INTRODUCTION

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Airway remodeling is a key feature of asthma. Part of this remodeling is characterized by an increase in the amount of airway smooth muscle (ASM) within the airway wall (1). This is likely to be an important etiologic factor in airway narrowing that accompanies asthma (4). Although there has been some uncertainty as to whether this is caused by hyperplasia or hypertrophy, studies indicate that both abnormalities occur, with a preponderance of hyperplasia (3). The cause of this hyperplasia is unknown, although many inflammatory mediators produce mitogenesis in a variety of animal and human ASM cells. However, these studies have been unable to address the differences between asthmatic and nonasthmatic cells because ASM cells from asthmatic patients were not available. Recently we and others have reported that exposure of nonasthmatic cells to serum from atopic asthmatic patients results in differences in cell cycling and cytokine production (5, 6). In addition, Naureckas and coworkers have found that cells from nonasthmatic individuals showed increased cell proliferation in the presence of bronchoalveolar lavage fluid (BALF) from asthmatic patients, an effect which was further accentuated when the BALF was obtained after allergen challenge (7). Thus the preceding studies have addressed the issue of whether asthmatic serum and lavage fluid are able to induce enhanced proliferation of nonasthmatic ASM cells.

In this study we have been able to culture ASM cells from asthmatic patients and thereby address the pivotal question as to whether asthmatic cells exhibit a different pattern of proliferation from that of cells obtained from nonasthmatic patients.

	METHODS

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Human ASM cells were obtained from 10 nonasthmatics and 12 asthmatics by methods adapted from those previously described (8). Approval for all experiments with human lung was provided by the human ethics committee of the University of Sydney and the Central Sydney Area Health Service. Nonasthmatic human ASM was obtained from bronchial airways of 10 patients undergoing resection for either lung transplantation or carcinoma. Asthmatic ASM was obtained from two patients undergoing resection for lung transplantation, one patient dying in status asthmaticus, and nine deep endobronchial biopsies obtained by flexible bronchoscopy (see Table 1 for patient details). Pure ASM bundles were dissected free from surrounding tissue with the aid of a dissecting microscope. The small pieces of muscle bundles were then plated in 12-cm² flasks as previously described (8). ASM cell characteristics were determined by immunofluorescence and light microscopy. Cells were stained with antibodies against -smooth muscle actin and calponin while omission of the primary antibody was used as a control (11). Sensitization status was assessed, as previously described (5) in two bronchial rings obtained from each of the lung specimens (excluding the endobronchial biopsy specimens).

Experimental Design

The ASM cells (passage 4 to 8) from the 22 patients were seeded at a density of 1 × 10⁴ cells/cm² for all experiments. Cells were plated in 5% fetal bovine serum (FBS) in Dulbecco's modified Eagle medium (DMEM) for 24 h and the medium was changed to 1% FBS in DMEM for a further 24 h to synchronize the cells. After 24 h in 1% FBS in DMEM, the cells were counted (designated Day 0); the medium was then changed to 5% FBS in DMEM.

Tritiated Thymidine Assay

ASM cells were seeded in triplicate in 24-well plates. After 24 h in 1% FBS in DMEM the medium was changed to either 1% or 5% FBS containing 1 µCi/well tritiated thymidine. After 24-h exposure, tritiated thymidine incorporation was assessed. Cells were washed with Dulbecco's phosphate-buffered saline (PBS), fixed with methanol: acetic acid (3:1), washed again with Dulbecco's PBS, then lysis performed with 0.5 M NaOH. The cell lysate (200 µl) was then added to 2 ml scintillant, the solution mixed, and counts per minute (cpm) were determined on a Beckman scintillation counter. Results were expressed as a percentage of the cpm in the presence of 1% FBS.

Flow Cytometric Analysis of DNA Content

ASM cells were seeded in duplicate in 6-well plates. After 24 h in 1% FBS in DMEM the medium was changed to 5% FBS. The ASM cells from the different patients were harvested from the wells using phenol red-free trypsin-ethylenediaminetetraacetic acid (EDTA) at 24 and 48 h. Cells were then immediately permeabilized and stained in the unfixed state using a solution of 0.5% wt/vol saponin and 0.1% wt/vol bovine serum albumin (BSA) in PBS containing propidium iodide, 50 µg/ ml, and ribonuclease A, 50 µg/ml. Data from the stained cell suspensions were acquired on a FACSCalibur Sort (Becton Dickinson, Sydney, Australia) using Cell Quest software (Becton Dickinson). The resulting DNA profiles were analyzed using FL2 peak area and width and Modfit software (Verity Software House Inc., Topsham, ME) to determine the percentage of cells in each phase of the cell cycle.

Cell Counting

ASM cells were seeded in triplicate in 12-well plates. After 24 h in 1% FBS in DMEM the medium was changed to 5% FBS and manual cell counting performed on Days 0, 3, 5, and 7. Results are expressed as a percentage of the cell counts on Day 0. Cell viability was determined by trypan blue exclusion as previously described (8).

Materials

The following compounds were obtained from the sources given in parentheses: DMEM, Dulbecco's PBS, penicillin, streptomycin, amphotericin B, trypan blue (Life Technologies, Melbourne, Australia); EDTA disodium salt, (Ajax, Sydney, Australia); FBS (CSL, Sydney, Australia); saponin, BSA, propidium iodide, ribonuclease A, fluorescein isothiocyanate (FITC)-conjugated monoclonal anti- smooth muscle actin (mouse IgG2 isotype), monoclonal anti-calponin (mouse IgG1), FITC-conjugated goat anti-mouse IgG, acetylcholine perchlorate, and chemicals to make Krebs-Henseleit solution (Sigma, Sydney, Australia); emulsifier safe (scintillant) (Canberra-Packard, Melbourne, Australia); antigen extracts of Dermatophagoides pteronyssinus standardized mite DP 30,000 BAU/ml, Timothy Phleum Pratense 1:20 wt/vol, Alternaria tenuis 1:10 wt/vol, and cat pelt 10,000 bioequivalent allergy units (BAU)/ml (Miles Laboratories, Inc., Elkhart, IN).

Statistics

Results from duplicate or triplicate wells from each individual patient were averaged and an overall mean and SE were calculated from values obtained from the 12 asthmatic and 10 nonasthmatic patients. Analysis of variance (ANOVA) using repeated measures and the Fisher projected least significant difference (PLSD) post test was performed on the results for tritiated thymidine, cell counts on individual days, and flow cytometric analysis of DNA content. Factorial ANOVA was performed on the proliferation curves comparing the asthmatic and nonasthmatic cells. In all cases a p value of less than 0.05 was considered significant.

	RESULTS

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Under light microscopy, both the asthmatic and nonasthmatic cells appeared spindle-shaped, with central oval nuclei containing prominent nucleoli, and displayed the typical "hill and valley" proliferation pattern in culture. Cells from both asthmatic and nonasthmatic patients showed uniform staining for both the smooth muscle-specific contractile proteins -smooth muscle actin (Figure 1A) and calponin (Figure 1B), indicating that these cells were ASM. Cells from asthmatic patients proliferated significantly faster than cells from control subjects as assessed by manual cell counting, tritiated thymidine incorporation, and fluorescent-activated cell sorter (FACS) analysis of DNA content. Cell numbers at Days 3, 5, and 7 were 238 ± 23, 301 ± 34, and 381 ± 38%, respectively of values for Day 0 in the asthmatic group. Corresponding values in control cells were significantly lower at each time point193 ± 24, 210 ± 21, and 229 ± 24%, respectively (p < 0.05) (Figure 2). Statistical analysis of the proliferation curves comparing the asthmatic cells with control cells revealed a significant difference between the curves (ANOVA factorial analysis) with the asthmatic proliferation curve lying to the left (Figure 2). The doubling time for the asthmatic cells was significantly increased compared with control, 2.9 ± 0.4 and 5.4 ± 1.3 d, respectively (p < 0.05). Cell viability as determined by trypan blue exclusion was greater than 99%. Tritiated thymidine incorporation was increased 3.2-fold in asthmatic cells compared with controls within the first 24 h (89 ± 13% nonasthmatic and 281 ± 54% asthmatic p < 0.05) (Figure 3). Flow cytometric analysis of DNA content revealed that a significantly greater percentage of asthmatic cells were in the G₂ + M phase than controls at 24 and 48 h (7 ± 2% nonasthmatic and 15 ± 1% asthmatic for 24 h [Figures 4A and 4B] and 9 ± 2% nonasthmatic and 14 ± 2% asthmatic for 48 h).

View larger version (133K): [in this window] [in a new window]   Figure 1.   Immunohistochemical staining of the airway smooth muscle cells for -smooth muscle actin (A) and calponin (B). Cells from both asthmatic and nonasthmatic patients showed consistent staining for both airway smooth muscle marker proteins. Cells were viewed under a 40× objective.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Cell number in response to exposure to 5% FBS in DMEM for Days 0, 3, 5, and 7. Results are expressed as a percentage of the cell number in the presence of 1% FBS in DMEM on Day 0. *Significant difference from nonasthmatic (p < 0.05, n = 12 asthmatic and 10 nonasthmatic).

View larger version (34K): [in this window] [in a new window]   Figure 3.   3H thymidine incorporation in response to 24-h exposure to 5% FBS in DMEM. Results are expressed as a percentage of the counts per minute in the presence of 1% FBS in DMEM. *Significant difference from nonasthmatic (p < 0.05, n = 3 asthmatic and 3 nonasthmatic).

View larger version (28K): [in this window] [in a new window]   Figure 4.   Cell cycle analysis after 2 d exposure to 5% FBS in DMEM. (A) Results are expressed as a percentage of the total number of cells. *Significant difference from nonasthmatic (p < 0.05, n = 3 asthmatic and 3 nonasthmatic). (B) DNA histogram from FACS cell analysis of a typical experiment. Filled peak represents the nonasthmatic response, unfilled peak the asthmatic response.

	DISCUSSION

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The cause of the increase in bulk of the ASM in the airways of asthmatic patients has been the focus of extensive research. Although many groups have identified mediators that have the potential to increase the proliferation of cultured ASM cells, no groups have reported on the proliferation of asthmatic ASM cells in culture. This study is the first to report that ASM cells obtained from asthmatic patients proliferate faster in culture than those obtained from nonasthmatic patients. In addition, this is the first report of the growth of human ASM cells from muscle bundles obtained from endobronchial biopsies.

Under light microscopy, cells from both groups looked similar. Cells from both groups showed uniform staining for the smooth-muscle specific contractile proteins -smooth muscle actin and the smooth muscle-specific protein, calponin (12). The only other cell with the same morphology which may have contaminated our cultures is the fibroblast; however, calponin is undetectable in fibroblasts (12) and thus the presence of this cell type in our cultures is unlikely. Moreover, Stewart has observed that when the proliferation of asthmatic fibroblasts obtained from endobronchial biopsies is compared with that of nonasthmatic control subjects, there is a relative decrease in the proliferation of the asthmatic fibroblasts, thus further highlighting the differences between fibroblasts and ASM cells (personal communication, Alastair Stewart 2000). There is a remote possibility that myofibroblasts may have contributed to the cell population; however, whether myofibroblasts can be distinguished from myocytes is controversial. Moreover, the site from which we obtain our muscle cells (i.e., the smooth muscle bundles) makes it unlikely that myofibroblasts were included, because these cells have been reported to be located immediately beneath the basement membrane and not within the ASM bundles.

Care must be taken when extrapolating from results of cells in culture to in vivo conditions; however, the fact that our results indicate that an inherent abnormality of the ASM proliferation exists suggests that this may be representative of the in vivo situation. Our results showed a substantial increase in the proliferation of the asthmatic cells in culture, which may translate to a small increase in vivo; however, the period over which the increase in the ASM in the airways of asthmatic patients occurs may be extended, suggesting that any alteration in the proliferation of the muscle over time may have profound effects on muscle bulk. Given that our results would indicate the presence of an intrinsic abnormality in the proliferation characteristics of the ASM from asthmatic patients, the nature of this abnormality would have to be such that it is maintained in culture conditions and through several passages. There are precedents for this phenomenon. Das and coworkers reported that fibroblasts obtained from chronic hypoxia-induced pulmonary hypertensive neonatal calves exhibited increased proliferation which was maintained in culture and indeed through several passages (13).

The exact nature of the abnormality producing the accelerated muscle proliferation in the cells from asthmatic patients remains to be elucidated. Recent evidence points to an active participation of the ASM in inflammatory responses, in that these cells can produce cytokines, express adhesion molecules for inflammatory cells and a variety of cell surface molecules including Fas (14), and can produce a number of extracellular matrix proteins (15), matrix metalloproteinases and their inhibitors (16). Many of the cytokines produced by the ASM can induce proliferation; thus, the ASM may respond in an autocrine manner to overproduction of one or more of these cytokines (5). Moreover, animal studies indicate that inflammatory mediators have the potential to induce hyperplasia of the ASM in vivo (17).

The cells used in this study were obtained from both resection specimens and from endobronchial biopsies. The question arose as to whether the source of the cells would influence proliferation patterns. The ASM cells obtained from biopsies exhibited a similar proliferation pattern to cells obtained from asthmatic resection specimens. Because the asthmatic patient group was younger than the nonasthmatic group, this could have influenced the proliferation pattern. However, a recent study of fibroblasts isolated from 150 patients indicates that donor age does not influence proliferation pattern of cells in culture (18). Moreover, when we analyzed a subset of our patients with a similar mean age of 45 ± 1 yr (mean ± SD, n = 3) for asthmatics and 51 ± 2 yr (mean ± SD, n = 3) for nonasthmatics, although the number for comparison was small we still observed significant changes in cell proliferation. Moreover, when we looked at the preoperative diagnosis of a subset of patients (all chronic obstructive pulmonary disease [COPD], n = 5), it was observed that proliferation was significantly less and the doubling time was significantly greater in the COPD group compared with the asthmatics (asthmatics 2.9 ± 04 and COPD 6.6 ± 2.1 d), highlighting the differences in pathophysiology of COPD and asthma.

Previous studies have used models of asthmatic airways to examine the proliferation of human ASM. Black and Johnson examined the effect of passive sensitization of nonasthmatic ASM and observed that antigen exposure increased the proliferation of the smooth muscle (5). Others have shown that bronchoalveolar lavage (BAL) from asthmatic patients contains substances that are mitogenic for ASM and that this mitogenesis is enhanced after allergen challenge (7). Whereas these studies point to the influence of allergic or asthmatic factors influencing the proliferation of the ASM, the present study shows for the first time that, regardless of its environment, the asthmatic ASM is intrinsically able to grow more rapidly. This abnormality may explain the consistent finding of increased ASM in asthmatic airways in vivo.