Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation.

BACKGROUND: The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. OBJECTIVES: To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. METHODS: TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. RESULTS: The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. CONCLUSION: The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention.

Dimethylfumarate inhibits CXCL10 via haem oxygenase-1 in airway smooth muscle.

CXCL10 stimulates mast cell infiltration into airway smooth muscle bundles and, thus, activate cytokine secretion and airway smooth muscle cell (ASMC) proliferation. Dimethylfumarate (DMF) reduces cytokine secretion by lymphocytes and ASMC proliferation through haem oxygenase (HO)-1. Therefore, we investigated the potency of DMF to inhibit tumour necrosis factor (TNF)-α- and interferon (IFN)-γ-induced CXCL10 secretion by human ASMCs. Human primary ASMCs were pre-incubated with DMF and/or fluticasone and/or glutathione ethylester before cells were stimulated with IFN-γ and/or TNF-α. DMF inhibited CXCL10 secretion and increased HO-1 levels, and p38 mitogen-activated protein kinase (MAPK) inhibition reduced DMF-dependent HO-1 expression. The DMF effect on CXCL10 secretion was abrogated by pre-treatment with HO-1 small interfering RNA (siRNA). Glutathione supplementation reversed all DMF effects on CXCL10 secretion and p38 MAPK phosphorylation. Importantly, combining DMF with fluticasone further reduced CXCL10 secretion. In addition, DMF inhibited IFN-γ-induced CXCL10 secretion. This effect was compensated by glutathione supplementation or by pre-treatment with HO-1 siRNA. In addition, DMF reduced TNF-α-induced granulocyte colony-stimulating factor (G-CSF) secretion but had no effect on INF-γ-induced G-CSF secretion. In human primary ASMCs, DMF inhibits CXCL10 secretion by reducing the cellular glutathione level and by activation of p38 MAPK and HO-1. Therefore, DMF may reduce airway inflammation in asthma by a glucocorticoid-independent pathway.

Mast cells produce novel shorter forms of perlecan that contain functional endorepellin: a role in angiogenesis and wound healing.

Mast cells are derived from hematopoietic progenitors that are known to migrate to and reside within connective and mucosal tissues, where they differentiate and respond to various stimuli by releasing pro-inflammatory mediators, including histamine, growth factors, and proteases. This study demonstrated that primary human mast cells as well as the rat and human mast cell lines, RBL-2H3 and HMC-1, produce the heparan sulfate proteoglycan, perlecan, with a molecular mass of 640 kDa as well as smaller molecular mass species of 300 and 130 kDa. Utilizing domain-specific antibodies coupled with N-terminal sequencing, it was confirmed that both forms contained the C-terminal module of the protein core known as endorepellin, which were generated by mast cell-derived proteases. Domain-specific RT-PCR experiments demonstrated that transcripts corresponding to domains I and V, including endorepellin, were present; however, mRNA transcripts corresponding to regions of domain III were not present, suggesting that these cells were capable of producing spliced forms of the protein core. Fractions from mast cell cultures that were enriched for these fragments were shown to bind endothelial cells via the α(2)ß(1) integrin and stimulate the migration of cells in "scratch assays," both activities of which were inhibited by incubation with either anti-endorepellin or anti-perlecan antibodies. This study shows for the first time that mast cells secrete and process the extracellular proteoglycan perlecan into fragments containing the endorepellin C-terminal region that regulate angiogenesis and matrix turnover, which are both key events in wound healing.

Thiazolidinediones inhibit airway smooth muscle release of the chemokine CXCL10: in vitro comparison with current asthma therapies.

BACKGROUND: Activated mast cells are present within airway smooth muscle (ASM) bundles in eosinophilic asthma. ASM production of the chemokine CXCL10 plays a role in their recruitment. Thus the effects of glucocorticoids (fluticasone, budesonide), long-acting ß2-agonists (salmeterol, formoterol) and thiazolidinediones (ciglitazone, rosiglitazone) on CXCL10 production by ASM cells (ASMC) from people with and without asthma were investigated in vitro. METHODS: Confluent serum-deprived cells were treated with the agents before and during cytokine stimulation for 0-24 h. CXCL10 protein/mRNA, IκB-α levels and p65 activity were measured using ELISA, RT PCR, immunoblotting and p65 activity assays respectively. Data were analysed using ANOVA followed by Fisher's post-hoc test. RESULTS: Fluticasone and/or salmeterol at 1 and 100 nM inhibited CXCL10 release induced by IL-1ß and TNF-α, but not IFNγ or all three cytokines (cytomix). The latter was also not affected by budesonide and formoterol. In asthmatic ASMC low salmeterol, but not formoterol, concentrations increased cytomix-induced CXCL10 release and at 0.01 nM enhanced NF-κB activity. Salmeterol 0.1 nM together with fluticasone 0.1 and 10 nM still increased CXCL10 release. The thiazolidinediones ciglitazone and rosiglitazone (at 25 and 100 µM) inhibited cytomix-induced CXCL10 release but these inhibitory effects were not prevented by the PPAR-g antagonist GW9662. Ciglitazone did not affect early NF-κB activity and CXCL10 mRNA production. CONCLUSIONS: Thus the thiazolidinediones inhibited asthmatic ASMC CXCL10 release under conditions when common asthma therapies were ineffective or enhanced it. They may provide an alternative strategy to reduce mast cell-ASM interactions and restore normal airway physiology in asthma.

RAGE: a new frontier in chronic airways disease.

Asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous inflammatory disorders of the respiratory tract characterized by airflow obstruction. It is now clear that the environmental factors that drive airway pathology in asthma and COPD, including allergens, viruses, ozone and cigarette smoke, activate innate immune receptors known as pattern-recognition receptors, either directly or indirectly by causing the release of endogenous ligands. Thus, there is now intense research activity focused around understanding the mechanisms by which pattern-recognition receptors sustain the airway inflammatory response, and how these mechanisms might be targeted therapeutically. One pattern-recognition receptor that has recently come to attention in chronic airways disease is the receptor for advanced glycation end products (RAGE). RAGE is a member of the immunoglobulin superfamily of cell surface receptors that recognizes pathogen- and host-derived endogenous ligands to initiate the immune response to tissue injury, infection and inflammation. Although the role of RAGE in lung physiology and pathophysiology is not well understood, recent genome-wide association studies have linked RAGE gene polymorphisms with airflow obstruction. In addition, accumulating data from animal and clinical investigations reveal increased expression of RAGE and its ligands, together with reduced expression of soluble RAGE, an endogenous inhibitor of RAGE signalling, in chronic airways disease. In this review, we discuss recent studies of the ligand-RAGE axis in asthma and COPD, highlight important areas for future research and discuss how this axis might potentially be harnessed for therapeutic benefit in these conditions.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement.

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1ß, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1ß, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1ß and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases.

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1ß (IL-1ß) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1ß, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

TLR2 ligand engagement upregulates airway smooth muscle TNFα-induced cytokine production.

Airway inflammation and respiratory infections are important factors contributing to disease exacerbation in chronic airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle (ASM) cells express Toll-like receptors (TLRs) and may be involved in the amplification of airway inflammatory responses during infectious exacerbations. We determined whether infectious stimuli (mimicked using Pam3CSK4, a synthetic bacterial lipopeptide that binds to TLR2/TLR1) further enhance ASM cell inflammatory responses to TNFα in vitro and the signaling pathways involved. Human ASM cells were pretreated for 1 h with Pam3CSK4 (1 µg/ml) in the absence or presence of TNFα (10 ng/ml), and IL-6 and IL-8 release was measured after 24 h. As expected, stimulation with Pam3CSK4 or TNFα alone induced significant IL-6 and IL-8 release. Furthermore, Pam3CSK4 significantly increased TNFα-induced IL-6 and IL-8 mRNA expression and protein release and neutrophil chemotactic activity. The potentiating effect of Pam3CSK4 on TNFα-induced inflammatory responses was not due to enhanced TLR2 expression nor did it involve augmentation of NF-κB or MAPK signaling pathways. Rather, Pam3CSK4 induced cAMP response element (CRE) binding protein phosphorylation and induced CRE-mediated transcriptional regulation, suggesting that Pam3CSK4 and TNFα are acting in concert to enhance ASM cytokine secretion via parallel transcriptional pathways. Our findings suggest that ASM cells may be involved in the amplification of airway inflammatory responses during infectious exacerbations in chronic airway disease.

The effect of asthma therapeutics on signalling and transcriptional regulation of airway smooth muscle function.

SCOPE OF THE REVIEW: Our knowledge of the multifunctional nature of airway smooth muscle (ASM) has expanded rapidly in the last decade, but the underlying molecular mechanisms and how current therapies for obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), affect these are still being elucidated. Our current knowledge has built on the pharmacology of human ASM contraction and relaxation established prior to that and which is reviewed in detail elsewhere in this issue. The advent of methods to isolate and culture ASM cells, especially human ASM cells, has made it possible to study how they may contribute to airway remodelling through their synthetic, proliferative, and migratory capacities. Now the underlying molecular mechanisms of ASM growth factor secretion, extracellular matrix (ECM) production, proliferation and migration, as well as contraction and relaxation, are being determined. A complex network of signalling pathways leading to gene transcription in ASM cells permits this functional plasticity in healthy and diseased airways. This review is an overview of the effects of current therapies, and some of those in development, on key signalling pathways and transcription factors involved in these ASM functions.

Cell density and serum exposure modify the function of the glucocorticoid receptor C/EBP complex.

The glucocorticoid receptor (GR) is a major control factor for proliferation, differentiation, and inflammation. Our knowledge about the GR is focused on its function as a transcription regulator. However, cells do not always respond to steroids in the same way or develop resistance. The mechanism underlying such a modified steroid response is not well understood, and may depend on the microenvironment of the cells or on the stage of their differentiation. Therefore, we studied the effect of cell density and inflammatory conditions on the expression, compartmentalization, activation, and the anti-proliferative function of the GR in primary human lung fibroblast cultures. In subconfluent cells the GR was located perinuclear, while in confluent cells it was ubiquitously expressed. Serum stimulation up-regulated the level of GR mRNA and protein under all conditions. In subconfluent cells dexamethasone activated the nuclear accumulation and DNA binding of the GR persistently, while in confluent cells its activity declined after 6 hours. In subconfluent cells, but not in confluent cells, the GR interacted with a 42-kD, but not the 30-kD C/EBP-alpha isoprotein, which resulted in an up-regulation of p21((Waf1/Cip1)) expression and suppression of proliferation. In confluent cells, glucocorticoids induced p27((Kip1)) expression via p38 mitogen-activated protein kinase and a 52-kD C/EBP-beta isoprotein. However, p27((Kip1)) did not mediate the antiproliferative effect of glucocorticoids, but simultaneous inhibition of p21((Waf1/Cip1)) and p27((Kip1)) unlocked contact inhibition in confluent cells. Our results indicate that cell density and serum exposure alter the localization and function of the GR.

S100A12 provokes mast cell activation: a potential amplification pathway in asthma and innate immunity.

BACKGROUND: The calcium-binding protein S100A12 might provoke inflammation and monocyte recruitment through the receptor for advanced glycation end products. OBJECTIVE: Because inflammation elicited by S100A12 in vivo had characteristics of mast cell (MC) activation, we aimed to define the mechanism. METHODS: Various MC populations were used to test S100A12 activation assessed on the basis of morphology, histamine release, leukotriene production, and cytokine induction. MC dependence of S100A12-provoked inflammation was tested in mice and on the rat microcirculation by means of intravital microscopy. Immunohistochemistry localized S100A12 in the asthmatic lung, and levels in sputum from asthmatic patients were quantitated by means of ELISA. Expression of the receptor for advanced glycation end products was evaluated by means of RT-PCR and Western blotting. RESULTS: S100A12 provoked degranulation of mucosal and tissue MCs in vitro and in vivo and amplified IgE-mediated responses. It induced a cytokine profile indicating a role in innate/T(H)1-mediated responses. S100A12-induced edema and leukocyte rolling, adhesion, and transmigration in the microcirculation were MC dependent. Eosinophils in airway tissue from asthmatic patients were S100A12 positive, and levels were increased in sputum. S100A12 responses were partially blocked by an antagonist to the receptor for advanced glycation end products, but MCs did not express mRNA or protein, suggesting an alternate receptor. CONCLUSION: This novel pathway highlights the potential importance of S100A12 in allergic responses and in infectious and chronic inflammatory diseases. CLINICAL IMPLICATIONS: MC activation by S100A12 might exacerbate allergic inflammation and asthma. S100A12 might provide a novel marker for eosinophilic asthma.

Effect of IL-6 trans-signaling on the pro-remodeling phenotype of airway smooth muscle.

Increased levels of IL-6 are documented in asthma, but its contribution to the pathology is unknown. Asthma is characterized by airway wall thickening due to increased extracellular matrix deposition, inflammation, angiogenesis, and airway smooth muscle (ASM) mass. IL-6 binds to a specific membrane-bound receptor, IL-6 receptor-alpha (mIL-6Ralpha), and subsequently to the signaling protein gp130. Alternatively, IL-6 can bind to soluble IL-6 recpetor-alpha (sIL-6Ralpha) to stimulate membrane receptor-deficient cells, a process called trans-signaling. We discovered that primary human ASM cells do not express mIL-6Ralpha and, therefore, investigated the effect of IL-6 trans-signaling on the pro-remodeling phenotype of ASM. ASM required sIL-6Ralpha to activate signal transducer and activator 3, with no differences observed between cells from asthmatic subjects compared with controls. Further analysis revealed that IL-6 alone or with sIL-6Ralpha did not induce release of matrix-stimulating factors (including connective tissue growth factor, fibronectin, or integrins) and had no effect on mast cell adhesion to ASM or ASM proliferation. However, in the presence of sIL-6Ralpha, IL-6 increased eotaxin and VEGF release and may thereby contribute to local inflammation and vessel expansion in airway walls of asthmatic subjects. As levels of sIL-6Ralpha are increased in asthma, this demonstration of IL-6 trans-signaling in ASM has relevance to the development of airway remodeling.

IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-alpha were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-kappaB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-alpha-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 +/- 0.01964, t(1/2) = 12.0 h), compared with vehicle (-0.01030 +/- 0.007963, t(1/2) = 67.3 h) [results are expressed as decay constant (means +/- SE) and half-life (t(1/2) in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle.

Mast cell microlocalization within the airway smooth muscle bundle is an important determinant of the asthmatic phenotype. We hypothesized that mast cells migrate toward airway smooth muscle in response to smooth muscle-derived chemokines. In this study, we investigated (1) chemokine receptor expression by mast cells in the airway smooth muscle bundle in bronchial biopsies from subjects with asthma using immunohistology, (2) the concentration of chemokines in supernatants from stimulated ex vivo airway smooth muscle cells from subjects with and without asthma measured by enzyme-linked immunosorbent assay, and (3) mast cell migration toward these supernatants using chemotaxis assays. We found that CXCR3 was the most abundantly expressed chemokine receptor on human lung mast cells in the airway smooth muscle in asthma and was expressed by 100% of these mast cells compared with 47% of mast cells in the submucosa. Human lung mast cell migration was induced by airway smooth muscle cultures predominantly through activation of CXCR3. Most importantly, CXCL10 was expressed preferentially by asthmatic airway smooth muscle in bronchial biopsies and ex vivo cells compared with those from healthy control subjects. These results suggest that inhibition of the CXCL10/CXCR3 axis offers a novel target for the treatment of asthma.

CD40 and OX40 ligand are increased on stimulated asthmatic airway smooth muscle.

BACKGROUND: Severe, persistent asthma is characterized by airway smooth muscle hyperplasia, inflammatory cell infiltration into the smooth muscle, and increased expression of many cytokines, including IL-4, IL-13, IL-1beta, and TNF-alpha. These cytokines have the potential to alter the expression of surface receptors such as CD40 and OX40 ligand on the airway smooth muscle cell. OBJECTIVE: To examine whether cytokines alter expression of CD40 and OX40 ligand on airway smooth muscle cells and identify any differences in response between asthmatic and nonasthmatic airway smooth muscle cells. METHODS: We used flow cytometry and immunohistochemistry to detect CD40 and OX40 ligand on airway smooth muscle cells cultured in the presence of TNF-alpha, IL-1beta, IL-4, or IL-13. Prostaglandin E 2 levels were assessed by ELISA. RESULTS: TNF-alpha increased expression of both CD40 and OX40 ligand on both asthmatic and nonasthmatic airway smooth muscle cells. The level of expression was significantly greater on the asthmatic cells. IL-1beta alone had no effect, but it attenuated the TNF-induced expression of both CD40 and OX40 ligand. The mechanism of inhibition was COX-dependent for CD40 and was COX-independent but cyclic AMP-dependent for OX40 ligand. IL-4 and IL-13 had no effect. CONCLUSION: Our study has demonstrated that TNF-alpha and IL-1beta have the potential to modulate differentially the interactions between cells present in the inflamed airways of a patient with asthma and therefore to contribute to the regulation of airway inflammation and remodeling.

IL-17A augments TNF-alpha-induced IL-6 expression in airway smooth muscle by enhancing mRNA stability.

BACKGROUND: IL-17A is implicated in the regulation of inflammation and is found in increased amounts in the asthmatic airway. Human airway smooth muscle (ASM) cells synthesize cell adhesion molecules and cytokines in response to inflammatory mediators. OBJECTIVE: In this study, we examined whether IL-17A modulated the synthetic function of ASM cells. METHODS: Primary ASM cultures were treated with IL-17A alone and in combination with the proinflammatory cytokines TNF-alpha and IL-1beta. Intercellular adhesion molecule 1 expression, GM-CSF, and IL-6 secretion were measured by ELISA. Examination of transcriptional regulation was performed via transient transfection of promoter constructs, whereas mRNA stability was assessed by actinomycin D chase and quantitative real-time PCR. RESULTS: Airway smooth muscle did not secrete IL-17A after stimulation of ASM with TNF-alpha and IL-1beta. Furthermore, IL-17A (0.1-10 ng/mL) had no effect on TNF-alpha-induced and IL-1beta-induced intercellular adhesion molecule 1 expression or GM-CSF secretion. However, IL-17A (10 ng/mL) significantly augmented TNF-alpha-induced IL-6 secretion 12-fold (TNF-alpha, 2.3 +/- 0.4 ng/mL; IL-17A and TNF-alpha, 27.5 +/- 4.8 ng/mL; P <.05) while having no effect on IL-1beta-induced IL-6. Although IL-17A had no effect on nuclear factor kappaB-mediated transcriptional regulation of IL-6 gene expression induced by TNF-alpha, IL-17A significantly augmented TNF-alpha-induced IL-6 mRNA stability. CONCLUSION: Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a posttranscriptional pathway, rather than transcriptional mechanisms, to augment TNF-alpha-induced secretion of IL-6 from ASM cells.

'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations.

The extension of airway smooth muscle cell (ASMC) functions, from just contractile, to synthetic and/or proliferative states, is an important component of airway remodelling and inflammation in asthma. Whereas all these functions have been demonstrated in ASM, currently, it is not known whether ASMC can be differentiated on the basis of their proliferative and synthetic functions. We used flow-cytometric techniques to determine, first, whether human ASMC are phenotypically heterogenous with regard to their secretory function, and second, the proliferative status of secretory cells. ASMC were induced to synthesize GM-CSF by stimulation with IL-1beta and TNF-alpha followed by 10% human serum. Flow-cytometric detection of intracellular GM-CSF revealed that only a proportion of cells in culture (approximately 20-60%) synthesize GM-CSF. To determine the proliferative status of GM-CSF producing cells, ASMC were pretreated with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), a fluorescein based dye used to track cell division, prior to cytokine/serum stimulation. Simultaneous analysis of intracellular GM-CSF and CFSE revealed that GM-CSF producing cells were present in both the divided and undivided ASMC populations. Thus, cytokine production and proliferation occurred in overlapping ASMC populations and prior progression through the cell cycle was not essential for ASMC cytokine production.