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The immuno-modulatory properties of airway smooth muscle have become of increasing importance in our understanding of the mechanisms underlying chronic inflammation and structural remodeling of the airway wall in asthma and chronic obstructive pulmonary disease (COPD). ASM cells respond to many cytokines, growth factors and lipid mediators to produce a wide array of immuno-modulatory molecules which may in turn orchestrate and perpetuate the disease process in asthma and COPD. Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified intracellular signaling pathways by which cytokines modulate or induce these cellular responses. In this review we provide an overview of the transcriptional mechanisms as well as intracellular signaling pathways regulating cytokine functions in ASM cells. The recent discovery of toll-like receptors in ASM cells represents a significant development in our understanding of the immuno-modulatory capabilities of ASM cells. Thus, we also review emerging evidence of the inflammatory response to toll-like receptor activation in ASM cells.
Cytokines and chemokines play a central role in regulating inflammatory and immune responses in chronic lung diseases such as asthma and COPD. Indeed, in vivo studies using selective inhibitors as well as neutralizing antibodies against various cytokines and chemokines demonstrate their importance in antigen-induced airway inflammation (leukocyte infiltration) and hyper-responsiveness in animal models of asthma [1-3]. Studies in knock-out or transgenic mice also illustrate the importance of cytokines in the abnormal airway changes induced by allergen challenge in sensitized animals . A potential site for the deleterious action of many cytokines in airways disease is the airway smooth muscle a primary effector tissue historically thought to only regulate bronchomotor tone. In human cultured ASM cells that retain physiological responsiveness, cytokines alter pro-inflammatory gene expression that in turn may play an important role in the pathogenesis of chronic inflammatory airways disease . Despite numerous studies of the cellular effects of cytokines on cultured ASM, few have identified downstream signaling cascades by which cytokines modulate or induce these cellular responses. In this review we discuss the role of three major intracellular signaling pathways: Mitogen-Activated Protein Kinase (MAPK), Nuclear Factor-kappa B (NF-κB), and Janus kinases and Signal Transducers and Activators of Transcription (STATs) in regulating cytokine functions, with a particular focus on inflammatory gene expression, in regulating ASM functions.
The capacity for ASM cells to respond to numerous cytokines has revealed the extensive immune-regulatory potential of these cells. In response to cytokines such as IL-1β, TNF-α and IFN-γ, ASM cells can be induced to express a host of cell-adhesion and co-stimulatory molecules that allow interactions between the ASM and inflammatory cells that infiltrate the airways. Moreover, ligation of ASM cell-surface molecules such as CD40 and OX40L by their respective counter-ligands leads to activation of ASM inflammatory responses. Further advances in understanding the immune-regulatory potential of ASM have come with the discovery that cytokines also up-regulate the expression of multiple toll-like receptors (TLRs) in ASM cells. These latter receptors are pattern-recognition receptors that mediate innate and adaptive immune and inflammatory responses to microbial infection, tissue injury or inflammation. Emerging evidence now suggests a role for TLRs in the development, perpetuation and exacerbation of chronic inflammatory airway disease . Thus, we also discuss the potential role of TLRs in the amplification of ASM inflammatory responses.
The MAPK signal transduction pathway consists of MAPK, MAPK kinase (MEK, MAPKK, or MKK), and MAPK kinase kinase (MEKK, MAPKKK, or MKKK). The MAPK cascade activation occurs by sequential phosphorylation of Thr-X-Tyr motifs. In mammalian cells, there are five distinct subfamilies including extracellular signal-regulated kinase (ERK), p38 MAPK (p38), c-Jun N-terminal kinase (JNK), ERK3/4 and ERK5. Among the five distinctive MAPK pathways, ERK, p38 MAPK and JNK have been extensively studied in ASM cells  (Figure 1).
ERK signaling induces downstream activation of different intracellular transcription factors such as Elk-1, c-fos, c-myc, Sap-1, and Tal, and consequently modulates DNA synthesis and cell proliferation . In ASM, activation of ERK signaling is elicited by various stimuli including platelet derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), endothelin-1 (ET-1), thrombin, oncostatin M, leukemia inhibitory factor (LIF), insulin-like growth factor I, and 5-hydroxytryptamine [9-14]. Cytokines are also important activators of ERK signaling. Phosphorylation of ERK1/2 by IL-1β leads to production of numerous inflammatory mediators including prostaglandin-E2 (PGE2), eotaxin, RANTES, and GM-CSF . ERK is also involved in mediating ASM eotaxin and IL-8 release in response to Th2 cytokines (IL-4, IL-9, IL-13) and the Th17 cytokine IL-17 [16-18]. The interleukin-17B receptor (IL-17BR) is also up-regulated in ASM cells in an ERK-dependent manner .
p38 signaling is activated in response to physical and chemical challenges including oxidative stress, UV irradiation, hypoxia, ischemia as well as various cytokines [20, 21]. The down-stream effectors of this cascade are transcription factors such as Elk-1, Sap-1, ATF-2, CREB, CHOP, and Max. p38 mediates bFGF-induced ASM proliferation  and ASM inflammatory gene expression in response to multiple stimuli. Indeed, p38 MAPK mediates IL-17A induced IL-6, IL-8 and eotaxin secretion [23-26] as well as bradykinin induced IL-6 secretion . Although there are no published reports of the MAPKs regulating IL-5 secretion, p38 regulates expression of the IL-5 receptor (IL-5R) in response to IL-1β, TNFα and IFN-γ . p38 MAPK appears to have both positive and negative regulatory effects on cytokine-induced inflammatory responses in ASM; it acts to augment TNF-α-induced IL-6 and RANTES release and IL-1β-induced eotaxin release, but inhibits TNF-α induced ICAM-1 expression and IL-1β induced GM-CSF release [15, 29]. This suggests a gene specific role of p38 MAPK in regulating specific transcriptional outcomes.
We recently made the novel finding that, under basal conditions, p38 negatively regulates IFN-β promoter activity (Damera et al., unpublished data). In line with this, treatment of ASM cells with the p38 inhibitor SB203580 showed a specific reduction in tonic p38 activity and enhanced IFN-β transcription and protein secretion. Functional studies using an IFN-β neutralizing antibody reversed the inhibitory effect of SB203580 on TNF-α-induced IL-8 secretion, indicating an important role of autocrine IFN-β in regulating p38-dependent inflammatory responses.
JNK signaling is activated by environmental stress, pro-inflammatory cytokines and genotoxic agents. Following activation of JNK, three Jun transcription factors (JunB, c-Jun and JunD), which are all members of the AP-1 family, are activated . These transcription factors modulate gene expression responsible for many biological responses, including migration, proliferation, differentiation and cell death . In murine studies, administration of the JNK inhibitor SP600125 after allergen challenge prevents T cell-mediated inflammation and ASM cell proliferation, indicating a role for JNK signaling in allergic airway inflammation and remodeling . Studies using the JNK inhibitor SP600125 implicated JNK in the regulation of IL-1β- and TNF-α-induced RANTES, GM-CSF, and IL-8 secretion in ASM cells . IFN-γ and TNF-α induced fractalkine expression also occurs through JNK dependent mechanisms .
While unique stimuli initiate the majority of cellular responses by specific signaling cascades, it is not uncommon to derive such responses by multiple and parallel signaling cascades. Exogenous addition of TGF-β1 to ASM cultures increases [3H]-thymidine incorporation and ASM cell proliferation via ERK, p38 and JNK-dependent pathways . Similarly, TNF-α-mediated induction of CD38, a potent modulator of calcium homeostasis and ASM tone, involves all MAPK cascade components . The induction of matrix metalloproteinase-9 (MMP-9) expression by cytokines also involves active participation of several MAPK pathways [37, 38]. Interestingly, in some instances, the induction of one MAPK pathway may antagonize another. Indeed, LPS-induced activation of p38 MAPK down-regulates changes in ASM responsiveness and IL-6 secretion associated with ERK1/2 activation .
Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed transcription factor that mediates the expression of many inflammatory mediators, including cytokines, adhesion molecules, chemokines, and growth factors . NF-κB-dependent pro-inflammatory genes are believed to play a central role in a variety of inflammatory diseases including chronic inflammatory airway diseases such as asthma. Increased markers of NF-κB pathway activity have been demonstrated in the airways of, or samples from, asthma patients [41-45] as well as in rodent models of asthma [46-49]. For this reason, the NF-κB signalling pathway is an attractive target for novel asthma therapies. Indeed, studies have shown that targeting NF-κB, using various molecular methodologies, inhibits aspects of the allergic response in rodent models of asthma [50-56].
NF-κB is activated in response to a number of stimuli, including physical and chemical stress, lipopolysaccharide (LPS), double-stranded RNA, T- and B-cell mitogens and proinflammatory cytokines . NF-κB induced gene expression is controlled by a complex series of enzymatic signalling events at multiple levels. An overview of the NF-κB activation cascade is depicted in Figure 2.
NF-κB is made up of a hetero- or homodimer of members of the DNA-binding Rel family of proteins which contains five known mammalian members: p50 (NF-κB1, precursor of which is p105), p65 (Rel A, NF-κB3), p52 (NF-κB2, precursor of which is p100), c-Rel and Rel B. The p65 and p50 subunits are ubiquitously expressed, whereas p52, c-Rel and Rel B are restricted to specific differentiated cell types . In resting cells, the majority of NF-κB is bound to I-κB inhibitory protein, which holds the complex in the cytoplasm. Upon cellular stimulation, the I-κB protein is phosphorylated, ubiquinated, and degraded by the proteosomal pathway. With the I-κB removed, NF-κB translocates to the nucleus and mediates gene transcription 
I-κB phosphorylation and activation of Rel proteins can occur via the classical (canonical) or non-classical (non-canonical) pathway. In the classical pathway, a critical phosphorylation of the I-κB protein is performed by the I-κB kinase (IKK) complex, which consists of at least three subunits, including two catalytic subunits IKK-α and -β, also known as IKK-1 and -2, and one regulatory subunit IKK-γ (also known as NEMO) . Of the two catalytic subunits, IKK-β is 20 fold more active than IKK-α in the phosphorylation of I-κB . It is also thought that IKK-β, not IKK-α, is critical for NF-κB activation [61-64] and hence attempts to target this pathway for therapeutic intervention have focused on inhibitors of this subunit [65, 66]. Stimuli of the classical pathway include the TLR/IL-1R family members, ligation of the T-cell receptor (TCR), and TNFR signalling (Figure 2). IKK-2 has been shown to be critical in NF-κB activation in ASM cells [67, 68].
In addition to the classical pathway, an alternative (non-canonical) pathway has been described mainly in B cells. This latter pathway can be activated by different stimuli such as lymphotoxin β, CD40 ligand, and receptor activator of NF-κB ligand [69, 70] The alternative NF-κB pathway is characterised by the inducible phosphorylation and processing of p100 to p52, and subsequent nuclear translocation of the heterodimer p52:Rel B is independent of IKKγ and IKKβ and only requires the IKKα subunit . This pathway is believed to play key roles in adaptive immunity .
The NF-κB pathway can be further controlled by post translational modifications, including the modulation of Rel protein interactions with other components of the transcriptional machinery. Altered activation of NF-κB can occur via its phosphorylation status, for example the phosphorylation of p65 enhances transcription, yet phosphorylation of p105 can reduce its processing into p50 and hence reduce activation . Acetylation of the Rel proteins also play a key role [74, 75]. Additionally, covalent modifications of the chromatin environment which regulates the access of transcription factors to gene promoters alter NF-κB-dependent transcription. This control is achieved by recruitment of protein complexes that alter chromatin structure via enzymatic modifications of histone tails and/or nucleosome remodelling. NF-κB activation requires several cofactor histone acetyltransferases, including CBP, p300, p/CAF, and SRC-1, of which p/CAF appeared to be relatively more important [74, 76].
A multitude of studies in ASM cells implicate a role for NF-κB in the regulation of inflammatory chemokines, cytokines, and adhesion molecules. Indeed, NF-κB is involved in IL-17-induced IL-8 release [23, 77]; IL-1β and TNF-α-induced GRO-α release ; neutrophil-derived elastase-induced TGF-β expression ; in the expression of cell adhesion molecules such as ICAM-1 and VCAM-1 induced by TNF-α, IL-1β and LPS [80-82]. As stated above, IKK-2 plays a crucial role in the classical NF-κB pathway and for this reason there has been considerable interest in studying and developing ways to manipulate this kinase in order to identify new therapeutics for the treatment of asthma. Data from ASM cells demonstrate that inhibition of IKK2 using the small molecule inhibitors TPCA-1, PS-1145 and ML120B, or molecular intervention using adenoviral approaches to knock down IKK2, demonstrate a role for this kinase in the expression of ICAM-1, cyclooxygenase-2, IL-6, IL-8, GM-CSF, RANTES, monocyte chemotactic protein-1 (MCP-1), GRO-α, neutrophil-activating protein-2 (NAP-2), and epithelial neutrophil activating peptide 78 (ENA-78), some of which are upregulated and play a role in asthma pathogenesis [67, 68]. Similarly, in rodent models of asthma, modulation of IKK-2 using parallel molecular techniques, have shown positive disease modifying data [83-86]. These data suggest that inhibition of IKK2 and hence the NF-κB pathway may have therapeutic implications for asthma treatment.
Of interest, TNF-α but not IL-1β activation of NF-κB signaling involves recruitment of the downstream transducer protein TRAF2 by TNF-α receptor 1 (TNFR1) via the receptor-associated death domain protein, TRADD [87, 88]. Similar findings were also reported in ASM cells from guinea pigs where TNFR1 activation with agonistic antibodies also induced NF-κB activation .
Recent work investigating pro-inflammatory stimuli on NF-κB activity with regard to phosphorylation and chromatin remodeling in ASM cells has emerged. TNF-α has been reported to phosphorylate both IKK-β  and the p65 subunit at Ser276 and Ser536 in ASM cells . In the latter study, the authors also demonstrated that TNF-α recruits the histone acetyl-transferase p/CAF to the CCL-11 (eotaxin) promoter to increase NF-κB mediated transactivation of this gene . p300/CBP acetylation is also required for NF-κB mediated TNF-α-induced VCAM-1 and ICAM-1 induction in ASM cells [80, 92, 93].
The classical components of the IFN signaling cascade include the Janus tyrosine kinases and signal transducers and activators of transcription (STATs) factors. Activation of each IFN receptor complex stimulates different receptor-associated tyrosine kinases, namely, JAK1 and Tyk2 by IFN-α/β (type I), or JAK1 and JAK2 by IFN-γ (type II) . JAKs-mediated phosphorylation of STAT proteins results in STAT assembly in dimeric or oligomeric forms, which translocate to the nucleus, where they can regulate gene expression via DNA binding motifs called either γ-activated sequence (GAS) elements (recognized by STAT1 homodimers) or IFN-stimulated response element (ISRE, recognized by STAT1-STAT2 heterodimers) [95, 96]. Up-regulation of STAT1 and STAT1-dependent genes such as ICAM-1 and IFN regulatory Factor-1 (IRF-1) are observed in asthmatic airways suggesting the potential contribution of IFN-associated JAK/STATs in the regulation of immuno-modulatory genes associated with asthma .
IFNs regulate many cellular responses in human ASM cells: IFN-γ induces the expression of ICAM-1 and VCAM-1 , the CysLT1 receptor  and the secretion of nerve growth factor in ASM cells . IFN-γ also synergizes with TNF-α to augment expression of CD38  and several chemokines including RANTES, IP-10 and fractalkine [34, 102, 103]. Most studies that used a combination of IFN-γ and TNF-α showed that the synergistic action involves several molecular mechanisms. In some instances, their co-operativity may be explained by the IFN-γ-induced up-regulation of TNF-α receptors  or vice-versa  (Figure 3). Furthermore, both cytokines may collaborate at the gene level by increasing promoter activation through a synergistic interaction between transcription factors activated by IFN-γ (STATs, IRF-1) and TNF-α (NF-κB) [106, 107] (Figure 3). These amplifying properties of IFN-γ may explain, at least in part, why viral infection, which increases production of IFNs, is an important trigger for asthma and chronic obstructive pulmonary disease exacerbation . Another mechanism of co-operation could be secondary induction of IFN-β, which has been shown to mediate TNF-α induced RANTES and CD38 expression [101, 109] (Figure 3) (see below).
In some instances, however, IFNs may antagonize TNF-α inflammatory responses by inhibiting the NF-κB pathway. Indeed, Keslacy and colleagues recently reported that IFN-γ potently inhibits TNF-α-induced NF-κB-dependent genes including IL-6, IL-8 and eotaxin in ASM cells . Multiple mechanisms underlying IFNs inhibitory effect on NF-κB pathways have been proposed including inhibition of NF-κB DNA binding, prevention of IκB degradation, or regulation of TNF-α receptor 1 via STAT interaction . Specifically, in ASM cells, IFN-γ inhibits the transcriptional activity of NF-κB by reducing the acetylation level of p65 .
In ASM cells, TNF-α is able to activate JAK1 and Tyk2, and STAT1- and STAT2-dependent gene expression via the autocrine action of IFN-β . Indeed, autocrine IFN-β regulates i) TNF-α-induced inflammatory gene expression, by suppressing IL-6 and promoting RANTES secretion and ii) TNF-α-associated airway hyper-responsiveness, by potentiating the ability for TNF-α to enhance GPCR-dependent contractile responses [111, 112].
The putative implication of IFN-β in lung diseases is supported by the heightened expression of IFN-β in the airways in mouse models of allergic asthma . We therefore propose that the functional cross talk between type I and II IFNs and TNF-α in lung structural cells, particularly the ASM, is a novel axis in the pathogenesis of lung diseases, although a similar phenomenon could also occur in other cell types (such as hemopoietic cells). A recent study by Ivashkiv and colleagues recently confirmed the inflammatory potential of IFN-β/TNF-α interaction in macrophages . This elegant study showed that IFN-β-mediated autocrine loops were essential for maintaining TNF-α-induced inflammatory genes that prime macrophages for augmented responses to additional stimulation by cytokines and toll-like receptors agonists. In previous studies performed in 3T3-L1 adipocytes, TNF-α was shown to induce phosphorylation of STAT1 by directly interacting with both JAK1 and JAK2 , whereas in Hela cells, STAT1 was shown to physically interact with TNFR1 and the adaptor proteins TNF receptor-associated death domain (TRADD), but not TRAF-2 . TNF-α also induces STAT1 phosphorylation at serine 727 in macrophages.
Most anti-inflammatory effects of steroids are mediated via the glucocorticoid receptor alpha isoform (GRα), which suppresses expression of inflammatory genes through mechanisms known as transactivation or transrepression . As a result of alternative splicing mechanisms, another glucocorticoid receptor isoform, namely GRβ, has been described . We and others recently showed that treatment of ASM cells with the specific combination of IFNs with TNF-α impairs the ability of steroids to inhibit the expression of various proinflammatory genes such as CD38, RANTES and ICAM-1 by a mechanism involving the up-regulation of GRβ isoform . Interestingly, steroids augment IFN-γ/TNF-α induced fractalkine and TLR2 expression in ASM [34, 119]: whether this involves similar mechanisms involved in the attenuation of corticosteroid activity by IFN-γ/TNF-α remains to be established. Although the pathological role of the GRβ isoform is not well understood, previous reports demonstrate a strong correlation between steroid resistance in individuals with asthma and the expression levels of GRβ . More importantly, increased GRβ in the airways has been detected in patients who died of asthma . Indeed, by its ability to act as a dominant-negative inhibitor of steroid action in other cell types , GRβ has been associated with steroid resistance in different inflammatory diseases . GRβ over-expression in ASM cells also prevents the capacity for steroids to induce transactivation activity and inhibit cytokine-induced pro-inflammatory gene expression .
Interestingly, short-term treatment of ASM cells with IFNs and TNF-α inhibits, in a GRβ-independent manner, the capacity for steroids to induce transactivation partially through the cellular accumulation of IRF-1 . IRF-1 is an early response gene involved in diverse transcriptional regulatory processes . Interestingly, a strong association was found between IRF-1 polymorphism and childhood atopic asthma . Early steroid dysfunction seen after short incubation with IFNs and TNFα could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1 which dose-dependently inhibited glucocorticoid response element (GRE)-dependent gene transcription . Consistently, reducing IRF-1 cellular levels using siRNA approach in TNF/IFN-treated ASM cells significantly restored steroid transactivation activities. These findings demonstrate for the first time that IRF-1 is a novel alternative GRβ-independent mechanism mediating steroid dysfunction induced by proinflammatory cytokines. The fact that different studies showed that the expression of IRF-1 was largely increased after viral infections  combined with the suppressive effect of IRF-1 on steroid signaling in ASM cells , may explain the reduced steroid responsiveness seen in asthmatic patients experiencing viral infections .
TLRs may be considered as a ‘sensing’ system that protects the host from infectious and non-infectious tissue injury and inflammation. TLRs also serve a homeostatic role to maintain tissue integrity and regeneration. TLRs ‘sense’ diverse molecules including microbial products and endogenous ligands generated in response to cell stress or injury. Currently, there are 10 known human TLRs named TLR1 through TLR10. TLR2 and TLR4, which primarily mediate recognition of bacterial cell wall components (eg LPS – the major ligand for TLR4) and endogenous ‘danger signals’ (eg heat shock proteins, extracellular matrix fragments) are the best studied of this receptor family. TLR3, TLR7 and TLR8 mediate recognition of viral RNA whilst TLR9 mediates recognition of bacterial DNA containing CpG motifs. Activation of TLRs triggers the activation of immune and inflammatory responses through NF-κB, IRF3/7 and MAP kinase dependent signaling pathways .
Epidemiological studies suggest that genetic polymorphisms in TLR genes, together with early-life exposure to environmental TLR stimulants (e.g. LPS in house dust, microbial exposure associated with certain farming activities, respiratory viral infections) are likely to be important, but also very complex, determinants of asthma incidence and severity. On the converse, emerging evidence shows that allergic airway inflammation impairs innate host-defense mechanisms, including TLR function, which results in impaired bacterial clearance [129, 130]. This may offer some explanation for increased bacterial colonization in asthmatic lungs and also provides some basis for infective exacerbations of asthma. Emerging evidence also indicates a role for TLR4 in the airway inflammatory response to cigarette smoke exposure, the primary causative factor of COPD [131-133].
The demonstration of functional TLR expression in human ASM cells over the past few years adds to the growing body of evidence of the immuno-modulatory capabilities of ASM cells. This has wide-ranging implications for the disease process in asthma and COPD, as activation of TLRs in ASM may exacerbate airway inflammatory responses by inducing expression of cell adhesion molecules and release of cytokines and chemokines, and may also amplify ASM-inflammatory cell interactions.
Human ASM cells in culture express TLR1 through TLR10 mRNA under basal conditions. TLR2, TLR3 and TLR6 are the most highly expressed, whilst TLR4 is the least expressed . Interestingly, in one study, constitutive expression of TLR7 or TLR8 was not demonstrated . Whether this was due to cell donor differences, type of ASM cells used (eg tracheal vs bronchial; distal vs proximal) or methodological issues remains to be resolved. In addition to evidence of TLR gene expression, cell surface and intracellular protein expression for both TLR2 and TLR3 has also been demonstrated [119, 135]. TLR2, TLR3 and TLR4 expression in ASM cells is up-regulated in response to inflammatory cytokines including IL-1β, TNF-α and IFN-γ, and microbial products including LPS and dsRNA. Combined stimulation with IFN-γ and TNF-α has synergistic and additive effects on TLR2 and TLR4 mRNA expression, respectively [119, 135].
Evidence of TLR expression in ASM has fuelled recent interest in ASM cell inflammatory responses to TLR ligands. Stimulation of ASM cells with synthetic TLR2 ligands, LPS or poly IC induces the production of various cytokines and chemokines [39, 119, 134-136]; Pam3CSK4 (a synthetic bacterial lipopeptide) and FSL-1 (S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Ser-Phe), which activate TLR2/TLR1 and TLR2/TLR6 heterodimers, respectively, induce IL-8 release; LPS induces expression of IL-6, IL-8 and eotaxin; and polyriboinosinc-polyribocytidylic acid (poly IC) induces expression of IL-6, IL-8, eotaxin, RANTES and IP-10. Stimulation of ASM cells with poly IC together with IL-1β or TNF-α has synergistic effects on IL-6, IL-8, IP-10 and RANTES release. Interestingly, poly IC induced eotaxin expression is inhibited in the presence of IL-1β or TNF-α, but is augmented by the Th2 cytokine IL-4.
The specific activation of TLR2 in mediating IL-8 release has not been confirmed in ASM cells, although anti-TLR2 or transfection with a dominant negative mutant form of TLR2 inhibits ERK1/2 signaling in response to the microbial derived TLR2 ligand lipoteichoic acid (LTA), thus providing functional evidence of TLR2 activation in ASM cells . Although ASM cells express TLR3 on the cell surface and in intracellular endosomes, specific activation of endosomal rather than surface TLR3 was shown to be responsible for poly IC mediated eotaxin release . The specific activation of TLR4 in mediating LPS-induced cytokine and chemokine release in ASM cells remains to be established.
In addition to inducing ASM cell cytokine and chemokine release, activation of TLRs in ASM cells may also amplify airway inflammatory responses by facilitating ASM-inflammatory cell interactions. This is demonstrated by studies showing that addition of TLR2, TLR4, TLR7 or TLR8 ligands to ASM cells in co-culture with peripheral blood mononuclear cells (PBMCs) leads to greater release of IL-6, IL-8 and CCL2 compared to TLR-activation of either cell type alone [134, 138]. IL-1β produced by LPS-activated monocytes was shown to be responsible, to some extent, for amplification of ASM-PBMC inflammatory responses . Poly IC and LPS may also promote ASM-inflammatory cell interactions via inducing the expression of cell adhesion molecules such as ICAM-1 and VCAM-1, respectively . Indeed, LPS has been shown to mediate VCAM-1-induced neutrophil adhesion in ASM cells .
In vitro infection of human ASM cells with respiratory viruses such as rhinovirus or respiratory syncytial virus leads to production of several cytokines and chemokines including IL-1β, IL-6, IL-8 and IL-11 [139-141]. The role of TLRs in mediating these responses has not as yet been addressed, although it is likely that viral-sensing TLRs as well as other intracellular viral recognition proteins such as protein kinase R, and cell-surface molecules such as ICAM-1 (which is a receptor for rhinovirus) are involved. Whether infection of ASM cells with respiratory viruses, or indeed other microbial pathogens that colonize the lungs in asthma and COPD, occurs in vivo is an important area of further investigation; especially given the potential impact of microbial-TLR interactions on ASM inflammatory responses.
Activation of TLRs in ASM occurs not only in response to microbial-derived products but may also occur in response to endogenous molecules present within the inflammatory milieu. Recently, it was shown that neutrophil-derived elastase (NE) activates ASM cells to synthesize TGF-β via a mechanism involving TLR4 and its associated down-stream signaling cascade. However, stimulation of TGF-β synthesis by NE was only partially inhibited by a TLR4-blocking antibody indicating that other mechanisms or perhaps TLRs may be involved. Interestingly, TLR4 protein expression on ASM was reduced following treatment with NE, indicating that NE-dependent TLR4 responses may require internalization of the receptor .
Although our understanding of the role of TLRs in the pathogenesis of asthma and COPD is only just evolving, evidence of their pro-inflammatory functions in ASM further extends the role of ASM as a critical mediator of the airway inflammatory response, potentially having the capacity to respond to environmental as well as endogenous molecules involved in the perpetuation and exacerbation of airway inflammatory disease. Studies of the expression and function of TLRs in ASM cells in vivo is an important area of future research.
Cytokines play a principal role in modulating inflammatory as well as immune responses in chronic inflammatory diseases such as asthma and COPD. Pro-inflammatory and immunomodulatory cytokines activate multiple signaling cascades in ASM cells that lead to amplification of ASM inflammatory responses. Research over the past decade has taken us forward in our understanding of MAPK, NF-κB and JAK/STAT signaling mechanisms involved in regulating ASM inflammatory gene expression and studies in animal models show that specific targeting of these pathways offer therapeutic potential for the treatment of chronic inflammatory airways disease [32, 68, 142-144]. Whilst there is some advantage in targeting these signaling pathways in isolation, further understanding of the cross-talk mechanisms and pathway interactions that exacerbate inflammatory responses or impair steroid responsiveness in ASM cells may provide novel targets or approaches for the future therapy of chronic inflammatory airways disease.
TLR ligands represent potentially exciting new therapeutic approaches for the treatment of asthma. Indeed, several studies published in the last five years demonstrate protective effects of TLR2, TLR3, TLR4, TLR7/8 and TLR9 ligands against allergic airway inflammation, airway hyperreactivity and airway remodeling in animal models of asthma [145-152]. The mechanisms that underlie protection against asthma in these models are slowly being unraveled and studies so far have focused on delineating immuno-modulatory pathways. However, evidence that the synthetic TLR7/8 ligand R-848 imparts some of its protection against airway remodeling by inhibiting ASM proliferation  indicates that the ASM is a potential target of immuno-modulatory therapy. An understanding of the signaling pathways regulating TLR-dependent inflammatory responses in ASM is an important area of further investigation.
Supported by National Institutes of Health grant 1 K99 HL089409-01 (to Dr. Tliba), American Lung Association grant RG-49342-N (to Dr. Tliba.). Dr Tliba is a Parker B. Francis Fellow in Pulmonary Research.