Is Airway Smooth Muscle the “Missing Link” Modulating Airway Inflammation in Asthma?^* FREE TO VIEW

Asthma, a chronic disease with a prevalence of 3 to 5% in the United States, manifests as airway hyperresponsiveness and inflammation. The precise mechanisms responsible for inducing airway inflammation and hyperresponsiveness in asthma patients remain elusive. Despite considerable efforts to characterize airway inflammation using measurements of bronchoalveolar levels of cytokines, chemokines, and inflammatory mediators, compelling evidence suggests that these inflammatory biomarkers may not correlate with asthma severity or with the degree of airway hyperresponsiveness. Indeed, the paradigm of a T-helper cell type 1-T-helper cell type 2 imbalance promoting the asthma diathesis may be simplistic. Several studies¹² in subjects with asthma as well as in murine models of allergen-induced airway hyperresponsiveness have suggested that airway inflammation may not correlate with airway hyperresponsiveness. Few will refute that asthma, in part, is a disease of airway inflammation, especially since anti-inflammatory drugs such as inhaled steroids are so therapeutically effective. New evidence, however, suggests that other airway compartments may play a role in the modulation and orchestration of airway inflammation. Attention has focused on the characterization of whether structural cells of the airway, namely, epithelium, myofibroblasts, and smooth muscle, may modulate airway inflammation. This review provides an overview of recent evidence suggesting that airway smooth muscle (ASM) may regulate airway inflammation and gives insight into new therapeutic targets in treating asthma. The immunomodulatory function of ASM will be addressed according to cell adhesion molecule (CAM) and chemokine expression, Toll-like receptor (TLR) activation (Fig 1 ), and the response of ASM to therapy with glucocorticoids (GCs) [Fig 2 ].

CAMs mediate leukocyte-endothelial cell interactions regulating cell recruitment and homing. New evidence³ suggests that infiltrating inflammatory cells interact through specific CAMs and bind to airway structural cells that ultimately perpetuate airway inflammation. Early studies⁴ suggested that anti-intercellular adhesion molecule (ICAM)-1 antibodies inhibited allergen-induced airway hyperresponsiveness and inflammation in mice. The mechanisms by which CAMs altered airway inflammation remained unclear. More recently, a focus on CAMs on ASM suggested that specific CAMs are expressed in ASM both in vivo and in vitro, and mediate cell-cell contact among leukocytes, mast cells, and ASM.

In situ hybridization and immunohistochemical analyses of lung tissue revealed the in vivo expression of CAMs in ASM.⁵⁶ Specifically, after lipopolysaccharide (LPS) stimulation of rat lungs, ASM markedly enhanced ICAM-1 expression both at the protein and messenger RNA levels.⁵ Using in vivo human bronchial tissue transplanted onto the flank of severe combined immunodeficiency (or SCID) mice, Lazaar and colleagues⁶ demonstrated a marked increase in ICAM-1 and vascular CAM (VCAM)-1 expression after the injection of tumor necrosis factor (TNF)-α, a cytokine that is produced in considerable quantities in asthmatic airways,⁷ into the bronchial lumen of the grafted tissue. Further in vitro studies confirmed the expression of ICAM-1 and VCAM-1 on cultured ASM that was inducible by a wide range of inflammatory mediators such as TNF-α, interleukin (IL)-1β, or interferon (IFN)-γ.⁶⁸ Although the function of CAMs in ASM remains incompletely defined, investigators⁶⁹¹⁰¹¹ have clearly demonstrated that the surface expression of CAMs in ASM may be pivotal in regulating ASM cell interactions with a variety of inflammatory cells relevant for the pathogenesis of asthma. Studies⁶ have suggested that activated T cells avidly adhere to cultured ASM, an interaction that is mediated through ICAM-1, VCAM-1, and CD44 cells. The latter interaction enhances T-cell binding that increases bronchoconstrictor responses to acetylcholine and impairs relaxation responses to isoproterenol.⁹ More recently, investigators¹² demonstrated that CD4⁺ T cells interact with ASM in vivo. Using adoptive transfer of CD4⁺ T cells from sensitized rats, there is a marked increase in the proliferation and inhibition of apoptosis of airway myocytes in naïve recipients on repeated allergen challenge. The consequence of the enhanced proliferation in ASM in vivo is an increase in ASM mass and airway hyperresponsiveness. Additionally, genetically modified CD4⁺ T cells expressing enhanced green fluorescent protein were localized by confocal microscopy to be juxtaposed to the ASM. This finding is clinically relevant and implies that CD4⁺ T cells may directly modulate ASM function through cell-cell interactions in vivo.¹² Furthermore, other inflammatory cells including eosinophils¹⁰ and recently neutrophils¹¹ also adhere to ASM in vitro. The attachment of such cells to ASM was inhibited by the presence of anti-ICAM-1 and VCAM-1 antibodies. Further, important studies¹³ showing mast cell-ASM interactions in vivo in subjects with asthma clearly have demonstrated that cell-cell attachment may modulate and alter stromal cell function. Collectively, these studies have suggested that direct interactions among leukocytes, inflammatory cells, and ASM via CAMs can directly contribute to the pathogenesis of asthma and, as such, therapeutic targets that will disrupt cell-cell adherence may provide new approaches to bronchodilatation while inhibiting the perpetuation of airway inflammation in the submucosa.

Chemokines play a central role in the recruitment and trafficking of inflammatory cells along diffusion gradients. After the initiation of injury or inflammation, chemokines provide the diffusion gradient for cell trafficking.¹⁴ Chemokines can be categorized on the basis of their molecular structure and exhibit a degree of selectivity for distinct inflammatory cell populations.¹⁵ For example, eotaxin; regulated on activation, normal T cells expressed and secreted (RANTES); and IL-5 primarily recruit eosinophils, although both eotaxin and RANTES have actions on other cell types. CXC chemokine ligand (CXCL)-8 markedly recruits neutrophils; monocyte chemotactic proteins (MCPs) recruit monocytes; thymus-regulated and activation-regulated chemokine recruits lymphocytes; and stem cell factor recruits mast cells. Many of the aforementioned chemokines, which act to recruit and activate leukocytes, are found in the BAL fluid and lung tissue of subjects with asthma. Using murine models of allergen-induced airway hyperresponsiveness, investigators¹⁶ showed that neutralizing MCP-5, eotaxin, RANTES, and MCP-1 dramatically reduced bronchial hyperresponsiveness as well as leukocyte migration. Others¹⁷ have shown that intranasal delivery of a recombinant poxvirus-derived viral CC-chemokine inhibitor protein dramatically improves pulmonary function and decreases inflammation of the airway and lung parenchyma. Recently, in a chronic allergen exposure murine model,¹⁸ the administration of CCR3 antagonist, a premier chemokine receptor involved in eosinophil infiltration, reduced eosinophil numbers in the airway wall tissue that was accompanied by a decrease in airway remodeling parameters. Together, these studies have demonstrated that in vivo chemokines promote and perpetuate airway inflammation during allergen exposure.

Although a variety of cells secrete chemokines, new evidence suggests that ASM may be a prominent source of chemokines in the submucosa. Immunohistochemical and in situ hybridization studies revealed that MCP-1, RANTES, and fractalkine are expressed in ASM bundles of bronchial biopsy specimens in subjects with asthma.¹⁹²⁰²¹ CXCL10, a potent chemokine for activated T cells, natural killer cells, and mast cells that bind to CXC chemokine receptor-3, is also expressed in ASM in subjects with asthma or COPD.²²²³ The concomitant expression of CXCL10 in ASM cells and CXC chemokine receptor-3 (the CXCL10 receptor) in mast cells was also found in ASM of subjects with asthma.²² In murine models of allergen-induced airway hyperresponsiveness, eotaxin, an eosinophil-specific chemokine mediator, is markedly expressed in ASM tissue.²⁴ The expression of chemokine receptors also exists in ASM, as has been demonstrated in subjects with asthma who express strong immunoreactivity for the eotaxin receptor (CCR3),²⁵ which has been previously linked to the pathogenesis of asthma.²⁶

To further understand the mechanisms by which chemokines are expressed, in vitro studies have demonstrated that, in response to specific inflammatory mediators, cultured ASM cells also express and secrete a variety of chemokines such as eotaxin, RANTES, CXCL8, MCP-1, MCP-2, MCP-3, and thymus-regulated and activation-regulated chemokine.²⁷ Although the precise physiologic relevance of chemokine receptor expression in ASM remains unclear, there is no doubt that the chemokine level increases in BAL fluid in subjects with asthma, and it is likely that, in part, the increased levels are mediated by ASM. The identification in 2002¹³ of the infiltration of mast cells into ASM bundles may suggest that mast cells migrate via the diffusion gradient of chemokines traffic to the submucosa. Activated ASM supernatant from subjects with asthma exhibits chemotactic activity for purified lung mast cells and subsequently elicits their migration toward ASM. The precise mechanisms by which this occurs remain unclear but can serve as a new therapeutic target in decreasing the airway infiltration of immunocytes and inflammatory cells in subjects with asthma. Importantly, blocking CXCL10 decreased mast cell migration into the ASM bundles.²² In parallel studies, El-Shazly and colleagues²⁰ demonstrated that fractalkine also mediates ASM-induced mast cell chemotaxis.

Collectively, compelling evidence suggests that ASM may serve as a source of chemokines in allergen-induced airway inflammation that ultimately recruits and retains inflammatory cells and may amplify proinflammatory markers. These new findings may provide unique therapeutic targets to decrease cell migration/infiltration, and ultimately reverse either airway remodeling or ongoing airway inflammation.

TLRs are molecules that recognize bacterial and viral components, and evoke inflammatory responses. Studies²⁸ have demonstrated that airway infections contribute to exacerbations of asthma. Such seminal observations prompted investigators to study whether activation of TLRs in the airways promotes airway inflammation. Several TLR and TLR ligands are associated with asthma.²⁹³⁰ Additionally, TLR function in ASM suggests that LPS, a major component of the membrane of Gram-negative bacteria, promotes airway hyperresponsiveness to contractile agonists.³¹

To date, 11 known TLRs that recognize different pathogen-associated molecular patterns have been identified. Although most of the work in this field has used cultured ASM cells, evidence suggests that most TLR isoforms at the messenger RNA level (TLR-1 to TLR-10) are expressed in muscle. At the protein level, there is a predominance of TLR-3 expression rather than of other receptors on the ASM.³² The expression of TLRs in cultured ASM is significantly up-regulated by inflammatory mediators such as TNF-α, IL-1β, and IFN-γ.³² The activation of TLR-2 receptor with Pam₃CSK₄ or PGN,TLR-3 receptor with polyriboinosinic polyribocytidylic acid (poly[I:C]), or double-stranded RNA, and the TLR-2/4 receptor with LPS dramatically increased the expression of proinflammatory mediators such as cytokines (ie, IL-6) or chemokines (ie, eotaxin, CXCL8, and CXCL10), as well as adhesion molecule expression (ie, ICAM-1).³²³³³⁴ Collectively, these studies demonstrate that TLRs modulate ASM at least in cultured cells.

In vivo studies using immunohistochemistry as well as reverse transcription-polymerase chain reaction analyses demonstrate that TLR-2, TLR-3, and TLR-4 are expressed in ASM from murine lungs.³⁵ The treatment of murine tracheal tissue with TLR-2/TLR-4 agonist (LPS) or TLR-3 agonist (poly[I:C]), enhance bradykinin-induced contractions. Simultaneous LPS and poly(I:C) treatment synergistically enhanced bradykinin-induced contraction.³⁵ Further, in a chronic model of ovalbumin-sensitized and ovalubumin-challenged rats, treatment with a TLR-7/TLR-8 ligand prevented the increase in ASM mass associated with airway remodeling after long-term allergen exposure.³⁶ Collectively, ASM expresses TLRs, and the activation of these specific receptors induces cytokine secretion and enhances bronchomotor responses to agonists. Whether these receptors are activated directly by viruses or bacteria or are adjuvantly activated through the ubiquitous exposure of LPS remains unclear. No doubt, however, as TLR antagonists are developed, it is likely that the effects of blocking TLRs directly on the muscle may mediate the antiinflammatory effects observed in asthma.

Inhaled steroids remain the cornerstone in the management of asthma. Most evidence suggests that inhaled steroids indirectly modulate inflammatory cytokine secretion and leukocyte transmigration into BAL fluid. The association of these changes with diminished airway hyperresponsiveness suggested that steroids may have a direct effect on the airway inflammatory diathesis. Despite these observations, the precise cells that are affected by GCs in asthma remain unclear. GCs mediate their effects through binding to the GC receptor (GR)-α isoform that suppresses the expression of inflammatory genes through mechanisms known as transactivation or transrepression.³⁷ Transactivation occurs via the direct binding of activated GR-α to DNA sequences termed GC-responsive elements that are present on the promoter of steroid-inducible genes. Transrepression defines a direct interaction of activated GR-α with different transcription factors, such as nuclear factor κB and activated protein-1, thus repressing the expression of proinflammatory genes.³⁸ The single GR gene found in humans, however, undergoes alternative splicing that can yield another GR isoform termed GR-β.³⁹ This isoform may act as a dominant negative inhibitor of steroid action and has been associated with steroid resistance in different inflammatory diseases.⁴⁰

Whether ASM is the target for steroids in patients with asthma remains controversial. Several in vitro studies^414243444546 have confirmed that GCs are effective in suppressing the induction of inflammatory genes including IL-6, eotaxin, RANTES, granulocyte macrophage-colony-stimulating factor (GM-CSF), ICAM-1, and CD38. Further, in situ hybridization and immunohistochemical studies⁴⁷ have revealed the in vivo expression of GR in ASM in both healthy subjects and those with asthma. Therefore, GCs, which are the most prominently used antiinflammatory agents for the treatment of asthma, may have a direct effect on ASM. Investigators⁴⁸ have shown that GC treatment induced GR nuclear translocation in bronchial smooth muscle cell lines from healthy subjects or subjects with asthma and those with emphysema. The activation of GR by GC through its binding to GC-responsive element DNA sequences has also been demonstrated in ASM.⁴⁴⁴⁸⁴⁹ Further, under certain inflammatory conditions, GR-β, the dominant negative receptor, can be expressed and inhibits the effects of steroids.⁴⁴

Although steroid effects on human ASM have been investigated,⁵⁰ their immunomodulatory effects on gene expression are complex and poorly understood. The effects of steroids appear to be gene-specific. For example, dexamethasone effectively inhibits cytokine-induced IL-6, RANTES, or eotaxin expression, while the same steroids have little effect on cytokine-induced ICAM-1 expression.⁴¹⁴²⁴³ Second, steroid-suppressive effects are time-dependent since dexamethasone partially abrogates cytokine-induced ICAM-1 expression at early time points, but has no effect at later time points.⁴² Finally, steroid inhibitory effects are also stimuli-specific. While dexamethasone significantly inhibits IL-1β-induced GM-CSF secretion, it only partially inhibits GM-CSF secretion induced by thrombin.⁴⁵

Using allergen-induced airway hyperresponsiveness in mice, GC treatment significantly reduced allergen-induced increases in peribronchial collagen deposition and levels of total lung collagen but failed to reduce allergen-induced increases in the thickness of the peribronchial smooth muscle.⁵¹ Similarly, Roth and colleagues⁴⁸ showed that GC failed to inhibit proliferative responses in bronchial smooth muscle from subjects with asthma. Further in vitro studies showed that the antiproliferative and antimigratory effects of GCs were impaired by the contact of ASM with collagen, but not with laminin,⁵² suggesting that in a collagen-rich microenvironment of inflamed and fibrotic airways, ASM may contribute to steroid resistance. Additionally, investigators⁴⁴ have shown that steroid-suppressive effects are dramatically impaired in ASM treated with the specific combination of TNF-α and IFN, but not with IL-1β or IL-13.

Collectively, these findings suggest that ASM may be a target for GC treatment. Exciting new evidence⁴⁴ suggests that combinations of cytokines, namely, TNF-α and IFN, may induce a transient steroid-resistant state. Even though steroids can inhibit the effects of TNF-α or IFN alone on ASM, the combination appears to promote a steroid resistance. The clinical importance of these molecular studies is demonstrated in Figure 2. It is commonly recognized by clinicians that patients will have exacerbations of their asthma that are typically due to potentially poor adherence to the inhaled steroids. The aforementioned studies suggest a new paradigm whereby the combinations of cytokines specifically released after ozone exposure or viral infections, namely, TNF-α and IFN, may induce a transient steroid-resistant state specifically in stromal cells that renders GCs ineffective. It is possible that over time with the resolution of the ozone exposure or the viral infection, GC sensitivity is reestablished with the decrement in TNF-α and IFN levels and thus the steroids may again be effective in abrogating allergen-induced airway inflammation. Further in vivo studies are needed to confirm this potentially important new observation and to develop new antiinflammatory approaches that are not inhibited in these states.

ASM, the most important cell in regulating bronchomotor tone, may serve another role in the promotion and orchestration of airway inflammation. The recognition that ASM and other structural cells may be important modulators of inflammation may provide new therapeutic targets in the treatment of asthma. Specifically, the recognition that in certain environments transient GC resistance occurs may lead to a better understanding and new therapies in the development of nonsteroidal antiinflammatory agents.