Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects

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Abstract

Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC20 methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC20 and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.

Introduction

Airway hyperresponsiveness and bronchial inflammation are two hallmarks of asthma. More recently, an airway remodeling process involving altered extracellular matrix deposition has been recognized as a prominent feature of asthma [1], [2]. These changes in the extracellular matrix may contribute to abnormal airway function by altering airway mechanics and modulating inflammatory and structural cell function. In addition, airway fibrosis may lead to airway hyperresponsiveness. Indeed, both our group and others found a significant correlation between airway responsiveness and the degree of bronchial subepithelial fibrosis in asthmatic subjects [3], [4], [5].

In chronically inflamed airway tissues, activated fibroblasts are likely to contribute to the excessive deposition of different extracellular matrix components [6]. Functionally, asthmatic bronchial fibroblasts can be distinguished from their normal counterparts regarding contraction of collagen lattices [7], in vitro life-span [8], and expression of metalloproteinases [9], [10]. Moreover, cytokine production by bronchial fibroblasts could contribute to the asthmatic airway inflammation by regulating recruitment and activation of inflammatory cells [11], and inhibiting eosinophil apoptosis [12].

The deposition of collagen and fibronectin in the bronchial subepithelial region and its association with increased numbers of activated fibroblasts are well documented in asthma [13]. In addition to structural collagens and fibronectin, the airway walls contain proteoglycans, a family of ubiquitously distributed glycosylated proteins covalently linked with glycosaminoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects including cell–matrix and matrix–matrix interactions, cell proliferation, and binding and storage of growth factors [14].

To our knowledge, there are only three reports on the immunolocalization of proteoglycans in the asthmatic bronchial mucosa. As part of a review paper, Roberts mentioned the localization of versican, a large chondroitin sulphate proteoglycan, biglycan and decorin, two small chondroitin/dermatan sulphate proteoglycans, as well as hyaluronan, a glycosaminoglycan not attached to a core protein, in the bronchial mucosa of individuals who died from severe asthma [1]. The other reports are somehow more interesting because of the mildest form of asthma studied. Redington and colleagues showed that the overall pattern of decorin immunolocalization was similar in biopsy specimens from asthmatic and normal subjects [15]. Others found an increased staining for lumican, biglycan and versican, but not decorin, in the subepithelial layer of the airways in asthmatic subjects in comparison with that in normal subjects [16]. In this latter study, proteoglycan immunostaining was correlated with the extent of airway responsiveness in asthmatic subjects supporting the hypothesis that altered proteoglycan deposition may alter airway properties in asthma [17]. In addition, altered proteoglycan structure and production may have profound effects on the properties of the extracellular matrix, which can affect cell growth and migration as well as organization of matrix fibers [6].

An abnormal constitutive metabolism of proteoglycans characterizes fibroblasts from different fibrotic conditions, such as systemic or localized sclerosis [18], inflamed periodontal tissues [19], and nasal polyposis [20] to name a few. However, the production of proteoglycans by bronchial fibroblasts has not yet been investigated. In the present study, we measured the production of proteoglycans by bronchial fibroblasts from normal and asthmatic subjects and showed that there is a correlation between proteoglycan production by these cells and airway responsiveness of donor subjects.

Section snippets

Chemicals

Except when mentioned, all chemicals were obtained from sources previously listed [21], [22].

Bronchial fibroblast lines

Primary bronchial fibroblast lines from six mild asthmatic and four normal subjects were used in this study (Table 1). Asthmatic subjects had airway responsiveness (PC20 methacholine) ranging from 0.03 to 12.78 mg/ml and were all atopic using only an inhaled β2-agonist on demand. None have ever used inhaled or systemic corticosteroids. Normal subjects were non-atopic with no history of asthma or systemic

Production of hyaluronan and total proteoglycans

Fibroblast cultures secreted the main part of hyaluronan and total proteoglycans into the medium. Only 3.4%±0.7% of the former and 10.6%±1.5% (mean±standard error) of the latter was detected in the cell layer. Cells from subjects with the most hyperresponsive airways (low PC20 methacholine) produced up to four-fold more total proteoglycans than cells from subjects with less hyperresponsive and normoresponsive airways (high PC20) as reflected by the significant correlation, rs=−0.723 (P=0.030),

Discussion

The present study shows that serum-stimulated fibroblasts from subjects with the most hyperresponsive airways produced conspicuously more proteoglycans than cells from subjects with less hyperresponsive and normoresponsive airways. This translates into a significant negative correlation between PC20 methacholine and production of total proteoglycans, perlecan, small HSPGs and biglycan, but not decorin, while there was a tendency for hyaluronan and versican. Such alteration of proteoglycan

Acknowledgements

This work was supported in part by grants to G.M.T. from the Association pulmonaire du Québec, and to G.W.-T. from the Swedish Medical Research Council (11550), Heart and Lung Foundation, Swedish Cancer Fund, Swedish Society for Medical Research, JA Persson, G Nilsson, Greta and John Kock, A Österlund and Anna-Greta Crafoord Foundations, Riksföreningen mot Rheumatism, Gustaf V.s 80 Årsfond and the Medical Faculty, Lund University. J.C. and G.M.T. are scholars from Fonds de la recherche en santé

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