Introduction

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The cysteinyl leukotrienes (CysLTs; LTC₄, LTD₄, and LTE₄) are lipid mediators synthesized de novo from membrane-associated arachidonic acid via the activity of 5-lipoxygenase and a membrane-bound 5-lipoxygenase activating protein. In the early 1980s, the CysLTs were identified as the active constituents of the "slow reacting substance of anaphylaxis" and were postulated to play a significant role in the pathophysiology of asthma (1). Subsequently, studies confirmed that CysLTs mimic many of the features of asthma, including mucus secretion, inflammatory cell recruitment, edema, and neuronal dysfunction, as well as bronchoconstriction, and thus may play a multidimensional role in its pathophysiology (1). Few studies have examined the effects of CysLTs on airways remodeling, with no reported findings in human airway smooth muscle (HASM) cells. Experiments in rat and rabbit lung in vitro and in vivo have provided some preliminary evidence that the CysLTs promote airway smooth muscle mitogenesis (5, 6).

Airway smooth muscle cell hyperplasia and subepithelial fibrosis are characteristic findings in the airways of patients with chronic severe asthma (7). Although the precise cellular mechanisms regulating smooth muscle cell growth remain unknown, recent evidence suggests that some, but not all, contractile agonists induce HASM cell proliferation (7). Interestingly, the airway smooth muscle contractile agonist endothelin-1 alone does not induce HASM cell growth but potentiates mitogen-induced proliferation (9). Despite subepithelial fibrosis being recognized as a common pathologic finding in airways of patients with mild asthma, only recently has this fibrosis been described as being due, in part, to deposition of types III and V collagen (10). Subepithelial fibrosis is more likely the result of increased secretion of these collagen proteins by mesenchymal cells such as myofibroblasts or airway smooth muscle cells, rather than by epithelial cells which produce type IV collagen (10). Little is known with regard to the substances that modulate collagen or matrix secretion in HASM cells. However, LTD₄ stimulates collagen synthesis in murine osteoblasts (11).

The goals of this study were: (1) to explore the actions of LTD₄, alone and in combination with the growth factors epidermal growth factor (EGF) and thrombin, on HASM proliferation and on expression of several extracellular matrix components; (2) to characterize, with the use of three structurally distinct CysLT receptor antagonists pranlukast (SB 205312; ONO-1078; Ultair^TM) (12), zafirlukast (ICI 204,219, Accolate^TM) (16), and pobilukast (SK&F 104353) (19, 20)the receptor responsible for the effects of LTD₄ on HASM growth; and (3) to compare the receptor population involved in LTD₄-induced contraction of HASM with that responsible for the effects on HASM cell proliferation.

	Materials and Methods

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Airway Smooth Muscle Proliferation Studies

Airway smooth muscle cell culture. Human trachea was obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the trachealis muscle was isolated; using this technique, approximately 0.5 g of wet tissue is obtained. The isolated muscle was then minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl₂, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min at 37°C in a shaking water bath. The cell suspension was then filtered through 105-mm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT). Aliquots of the cell suspension were plated at a density of 1.0 × 10⁴ cells/cm². Ham's F12 medium was supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 mg/ml amphotericin B, and was replaced every 72 h. Cell counts were obtained using a Coulter counter (Coulter Electronics, Hialeah, FL) in triplicate wells with 0.5% trypsin in 1 mM ethylenediamenetetraacetic acid (EDTA) solution.

[³H]Thymidine and cell proliferation assays. Proliferation studies were performed on confluent, growth-arrested HASM cells. Cells were growth-arrested by incubating the cultures at Day 10 in serum-free media consisting of F12 medium with 5 ng/ml insulin, and 5 ng/ml transferrin (9, 21). Confluent, growth-arrested cells were used because cells can be synchronized in the G₀/G₁ phase of the cell cycle and, at this baseline, incorporation of [³⁵S]-methionine and [³H]thymidine is minimal (21). After 24 h in serum-free media, the cells were stimulated with either EGF (1 ng/ ml) (Sigma Chemical Co., St. Louis, MO), 10% FBS, LTD₄ (0.1-10 µM), or a combination of EGF and LTD₄. In a separate series of experiments, cells were pretreated with a CysLT receptor antagonist (pranlukast, pobilukast, or zafirlukast) for 20 min, and DNA synthesis was measured after stimulation with agonists as described below. After 16 to 18 h of stimulation with mitogen, HASM cells were incubated in the presence of 1.0 µCi/ml of [methyl-³H]- thymidine (40-60 Ci/mmol; Amersham, Arlington Heights, IL) for 12 to 18 h. The cells were then scraped off the plate and lysed, and the protein/DNA was precipitated with 12% trichloroacetic acid; the precipitant was aspirated onto glass filters, extensively washed, and dried, and the radioactive incorporation was determined using liquid scintillation counting (21). [³H]Thymidine incorporation experiments were performed in parallel with cell proliferation assays. Cell number was quantified by removing cells from wells with 0.5% trypsin-1 mM EDTA solution. Cell counts were then obtained from each well using a Coulter Counter.

Extracellular Matrix Component Expression Studies

Effects of LTD₄ and tumor growth factor-. HASM cells were grown in F12 medium with 10% serum until confluence and then quiesced for 48 h in serum-free media containing insulin (5 µg/ml), transferrin (5 µg/ml), and ascorbic acid (50 µg/ml). Monolayers were then stimulated with LTD₄ (0.1 and 10 µM) and/or tumor growth factor- (TGF-), and messenger RNA (mRNA) expression was examined as described below; TGF-, which stimulates collagen and extracellular protein expression in vascular smooth muscle cells (22), served as a positive control in these experiments. Vehicle-treated control cells without LTD₄ or TGF- were also examined.

Complementary DNA probes. The human complementary DNA (cDNA) probes that were employed in Northern transfer analysis included: a 1.5-kb pro-1(I) (HF-677) cDNA of the human pro-1(I) chain of type I procollagen (23); a 2.6-kb 1(IV) (HT-21) cDNA corresponding to the helical portion of human 1(IV) chain procollagen (24); a 1.7-kb fibronectin (HF-771) cDNA (25); a 1.6-kb decorin (PG-40) cDNA (26, 27); a 1.6-kb biglycan cDNA (P16) (27) (both decorin and biglycan were generously provided by Dr. Larry W. Fisher, Bone Research Branch, NIDR, NIH, Washington, DC); a 1.3-kb human versican proteoglycan (PG-350) cDNA (28); and a 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase cDNA (pRGAPDH13) (29).

Isolation of RNA and Northern transfer analysis. Total RNA was isolated from the cell cultures as described previously (30, 31). The amount of RNA was quantitated by spectrophotometric absorbance at 260/280 nm.

Airway Smooth Muscle Contraction Studies

Tissue preparation. Human lung tissue from organ donors who had no known history of respiratory disorders was obtained from the International Institute for the Advancement of Medicine (IIAM, Exton, PA) and the National Disease Research Interchange (NDRI, Philadelphia, PA); lungs were received within 24 h of their removal. Bronchial tissue was removed from the lung by carefully placing a glass probe within individual segments and dissecting away lung parenchymal, fat, connective, and vascular tissues; no attempts were made to remove the epithelium. First- to fifth-generation bronchial strips (approximately 4-15 mm diameter) were prepared; using this classification, the main bronchus is regarded as the first-generation airway.

Concentration-response curves. After the equilibration period and before construction of LTD₄ concentration- response curves, tissues were exposed to 10 µM carbachol to test for tissue viability. Following plateau of this contraction, tissues were washed several times over 30 to 60 min until the tension returned to baseline level. The preparations were then left for at least 30 min before the start of the experiment. LTD₄ concentration-response curves were obtained by their cumulative addition to the organ bath in 3-fold increments; each drug concentration was left in contact with the preparation until the response reached a plateau before addition of the subsequent agonist concentration. At the end of the experiment, tissues were exposed again to 10 µM carbachol, and agonist-induced responses in each tissue were expressed as a percentage of this reference contraction ("% post-carbachol maximal"). The antagonist under study (pranlukast, pobilukast, or zafirlukast) was added to preparations 30 min before initiation of LTD₄ concentration-response curves. In each tissue the response to 10 µM carbachol at the end of the experiment was larger than that obtained at the start of the study ("pre-carbachol maximum"). However, none of the antagonists had an effect on the ratio of post-carbachol maximum to pre-carbachol maximum, indicating that they do not exert a nonselective effect on smooth muscle tone (data not shown). Experiments were conducted in the presence of 1 µM sodium meclofenamate, a cyclooxygenase inhibitor, which was added 45 min before the start of construction of agonist concentration-response curves. Only one agonist curve was generated per tissue.

Statistics

For the proliferation studies, the results are expressed as [³H]thymidine uptake (dpm or fold-increase) and represent the mean ± SEM from a minimum of four separate experiments; statistical significance was assessed by one-way analysis of variance (ANOVA) (Bonferroni-Dunn test or Fischer's protected least-squares difference and Dunnets) with P < 0.05 regarded as significant. The proliferation assays were performed with a minimum of three different airway smooth muscle cell lines.

For the contraction studies, agonist-induced responses for each tissue were expressed as a percentage of the reference contraction obtained at the end of the experiment ("post-carbachol"). Geometric mean EC₅₀ values (pD₂s) were calculated from linear regression analyses of data. Where appropriate, antagonist potencies were calculated and expressed as pK_B; pK_B = log [antagonist]/X  1, where X is the ratio of agonist concentration required to elicit 50% of the maximal contraction in the presence of the antagonist compared with that in its absence. Results from control and treated tissues were analyzed for differences in both the EC₅₀s and also the maximum contractile responses. All data are reported as the mean ± SEM. Statistical analysis was conducted using ANOVA or two-tailed Student's t test for paired samples where appropriate with P < 0.05 regarded as significant.

Drugs

The following drugs were used: LTD₄; pranlukast (SB 205312, ONO-1078, Ultair^TM; (4-oxo-8-[4-phenyl-butoxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate); pobilukast (SK&F 104353; 2(S)-hydroxy-3(R)-[2-carboxyethyl)thio]-3-[2-(8-phenyloctyl)phenyl]-propanoic acid); and zafirlukast (ICI 204219, Accolate^TM; 4-(5-cyclopentyloxycarbonylamino-1-methylindol-3-ylmethyl)-3-methoxy-N-o-tolylsulfonylbenzamide); all of which were synthesized in the Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Sodium meclofenamate was a generous gift from Warner Lambert (Ann Arbor, MI). All other reagents, unless specifically stated, were obtained from Sigma.

	Results

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Airway Smooth Muscle Proliferation

Effects of LTD₄ and EGF. LTD₄ (0.1-10 µM) alone had no effect on DNA synthesis of HASM cells, assessed by measuring [³H]-thymidine incorporation (Figure 1A). The same concentrations, however, produced a concentration-dependent potentiation of DNA synthesis induced by EGF (1 ng/ml) (Figure 1A). For example, 1 and 10 µM LTD₄ produced about a 2- and 4-fold potentiation of EGF-induced DNA synthesis, respectively (Figures 1A and 1B).

View larger version (16K): [in this window] [in a new window]   Figure 1.   (A) The effects of LTD4 alone (filled circle) and the influence of increasing concentrations of LTD4 on EGF (1.0 ng/ ml)-induced HASM cell proliferation (open square); (B) Influence of LTD4 1.0 µM (open square) on EGF-induced HASM cell proliferation (filled circle). The effects of 1.0 µg/ml EGF (A) and 1.0 µM LTD4 (B) alone are shown in the histograms. The data are expressed as [3H]thymidine incorporation and are the mean ± SEM of six experiments; each condition requires six replicates. *Significant compared with EGF alone, P < 0.05.

View larger version (39K): [in this window] [in a new window]   Figure 2.   The effects of LTD4 (10 µM) and EGF (1 ng/ml), either alone or in combination, on HASM cell proliferation, assessed by measuring cell counts. The effect of pranlukast (Pran; 1 µM) was also examined. The data are expressed as cell number/well and are the mean ± SEM of five experiments; there are six replicates per treatment in each experiment. *Significant compared with control, P < 0.05.

Effects of receptor antagonists. A series of experiments was conducted to investigate the effects of the receptor antagonists pranlukast, pobilukast, and zafirlukast, on LTD₄-induced potentiation of DNA synthesis induced by EGF (Figures 3-5). The diluent used in these studies (0.1% dimethyl sulfoxide; equivalent to 10 µM antagonist) was without effect on DNA synthesis (data not shown). Pranlukast at 0.1 µM was without effect (data not shown), whereas a higher concentration, 1 µM, essentially abolished the LTD₄-induced potentiation of DNA synthesis elicited by EGF (Figure 3). Similar findings were obtained with pobilukast, although higher concentrations were required; thus, 3 µM pobilukast was without effect (data not shown) and 30 µM abolished the augmentation of EGF-induced DNA synthesis induced by LTD₄ (Figure 4). In contrast, zafirlukast (0.1 and 1.0 µM) had no effect on LTD₄-induced potentiation of DNA synthesis stimulated by EGF (Figure 5); the effects of higher concentrations could not be assessed since 10 µM zafirlukast produced detachment of cells.

View larger version (23K): [in this window] [in a new window]   Figure 3.   Effects of pranlukast (1 µM) against LTD4-induced augmentation of HASM proliferation elicited by EGF (1.0 ng/ ml): filled circle, LTD4 alone; open square, LTD4 + EGF; open triangle, LTD4 + EGF + pranlukast. The histograms show the effects of EGF alone or in combination with pranlukast (1 µM) on DNA synthesis. The data are expressed as [3H]thymidine incorporation and are the mean ± SEM of six experiments; each condition requires six replicates. *Significant compared with EGF alone, P < 0.05.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Effects of pobilukast (30 µM) against LTD4-induced augmentation of HASM proliferation elicited by EGF (1.0 ng/ ml): filled circle, LTD4 alone; open square, LTD4 + EGF; open triangle, LTD4 + EGF + pobilukast. The histograms show the effects of EGF alone or in combination with pobilukast (30 µM) on DNA synthesis. The data are expressed as [3H]thymidine incorporation and are the mean ± SEM of six experiments; each condition requires six replicates. *Significant compared with EGF alone, P < 0.05.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Effects of zafirlukast (1 µM) against LTD4-induced augmentation of HASM proliferation elicited by EGF (1.0 ng/ ml): filled circle, LTD4 alone; open square, LTD4 + EGF; open triangle, LTD4 + EGF + zafirlukast. The histograms show the effects of EGF alone or in combination with zafirlukast (1 µM) on DNA synthesis. The data are expressed as [3H]thymidine incorporation and are the mean ± SEM of six experiments; each condition requires six replicates; *P < 0.05 significance compared with EGF or LTD4 alone. LTD4 alone had no effect on baseline [3H]thymidine incorporation (data not shown).

Extracellular Matrix Components

Constitutive expression of genes encoding extracellular matrix proteins by HASM. Cultured HASM cells express genes that encode a range of extracellular matrix proteins: pro-1(I) type I collagen, 1(IV) type IV collagen, fibronectin, decorin, and biglycan genes (Figure 6A). The abundance of the 1(IV) collagen transcripts is, however, considerably less than that of the pro-1(I) collagen in airway myocytes. Interstitial collagenase gene expression was not detected in these cells.

View larger version (63K): [in this window] [in a new window]   Figure 6.   Effects of LTD4 and TGF- on total mRNA expression of extracellular matrix proteins in HASM cells. Left-hand panel: Confluent, growth-arrested cells were treated with 0.1 µM LTD4 (2), 10 µM LTD4 (3), 10 ng/ml TGF- (4), or diluent alone (1), and total mRNA expression was determined as described in MATERIALS AND METHODS. Right-hand panel: Confluent, growth-arrested cells were treated with diluent (1 and 2), 0.1 µM LTD4 (3), 10 µM LTD4 (4), 10 ng/ml TGF- (5), 0.1 mM LTD4 and 10 ng/ml TGF- (6), or with 10 µM LTD4 and 10 ng/ml TGF- (7). Total mRNA expression to fibronectin, 1(IV) collagen, pro-1(I) collagen, elastin, biglycan, and decorin was examined. Equivalent mRNA loadings were determined by using a GAPDH cDNA probe. These are representative Northern analyses of three separate experiments.

Effects of LTD₄ on expression of various extracellular matrix genes. LTD₄, 0.1 or 10 µM, did not alter pro-(I) type I or 1(IV) type IV collagen mRNA levels in HASM cells when compared with parallel control cultures treated with diluent alone (Figure 6A). Cells treated with 10 ng/ml TGF- showed substantial increases in the mRNA content of both types of collagens (Figures 6A and 6B; Table 1). The TGF- (10 ng/ml)-stimulated increases in pro-1(I) and 1(IV) collagen gene expression were the same in the presence or absence of LTD₄ (0.1 or 10 µM); e.g., type I collagen: +TGF- = 1.8-fold increase; +TGF- and LTD₄ (10 µM) = 1.75-fold increase; type IV collagen: +TGF- = 3.5-fold increase; +TGF- and LTD₄ (10 µM) = 3.6-fold increase (Figure 6B; Table 1).

Airway Smooth Muscle Contraction

LTD₄ (0.001-1 µM) potently and effectively contracts human isolated bronchial smooth muscle preparations: pD₂ = 8.1 ± 0.1; maximum contraction (% 10 µM carbachol) with 10 µM LTD₄ = 85.2 ± 3.3% (n = 7) (Figure 7). Pranlukast (3 µM; n = 6), zafirlukast (3 µM; n = 6), or pobilukast (3 µM; n = 3) antagonized LTD₄-induced contractions, producing a marked shift to the right in agonist concentration-response curves; there was no effect of the antagonists on the maximum response produced by LTD₄ (Figure 7). The respective pK_Bs for pranlukast, zafirlukast, and pobilukast were 6.9, 6.5, and 7.0, indicating that the three compounds have a similar potency for antagonizing LTD₄- induced contractions in human bronchus.

View larger version (21K): [in this window] [in a new window]   Figure 7.   Effects of pranlukast, zafirlukast, or pobilukast on LTD4 concentration-response curves in human bronchus. Results are expressed as a percentage of the response to 10 µM carbachol added at the end of the experiment and are given as the mean ± SEM of three to seven experiments: filled circle, LTD4 alone; open square, +3 µM pranlukast; open triangle, +3 µM zafirlukast; open circle, +3 µM pobilukast.

	Discussion

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To our knowledge this is the first report of the effects of the CysLTs on mitogenesis and matrix production in HASM cells. The major findings are: (1) LTD₄ alone has no effect on mitogenesis of HASM cells but markedly augments DNA synthesis induced by the growth factors, EGF and thrombin; (2) LTD₄, either alone or in combination with TGF-, was without effect on the expression of various extracellular matrix components of HASM cells; (3) the potentiating influence of LTD₄ (1 µM) on DNA synthesis of HASM cells was differentially sensitive to three structurally distinct receptor antagonists: abolished by pranlukast (1 µM) and a higher concentration of pobilukast (30 µM), but unaffected by zafirlukast (1 µM); and (4) LTD₄-induced contraction in human bronchus was equally sensitive to all three compounds.

The CysLTs have been implicated as significant contributors to the pathophysiology of asthma via multiple mechanisms, including mucus secretion, inflammatory cell recruitment, edema, and neuronal dysfunction, in addition to bronchoconstriction, which is the most widely recognized activity of the CysLTs (1). Although the CysLTs mimic many of the features of asthma, little is known about the influence of the CysLTs on airway smooth muscle proliferation and extracellular matrix protein expression. In a rat model of asthma, repeated challenge with ovalbumin in ovalbumin-sensitized animals produced increases in smooth muscle mass in airways > 2 mm diameter, which was partially inhibited by the CysLT receptor antagonist MK-571, suggesting a role of the CysLTs in this phenomenon (5). LTD₄ potentiated insulin-like growth factor-I-induced proliferation of rabbit tracheal smooth muscle cells (6). In HASM cells, LTD₄ was demonstrated to release matrix metalloproteinase-1 (MMP-1), which is proposed to be an IGF binding protein protease (34). These preclinical results in animals provide preliminary evidence in support of an influence of the CysLTs on lung remodeling. The present study demonstrates that although LTD₄ alone has no effect on proliferation of HASM cells, it produces marked potentiation of mitogenesis elicited by the growth factors EGF and thrombin. The inhibition of the proliferative effects of LTD₄ by the chemically distinct CysLT receptor antagonists pranlukast and pobilukast indicates that this is a receptor-mediated phenomenon. Furthermore, the lack of influence of LTD₄ on expression of collagen or extracellular matrix protein mRNAs suggests that the LTD₄-mediated effect on HASM proliferation is specific and does not represent a generalized activation of these cells.

The effects of the CysLTs in various systems are mediated via two G protein-coupled receptor subtypes, designated CysLT1 and CysLT2 (35). Most CysLT receptor antagonists, including the pranlukast, zafirlukast, and pobilukast used in this study, appear to be more potent CysLT1 rather than CysLT2 receptor antagonists. In support of this characterization, in the present study LTD₄- induced contractions in human bronchus were inhibited by pranlukast, zafirlukast, or pobilukast. In contrast to the similar potencies of the compounds for inhibition of the contractile responses elicited by LTD₄, marked differences were noted in their ability to inhibit the LTD₄-induced potentiation of proliferation of HASM cells elicited by EGF. Thus, the augmentation elicited by LTD₄ was abolished by pranlukast (1 µM) and pobilukastalthough a significantly higher concentration, 30 µM, was requiredwhereas zafirlukast, at a concentration of 1 µM, was without effect; note that a higher concentration of zafirlukast, 10 µM, caused detachment of the HASM cells. The differences in the relative potencies of the compounds for inhibition of LTD₄-induced contraction and the potentiation of growth factor-elicited DNA synthesis provides preliminary evidence to suggest that different CysLT receptors may mediate these activities; both of the putative receptors are sensitive to pranlukast, whereas that mediating the mitogenic effect is much less sensitive to pobilukast and zafirlukast. However, additional research is required to confirm this postulate. The hypothesized existence of two CysLT receptor subtypes in human airways is supported by observations in guinea-pig trachea, where there is evidence for at least two CysLT receptors. It has been proposed that a structural analog of zafirlukast, ICI 198615, was a noncompetitive antagonist of LTD₄-induced contraction in this tissue and inhibited only one of the two receptors mediating this response (39).

Smooth muscle cells express a wide variety of extracellular matrix macromolecules in vitro (10). To date, little is known regarding HASM cell expression of extracellular matrix or collagen. Our study demonstrated the expression of types I and IV collagens, fibronectin, elastin, small dermatan/chondroitin sulfate proteoglycansnamely, biglycan and decorinin cultured HASM cells. The matrix macromolecules, collagen types III and IV, constitute the bulk of the connective tissue in the arteries and airways of the lungs. It is well known that the extracellular matrix influences the migration and proliferation of many cell types. The expression of genes that encode the collagens and noncollagen matrix proteins are regulated by several cytokines, including TGF-. The results of our study show that TGF-, but not LTD₄, upregulates expression of several of the extracellular matrix genes, in particular types I and IV collagen, in HASM cells. There were also several-fold increases in elastin, fibronectin, and biglycan mRNAs induced by TGF-. In contrast to the marked reduction in decorin mRNA levels elicited by TGF- in HASM cells observed in the present study, decorin mRNA and protein levels were reported to be unaltered by TGF- in lung fibroblasts (40). Although LTD₄ does not appear to directly, or in conjunction with TGF-, modulate the production of extracellular matrix molecules by HASM cells, the potentiating influence of LTD₄ on EGF-induced increases in HASM cell number may yield a net increase in these molecules at the tissue level.

Airways remodeling is a feature of asthma, and is manifest as hypertrophy and hyperplasia of the airway smooth muscle, thickening of the basement membrane, and collagen deposition (7, 22). Although these characteristics are more striking in disease of a severe and chronic nature, and contribute to the irreversible component of asthma, they are also present in patients with mild forms of the disorder. The ability of LTD₄ to potentiate growth factor- induced mitogenesis of HASM cells introduces the possibility that the CysLTs, which are present in the lung in large quantities and are released by exposure to antigen (1), may contribute to the structural changes that occur in asthma via a mechanism that is sensitive to some CysLT receptor antagonists, such as pranlukast. Recent large-scale clinical trials with pranlukast and zafirlukast in patients with asthma have demonstrated their general therapeutic benefit (14, 15, 18). However, the effects of these compounds on airways remodeling have not been evaluated. Further study is needed to assess directlyfor example, using biopsy specimensthe impact of CysLT receptor antagonists on the changes in the lung architecture that occur in asthma. This will clarify whether the observed differences in the ability of CysLT receptor antagonists to inhibit LTD₄-induced potentiation of DNA synthesis elicited by mitogens in HASM cells are clinically significant, with respect to effects on airways remodeling.

In summary, the results of the present study demonstrate that although LTD₄ alone has no effect on mitogenesis of HASM cells or on the expression of various extracellular matrix components, it markedly augments DNA synthesis induced by the growth factors EGF and thrombin. The mechanism(s) underlying this potentiating influence of LTD₄ are not known, although the results indicate that it is a receptor-mediated phenomenon. The differential effects of the three structurally distinct CysLT receptor antagonists provide preliminary evidence that LTD₄-induced augmentation of HASM cell mitogenesis may be mediated by a CysLT receptor distinct from that which mediates LTD₄-induced contraction in human airways. The ability of CysLTs to influence airway smooth muscle proliferation may represent another important manner whereby this group of mediators play a significant role in the pathophysiology of asthma, via a mechanism which is sensitive to some CysLT receptor antagonists.