Effect of IL-1beta on CRE-dependent gene expression in human airway smooth muscle cells

IL-1beta inhibits isoproterenol (ISO)-induced relaxation of cultured human airway smooth muscle (HASM) cells. The purpose of this study was to determine whether IL-1beta can also suppress ISO-induced cAMP response element (CRE)-dependent gene expression. ISO (10 microM) caused a marked increase in CRE-binding protein (CREB) phosphorylation, which was attenuated by IL-1beta (2 ng/ml). This effect of IL-1beta was abolished by the cyclooxygenase (COX) inhibitor indomethacin. To examine CRE-driven gene expression, we transiently transfected HASM cells with a construct containing CRE upstream of a luciferase reporter gene. ISO (6 h) caused a sixfold increase in luciferase activity. IL-1beta (24 h) alone also increased luciferase activity, although to a lesser extent (2-fold). However, the ability of ISO to elicit luciferase expression was markedly reduced in cells treated with IL-1beta. Indomethacin, the MEK and p38 inhibitors U-0126 and SB-203580, the protein kinase A inhibitor H-89, and dexamethasone each completely abolished the ability of IL-1beta to induce CRE-driven gene expression but only slightly increased the ability of ISO to induce CRE-driven gene expression in IL-1beta-treated cells. IL-1beta also attenuated dibutyryl cAMP-induced CRE-driven gene expression, but not dibutyryl cAMP-induced CREB phosphorylation. Tumor necrosis factor-alpha (10 ng/ml) also attenuated ISO-induced CRE-driven gene expression, even though it was without effect on ISO-induced cAMP formation or ISO-induced CREB phosphorylation. The results suggest that IL-1beta and tumor necrosis factor-alpha may attenuate the ability of beta-agonists to induce expression of genes with CRE in their regulatory regions at least in part through events downstream of CREB phosphorylation.     

Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype

Background

A major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.

Methods

To this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.

Results

Both CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.

Conclusions

Direct exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.     

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.     

IL-17 enhances IL-1beta-mediated CXCL-8 release from human airway smooth muscle cells

Recent studies into the pathogenesis of airway disorders such as asthma have revealed a dynamic role for airway smooth muscle cells in the perpetuation of airway inflammation via secretion of cytokines and chemokines. In this study, we evaluated whether IL-17 could enhance IL-1beta-mediated CXCL-8 release from human airway smooth muscle cells (HASMC) and investigated the upstream and downstream signaling events regulating the induction of CXCL-8. CXCL-8 mRNA and protein induction were assessed by real-time RT-PCR and ELISA from primary HASMC cultures. HASMC transfected with site-mutated activator protein (AP)-1/NF-kappaB CXCL-8 promoter constructs were treated with selective p38, MEK1/2, and phosphatidylinositol 3-kinase (PI3K) inhibitors to determine the importance of MAPK and PI3K signaling pathways as well as AP-1 and NF-kappaB promoter binding sites. We demonstrate IL-17 induced and synergized with IL-1beta to upregulate CXCL-8 mRNA and protein levels. Erk1/2 and p38 modulated IL-17 and IL-1beta CXCL-8 promoter activity; however, IL-1beta also activated the PI3K pathway. The synergistic response mediating CXCL-8 promoter activity was dependent on both MAPK and PI3K signal transduction pathways and required the cooperation of AP-1 and NF-kappaB cis-acting elements upstream of the CXCL-8 gene. Collectively, our observations indicate MAPK and PI3K pathways regulate the synergy of IL-17 and IL-1beta to enhance CXCL-8 promoter activity, mRNA induction, and protein synthesis in HASMC via the cooperative activation of AP-1 and NF-kappaB trans-acting elements.     

Effect of extracellular matrix proteins on vascular smooth muscle cell phenotype

The effect on phenotypic expression of rabbit vascular smooth muscle cells (SMC) of the interstitial matrix proteins collagen I and fibronectin, the basal lamina proteins collagen IV and laminin, and the serum adhesion protein vitronectin was examined in culture. Experiments were performed in foetal calf serum stripped of fibronectin and vitronectin to eliminate their confounding effects. All the proteins promoted adhesion to the plastic culture dish (in a concentration dependent manner) of SMC freshly isolated from the artery wall. These cells had a high volume density of myofilaments (V_vmyo) in their cytoplasm. Laminin was best at maintaining SMC with a high V_vmyo (V_vmyo = 49.8%) followed by collagen IV (41.7%). Cells plated on vitronectin showed the lowest V_vmyo (31.3%). The results support the concept that the SMC basal lamina has a role in maintaining cells in the high V_vmyo phenotype.     

Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation

Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. The DGC is expressed in smooth muscle tissue, but its functional role is not fully established. We tested whether contractile phenotype maturation of human ASM is associated with accumulation of DGC proteins. We compared subconfluent, serum-fed cultures and confluent cultures subjected to serum deprivation, which express a contractile phenotype. Western blotting confirmed that beta-dystroglycan, beta-, delta-, and epsilon-sarcoglycan, and dystrophin abundance increased six- to eightfold in association with smooth muscle myosin heavy chain (smMHC) and calponin accumulation during 4-day serum deprivation. Immunocytochemistry showed that the accumulation of DGC subunits was specifically localized to a subset of cells that exhibit robust staining for smMHC. Laminin competing peptide (YIGSR, 1 microM) and phosphatidylinositol 3-kinase (PI3K) inhibitors (20 microM LY-294002 or 100 nM wortmannin) abrogated the accumulation of smMHC, calponin, and DGC proteins. These studies demonstrate that the accumulation of DGC is an integral feature for phenotype maturation of human ASM cells. This provides a strong rationale for future studies investigating the role of the DGC in ASM smooth muscle physiology in health and disease.     

IL-13 may mediate allergen-induced hyperresponsiveness independently of IL-5 or eotaxin by effects on airway smooth muscle

IL-13 is a mediator of allergen-induced airway hyperresponsiveness (AHR). The aim of this study was to evaluate whether eotaxin and IL-5 were implicated in the effects of IL-13 on allergen-induced AHR or whether IL-13 may exert its effects through direct actions on airway smooth muscle (ASM). To study this question airway inflammation and AHR were induced in mice by sensitization and subsequent challenge on three successive days with ovalbumin. A monoclonal anti-IL-13 antibody administered before each challenge significantly reduced AHR without affecting airway eosinophilia. No changes of mRNA in BAL and lung tissues or protein levels in BAL of IL-5 or eotaxin were found following anti-IL-13 treatment. Combined injection of monoclonal anti-IL-5 and antieotaxin antibodies before each antigen challenge blocked airway eosinophilia but failed to reduce AHR. IL-13 induced calcium transients in cultured murine ASM cells and augmented the calcium and contractile responses of these cells to leukotriene D4. These results suggest that IL-13 plays an important role in allergen-induced AHR and is important in the early phases of the inflammatory process. Its effects on AHR are mediated independently of IL-5 and eotaxin and may involve a direct effect on ASM to augment its responsiveness.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to na?ve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.     

Role of IL-6 trans-signaling in CCl4 induced liver damage

Interleukin-6 (IL-6) plays an important role in liver regeneration and protection against liver damage. In addition to IL-6 classic signaling via membrane bound receptor (mIL-6R), IL-6 signaling can also be mediated by soluble IL-6R (sIL-6R) thereby activating cells that do not express membrane bound IL-6R. This process has been named trans-signaling. IL-6 trans-signaling has been demonstrated to operate during liver regeneration. We have developed methods to specifically block or mimic IL-6 trans-signaling. A soluble gp130 protein (sgp130Fc) exclusively inhibits IL-6 trans-signaling whereas an IL-6/sIL-6R fusion protein (Hyper-IL-6) mimics IL-6 trans-signaling. Using these tools we investigate the role of IL-6 trans-signaling in CCl₄ induced liver damage. Blockade of IL-6 trans-signaling during CCl₄ induced liver damage led to higher liver damage, although induction of Cyp4502E1 and thus bioactivation of CCl₄ was unchanged. Depletion of neutrophils resulted in reduced liver transaminase levels irrespective of IL-6 trans-signaling blockade. Furthermore, IL-6 trans-signaling was important for refilling of hepatocyte glycogen stores, which were depleted 24 h after CCl₄ treatment. We conclude that IL-6 trans-signaling via the soluble IL-6R is important for the physiologic response of the liver to CCl₄ induced chemical damage.     

Effect of length oscillations on airway smooth muscle reactivity and cross-bridge cycling

Excessive airway narrowing due to airway smooth muscle (ASM) hyperconstriction is a major symptom in many respiratory diseases. In vitro imposition of length oscillations similar to those produced by tidal breathing on contracted ASM have shown to reduce muscle active forces, which is usually attributed to unconfirmed disruption of actomyosin cross-bridges. This research focuses on an in vitro investigation of the effect of mechanical oscillations on ASM reactivity and actomyosin cross-bridges. A computerized organ bath system was used to test maximally precontracted bovine ASM subjected to length oscillations at frequencies in the range of 10-100 Hz superimposed on tidal breathing oscillation. Using an immunofluorescence technique, two specific antibodies against the phospho-serine19 myosin light chain and the α-smooth muscle actin were used to analyze the colocalization between these two filaments. Data were processed using the plug-in "colocalization threshold" of ImageJ 1.43m software. The results demonstrate that both tidal and superimposed length oscillations reduce the active force in contracted ASM for a relatively long term and that the latter enhances the force reduction of the former. This reduction was also found to be frequency and time dependent. Additionally colocalization analysis indicates that length oscillations cause the detachment of the actomyosin connections and that this condition is sustained even after the cessation of the length oscillations.     

Effect of interleukin-6 (IL-6) on the vascular smooth muscle contraction in abdominal aorta of rats with streptozotocin-induced diabetes

Patients with type 1 diabetes are at a risk of hypertension. However, the mechanisms behind the findings are not completely known. The aim of the present study was to investigate involvement of interleukin-6 (IL-6) on the contraction of abdominal aorta in rats with type 1 diabetes. IL-6 levels in the plasma of rats with streptozotocin (STZ)-induced diabetes were determined by ELISA. The abdominal aorta was dissected free of fat and connective tissues and then cut into spiral rings. The endothelium-denuded strip was vertically suspended in tissue chambers containing 5 ml Krebs solution at 37 degrees C and bubbled continuously with 95% O2-5% CO2. The effects of phenylephrine (Phe) on the contractile responses of abdominal aorta were recorded. The effects of IL-6 and anti-rat IL-6 antibody on the Phe-induced response were also examined. Plasma levels of IL-6 increased time-dependently in rats with STZ-induced diabetes. Phe caused concentration-dependent contraction in aortic rings. Phe-induced contractions were higher in vascular strips of STZ-induced diabetic rats than that of control rats. Pretreatment of vascular strips with IL-6 for 1 h did not cause contraction but enhanced the contraction in response to Phe. Treatment of the vascular strips with an anti-IL-6 antibody for 1 h decreased the Phe-induced contractions. These results suggest that IL-6 causes vascular smooth muscle contraction in abdominal aorta of rats with type 1 diabetes.