INTRODUCTION

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Glucocorticoids are the most effective therapy currently available for the treatment of asthma. Glucocorticoids act primarily as anti-inflammatory agents in asthma and partially reduce the characteristic airway hyperresponsiveness present in this condition (1). Their main action in the airways of subjects with asthma is believed to be inhibition of recruitment of inflammatory cells and inhibition of release of pro-inflammatory mediators and cytokines from activated inflammatory and airway epithelial cells (2). However, glucocorticoids may have several other actions and cellular targets in the airways that contribute to their therapeutic efficacy in asthma management. Prominent among the cellular targets of inhaled steroids in the lung are airway smooth muscle cells, but little is known of the action of glucocorticoids on the function of airway smooth muscle. This review brings together a small amount of available information examining the action of glucocorticoids on several aspects of airway smooth muscle function likely to be relevant to remodeling of the airways in asthma. These are contraction, proliferation, and production of pro-inflammatory mediators and cytokines.

Traditionally, the role of the smooth muscle cell in airway inflammation has been regarded to be passive, contributing to the pathogenesis of asthma solely by its contractile properties. Several reports have shown that in addition to its contractile function in asthma, airway smooth muscle can undergo hyperplasia and/or hypertrophy (3, 4) leading to structural changes in the airway wall that contribute to the development of persistent airway obstruction and increased nonspecific airway hyperresponsiveness (5, 6). Additional reports from cell culture-based studies are emerging to suggest a further role for airway smooth muscle in airway inflammation by acting as an important source of pro-inflammatory and bronchoprotective mediators (7, 8). This apparent functional diversity of airway smooth muscle has prompted interest in the possibility that there is plasticity in its function that may be related to the severity of the tissue remodeling process during chronic inflammation of the airway wall (7, 9). In this article we review what is known about the direct actions of glucocorticoids on airway smooth muscle with particular emphasis on their action on the proliferative or "synthetic" phenotype.

	CONTRACTILE FUNCTION

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Few studies have addressed the action of glucocorticoids on airway smooth muscle contractile function. One study demonstrated that pretreatment with dexamethasone inhibited the contraction of sensitized guinea pig tracheal ring preparations exposed to antigen (10). The mechanism was presumed to reflect the inhibitory action of glucocorticoids on antigen-induced release of airway smooth muscle spasmogens in the preparation, rather than a direct effect on the smooth muscle, since in the same study inhibition of the release of a range of cyclooxygenase-derived spasmogens was also demonstrated (10). More recent studies have questioned this interpretation, as glucocorticoids have been found to impinge directly on mechanisms within airway smooth muscle cells that lead to contraction.

Effects of Glucocorticoids on Mechanisms Leading Directly to Contraction

We are aware of only one study that has examined directly the effect of glucocorticoids on contraction of airway smooth muscle. In this study Nabishah and colleagues (11) found that treatment of rats for 7 d with either dexamethasone or cortisone reduced the contractile response of isolated bronchial smooth muscle preparations to acetylcholine. Treatment with deoxycortisone, however, had no effect. In a subsequent study where rats were treated with dexamethasone for 5-10 d, the reduction in bronchial smooth muscle contraction to acetylcholine was found to correlate with reduced total muscarinic receptor number (12). More recently, studies of canine airway smooth muscle from animals treated in vivo with methylprednisolone for 3 d have shown the reduction in total muscarinic receptor number to involve both the M₂ and M₃ subtypes. Surprisingly, this effect could not be repeated when the airway smooth muscle preparations were treated in vitro with methylprednisolone prior to radioligand binding analysis. In light of this observation, the mechanism whereby glucocorticoids might protect against bronchospasm, by at least in part reducing cholinergic hypersensitivity, most likely does not involve a direct action on transcription of the M₂ or M₃ receptor genes (13). Since steroid hormone response elements have not been identified within their genes, the mechanism more likely depends on altered gene transcription of some other unknown factor or intermediateperhaps from another cell type in the lung which was not present when airway smooth muscle preparations were incubated in vitro with methylprednisolone (13). Another possibility, that methylprednisolone might also uncouple muscarinic receptors under these conditions, was not investigated.

A similar protective effect of glucocorticoids was reported in cultures of human bronchial smooth muscle by Hardy and coworkers (14) where prolonged exposure (22 h) to dexamethasone inhibited histamine-stimulated inositol phosphate formation, suggesting either downregulation or uncoupling of the histamine H₁ receptor. In another study that examined the effect of dexamethasone on histamine-induced responses, dexamethasone augmented histamine H₂ receptor-stimulated increases in cyclic AMP content of guinea pig airway smooth muscle in culture in the absence of any detectable change in H₂ receptor expression determined by radioligand binding (15). An increase in cellular cyclic AMP concentration might be expected to counter any H₁-mediated effect and could contribute to the apparent uncoupling of this receptor reported by Hardy and coworkers (14), since these experiments were performed in the absence of an H₂ receptor antagonist, which might otherwise prevent H₂ receptor-mediated elevation in cellular cyclic AMP levels. Another study examined the effect of glucocorticoids on agonist-induced changes in intracellular calcium ion concentration in airway smooth muscle cells and found that pretreatment with dexamethasone for 24 h reduced the subsequent calcium-mobilizing response to bradykinin (16). A similar effect has been described on intracellular calcium in cultured vascular smooth muscle following stimulation with endothelin-1 or platelet-activating factor (17). The suppressive effect of glucocorticoids on agonist-stimulated increases in intracellular calcium concentration reported in these studies may involve reduced receptor affinities or numbers or increase the amount of stimulatory G proteins or adenylate cyclase activity (18, 19), since evidence now exists for each of these mechanisms in smooth muscle (13).

Effects of Glucocorticoids on Mechanisms Augmenting Relaxation

Increased relaxation of airway smooth muscle by glucocorticoids may involve either direct upregulation of -adrenergic- dependent or -independent pathways linked to relaxation, or downregulation of an opposing mechanism such as the muscarinic pathway (as discussed above), mediating either contraction or inhibition of relaxation.

In a recent study by Schramm and Grunstein (20) methylprednisolone was found to increase rabbit airway smooth muscle relaxation by potentiating the electrogenic Na⁺/K⁺ ATPase pump. This effect was rapid in onset, occurring within 1 h of incubation with methylprednisolone, and was therefore unlikely to be due to glucocorticoid-induced transcription of pump enzymes. Although the effects of protein synthesis inhibitors were not examined, it is more likely that changes in pump kinetics were involved, as reported elsewhere (21). The potentiating effect of methylprednisolone on airway smooth muscle relaxation was noted to be increased further in immature airways.

In asthma, glucocorticoid administration enhances lung responses to -adrenoceptor agonists (22) and reverses -adrenoceptor dysfunction (23). The cellular mechanisms that underlie these effects include increased ₂-adrenoceptor proteins in human lung (18, 24) as well as direct modulation of the activities of adenylate cyclase and cyclase and cyclic AMP-dependent protein kinase (25). Recently it has been demonstrated that the potentiating effect of glucocorticoids on ₂-adrenoceptor number and function occurs directly in airway smooth muscle (13, 26).

Glucocorticoids also interact with the ₂-adrenoceptor to prevent its downregulation following chronic administration of ₂-adrenoceptor agonists (18). Autoradiographic mapping studies of rat lung indicate that in vivo, glucocorticoids upregulate ₂-adrenoceptors and protect against downregulation of ₂-adrenoceptors in all cell types, including airway smooth muscle (27). This contrasts with the findings of Hall and associates (28) using human cultured airway smooth muscle cells, where treatment with dexamethasone for 16 h failed to prevent isoprenaline-induced desensitization of the -adrenergic pathway measured by cyclic AMP accumulation.

Implications

In general, the effect of glucocorticoids has been examined only on individual elements of the contractile machinery, and inferences must therefore be made about their likely effect on subsequent airway smooth muscle contractility. With this in mind, one effect of glucocorticoids on airway smooth muscle appears to be to limit those mechanisms that would otherwise lead directly to contraction (e.g., reduction of intracellular calcium concentration, reduced expression of cholinergic muscarinic receptors, and uncoupling of histamine H₁ receptors), while augmenting those mechanisms associated with relaxation of airway smooth muscle (e.g., increased ₂-adrenoceptor number, reduced desensitization of ₂-adrenoceptors, increased adenylate cyclase activity, increased Na⁺/K⁺ electrogenic pump activity).

	PROLIFERATIVE FUNCTION

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It is well established that patients with chronic severe asthma often develop irreversible airflow obstruction that is resistant to bronchodilator and anti-inflammatory therapy. This is thought to be the consequence of persistent structural changes in the airway wall, which are due in part to a marked increase in the smooth muscle mass (3, 4). Both hyperplasia (an increase in cell number) and hypertrophy (an increase in cell size) of airway smooth muscle contribute to this process of airway wall remodeling (4, 29), which has important functional implications related to increased airway narrowing for a given amount of airway smooth muscle shortening (5, 30). Patients with asthma also have increased maximal airway narrowinganother feature that can be explained by increases in airway wall thickening (30). Further, mathematic models indicate that the increase in airway smooth muscle mass found in asthma is sufficient to explain a major part of the bronchial hyperresponsiveness associated with persistent severe asthma (31). For these reasons, an increase in smooth muscle content may be the most important structural abnormality present in the airway wall in asthma (5, 31).

Effects of Glucocorticoids on Airway Remodeling In Vivo

Although many studies have addressed the effects of glucocorticoids on many aspects of airway inflammation (32), the impact of glucocorticoids on remodeling of airway smooth muscle proliferation has not been fully explored. Several studies have examined the effects of inhaled glucocorticoid treatment on airway morphology and hyperreactivity. Laursen and coworkers (35) reported that connective tissue atrophy was not associated with nearly 1-yr of treatment of patients with severe asthma with inhaled budesonide. Jeffery and coworkers (36) showed that 4-wk treatment of patients with atopic asthma with budesonide reduced airway inflammation, but despite prolonged treatment (up to 3.7 yr), the increase in reticular basement membrane thickening was not prevented. Similarly, in patients with birch pollen-sensitive seasonal asthma, no change in collagen deposition was detected following inhaled budesonide (37). In a 10-yr follow-up study, inhaled steroid treatment did not cause any significant reduction in airway hyperreactivity (38). Together, one implication of these reports is that remodeling of the airways is poorly reversible and is the result of persistent changes in airway structure.

Effects of Glucocorticoids on Proliferation of Cultured Airway Smooth Muscle

Several studies have sought to examine the effects of glucocorticoids in vitro directly on airway smooth muscle proliferation. Stewart and associates (39, 40) found that pretreatment of human airway smooth muscle cells in culture with glucocorticoids such as dexamethasone, methylprednisolone, or hydrocortisone inhibited thrombin-stimulated proliferation. This effect was not observed with the androgenic mineralocorticosteroid aldosterone or with progesterone or 17-estradiol. In addition to thrombin, glucocorticoids were also effective against several other mitogens, including basic fibroblast growth factor and fetal calf serum, but were poorly effective against epidermal growth factor. In addition, beclomethasone and cortisol have also been found to inhibit proliferation of bovine tracheal smooth muscle cells in culture (41).

Schramm and colleagues (42) have reported that the inhibitory effect of methylprednisolone on rabbit tracheal airway smooth muscle proliferation is additive to that of isoprenaline. Experiments performed in the presence of the cyclic AMP- dependent protein kinase antagonist, Rp-cyclic AMPS, demonstrated that the antiproliferative effect of methylprednisolone did not involve activation of adenylate cyclase, as has been suggested for contraction (see above). The authors concluded that the growth inhibitory effects of isoprenaline and methylprednisolone were additive but were mediated through distinct mechanismsinhibition produced by isoprenaline was dependent on cyclic AMP-dependent protein kinase activation, whereas the inhibitory effect of methylprednisolone was independent or downstream of cyclic AMP-dependent protein kinase activation (42).

Studies in HeLa cells suggest that for glucocorticoids to be effective in preventing cellular proliferation they must be present in late G1 and S phases of the cell cycle; they are ineffective in late G2, M, and early G1 (43). This may be related to availability of glucocorticoid receptors whose phosphorylation and glycosylation state, and therefore affinity for the glucocorticoid ligand (44), may be controlled by cell cycle-dependent protein kinases (45). This is consistent with the findings of Stewart and colleagues (39) that cell cycle-synchronized human airway smooth muscle in culture became refractory to the antiproliferative effect of dexamethasone when added 21 h or more after addition of the initial mitogen, when cells have already progressed through S phase and entered the G2 or M phases of the cell cycle.

Implications

The antiproliferative effect of glucocorticoids on airway smooth muscle proliferation, at least in vitro, raises the possibility that one of the anti-asthma effects of these compounds may be prevention of the airway wall thickening present in asthma. However, this does not accord well with the low therapeutic efficacy of inhaled glucocorticoids on airway morphology and bronchial hyperresponsiveness reported by others (36), and may reflect the possibility that more chronic or prophylactic treatment with glucocorticoids is required in order to protect airway function (and presumably structure). Haahtela and associates (46) have suggested that maximum therapeutic effectiveness may take as long as 1 yr. Further, their study also demonstrated that the potential to reverse airflow obstruction and bronchial hyperreactivity was impaired in patients in whom anti-inflammatory therapy was delayed. Clinical observations such as these are consistent with the possibility that the resolution of established changes in airway wall structure by glucocorticoids is only slowly achieved, compared with the anti-inflammatory effects of glucocorticoids, and that these changes may be more difficult to reverse than to prevent with prophylactic therapy. Another possibility contributing to the apparent ineffectiveness of glucocorticoids in resolving some aspects of the airway remodeling process is suggested by the observations of Stewart and colleagues (40) that high concentrations of tumor necrosis factor (TNF)- reduce the mitogenic response of human airway smooth muscle cells in culture and that this inhibitory effect of TNF- occurs through a dexamethasone-sensitive pathway that is unrelated to the anti-inflammatory effects of the steroid (40). Thus, the antiproliferative effects and potential protective effects of TNF- in the inflamed asthmatic airway may be compromised by treatment with anti-inflammatory steroids.

	SECRETORY FUNCTION

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The contribution made by proliferation of airway smooth muscle to the pathogenesis of chronic asthma may not be limited simply to geometric obstruction to airflow due to the increased airway wall thickening. Although little or no supporting in vivo evidence is currently available, several reports from cell culture-based studies suggest that airway smooth muscle may also be an important source of pro-inflammatory and bronchoprotective mediators (8). This may be especially relevant in the diseased lung where the content of airway smooth muscle as a fraction of the total cells in the airway wall is already increased. Several preliminary studies have examined the effects of glucocorticoids on cytokine production by airway smooth muscle cells and have suggested that these previously considered structural cells may be an important target for the anti-inflammatory efficacy of inhaled steroids.

Effects of Glucocorticoids on Arachidonic Acid Metabolite Production

Treatment of human airway smooth muscle cells in culture with interleukin (IL)-1 and TNF- increases their expression of cyclooxygenase (COX)-2 (47), the inducible isoform of the major isoenzyme associated with inflammation. Although low levels of other arachidonic acid metabolites such as 6-keto-PGF₁ (a stable product of prostaglandin [PG]I₂), PGD₂, PGF₂, and thromboxane (TX)B₂ (a stable product of TXA₂), were produced, the major metabolite produced as a result of COX-2 induction was PGE₂. PGE₂ is a major bronchoprotective metabolite in the airways (50), and studies using cultured airway smooth muscle and epithelial cells suggest that airway smooth muscle could also be a major source of PGE₂ production in the airway (51, 52). It is unclear whether the consequences of COX-2 induction and prostaglandin production by pro-inflammatory cytokines in airway smooth muscle would be beneficial or deleterious in asthma, although PGE₂, the major COX-2 metabolite in these cells, exerts several anti-inflammatory and anti-spasmogenic effects in the lung and inhibits airway smooth muscle proliferation (53, 54). Despite this confusion, the effect of dexamethasone in each of the studies in airway smooth muscle was complete abolition of PGE₂ production, COX activity, and COX-2 induction (47, 55).

Effects of Glucocorticoids on Cytokine Production

Other studies of human airway smooth muscle stimulated with pro-inflammatory cytokines have shown increased expression and release of RANTES (56), IL-8 (57, 58), eotaxin (56, 59), and other cytokines, such as IL-6 (60). RANTES, IL-8, and eotaxin are important chemokines for activation of eosinophils, critical effector cells in the pathogenesis of asthma. RANTES is a potent chemoattractant for eosinophils as well as for other cell types observed in allergic inflammation, including monocytes and memory T lymphocytes. IL-8, in addition to its action on neutrophils, is also a potent eosinophil chemoattractant. Eotaxin, however, is a highly selective chemoattractant for eosinophils. Production by airway smooth muscle of RANTES, IL-8, and eotaxin implies a role for these structural cells to participate directly in the inflammatory process through recruitment and activation of eosinophils and neutrophils in the airways. In addition to recruitment of eosinophils by chemoattractants, enhanced survival of infiltrating eosinophils is also thought to contribute to airway inflammation in asthma (61). In our own studies using conditioned medium from human airway smooth muscle cells stimulated with IL-1 or TNF- and subsequently examined on eosinophil survival in vitro, we have found that cultured human airway smooth muscle cells stimulated with IL-1, but not TNF-, produce an eosinophil survival-enhancing activity that is functionally indistinguishable from granulocyte macrophage colony-stimulating factor (GM-CSF) (62).

Pretreatment of cytokine-treated airway smooth muscle cells with dexamethasone resulted in reduced expression of the RANTES gene and inhibition of its protein product (56), indicating that dexamethasone produced its effect at the level of gene transcription, as has been reported for this and other glucocorticoids on cytokine gene expression in other cell systems. Similarly, dexamethasone is reported to inhibit production of IL-8 from human airway smooth muscle cells stimulated with either TNF- or IL-1 (58), and in our own studies was found to abolish IL-1-stimulated GM-CSF production from these cells (62). These findings are consistent with those of another study of human airway smooth muscle cells showing that dexamethasone inhibited the GM-CSF production stimulated by a mixture of cytokines (63).

	CONCLUSIONS

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Changes in airway smooth muscle contractility and proliferation are implicated in the development of poorly reversible airways obstruction and bronchial hyperresponsiveness in asthma. Glucocorticoid therapy offers some clinical improvement in airway function, but the mechanisms by which glucocorticoids improve bronchial hyperresponsiveness are incompletely understood. It is becoming clear that airway smooth muscle can potentially contribute to the pathogenesis of asthma through increased contraction and proliferation, and perhaps by expressing and secreting a number of pro-inflammatory cytokines and mediators that in turn may lead to the activation and recruitment of key inflammatory cells in the airways. Glucocorticoids, either directly or indirectly, modulate these diverse functions of smooth muscle by inhibiting specific elements of the cellular processes in airway smooth muscle that otherwise lead to contraction, proliferation, and secretion of specific cytokines. As we begin to understand and speculate more about the likely role of airway smooth muscle and the diversity of its function in the pathogenesis of asthma, it may be necessary to reconsider airway smooth muscle as an important cellular target for the action of glucocorticoids in the treatment of asthma.