American Journal of Respiratory and Critical Care Medicine

Asthma as well as bronchiolitis obliterans and chronic bronchitis are chronic lung diseases characterized by airflow obstruction and airway inflammation. Although asthma typically induces reversible airflow obstruction, in some patients airflow obstruction becomes irreversible. The irreversible airway remodeling that occurs in asthma has been attributed in part to increased smooth muscle mass. However, the precise mechanisms regulating increases in myocyte number in vivo remain unknown. Frequent stimulation of airway smooth muscle by contractile agonists, inflammatory mediators, and growth factors may contribute to stimulation of myocyte proliferation. The recognition of phosphatidylinositol 4,5-bisphosphate hydrolysis as a ubiquitous receptor-activated signaling mechanism has led to the discovery that phospholipids, in particular 3-PI, may play a critical role in signaling cell growth. Infiltrating inflammatory cells may indirectly stimulate myocyte growth and activate PI 3-kinase through secretion of agonists and growth factors. In addition, new evidence reveals that direct cell– cell interaction between immunocytes and airway smooth muscle may also modulate airway smooth muscle cell function. Studies have shown that activated T lymphocytes can adhere to cultured airway smooth muscle, and the functional consequence of this adherence is the upregulation of cell adhesion molecules and the stimulation of DNA synthesis in human airway smooth muscle cells. The identification of the critical regulatory sites that mediate airway smooth muscle cell proliferation or modulate cell adhesion molecule expression in these cells may improve our understanding of the mechanisms that regulate airway inflammation and possibly provide new therapeutic approaches to alter airway remodeling seen in patients with chronic airflow obstruction. Panettieri RA, Jr. Cellular and molecular mechanisms regulating airway smooth muscle proliferation and cell adhesion molecule expression.

The pathophysiologic features of asthma include exaggerated airway narrowing to bronchoconstrictor agents and attenuated relaxation to beta-adrenoceptor stimulation. These effects are associated with inflammation and airway remodeling, which include an increase in airway smooth muscle (ASM) cell mass, disruption of the airway epithelium, and alterations in the airway tissue extracellular matrix. The precise mechanisms regulating these processes remain unknown. Compelling evidence, however, supports two fundamental concepts. First, mast cells, eosinophils, lymphocytes, and neutrophils significantly contribute to the initiation and the perpetuation of the changes in airway structure and function in asthma. These immunocytes mediate their effects primarily by secreting inflammatory mediators and growth factors and by trafficking to the airways via cytokine-induced expression of cell adhesion molecules (CAM). Second, the structural remodeling of the airways, which, in part, is due to ASM cell hyperplasia and hypertrophy, contributes to the airway hyperresponsiveness to contractile agonists and to the airflow limitation seen in asthma. Classically, airway smooth muscle cells were considered the major end-effector cells mediating bronchomotor tone in health and disease. Recent evidence, however, suggests that airway myocytes also express CAMs and secrete cytokines and extracellular matrix. These newly described myocyte functions may play a role in perpetuating and orchestrating acute and chronic inflammatory responses in the asthmatic airway as summarized in Figure 1. Further characterization of ASM cells as effector cells in the modulation of airway inflammation may lead to new therapeutic approaches in the treatment of this disease.

This review examines the cellular and molecular signaling pathways that mediate mitogen-induced cell proliferation and that stimulate expression of cell adhesion molecule expression in airway myocytes. Since recent articles have extensively reviewed important signaling pathways that mediate ASM cell proliferation (1, 2), this discussion will specifically examine the newly identified role of inositol phospholipids in the regulation of ASM growth. In addition, cytokine-induced CAM expression in ASM cells will also be reviewed as it relates to stimulation of myocyte growth or to alteration of myocyte function.

Signaling Via Extracellular Stimuli

Many studies have characterized the stimulation of ASM growth in response to mitogenic agents such as polypeptide growth factors, inflammatory mediators, and cytokines, as summarized in Figure 2. Other trophic factors, such as alterations in extracellular matrix and mechanical stress, have also been identified. The observation that contractile agonists induce smooth muscle cell proliferation may be a critical link between the chronic stimulation of muscle contraction and myocyte proliferation (1). Although the mechanisms by which agonists induce cell proliferation are unknown, similarities exist between signal transduction processes activated by these agents and those of known growth factors, which can also stimulate smooth muscle contraction. These diverse extracellular stimuli appear to induce cell growth, at least in part, by activating certain common intracellular signal transduction pathways. The complex interaction between signaling pathways that induce myocyte proliferation and those that inhibit cell growth by stimulation of apoptosis may promote airway remodeling, as seen in the bronchi of patients with asthma, bronchiolitis obliterans, or chronic bronchitis.

Smooth muscle cell proliferation is stimulated by mitogens that fall into two broad categories: (1) those that activate receptors with intrinsic tyrosine kinase activity (RTK); and (2) those that mediate their effects through receptors coupled to heterotrimeric GTP-binding proteins (G proteins) and activate non–receptor-linked tyrosine kinases found in the cytoplasm (Figure 3). Although both pathways increase cytosolic calcium through activation of phospholipase C (PLC), different PLC isoenzymes appear to be involved. Activated PLC hydrolyzes phosphatidylinositol bisphosphate (PIP2) to phosphatidylinositol trisphosphate (IP3) and diacylglycerol (DAG). These second messengers activate other cytosolic tyrosine kinases as well as serine and threonine kinases (protein kinase C, G, and N) that have pleotrophic effects, including the activation of proto-oncogenes. Proto-oncogenes, which are a family of cellular genes (c-onc) that control normal cellular growth and differentiation, were characterized initially from viral genes (v-onc) that induced cellular transformation in eukaryotic cells. The protein products of proto-oncogenes play a critical role in transducing growth signals from the cell surface to the nucleus and in regulating gene transcription.

Inositol Lipids in the Regulation of Cell Proliferation

Phospholipase C activation and inositol trisphosphate. The recognition of phosphatidylinositol 4,5-bisphosphate hydrolysis as a ubiquitous receptor-activated signaling mechanism in eukaryotic cells was one of the major achievements of the 1980s. Recent studies have focused on other aspects of phospholipid metabolism in signaling cell growth, including phosphatidylinositol 3,4,5-trisphosphate formation by inositol lipid 3-kinases and phospholipase D–catalyzed phosphotidylcholine hydrolysis (3). Although there is little doubt that inositol lipids are important in cell signaling, the precise mechanism by which these molecules modulate cell proliferation remains largely unknown.

To date, two phosphoinositide pathways have been characterized. In the canonical PI pathway, activation of phosphatidylinositol-specific phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5,-trisphosphate and DAG. In the 3-phosphoinositide pathway, activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase), which involves protein tyrosine kinase-mediated recruitment, phosphorylates phosphatidylinositides at the D3 position of the inositol ring, leading to the formation of phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3) (Figure 4) (3-6).

Receptors with intrinsic tyrosine kinase activity and those coupled to G proteins both activate specific phospholipase C isoforms. These phosphoinositidases are the critical regulatory enzymes in activation of the PI pathway. The γ family of PLC contains src-homology SH2 and SH3 domains and is regulated by tyrosine phosphorylation. In ASM cells, some growth factors that activate receptors with intrinsic tyrosine kinases have been identified. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) in human ASM cells (7-9) and insulin-like growth factor (IGF)-1 in bovine and rabbit ASM cells (10-12) have been shown to induce myocyte proliferation. However, the role of PLC-γ1 activation in modulating ASM cell growth remains unknown.

Other PLC isoforms are controlled by G proteins and/or Ca2+. In recent years, our understanding of the regulation of PLC by G proteins has grown remarkably. Although the role of PLC activation in mediating G protein–dependent cell growth is complex, G protein activation is critically important in transducing contractile agonist-induced cell growth. G proteins are composed of three distinct subunits: α, β, and γ, with the latter two existing as a tightly associated complex (13). Although α subunits were considered the functional components important in downstream signaling events, recent evidence suggests that βγ subunits also play a critical role in modulating cell function (14). To date, there exist 15 distinct genes that encode mammalian Gα subunits, which are grouped into four classes (Gs, Gi, Gq, and G12) based on their amino acid sequence (15). On the basis of the ADP-ribosylation of certain α subunits by pertussis toxin (PTX), G proteins may also be classified as PTX-sensitive and as PTX-insensitive. The subunits Giα1, Giα2, Giα3, Goα1, and Goα2 are PTX-sensitive, whereas all other α subunits are PTX-insensitive.

Advances in single-cell microinjection techniques in combination with the development of neutralizing antibodies to specific Gα subunits have enabled investigators to characterize the role of G protein activation in cell proliferation. Using these techniques, studies with 3T3 fibroblasts have determined that while both thrombin and bradykinin required Gq activation to mobilize cytosolic calcium, to generate IP3, and to induce mitogenesis, thrombin, but not bradykinin, appeared also to require Gi2 in addition to Gq to stimulate cell growth (16). These studies determined that a single mitogen may require functional coupling to distinct subtypes of G proteins in order to stimulate cell growth. This also provides a mechanism to explain why some, but not all, agonists induce cell proliferation while mobilizing comparable levels of cytosolic calcium.

Recently, the role of PLC activation and inositol trisphosphate in mediating contractile agonist-induced ASM cell growth has been explored. Several contractile agonists, which mediate their effects through G protein–coupled receptors, induce ASM cell proliferation. Studies have determined that histamine (17) and serotonin (18) induce canine and porcine ASM cell proliferation. Endothelin-1, leukotriene D4 (LTD4), and U-46619, a thromboxane A2 mimetic, induce rabbit ASM cell growth (19), and thrombin induces mitogenesis in human ASM cells (9). Although the mechanisms that mediate these effects are unknown, agonist-induced cell growth probably is modulated by activation of G proteins in a manner similar to that described in vascular smooth muscle.

Using human ASM cells, Panettieri and colleagues (9) recently examined whether contractile agonist-induced human ASM cell growth was dependent on PLC activation and inositol trisphosphate formation. These investigators examined the relative effects of bradykinin and thrombin on myocyte proliferation and PI turnover. Thrombin, but not bradykinin, stimulated ASM cell proliferation despite a fivefold greater increase in [3H]inositol phosphate formation in cells treated with bradykinin as compared with those treated with thrombin. These investigators also determined that inhibition of PLC activation with U-73122 had no effect on thrombin- or EGF-induced myocyte proliferation. In addition, pertussis toxin completely inhibited thrombin-induced ASM cell growth but had no effect on PI turnover induced by either thrombin or bradykinin (9). Taken together, these studies suggest that thrombin induced human ASM cell growth by activation of a pathway that was pertussis toxin–sensitive and independent of PLC activation or PI turnover.

Compared to RTK-dependent growth factors, contractile agonists, with the exception of thrombin, appear to be less effective human ASM mitogens (20). In cultured human ASM cells, 100 μM histamine or serotonin induces two- to threefold increases in [3H]thymidine incorporation as compared with that obtained from unstimulated cells. EGF, serum, or phorbol esters, which directly activate protein kinase C, induce 20- to 30-fold increases in [3H]thymidine incorporation (18, 21, 22). Interestingly, histamine is as effective as serum in stimulating cell growth and c-fos expression in canine ASM cells (17). In rabbit ASM cells, endothelin-1 induces cell proliferation by activating phospholipase A2 and by generating thromboxane A2 and LTD4 (19, 23). In human ASM cells, however, endothelin-1, thromboxane A2, and LTD4 appear to have little effect on ASM cell proliferation, despite these agonists inducing increases in cytosolic calcium (9, 22, 24). Clearly, interspecies variability exists with regard to contractile agonist- induced cell proliferation. These models, however, may prove useful in dissecting downstream signaling events that modulate the differential effects of contractile agonists on ASM cell proliferation.

3-Phosphorylated inositol lipids. Recently, these phospholipids have been recognized as a new class of second messengers (6, 25, 26). Based on a number of studies, PtdIns 3,4,5-P3 appears to be the critical signaling 3-phospholipid (Figure 5). This assumption is supported by the time course of accumulation and the subsequent metabolism of the individual 3-phosphoinositides following agonist stimulation (3, 27). Although the metabolic routes of these lipids are poorly understood when compared with those of the canonical phosphoinositides, some important features have emerged. The 3-phosphoinositides are not substrates for any known PLC and are not components of the canonical phosphoinositide turnover pathway. Rather, their rapid increase upon growth factor stimulation suggests that the lipids themselves may act as second messengers, mediating PtdIns 3-kinase mitogenic signals (5). The recent identification that 3-phosphoinositides directly activate the ζ isoform of protein kinase C may have important implications in understanding mechanisms that induce smooth muscle cell proliferation, since myocyte growth is thought to be protein kinase C–dependent (28).

Compelling evidence suggests a role for PtdIns 3-kinase and its lipid products in regulating various cellular functions that include mitogenesis (5). PtdIns 3-kinase, which consists of an 85-kilodalton (kD) regulatory subunit (p85) and a 110-kD catalytic subunit (p110), is required for DNA synthesis induced by some, but not all, growth factors (29). In 3T3 fibroblasts, microinjection of cells with a neutralizing antibody to the p110 catalytic subunit of PtdIns 3 kinase was found to completely inhibit PDGF- and EGF-induced mitogenesis (29). In some cells, however, bombesin and LPA, which induce cell proliferation by activating receptors coupled to G proteins, stimulated cell growth in the absence of PtdIns 3-kinase activity (5, 29). Taken together, these studies suggest that mitogens may activate different intracellular signaling pathways in a cell-specific manner.

Few investigators have examined the role of PtdIns 3-kinase activation in modulating human ASM cell proliferation. A recent study examined whether PtdIns 3-kinase mediated ASM cell growth or modulated calcium transients induced by contractile agonists. Thrombin-induced increases in cytosolic calcium were examined in fura-2 loaded cells pretreated with wortmannin, a PtdIns 3-kinase inhibitor (2, 30). Inhibition of PtdIns 3-kinase did not alter calcium transients induced by thrombin (30). In parallel experiments, ASM cells were pretreated with wortmannin and then stimulated with thrombin or EGF. DNA synthesis was then measured by [3H]thymidine incorporation. In a dose-dependent manner, wortmannin inhibited thrombin and EGF-induced DNA synthesis but had no effect on basal levels of [3H]thymidine incorporation as compared with cells treated with diluent alone. If wortmannin was added 6 h after the cells were stimulated with mitogens, proliferation was no longer inhibited. These data suggest that PtdIns 3-kinase may mediate early signaling events that modulate myocyte growth as depicted in Figure 5. Collectively, these studies suggest that PtdIns 3-kinase activation may play an important role in modulating ASM cell proliferation induced by growth factors and contractile agonists.

Many of the agonists and growth factors, which stimulate myocyte growth, are secreted by infiltrating inflammatory cells. New evidence, however, reveals that direct cell–cell interaction between immunocytes and ASM can directly modulate ASM function. These recent studies may improve our understanding of processes that regulate ASM cell proliferation

Cytokine-induced Cell Adhesion Molecule Expression in ASM Cells

The variety of cell types that reside in or infiltrate the inflamed submucosa present the potential for many important cell–cell interactions. Eosinophils, macrophages, and, particularly, lymphocytes, are postulated as also being critical in the initiation and perpetuation of the asthmatic response. One mechanism by which immunocytes exert their effects is the production of proinflammatory mediators that may act directly or indirectly on ASM. The presence of several cytokine mRNAs have been detected within the airways of asthmatic subjects, including tumor necrosis factor (TNF), interleukin (IL)-1, IL-3, IL-4, IL-5, and granulocyte macrophage colony-stimulating factor (31-33). In addition to these cytokines, IL-2 and IL-6 protein were detected (34-36). A second potential mechanism may involve direct contact of immunocytes with airway myocytes. Although many cell–cell interactions likely contribute to airway hyperresponsiveness in asthma, recent evidence supports that T cells and ASM, two important effector cells in this disease, can directly interact via cell adhesion molecules (CAMs).

Mechanisms of Leukocyte Recruitment to Inflammatory Sites

Cell adhesion molecules mediate leukocyte–endothelial cell interactions during the process of cell recruitment and homing (37). The expression and activation of a cascade of CAMs that include selectins, integrins, and CD31, as well as the local production of chemoattractants, lead to leukocyte adhesion and transmigration into lymph nodes and sites of inflammation involving non-lymphoid tissues. The mechanisms utilized for extravasation of leukocytes from the circulation during the establishment of a local inflammatory response are rapidly being delineated. However, the subsequent interactions of the infiltrating leukocytes with other cell types in the bronchial submucosa or with the extracellular matrix that may be important for sustaining the inflammatory response have not been defined. In addition to mediating cell contact, some of the CAMs may also function as co-stimulatory molecules that contribute to the activation of cells (38-40).

Recent attention has been directed toward the role of CAMs in mediating the asthmatic response (41). Previous studies showed increased expression of the α1 and α2 integrins, VLA-4 and LFA-1, in bronchial biopsies of patients with asthma (42-44). Further, Wegner and coworkers (45) demonstrated that antibodies against intercellular adhesion molecule (ICAM)-1 decreased eosinophil infiltration and attenuated bronchial hyperresponsiveness in a primate model of asthma, while Nakajima and colleagues (46) demonstrated that antibodies to VLA-4 and vascular cell adhesion molecule (VCAM)-1, but not LFA-1 and ICAM-1, block eosinophil entry into lungs of mice challenged with aerosolized antigen (46).

CAMs Mediate Cell–Cell and Cell–Matrix Interactions in Inflammation: What Is Known about CAM Expression on Smooth Muscle?

In addition to mediating leukocyte extravasation and transendothelial migration, CAMs mediate submucosal or subendothelial contact with cellular and extracellular matrix components and serve as co-stimulatory molecules in the activation of leukocytes. Infiltrating leukocytes are in close proximity to the ASM. However, few studies have examined the expression or function of adhesion molecules on smooth muscle cells. Couffinhal and associates (47) found that aortic vascular smooth muscle (VSM) treated with TNF expressed functionally active ICAM-1 that mediated monocyte binding. In addition, Stemme and coworkers (48) demonstrated that ICAM-1 expression on VSM could be upregulated by IL-1, interferon gamma (IFN-γ), and lipopolysaccharide (LPS) and was accompanied by an increase in T-lymphoblast binding. This binding was partially inhibited by monoclonal antibodies (mAb) specific for ICAM-1, LFA-1, and CD29, implicating these receptors in the adhesion of T cells to VSM. The fact that blocking by these mAbs was incomplete suggested that additional adhesion pathways might also be involved.

Some evidence suggests that α4 integrins may play an important role in mediating airway hyperresponsiveness in an antigen-sensitized ovine model. Abraham and colleagues (49) found that antibodies to the α4 component of integrin receptors blocked antigen-induced airway hyperreactivity in sheep but without any apparent effect on leukocyte recruitment. These findings implicate α4 integrins in processes that lead to persistent airway hyperresponsiveness after antigen challenge that cannot be totally accounted for by inhibition of leukocyte recruitment. Despite the attempt to link airway hyperreactivity to leukocyte recruitment, these data suggest that α4 integrins may modulate cell–cell or cell–matrix interactions apart from those promoting leukocyte infiltration. Whether T cell– myocyte interactions in this model could explain these findings remains unknown.

Based on studies of VSM showing that the expression of CAMs such as ICAM-1 and VCAM-1 could be upregulated in atherosclerosis (50-53), investigators have examined whether ASM cells could likewise express CAMs. Analysis of immunoprecipitates of extracts of 35S-methionine-labeled cells demonstrated constitutive expression of a 90-kD form of CD44 that was not changed by treating the ASM with TNF, as shown in Figure 6A. In contrast, ICAM-1 and VCAM-1 were detected at low and barely detectable levels, respectively, on untreated ASM but were upregulated following treatment with TNF for 24 h. TNF-induced expression of ICAM-1 was dose dependent and was detectable by 4 h and maximal at 36 h. IL-1, LPS, and IFN also increased ICAM-1 expression, while VCAM-1 expression was most notably increased by TNF and IL-1 (54). Using a quantitative assay of T-cell binding to ASM, investigators determined that activation of T cells is required for adhesion to ASM, as shown in Figure 6B. Furthermore, upregulated expression of cell adhesion receptors by the ASM is not sufficient for resting T-lymphocyte adhesion, but enhanced adhesion of activated T cells.

Adhesion of Mitogen-activated T Cells to ASM Is Mediated by LFA-1/ICAM-1, VLA-4/VCAM, and CD44/HA

The role of specific adhesion receptors in mediating lymphocyte–ASM binding was determined using blocking mAbs. When activated T cells were pretreated with mAbs against LFA-1 or when the ASM was pretreated with antibodies to ICAM-1, T-cell binding to ASM was partially abrogated. Antibodies specific for VLA-4 or one of its ligands, VCAM-1, had little inhibitory effect on adhesion of activated T cells to ASM (54). However, mAbs against LFA-1 and VLA-4 together, or ICAM-1 and VCAM-1 in combination, inhibited binding to a greater extent than anti-LFA-1 or anti-ICAM-1 alone. Since significant adhesion occurred even in the presence of these combinations of mAbs, the potential role for a third adhesion pathway mediated by CD44-HA interactions was studied. Pretreatment of T cells with mAbs specific for the HA binding site of CD44 alone did not cause a decrease in activated T-lymphocyte adhesion to TNF-stimulated ASM. Pretreatment of the ASM monolayer with hyaluronidase also failed to decrease adhesion of activated T cells. However, the combination of mAbs against LFA-1, VLA-4, and CD44 acted synergistically, reducing the binding of activated T cells to the level observed for resting T cells (54). In addition, LFA-1/ VLA-4–independent adhesion was also sensitive to hyaluronidase treatment. These studies indicate that CD44 is important in mediating T-cell adhesion to ASM and accounts for the majority of the LFA-1/ICAM-1, VLA-4/VCAM-1–independent adhesion (54).

Antigen-activated T Cells Derived from the Peripheral Blood of Atopic Donors Adhere to ASM

Although some evidence exists that T cells activated with phorbol esters and ionomycin adhere to human ASM cells, do T cells activated in vivo also adhere to ASM cells? Lazaar and coworkers (55) recently determined that anti-CD3–stimulated peripheral blood T cells also adhere to ASM and markedly upregulate ICAM-1 expression and induce expression of major histocompatibility complex (MHC) class II on ASM. Interestingly, the induction of HLA-DR was completely inhibited, and the induction of ICAM-1 partially inhibited by a neutralizing antibody against IFN-γ. Using bronchoalveolar lavage– derived T cells isolated from atopic donors following local antigen challenge, these investigators found that T cells adhered to ASM and upregulated ASM expression of ICAM-1 and HLA-DR similar to that seen with in vitro activated T cells.

Physical approximation of T cells to an antigen-presenting cell exhibiting antigen in the context of MHC class I or II molecules is required for T-cell activation via the T-cell receptor. Co-stimulatory molecules, including some of the CAMs discussed above, provide the additional signals that are required for optimal T-cell stimulation (38-40). Although smooth muscle cells have not previously been focused on as participants in the immune response, it has been demonstrated that VSM can express class II antigens in response to IFN-γ. Stemme and associates (56) described increases in HLA-DR expression in arterial smooth muscle cell treated with IFN-γ, which was enhanced by treatment with TNF. Similarly, Fabry and colleagues (57) reported that smooth muscle cell derived from murine cerebral microvessels expressed MHC class II antigens in response to IFN-γ and were able to present antigen to T-cell hybridomas. Recently, ASM cells were also reported to express class II antigens when stimulated with IFN-γ (55). In this study, the ASM cells were not capable of presenting alloantigen to CD4+ T cells.

Taken together, these studies suggest that, similar to normal donors, adhesion of peripheral blood T cells from allergic patients requires activation and furthermore indicates that antigen and accessory cells are sufficient to provide all the signals required to induce T-cell adhesion to ASM.

Intracellular Signaling and CAM Expression in ASM Cells: A-Kinase Activation Inhibits TNF-induced ICAM-1 Expression

The intracellular signals by which agents such as TNF regulate adhesion molecule expression remain unknown. Several investigators have examined the role of protein kinase C and cyclic AMP (cAMP)–dependent kinase (A-kinase) in TNF-induced expression of ICAM-1 and VCAM-1 on endothelial cells (58, 59). Direct activation of protein kinase C can mimic the effects of TNF on endothelial cells by inducing ICAM-1 expression (58, 60); other studies have shown that A-kinase activation inhibits TNF-induced increases in VCAM-1 but not ICAM-1 (59).

In human ASM cells, investigators recently reported the effects of activation of A-kinase on ICAM-1, VCAM-1, or CD44 expression in airway myocytes treated with cytokines. ASM cells were pretreated with 5 μM forskolin and then stimulated with either control media or 1,000 U/ml TNF. Forskolin directly stimulates adenylyl cyclase and increases [cAMP]i, which in turn activates A-kinase. As determined by cell surface expression, cells pretreated with forskolin and stimulated with TNF had markedly reduced ICAM-1 expression, while VCAM-1 expression was less affected and CD44 expression appeared unchanged as compared with those that were treated with TNF alone. Forskolin alone had no effect on basal levels of CAM expression. Taken together, these studies suggest that signaling pathways that increase [cAMP]i and activate A-kinase appear to inhibit cytokine-induced expression of ICAM-1 and VCAM-1 in human ASM cells (61).

Functional Consequences of T-cell Adhesion to ASM Cells

Smooth muscle cell hypertrophy and hyperplasia are hallmarks of asthma. Inflammatory mediators or contractile agonists can induce changes in DNA synthesis and cellular proliferation (23). The mechanisms that mediate these growth responses, however, remain unknown. Lazaar and coworkers (54) recently studied the effect of T-lymphocyte adherence on DNA synthesis in ASM as measured by uptake of bromodeoxyuridine (BrdU). Irradiated activated T cells adhered and induced approximately a 30-fold increase in BrdU incorporation by subconfluent ASM as compared with untreated control cells. Immunohistochemical analysis with an antibody against BrdU (62) revealed fluorescence in 8.8 ± 1.1% of growth-arrested ASM associated with activated T lymphocytes, compared to less than 0.3 ± 0.2% in ASM maintained in serum-free media, and greater than 30 ± 2.4% in ASM treated with 10% fetal bovine serum, a potent ASM mitogen. Neither unstimulated T cells nor the conditioned media from the activated T cells induced DNA synthesis. In addition, the supernatant from ASM–T cell co-cultures did not induce DNA synthesis in ASM. These results suggest that cell contact may be required to induce ASM DNA synthesis.

Asthma as well as bronchiolitis obliterans and chronic bronchitis are chronic lung diseases characterized by airflow obstruction. Despite considerable research effort, primary defects that underlie airway obstruction remain unknown, although an intrinsic abnormality of airway smooth muscle has been postulated.

Although asthma typically induces reversible airway obstruction, in some asthmatics airflow obstruction can become irreversible. Such obstruction may be a consequence of persistent structural changes in the airway wall due to the frequent stimulation of airway smooth muscle by contractile agonists, inflammatory mediators, and growth factors. Increased smooth muscle mass, which has been attributed to increases in myocyte number, is a well-documented pathologic finding in the airways of patients with chronic severe asthma.

Although the mechanisms by which mitogens induce cell proliferation are unknown, similarities exist between signal transduction processes activated by these agents and those of known growth factors. Diverse extracellular stimuli induce cell growth in part by activating common intracellular signal transduction pathways.

Recent studies have also determined that ASM cells may directly interact with infiltrating immunocytes. The functional consequences of immunocyte–myocyte adherence may modulate inflammatory responses in the asthmatic airway. Studies have shown that activated T lymphocytes can adhere to cultured ASM. In addition, airway myocytes upregulated expression of ICAM-1 and VCAM-1 in response to inflammatory cytokines, in both in vitro and in situ models of transplanted human bronchial tissue into SCID mice. Further, ASM constitutively expresses another CAM, CD44, and blocking antibodies to ICAM-1, VCAM-1, and CD44 in combination completely abolished adhesion of activated T cells to ASM. Finally, adherence of activated T cells to ASM induces DNA synthesis in quiescent myocytes. The identification of the critical regulatory sites that mediate ASM cell proliferation or modulate CAM expression on airway myocytes may provide new therapeutic approaches to alter the airway remodeling seen in patients with chronic airflow obstruction.

The writer thanks the following individuals for review and helpful comments concerning the manuscript: Mr. Andrew Eszterhas, Dr. Vera Krymskaya, Dr. Yassine Amrani, and Dr. Aili Lazaar; and Mary McNichol for assistance in the preparation of the manuscript.

Supported by grants from the National Institutes of Health (R01-HL55301), the National Aeronautics and Space Administration (NRA-94-OLMSA-02), and a Career Investigator Award from the American Lung Association.

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Correspondence and requests for reprints should be addressed to Reynold A. Panettieri, Jr., M.D., Pulmonary and Critical Care Division, Room 805 East Gates Building, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail: .

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