIONS
tion of disease development.
If there is a T-cell-mediated compo- This group of skin diseases includes pem-
nent to this disease coupled with a genetic phigus vulgaris, bullous pemphigoid, der-
susceptibility, what antigens stimulate matitis herpetiformis, and several other rel-
this T-cell activation? Among the recent atively rare blistering disorders. Although
hypothesis has been the suggestion that uncommon, these are serious, sometimes
the autoantigen in AA is the melanocyte. fatal, skin diseases. The group is unified by
Evidence that supports this conclusion production of autoantibodies to different
includes the clinical observation that with adhesion proteins/structures within the
disease activity pigmented hairs are lost epidermis or basement membrane zone at
more quickly then nonpigmented/white the epidermaldermal junction.
hairs. Second, melanocytes are a significant
component of the hair bulb, which is the
Pemphigus Vulgaris
site of the immunological attack. Finally,
there is an association of AA with vitiligo This is the most serious of these disorders,
a disease of focal melanocyte ablation. Sup- and before the introduction of steroids, it
portive evidence can also be found in the was often fatal. The usual age of onset is

Immunological Aspects of Skin Diseases 171

between forty and sixty years old, but it must be high to suppress new lesions and
may occur in any age group. Clinically, may reach 100 mg/day. Once new blister
it often begins with ulceration of the oral formation is under control, the dose can be
mucosa followed by formation of wide- attenuated. Other forms of antibody sup-
spread flaccid weepy bullae. Most patients pressive treatments have been used, such
with Pemphigus vulgaris have circulating as azathioprine and low doses of metho-
antibodies to desmosomal adhesion mole- trexate, and even plasmapheresis has been
cules such as desmoglein 3, which disrupts successful in removing circulating anti-
intercellular connections within the epider- bodies, especially if the disease becomes
mis. This results in the loss of normal epi- steroid resistant. More recently, deple-
thelium and detachment of cells. The titer tion of B cells with the monoclonal CD20
of this antibody often but not always cor- antibody rituximab has shown efficacy in
relates with disease activity. difficult cases.
Direct immunofluorescence of the per-
ilesional skin is diagnostic and the IgG
Bullous Pemphigoid
class of antibodies and C3 complement are
seen at the site of the lesions (Figure 10.3). The condition is clinically similar to pem-
Further support for the direct role of auto- phigus, but the blisters are subepidermal
antibodies in this disease is the fact that not intraepidermal. This disease occurs
sera from pemphigus patients will pro- more often in an older age group (usually
duce pemphigus-like lesions in monkeys sixty years or older) and is characterized
and mice. Furthermore, IgG fractions from by large tense bullae on thighs, arms, and
these sera will induce epithelial cell detach- abdomen. IgG immune complexes and C3
ment in human skin cultures. are visualized by indirect immunofluores-
The main treatment has been the use of cence as a continuous linear band along the
systemic corticosteroids. The initial dose basement membrane, detectable in 7590
percent of patients with active disease.
Although it is believed that autoantibod-
ies to a basement membrane protein cause
these blisters, experimental demonstration
of this association is more difficult than with
pemphigus. Sera from these patients do not
produce pemphigoid lesions in monkeys
and mice. Second, the antibodies remain
detectable during remission, and the titers
do not correlate with disease activity. Treat-
ment is similar to that described for pem-
phigus, but the doses needed to suppress
Figure 10.3 Immunofluorescent photograph of lesions are usually lower.
a frozen section of psoriasis plaque using serum
from a patient with pemphigus vulgaris. Note the
clear binding of anti-desmoglein-3 antibodies Dermatitis Herpetiformis
in the patients serum binding to desmoglein-3
protein that is present in desmosomes cementing The lesions in this condition are usually
epidermal cells together. smaller than those seen in pemphigus,

172 Immunological Aspects of Skin Diseases

are extremely itchy, and are seen on exte- which basic research frequently translates
rior surfaces of elbows, knees, buttocks, into clinical applications. Accessibility
necks, and shoulders. Visually, the lesions of human skin biopsies and intersection
resemble herpetic papules; however, the with biopharma production of immune-
herpes virus is not involved in this condi- modulating therapies increases research
tion. The age group is quite different from velocity and immediacy. Current hot top-
that of pemphigus (twenty-five to forty ics for researchers studying psoriasis, for
years old) but it may occur at any age. IgA example, include the newly defined TH17
is the major class of antibody detected, T cell, and immunomodulating effects of
deposited in a granular fashion in the tips innate antimicrobial peptides contribut-
of the dermal papillae, forming subepi- ing to disease initiation and maintenance.
thelial bullae. However, unlike pemphi- Since the advent of effective immune-
gus, no circulating autoantibodies to skin modulating therapies targeting T cells, and
are seen in the sera of these patients. An the discovery of a large IFN- signature in
interesting and as yet unexplained obser- psoriatic lesions, psoriasis has been known
vation is that many of the DH patients as a TH1-mediated disease. TH17 cells pro-
have a concomitant enteropathy similar ducing IL-17 and IL-22, first discovered in
to celiac disease. Clinically, they do not animal models of autoimmunity, have now
have the symptoms of celiac disease and been found to promote psoriasis disease
the associated condition is usually only pathogenesis, both through IL-17-depen-
demonstrable by jejunal biopsy. About 30 dent leukocyte migration, and through IL-
percent of these patients have antibodies 22-dependent keratinocyte hyperprolifera-
to gliadin and endomysium in their sera. tion. Current questions in the field include
They also have an increased risk of lym- defining the relationship between TH1 and
phoma (like celiac disease patients) and a TH17 cells in humans: Are they synergistic
genetic component exhibited by a mark- or antagonistic? Are they both involved in
edly increased inheritance of HLA-B8, initiation and maintenance and is block-
DR3-DQ2 haplotypes. ing one or both subsets most effective for
Dermatitis herpetiformis responds to patient therapy? Another current topic of
a strict gluten-free diet, which may need research is the role of innate antimicrobials,
to be in force for several years before the including -defensins and cathelicidins,
lesions heal. Reintroduction of gluten as upstream versus downstream inflam-
results in the reappearance of the lesions, matory mediators. LL-37 cathelicidin was
indicating the important role of gluten recently found to bind ribonucleic acids
in the condition. Yet the presence of IgA and initiate plasmacytoid DC activation
deposits in the skin and their immunologi- through TLR9. Activated plasmacytoid
cal basis remain unknown. DCs produce large quantities of IFN-,
which leads to downstream myeloid DC
activation and subsequent T-cell activation.
FUTURE DIRECTIONS However, both IL-17 and IL-22 are potent
FOR RESEARCH inducers of keratinocyte-elaborated anti-
microbial peptides, forming a potential for
Inflammatory diseases of the skin are an positive feedback and continued inflam-
exciting and evolving field of research in mation. Unanswered questions include

Immunological Aspects of Skin Diseases 173

the temporal relationship between innate inflammatory gene expression. Trends
antimicrobial peptides and T-cell adaptive Immunol. 2004;25:295305.
immunity, as well as the genetic basis for
this breakdown in tolerance and immune LANDMARK PAPERS
regulation. Asadullah K, Sterry W, Stephanek K,
In summary, inflammatory diseases of et al. IL-10 is a key cytokine in psoriasis.
the skin are an important and exciting area Proof of principle by IL-10 therapy: A
of research. Working with other research- new therapeutic approach. J Clin Invest.
ers in both academia and industry rap- 1998;101(4):783794.
idly moves the field from the bench to the Cicardi M, Agostoni A. Hereditary angio-
bedside. Researching inflammatory skin edema. N Engl J Med. 1996;334(25):
diseases also allows for collaboration with 16661667.
other related fields where the immune Gilhar A, Ullmann Y, Berkutzki T, Assy B,
system is deviated as is the case for skin Kalish RS. Autoimmune hair loss (alo-
cancer. pecia areata) transferred by T lympho-
cytes to human scalp explants on SCID
mice. J Clin Invest. 1998;101:6267.
BIBLIOGRAPHY Gottlieb SL, Gilleaudeau P, Johnson R, et al.
Response of psoriasis to a lymphocyte-
selective toxin (DAB389IL-2) suggests
Chamian F, Krueger JG. Psoriasis vulgaris:
a primary immune, but not keratino-
an interplay of T lymphocytes, den-
cyte, pathogenic basis. Nat Med. 1995;1:
dritic cells, and inflammatory cytokines
in pathogenesis. Curr Opin Rheumatol.
Kalish RS, Johnson KL, Hordinsky MK.
Alopecia areata: autoreactive T cells are
Kalish RS, Gilhar A. Alopecia areata:
variably enriched in scalp lesions rela-
autoimmunity the evidence is com-
tive to peripheral blood. Arch Dermatol.
pelling. J Investig Dermatol Symp Proc.
McElvee KJ, Boggess D, King LE Jr, Sund-
Krueger JG. Treating psoriasis with biolog-
berg JP. Experimental induction of alo-
ical agents. Sci Med. 2002;8:149161.
pecia areata-like hair loss in C3H/HeJ
Lew W, Bowcock AM, Krueger JG. Psoria-
mice using full-thickness skin grafts. J
sis vulgaris: cutaneous lymphoid tissue
Investig Dermatol. 1998;11:797803.
supports T-cell activation and type 1

174 Immunological Aspects of Skin Diseases

11. Experimental Approaches to the Study of Autoimmune
Rheumatic Diseases

Dalit Ashany, M.D., and Mary K. Crow, M.D.

INTRODUCTION and lower mammals, current data from the

analysis of the genomes of mice and man,
The ultimate objective of biomedical including study of disease susceptibility
research is to gain new understanding loci, indicate that important hints regard-
of the mechanisms of disease such that ing disease mechanisms can be gleaned
advances in treatment, and ultimately pre- from study of animal models. This chapter
vention and cure, are applied to that dis- will review the major animal models inves-
ease. However, progress is complicated by tigators are using to explore the mecha-
the inherent heterogeneity of human popu- nisms of autoimmune rheumatic diseases,
lations, even among those diagnosed with a emphasizing the degree of fidelity of those
particular disease. Even when appropriate models to the relevant human disease.
study populations can be defined, access The most frequently studied mammal,
to the most relevant tissues and respect the mouse, will be the focus of discussion.
for patient privacy and autonomy present
challenges to unfettered human-subject
research. Although recent large-scale TYPES OF ANIMAL MODELS
genomewide association studies in patients
with systemic autoimmune diseases have
identified candidate disease susceptibility Mouse strains that exhibit spontaneous
genes, in vitro systems and experimental development of syndromes that closely
models are required to characterize the resemble human autoimmune rheumatic
immunopathogenic significance of those diseases have been identified by experi-
genes and their products. Once targets for enced investigators carefully observing
potential new therapies are identified, ini- mouse colonies. In addition, some efforts
tial testing for toxicities of those therapeu- to induce mutation using chemical muta-
tics in humans is rarely appropriate. These gens have generated mice that express
limitations are among those that have led immune system defects or pathology that
investigators to identify and to develop provide clues to human diseases with simi-
useful animal models that closely mimic lar problems. Advantages of spontaneous
human disease. Such models have been a models of disease are that the phenotype
particular focus of research relevant to the is generally quite reproducible from ani-
autoimmune rheumatic diseases. Although mal to animal (although, in some cases,
the use of animal models will always be the disease is only partially penetrant)
limited by the likelihood of genetic and and development of the disease does not
molecular distinctions between humans require intervention by the investigator.

A disadvantage of spontaneous models overexpress or are deficient in a single
is that the disease often takes months to gene to investigate the relevance of that
develop, slowing the pace of investigation. gene product to development of a dis-
ease phenotype. This approach has been
particularly fruitful in the study of lupus,
with transgenic and knockout mice dem-
Some of the most useful animal models are onstrating that any number of genetic
those that are induced by the investigator modifications that alter self-antigen acces-
by administration of a drug, an antigen, an sibility or threshold for immune system
adjuvant, an antibody, or through surgi- activation can lead to production of the
cal manipulation of the immune system. classic lupus autoantibody, anti-DNA
For example, demethylating drugs alter antibody, and deposition of those autoan-
the structure of chromatin, resulting in tibodies in the kidney, another character-
increased accessibility of positive or nega- istic of human lupus. Deficiencies in the
tive regulatory elements in gene promoters complement system, members of which
or enhancers and subsequent alteration of help to clear apoptotic debris and solu-
the gene expression profile of the animal, bilize immune complexes; increased or
sometimes resulting in disease. Removal decreased expression of cell surface mol-
of the thymus at day 3 after birth has been ecules, kinases, phosphatases, or adaptor
demonstrated to promote development molecules relevant to intracellular signal-
of several organ-targeted autoimmune ing and that modulate the threshold for
diseases, most likely based on removal of lymphocyte activation; and altered expres-
an important regulatory T-cell population. sion of death pathway molecules, such as
Induction of immune system activation, as Fas, can result in increased targeting of
in the pristane model of lupus, can lead to the immune response toward self compo-
altered patterns of cytokine production and nents and manifestations of autoimmu-
autoimmunity. In some cases, autoimmune nity. An advantage of these sophisticated
disease can be simply transferred from one animal systems is that the modification
animal to another by administration of an is often restricted to one gene, or at least
autoantibody or autoreactive T-cell popu- a small genomic region adjacent to the
lation. Under these circumstances, the cells gene of interest, allowing the study of the
and molecules of the immune system that impact of that particular molecular prod-
are required for disease expression can be uct and its relevant pathway on disease
identified and the investigation narrowed expression. However, there are inherent
to one or at least a more narrow range of dangers in relying solely on transgenic
immune system components. The contribu- and knockout models. That the gene of
tion of background genetic factors to devel- interest is absent during embryonic and
opment of a disease phenotype can also be fetal development means that other gene
studied in inducible animal models. products can take over some of the func-
tions of the modified gene, obfuscating the
predicted phenotype. The role of a gene in
Transgenic and Knockout
development of a disease can also be over-
Many investigators have used the tech- estimated if background genomic factors
nology that generates animals that either in the neighborhood of the modified gene

176 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

also contribute to disease. Finally, the fact Certain populations, such as the Pima
that overexpression or deletion of a given Indians in North America, have a much
gene results in a disease phenotype does higher incidence of RA (about 5 percent)
not necessarily indicate that the same than the general population. RA is one of
human gene is altered in the correspond- many chronic inflammatory diseases that
ing human disease. In fact, few of the predominate in women. The ratio of female
genes that result in disease when modified to male patients is estimated to be from
in murine systems have been confirmed as 2:1 to 4:1.
disease genes in humans. Nonetheless, the
transgenic and knockout approaches help
Etiology and Pathogenesis
to identify important pathways for further
study and that may be eventually targeted The etiology of RA has not been elucidated
therapeutically. although a variety of studies suggest that a
combination of environmental and genetic
Congenic factors is responsible; a contribution of
either one is necessary but not sufficient
Like the transgenic and knockout
for full expression of the disease. A genetic
approaches, congenic mice have proved
component is demonstrated by studies of
very useful in studying the specific con-
monozygotic twins, in whom the concor-
tributions of narrow genomic regions to
dance rate is perhaps 30 to 50 percent when
aspects of disease pathology. The util-
one twin is affected, compared with 1 per-
ity of congenic mouse strains, in which a
cent for the general population. The immu-
segment of a chromosome is bred into a
nogenetics is under intense study although
desired genetic background, is nicely illus-
there is a clear risk factor in the class II
trated by the NZM 2410 strain. That mouse
MHC haplotype of an individual. Addi-
model has allowed identification of at least
tional genes associated with RA include
three chromosomal loci that confer distinct
PTPN22 and STAT4.
immune system alterations, which when
The major pathology of RA is local-
present together result in severe lupus-like
ized to the joints. The synovial membrane
disease. As in the case of the transgenic
surrounding the joints becomes highly
and knockout models, gene sequences in
vascular and infiltrated with virtually all
the chromosomal segment of interest can
cellular components of the immune sys-
interact with background genes to compli-
tem. T and B lymphocytes are highly rep-
cate interpretation of the role of gene prod-
resented and form lymphoid aggregates
ucts in the congenic region.
where B cells differentiate to antibody-
forming cells. Poorly controlled produc-
tion of pro-inflammatory cytokines, such
RHEUMATOID ARTHRITIS as tumor necrosis factor (TNF) and inter-
leukin-1 (IL-1), promote recruitment of
Introduction and Epidemiology
additional inflammatory cells, and pro-
Rheumatoid arthritis (RA) is the most com- duction of metalloproteinases contributes
mon inflammatory arthritis. Its incidence to tissue damage. The presence of immune
worldwide is estimated to be between 0.5 complexes in joint fluid and direct deposi-
and 1 percent of the general population. tion of those complexes on the surface of

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 177

cartilage support a direct role for autoan- of the disease and is related to the accu-
tibodies in some aspects of RA inflamma- mulation of edema fluid within inflamed
tion. Among the autoantibody specificities tissues during sleep. The joints most com-
that have been implicated in RA patho- monly involved first in RA are the small
genesis are rheumatoid factor, an anti- joints of the hands and feet, including
body with specificity for the Fc portion the metacarpophalangeal joints, proximal
of IgG, and anticyclic citrullinated pep- interphalangeal joints, metatarsophalan-
tide antibodies, which have been shown geal joints, and the wrists. Larger joints
in some cases to precede development of generally become symptomatic after small
clinical disease by years. joints although patients can also present
initially with large-joint involvement.
Although RA is primarily a disease
Clinical Features
of the joints, it is also clearly a systemic
RA usually has a slow onset over the disease that can cause a variety of extra-
course of weeks to months. The initial articular manifestations involving major
symptoms may be systemic with indi- organ systems. A full discussion of these is
viduals presenting with fatigue, malaise, beyond the scope of this chapter; however,
puffy hands, or diffuse musculoskeletal they include the development of rheuma-
pain with joints becoming involved later. toid nodules, subcutaneous nodules occur-
Articular involvement is usually symmet- ring on the extensor surfaces; pulmonary
ric although an asymmetric presentation involvement, including pleuritis, bronchi-
with more symmetry developing later olitis obliterans, or interstitial fibrosis; and
in the course of disease is not unusual ocular involvement, including scleritis,
(Figure 11.1). Morning stiffness that per- episcleritis, and vasculitis. Extra-articular
sists for at least one hour is characteristic manifestations of RA occur most often in


Figure 11.1 Metacarpalphalangeal joint swelling and ulnar deviation in

a patient with rheumatoid arthritis. 19722004 American College of
Rheumatology Clinical Slide Collection. Used with permission.

178 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

seropositive patients with more severe ANIMAL MODELS OF RHEUMATOID
joint disease. ARTHRITIS

No animal model is regarded as a high-

Treatment of RA
fidelity replicate of human RA. However,
Patients with RA have variable courses, several models that variably use T cells
ranging from those who progress rapidly or antibodies that target the joint, or that
to complete destruction of joints if left induce monocyte activation and inflam-
untreated, to those who, with few phar- mation, have been used to study distinct
macologic interventions, smolder along aspects of the disease phenotype.
with minimal disease that leaves cartilage Animal models of arthritis can be used
functional and activities of daily living to understand elements of the arthritic
unchanged from the premorbid state. The process in patients. Recent therapeutic
challenge for physicians is to determine approaches in patients with RA, includ-
into which category a patient falls. Treat- ing the use of biological agents, are based
ment choices include nonsteroidal anti- on initial findings in murine models of
inflammatory drugs (NSAIDs), which are experimental arthritis, although final
useful to help control some of the inflam- proof of concept must come from clini-
matory signs of RA but do not prevent cal studies. Animal models are powerful
progression of the disease. A large class tools for studying pathological changes
of drugs known as DMARDs (disease- in articular cartilage and bone in great
modifying antirheumatic drugs) includes detail and can be used to evaluate mecha-
hydroxycholoroquine, an agent that has nisms of erosive processes. Although, in
been shown to have anti-inflammatory general, more inflammation drives more
properties in the disease but does not destruction, the uncoupling of inflamma-
prevent erosive arthritis. Other DMARDs tion and erosion can be seen as well, and
include those that have been shown to different mediators are involved in these
prevent joint destruction, which include processes.
sulfasalazine, methotrexate, leflunomide,
and azathioprine. In recent years, the COLLAGEN-INDUCED ARTHRITIS
development of biologics targeting spe- Collagen-induced arthritis (CIA) is an
cific cytokines, immune cells, and immune example of an inducible animal model for
receptors have provided additional potent human RA. In this model, the disease is
therapeutics with which to treat RA. In induced in susceptible strains of animals
this category are three different drugs by immunization with cartilage type II
that target the TNF cytokine (etancercept, collagen. The resulting disease is a T-cell-
adalimumb, and infliximab), an IL-1 recep- dependent, antibody-mediated autoim-
tor antagonist (anakinra), an anti-CD20 mune disease directed against the type II
antibody that selectively depletes B cells collagen immunogen. Similar to RA, CIA
(rituximab), and a selective co-stimulation is characterized by massive infiltration
modulator that inhibits T-cell activation of synovial joints by inflammatory cells
by binding to CD80 and CD86, thereby and hyperplasia of the synovial mem-
blocking CD28 interaction (abatacept). brane (pannus). In addition, the disease is

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 179

characterized by both cartilage and bone rather than synovitis, and massive inflam-
destruction as seen in RA. mation in the bone marrow. Immune-
Both collagen-specific T and B lym- complex deposition in the cartilage is
phocytes are involved in the induction of not a characteristic feature, and cartilage
CIA. The effector mechanisms that lead to destruction is limited in early disease.
joint tissue destruction are less well under- Adjuvant arthritis has a pure T-cell-
stood, and a number of inflammatory cell driven pathogenesis, and passive trans-
types have been implicated. These include fer of T cells from diseased animals can
fibroblast-like synoviocytes, bone mar- induce arthritis. The joint inflammation is
rowderived macrophages, granulocytes, thought to reflect the generation of a T-cell
and dendritic cells as well as lymphocytes. reaction to bacterial epitopes cross-react-
Pro-inflammatory cytokines (IL-1, TNF) ing with endogenous bacterial fragments
and various chemokines are involved in continuously present in synovial tissues or
the pathogenesis of the immune-mediated with cartilaginous antigens. Similar to RA,
joint damage observed in both CIA and RA. TNF and IL-1 have been shown to play a
Another similarity between RA and CIA is role in pathogenesis. Treatment with anti-
that the development of synovial inflam- TNF or anti-IL-1 therapy blocks inflamma-
mation is associated with pronounced tion and tissue destruction in the model,
angiogenesis with growth of high endothe- with the most optimal blockade occurring
lial venules. with combination therapy. As seen in CIA,
In contrast to RA, where large num- NSAIDs are effective inhibitors of cartilage
bers of T lymphocytes accumulate in the and bone destruction in this model and
inflamed synovium, few T cells are found this represents a significant difference from
in the synovium of CIA. Another differ- human RA.
ence between collagen arthritis and RA is Adjuvant-induced arthritis is severe but
that the former is more highly destructive self-limited, and the rat recovers within a
than RA and in contrast to RA has a marked few months. This is a general shortcoming
sensitivity to suppression of inflammation of most animal models when compared
and joint destruction by NSAIDs such as with the chronic process of human RA.
indomethacin. In contrast to the female An additional feature of adjuvant-induced
preponderance in RA, males are more sus- arthritis is that once the rats undergo
ceptible than females in murine CIA. spontaneous remission the animal can no
longer be reinduced to develop disease.
ADJUVANT ARTHRITIS This resistance has made the model suit-
Adjuvant arthritis is the oldest stud- able for studies in regulation of T-cell
ied model of polyarthritis. It is induced by tolerance.
intradermal injection of complete Freunds
adjuvant, containing heat-killed myco- ANTIGEN-INDUCED ARTHRITIS
bacteria, in susceptible rat strains, mostly Antigen-induced arthritis is a model
Lewis rats. Arthritis develops within two of RA that is induced by immunizing
weeks. The active component in bacteria is animals with a foreign antigen, usually
the cell wall peptidoglycan. bovine serum albumin, and subsequently
Histopathological features are primar- injecting the same antigen into a joint. As
ily a periarthritis, with marked periostitis a result, a pronounced T-cell-dependent

180 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

immune complex-mediated arthritis devel- this form of arthritis. Elimination of IL-1,
ops that is severe but self-limited. The however, did yield impressive protection
advantage of this model is that a defined against cartilage destruction.
part of the pathogenesis leading to arthritis The model of antigen-induced arthritis
is addressed and the arthritis remains con- is most suited to studies into the mecha-
fined to the injected joint, enabling com- nism of cartilage destruction, as induced
parison with a contralateral control joint of by a mix of immune complexes and T-cell
the same animal. reactivity. The model is useful for the study
The histopathology of antigen-induced of the regulation of local T-cell hyperreac-
arthritis has many characteristics of tivity against a retained foreign antigen in
human RA. These include granulocyte- comparison with similar events against self
rich exudates in the joint space, thicken- antigens as seen in CIA.
ing of the synovial lining layer, and at
later stages a predominantly mononuclear K/B X N (KRN) MODEL
infiltrate in the synovium, which later This spontaneous mouse model of
includes numerous T cells and clusters arthritis was generated by crossing the
of plasma cells. Intense immune complex T-cell receptor (TCR) transgenic mouse
formation is seen in superficial layers of line (known as KRNxC56Bl/6) with the
the articular cartilage, which may con- nonobese diabetic mouse strain. Autoan-
tribute to localized cartilage destruction. tibodies that arise in the K/BxN mouse
Early loss of proteoglycan, followed by recognize the ubiquitously expressed
pannus formation and cartilage and bone intracellular enzyme GPI. The novel fea-
erosion, is a common finding, and these ture of this model is the development of
characteristics are close to those found in a small-joint arthritis where the driving
human RA. antigen has been elucidated and where
The arthritis produced is chronic the autoantibodies generated are patho-
and is thought to be due to prolonged genic and transfer disease to other strains
antigen retention in the joint tissue of mice.
in combination with antigen-specific The antibodies recognize endogenous
T-cell-mediated delayed hypersensitivity. GPI, which seems to associate preferen-
Prolonged antigen retention in the joint tially with the cartilage surface. This may
occurs by antibody-mediated trapping and explain the dominance of joint pathology
charge-mediated binding. This principle is in these mice, although GPI is also abun-
also of importance in the recently devel- dant at other sites of the body. Activation of
oped KRN model of arthritis in which complement through the alternative path-
anti-glucose-6-phosphate isomerase (GPI) way is crucial in these mice, and variable
antibodies stick to GPI antigen trapped at susceptibility in different mouse strains
cartilage surfaces and contribute to chro- may largely be attributed to varying activ-
nicity and destruction. ity of complement and level of expression
Unlike the other mouse models of Fc receptors on phagocytes among the
described so far, elimination of TNF and different strains.
IL-1 is poorly effective in suppressing joint Although anti-GPI antibodies are found
inflammation, pointing to a substantial in some RA patients in moderate lev-
role for other mediators of inflammation in els, the role of these antibodies in disease

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 181

pathogenesis remains to be defined. The factors. Recent genomewide association
role of IL-1 and TNF in the pathogenesis studies of single-nucleotide polymor-
of KRN arthritis is similar to other murine phisms have identified gene variants that
models. IL-1-deficient mice do not develop show a significant association with SLE
arthritis and TNF-deficient mice have a in patients of European descent. These
more variable course. include HLA-DRB1, ITGAM, IRF5, PXK,
BANK1. While the functional correlates of
SYSTEMIC LUPUS ERYTHEMATOSUS these gene variants have yet to be charac-
terized, most components of the innate and
Introduction and Epidemiology
adaptive immune responses contribute to
Systemic lupus erythematosus (SLE) is an lupus pathogenesis. Current understand-
autoimmune disease in which the immune ing of the disease implicates self-reactive B
system targets intracellular particles that cells and helper T cells in the production
contain both nucleic acids and nucleic acid- of autoantibodies, with cytokines, particu-
binding proteins. Virtually all components larly IFN-, promoting augmented anti-
of the immune system have been impli- gen-presenting cell function and immune
cated in SLE, with B-cell hyperactivity, system activation.
antigen-presenting cell activation, T-cell One proposed mechanism for the
dysfunction, and altered cytokine pro- development of autoantibodies involves a
files documented. The consequence of this defect in apoptosis or clearance of apoptotic
broad scope of autoimmune manifestations cells, leading to a disturbance in immune
is a multisystem disease with the potential tolerance. The redistribution of cellular
for affecting many organs, including kid- antigens during apoptosis leads to a dis-
ney, brain, heart, skin, and joint. Studies play of cytoplasmic and nuclear antigens
exploring the prevalence of SLE in the con- on the cell surface, enhancing immune
tinental United States have reported rates reactivity to antigens, which are normally
of disease ranging between 14.6 and 50.8 protected intracellularly. Activation of
per 100,000. The disease occurs predomi- antigen-presenting cells by IFN- might
nantly in women of childbearing age, with promote presentation of autoantigens to
a female to male ratio of approximately self-reactive T cells. Immune complexes
10:1, suggesting a role for hormonal fac- form in the microvasculature, leading to
tors in the pathogenesis of the disease. The complement activation and inflammation.
incidence of disease also appears to vary Antibodyantigen complexes deposit on
among different races. In the United States, the basement membranes of skin and kid-
for example, African American women neys. In active SLE, this process has been
have an increased relative risk of disease confirmed based on the presence of com-
compared with Caucasian women. plexes of nuclear antigens such as DNA,
immunoglobulins, and complement pro-
teins at these sites. Immune complexes con-
Etiology and Pathogenesis
taining nucleic acids also play an important
Multiple factors are associated with the role in lupus pathogenesis based on their
development of SLE, including genetic, capacity to stimulate toll-like receptors
racial, hormonal, and environmental and amplify production of IFN-. Gene

182 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

targets of IFN- include pro-inflammatory
mediators, chemokines, and cytokines, and
activation of those mediators can result in
recruitment of inflammatory cells to target
tissue, resulting in target organ damage.

Clinical Features
Systemic lupus erythematosus is a chronic
autoimmune disease that can affect almost
any organ system. Its presentation and
course are highly variable, ranging from
indolent to fulminant. Constitutional
symptoms, including fatigue, fever, and
arthralgia are often present during pre-
sentation or recurrent SLE flares. Arthri-
tis of the small joints of the hands, wrists,
and knees may occur. Multiple cutaneous
manifestations of SLE include malar rash,
an erythematous rash over the cheeks and Figure 11.2 Malar rash in a patient with
nasal bridge; photosensitivity; and dis- systemic lupus erythematosus. 19722004
American College of Rheumatology Clinical
coid lesions, which are plaquelike erup-
Slide Collection. Used with permission.
tions with follicular plugging and scarring,
among others (Figure 11.2).
The kidney is the most commonly manifestations of SLE include delirium,
involved visceral organ in SLE. Glomerular seizures, strokes, and transient ischemic
disease usually develops within the first few attacks and movement disorders. The
years after onset and is usually asymptom- etiology of these is not always clear but
atic. Acute nephritic disease may manifest may be related to vasculitis or presence of
as hypertension and hematuria. Nephrotic antiphospholipid antibodies.
syndrome may cause edema, weight gain, Other organ systems that may be
or hyperlipidemia. Acute or chronic renal involved in SLE include the cardiopulmo-
failure may cause symptoms related to ure- nary system, with episodes of pleuritis and
mia and fluid overload. pericarditis being the most common mani-
Neuropsychiatric manifestations of festations, as well as the hematopoietic
SLE are widely variable. Headache is the system in which various cytopenias due to
most common neurological symptom, antibody-mediated peripheral cell destruc-
often with migraine or complex migraine tion occur.
features. Cognitive disorders may be vari-
ably apparent in patients with SLE. Formal
Treatment of SLE
neuropsychiatric testing reveals deficits in
2167 percent of patients with SLE. Psycho- Managing SLE is challenging because no
sis related to SLE may manifest as paranoia interventions can result in cure, exacerba-
or hallucinations. Other neuropsychiatric tions of disease can occur after months of

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 183

stable maintenance treatment, and unde- NZB X NZW F1 (B/W F1) LUPUS MODEL
sirable side effects of the therapies can be The F1 hybrid is the earliest recognized
significant. Careful and frequent monitor- spontaneous model of lupus. The mouse
ing of patients is important in selecting was developed from a cross between NZB
management plans, monitoring efficacy, mice, which develop a spontaneous auto-
and changing treatments. The physician immune disease resembling hemolytic
must first decide whether a patient needs anemia, and NZW mice, which are phe-
treatment and, if so, whether conserva- notypically normal. The B/W F1 strain is
tive management is sufficient or aggres- one of the best-studied models of human
sive immunosuppression is necessary. For SLE and is considered by many to be the
manifestations that are not life or organ murine model most closely resembling
threatening such as rash, musculoskel- human SLE. The disease is characterized
etal disease, or mild serositis, conserva- by autoantibodies, including antinuclear
tive management with NSAIDs, topical antibodies (ANAs), IgG autoantibodies to
or low-dose glucocorticoids, or hydroxy- double-stranded DNA (dsDNA), as well
chloroquine are mainstays of treatment. as lupus erythematosus (LE) cells, repre-
For severe manifestations of disease such senting phagocytic cells that have engulfed
as renal or central nervous system disease, antibody-opsonized cell debris. Death
immunosuppression with high-dose ste- results from a severe glomerulonephri-
roids and cytotoxic agents such as cyclo- tis (GN) due to the deposition of immune
phosphamide, mycophenolate mofetil, or complexes, including those containing
azathioprine is a therapeutic alternative. anti-DNA antibodies. The disease is more
Most recently, studies investigating the severe and occurs earlier in female mice
role of the anti-CD20 monoclonal antibody than in their male counterparts.
rituximab, co-stimulatory blockade with The B/W F1 is a good representative of
abatacept and blockade of the cytokines human SLE because female B/W F1 mice
IFN-, IL-6, and B-lymphocyte stimulator manifest similar autoantibody profiles,
in controlling various manifestations SLE and the disease develops in the absence
are being actively pursued in clinical trials. of any known disease-accelerating gene,
unlike other mouse models. Production of
anti-dsDNA antibodies in this mouse, as in
Animal Models of SLE
human SLE, is thought to be T-cell depen-
Several murine strains have served as clas- dent as suggested by the IgG isotype, by
sic models of lupus-like disease, with each somatic mutations in the antibody genes,
having many features that mimic immuno- and by experiments in which depletion of
logical and pathological aspects of human T cells suppresses antibody production and
lupus. Although several spontaneous ameliorates disease. As is true for humans
mouse models of lupus have striking simi- with lupus, the correlation between nephri-
larities to human SLE, none of the animal tis and anti-DNA antibody levels is not
models reproduces human SLE perfectly. universal. In addition to anti-dsDNA anti-
What makes these animal models particu- bodies, the mice develop other autoanti-
larly interesting to study is that the dif- bodies found in human disease, including
ferences may reflect different forms of the those that bind single-stranded (ssDNA),
human disease. transfer RNA (tRNA), polynucleotides,

184 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

histones, and nucleic acidprotein com- bodies was not required for renal disease.
plexes. Anti-erythrocyte antibodies occur Mice that expressed a mutant transgene
in 3557 percent of B/W F1 females, but that did not permit secretion of circulat-
the mice rarely develop hemolytic anemia. ing immunoglobulin still developed renal
Consistent with the important pathogenic disease, suggesting that immunoglobulin-
role for IFN- in human lupus, disease and expressing B cells might also participate
death are accelerated in B/W F1 mice who in the disease process either as antigen-
receive IFN-. presenting cells or as part of the local
inflammatory process.
MRL/LPR LUPUS MODEL In contrast to B/W F1 mice, both male
MRL/MpJ-Faslpr (MRL/lpr) is another and female MRL/lpr mice develop high
spontaneous model of SLE. The MRL/lpr serum levels of immunoglobulins, ANAs,
mouse has a genetic mutation, termed lpr and immune complexes, as well as dis-
(lymphoproliferation), which leads to mas- ease. Other antibodies in their repertoire
sive lymphoid organ enlargement and include IgG2a anti-chromatin, anti-RBCs,
severe early-life lupus-like disease. The anti-thyroglobulin, antilymphocyte, anti-
congenic MRL/+/+ strain lacks the muta- ribosomal P, and anti-RNA polymerase I.
tion and develops a mild lupus-like dis- Polyarthritis occurs in MRL/lpr mice
ease. MRL/lpr mice exhibit many clinical in some but not all colonies. The preva-
manifestations found in human SLE. Auto- lence varies between 15 and 20 percent.
antibodies produced by these mice are The arthritis is a destructive arthropathy,
similar in spectrum to those seen in human characterized by synovial cell proliferation
lupus, including anti-dsDNA and anti-Sm with early subchondral bone destruction
antibodies. The mice also make rheuma- and marginal erosions. This is unlike the
toid factors. MRL/lpr mice develop pro- nonerosive arthritis that occurs in some
liferative GN at an early age (45 months), patients with SLE.
and renal failure is the presumed primary Unlike human SLE, MRL/lpr mice are
cause of death in these mice. characterized by massive lymphadenopa-
Although the exact mechanisms thy and splenomegaly, which is due to a
involved in the pathogenesis of lupus defect in the fas gene, a key mediator of
nephritis are as yet unclear, there is gen- apoptosis. The fas defect alone is sufficient
eral agreement that disease is mediated by to induce autoantibody production but is
glomerular deposition of autoantibodies not sufficient to induce renal disease. This
as immune complexes formed in situ or by is demonstrated in experiments in which
direct binding of the antibodies to an intrin- the lpr gene is bred onto a normal back-
sic renal antigen or a self-antigen deposited ground, resulting in congenic lpr mice that
in the kidney. This deposited immunoglob- produce autoantibodies but do not develop
ulin then induces renal injury primarily renal disease. Thus, genes in the MRL back-
through complement activation, leading ground, independent of fas, are necessary
to recruitment and activation of inflamma- for disease development, including renal
tory mediators. These mechanisms appear disease, in MRL/lpr mice. Fas defects in
to exist in both human and murine lupus. humans are generally not seen in patients
However, a recent study in MRL/lpr mice with SLE, although a population of patients
suggested that the production of autoanti- with genetic defects in the fas gene develop

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 185

a disease known as autoimmune lympho- by immune complex GN. Histologically,
proliferation syndrome, characterized by the disease is more exudative than in other
massive lymphadenopathy along with mouse models, with neutrophils invading
some autoimmune features. glomeruli along with IgG and C3 deposi-
tion, proliferative changes in mesangia and
BXSB LUPUS MODEL endothelial cells, and basement membrane
The BXSB mouse is a recombinant thickening. The progression from nephritis
inbred strain that spontaneously devel- to death is rapid. Of the most widely stud-
ops an autoimmune syndrome similar to ied SLE mouse models, the BXSB has the
human SLE. This mouse model is charac- most fulminant disease.
terized by the production of autoantibod-
ies, hypergammaglobulinemia with class NZM2410 MODEL
switching to IgG3 and IgG2b, hypocomple- The NZM2410 mouse is another spon-
mentemia, splenomegaly, and GN. BXSB taneous model of SLE and is a congenic
mice develop a wide range of autoantibod- recombinant inbred strain (termed NZM
ies to nuclear components, typical of SLE, for New Zealand Mixed) produced by
including ANAs, anti-dsDNA, anti-ssDNA, inbreeding (NZB NZW)F1 NZW back-
and antichromatin antibodies, with accom- cross progeny. This mouse strain displays
panying splenomegaly and lymphadenop- systemic autoimmunity with a highly
athy. In addition, a small proportion make penetrant, early-onset acute GN. The mice
anti-erythrocyte antibodies. develop GN with a penetrance of about
The unique features of BXSB mice are 85 percent and a 50 percent mortality at
that their disease is much worse in the about six months of age. This represents
male than the female, and the disease- an earlier onset of disease than that of B/W
accelerating gene responsible for that dif- F1 mice.
ference (called Yaa for Y chromosome auto- The NZM2410 mouse strain differs sig-
immunity accelerator) is located on the Y nificantly from the classical B/W F1 model
chromosome and manifests in a male mor- and human SLE, where there is a strong
tality of 50 percent by the age of six months. female preponderance, in that both males
These mice have recently been documented and females develop the disease equally. It
to have a duplicated segment of the X chro- is possible either that the genomic regions
mosome that has been translocated to the Y responsible for the strong gender dimor-
chromosome. That segment includes TLR7, phism in B/W F1 were not included in
encoding a toll-like receptor that responds NZM2410 or that NZM2410 contains a
to ssRNA. The female BXSB mouse gets collection of homozygous susceptibil-
late-life lupus with death occurring at four- ity alleles that are so severe that they can
teen months. This suggests that additional override the effects of sex hormones on the
genes contribute to disease in female mice. immune system.
By three months of age the mice have Linkage analysis of susceptibility to
elevated levels of circulating immune GN and ANA production in the NZM2410
complexes and hypocomplementemia. strain by Morel and Wakeland (2000) have
They are the only lupus mouse strain that identified three prominent loci termed
has serum levels of C4 that diminish as Sle1, 2, and 3 that are strongly associated
clinical disease appears. Death is caused with lupus susceptibility. Subsequent

186 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

congenic strains made by moving each appear to be conserved between mice and
locus onto the lupus-resistant C57Bl/6J humans, supporting approaches to look
(B6) background have determined (1) that for candidate human SLE-associated genes
Sle1 mediates a spontaneous loss of immu- based on mouse studies.
nological tolerance to nuclear antigens; (2)
that Sle2 lowers the activation threshold of
the B-cell compartment and mediates poly-
clonal/polyreactive antibody production; Introduction and Epidemiology
and (3) that Sle3 mediates a dysregulation
Primary Sjgrens syndrome (SS) is a sys-
of the T-cell compartment that potentiates
temic autoimmune disease that targets the
polyclonal IgG antibody production and
exocrine glands (Figure 11.3). It is character-
decreases activation-induced cell death
ized by xerostomia (dry mouth), xerophthal-
in CD4 T cells. Sle1 and Sle3 in combina-
mia (dry eyes), and is usually accompanied
tion revealed that these two susceptibility
by production of autoantibodies specific
genes are sufficient to mediate the develop-
for the Ro RNA-binding protein. SS is a
ment of severe humoral autoimmunity and
common autoimmune disorder. Prevalence
fatal lupus nephritis on the B6 background.
estimates range from approximately 0.5 to
B6 bicongenic mice with these two alleles
5 percent. Approximately, one-half of all
spontaneously develop high titers of auto-
cases of SS are primary and the remain-
antibody directed against a broad spec-
der occur as secondary SS. Secondary SS is
trum of nuclear chromatin autoantigens
defined as the former definition of primary
and die of kidney failure (penetrance >55
Sjgrens in the presence of another auto-
percent) due to autoimmune GN within
immune connective tissue disorder such
twelve months of age.
as RA or SLE. Similar to most autoimmune
The lethal phenotype produced by the
disorders, most cases (approximately 90
combination of Sle1 and Sle3 on the B6
percent) occur in women. The majority of
background was somewhat surprising, in
cases occur in midlife; however, the disor-
that both of these genes are derived from
der is also seen in children and the elderly.
the relatively unaffected NZW strain. This
suggested that their severe autoimmune
phenotypes must be suppressed in some
manner by the NZW genome. Genetic anal-
ysis of this epistatic suppression in NZW
identified the presence and locations of
four SLE suppressor loci (designated Sles1
through Sles4) that account for the absence
of fatal disease in NZW.
Murine chromosome 1 contains the
Sle1 locus as well as other loci of lupus
susceptibility. This region in humans, spe- ACR
cifically 1q41-q42, also shows evidence of
Figure 11.3 Parotid gland swelling in a patient
linkage with SLE, including the production
with Sjgrens syndrome. 19722004
of antichromatin antibodies. Thus, impor- American College of Rheumatology Clinical Slide
tant susceptibility genes for autoimmunity Collection. Used with permission.

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 187

Etiology and Pathogenesis properties of saliva in the dry mouth accel-
erate tooth decay. Unexplained rampant
Sjgrens syndrome has a strong immu-
dental caries may, in fact, be the first sign
nogenetic component. Like many other
of a dry mouth. Other xeroses such as dry
autoimmune disorders, HLA studies have
skin and dry vaginal mucosa leading to
identified associations with MHC haplo-
irritation and dyspareunia may also occur.
types. Studies have demonstrated that -
Extraglandular manifestations of SS
DR2 and -DR3 associations in Sjgrens are
include musculoskeletal symptoms, with
secondary to linkage disequilibrium with
arthralgias and transient synovitis occur-
HLA-DQ alleles. Genetically defined allelic
ring in 54 to 84 percent of patients. Pulmo-
markers have subsequently identified a
nary involvement, which most commonly
large number of polymorphisms involving
is cough due to xerotrachea, occurs in
the HLA DRB1/DQA1/DQB1 haplotype,
approximately 50 percent of patients. Mild
which adds to the complexity of the genetic
autoimmune hepatitis has been reported
background of SS. These polymorphisms
to occur in approximately one-quarter of
have been shown to vary with ethnicity,
primary SS patients, with smooth muscle
clinical manifestations, and, importantly,
antibodies seen in 7 to 33 percent. Antimi-
with the autoantibody response. Similar to
tochondrial antibodies have been reported
the situation in SLE, IFN- has been impli-
in 7 to 13 percent of patients, suggesting
cated in SS pathogenesis based on expres-
an association of primary SS with primary
sion of interferon-induced genes in salivary
biliary cirrhosis (PBC).
gland tissue and increased type I interferon
Neurologic disease is perhaps the most
activity in serum.
common significant extraglandular mani-
Salivary gland pathology shows infil-
festation of SS and can involve the cranial
tration with perivascular collections of
nerves, peripheral nerves, and rarely the
T and B lymphocytes. The cells form dis-
central nervous system. Clinical reports
tinct aggregates, termed foci, in periduc-
suggest that half of SS patients have some
tal and periacinar locations that can result
form of neurological involvement, with
in replacement of epithelial structure. In
estimates ranging from 22 to 76 percent.
rare cases, patients with SS develop non-
Hodgkins B-cell lymphoma.
Treatment of Sjgrens Syndrome
For the majority of patients, the main-
Clinical Features
stay of treatment for Sjgrens disease
The most common clinical manifestations is external replacement of moisture.
of SS are ocular and oral. Patients most Patients should be encouraged to use tear
often perceive xerophthalmia as a foreign substitutes often, and a variety of over-
body-type sensation manifested by scratch- the-counter preparations are available. For
iness, grittiness, or irritation. Patients with xerostomia, artificial salivas are available,
xerostomia will complain of a parched sen- but these are generally not very success-
sation in the mouth, which often extends to ful and most patients find them unpalat-
the throat. Eating can be difficult without able. Patients whose symptoms of dryness
supplemental fluids. The reduction in sali- are not optimally controlled by mois-
vary volume and loss of the antibacterial ture replacement should be considered

188 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

for treatment with secretory stimulants. other autoimmune conditions, such as
There are two approved agents available lupus, inflammatory arthritis, or insulitis.
for use as secretagogues in SS: pilocar- IQI mice spontaneously develop autoim-
pine (Salagen) and cevemiline (Evoxac). mune infiltration of lymphocytes into the
Both agents have been shown, in con- lacrimal and salivary glands, leading to
trolled clinical trials, to increase salivary dacryoadenitis and sialoadenitis. The inci-
flow rate significantly in SS. There are no dence of the disease is higher in females
disease-modifying drugs that have been than in males. Sialoadenitis in female
demonstrated to have specific efficacy for mice can be detected from two months of
SS. Minor musculoskeletal symptoms usu- age onward and significant progression of
ally respond to NSAID therapy. Because the lesions is observed after nine months
erosive joint disease is rare, therapy with of age. Inflammatory lesions occurring at
DMARDs is usually unnecessary; how- young ages mainly consist of CD4+ T cells
ever, hydroxychloroquine has been used with lesser abundance of CD8+ T cells,
to treat fatigue, arthralgia, and myalgia in B cells, and macrophages, and the pro-
primary SS. Cranial and peripheral neu- portions of B cells and plasma cells are
ropathy may be treated with low-dose elevated in accordance with increasing
tricyclic antidepressants or gabapentin. magnification of the lesions. Production of
Symptomatic cases resistant to the previ- ANAs, one of the prominent pathophysi-
ously mentioned therapies may be treated ological features in patients with SS, is
with intravenous gammaglobulin. Central also observed in old IQI mice. Moreover,
nervous system manifestations, which IQI mice develop inflammatory lesions in
are felt to be due to Sjgrens, should the lung, pancreas, and kidney in addi-
be treated aggressively with high-dose tion to the lacrimal and salivary glands
oral or pulse intravenous corticosteroid that are similar to those in patients with
and daily or monthly intravenous pulse primary SS. Thus, this murine model may
cyclophosphamide. be the most suitable for the investigation
of the pathogenesis of SS, with progression
from oral and ocular disease to a systemic
Animal Models of Sjgrens Syndrome
Nonobese diabetic mice have been used
predominantly as a model of autoimmune
diabetes mellitus, but they also develop an PROGRESSIVE SYSTEMIC
autoimmune exocrinopathy characterized SCLEROSIS
by hyposecretion of saliva and acinar cell
Introduction and Epidemiology
MRL+/+ and MRL/lpr mice spontane- Progressive systemic sclerosis (PSS) is a
ously develop lacrimal and salivary gland disorder of connective tissue clinically
inflammation and have been used as mod- characterized by thickening and fibro-
els for SS. sis of the skin (scleroderma) and by dis-
IQI/Jic (IQI) mice are a model of pri- tinctive forms of involvement of internal
mary SS, unlike the animal models above organs, including the heart, lungs, kid-
that are autoimmune-prone mice that neys, and gastrointestinal tract. The hall-
develop SS-like pathology associated with marks of the disease are autoimmunity

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 189

and inflammation, widespread vascu- reactive oxygen radicals generated during
lopathy (blood vessel damage), affecting ischemia/reperfusion.
multiple vascular beds, and progressive
interstitial and perivascular fibrosis. The
etiology is unknown. The incidence of sys-
Clinical Features
temic sclerosis is estimated to be between Patients with systemic sclerosis have a
eighteen and twenty individuals per one wide array of symptoms and difficulties,
million persons per year. All age groups ranging from complaints related to spe-
may be affected, but the onset of disease cific internal organ involvement as well
is highest between the ages of thirty and as the symptoms of a chronic catabolic
fifty years old. Systemic sclerosis is three to illness. Raynauds phenomenon is the
four times more common in women than initial complaint in approximately 70 per-
in men, with women of childbearing age cent of patients with systemic sclerosis.
at peak risk. Peripheral vasoconstriction in response
to cold is physiologic, and individu-
als with Raynauds, no matter the cause,
Etiology and Pathogenesis
have undue intolerance to environmental
The pathogenesis of systemic sclerosis cold. The digital arteries of patients with
involves an interplay between oblitera- systemic sclerosis exhibit marked intimal
tive vasculopathy in multiple vascular hyperplasia as well as adventitial fibro-
beds, inflammation, and autoimmunity, sis. Severe narrowing (more than 75 per-
and progressive fibrosis. Vascular injury cent) of the arterial lumen results, which
and activation are the earliest and possibly may be sufficiently severe to account for
primary events in the pathogenesis of PSS. Raynauds phenomenon. The skin thick-
This is suggested by histopathological evi- ening of systemic sclerosis begins on the
dence of vascular damage that is present fingers and hands in nearly all cases.
before fibrosis, and clinical manifestations, The skin initially appears shiny and taut
such as Raynauds phenomenon, an epi- and may be erythematous at early stages
sodic and reversible cold-induced vaso- (Figure 11.4). The skin of the face and neck
spasm of the fingers and toes, that precede is usually involved next and is associated
other disease manifestations. Additional with an immobile and pinched facies. The
manifestations of PSS-associated vascu- skin change may stay restricted to fingers,
lopathy include cutaneous telangiectasia, hands, and face and may remain rela-
nail-fold capillary alterations, pulmo- tively mild. Extension to the forearms is
nary arterial hypertension, gastric antral often followed by rapid centripetal spread
vascular ectasia, and scleroderma renal to the upper arms, shoulders, anterior
crisis with malignant hypertension. In chest, back, abdomen, and legs (diffuse
late-stage PSS, there is a striking paucity scleroderma).
of small blood vessels in lesional skin and Involvement of the gastrointestinal tract
other organs. Endothelial cell injury might is the third most common manifestation of
be triggered by granzymes, endothe- systemic sclerosis. Impaired function of the
lial cell-specific autoantibodies, vasculo- lower esophageal sphincter is associated
tropic viruses, inflammatory cytokines, or with symptoms of intermittent heartburn,

190 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases


Figure 11.4. Skin tightening and distal necrosis in a patient with systemic
sclerosis. 19722004 American College of Rheumatology Clinical Slide
Collection. Used with permission.

typically described as a retrosternal burn- sclerosis include the musculoskeletal sys-

ing pain. Complications of chronic esoph- tem, with arthralgias, arthritis, and myo-
ageal reflux include erosive esophagitis sitis as potential manifestations, as well as
with bleeding, Barretts esophagus, and the heart and kidney.
lower esophageal stricture. Small-bowel
involvement is encountered in patients
with longstanding disease. Symptoms Treatment of Progressive Systemic
include intermittent bloating with abdom- Sclerosis
inal cramps, intermittent or chronic
No drug therapies are available for the
diarrhea, and presentations suggestive of management of systemic sclerosis that
intestinal obstruction. have been proven to enhance survival, pre-
Pulmonary involvement is the leading vent internal organ involvement, or slow,
cause of mortality and a principal source of halt, or improve deterioration of function
morbidity in systemic sclerosis. Pathologi- of involved organs, including the skin. In
cal processes operative in scleroderma lung the absence of such agents, management
include combinations of vascular oblitera- is directed at treating manifestations of
tion, fibrosis, and inflammation. Patients the disease. NSAIDs are generally useful
with diffuse scleroderma are at risk for in managing arthralgias and myalgias,
progressive interstitial fibrotic lung disease although occasional patients require low-
and pulmonary arterial hypertension. The dosage oral glucocorticoids. Symptoms of
clinical onset of pulmonary involvement reflux esophagitis are typical of systemic
is frequently insidious and characterized sclerosis but generally amenable to ther-
by progressive dyspnea on exertion, lim- apy with H2 blockers and protein pump
ited effort tolerance, and a nonproductive inhibitors. Pulmonary involvement has
cough. Other organs involved in systemic generally not been considered amenable

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 191

to therapy. A patient in whom there is evi- mal models have been investigated as
dence of pulmonary interstitial inflamma- spontaneous or inducible models for
tion might be treated with glucocorticoids SSC. Although none of them reproduce
and immunosuppressive agents. A recent all three pathogenetic components of
double-blind, randomized, placebo-con- the disease, some models do recapitulate
trolled trial to determine the effects of oral selected phenotypic features. Two useful
cyclophosphamide on lung function and animal models of PSS include bleomy-
health-related symptoms in patients with cin-induced skin fibrosis and the chronic
evidence of active alveolitis and sclero- graft-versus-host disease system, which
derma-related interstitial lung disease results in skin fibrosis. In addition, the
reported a significant but modest ben- tight skin (Tsk-1) mouse has been pro-
eficial effect on lung function, dyspnea, posed as a model for PSS based on skin
thickening of the skin, and health-related pathology that is similar to that of the
quality of life. Pulmonary hyperten- human disease, with increased accumula-
sion has emerged as a principal cause of tion of collagen and glycosaminoglycans
morbidity and mortality in late systemic in the skin and production of serum auto-
sclerosis. Centrally infused prostacyclin antibodies. The gene mutated in the Tsk-1
(epoprostenol) improves both short- and mouse is fibrillin-1, whose product can
long-term hemodynamics, as well as the form a part of elastic fibers.
quality of life and survival. Studies have
demonstrated that treatment with the oral
endothelin receptor anatagonist bosen- POLYMYOSITIS
tan is well tolerated in PSS patients with
Introduction and Epidemiology
interstitial lung disease and is effective for
treatment of severe pulmonary hyperten- Polymyositis (PM) is an inflammatory
sion in study patients. Scleroderma renal muscle disease characterized by injury and
crisis demands prompt recognition of death of muscle cells and resulting in severe
the diagnosis and aggressive treatment weakness. Polymyositis is considered to be
of the accompanying accelerated hyper- an autoimmune disease based on the pres-
tension. Angiotensin-converting enzyme ence of autoantibodies with characteristic
inhibitors such as captopril and enala- specificities, including those reactive with
pril are mechanistically ideal to treat the tRNA synthetases. In addition, infiltration
hyperreninemic hypertension of sclero- of T cells, predominantly CD8+ cytotoxic
derma renal crisis and are the treatment of T cells, may contribute directly to muscle
choice. damage. PM is a rare disease and only a
few epidemiological studies have been
published. The reported incidence ranges
Animal Models of Progressive
between two and eight new cases per mil-
Systemic Sclerosis
lion people per year.
The pathogenesis of PSS involves a triad
of small-vessel vasculopathy; inflamma-
Etiology and Pathogenesis
tion, and autoimmunity; and interstitial
and vascular fibrosis in the skin, lungs, The idiopathic inflammatory myopa-
and multiple other organs. Various ani- thies, including PM, are believed to be

192 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

immune-mediated processes possibly oropharyngeal muscles or involvement
triggered by environmental factors in of the striated muscle fibers of the upper
genetically susceptible individuals. This esophagus. Pulmonary symptoms are
hypothesis is based on the recognized due to weakness of the thoracic muscles
association with other autoimmune and or interstitial lung disease. Fibrosis is
collagen vascular diseases, the preva- radiologically demonstrated in 510 per-
lence and type of circulating autoanti- cent of cases and is often associated with
bodies, animal models, immunogenetic anti-tRNA synthetase antibodies such as
observations, specifics of the inflamma- anti-Jo-1.
tory changes in muscle, and the treat-
ment response to immunosuppressive
Treatment of Polymyositis
agents. Autoantibodies associated with
PM include the anti-tRNA synthetase Glucocorticoids are the standard first-line
antibodies (anti-Jo-1 and others) as well medication for patients with idiopathic
as antibodies directed against the sig- inflammatory myopathy. Initially, predni-
nal recognition particle. In PM, CD8 + sone is given in a single dose of 1 to 2 mg/kg
cytotoxic T cells invade muscle fi bers per day. Regular evaluations of muscle
that express MHC class I antigens, lead- strength and serum enzymes should be
ing to fi ber necrosis via the perforin performed during treatment. Ideally, the
pathway. initial steroid dosage is maintained until
strength and creatine kinase values have
returned to normal and have remained
Clinical Features
normal for four to eight weeks and then
The main symptoms of PM are weakness tapered. If a patient fails to respond to
affecting primarily the proximal muscles. glucocorticoid therapy or if a patient has
The onset is typically subacute, occurring had some improvement but the level of
over several weeks, with patients com- strength has reached a plateau, additional
plaining of increasing fatigue in rising therapy with either the immunosuppres-
from a chair, lifting their arms, climbing sive agent azathioprine or methotrexate is
steps, or combing their hair. The neck added. Another therapeutic option is intra-
extensor muscles may be involved caus- venous immune globulin, which is being
ing difficulty in holding up the head. In used increasingly to treat inflammatory
severe advanced cases, the respiratory myopathies. This therapy is associated with
and oropharyngeal muscles are involved little toxicity but is extremely expensive.
causing dysphagia and respiratory dif- Controlled trials involving larger numbers
ficulties. Myalgias are less frequent than of patients are needed to prove a benefit for
muscular weakness, and when present, this and other therapies in PM.
accompany the weakness. The most com-
mon clinical signs are decreased strength
Animal Models of Polymyositis
in the proximal muscles, contractures, and
late in the course of the disease, muscular Several animal models have been devel-
atrophy, which occurs in up to 40 percent oped that may prove useful for understand-
of cases. Dysphagia occurs in one-third ing the pathogenesis of PM. Experimental
of cases and is due to involvement of the autoimmune myositis (EAM) can be

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 193

induced by immunization of rodents with SERONEGATIVE
skeletal muscle homogenate and adju- SPONDYLOARTHROPATHY
vant. Similarly, Lewis rats immunized
with purified skeletal muscle myosin
Introduction and Epidemiology
develop EAM with the same pattern and Ankylosing spondylitis (AS) and related
severity as EAM induced by whole-rabbit spondyloarthropathies are characterized
skeletal-muscle homogenate. Multiple by sacroiliitis and inflammation of the
inflammatory lesions are detected histo- intervertebral discs in the lumbar spine, as
pathologically in various muscle groups well as an enthesitis at sites of ligamentous
in animals immunized with either prepa- insertions into bone. Patients with AS
ration. Dendritic cells presenting pyruvate develop calcification of the ligamentous
kinase M1/M2 peptide 464-472 to T cells insertions, back stiffness, and pain. The
have also been effective in inducing EAM immunologic basis of the specific loca-
in BALB/c mice, with >40 percent of the tions of inflammation in this disorder is
mice developing pathological changes in not understood, but a strong association
skeletal muscle similar to those seen in of the disease with particular HLA alleles,
human PM. particularly HLA-B27, suggests a possible
PM can also be induced in mice by role for antigen presentation to T cells in
viral infection. Intraperitoneal inoculation the immunopathogenesis. The prevalence
of Coxsackie B1 virus, Tucson strain, into of AS closely parallels the frequency of
young mice can result in proximal hind- HLA-B27. Among whites, the estimated
quarter weakness that persists for more prevalence rate of AS is 197 per 100,000 in
than ten weeks. The myositis persists long the United States. AS, in general, is diag-
after the virus is cleared and in the pres- nosed more frequently in males. Females,
ence of neutralizing antibody, suggesting however, may have milder or subclinical
that the immunological response is con- disease. AS is more common in males with
tributing to pathology. a male to female ratio of 3:1. The age of
A possible murine model of inflamma- onset of AS is usually from the late teens
tory myositis has resulted from a genetic to age 40.
deficiency in a member of the synapto-
tagmin family, comprising Ca2+ sensors
Etiology and Pathogenesis
involved in cell membrane fusion. Syt VII
is a ubiquitously expressed synaptotagmin The exact etiology of AS is unclear. The
previously implicated in plasma membrane strong association with most subtypes of
repair and Trypanosoma cruzi invasion, HLA-B27 supports the hypothesis that the
events mediated by the Ca2+-regulated disease is due to a genetically determined
exocytosis of lysosomes. Mice that are immune response to environmental fac-
Syt VII deficient have an inflammatory tors in susceptible individuals. HLA-B27 is
myopathy, with muscle-fiber invasion by present in 80 to 98 percent of white patients
leukocytes and endomysial collagen depo- with AS, in contrast to only about 8 percent
sition. This pathological picture is associ- of the general population. The arthritogenic
ated with elevated creatine kinase release peptide hypothesis postulates that AS
and progressive muscle weakness as well results when external antigenic challenge
as a strong ANA response. activates autoreactive T cells that recognize

194 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

endogenous peptides presented by HLA- including uveitis, occurring in 2535 per-
B27. In normal situations, the HLA-B27 cent of patients during the course of the
molecule on the surface of the antigen- disease. Manifestations of cardiac involve-
presenting cells presents endogenously ment include ascending aortitis, aortic
derived peptides to CD8+ T cells. These valve incompetence, conduction abnor-
peptides, which are usually nine amino malities, cardiomegaly, and pericarditis.
acids long and have arginine in position 2, In rare situations, aortitis may precede
are mostly self-derived, but they may other features of AS. Aortic incompetence
also be of viral or bacterial origin. Recent was noted in 3.5 percent of patients who
studies also suggest that the intracellu- had the disease for fifteen years and in 10
lar handling of molecular complexes that percent after thirty years. Cardiac conduc-
include HLA-B27 molecules may activate tion disturbances are seen with increasing
an inflammatory stress response and con- frequency with the passage of time, occur-
tribute to disease. ring in 2.7 percent of those with disease of
fifteen years duration and in 8.5 percent
after thirty years.
Clinical Features
Back pain is the most common symptom
Treatment of Ankylosing Spondylitis
and the first manifestation in approxi-
mately 75 percent of patients with AS. AS is a relatively mild disease for most
The inflammatory back pain of AS has patients with a good functional prognosis.
particular features that differentiate it The majority of patients do not experience
from mechanical back pain. These include significant extraskeletal manifestations,
insidious onset of the pain occurring over except for acute anterior uveitis. Usu-
months or years, generally with at least ally, the eye disease can be well managed
three months of symptoms before presenta- with eye drops containing corticosteroids
tion. Symptoms include morning stiffness to reduce inflammation and with pupil-
lasting at least thirty minutes, improve- dilating, atropine-like agents to prevent
ment of symptoms with moderate physical or diminish synechiae. The objectives for
activity, and diffuse nonspecific radiation treatment of AS are to relieve pain, stiff-
of pain into both buttocks. Patients often ness, and fatigue and to maintain good pos-
experience stiffness and pain that awakens ture and good physical and psychosocial
them in the early morning hours, a dis- functioning. Physiotherapy provided as
tinctive symptom not generally found in exercises is effective in improving thoraco-
patients with mechanical back pain. Ten- lumbar mobility and fitness, at least in the
derness at tendon insertion points due to short term (up to one year). Until recently,
enthesitis, an inflammatory reaction, is a no drug had been shown to significantly
common complaint. Typical tender sites influence the course of spinal disease and
include the costosternal junctions, spinous retard the process of ossification in AS.
processes, iliac crests, greater trochan- Similarly, there was no evidence to suggest
ters, ischial tuberosities, tibial tubercles, that any of the conventional DMARDs,
or heels (Achilles tendinitis or plantar including sulfasalazine and methotrexate,
fasciitis). AS is also characterized by a altered or inhibited the inflammation seen
number of extra-articular manifestations in the spine and entheses in AS. Recently,

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 195

the introduction of anti-TNF agents in the of BALB/c mice with versican results in
treatment of AS can be regarded as a defi- an AS-like pathology, including sacroi-
nite advantage in the therapy of this dis- liitis, enthesitis, and discitis. Those mice
ease. Both infliximab and etancercept have develop an ankylosis of the spine similar to
been shown to improve signs and symp- that seen in AS. It has also been noted that
toms of disease. Continuous treatment is immunity to versican can induce uveitis, a
necessary in most patients. More than two- clinical feature of the human disease.
thirds of the patients stay on therapy after
one year. Guidelines for the use of anti-
Future Directions of Research
TNF therapies have recently been devel-
oped. The new therapeutic modalities Significant progress in characterizing the
identify important clinical questions to be underlying immunopathogenic mecha-
answered by further research. Clearly, inf- nisms of rheumatic diseases is being stim-
liximab and etanercept have disease-modi- ulated by accelerated success in defining
fying properties, but their long-term safety disease susceptibility genes using high-
and disease-controlling effects in terms throughput single-nucleotide polymor-
of improvement or maintenance of func- phism analysis. In addition, increased focus
tion, as well as the prevention of structural on the activation of the innate immune
damage, still have to be demonstrated. response, resulting in activation of molecu-
lar pathways involved in either production
of type I interferon or TNF, is leading to
Animal Models of Seronegative
new understanding of the effect of those
cytokines on altered immunoregulation
HLA-B27 transgenic rats and strains of and inflammation and has substantially
HLA-B27 transgenic 2-microglobulin- advanced development of new targeted
deficient mice develop a multisystem therapies.
inflammatory disease affecting the joints, Future advances in rheumatic disease
skin, and bowel with strong similarity research are likely to derive from func-
to human spondyloarthritis. This model tional studies related to disease-associated
supports a direct mechanistic role for that genes identified in the large-scale studies.
MHC product in the disease, although the The regulation of innate immune system
details of that mechanism have not been activation, alterations in function of cell
elucidated. surface molecules, and signaling pathways
As is the case for other autoimmune that control the threshold for lymphocyte
rheumatic diseases, antigen-induced activation and regulation of the comple-
murine models are proving useful in study ment system are areas for further study
of AS. Aggrecan and versican are large pro- that may identify functional correlates of
teoglycan molecules that are present in the the genes characterized in recent collab-
intervertebral disc and hyaline cartilages of orative projects. The regulation of cytokine
the sacroiliac joint, as well as in entheses. production, with a particular emphasis on
Versican is generally absent from cartilage the type I interferon pathway, will be an
tissue except in the sacroiliac joint but is important topic that can link genetics and
concentrated in ligaments and the annulus translational research that is based on care-
of the intervertebral disc. Immunization ful clinical characterization in association

196 Experimental Approaches to the Study of Autoimmune Rheumatic Diseases

with laboratory investigations. The use of GS, Panayi GS, Wollheim FA, eds.
the experimental murine models described Rheumatoid Arthritis New Frontiers in
in this chapter will represent an important Pathogenesis and Treatment. New York,
approach to elucidating the mechanisms NY: Oxford University Press; 2000:39.
through which gene variants and altered Jorgensen TN, Gubbels MR, Kotzin BL.
immune function result in tissue damage New insights into disease pathogenesis
and disease consistent with the pathol- from mouse lupus genetics. Curr Opin
ogy that occurs in humans. Future studies Immunol. 2004;16:787793.
should be aimed at identifying molecular Kono DH, Theofilopoulos AN. Genetics
targets that can be practically modulated of systemic autoimmunity in mouse
using new therapeutics, with animal stud- models of lupus. Int Rev Immunol.
ies serving to speed translation to humans. 2000;19:367387.
Lauwerys BR, Wakeland EK. Genetics of
lupus nephritis. Lupus. 2005;14:212.
Morel L, Wakeland EK. Lessons from the
SUGGESTED READING NZM2410 model and related strains.
Briani C, Doria A, Sarzi-Puttini P, Int Rev Immunol. 2000;19:423446.
Dalakas MC. Update on idiopathic Nagaraju K, Plotz PH. Animal models of
inflammatory myopathies. Autoimmu- myositis. Rheum Dis Clin North Am.
nity. 2006;39(3):161170. 2002;28:917933.
Christner PJ, Jimenez SA. Animal models Theofilopoulos AN, Dixon FJ. Murine
of systemic sclerosis: insights into sys- models of systemic lupus erythemato-
temic sclerosis pathogenesis and poten- sus. Adv Immunol. 1985;37:269390.
tial therapeutic approaches. Curr Opin Van den Berg WB. 2004. Animal models. In:
Rheumatol. 2004;16:746752. St. Clair EW, Pisetsky DS, Haynes BF,
Crow MK. Collaboration, genetic associa- eds. Rheumatoid Arthritis. Philadelphia,
tions, and lupus erythematosus. N Engl PA: Lippincott Williams & Wilkins;
J Med. 2008;358(9):956961. 2004.
Hahn BH. Animal models of systemic Varga J, Abraham D. Systemic sclero-
lupus erythematosus. In: Wallace DJ, sis: a prototypic multisystem fibrotic
Hahn BH, eds. Dubois Lupus Erythema- disorder. J Clin Invest. 2007;117(3):
tosus. 6th ed. Philadelphia, PA: Lippin- 557567.
cott Williams & Wilkins; 2002:339. Wakeland EK, Liu K, Graham RR, Behrens
Holmdahl R, Andersson M, Goldschmidt TW. Delineating the genetic basis of
TJ, Gustafsson K, Jansson L, Mo JA. systemic lupus erythematosus. Immu-
Type II collagen autoimmunity in ani- nity. 2001;15:397408.
mals and provocations leading to arthri- Zhang Y. Animal models of inflamma-
tis. Immunol Rev. 1990;118:193232. tory spinal and sacroiliac joint diseases.
Holmdahl R. Experimental models for Rheum Dis Clin North Am. 2003;29:
rheumatoid arthritis. In: Firestein 631645.

Experimental Approaches to the Study of Autoimmune Rheumatic Diseases 197

12. Immunological Aspects of Cardiac Disease

John B. Zabriskie, M.D., Allan Gibofsky, M.D., J.D., Wesley C. Van Voorhis, M.D.,
Ph.D., Frederick S. Buckner, M.D., and Noel R. Rose, M.D., Ph.D.

INTRODUCTION concomitant clinical and laboratory signs

of carditis and valvulitis, or carditis and
Although the vast majority of cardiac dis- valvulitis may be the only signs of an acute
eases relate to the presence of atheroscle- episode. (3) There may be involvement of
rosis in the vessels supplying blood and the central nervous system, manifesting
oxygen to the heart, there are certain con- itself as Sydenhams chorea. The clinical
ditions in which immunological events episodes are self-limiting but damage to
play an important role. This chapter will the valves may be chronic and progressive,
concentrate on the immunological aspects resulting in cardiac decompensation and
of these diseases. We will also discuss the death.
animal models associated with each of Although both the severity and mor-
these disorders with particular emphasis tality of the disease have dramatically
on whether these models help us to under- declined since the turn of the century,
stand further the immunological aspects of recent reports indicate a resurgence in this
these conditions. country (Veasy et al. 1987) and in many
military installations in the world, remind-
ing us that ARF remains a public health
RHEUMATIC FEVER problem even in developed countries. In
addition, the disease continues essentially
Acute rheumatic fever (ARF) is a delayed, unabated in many of the developing coun-
nonsuppurative sequela of a pharyngeal tries. Estimates suggest 1020 million new
infection with the group A streptococcus. cases per year in those countries where
A latent period of two to three weeks fol- two-thirds of the worlds population lives.
lows the initial streptococcal pharyngitis.
The latent period remains the same for
each individual patient in the event of a
recurrence. This suggests that the patient The incidence of ARF actually began to
has already been exposed to more than decline long before the introduction of
one streptococcal infection in the past. The antibiotics into clinical practice, decreasing
onset of disease is usually characterized by from 250 to 100 patients per 100,000
an acute febrile illness, which may mani- population from 1862 to 1962 in Denmark
fest itself in one of three classical ways: (1) (see Figure 12.1). Why the decline occurred
The patient may present with migratory is not entirely clear, but better hygiene,
arthritis predominantly involving the large improved economic conditions, and less
joints of the body. (2) There may also be crowding were certainly important factors.



Morbidity Per 100,000



1860 70 80 90 1900 10 20 30 40 50 60 70
Figure 12.1 Data demonstrating the decline of mortality rates of rheumatic
fever long before the advent of penicillin and then the precipitous drop after
its introduction.

The introduction of antibiotics in 1950 rap- unresolved. What is true, however, is that
idly accelerated this decline, until by 1980, a streptococcal strain capable of causing a
the incidence ranged from 0.23 to 1.88 well-documented pharyngitis is generally
patients per 100,000, primarily in children capable of causing ARF.
and teenagers in the United States. A nota-
ble exception has been in the Australian
aborigines (125/100,000) and the native
Hawaiian and Maori populations (both of Although there is little evidence for the
Polynesian ancestry), where the incidence direct involvement of group A streptococci
continues to be 13.4/100,000 hospitalized in the affected tissues of ARF patients, a
children per year. large body of epidemiological and immu-
Whether certain M-type strains are nological evidence indirectly implicates
more rheumatogenic than others remains the group A streptococcus in the initiation
controversial. Certain investigators have of the disease process: (1) It is well known
stated that M protein types 5, 14, 18, and that outbreaks of ARF closely follow epi-
24 are mainly associated with outbreaks, demics of either streptococcal sore throats
but in the large series collected in the Great or scarlet fever. (2) Adequate treatment of
Lakes Naval Station of ARF during World a documented streptococcal pharyngitis
War II, the main strains were types 17, markedly reduces the incidence of
19, and 30. In the Caribbean and in Latin subsequent ARF. (3) Appropriate anti-
America, the main ARF strains are 11 and microbial prophylaxis prevents the
41. Thus, whether certain strains are more recurrences of disease in known patients
rheumatogenic than others remains with acute ARF.

200 Immunological Aspects of Cardiac Disease

A note of caution is necessary concerning is whether any or some of these antibodies
documentation (either clinically or micro- play a role in initiating the disease process.
biologically) of an antecedent streptococ- This will be discussed in more detail in the
cal infection. The frequency of isolation of etiology section. For a more detailed dis-
group A streptococci from the oropharynx cussion of these cross-reactions, the reader
is extremely low during the acute stage of is referred to several excellent reviews of
rheumatic fever even in populations with this subject matter.
limited access to antibiotics. Further, there Although, numerous attempts to pro-
appears to be an age-related discrepancy duce antibodies to this capsule have been
in the clinical documentation of an ante- unsuccessful, more recently reports have
cedent sore throat. In older children and emerged demonstrating high antibody
young adults, the recollection of a strepto- titers to hyaluronic acid using techniques
coccal sore throat approaches 70 percent; in designed to detect nonprecipitating anti-
younger children, this rate approaches only bodies in the sera of immunized animals.
20 percent. Thus, it is important to have a Similar antibodies have been noted in
high index of suspicion of ARF in children humans. The data establishing the impor-
or young adults presenting with signs of tance of this capsule in human infections
arthritis or carditis even in the absence of have been almost nonexistent, although
a clinically and microbiologically docu- Stollerman has commented on the pres-
mented sore throat. ence of a large mucoid capsule as being
Another intriguing, yet unexplained one of the more important characteristics
observation has been the invariable asso- of certain rheumatogenic strains.
ciation of ARF only with streptococcal
pharyngitis. Although there have been
many outbreaks of streptococcal skin infec-
tions (impetigo), ARF almost never occurs That ARF might be the result of a host
following infection with these strains. genetic predisposition has intrigued inves-
Furthermore, in Trinidad, where both tigators for over a century. It has been
impetigo and ARF are concomitant infec- variously suggested that the disease gene
tions, the strains colonizing the skin are dif- is transmitted in an autosomal dominant
ferent from those associated with ARF and fashion, in an autosomal recessive fashion
did not influence the incidence of ARF. with limited penetrance, or that it is pos-
sibly related to the genes conferring blood-
group secretor status.
Group A Streptococcus
Renewed interest in the genetics of ARF
Figure 12.2 is a schematic cross section of occurred with the recognition that gene
the group A streptococci. Many of the anti- products of the human MHC (major his-
gens present in the group A streptococcus tocompatibility complex) were associated
cross-react with antigens present in mam- with certain clinical disease states. Using
malian tissues: a concept we call molecular an alloserum from a multiparous donor, an
mimicry. Thus, antibodies present in the increased frequency of a B-cell alloantigen
sera of ARF patients bind to cardiac tissue was reported in several genetically distinct
or cells in the caudate nucleus or to human and ethnically diverse populations of ARF
kidney tissues. The controversial question individuals and was not MHC related.

Immunological Aspects of Cardiac Disease 201


CELL WALL Myocardium

Protein (M, R, T)
Group Carbohydrate Valvular

Vascular Intima
Lipoteichoic Acid Myocardial Antigen




HLA Antigen
B Cell Antigen

Figure 12.2 Schematic representation of the various structures of the group A streptococcus. Note
the wide variety of cross-reactions between its antigens and mammalian tissues.

Most recently, a monoclonal antibody the HLAs, depending on the racial fea-
(D8/17) was prepared by immunizing mice tures of the patient group. These seem-
with B cells from an ARF patient. A B-cell ingly conflicting results concerning HLA
antigen identified by this antibody was and RF susceptibility prompt speculation
found to be expressed on increased num- that these reported associations might be
bers of B cells in 100 percent of rheumatic of class II genes close to (or in linkage dis-
fever patients of diverse ethnic origins and equilibrium with) but not identical to the
only in 10 percent of normal individuals. putatative RF susceptibility gene. Alter-
The antigen defined by this monoclonal natively, and more likely, susceptibility to
antibody showed no association with or ARF is polygenic, and the D8/17 antigen
linkage to any of the known MHC hap- might be associated with only one of the
lotypes, nor did it appear to be related to genes (i.e., those of the MHC encoding for
B-cell activation antigens. the D-related [DR] antigens) conferring
These studies are in contrast to other susceptibility. Although the full explana-
reports in which an increased frequency of tion remains to be determined, the pres-
certain human leucocyte antigens (HLAs) ence of the D8/17 antigen does appear
was seen in ARF patients. There are marked to identify a population at special risk of
differences in the increased frequency of contracting ARF.

202 Immunological Aspects of Cardiac Disease

response (both humoral and cellular) in the
Etiologic Considerations
genetically susceptible individual to those
A large body of both immunological and streptococcal antigens cross-reactive with
epidemiological evidence has implicated mammalian tissues. The evidence support-
the group A streptococcus in the induction ing this theory can be divided into three
of the disease process. Yet, the precise broad categories:
pathological mechanisms involved in the
process remain obscure. At least three main 1. Employing a wide variety of methods,
theories have been proposed. numerous investigators have docu-
The first theory is concerned with the mented the presence of heart-reactive
question of whether persistence of the antibodies (see Figure 12.3) in ARF
organism is important. Despite several sera. The prevalence of these antibodies
controversial reports, no investigators have has varied from a low of 33 percent to
been able to consistently and reproducibly a high of 85 percent in various series.
demonstrate live organisms in rheumatic Although these antibodies are seen in
fever joints, cardiac tissues, or valves. other individuals (notably those with
The second theory focuses on whether uncomplicated streptococcal infections
deposition of toxic products is required. that do not go on to rheumatic fever
Although an attractive hypothesis, little or and patients with poststreptococcal glo-
no experimental evidence has been obtained merulonephritis), the titers are always
to support this concept. Renewed interest lower than that seen in rheumatic fever
in extracellular toxins has recently emerged and decrease with time during the con-
with the observation that certain strepto- valescent period (Figure 12.4).
coccal pyrogenic exotoxins (A and C) may In terms of diagnosis and progno-
act as superantigens. These antigens may sis, it is important to observe that these
stimulate large numbers of T cells through heart-reactive antibody titers decline
their unique bridging interaction with over time. By the end of three years,
T-cell receptors of specific V types and these titers are essentially undetect-
class II MHC molecules. This interaction is able in patients who had only a single
clearly distinct from conventional antigen attack (see Figure 12.4) This pattern is
presentation in the context of MHC. Once consistent with the well-known clinical
activated, these cells elaborate tumor necro- observation that recurrences of rheu-
sis factor, gamma interferon, and a number matic fever most often occur within the
of interleukin moieties, thereby contribut- first two to three years after the initial
ing to the initiation of pathological damage. attack and become rarer five years after
Furthermore, it has been suggested that in an initial episode.
certain disease states such as rheumatoid As illustrated in Figure 12.5, this pat-
arthritis, autoreactive cells of specific V tern of titers also has prognostic value.
lineage may home to the target organ. During the two- to five-year period after
Although this is an attractive hypothesis, no the initial attack, patient M.P.s heart-
data concerning the role of these superanti- reactive titers dropped to undetectable
gens in ARF have been forthcoming. levels. However, with a known break in
Perhaps the best evidence to date favors prophylaxis starting in year 6, at least
the theory of an abnormal host immune two streptococcal infections occurred,

Immunological Aspects of Cardiac Disease 203

Figure 12.3 Photomicrograph of immunofluorescent staining of heart
sections with a serum from an acute rheumatic fever patient followed
by a fluorescein-tagged antihuman IgG goat serum. Note the intense
sarcolemmal staining pattern of the serum. Identical results were obtained
with rabbit sera immunized with Group A streptococcal cell walls but not
Group D walls.

3 1:5 dilutions
2 1:10 dilutions
1 1:20 dilutions
1 2 3 4 5 6

3 1:5 dilutions
2 1:10 dilutions
1 1:20 dilutions
1 2 3 4 5 6

Figure 12.4 Serial heart-reactive antibody titers in forty

patients with documented acute rheumatic fever. Note the
slow decline of these titers over the first two years after
the initial episode and the absence of these antibodies five
years after the initial attack.

as evidenced by rise in antistreptolysin infection was followed by a clinical

O (ASO) titers during that period. recurrence of classical rheumatic cardi-
Of note was the concomitant rise in tis complete with isolation of the organ-
heart-reactive antibody titers. The final ism, elevated heart-reactive antibodies,

204 Immunological Aspects of Cardiac Disease

Figure 12.5 Heart-reactive antibody titers and laboratory data obtained from a patient with rheumatic
fever who had two well-documented acute attacks eleven years apart. Note absence of the heart-
reactive antibody during years 2 to 5 and its reappearance during years 6 to 10 after evidence of two
intercurrent streptococcal infections secondary to breaks in penicillin prophylaxis (see ASO titers). High
titers of heart-reactive antibody appeared with the second attack. CRP, C reactive protein. Reprinted
from Kelleys Textbook of Rheumatology, chapter 103, copyright Elsevier 2006.

and acute-phase reactants eleven years those streptococcal antigens of the

after the initial attack. M protein that share homology with
2. Sera from patients with ARF also con- human myosin, tropomyosin, keratin,
tain increased levels of antibodies to and so on.
both myosin and tropomyosin, as com-
pared with sera from patients with pha- At a cellular level, there is now ample
ryngeal streptococcal infections that do evidence for the presence of both lym-
not develop ARF. These myosin-affinity phocytes and macrophages at the site of
purified antibodies also cross-react with pathological damage in the heart valves
M protein moieties (known to share in patients with ARF (see Figure 12.6).
amino acid homology with myosin), The cells are predominantly CD4+ helper
suggesting this molecule could be the lymphocytes during acute stages of the
antigenic stimulus for the production disease (4:1). The ratio of CD4+/CD8+ lym-
of myosin antibodies in these sera. phocytes (2:1) more closely approximates
3. Finally, as indicated earlier, autoim- the normal ratio in chronic valvular speci-
mune antibodies are a prominent find- mens. A majority of these cells express DR
ing in another major clinical manifes- antigens. A potentially important finding
tation of ARF, namely, Sydenhams has been the observation that macrophage-
chorea, and these antibodies are like fibroblasts present in the diseased
directed against the cells of the caudate valves express DR antigens and might be
nucleus. The titers of this antibody cor- the antigen-presenting cells for the CD4+
respond with clinical disease activity. lymphocytes. Increased cellular reactiv-
4. In all cases in which autoreactive anti- ity to streptococcal antigens has also been
bodies are seen (heart, brain, cardiac noted in the peripheral blood mononuclear
valves, kidney), they can be absorbed cell preparations of ARF patients when
with streptococcal antigens, notably compared with these cells isolated from

Immunological Aspects of Cardiac Disease 205

Composition of the Mononuclear Celllular Infiltrates in
Acute and Chronic Active Rheumatic Valvulitis

Percentage of
Type of Type of Percentage of mononuclear cells Leu 3a/
Patient Leu 4 + T cell
valve valvulitis" Leu 2a ratio
HLA-DR+ 63D3+ Leu 16+ Leu 4+ Leu 3a+ Leu 2a+
Acute valvulitis
1 Mitral Acute 58.9 42.6 5.1 49.5 75.6 23.9 3.1
2 Mitral Acute 49.8 43.1 6.9 43.1 58.7 34.3 1.9
Aortic Acute 52.7 51.0 3.9 38.1 65.9 26.5 2.3
3 Mitral Acute 63.9 42.0 5.5 52.4 75.4 18.9 4.0
4 Aortic Acute 68.1 56.0 7.4 33.7 71.6 22.0 3.3
Chronic valvulitis
4 Mitral Chronic active 49.4 47.4 7.4 44.3 53.7 38.8 1.4
5 Mitral Chronic active 48.8 39.1 1.4 53.9 45.2 51.5 0.9
Aortic Chronic active 67.8 35.0 4.0 36.8 47.5 49.1 1.0
6 Mitral Chronic active 41.8 23.4 8.0 65.9 57.3 33.3 1.7
Aortic Chronic active 69.6 48.7 6.2 30.1 58.2 32.6 1.8
7 Mitral Chronic active 55.4 24.2 8.1 59.8 64.9 24.7 2.6
8 Mitral Chronic active 80.4 34.1 13.4 44.4 44.8 50.9 0.9
9 Mitral Chronic active 46.1 29.6 0.8 65.6 61.6 33.3 1.8
"Determined in the frozen valve samples studies

Figure 12.6 Composition of the mononuclear cellular infiltrates in acute and chronic rheumatic
valvulitis. Note the increased CD4/CD8 ratios in acute cases of ARF compared with the more normal
CD4/CD8 ratios in chronic more inactive disease patients. Reprinted from Kemeny, et al. (1989)
with permission from Elsevier. Kemeny E, Grieve T, Marcus R, Sareli P, Zabriskie JB. Identification
of mononuclear cells and T cell subsets in rheumatic valvulitis. Clin Immunol Immunopathol.
1989;52:225237, table 3, p. 231.

nephritis patients. This abnormal reactiv- rheumatic feverassociated strain are also
ity peaks at six months after the attack but cytotoxic for myofibers, but specificity
may persist for as long as two years after solely for cardiac cells was lacking in the
the initial episode. Once again, the reactiv- human studies. Similar studies have not
ity was specific only for those strains asso- yet been performed using lymphocytes
ciated with ARF, suggesting an abnormal from active ARF patients.
humoral and cellular response to strepto- A new and potentially interesting
coccal antigens unique to rheumatic fever chapter is unfolding concerning this mani-
associated streptococci. festation of rheumatic fever. It has been
The observation that lymphocytes known for years that often the early symp-
obtained from experimental animals sensi- toms of chorea may present as emotional
tized to cell membranes but not cell walls or behavioral changes in the patient and
are specifically cytotoxic for syngeneic only later do the choreiform motor symp-
embryonic cardiac myofibers in vitro fur- toms appear. Years after the choreiform
ther strengthens support for the potential symptoms subsided a number of chorea
pathologic importance of these T cells. In patients presented with behavioral disor-
humans, normal mononuclear cells primed ders such as tics or obsessive-compulsive
in vitro by M protein molecules from a disorders (OCD).

206 Immunological Aspects of Cardiac Disease

Figure 12.7 Immunofluorescent micrograph of frozen sections of normal
human caudate nucleus showing positive staining of large neurons. The
serum used was obtained from a child with prolonged active chorea.

These earlier observations coupled with differential diagnosis of ARF from other
the known presence of antibrain antibodies disorders. In our hands, all rheumatic
in the sera of Sydenhams chorea patients fever patients express abnormal levels of
(see Figure 12.7) raised the question of D8/17-positive B cells, especially during
whether a prior streptococcal infection the acute attack. In those cases where the
(or infection with other microbes) might diagnosis of ARF has been doubtful, pres-
induce antibodies cross-reactive with ence of elevated levels of D8/17-positive
brain antigen(s) involved in neural path- B cells has proven to be helpful in estab-
ways associated with behavior. Two recent lishing the correct diagnosis.
papers indicate a strong association of the
D8/17 B-cell marker with children with
Animal Models of Rheumatic Fever
OCD. While Swedo and colleagues selected
patients on the basis of a strong history of Although investigators over the past sixty
prior streptococcal infections, Murphy and to seventy years have tried to understand
colleagues noted a strong association of the the pathogenetic mechanisms involved in
marker with OCD patients without a his- the initiation and progression of ARF, to
tory of streptococcal infections. These pre- date we still do not have a clear picture of
liminary studies suggest that streptococci the factors involved in the microbe-host
and probably other microbes may induce relationship.
antibodies, which functionally disrupt the The most significant drawback to
basal ganglia pathways leading not only our understanding is that the relation-
to classical chorea but also to other behav- ship of the group A streptococcus to this
ioral disorders in these children without disease is primarily human, and animal
evidence of classical chorea. models have not been helpful to date.
Second, the use of the D8/17 monoclo- As elegantly pointed out by Krishna and
nal antibody has also proved helpful in the Iyer in a recent chapter devoted to these

Immunological Aspects of Cardiac Disease 207

models, the design of the animal mod- lating a large release of cytokines that could
els has closely paralleled the prevailing initiate the ARF process. Furthermore they
hypothesis at any given time. These have are even capable of deleting certain TCR
involved (1) whether persistent and sub- alleles, possibly inducing loss of self-tol-
clinical infection by the organism was a erance and the induction of self-reactive
factor, (2) whether direct injury to the myo- autoantibodies.
cardium or valves by streptococcal toxins All of these observations coupled
was involved, or (3) whether streptococcal with the unique association of group A
antibodies cross-reactive with human tis- streptococcal infections in humans ver-
sues can initiate immune-mediated injury. sus animals suggest that introduction
Unfortunately, none of these approaches of human genes in mice or rats to create
have yielded any unequivocal evidence transgenic animals may be more helpful in
of the classic lesions of ARF. Further- understanding the human rheumatic fever
more, none have clearly demonstrated the process. These animals are now available
known clinical features of the human dis- for study mainly based on the long-term
ease such as a prior streptococcal pharyn- commitment to transgenic mice by Dr.
gitis followed by a latent period followed Chella David. These mice contain the gene
by clinical and pathological signs of an for human CD4+ cells and genes for several
ARF episode. different MHC molecules. These mice are
However, since the 1970s, observation more susceptible to the lethal efforts of the
in ARF patients both in circulatory blood superantigen toxins (similar to humans)
samples and serum as well as studies in tis- when compared with nontransgenic
sues removed from ARF and RHD patients littermates, but to date, no studies have
have strengthened the concept that an been carried out to determine how these
abnormal response to streptococcal anti- mice might react to oral infection with
gens at a cellular and humoral level in the several different group A streptococcal M
genetically susceptible host results in the types, particularly with respect to myo-
clinical and laboratory signs of ARF. Fur- cardial and valvular damage. The gene
ther studies have revealed the following: for human B-cell production may also be
(1) heart-reactive antibodies cross-reactive needed to complete the pathological find-
with streptococcal antigens are present in ings seen in human disease.
the sera of these patients; (2) the number of Whether these human gene introduc-
CD4+ cells are increased both in the plasma tions are sufficient to mimic the human
and tissues of ARF patients compared disease is not known. But the creation of
with CD8; (3) the total number of B cells these and other transgenic models may
is increased compared with age-matched well be more fruitful since all natural mod-
controls; (4) the inflammatory cytokines els tested so far have had little success.
such as TNF-, IL-2, and IL-1 are elevated
in the acute phase and return to normal
Streptococcal Vaccine Candidates
levels after the episode; and (5) group
A streptococci secrete several exotoxins As early as the 1930s, researchers were
called SPEA, SPEB, and SPEC, which are pursuing the study of streptococcal vac-
called superantigens because they bind to cinations, with the injection of whole-
the lateral side of TCR and the MHC stimu- killed group A streptococci and cell walls,

208 Immunological Aspects of Cardiac Disease

thereof culminating in injections of par- (1989) produces protection against oral
tially purified M protein extracts in the colonization of the throat by group A
1970s. However, all interest, especially streptococci.
by the pharmaceutical companies, ceased 3. A surface protein called C5 peptidase,
at that point because the U.S. Food and which is present on the surface of all
Drug Administration (FDA) proclaimed group A and group B streptococci,
that one could work on streptococcal vac- produces antibodies that block both
cines as long as no streptococcal compo- the colonization of group A and group
nent was used! The FDA was afraid that B streptococci in oral colonization
induction of antibodies cross-reactive studies.
with human tissues, especially cardiac tis- 4. The streptococcal group A carbohydrate
sues, could be detrimental to the vaccinee. (CHO) as proposed by Zabriskie and
However, it was soon apparent that many colleagues has been purified and used
individuals had antibodies cross-reactive as an immunogen to protect against
with a variety of human tissues and were streptococcal infections. This vaccine
perfectly normal. In the past decade, the candidate promotes phagocytosis of
restriction was removed on the condition group A streptococci of several different
that toxicity studies in animals did not M types, will protect against infection
reveal any deletion effects in the animals using passive and active immunization
when injected with a streptococcal vaccine studies, and also protects against oral
candidate. colonization. Table 12.1 summarizes all
This ushered in the search for an effec- the studies on these four candidates and
tive, safe, and inexpensive group A strep- it can be seen that the variable region of
tococcal vaccine, and there are now at least the M protein and the CHO molecule
four prominent candidates with more offer the most convincing evidence for
in the pipeline. This area has recently a protective vaccine against group A
been reviewed in New Generation Vaccines streptococcal infections.
(chapter 57) and will only briefly be sum-
marized here.
Directions for Future Research
1. Perhaps the most advanced candidate Obviously, the most active area for strep-
is by Dale and colleagues (1996), which tococcal research in the future will be the
is synthetic peptide sequences of a vari- development of a good group A strepto-
ety of M protein types taken from the coccal vaccine. Several good candidates
variable region of the M protein and are present and more will be studied and
hooked together by linkers. This has evaluated in the future. There are also
induced protective immunity in ani- candidates such as the hyaluronic acid
mals to a number of different M protein capsule (see Figure 12.2) and many other
types and is safe for use in humans. surface antigens, including the T antigen
Clinical trial of its efficacy to prevent (similar in many cases but not identical to
streptococcal infections is currently the M antigen) and others still unknown.
under way. It is an exciting area and the production of
2. The use of the C-repeat constant region a safe and inexpensive efficacious vaccine
of the M protein advanced by Fischetti will be the desired goal, especially in

Immunological Aspects of Cardiac Disease 209

Table 12.1 Summary of Functional Properties of in developing countries (Africa, South
4 Group A Streptococcal Vaccine Candidates America, India, China, etc.), but careful and
Candidates I II III IV well-funded collaborations with clinicians
Variable + + + + and researchers in these countries could
Region M be rewarding. Another fascinating chal-
lenge has been the obvious change both in
Region the organisms ability to cause these com-
C5 Peptidase + ND ND
plications but also in the severity of the
disease. One might argue that this is sec-
Group + + + +
A CHO ondary to better health care and medical
I. Phagocytosis care and better nutrition in these countries.
II. Prevention of colonization However, in many poor countries, there
III. Passive Protection really is no health care system in place nor
IV. Active Protection has nutrition improved significantly in the
past sixty years; yet, the disease is milder.
In some ways, it is similar to diseases such
countries that do not have a well-developed as measles, which is not a particular prob-
health care system. lem in most populations but can be devas-
Yet our understanding of the basic tating in those populations that have never
mechanisms involved in the transition from been exposed to the agent. Over time,
a relatively mild pharyngeal infection with even in these populations, the disease has
group A streptococci followed by a latent decreased in severity as herd immunity to
quiescent period and then followed by ARF the agent increases.
with or without carditis or the appearance Finally, one of the most intriguing,
of Sydenhams chorea is still an important unresolved questions has been the possible
goal because the appearance of the arthri- association of the group A streptococcus
tis or acute damage to the valvular tissue with behavioral disorders such as OCD,
is clearly associated with infection of the tics, Tourettes syndrome, and anorexia
group A streptococcus. It is the only infec- nervosa. Even as early as the 1890s, Osler
tious disease of its type in which we at least observed in his textbook of medicine that
know the causative organism. Do superan- many of his former Sydenhams chorea
tigens play a role in this transformation? patients (a well-known neurological mani-
Are particular cytokines and lymphokines festation of a streptococcal infection) often
present during the quiescent period? Are were later seen as patients in the behav-
cellular mechanisms at work, and if so, ioral disorder clinics. One can also expand
what cells are involved? These questions these concepts to other organisms in which
are important because the answers to those there may be shared antigenicity between
questions in ARF may hold keys to unlock- microbial and mammalian antigens.
ing other rheumatic diseases (see Chapter
11) as well as our basic understanding of
host-microbial relationships in other pre-
sumed autoimmune diseases. ARF remains one of the few autoimmune
Those are the difficulties in these stud- disorders known to occur as a result of
ies. The disease is now seen most frequently infection with a specific organism. The

210 Immunological Aspects of Cardiac Disease

confirmed observation of an increased deposits T. cruzi as well. The individual
frequency of a B-cell alloantigen in several then becomes infected either by the feces
populations of rheumatics suggests that it deposited at the site of the puncture or
might be possible to identify individuals inadvertent contamination of a mucous
susceptible to ARF at birth. If so, then from membrane of the human secondary to
a public health standpoint, these individu- hand contamination with organisms. The
als would be prime candidates for immu- distribution of the arthropod vectors is
nization with any streptococcal vaccine limited to the Western Hemisphere and is
that might be developed in the future. In widely distributed in the southern United
addition, careful monitoring of streptococ- States, Mexico, and Central America. The
cal disease in the susceptible population reduviid vectors are common in most
could lead to early and effective antibiotic South American countries, especially
strategies, resulting in disease prevention. Brazil, Argentina, Bolivia, and Chile.
Finally, in individuals previously infected, Chagas is a disease primarily of the poor
who later present with subtle or nonspecific largely because the reduviid bugs favor
manifestations of the disease, the presence living in adobe mud or enter poor housing
or absence of the marker could be useful in through cracks or holes in the walls and
arriving at a diagnosis. thatched roofs.
The continued study of ARF as a para-
digm for microbial-host interactions also
Mode of Transmission and
has important implications for the study
of autoimmune diseases in general and
rheumatic diseases in particular. Further An estimated 20 million people are cur-
insights into this intriguing hostparasite rently infected with T. cruzi, and approxi-
relationship may shed additional light into mately 100 million people are at risk of
those diseases in which the infection is infection. The endemic area of Chagas
presumed but has not yet been identified. disease is huge, stretching from Mexico to
southern Argentina, but is limited to the
Western Hemisphere. The incidence, prev-
CHAGAS DISEASE alence, and severity of Chagas disease and
T. cruzi infection seem to be higher in South
Chagas disease is named after the America than Central America; this may
Brazilian physician Carlos Chagas who be due to differences in vector behavior or
first discovered the parasite responsible strain variation of T. cruzi or differences in
for the signs and symptoms of the disease. disease recognition and reporting.
The causative agent is the hemoflagellate T. cruzi is transmitted by blood-feed-
protozoan Trypanosoma cruzi. This proto- ing reduviid insect vectors, vertically from
zoan parasite is transmitted to the blood- mother to child, or by blood transfusions.
sucking reduviid bug vector when the Vector transmission is responsible for
reduviids take a blood meal from animals most of T. cruzi infections, and most of this
or humans who are infected with circulat- transmission is secondary to the deposit
ing T. cruzi. When the infected bug takes of infected feces and urine by the reduviid
a subsequent blood meal, it usually def- bug at the time of the blood meal. As an
ecates on the skin of the next victim and alternative, humans have been reported to

Immunological Aspects of Cardiac Disease 211

be infected by accidental oral ingestion of transmission in the northern part of South
reduviid bugs, which presumably release America and in Central America.
metacyclic trypomastigotes to invade the
oral mucosal cells. Parasites persist in the
HostParasite Interaction and Host
blood of seropositive asymptomatic indi-
viduals, and blood from these individuals
can transmit infection in approximately 12 INNATE IMMUNE RESPONSE
percent to 25 percent of transfusions. Blood Upon inoculation into tissues of the
transfusion transmission has become less mammalian host, T. cruzi infects local
frequent as blood banks in endemic areas cells where it multiplies intracellularly
screen blood with serologic tests to deter- as amastigotes. At this early point in
mine whether the blood is likely to be con- infection, the host depends on its innate
taminated with T. cruzi. However, some immune response to microorganisms,
areas do not routinely screen donors for which is primarily mediated by macro-
T. cruzi infection. Congenital transmission phages and natural killer (NK) cells. Unac-
occurs in approximately 1 percent of preg- tivated (resting) macrophages, however,
nancies of infected women, but the inci- have little ability to kill T. cruzi and serve
dence is higher if the woman is infected as important host cells for parasite repli-
during pregnancy. Infection of the fetus cation. Within two hours of macrophage
probably occurs transplacentally in months invasion, the majority of T. cruzi have
5 to 9 of pregnancy. escaped the phagosome and exist free
The reduviid vectors that transmit in the host cytoplasm. It is assumed that
T. cruzi are members of the order by rapidly escaping the phagolysosome,
Hemiptera, family Reduviidae, and sub- T. cruzi is able to avoid degradation by
family Triatominae. Although there are intracellular microbicidal enzymes in the
more than 100 species of triatomines, few unprimed macrophage. A variety of stim-
are epidemiologically significant as vectors uli are capable of priming macrophages
of T. cruzi to humans. Species that colonize for antitrypanosomal activity, with IFN-
housing (domiciliary app.) are important being particularly potent. The addition of
in transmission to humans in the south- a complex mixture of cytokines, including
ern part of South America. Triatoma infes- interleukins (IL-2, IL-3, IL-4, and IL-5) to
tans is the principal vector responsible for IFN- leads to even greater macrophage
the most transmissions in these southern trypanocidal activity. Anti-IL-4 antibodies
cone countries of South America. added to the mixture were capable of neu-
Many reduviid species live in wild tralizing the effect, but the combination of
habitats and do not invade housing; thus, IFN- and purified IL-4 was no better than
they do not come in contact with humans IFN- alone.
and are not important in transmission to Lipopolysaccharide significantly in-
humans, although they are important in creases the trypanocidal effect of IFN-
the propagation of T. cruzi in nature. Some on macrophages, apparently through a
sylvatic species (e.g., Triatoma dimidiata mechanism not solely mediated by the
and Rhodnius prozlixus) can inhabit both stimulation of TNF production. Another
wild habitants and housing, and these cytokine that is effective at priming macro-
sylvatic species are important in human phages for trypanocidal activity in vitro is

212 Immunological Aspects of Cardiac Disease

granulocyte-macrophage colony-stimulat- also bound human C4b, a component of
ing factor. the classical pathway C3 convertase, and
Serum taken from an uninfected therefore may restrict classic complement
human does not lyse trypomastigotes, but activation. The T. cruzi complement regu-
serum taken from infected patients does latory protein is stage specific in that it is
lyse trypomastigotes. Thus, the comple- only expressed by mammalian forms of
ment system requires the participation of the parasite. A recent study reported that
the acquired immune response to T. cruzi stable transfection of T. cruzi epimastigotes
to become operational. Parasite lysis by (which are susceptible to complement lysis
immune serum occurs primarily via the by normal serum) with the complementary
alternative complement pathway. This DNA for the T. cruzi complement regula-
means that the complement cascade is ini- tory protein conferred complement resis-
tiated by direct association of complement tance.
components (C3) with the parasite surface,
rather than by fixation of complement by TISSUE TROPISM
antibody Fc receptors. In fact, Fab and Why does T. cruzi preferentially para-
F(ab)2 prepared from chagasic patients sitize muscle and nerve tissue? Arguing
sera (which are incapable of fixing com- teleologically, the host immune response
plement) were nearly as efficient as intact needs to control the parasite infection but
immunoglobulin G (IgG) in complement not at the cost of destroying vital organs
lysis assays with trypomastigotes. This (i.e., heart and peripheral nerves). The
implied that the effect of immune anti- down-regulatory immune responses may
bodies on complement lysis of trypomas- be more exuberant in these critical tissues
tigotes was a function of the specificity where even minor injury can be fatal and
of the antigen-binding sites, rather than regenerative capacity is limited (e.g., the
complement fixation by bound antibod- cardiac conduction system). T. cruzi may
ies. In other words, there was a surface have evolved to preferentially infect these
component on the parasites that, when tissues as relatively immune-privileged
neutralized by immune sera, rendered the sites. Several other parasites that preferen-
parasites susceptible to complement lysis. tially infect muscle tissue or nervous tissue
Antibodies directed to a 160-kDa pro- include toxoplasmosis, trichinella, Taenia
tein correlated with the capacity of the species (beef and pork tapeworms), and sar-
serum for complement-mediated lysis of cocystis. Each of these species depends on
trypomastigotes. Subsequent work led to a carnivorous-definitive host ingesting the
the [purification and characterization of the meat of an intermediate host to complete
specific parasite product, gp160. This glyco- its life cycle. There is no evidence, how-
protein bound the complement component ever, that consumption of T. cruziinfected
C3b and inhibited C3 convertase formation, tissues plays a role in this parasites life
thus inhibiting activation of the alterna- cycle. Thus, the predilection for growth in
tive complement pathway. It is membrane muscle and nervous tissue is more likely
bound and shares genetic and functional to be related to a strategy for long-term
similarities to the human complement reg- parasite survival in the host, rather than a
ulatory protein, decay accelerating factor. strategy related to direct transmission to
Further work indicated that this protein secondary hosts.

Immunological Aspects of Cardiac Disease 213

IFN- induction of macrophage try- the IFN- production. Since IFN- acti-
panocidal activity is associated with the vates macrophages to kill T. cruzi, the role
production of hydrogen peroxide. How- of NK cells in T. cruzi infection would be
ever, treatment of activated macrophages expected to be protective. In fact, a rela-
with catalase, superoxide dismutase, or tively T. cruziresistant strain of mouse
sodium benzoate to scavenge respira- was rendered highly susceptible to infec-
tory burst metabolites failed to inhibit tion by pretreatment with anti-NK1.1 anti-
trypanocidal activity in vitro, suggesting bodies. Thus, NK cells appear to be an
an oxygen-independent mechanism of T. early source of IFN- that helps control
cruzi destruction. Subsequent studies have parasite replication before the acquired
revealed the importance of nitric oxide immune response becomes predominant.
(NO) production in the killing mechanism NK cells may also be involved in the
in murine systems. Macrophages primed immune response later in infection, since
with IFN- produce NO, and the addition they are present in inflammatory lesions
of an inhibitor to NO synthase blocks the of muscle in experimental mice 270 days
trypanocidal activity. Levels of extracellu- after infection.
lar l-arginine (the substrate for NO produc-
tion) modulate the trypanocidal activity of ANIMAL MODELS
macrophages. NO may be directly toxic to Trypanosoma cruzi multiplies and dis-
T. cruzi; however, it reacts with superoxide seminates throughout the host before a
(O2) to yield peroxynitrite (ONDO), which specific antiparasitic immune response is
is highly toxic to T. cruzi. In vivo, inducible mounted. In mice, parasites first become
nitric oxide synthase (iNOS) is induced at apparent in blood approximately five
the protein and messenger RNA levels, to seven days after infection and rise in
and NO is released during acute infection numbers until three or four weeks into
in coincidence with secretion of IFN- and infection when the mice either die or the
TNF. The administration of inhibitors of infection is controlled by the immune
NO production leads to greater suscepti- response (Figure 12.1). RAG knockout
bility of T. cruziinfected mice. Mice carry- mice, which are deficient in both B- and
ing disruption of the iNOS genes are highly T-cell function, have similar levels of par-
susceptible to T. cruzi infection. The IFN- asitemia compared with wild-type mice
induced, NO-dependent mechanism of until day 13 of infection, at which point
macrophage killing of T. cruzi can be inhib- the parasitemia level becomes higher in
ited by the addition of interleukin-10 or the RAG knockouts. This indicates that
transforming growth factor- in vitro. the acquired immune response has little
NK cells participate in the innate effect until about two weeks into the infec-
immune response to T. cruzi infection, and tion. Although antiT. cruzi antibodies are
NK cells secrete IFN- after incubation detectable at about day 7 in murine mod-
with T. cruzi in vitro. This was shown by els, protective antibodies are not present
culturing splenocytes from athymic nude until several weeks later. This was shown
mice with T. cruzi and detecting the secre- in experiments in which antibodies taken
tion of IFN-, then demonstrating that from acutely infected mice were not protec-
the pretreatment of the splenocytes with tive against T. cruzi infection in immune-
anti-NK1.1 monoclonal antibody blocked nave mice. However, experimental mice

214 Immunological Aspects of Cardiac Disease

or rats that received sera from animals that CD8+ T cells. Nickell and colleagues iso-
survived acute infection experience a sig- lated CD4+ T cells from spleens of infected
nificant decrease in parasitemia level and mice that proliferate to T. cruzi antigen in
mortality following a challenge with viru- a HLA-restricted fashion. The CD4+ T-cell
lent parasites. line recognized an undefined trypomasti-
The importance of IgG in the humoral gote antigen(s), did not cross-react with
immune response was first demonstrated Leishmania spp. or Toxoplasma gondii, and
by Castelo Branco. The protective compo- was able to passively protect syngeneic
nent of serum from chronically infected recipients from lethal T. cruzi challenge
mice could be removed by staphylococcal infection. Kahn and Wleklinski isolated and
protein A (which absorbs IgG). Further- cloned CD4+ T cells from T. cruziinfected
more, the purified IgG component from mice that proliferate and secrete cytokines
whole serum was capable of conferring in response to the surface protein, SA-85,
protection. Both IgG subclasses 1 and 2 a member of the sialidase superfamily.
are capable of clearing T. cruzi. Antibodies The MHC class II epitope in this protein
mediate protection from T. cruzi by was mapped to a 20-amino acid sequence.
opsonization, complement activation, and Nickell and associates were able to isolate
antibody-dependent cellular cytotoxicity. CD8+ T cells from infected mice that lysed
T cells have a protective role in the parasite-infected target cells in an MHC-
acquired immune response to T. cruzi. restricted manner. The parasite antigens
Experiments in mice showed that T-cell involved in the stimulation of CD8+ T cells
activation correlated with resistance to were not characterized. However, Wizel
infection. Passive transfer of T cells from and colleagues detected class Irestricted
mice immunized against T. cruzi conferred CD8+ T cells from spleens of infected mice
resistance in mice challenged with T. cruzi. that lyse target cells, presenting epitopes
Deficient T-cell function is associated with from the trans-sialidase family of proteins.
increased sensitivity to infection. This These cytotoxic lymphocytes passively
has been shown in nude mice; thymec- transferred protection against challenge
tomized mice; mice treated with cyclo- infection.
sporine A, anti-CD4, and anti-CD8; and The inflammatory infiltrates in T.
mice genomically deleted of CD4, CD8, cruziinfected experimental animals have
2-microglobulin, TAP-1, or MHC mol- been analyzed for cellular surface mark-
ecules. The increased susceptibility associ- ers. Mirkin and colleagues studied tis-
ated with deficient T-cell function is most sues from C3H/HeN mice during acute,
apparent during acute infection. Mice that early chronic, and late chronic infection.
are depleted of T cells (CD4+ or CD8+) after The inflammatory infiltrates consisted
they have survived the acute infection do mainly of lymphocytes (6090 percent)
not have altered parasitemia or longevity. and macrophages (1040 percent). The
The declining role of T cells and the impor- lymphocytes primarily carried the T-cell
tance of humoral immunity in controlling marker, Thy1.2. Both CD4+ and CD8+
infection after the acute stage are schemati- T cells were present in the infiltrates of
cally illustrated (Figure 12.1). skeletal muscle, sciatic nerve, and spinal
The presence of parasite-specific T cells cord. The general trend was for a slight
has been demonstrated for both CD4+ and predominance of CD8+ cells over CD4+,

Immunological Aspects of Cardiac Disease 215

particularly during the early chronic and cytolytic pathways had parasitemia and
late chronic stages. Sun and Tarleton also mortality rates similar to wild-type mice,
found that Thy1.2+ cells were the major suggesting that cytolytic function, in fact,
lymphocyte population in tissues (cardiac may not be the protective effector mecha-
and skeletal muscle) during acute infec- nism of CD8+ T cells. Wizel and cowork-
tion. CD8+ T cells (4759 percent) domi- ers showed that T. cruzispecific CD8+
nated over CD4+ T cells (919 percent). T cells produce IFN- and TNF- upon
B cells and macrophages each repre- stimulation, thus raising the possibility
sented less than 1 percent of the cells in that these cells mediate their effects by
the inflammatory infiltrates. Tissues from cytokine release.
humans with chronic chagasic cardiomy-
opathy were similarly analyzed. As with
Pathogenesis and Modulation of
mice, T cells were the primary cell type in
Immune Function
the inflammatory infiltrates, with a greater
proportion of CD8+ cells than CD4+. Many PATHOGENESIS
of the CD8+ T cells expressed granzyme The pathogenesis of acute Chagas dis-
A. The extent that the tissue lymphocytes ease is not in dispute. On standard histo-
are directed toward parasites and para- pathological study, inflammatory lesions
site antigen as opposed to self-targets (i.e., are found to co-localize with abundant
autoimmunity) has not been fully clari- parasites, demonstrating that inflamma-
fied (see Pathogenesis and Modulation of tory damage is directed to the parasite.
Immune Function section). The pathophysiology of chronic Chagas
T cells in T. cruzi infection perform a disease is still in question, but the compet-
variety of antiparasitic functions. They ing (nonexclusive) views are that disease
provide helper T-cell function by stimu- results from an autoimmune response
lating B cells to produce parasite-specific directed at the affected organ systems or
antibody. Nickell and colleagues showed from damage resulting from inflammation
that a T-cell line derived from the spleens related to the persistence of the parasite.
of T. cruziinfected mice was able to Supporting the autoimmune hypothesis
induce normal spleen cells to produce are observations that live T. cruzi para-
parasitic-directed antibodies when stimu- sites have been difficult to demonstrate in
lated in vitro with T. cruzi antigen. The involved organs by conventional histologi-
activation of helper T-cell function is sup- cal study and that autoimmune T cells and
ported by the predominance of IgG2a antibodies develop during infection with
and IgG2b antibodies in T. cruzi infection, T. cruzi. Furthermore, autoimmune anti-
which are typical of CD4+ T-cell-depen- bodies and autoimmune T cells have been
dent responses. T cells produce cytokines associated with chronic Chagas disease or
in T. cruzi infection, which mediate impor- lesions. Some T. cruzi antigens molecularly
tant antiparasitic functions. CD8+ T cells mimic affected host tissues. It is hypothe-
lyse T. cruziinfected host cells, which pre- sized that the chronic infection leads to loss
sumably interrupts the parasite life cycle, of tolerance and, combined with antigenic
thus limiting its replication. Interestingly, mimicry, results in specific autoimmune
mice genetically deficient for genes con- attack of cardiac, gut, and peripheral ner-
trolling perforin or granzyme Bmediated vous tissues.

216 Immunological Aspects of Cardiac Disease

The other hypothesis is that progres- additional specificity of Chagas disease
sive inflammation directed at parasites is imposed during infection, perhaps by
that reside in target organs cause the the particular autoimmune response that
pathologic damage of chronic infection. is generated by chronic T. cruzi infection.
Studies that support this hypothesis have Fourth, the long lag time from infection to
found that parasite antigens and DNA can disease could be necessary to generate dis-
be detected in many chronic inflammatory ease by autoimmunity. Fifth, the presence
infiltrate and that parasitologic treatment of autoimmune T cells and antibodies in
of chronically infected animals or humans infected individuals, especially when they
tends to lead to improvement and resolu- are associated with disease, is evidence
tion of disease. The evidence for the com- that autoimmunity may play a role in
peting theories of chronic Chagas disease pathogenesis. It should also be noted that
pathogenesis is discussed in detail in the each of these properties of chronic Chagas
next section. Since the autoimmune and disease could be due to parasite-directed
parasite-directed pathogenesis theories pathogenesis, as discussed below.
are not mutually exclusive and since evi- Autoimmune antibodies are easily
dence exists to support each of them, it demonstrated in T. cruzi-infected indi-
seems likely that both mechanisms could viduals. Antibodies to myocardium and
be pathogeneic in chronic Chagas disease. nervous tissues are found in high levels
in persons and mice infected with T. cruzi
AUTOIMMUNITY AND PATHOGENESIS OF compared with those that are not. Since the
CHAGAS DISEASE myocardium and nervous tissues are key
The theory of autoimmunity leading to organs that are damaged in chronic Cha-
damage in chronic Chagas disease is sup- gas disease, finding antibodies to these
ported by a number of observations. First, tissues could mean that these antibodies
few parasites can be demonstrated in the cause autoimmune damage to these tis-
inflammatory lesions by conventional his- sues. As an alternative, antigens from myo-
tological study. This observation suggests cardium and nervous tissues are probably
the disease may not be driven solely by reac- exposed by the damage of infection of these
tion to the parasite. Second, only 10 percent organs, and antibodies may be generated
to 30 percent of chronically infected people to these tissues without being pathogenic.
develop Chagas disease, although most Many of the autoantibodies found in the
can be shown to have chronic parasitemia. sera of T. cruziinfected persons and mice
This suggests that, in addition to chronic are so-called natural autoantibodies, which
T. cruzi infection, some other factor(s) can be found in low levels in the serum of
determines which individual develops dis- normal humans and mice. The natural
ease. Susceptibility to autoimmune disease autoantibodies are directed against pro-
could be the host factor that determines teins that are highly conserved in evolu-
who develops disease. Third, chronic Cha- tion and are not necessarily an indication
gas disease is very organ specific, gener- of autoimmune disease. The high levels of
ally limited to the heart, nervous tissues, natural autoantibodies after T. cruzi infec-
or innervation of the gut. Since the parasite tion may be the result of the polyclonal lym-
can reside in almost any cell type, the organ phocytic proliferative response that occurs
specificity of Chagas disease suggests that during acute infection. Levels of natural

Immunological Aspects of Cardiac Disease 217

autoantibodies do not correlate with dis- these patients. In addition, T-cell responses
eases in individuals who are chronically to this protein have not been seen in indi-
infected with T. cruzi. viduals with Chagas disease, and passive
transfer of these immune T-cells was not
Molecular mimicry of host antigens by Another peptide of interest has been
parasite antigens has been found to gener- the ribosomal Po protein (R13 peptide),
ate autoantibodies that are found in chronic which has been shown to cross-react with a
T. cruzi infection (See table 12.2). Autoim- functional protein on human B1-adenergic
mune pathogenesis by molecular mimicry receptors. Antibodies to this receptor via
requires breakdown in tolerance of the immunization with the R13 peptide have
immune system to self-antigens. A break- led to electrocardiographic (ECG) changes
down in tolerance could occur in response in mice that are similar to those seen in
to chronic T. cruzi infection or because of chronic Chagas carditis but no changes in
the polyclonal lymphocyte proliferative digestive symptoms have been noted.
response that occurs early after infection. Antibodies to an interesting T. cruzi
Although many molecular mimicry epit- protein called B13 is seen in the sera of
opes have been described, only a few have all patients with chronic Chagas cardio-
been shown to correlate with disease in myopathy and this antibody cross-reacts
chronic T. cruzi infection. with human cardiac myosin but in only
For example, high levels of antibod- 17 percent of asymptomatic individu-
ies to rodent endocardium, blood vessels, als. Furthermore, T cells directed to both
and interstitium (EVI) have been found B13 and myosin have been detected in
in the sera of T. cruziinfected individu- biopsy specimens from persons with
als and the mammalian host and were chronic Chagas cardiomyopathy. These
absorbed by epimastigote antigens, thus results suggest that antibody and T cells
demonstrating cross-reactivity. However, directed to cardiac myosin may be patho-
these EVI antigens shown to be directed genic in chronic Chagas cardiomyopathy.
against -galactose epitopes were spe- However, in animal models transfer of anti-
cific for murine laminin and not human body and T cells directed to myosin have
laminin. Furthermore, anti-EVI antibodies not been reported to lead to pathogenic
(-galactose/laminin) or other autoanti- changes. Perhaps the mouse model may
bodies have not been shown to transfer not be correct and as in rheumatic fever
disease to noninfected animals. the introduction of myosin in the Lewis rat
Perhaps more interesting (in terms of the may be a better animal model.
organs infected) has been a protein found
on the surface of trypomastigotes in asso- PARASITE-DIRECTED PATHOGENESIS
ciation with the flagellum, Fl-160. This pro- Since the studies reporting that autoim-
tein molecularly mimics myenteric plexus munity may be involved in the inflamma-
and peripheral neurons. These antibodies tory infiltrates of chronic Chagas disease
are found in 30 percent of individuals with are not conclusive, the alternative theory
chronic Chagas disease; yet, they do not is that parasites residing in chronically
appear to correlate with disease activity in infected host tissue may cause chronic

218 Immunological Aspects of Cardiac Disease

Table 12.2 Molecular Mimicry Antigens of Trypanosoma Cruzi

Cross-reactive Pattern of
Mammalian Mammalian Mammalian Relationship to
T. Cruzi Antigen Antigen Reactivity Species Disease

(1-3)-galactose Murine laminin Endocardium, Rodent, not None

blood vessels, human
and interstitium
Sulfated Sulfagalactosyl- Neurons, Mouse Not tested
glycolipids ceramide astrocytes
Neutral Cardia-neutral- Heart muscle Mouse Not tested
glycosphingolipids glycosphingolipids
GP50/55 kDa P28 kDa Activated T and Mouse, Not tested
B lymphocytes human
Amastigote cell- P60, p32 kDa Central and Rodent, Not tested
surface and peripheral human
epimastigote neurons
cytoplasmic (CE5
p58, p35 kDa (5H7 P58, p37 Central and Rodent Not tested
& 3H3 Mabs) peripheral
neurons, glia
150-kDa surface Striated and Human, Not tested
trypomastigote smooth muscle mouse
antigen including
smooth muscle
of cardiac

FL-160, p48 kDa Myenteric Rodent, Antibodies to

160-kDa flagellar- plexus and human FL-160 found in
associated peripheral nerve 30 percent
surface protein of axons of those infected
trypomastigotes with T. cruzi
but no
with disease
Microtubule- Microtubule- Fibroblasts, Mouse, Not tested
associated protein associated protein brain cells bovine
B13, 140, and Cardiac myosin Heart ventricle Human, Antibodies
116-kDa proteins mouse correlate with
cardiac disease,
T cells from
cardiac lesions
Ribosomal P Ribosomal P All mammalian Human High levels of
protein protein cells antibodies to
R13 peptide
with Chagas


Immunological Aspects of Cardiac Disease 219

Table 12.2 (continued)

Cross-reactive Pattern of
Mammalian Mammalian Mammalian Relationship to
T. Cruzi Antigen Antigen Reactivity Species Disease

Ribosomal PO 1-adrenergic Heart Human Not tested,

protein (R13 receptor though
peptides) antibodies found
in 47 percent
of chagasic
heart disease
patients and
levels of anti-R13
correlate with
Ribosomal 23-kDa Ribosomal 23-kDa Human No correlation of
protein protein antibody levels
with cardiac
p25-kDa Heart sarcolemma Heart Human, Antibody levels
Epimastigote rodent with cardiac
protein disease
Microsomal p71, 65, 59, 45, Myocardium Human, Not tested
and membrane 44, 34, 3027 kDa and skeletal hamster
antigens muscle

Adapted with permission from Buckner FS, Van Voorhis WC. Immune response to Trypanosoma
cruzi: control of infection and pathogenesis of Chagas disease. In: Cunningham MW, Fujinami RS,
eds. Effects of Microbes on the Immune System. Philadelphia, PA: Lippincott-Raven Press; 2000:5

Parasite Burden
CD4 and CD8
B cells and Ig

Activated M'phages
NK cells

0 4 8 12
Weeks (post-infxn)
Figure 12.8 Relative magnitude of immune response mediators and
parasite burden during acute Trypanosoma cruzi infection in the mouse.
Adapted with permission from Buckner FS, Van Voorhis WC. Immune
response to Trypanosoma cruzi: control of infection and pathogenesis
of Chagas disease. In: Cunningham MW, Fujinami RS, eds. Effects of
Microbes on the Immune System. Philadelphia, PA: Lippincott-Raven
Press; 2000:5 69591.

220 Immunological Aspects of Cardiac Disease

disease by either damaging tissues directly cruzi infection could be cured with chemo-
or focusing the inflammatory response therapy would provide a model to evalu-
in host tissues. Many of the properties of ate whether parasites need to persist for
chronic Chagas disease cited in support of the chronic pathology to occur. In humans,
the autoimmune hypothesis may also be nonrandomized study of patients treated
possible with parasite-directed pathogen- with benznidazole indicates that cardiac
esis. Parasites may be difficult to demon- complications are reduced compared with
strate in the lesions but that may be due to untreated individuals, suggesting that the
limitations of present techniques. We do pathology may be parasite driven. A pro-
know that chronically infected individuals spective randomized trial is needed to
harbor parasites, and the fact that not all cure humans of parasites and then observe
chronically infected individuals develop whether cardiac disease remits or fails to
chronic disease may reflect parasite bur- develop. For this to work, better drugs and
den, strain differences, or host variation in better ways to detect chronic infection and
the immune response. The organ specific- to assess the cure of infection are required.
ity may be due to tropism of the parasite In addition, in humans, better correlations
for cardiac and neuronal tissues. Thus, the between clinical cardiac disease and auto-
long lag time to develop disease may be immune phenomena are needed. Finally,
due to the slowly progressive damage that therapeutic trials to interrupt autoimmune
is required to generate clinical disease. cardiac damage are needed, but this will
Two other factors support the para- require further research to learn the best
site-directed damage. First, immunosup- way to intervene.
pression exacerbates, not ameliorates, the
disease process and causes worsening Conclusions
of the pathologic lesions of Chagas dis-
ease. However, one could argue that loss These findings support the importance of
of the immune response permits contin- the parasite in generating chronic Chagas
ued growth of the organism and damage. disease and the search for more effective
The second factor is that treatment with and less toxic antiparasitic therapeutics.
antiparasitic drugs in early Chagas dis- However, autoimmune mechanisms may
ease markedly lessened the progression potentiate or may even be necessary for the
to chronic disease, although the number parasite-directed pathogenesis of chronic
of patients assigned to treatment versus T. cruzi infection. Further understanding
placebo groups was small. of these autoimmune mechanisms could
suggest immunomodulatory therapy that
could act as an adjunct to antiparasitic ther-
Future Directions of Research apy to help reduce the damage of chronic
Clearly, more evidence is needed to sup- Chagas disease.
port or refute the autoimmune hypothesis
of Chagas disease. In animal models, trans- OTHER IMMUNE-MEDIATED
fer of immune cells or sera without para- MYOCARDIAL DISEASES
sites and recapitulation of cardiac disease
would be one convincing line of evidence. A suggestive antecedent viral infection
Animal experiments in which chronic T. leads to immune-mediated heart-muscle

Immunological Aspects of Cardiac Disease 221

disease. These