Clin Chest Med 27 (2006) 53 69

Airway Smooth Muscle as a Regulator of Immune Responses and Bronchomotor Tone

Aili L. Lazaar, MDT, Reynold A. Panettieri, Jr, MD
Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania Medical Center, BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA

Asthma, a chronic disease with a prevalence of 3% to 5% in the United States, affects millions of people worldwide. Complex genetic and environmental factors contribute to the development of asthma, which is characterized by reversible airway obstruction, airway inflammation, and airway smooth muscle (ASM) cell hyperplasia. The traditional view that in asthma ASM is a purely contractile tissue seems to be inadequate. Compelling evidence now suggests that ASM plays an important role in regulating bronchomotor tone, in perpetuating airway inflammation, and in remodeling of the airways. This article reviews three distinct functions of ASM cells: the process of excitation contraction coupling, with a particular focus on the role of cytokines in modulating calcium responses; the processes of smooth muscle cell proliferation and migration; and the synthetic and immunomodulatory function of ASM cells. It also discusses how altered synthetic function contributes to airway remodeling.

Airway smooth muscle shortening and airway hyperresponsiveness Airway smooth muscle bronchomotor tone and asthma Smooth muscle cell shortening regulates airway luminal caliber. Many studies have examined whether

T Corresponding author. E-mail address: Aili.L.Lazaar@gsk.com (A.L. Lazaar).

the nonspecific airway hyperresponsiveness (AHR) characterizing asthma can be attributed to increased ASM force generation as a consequence of altered receptor ligand interactions or altered signal transduction pathways. For example, expression of the bradykinin B2 receptor is increased in ASM exposed to interleukin (IL)-1b, tumor necrosis factor-alpha (TNFa), or transforming growth factor-beta (TGFb), whereas the bradykinin B1 receptor is increased by treatment with IL-4 [1 5]. In contrast, TNFa dramatically decreases muscarinic receptor density but enhances acetylcholine-induced hyperresponsiveness because of altered downstream signal transduction pathways [6,7]. Despite evidence that ASM derived from patients who have asthma may not exhibit a greater increase in isometric force generation than in nonasthmatic persons, exposure to inflammatory mediators clearly enhances force generation of normal tissue to contractile agonists. Early studies demonstrated that passive sensitization of human ASM with asthmatic serum nonspecifically increases smooth muscle cell responsiveness [8 11]. TNFa and IL-1b induce bronchial hyperreactivity in both humans and animals, whereas in vitro cytokines and other mediators, including TNFa, IL-1b, IL-13, IL-5, lysophosphatidic acid, and phospholipase A2 also prime ASM to become hyperresponsive to contractile agonists [12 16]. Leukotrienes (LTs) are potent bronchoconstrictors in normal and asthmatic persons, and ASM cells express both receptors for the cysteinyl leukotrienes, LTC4, LTD4, and LTE4 [17,18]. Expression of the CysLT1 receptor is increased by exposure to interferon (IFN)-g and results in an increase in LTD4-

0272-5231/06/$ see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ccm.2005.10.003

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mediated force generation in cultured ASM cells [19]. Conversely, TNFa, IL-1b, and IL-13, but not IL-4, reduce b-adrenergic relaxation responses [20]. Together, these studies suggest that proinflammatory mediators promote airway hyperresponsiveness by enhancing ASM contraction or altering ASM relaxation. Modulation of calcium homeostasis in airway smooth muscle cells In human ASM cells, contractile agonists bind Gq- or G0-protein coupled receptors (GPCR) and activate phospholipase C. The subsequent hydrolysis of phosphatidylinositol 4,5 bisphosphate into inositol trisphosphate and diacylglycerol ultimately increases cytosolic calcium [21]. Because most inflammatory agents evoke neither a calcium response or nor phosphoinositide (PI) hydrolysis in human ASM, modu-

lation of agonist-induced increases in intracellular + calcium (Ca2 ) by extracellular stimuli probably i modulates downstream GPCR signaling. TNFa increases the expression as well as the activity of G-proteins in several cell types, including ASM [20]. The effect of cytokines on agonist-evoked calcium responses seems to be stimulus specific, however, because bradykinin-evoked PI accumulation is significantly enhanced by TNFa and IL-1b, whereas IL1b diminishes histamine-induced PI metabolism [1,22,23]. + The level of Ca2 i , in ASM modulates excitation contraction coupling and force generation (Fig. 1) + [24]. Agonist-induced elevations in Ca2 activate i the calcium-/calmodulin-sensitive myosin light chain kinase (MLCK), leading to phosphorylation of the regulatory myosin light chain (MLC20). Phosphorylation of MLC20 initiates cross-bridge cycling between myosin and actin and sustains ATP binding,

Fig. 1. Cytokine effects on excitation contraction coupling in ASM cells. Cytokines binding to their receptors influence intracellular signaling as well as the function and expression of GPCRs and CD38. Alterations in intracellular signaling affect calcium homeostasis and calcium sensitization. cADPR, cyclic ADP ribose; CaM, calmodulin; DAG, diacylglycerol; GEF, guanine exchange factor; GPCR, G-protein coupled receptor; IP3, inositol 3-phosphate; MLC, myosin light chain; MLCK, myosin light chain kinase; PLC, phospholipase C; R, cytokine receptor; RyR, ryanodine receptor; SR, sarcoplasmic reticulum.

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hydrolysis and ADP release. Dephosphorylation by the MLC phosphatase terminates cross-bridge cycling and relaxes smooth muscle [21]. Evidence now suggests that abnormalities in these two major regulatory + events (increases in Ca2 and MLC phosphorylation) i induce bronchial hyperresponsiveness in asthma [12]. Agonist-evoked calcium responses and calcium sensitization Calcium homeostasis in cultured ASM cells is altered by inflammatory mediators. Cytokines such as TNFa, IL-1b, or IL-13 enhance cytosolic calcium responses in human ASM induced by carbachol, bradykinin, or thrombin [1,15,25]. In contrast, IL-4 suppresses carbachol-induced calcium signals [26,27]. Other stimuli may directly enhance agonist-induced calcium signaling in cultured ASM cells, including eosinophil-derived polycationic proteins or major basic protein, the aldehyde pollutant acrolein, or phospholipase A2 [11,28,29]. More recently, a phenomenon known as calcium sensitization has been described that involves calcium-independent mechanisms for maintaining MLC phosphorylation and smooth muscle contraction [30]. The signaling pathways for calcium sensitization involve inhibition of MLC phosphatase and, thus, inhibition of MLC dephosphorylation, most notably by Rho/Rho kinase and integrin-linked kinase [30]. Ultimately, enhanced MLC phosphorylation increases actin myosin interactions. Aberrant RhoA/Rho kinase activation has been implicated in a variety of disease states [31]. In ASM cells, carbachol- and TNF-induced calcium sensitization is mediated through activation of RhoA/ Rho kinase [32,33]. Additionally, animal models of allergen-induced AHR and airway inflammation demonstrate increased RhoA activation; in turn, antigen-induced AHR is abrogated by pharmacologic inhibition of Rho kinase [34,35]. Allergen sensitization also increases lung expression of Rho and directly impugns Rho kinase in agonist-induced ASM contraction [36]. Thus, pathways that modulate Rho and Rho kinase activation, particularly dysregulated contractile responses, may play an important role in asthma pathogenesis. CD38 Recent studies show that CD38, a membranebound protein expressed on ASM cells, modulates cyclic adenosine diphosphate ribose (cADPR) levels [37]. cADPR, a b-nicotinamide adenine dinucleotide metabolite, contributes to agonist-induced elevation + of Ca2 concentration in ASM cells by activating i

ryanodine receptors [38,39]. Recently, TNFa and IL-13 have been shown to increase expression of CD38 on ASM cells [40,41]. Furthermore, a cADPR antagonist inhibited agonist-induced increases in + Ca2 [40,42]. Finally, agonist-induced increases in I lung resistance are attenuated in CD38-deficient animals [43], suggesting that CD38 expression on ASM plays a significant role in maintaining calcium homeostasis and contributes to AHR. Alterations in the biophysical properties of airway smooth muscle can modulate airway smooth muscle shortening Proinflammatory mediators may also alter the internal resistance to shortening of the muscle, thereby affecting force generation. For example, MLCK content is increased in sensitized airways [44], which may contribute to the enhanced shortening velocity of ASM from asthmatic patients [45]. Alternatively, TNFa and contractile agonists such as acetylcholine can induce reorganization of the actin cytoskeleton, a process mediated by the Rho family of guanosine triphosphatases (GTPase), RhoA and cdc42 [46,47]. Inflammatory mediators and agonists also activate ribosomal S6 kinase, p38MAPK. Inhibition of p38MAPK reduces agonist-induced force generation, possibly through effects on heat shock protein 27, an actin-capping protein [48,49]. A more detailed discussion of the effects of mediators on the actin cytoskeleton has recently been published [50]. Proinflammatory cytokines affect ASM contractility on many levels. Alterations in calcium homeostasis and sensitivity, as well as contractile agonist receptor expression and signal transduction pathways, have profound effects on airway hyperreactivity. The identification of the fundamental signaling pathways promoting ASM excitation contraction coupling will provide new therapeutic targets to modulate ASM function in asthma.

Cellular and molecular mechanisms regulating airway smooth muscle cell growth Early studies of the histopathology of asthma described increases in both size and number of airway smooth muscle cells [51,52] and a correlation between airway hyperreactivity and increased airways resistance. During the past decade, significant advances have been made in identifying the many diverse mitogens and signal transduction pathways that modulate ASM growth (reviewed in [53]). This

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section examines the critical signaling pathways that regulate ASM growth. Hyperplasia Animal models of allergic airway inflammation have shown clear indications of in vivo ASM cell proliferation, using various markers such as bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA) [53]. Studies of bronchial biopsies from patients who had asthma, however, have yielded conflicting results regarding the presence of ASM proliferation in vivo [54 56]. These differences may be caused by sampling error or compartmentalization of the proliferative response [53]. Mediators Many inflammatory mediators are increased in bronchoalveolar lavage (BAL) fluid from airways of patients who have asthma, and such mediators induce ASM mitogenesis in vitro [57]. To date, mitogenic stimuli include growth factors such as epidermal growth factor (EGF), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF) isoforms BB and AB, and basic fibroblast growth factor; plasma- or inflammatory cell-derived mediators, such as lysosomal hydrolases (b-hexosaminidases and b-glucuronidase), a-thrombin, tryptase, and sphingosine 1-phosphate; and contractile agonists, such as histamine, endothelin-1, substance P, phenylephrine, serotonin, thromboxane A2, and LTD4. TGFb inhibits mitogen-induced ASM cell proliferation [58] but also increases expression of the leukotriene receptor cysLT1R, thereby rendering cells responsive to the proliferative effects of LTD4 [59]. Expression of peroxisome-proliferator activated receptor gamma (PPAR-g) is increased in the smooth muscle of patients who have asthma [55]; PPAR-g ligands inhibit mitogen-induced ASM mitogenesis by preventing cell cycle progression [60]. Although IL-1b, IL-6, and TNFa are also increased in the BAL fluid of asthmatics [61], whether these cytokines stimulate ASM proliferation in vitro remains controversial. IL-1b and IL-6 may induce hyperplasia and hypertrophy of cultured guinea pig or rat ASM cells [62,63], but other studies have shown that IL-1b [64] and IL-6 [65] are nonmitogenic for human ASM cells. The effects of TNFa on ASM proliferation are controversial. McKay and colleagues [65] reported that TNFa had no immediate mitogenic effect on human ASM cells, in contrast to the findings of Stewart and colleagues [66] that the proliferative effect of TNFa on human ASM cells seemed to be biphasic [65,66]. New evidence reveals

that the inhibitory effect of TNFa on ASM proliferation is caused, in part, by autocrine release of IFNb [67] as well as by cytokine-induced production of cyclo-oxygenase 2 dependent prostanoids, such as prostaglandin E2 (PGE2), which inhibit DNA synthesis [64]. Agonist-induced release of PGE2 is significantly lower in smooth muscle derived from asthmatics than in smooth muscle obtained from nonasthmatic persons [68]. Therefore, cytokineinduced proliferative responses in ASM may be enhanced under conditions of cyclo-oxygenase inhibition, in which the expression of growth-inhibitory prostanoids, such as PGE2, is limited [62,64,66]. Extracellular matrix Extracellular matrix (ECM) modulates mitogeninduced ASM cell growth. Collagen types I, III, and V, fibronectin, tenascin, hyaluronan, versican, and laminin are increased in the airways of asthmatics [69]. In vitro, fibronectin and collagen I increase human ASM cell mitogenesis in response to PDGFBB or a-thrombin, whereas laminin inhibits proliferation [70]. In addition, the increase in cell proliferation is accompanied by decreased expression of smooth muscle cell contractile proteins such as a-actin, calponin, and myosin heavy chain, suggesting that matrix may also modulate smooth muscle phenotype [70]. ECM composition also affects ASM cell survival. Fibronectin, laminin, and collagens I and IV were identified as important antiapoptotic elements, whereas elastin promoted ASM apoptosis. Recently, human ASM cells were shown to secrete ECM proteins in response to asthmatic sera [71], identifying ASM as a cellular source for ECM deposition in airways and suggesting a novel mechanism by which ASM cells may modulate autocrine proliferative responses. ASM cells from asthmatic patients secrete significantly more perlecan, collagen I, and chondroitin sulfate and less laminin a1 and collagen IV than ASM cells from nonasthmatic persons [72]. In addition, ECM derived from ASM of asthmatics induces greater cell proliferation than that obtained from nonasthmatics. In contrast, conditioned media from either asthmatic or nonasthmatic ASM promote a similar degree of cell proliferation. Mitogenic signaling pathways ASM mitogens may act through different receptor-operated mechanisms. Growth factors induce ASM cell mitogenesis by activating receptors with intrinsic receptor tyrosine kinase (RTK) activity, whereas contractile agonists released from inflammatory cells mediate their effects by activation of GPCRs.

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Despite disparate receptor-operated mechanisms, recent evidence suggests that the small GTPase, p21ras, acts as a point of convergence for diverse extracellular signal-stimulated pathways in ASM cells (Fig. 2) [73]. In their active state, p21ras proteins interact with downstream effectors, namely, Raf-1 and phosphatidylinositol 3-kinase (PI3K). By recruiting Raf-1 to the plasma membrane, guanosine triphosphate bound p21ras activates the extracellular signal-regulated kinase (ERK) pathway. p21ras also binds and activates PI3K [74]. Synergy can occur between RTK and GPCRs to promote human ASM mitogenesis and p21ras activation [75]. Although alternative pathways do exist (as discussed later), PI3K and ERK activation seems to be the dominant signal transduction pathways for RTK-, GPCR- or cytokine-stimulated growth of ASM cells. PI3K phosphorylates membrane phosphoinositides, which function as second messengers and activate downstream effector molecules, such as 70-kd ribosomal S6 kinase (p70S6k), Rac1, or Cdc42 to regulate cell cycle protein expression and thus modulate cell cycle traversal [76]. Activation of PI3K is

Fig. 2. Intracellular pathways regulating ASM cell mitogenesis. Ras serves as a point of convergence for growth factor receptor tyrosine kinase (RTK) and G-protein coupled receptor (GPCR) signaling. Ras activates the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), and ultimately leads to activation of cyclins and cyclin-dependent kinases (CDKs). MEK, mitogen-activated protein kinase/ERK kinase; p70S6K, ribosomal S6 kinase; ROS, reactive oxygen species.

critical for ASM cell cycle progression [77 79]; however, although active PI3K is sufficient to stimulate ASM DNA synthesis, other signaling events are also necessary to promote maximal ASM growth responses [80]. As mentioned previously, Raf-1 activation induces phosphorylation and activation of mitogenactivated protein (MAP) kinase/ERK kinase (MEK1). Activated MEK1 directly activates the 42-kd ERK2 and 44-kd ERK1, collectively referred to as p42/p44 MAP kinases. Inhibition of MEK1 and ERK activity attenuates mitogen-induced DNA synthesis in ASM cells, suggesting that activation of MEK1 and ERK is required for proliferation [81,82]. Studies such as these suggest that the ERK pathway is a key signaling event mediating mitogen-induced ASM proliferation. D-type cyclins (cyclins D1, D2, and D3) are key regulators of G1 progression in mammalian cells; consequently, cyclin D1 has been the most widely studied cyclin in ASM biology. Another critical protein is p27Kip1, a cell cycle inhibitor [83]. A coordinated increase of cyclin D1 expression promotes complexing of unbound p27Kip1 molecules with cyclin D-dependent kinases, thereby facilitating cyclin E cyclin-dependent kinase-2 activation later in the G1 phase (Fig. 3) [83]. Recent studies also have implicated the Rho GTPases, Rac1, Cdc42, and RhoA in cyclin D1 regulation and ASM growth. Over expression of the catalytically active subunit of PI3K was sufficient to activate the cyclin D1 promoter and could be attenuated by inhibitors of Rac1 signaling [84], suggesting that Rac1 may be downstream of PI3K; further study is necessary to confirm this observation. Other studies using overexpression constructs of Cdc42, RhoA, and Rac1 also showed that overexpression of Cdc42 or Rac1, but not of RhoA, induced transcription from the cyclin D1 promoter in an ERK-independent manner [85,86]. Although cyclin D1 expression may be necessary for cell growth, it is not sufficient. Amrani and colleagues [87] demonstrated that IFNg inhibits ASM growth without modulating the growth factor induced increased expression of cyclin D1. In human ASM, corticosteroids reduce mitogen-stimulated increases in cyclin D1 protein and attenuate pRb phosphorylation [88]. Corticosteroid binding to the glucocorticoid receptor results in activation of the transcription factor C/EBPa and subsequent increased expression of cyclin-dependent kinase inhibitor p21WAF/Cip1, thus providing a potential mechanism for the antiproliferative effect of glucocorticoids on ASM cell growth [89 91]. A recent study suggests that ASM cells derived from patients

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Fig. 3. Activation of cell-cycle progression in ASM cells. Activation of cyclin D1 by growth factors or G-protein coupled receptor (GPCR) agonists leads to phosphorylation of retinoblastoma protein (Rb) and cell cycle progression. E2F, elongation factor 2; RTK, growth factor receptor tyrosine kinase.

who have asthma lack expression of C/EBPa and are therefore resistant to the effects of glucocorticoids on proliferation [92]. Despite evidence suggesting that oxidative stress plays an important part in regulating vascular smooth muscle [93], less is known about the role of reactive oxygen species in ASM cell function. Brar and colleagues [94] demonstrated that ASM cells express several components of the phagocyte NADPH, including gp22phox, gp91phox, and gp67phox. Reactive oxygen species can modulate mitogen-induced ASM cell proliferation [86,95,96]. Antioxidants inhibit mitogen-induced growth [95,96], whereas expression of a constitutively active Rac1 (another component of the NADPH oxidase complex) results in cyclin D1 expression [86]. Decreased expression of gp22phox by antisense oligonucleotides also inhibits ASM cell proliferation [94]. Stat3 also has been shown to be important in PDGF-induced pro-

liferation of human ASM cells. Activation of the JAK2 and Stat3 by PDGF seems to be redox dependent and affects the proliferative response to mitogen, independent of MAP kinase activation [97]. IFNg and IFNb activate Stat1/2, JAK1, and Tyk2 in ASM; these cytokines, however, are potent inhibitors of mitogen-induced proliferation, in part through increased expression of interferon-gamma-inducible protein 16 (IFI 16) [67,87]. Hypertrophy There is renewed attention to the role of increased myocyte size or hypertrophy as a mechanism of increased ASM cell mass in asthma. Some mediators, such as IL-1b, IL-6, TGFb, angiotensin II, and cardiotrophin I, induce cellular hypertrophy in vitro, although the mechanisms remain unclear [62,98,99]. In a primate model of asthma, the airways of mon-

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keys sensitized to house dust mite antigen display an increase in smooth muscle mass as well as in smooth muscle bundle size [100]. Morphologic studies confirm the presence of ASM cell hypertrophy in some, but not all, patients who have asthma [51]. Increased cell size seems to correlate negatively with postbronchodilator forced expiratory volume in 1 second and distinguishes between severe persistent asthma and milder disease [101]. In contrast, Woodruff and colleagues [56] studied ASM cell gene expression and cell morphology in bronchial biopsies obtained from patients who had mild to moderate asthma. These investigators found no evidence for ASM cell hypertrophy, although they did confirm the presence of ASM hyperplasia. More work is needed in this particular area of ASM cell biology. Smooth muscle migration Hyperplasia and hypertrophy are important processes regulating increased smooth muscle mass in asthma. Analogous to vascular smooth muscle migration in atherosclerosis, however, ASM cell migration probably promotes airway remodeling in chronic asthma. Evidence suggests that proliferating smooth muscle cells migrate along chemotactic gradients. During chronic inflammation, myocyte migration would be promoted by exposure of cells to a variety of cytokines and growth factors, as well as to an altered ECM. Structurally, myofibroblasts display a phenotype intermediate between fibroblasts and smooth muscle cells, express a-smooth muscle actin, and have the ability to secrete matrix proteins such as collagen and hyaluronan. In addition, myofibroblasts secrete chemokines and prolong eosinophil survival [102]. Although myofibroblasts are found in the lamina reticularis, the origin of these cells remains unknown. Possibly the cells exist in small numbers within the lamina reticularis and proliferate locally following stimulation with inflammatory mediators or growth factors. Alternatively, under the influence of similar stimuli, smooth muscle cells might migrate from the periphery of the smooth muscle bundle into the submucosa. A final possibility is that cells are recruited from a circulating pool of fibrocytes, as has been described by Schmidt and colleagues [103]. In this study, CD34+ cells expressing procollagen I and a-smooth muscle actin were increased in the bronchial mucosa following allergen challenge in patients who had asthma. Using a murine model of chronic allergic airway inflammation, the investigators demonstrated that these cells were recruited from a circulating population of CD34+ fibrocytes.

The potential for mesenchymal cells such as myofibroblasts to migrate into the airway wall is well established in patients who have asthma [104,105]. BAL-derived fibroblasts from patients who have mild asthma have enhanced migratory capacity compared with tissue fibroblasts isolated from bronchial biopsies [106]. In vitro, many studies support the capacity of ASM cells to migrate. Increased chemotaxis of ASM cells has been demonstrated to PDGF, TGFb, basic fibroblastic growth factor, and IL-1b. Urokinase has been shown to increase chemotaxis in some, but not all, studies [107,108]. Cysteinyl-leukotrienes alone promote chemokinesis only; however, in combination with PDGF, increased chemotaxis is observed over PDGF alone [109]. Vascular endothelial growth factor (VEGF), a potent inducer of vascular smooth muscle migration, has no effect on ASM cell chemotaxis [110,111]. Similarly, not all mitogens are promigratory; recent data demonstrate that only PDGF, but not EGF or thrombin, induce migration in ASM cells [112]. The ECM also regulates the migratory phenotype, but its role in ASM migration has not been extensively characterized. Parameswaran and colleagues [113] recently demonstrated that ASM cells migrate more on collagen III and V and fibronectin than on collagen I, elastin, or laminin. The signaling pathways regulating ASM cell migration are beginning to be elucidated. Early studies suggested that p38 MAP kinase and heat shock protein 27 are important regulators of growth factor and cytokine-induced migration [114]. Activation of p38 MAP kinase seems to occur, in part, through p21-activated protein kinases [115] and is important for phosphorylation of caldesmon, which acts to regulate actin myosin interactions [116]. Subsequent studies have demonstrated that PI3K also regulates PDGF-induced smooth muscle cell migration [110]. PI3K is activated in part through activation of Src kinase, because inhibition of Src attenuates PI3K activity [112]. Others have shown that ASM migration on collagen I, but not on elastin or laminin, is associated with activation of Src [113]. These observations suggest that PDGF-induced migration optimally requires activation of both Src and p38 MAP kinase.

Synthetic function of airway smooth muscle The previous discussion has focused on the influence of cytokines and growth factors on ASM contractility and proliferation, but ASM cells also express a variety of secreted cytokines, chemokines,

60 Table 1 Airway smooth muscle cell derived factors Cytokines IL-1b IL-6 IL-5 IL-11 TNFa IFNb LIF Chemokines Eotaxin IL-8 MCP-1, 2, 3 GM-CSF RANTES TARC SCF Fractalkine IP-10 Growth factors PDGF TGFb VEGF CTGF IGF Cardiotrophin-1

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Cell adhesion molecules Integrins ICAM-1 VCAM-1 CD44

Extracellular matrix Hyaluronan Collagen types I, III, V Laminin Tenascin Fibronectin Perlecan Versican Decorin Thrombospondin Proteoglycan

Matrix metalloproteinase MMP-1 MMP-2 MMP-3 MMP-9 MT-MMP1 TIMP-1 TIMP-2 ADAM33

Other CD40 MHC II PGE2 NOS

Abbreviation: IP-10, interferon-inducible protein 10.

and immunomodulatory proteins; this expression is termed synthetic function (Table 1). The synthetic function of ASM may play a considerable role in modulating airway inflammation and remodeling in asthma.

Extracellular matrix ECM proteins are critical for maintaining the structure and function of the airways. ASM cells, by producing ECM components as well as matrixmodifying enzymes, may contribute to airway remodeling. Alterations in the ECM, in turn, modify the growth and synthetic function of ASM cells. The composition of the ECM is tightly controlled and involves a dynamic process of matrix deposition and degradation. In inflammatory processes such as asthma, this balance is disturbed, resulting in an abnormal amount of matrix deposition and also in an altered composition of matrix components. ASM cells secrete a wide variety of matrix proteins, including fibronectin, collagen, hyaluronan, laminin, and versican. In asthma, there is an increase in hyaluronan, fibronectin, tenascin, versican, laminin, and collagen types I, III, and V [117,118]. In vitro, serum from asthmatic patients increases smooth muscle cell release of fibronectin, laminin, perlecan, and chondroitin sulfate [71]. Treatment of cells with corticosteroids had no effect on the production of matrix proteins by ASM cells [71]. This finding has been borne out in human studies in which inhaled corticosteroids had minimal effect on altering ECM composition in asthmatics [119]. TGFb is secreted from ASM cells and, in turn, induces smooth muscle cell synthesis of hyaluronan and collagen [98,120,121]. The mechanism of this

effect probably involves induction of connective tissue growth factor (CTGF) by TGFb [122]. Recent studies have demonstrated that ASM cells also secrete VEGF, a potent endothelial mitogen [111,123]. In vitro, VEGF increases fibronectin expression by ASM but has no effect on migration or proliferation [111]. Finally, LTD4 and EGF increase expression of versican and fibronectin in ASM cells [124]. Increased matrix deposition seen in asthma is probably caused by increased secretion by mesenchymal cells as well as by an imbalance between matrix-degrading enzymes and inhibitors of these proteases. One class of proteins that has been intensively studied is the matrix metalloproteinase (MMP) family. MMP-1 expression is elevated in the smooth muscle cells of patients who have asthma, and studies have shown that LTD4 increases expression of MMP-1, which acts to degrade insulin-like growth factor binding protein, a growth inhibitor [125]. Progelatinase A (MMP-2) is constitutively released by ASM cells but remains inactive because of high levels of tissue inhibitor of metalloproteinases (TIMP)-2 on the cell membrane [126], although thrombin has been shown to activate MMP-2 [127]. In contrast, TIMP-1 is secreted in large amounts into the conditioned media of ASM cells [126]. Membrane type 1 MMP is also found on ASM [126,127]; this proteinase can activate MMP-2 and has been shown to cleave CD44 from the cell surface and promote cell migration [128]. ASM cells also express pro- and active MMP-3 [127], whereas TNFa induces the release of MMP-9, which can degrade matrix but also plays a critical role in cleaving latent TGF-b to its active form [129]. ADAMs (a disintegrinase and metalloproteinase) are a subfamily of metalloproteinases located on the cell surface. Polymorphisms in the ADAM-33 gene have been asso-

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ciated with airway hyperresponsiveness and asthma [130]. ADAM-33 is expressed by ASM cells and lung fibroblasts [130], although there does not seem to be a significant difference in ADAM-33 protein in persons who have asthma and controls [131]. The composition of the ECM also influences ASM cell function. For example, PDGF-induced proliferation is enhanced by monomeric collagen I, fibronectin, and vitronectin, whereas other ECM components, such as fibrillar collagen I, collagen III, and tenascin-C, have no effect [132]. Others have shown that ECM proteins secreted by asthmatic ASM differ from nonasthmatic ASM. Cells plated on the ECM derived from asthmatic ASM displayed greater rates of proliferation; this finding was true for both nonasthmatic and asthmatic ASM cells [72]. In a study examining the effect of ECM composition on b2adrenoceptor signaling in human ASM cells, the investigators demonstrated that fibronectin increased, whereas collagen V and laminin decreased, accumulation of the second messenger cyclic AMP when compared with collagens I or IV [133]. This finding suggests that altered ECM might affect responses to bronchodilator therapy in patients who have asthma.

motes airway hyporesponsiveness, suggesting a complex role for IL-6 in modulating local inflammation and regulating airway reactivity [142,143]. Other IL-6 family cytokines, such as leukemia inhibitory factor (LIF) and IL-11 but not oncostatin M, are secreted following exposure of ASM cells to viral particles [144]. T-helper (Th)-2 cytokines IL-4, IL-13, and to some extent IL-9 have profound effects on ASM cell synthetic function through induction of a number of signaling molecules and contractile proteins [145,146]. The Th2 cytokines IL-4 and IL-13 induce eotaxin and, in combination with TNFa, TARC expression [69]. In contrast, IL-4 and -13 have no effect on GM-CSF, RANTES, or IL-8 secretion by ASM [147]. IL-5 and other Th2 cytokines may also play a role in sensitizing ASM cells to the effects of contractile agonists, independent of their effects on airway eosinophilia [146]. More controversial, perhaps, is the potential for ASM cells to secrete IL-5 and IL-13 [13].

Immunomodulatory proteins Cell adhesion molecules (CAMs) mediate leukocyte endothelial cell interactions during the process of cell recruitment and homing [148]. Evidence suggests that CAMs mediate inflammatory cell stromal cell interactions that may contribute to airway inflammation. ASM cells express intracellular adhesion molecule-1 (ICAM-1) and VCAM-1, which are inducible by a wide range of inflammatory mediators, and constitutively express CD44, a hyaluronan receptor [149]. Recent data suggest that ASM cells express variable levels of integrin subunits, with the av, a5, and b1 subunits predominating [150]. b1 subunits are important in mediating enhanced chemokine release by ASM cells adherent to fibronectin or collagen I [151]. CAMs also function as accessory molecules for leukocyte activation [152]. Whether CAMs expressed on smooth muscle serve this function remains controversial, however. Although ASM cells do express major histocompatibility complex (MHC) class II and CD40 following stimulation with IFNg [153,154], and a recent study suggests that coculture of T cells with ASM increases T-cell expression of CD25 [155], the physiologic relevance of these findings remains unknown, because ASM cells are unable to present alloantigen to CD4 T cells [153]. Functionally, however, adhesion of stimulated CD4 T cells induces smooth muscle cell DNA syn+ thesis [149,156] Ligation of CD40 increases Ca2 , i

Chemokine and cytokine release Many chemokines, which act to recruit and activate leukocytes, are found in the BAL fluid and lung tissue of patients who have asthma. ASM cells have been shown to secrete RANTES (regulated on activation, normal T-cell expressed and secreted), eotaxin, IL-8, monocyte chemoattractant protein (MCP)-1, -2, and -3, thymus and activation-regulated chemokine (TARC), and granulocyte macrophage colony-stimulating factor (GM-CSF) in response to TNFa and IL-1b (see Table 1). ASM cells may play a role in promoting both the recruitment and survival of eosinophils by secretion of GM-CSF and IL-5 [134 136]. ASM infiltration by mast cells also has been shown in patients who have asthma [137]. In addition to eotaxin, ASM cells secrete stem cell factor (SCF) [138], which acts to recruit and retain mast cells within the smooth muscle layer. Release of lipid mediators and enzymes by activated mast cells probably promotes AHR and either inhibits (chymase) or promotes (tryptase) ASM cell proliferation [139 141]. IL-6 secretion is inducible by multiple stimuli, including TNFa, IL-1b, and bradykinin, and has autocrine effects on smooth muscle cell hyperplasia [62]. Transgenic expression of IL-6 in the murine lung induces peribronchiolar inflammation but pro-

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whereas engagement of VCAM-1 on ASM cells augments growth factor induced ASM cell proliferation [154,157]. Enhancement of PDGF-induced proliferation by either collagen I or fibronectin requires a2b1, a4b1, and a5b1 integrins [132]; similarly, blocking a5b1 integrin on ASM cells enhances apoptosis [150]. Investigators have demonstrated that ASM cells express Fas in vivo and in vitro, and crosslinking of Fas also induces smooth muscle cell apoptosis [158]. Adhesion of activated T cells also seems to alter smooth muscle cell contractility in a contactdependent manner [155], potentially because of the autocrine secretion of IL-5 and IL-1b [159]. Others have shown that ASM cells cultured with activated T cells show increased calcium mobilization responses to LTD4 and serotonin [160]. These studies highlight the finding that direct interactions between leukocytes and smooth muscle cells through immune or adhesion receptors contribute to the modulation of the local milieu resulting in smooth muscle cell activation and growth. Increased amounts of exhaled nitric oxide have been detected in patients who have asthma [161]. Nitric oxide seems to have a selective suppressive effect on the Th1 subset of Th cells, suggesting that increased levels of nitric oxide may therefore lead to the predominantly Th2-type response associated with asthma. Nitric oxide synthase (NOS) has been demonstrated in cultured ASM cells, where it results in an inhibition of ASM cell proliferation [162,163], and has been localized to ASM cells in patients who have asthma [164]. In an animal model, expression of neural nitrous oxide synthase is decreased following allergen challenge [165]. ASM cells also secrete PGE2 and, to a lesser extent, other prostanoids following stimulation with proinflammatory cytokines [166]. PGE2, a potent bronchodilator, also has significant immunologic effects. For example, PGE2 can decrease expression of CD23 (FcgRII), and may have a role as a negative regulator of airway inflammation and hyperresponsiveness [167 169]. PGE2 inhibits cytokine-induced secretion of GM-CSF, RANTES, and CAMs in vitro [170 172] and allergen-induced release of PGD2 in patients who have asthma [173]. In contrast, PGE2 increases IL-6 secretion by ASM cells [174], primes dendritic cells toward a Th2-promoting capacity, and synergizes with IL-4 to induce IgE synthesis [175,176]. Receptors for the complement-derived anaphylatoxin peptides C3a and C5a have also been described on ASM cells. C3a stimulates enhanced mast cell degranulation following cell cell contact with ASM

cells [177]. These peptides may play an important role in the pathogenesis of asthma by altering airway hyperresponsiveness rather than airway inflammation [178,179]. Finally, new data suggest that immunostimulatory DNA sequences containing the CpG motif induce Th1-type inflammation, thus suppressing Th2-type responses that are characteristic of asthma (reviewed in Ref. [180]). These DNA sequences bind to Toll-like receptors, part of the innate immune system, and show promise as potential immunotherapy for airway remodeling [180]. ASM cells express several Toll-like receptors, including Toll-like receptor 9, the receptor for CpG (A. Lazaar, unpublished data), suggesting that ASM cells play a role in innate immunity. ASM cells provide a rich source of cytokines and chemokines and, under certain conditions, can express a wide variety of adhesion receptors, costimulatory molecules, and other immunomodulatory proteins (see Table 1). Taken together, these data provide strong support for the potential role of ASM cells in perpetuating airway inflammation and in leukocyte activation.

Summary The biology of ASM is complex and fascinating. The myriad pathways regulating cell growth and proliferation, combined with the expanding understanding of the role of ASM as a modulator of inflammation, provide a rich area of investigation. Future studies will focus on mechanisms regulating acute inflammation and the chronic repair processes resulting in airway remodeling. These studies may provide the basis for the development of new therapeutic agents aimed at modulating the pathophysiological changes associated with asthma.

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