Interleukin-13 inhibits proliferation and enhances contractility of human airway smooth muscle cells without change in contractile phenotype.

IL-13 is an important mediator of allergen-induced airway hyperresponsiveness. This Th2 cytokine, produced by activated T cells, mast cells, and basophils, has been described to mediate a part of its effects independently of inflammation through a direct modulation of the airway smooth muscle (ASM). Previous studies demonstrated that IL-13 induces hyperresponsiveness in vivo and enhances calcium signaling in response to contractile agonists in vitro. We hypothesized that IL-13 drives human ASM cells (ASMC) to a procontractile phenotype. We evaluated ASM phenotype through the ability of the cell to proliferate, to contract, and to express contractile protein in response to IL-13. We found that IL-13 inhibits human ASMC proliferation (expression of Ki67 and bromodeoxyuridine incorporation) in response to serum, increasing the number of cells in G0/G1 phase and decreasing the number of cells in G2/M phases of the cell cycle. IL-13-induced inhibition of proliferation was not dependent on signal transducer and activator of transcription-6 but was IL-13Rα2 receptor dependent and associated with a decrease of Kruppel-like factor 5 expression. In parallel, IL-13 increased calcium signaling and the stiffening of human ASMC in response to 1 µM histamine, whereas the stiffening response to 30 mM KCl was unchanged. However, Western blot analysis showed unchanged levels of calponin, smooth muscle α-actin, vinculin, and myosin. We conclude that IL-13 inhibits proliferation via the IL-13Rα2 receptor and induces hypercontractility of human ASMC without change of the phenotypic markers of contractility.

Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma.

BACKGROUND: The origin of the excess airway smooth muscle in asthma and when in the course of the disease it is acquired are uncertain. OBJECTIVES: We examined the relative sensitivities of 2 markers of proliferation, proliferating cell nuclear antigen (PCNA) and Ki 67, in airway smooth muscle in vivo and in vitro. We then studied whether muscle remodeling is a dynamic process in asthma by quantifying proliferation rate and area. Finally we examined heparin-binding epidermal growth factor as a biomarker of remodeling. METHODS: We obtained bronchoscopic biopsies from subjects with moderate or severe asthma and healthy controls (n = 9/group). For in vitro studies, airway smooth muscle cells were cultured from tracheas of transplant donors. The proliferation rate was quantified from PCNA and Ki 67, co-localized to smooth muscle-specific alpha-actin cells in vivo. Muscle area was assessed morphometrically. We examined the expression of heparin-binding epidermal growth factor on tissues by in situ hybridization and by immunohistochemistry and in cells in culture by RT-PCR. RESULTS: Proliferating cell nuclear antigen and Ki 67 were highly correlated, but PCNA was a significantly more sensitive marker both in vivo and in vitro. Muscle area was 3.4-fold greater and the fraction of PCNA(+) nuclei in muscle was 5-fold greater in severe asthma than in healthy subjects. Heparin-binding epidermal growth factor was upregulated in proliferating muscle cells in culture and in airway smooth muscle in severe asthmatic tissues. CONCLUSION: Proliferating cell nuclear antigen is a highly sensitive marker of proliferation and heparin-binding epidermal growth factor is a potential biomarker during active remodeling of ASM in severe asthma.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells.

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca(2+) release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca(2+) response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca(2+) responses to histamine. The augmentation of Ca(2+) signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca(2+) responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

Expression and regulation of CCR1 by airway smooth muscle cells in asthma.

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-alpha and, to a lesser extent, IFN-gamma resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.

IFN-gamma, IL-4 and IL-13 modulate responsiveness of human airway smooth muscle cells to IL-13.

BACKGROUND: IL-13 is a critical mediator of allergic asthma and associated airway hyperresponsiveness. IL-13 acts through a receptor complex comprised of IL-13Ralpha1 and IL-4Ralpha subunits with subsequent activation of signal transducer and activator of transcription 6 (STAT6). The IL-13Ralpha2 receptor may act as a decoy receptor. In human airway smooth muscle (HASM) cells, IL-13 enhances cellular proliferation, calcium responses to agonists and induces eotaxin production. We investigated the effects of pre-treatment with IL-4, IL-13 and IFN-gamma on the responses of HASM cells to IL-13. METHODS: Cultured HASM were examined for expression of IL-13 receptor subunits using polymerase chain reaction, immunofluorescence microscopy and flow cytometry. Effects of cytokine pre-treatment on IL-13-induced cell responses were assessed by looking at STAT6 phosphorylation using Western blot, eotaxin secretion and calcium responses to histamine. RESULTS: IL-13Ralpha1, IL-4Ralpha and IL-13Ralpha2 subunits were expressed on HASM cells. IL-13 induced phosphorylation of STAT6 which reached a maximum by 30 minutes. Pre-treatment with IL-4, IL-13 and, to a lesser degree, IFN-gamma reduced peak STAT6 phosphorylation in response to IL-13. IL-13, but not IFN-gamma, pre-treatment abrogated IL-13-induced eotaxin secretion. Pre-treatment with IL-4 or IL-13 abrogated IL-13-induced augmentation of the calcium transient evoked by histamine. Cytokine pre-treatment did not affect expression of IL-13Ralpha1 and IL-4Ralpha but increased expression of IL-13Ralpha2. An anti-IL-13Ralpha2 neutralizing antibody did not prevent the cytokine pre-treatment effects on STAT6 phosphorylation. Cytokine pre-treatment increased SOCS-1, but not SOCS-3, mRNA expression which was not associated with significant increases in protein expression. CONCLUSION: Pre-treatment with IL-4 and IL-13, but not IFN-gamma, induced desensitization of the HASM cells to IL-13 as measured by eotaxin secretion and calcium transients to histamine. The mechanism of IL-4 and IL-13 induced desensitization does not appear to involve either downregulation of receptor expression or induction of the IL-13Ralpha2 or the SOCS proteins.

Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist.

Complex rheology of airway smooth muscle cells and its dynamic response during contractile stimulation involves many molecular processes, foremost of which are actomyosin cross-bridge cycling and actin polymerization. With an atomic force microscope, we tracked the spatial and temporal variations of the viscoelastic properties of cultured airway smooth muscle cells. Elasticity mapping identified stiff structural elements of the cytoskeletal network. Using a precisely positioned microscale probe, picoNewton forces and nanometer level indentation modulations were applied to cell surfaces at frequencies ranging from 0.5 to 100 Hz. The resulting elastic storage modulus (G') and dissipative modulus (G'') increased dramatically, with hysteresivity (eta = G''/G') showing a definitive decrease after stimulation with the contractile agonist 5-hydroxytryptamine. Frequency-dependent assays showed weak power-law structural damping behavior and universal scaling in support of the soft-glassy material description of cellular biophysics. Additionally, a high-frequency component of the loss modulus (attributed to cellular Newtonian viscosity) increased fourfold during the contractile process. The complex shear modulus showed a strong sensitivity to the degree of actin polymerization. Inhibitors of myosin light chain kinase activity had little effect on the stiffening response to contractile stimulation. Thus, our measurements appear to be particularly well suited for characterization of dynamic actin rheology during airway smooth muscle contraction.

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.

LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] affects calcium signaling in airway smooth muscle cells independently of phosphoinositide 3-kinase inhibition.

Phosphoinositide 3-kinase (PI3K) may potentially influence intracellular [Ca(2+)](i) concentration by several mechanisms. We have investigated the effects of phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] on Ca(2+) signaling in rat airway smooth muscle (ASM) cells using fura-2 and imaging methodology. Wortmannin (1 microM) and LY-294002 (1 and 10 microM) had opposite effects: wortmannin caused a small increase, whereas LY-294002 caused a significant decrease of peak Ca(2+) responses to serotonin (5-HT). LY-294002 (10 microM) diminished 5-HT-induced ASM cell contraction, measured as a change in cell surface area, and inositol phosphate formation, measured by anion exchange chromatography. Thin layer chromatography revealed that the levels of phospholipase C (PLC) substrate phosphatidylinositol 4,5-bisphosphate were not affected. SDS polyacrylamide gel electrophoresis and Western blotting have shown that both wortmannin and LY-294002 inhibited platelet-derived growth factor-induced PI3K activation. However, PI3K activation could not be detected after 5-HT stimulation. The specific casein kinase-2 (CK2) inhibitor 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (10-40 microM) reduced 5-HT-triggered responses to a similar extent as LY-294002. We conclude that LY-294002 modulates Ca(2+) signaling in rat ASM independently of its action on PI3K by acting on, or upstream of, PLC, possibly by inhibiting CK2.

Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate.

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca(2+) signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP(2)). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca(2+) mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1). Peak Ca(2+) responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca(2+) responses to 5-HT and Ca(2+) influx resulting from emptying of Ca(2+) stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP(2) in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca(2+) signaling by regulating PIP(2) levels and Ca(2+) influx.