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Rhinovirus (RV) infections are the major cause of asthma exacerbations in children and adults. Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled beta(2)-adrenergic receptor (beta(2)AR) agonists. During virus-associated exacerbations, an impaired response to beta(2)AR agonists is observed; the reason for this is not known. The objective of this study was to determine the effect of RV infection on airway smooth muscle beta(2)AR function. The human cell line Beas-2B and primary human bronchial epithelial cells (HBECs) were infected with RV (multiplicity of infection = 1). After 1 or 5 days for primary and Beas-2B cells, respectively, cell culture supernatants were harvested, UV-irradiated to inactivate RV, and applied to human airway smooth muscle cells for 3 days to assess modifications of beta(2)AR function. RV conditioned medium from Beas-2B and HBECs decreased beta(2)AR agonist-induced cAMP by 50 and 65%, respectively (n = 5; P < 0.05). When cAMP was induced independently of the beta(2)AR using forskolin, no impairment was found. Using flow cytometry, we demonstrated that this decrease was likely the result of beta(2)AR desensitization because membrane but not total cell receptor beta(2)AR was decreased. Pretreatment of HBECs and Beas-2B cells but not human airway smooth muscle cells with the corticosteroids dexamethasone or fluticasone abolished virus-mediated beta(2)AR loss of function. This study shows that epithelial infection with RV induces a decrease of beta(2)AR function on airway smooth muscle cells, potentially explaining the clinical observation of loss of beta(2)AR agonist function during RV-induced asthma exacerbations.

Transforming growth factor (TGF)-beta and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-beta increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-beta and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-beta and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase-dependent in both cell types but phosphoinositide-3 kinase-dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-beta induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.

There is strong evidence to implicate transforming growth factor-beta in the remodeling that occurs in asthma, as levels are increased in bronchial lavage fluid and gene expression is increased in bronchial tissue. Transforming growth factor-beta is also known to increase the release of collagen from airway smooth muscle. Here we identify for the first time a possible mechanism for the effects of transforming growth factor-beta. Transforming growth factor-beta specifically induces mRNA and protein for connective tissue growth factor in airway smooth muscle, and moreover, we report that the connective tissue growth factor response is greater in airway smooth muscle cultured from patients with asthma compared with patients without asthma. This occurs at both the level of mRNA (37.53 +/- 11.62- and 13.59 +/- 3.12-fold increase at 24 hours compared with time 0, respectively, p < 0.02) and protein production (67.57 +/- 27.80- and 3.58 +/- 0.6-fold increase at 24 hours compared with time 0, respectively, p < 0.03). The differential connective tissue growth factor response to transforming growth factor-beta in asthmatic airway smooth muscle identifies a potential role for connective tissue growth factor in the remodeling that is characteristic of severe persistent asthma.

Increased airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely to be the result of increased muscle proliferation. We have for the first time been able to culture ASM cells from asthmatic patients and to compare their proliferation rate with that of nonasthmatic patients. Asthmatic ASM cell cultures (n = 12) were established from explanted lungs and endobronchial biopsies. Nonasthmatic ASM cells (n = 10) were obtained from explanted tissue from patients with no airway disease, emphysema, carcinoma, and fibrosing alveolitis. Cell counts, tritiated thymidine incorporation, and cell cycle analysis were conducted over 7 d. Asthmatic ASM cell numbers at Days 3, 5, and 7 were significantly higher than corresponding values for nonasthmatic cells (p0.05). Tritiated thymidine incorporation was increased 3.2-fold in asthmatic cells compared with nonasthmatic cells within the first 24 h (p = 0.026). Flow cytometric analysis of DNA content on Days 1 and 2 revealed that a significantly greater percentage of asthmatic ASM cells were in the G2 + M phase (p0.05). This study shows for the first time that proliferation of ASM cells is increased in patients with asthma and provides evidence for an intrinsic abnormality in the ASM cell in this disease.asthma; human airway smooth muscle; cell culture; cell proliferation; hyperplasia

Airway remodeling is a key feature of persistent asthma. Part of the remodeling process involves the laying down of extracellular matrix (ECM) proteins within the airways. In this study we compared the production of ECM proteins by human airway smooth-muscle (ASM) cells in culture after exposure to 10% serum from an asthmatic individual or 10% serum from a nonasthmatic individual with or without beclomethasone (0.01 to 100 nM). Enzyme-linked immunosorbent assays were done with antibodies to human fibronectin; perlecan; elastin; the laminin beta(1), gamma(1), beta(2), alpha(1) chains; thrombospondin; chondroitin sulfate; collagen types I, III, IV, and V; versican; and decorin. Serum from the asthmatic individual, when compared with that from the nonasthmatic individual, caused a significant increase in the production of fibronectin, perlecan, laminin gamma(1), and chondroitin sulfate. Beclomethasone caused a significant reduction in the number of cells exposed to serum from either the asthmatic or nonasthmatic individual, but did not reverse the increase in ECM protein induced by the former. These results suggest an interaction between the ASM and the allergic process that may alter components of the airway wall in asthma, and that corticosteroids may not prevent the fibrosis induced by resident cells within the airways.

Rationale: We have previously shown cigarette smoke extract (CSE) induces the production of the extracellular matrix proteins fibronectin and perlecan in human lung fibroblasts (Krimmer et al AJRCMB 2011). The proteins were also induced by transforming growth factor β (TGFβ); however the signal transduction pathways activated by CSE were different to those used by TGFβ. These differences lead us to speculate that if we were to look at an increased number of molecules involved in the development of fibrosis, we would find differential upregulation by TGFβ and CSE. Aim To compare fibrosis related genes upregulated by TGFβ or CSE in primary human bronchial epithelial (HBE) cells. Methods: HBE cells were grown to confluence in BEGM in the absence or presence of 5ng/ml of TGFβ1 or 5% CSE. Total RNA were collected after 72 hours. The expression of fibrosis related genes was measured by real time PCR. In total we examined 85 fibrosis related genes. A cut off of a minimum of 1.5 fold induction was used to indicate upregulation of a gene. Results: TGFβ upregulated 49 different fibrosis associated genes, CSE upregulated only 16, and of all the upregulated genes, 4 were uniquely upregulated by CSE (catenin β1, extracellular matrix protein 1, and TIMP 1 and 2). As expected, TGFβ upregulated a variety of ECM proteins (eg collagen I, IV-VIII, fibronectin); integrins (eg integrin α2, α4-6), and degradative enzymes (eg matrix metalloproteinase (MMP) 1-3, 9-16). In contrast, CSE upregulated only two collagens (XI and XIV) and MMP9. Conclusion: As CSE selectively upregulated a subset of TGF-β induced genes, elucidation of the specific mechanisms involved is likely to give novel insight into the pathophysiology of COPD.