INTRODUCTION

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Asthma is a common, sometimes fatal disease, characterized by episodic airway obstruction, airway hyperresponsiveness, and chronic airway inflammation. Ongoing inflammation may result in structural remodeling and thickening of the airway wall (1, 2). This airway wall thickening may contribute to the exaggerated airway narrowing observed in asthmatics; not only is the actual airway caliber affected, but the decrease in airway caliber for a given degree of airway smooth muscle shortening may also be enhanced (3, 4). Controversies have emerged recently about the association between airway inflammation and airway hyperresponsiveness in asthma (5). Haley and Drazen (7) have recently pointed out that airway wall remodeling, rather than inflammation per se, may be the major determinant.

The nature of the airway wall thickening seen in asthma is not completely understood. A number of studies have reported an increase in the subepithelial layer resulting from excess interstitial collagen and fibronectin deposition (1, 8). However, relatively little is known about changes in the other extracellular matrix (ECM) components, i.e., proteoglycans (PGs). PGs, which are involved in many aspects of ECM pathophysiology (9), are macromolecules composed of a protein core and glycosaminoglycans (GAG) side chains. Roberts (10) has reported a marked deposition of versican and hyaluronan in airway walls in postmortem tissue from patients with severe asthma. His findings suggest the possibility that changes in PG metabolism may contribute to altered ECM properties in the asthmatic airway wall.

The purpose of this study was to examine bronchial biopsies from patients with mild atopic asthma and nonatopic, nonasthmatic control subjects in order to compare PG deposition in the airway wall. We hypothesized that the deposition of PGs would be increased in asthmatic airways, and that this change would be correlated with airway hyperresponsiveness.

	METHODS

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Seven atopic asthmatic subjects and six nonatopic normal subjects were recruited from the Asthma Clinic of the Montreal Chest Institute, McGill University (Montreal, PQ, Canada). Clinical and demographic characteristics of the subjects are shown in Table 1. Asthmatic subjects studied were in stable condition and had clinically mild asthma requiring only occasional ₂-agonist therapy. None of the asthmatics had received inhaled (> 1,000 µg/d) or oral steroid therapy in the 3 mo prior to the study, nor inhaled steroid therapy ( 1,000 µg/d) in the 30 d prior to the study. Baseline FEV₁ was greater than 60% of predicted value. All asthmatic subjects had increased bronchial reactivity to inhaled methacholine (i.e., provocative concentration required to decrease FEV₁ by 20% of its baseline value [PC₂₀] less than 4 mg/ml). Atopy was demonstrated by a positive skin-prick test (> 3-mm skin wheal) to one or more of 15 common aeroallergens. All the subjects were nonsmokers, had no evidence of any other pulmonary disease, and had not experienced an upper respiratory tract infection during the 2 mo preceding the study. The study was approved by the Ethics Committee of the Montreal Chest Institute, and all subjects gave written informed consent.

Fiberoptic bronchoscopy was performed following American Thoracic Society guidelines. Three to five biopsies per patient were taken from the segmental divisions of the right middle and right lower lobe bronchi using alligator forceps. Endobronchial biopsies were embedded in OCT (Tissu-Tek; Miles Inc., Elkhart, IN), snap-frozen in isopentane (Fisher Scientific, Montreal, PQ, Canada) cooled in liquid nitrogen, and cut with a cryostat in 5-µm sections. Sections were fixed in acetone/methanol and stored at 80° C.

Sections were incubated with primary rabbit antihuman polyclonal antibodies (IgG) for lumican, biglycan, decorin, and fibromodulin (generously provided by Dr. Peter Roughley, Shriners Hospital for Crippled Children, McGill University), and mouse antihuman monoclonal antibody (IgG₁) for versican (Seikagaku American Inc., Ijamsville, MD). The tissue was then incubated with biotinylated swine antirabbit IgG (lumican, biglycan, decorin, and fibromodulin) or biotinylated rabbit antimouse IgG (versican) (Dako Canada, Inc., Mississauga, ON, Canada), washed again, and incubated with alkaline phosphatase-conjugated avidin (Dako). Sections were developed with Fast Red (Sigma). Control sections were processed in the absence of the primary antibody. We evaluated one biopsy specimen from each patient.

A semiquantitative analysis was done by calculating the area of positive staining (mm²) in the subepithelial layer, i.e., between the epithelium and the smooth muscle layer, correcting for basement membrane length (mm). The area of positive staining and length of basement membrane were traced using a conventional light microscope with a drawing tube attachment. The trace was then read from a digitizing table (Jandel Scientific, Corte Madera, CA), and the area and basement membrane length were calculated by a Sigma-Scan computer software package (Jandel Scientific). All sections were coded and studied in a blinded fashion by a second observer; interobserver coefficient of variation was less than 5%. (Values reported are those obtained by the first observer.)

Comparisons between PG deposition in atopic asthmatics and normal control subjects were performed using Student's t test. Correlation between PG deposition and pulmonary function variables was performed using linear regression. Values of p < 0.05 were accepted as statistically significant.

	RESULTS

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Lumican, biglycan, and versican immunoreactivity was identified in all bronchial biopsies from atopic asthmatics and normal control subjects (Figure 1). These molecules were localized predominantly in the subepithelial layer, although some staining was also observed in and around smooth muscle bundles. No staining was found in the epithelium. Only minimal staining for decorin was observed in one of seven atopic asthmatics and one of six normal control subjects. Staining for fibromodulin was not observed in the subepithelial or smooth muscle layer in any of the subjects (Figure 1). (In one subject bronchial cartilage was sampled in the biopsy specimen; the cartilage stained intensely positive for fibromodulin.)

View larger version (120K): [in this window] [in a new window]   Figure 1.   Representative photomicrographs (original magnification: ×200) of immunostaining in bronchial biopsies from an asthmatic (A: lumican; C : biglycan; E: fibromodulin) and a normal control subject (B: lumican; D: biglycan; F : fibromodulin).

Lumican, biglycan, and versican staining was significantly increased in the subepithelial layer of the airway wall in asthmatics as compared with that in normal control subjects (Figure 2). In addition, a significant correlation was found between PG deposition in the subepithelial layer and airway responsiveness to methacholine in asthmatics. The amount of both lumican and biglycan deposition correlated inversely with the methacholine PC₂₀ value (p < 0.05). Although the versican deposition also showed an inverse correlation with PC₂₀, this value did not reach statistical significance (p = 0.06) (Figure 3). There was no correlation between lumican, biglycan, or versican deposition and FEV₁ or FEV₁/FVC ratio (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Area of immunostaining for lumican, biglycan, and versican in the bronchial biopsies of asthmatics and normal control subjects. *p < 0.05, **p < 0.01, compared with control subjects. BM = basement membrane.

View larger version (11K): [in this window] [in a new window]   Figure 3.   Correlation between (A) subepithelial lumican and PC20, (B) subepithelial biglycan and PC20, and (C ) subepithelial versican and PC20 in asthmatics (n = 7). There were significant correlation between lumican and PC20 (p < 0.05) and between biglycan and PC20 (p < 0.05). BM = basement membrane.

	DISCUSSION

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In this study we observed prominent staining for lumican, biglycan, and versican in the subepithelial layer of the airway wall in atopic asthmatics. Deposition of these PGs was significantly increased in the airways of asthmatics as compared with control subjects. Moreover, in asthmatics, a significant correlation was found between the amount of PG in the airway wall and airway responsiveness.

Alteration in PG deposition in asthmatic airways has not been well studied, although other matrix proteins such as collagen have been found to be increased in asthma (1, 8). Roche and coworkers (1), for example, found that the thickness of the collagenous matrix band of the basement membrane was almost doubled in bronchial biopsies from a group of asthmatics when compared with normal subjects. It has been postulated that the increase in subepithelial collagen is a part of the inflammatory response to the offending agent (11) and represents a marker of structural changes in the airway wall related to the chronic nature of the disease (12). There is less information available on the role of PGs in airway wall remodeling in asthma. Roberts (10) has reported that versican, biglycan, decorin, and hyaluronan were localized in airways in postmortem lung tissue from six patients who had severe asthma and for whom asthma was considered to be either the cause of, or a significant contributor to, death. We performed immunostaining for these specific PGs, as well as fibromodulin, a small PG of the same family as biglycan and decorin, and lumican, again a small, leucine-rich repeat PG, which we have recently shown to be present in the peripheral airway wall in lung tissue from humans undergoing tumor resections (13). In addition, we compared airways from asthmatics with those of normal control subjects.

PGs are complex macromolecules that consist of a protein core and one or more covalently bound GAG sidechains (9). Nearly 30 individual PGs have been identified in the ECM, on the cell surface, or inside cells. Among the PGs in ECM, lumican, biglycan, decorin, and fibromodulin belong to the family of relatively small, leucine-rich repeat PGs. Versican is a large chondroitin sulfate PG that contains a C-terminus homologous to that of the selectins (14). The upregulation of PGs in asthmatic airway wall may be driven by inflammatory mediators such as TGF- (8) or by mitogens such as platelet-derived growth factor (PDGF) (15). Versican has been shown to be upregulated in smooth muscle cells exposed to TGF- and PDGF (16). The functional role of increased PG deposition in the airway wall of asthmatics is unknown. PGs bind with fibrillar collagen and influence the interaction of collagen fibrils and their assembly (9, 17). They also bind with other components of ECM and may affect cell adhesion (9, 17).

A principal feature that distinguishes asthmatics from normal subjects is that the airways of asthmatics narrow excessively in response to a contractile stimulus. There have been various mechanisms suggested to explain this excessive airway narrowing in asthma (3, 4). Increased smooth muscle force caused by either an increase in the amount or contractility of ASM, could result in increased smooth muscle shortening for a given dose of agonist and, therefore, an enhanced contractile response (18). A recent study by Thompson and colleagues (19), however, reported no differences in the proportion of smooth muscle, the ECM between smooth muscle, or the connective tissue within smooth muscle bundles in the airways of asthmatics and normal control subjects. Similarly, conflicting data have been reported in the literature regarding altered contractility of asthmatic ASM. Some studies have reported that asthmatic smooth muscle demonstrates enhanced contractility (20), others have reported normal contractility (21), and still others, decreased contractility (22). Airway wall thickening as a result of chronic inflammation is another important mechanism that could lead to exaggerated airway narrowing in asthma. Wiggs and coworkers (4), in a modeling study, showed that small changes in airway wall thickness, which had little effect on baseline airflow resistance, could dramatically accentuate the airway narrowing caused by "physiologic" amounts of agonist-induced smooth muscle shortening. Although these investigators ascribed responsiveness in their model to changes in the peripheral airways, it is not unreasonable to postulate a similar effect might apply in more central airways.

We found that there was a significant correlation between the enhanced subepithelial deposition of PG in the airway wall and airway responsiveness to methacholine in patients with mild asthma. Recently, Haley and Drazen (7) have called into question the implied relationship between the presence of inflammatory cells in the airway and airway hyperresponsiveness. They comment on the findings of Crimi and colleagues (6) that failed to show a clear relationship between the presence of inflammatory cells in the airway and enhanced airway responsiveness in asthmatics. Inflammatory cells in asthmatic airways release not only short-lived mediators such as histamine, leukotrienes, and platelet-activating factor, they also release cytokines and chemokines, which effects may occur on a more chronic basis (7). It is possible that these latter factors contribute to airway hyperresponsiveness as a result of the chronic airway wall remodeling they induce (7). Our data show a relationship between the amount of PGs in the airway wall and methacholine responsiveness support such an hypothesis.

In conclusion, this study demonstrated that the deposition of both small PGs, i.e., lumican, and biglycan, and the large PG, versican, was significantly increased in the airway wall of patients with mild atopic asthma. The increase in each PG was correlated with increased airway responsiveness. Our results suggest that PGs may play a role in airway wall remodeling and airway mechanics in mild asthma. Their role in more severe asthma, and their sensitivity to modulation by anti-inflammatory agents such as steroids, needs to be assessed. Studies are also needed to explore the mechanisms by which PGs are upregulated and precisely how they contribute to the airway wall remodeling observed.