INTRODUCTION

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AIRWAY REMODELING IN BRONCHIAL asthma has been implicated in the development of poorly reversible airway narrowing and in the persistence of nonspecific bronchial hyperresponsiveness (42). Three-dimensional morphometric studies of asthmatic airways have indicated that hyperplasia of airway smooth muscle cells (SMCs) is a prominent feature of the chronic disease process (16). Airway SMCs are likely to be exposed to a range of mediators or growth factors released from inflammatory cells within the bronchial mucosa, but the key signals for cell proliferation have been little investigated.

An increased degree of mast cell activation has long been recognized in asthmatic airways (32, 39), and the number of mast cells has been recognized to increase within the smooth muscle layer (1). The mast cell can secrete a number of potent mediators, several of which have profound effects on smooth muscle. Mast cell-derived histamine and products of arachidonic acid metabolism have attracted attention for their important roles in mediating bronchoconstriction (17, 41), and histamine is a mitogen for SMC (39). The major product of human mast cell activation, however, is the trypsin-like serine proteinase tryptase (EC 3.4.21.59). This enzyme is emerging as a major mediator of allergic inflammation and tissue remodeling (43), and its potential contribution to airway SMC hyperplasia deserves further study.

Asthmatic subjects have appreciably higher levels of tryptase in bronchoalveolar lavage fluid than nonasthmatic controls, even during asymptomatic periods (6, 21), and concentrations may be further increased following allergen challenge (46). Relatively few natural substrates have been identified, although tryptase can degrade certain regulatory peptides, including vasoactive intestinal peptide and calcitonin gene-related peptide (39, 45), implicating this enzyme in neurogenic inflammation. Tryptase can activate matrix metalloprotease-3 (18) and urokinase plasminogen activator (38), suggesting a contribution in tissue remodeling. Addition of tryptase to isolated bronchial tissue from dogs (36) or from spontaneously sensitized (22) or nonsensitized patients (2) can induce hyperresponsiveness to histamine. Consistent with these findings are the results of studies in vivo with a sheep model, in which administration of this enzyme by inhalation also induced bronchial hyperresponsiveness (25), whereas inhibitors of tryptase, such as APC-366, reduced allergen-induced early and late-phase bronchoconstriction and hyperresponsiveness (10). A potential role in tissue remodeling has been underscored by the finding that purified human lung tryptase can act as a potent mitogen for fibroblasts (9, 19), epithelial cells (8), and dermal vascular endothelial cells (3); however, this protease has failed to stimulate the proliferation of human umbilical vein endothelial cells (12). The observation of a mitogenic effect of dog tryptase on canine airway SMC by Brown and colleagues (7) is of some interest in this context, but this has not been investigated with tryptase and SMC of human origin.

The mechanism whereby tryptase may alter cell function is not well understood. However, the identification of a series of proteinase-activated receptors (PARs) may provide some clues. The PARs are seven-transmembrane-domain G protein-coupled receptors whose activation involves proteolytic cleavage at a site on the NH₂ terminus to expose a "tethered ligand" that binds to and activates the receptor. PAR-2, the second of the series to be identified, has been found to be activated by both trypsin (28) and tryptase (13, 26). A synthetic peptide with a sequence corresponding to the tethered ligand domain of PAR-2 (SLIGKV in humans) has been employed experimentally to activate PAR-2 without proteolytic cleavage (4). PAR-2 expression has been detected immunocytochemically in a diverse range of tissues including the airways, where it has been localized in particular to smooth muscle and the epithelium (14). Thrombin and SFLLR, the peptide agonist of the thrombin receptor PAR-1, have been found to induce airway SMC proliferation (29), whereas trypsin and SLIGKV can exhibit mitogenic actions on human aortic SMC (5). However, the effects of human tryptase and agonists of PAR-2 on human airway SMC proliferation have not been examined. The aims of the present study were to investigate the ability of human lung tryptase to induce the proliferation of human airway SMC and to evaluate the potential role of PAR-2.

	METHODS

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Purification and characterization of tryptase. Human mast cell tryptase was purified from lung tissue obtained post mortem. The tissue (400-500 g) was finely chopped, homogenized, and first incubated in a low-salt buffer and then subjected to a high-salt extraction procedure, as described previously (44). The supernatant was filtered through a microfiber membrane, dialyzed against distilled water (24 h, 4°C), and subjected to heparin-agarose affinity chromatography equilibrated with a low-salt buffer. Fractions were eluted using a NaCl gradient between 0.4 and 1.5 M in 10 mM MES (Sigma Chemical, Poole, UK) buffer. Tryptase-rich fractions were then subjected to a benzamidine-agarose affinity chromatography equilibrated with a high-salt buffer (2 M NaCl, 10 mM MES). Fractions were eluted with 0.15 M benzamidine and concentrated using an Amicon concentrator with a YM30 membrane that separated tryptase from benzamidine. Filtrates were then applied to a Sephacryl S-300 gel filtration column equilibrated with a high-salt buffer (2 M NaCl, 10 mM MES). Tryptase-rich fractions were again concentrated, passed through a 0.22-µm membrane filter, and stored at 80°C. The purity of the tryptase samples was confirmed by SDS-PAGE with 10% reducing gels. The preparation employed in these studies appeared as a single band on silver staining with a molecular weight of ~34 kDa. The identity of the purified protein was confirmed by immunoblotting with the tryptase-specific monoclonal antibody AA5 (44).

Enzyme assay. Tryptase activity was determined using the synthetic peptide substrate N-benzoyl-DL-Arg-p-nitroanilide (BAPNA), adding 10 µl of enzyme to 90 µl of 20 mM Tris buffer containing 1 M glycerol and 7.77 mM BAPNA. Absorbance was measured at 410 nm in an ELISA plate reader. Protein concentration was measured spectrophotometrically at 280 nm, using the coefficient of extinction of Smith et al. (37). The specific activity of the tryptase preparation employed was 2.9 U/mg, where 1 unit represents the amount of tryptase required to hydrolyze 1 µmol substrate per minute at 25°C. The enzyme preparation was found to be 100% active by titration with the substrate 4-methylumbelliferyl p-guanidinobenzoate (Sigma Chemical). No chymase or elastase activity was found in these samples, using the chromogenic substrates N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide and N-succinyl-L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide (both from Sigma Chemical), respectively.

Tissue preparation. Tissues were collected following lung resection for bronchial carcinoma and immediately transferred to the laboratory in DMEM (GIBCO BRL Life Technologies, Paisley, UK). From a macroscopically tumor-free part of the specimen, segments of human bronchus (3rd to 4th division) were carefully dissected under a dissecting microscope. After removal of adhering fat, parenchyma, epithelium, and submucosal tissue, the smooth muscle bands were cut into squares measuring 1-2 mm² and cultured in six-well culture plates in a humidified atmosphere at 37°C with 5% CO₂. Human airway SMCs were maintained in DMEM containing 10% (vol/vol) FCS (GIBCO), supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (Sigma Chemical), 1% (vol/vol) nonessential amino acid mixture (Sigma Chemical), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antimycotic-antibiotic solution; GIBCO). The medium was changed every 48-72 h. After 4-6 wk, confluent cells were rinsed twice with Hanks' balanced salt solution (GIBCO) and then passaged with trypsin-EDTA (GIBCO). Only cells at passages 2-4 were used for this study.

Immunocytochemistry. To assess purity of the cultured cells, an immunocytochemical method was employed using an indirect immunofluorescence technique. Cells of varying passage number were grown to 70-80% confluence on eight-well chamber slides (GIBCO). Growth was arrested using serum-free DMEM supplemented with 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 0.5 µg/ml BSA, 4.7 µg/ml linoleic and oleic acid (ITS solution, Sigma Chemical), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% (vol/vol) nonessential amino acid mixture, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antimycotic-antibiotic solution). After 24 h, cells were rinsed twice in PBS (GIBCO) and fixed with cold methanol for 20 min. Nonspecific staining was blocked using PBS containing 3% BSA (GIBCO) for 30 min. Monoclonal antibodies diluted in PBS with 1% BSA including anti--smooth muscle actin (1:200, Sigma Chemical), anti-smooth muscle myosin (1:200, Sigma Chemical), anti-cytokeratin 18 (1:500, Sigma Chemical), anti-factor VIII (1:25, Dako), or anti-fibroblast surface protein (1:100, Sigma Chemical) were incubated for 1 h. After cells were rinsed with PBS containing 0.05% Tween 20 (Sigma Chemical), the cells were incubated for 1 h with FITC-conjugated anti-mouse immunoglobulins (Dako), diluted 1:20. Counterstaining was performed using 2 µg/ml propidium iodide (Sigma Chemical). Slides were mounted with a drop of 10% glycerol in PBS and observed under a Diastar fluorescence microscope.

Mitogenesis and cell proliferation assays. Cells were seeded in 96-well microtiter plates at a density of 10⁵ cells/ml. When close to confluence (70-80%), cells were rinsed twice with Hanks' balanced salt solution and growth was arrested using serum-free DMEM (described above). Cells that achieved complete confluence were not used because of the potential for contact inhibition of cell growth and decreased cell viability. After 24 h of serum deprivation, various stimuli were added. Cells were incubated with purified lung tryptase, added in the absence or presence of heparin (in a weight ratio of 1:1 to stabilize enzymatic activity) (34) or with recombinant  II tryptase isolated from a Pichia pastoris expression system (27) (a kind gift from Dr. Mary Haak-Frendscho and Andrew Niles, Promega, Madison, WI). To investigate the dependency on an intact catalytic site, tryptase (with added heparin) was incubated in the presence or absence of specific inhibitors such as leupeptin (100 µM, Sigma Chemical), benzamidine (100 µM, Sigma Chemical), and APC-366 (12) (100 µM, a kind gift from Axys Pharmaceuticals, South San Francisco, CA). The effect of heat treatment was also investigated, by heating tryptase at 56°C for 60 min. The potential involvement of PAR-2 in airway SMC proliferation was investigated using the agonist peptide SLIGKV-NH₂ and, as a control, the reverse peptide VKGILS-NH₂ (both synthesized by MWG-Biotech). Bovine trypsin, a serine protease that cleaves PAR-2, was also tested. FCS (10%) and PDGF (15 ng/ml) acted as positive controls.

Statistical analysis. Proliferation assays were performed with cells derived from tissue from a minimum of four different patients. Experiments from each patient were conducted in triplicate, and data represented the mean of three wells. The relative degree of [³H]thymidine incorporation was assessed in each group of samples as a percentage of the control with buffer alone. Comparisons between different groups were made by one-way ANOVA; when significant, Student's t-tests were performed with Bonferroni correction for multiple comparisons. P < 0.05 was taken as significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell preparations. All cells stained positively for smooth muscle actin and myosin, and there was no apparent variation in staining intensity between cells of different passage numbers. No immunostaining was seen with antibodies specific for cytokeratin, factor VIII, or fibroblast surface protein (data not shown).

Effects of tryptase and trypsin on DNA synthesis and SMC proliferation. Tryptase stimulated a concentration-dependent increase in DNA synthesis in quiescent human airway SMCs (Fig. 1A). In the presence of heparin (added to stabilize tryptase activity), the mitogenic effect of tryptase was maximal at 30 mU/ml (EC₅₀ of 6.0 ± 2.0 mU/ml). For each concentration, the addition of heparin to tryptase appeared to increase the degree of thymidine incorporation. Nonetheless, heparin alone in the absence of tryptase inhibited thymidine uptake to 65.2 ± 8.5% of control values. The mitogenic effect of purified tryptase was associated with an increase in cell number, as determined by the MTS assay, and SMC proliferation was stimulated to a similar extent with recombinant tryptase at 30 mU/ml (Fig. 1B).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   DNA synthesis and proliferation of human airway smooth muscle cells (SMCs) stimulated by tryptase. A: [3H]thymidine incorporation in growth-arrested human airway SMCs incubated for 24 h with various concentrations of purified human lung tryptase in the absence (shaded bars) or presence (solid bars) of heparin (1:1, wt/wt). Values, expressed as a percentage of the control (hatched bar), are means ± SE from 6 separate experiments each performed in triplicate. *P < 0.05 compared with control. B: cell number was assessed by the MTS assay (see text) 48 h after addition of heat-inactivated tryptase (Heat T), 30 mU/ml of purified (T30) or recombinant (r-T30) tryptase, 15 ng/ml platelet-derived growth factor (PDGF), or 10% FCS, in the absence (solid bars) or presence (open bars) of 100 µM leupeptin. Values are means ± SE from 5 separate experiments each performed in triplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Trypsin-induced DNA synthesis in human airway SMCs. [3H]thymidine incorporation was measured in growth-arrested human airway SMCs incubated for 24 h with various concentrations of trypsin (solid bars). Values, expressed as a percentage of the control (hatched bar), are means ± SE from 5 separate experiments each performed in triplicate. *P < 0.05 compared with control.

Effects of peptide agonist of PAR-2 on DNA synthesis and SMC proliferation. To investigate the potential role of PAR-2 activation in SMC mitogenesis, the agonist peptide SLIGKV was added for 24 h at concentrations ranging from 10⁶ to 10⁴ M. SLIGKV at 10⁴ M significantly enhanced thymidine incorporation compared with that with the same concentration of the reverse peptide VKGILS (Fig. 3A). The enhancement with SLIGKV in thymidine uptake was associated with cell proliferation, as indicated by the MTS assay after 48 h (Fig. 3B).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   DNA synthesis and proliferation of human airway SMC stimulated with proteinase-activated receptor-2 (PAR-2) agonist peptide. A: [3H]thymidine incorporation was measured in growth-arrested human airway SMCs incubated for 24 h with various concentrations of the agonist SLIGKV (solid bars) or the reverse peptide control VKGILS (open bars). Values, expressed as a percentage of the control (hatched bar), are means ± SE from 6 separate experiments each performed in triplicate. B: cell number was assessed by the MTS assay after 48-h challenge with SLIGKV (black bar) or VKGILS (white bar). Values are means ± SE from 5 separate experiments each performed in triplicate. *P < 0.05 compared with control.

Time course of mitogenic responses. The effect of purified, recombinant, and heat-inactivated tryptase on thymidine uptake was investigated after 12, 24, and 48 h (Fig. 4A). At 24 and 48 h, both purified and recombinant tryptase induced a significant increase in thymidine incorporation, whereas the heat-inactivated tryptase was without effect. The PAR-2 agonist SLIGKV also stimulated increased thymidine incorporation at 24 and 48 h (Fig. 4B). The reverse peptide VKGILS added as a control did not induce mitogenesis.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Time course of tryptase and PAR-2-mediated DNA synthesis in human airway SMCs. [3H]thymidine incorporation was measured in growth-arrested human airway SMCs incubated for 12 (n = 4), 24 (n = 6), or 48 h (n = 5) with heat-inactivated tryptase () or 30 mU/ml of purified () or recombinant () human tryptase (A) or with the synthetic peptides VKGILS () or SLIGKV () (B). Values are means ± SE from separate experiments each performed in triplicate. *P < 0.05 compared with control cells at each time point.

Signal transduction mechanisms. To investigate the signal transduction pathways involved in tryptase-induced DNA synthesis, SMC were preincubated with the various inhibitors listed in Table 2. Pertussis toxin, a G protein inhibitor, inhibited completely the thymidine uptake induced by tryptase. Both the PKC inhibitor calphostin C and the tyrosine kinase inhibitor genistein abolished tryptase-stimulated-DNA synthesis. The phorbol ester PKC activators phorbol 12-myristate 13-acetate at 10 nM and phorbol 12,13-dibutyrate at 100 nM increased thymidine uptake by SMC (945 ± 234% and 1,360 ± 251% of control values, respectively).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that human tryptase can provide a potent stimulus for both DNA synthesis and cell proliferation in human airway SMC. Similar responses were evoked with trypsin and the peptide agonist of PAR-2, SLIGKV, consistent with the idea that tryptase may be acting through this receptor. Our studies also indicate some of the signal transduction mechanisms that could be involved in tryptase-induced proliferation of SMC.

Purified lung tryptase at a concentration of 30 mU/ml increased by more than twofold the degree of [³H]thymidine incorporation in primary cultures of SMC at 24 or 48 h, and this was reflected in an increase in cell number. The tryptase preparation employed was of high purity, appearing as a single band on silver-stained gels. Recombinant tryptase also induced SMC proliferation and to a similar extent, providing compelling supporting evidence that tryptase was indeed responsible for the actions of the purified preparation. We found that, as a growth factor for SMC, human tryptase was as effective as either FCS or PDGF when added at concentrations optimal for cell proliferation.

The concentrations of tryptase that stimulated airway SMC proliferation are likely to be achieved in allergic airway diseases. Mast cells in lung tissue contain ~10 pg tryptase per cell (35). After allergen challenge, tryptase concentrations of 10 ng/ml or more have been recorded in bronchoalveolar lavage fluid (46), and this is likely to represent a dilution of the extracellular fluid of more than 100-fold (32). Moreover, there will be high local concentrations of tryptase where this protease binds to proteoglycans in the extracellular fluid, or on cells or tissue structures, thus rendering it enzymatically more stable (34).

The actions of tryptase appeared to be dependent on an intact catalytic site. Tryptase-induced DNA synthesis and SMC proliferation were inhibited by the synthetic tryptase inhibitor APC-366 and also by leupeptin and benzamidine, both of which have been shown previously to inhibit tryptase-induced proliferation of fibroblasts (9, 33) and epithelial cells (8). In the present study, leupeptin reduced thymidine incorporation by 113%, benzamidine by 81%, and APC-366 by 79%. The inactivation of the enzymatic activity by heating also abolished the ability of tryptase to stimulate DNA synthesis and SMC proliferation.

Because purified tryptase is enzymatically unstable in physiological solutions but may be stabilized by heparin in a 1:1 ratio (35), the actions of tryptase on SMC were compared in the presence or absence of heparin. The addition of heparin did indeed enhance the mitogenic effect of tryptase on SMC, consistently increasing the degree of thymidine incorporation. There was a bell-shaped curve in both cases, but the maximal thymidine incorporation was achieved at a lower concentration of tryptase when heparin was absent. We found that heparin was able to inhibit by about one-third the degree of thymidine incorporation in unstimulated cells, in accord with previous investigations of the effect of heparin on SMC mitogenesis (23). It seems likely that in the present studies heparin will have had a dual effect, enhancing the ability of tryptase to stimulate cell proliferation by stabilizing this enzyme but also interacting with SMC to inhibit cell proliferation.

Our studies strongly suggest that tryptase-induced SMC proliferation is mediated through the activation of PAR-2. Trypsin, which like tryptase (26) is able to activate this receptor (28), was found to stimulate an increase in thymidine incorporation in a manner very similar to that elicited with tryptase. The maximal response was of a similar order for both proteases. Moreover, an increase in cell number was induced, and once again the response was inhibited by adding the enzyme in the presence of the protease inhibitor leupeptin. Both tryptase and trypsin can cleave the extracellular chain of PAR-2 at the site (R³⁶-S³⁷) to expose a "tethered ligand" with the sequence SLIGKV, which appears to bind to the second extracellular loop, activating the receptor (26). We found that the synthetic peptide SLIGKV, which can act as a full agonist of PAR-2 without proteolysis (4), was able to induce both DNA synthesis in airway SMC and cell proliferation. A peptide with the reverse sequence (VKGILS), which fails to activate PAR-2 (4), had no effect on SMC over the same range of concentrations. It has been reported previously that tryptase is able to stimulate the proliferation of a lymphoid cell line expressing functional PAR-2 but not when a noncleavable mutant of PAR-2 (R³⁶-S³⁶) was expressed (24). The advent of a selective antagonist of PAR-2 should allow further investigations of the degree to which this receptor contributes to the cellular actions of tryptase.

Studies of signal transduction mechanisms that follow activation of PAR-1, and to a much lesser extent PAR-2, have been conducted in several other cell types, including enterocytes, keratinocytes, and transfected cells (15). Two major pathways have been identified: 1) a pertussis toxin-insensitive G protein linked to a phospholipase C, leading to inositol trisphosphate formation, that releases intracellular Ca²⁺ and is linked to diacyl glycerol that activates PKC and 2) a pertussis toxin-sensitive G protein linked to cytosolic tyrosine kinases such as Src. However, these two pathways are not independent because Ca²⁺-dependent kinases also activate Src and the mitogen-activated protein kinases can be activated by both PKC and tyrosine kinase (15). We have investigated the mechanisms underlying the mitogenic activity of tryptase on SMCs using several inhibitors of signal transduction pathways. Tryptase-induced thymidine incorporation was abolished by pretreatment of cells with pertussis toxin, implicating a pertussis toxin-sensitive G protein in the SMC proliferation induced. A role for PKC was also indicated, with the PKC inhibitor calphostin C completely abolishing the tryptase-induced SMC proliferation; in separate experiments, it was found that phorbol esters, which can activate PKC by nonphysiological means, significantly increased thymidine incorporation. Evidence for the participation of tyrosine kinases was provided by the finding that the specific inhibitor genistein abolished tryptase-induced DNA synthesis. Transduction mechanisms could thus involve a pertussis toxin-sensitive G protein, linked to the phosphatidylinositol 3-kinase, which could stimulate PKC and tyrosine kinase.

In conclusion, we have found that mast cell tryptase can provide a potent stimulus for DNA synthesis and the proliferation of human airway SMCs in culture. The actions of tryptase were dependent on an intact catalytic site, and the similarities in findings with tryptase and other agonists of PAR-2 suggest that the activation of this receptor could be central to an understanding of the mechanism of tryptase-induced SMC mitogenesis. Other potential actions of tryptase and other PAR-2 agonists on airway SMCs deserve investigation, but the ability to contribute to airway remodeling could be particularly important in asthma, a condition associated with hyperplasia of airway SMC (16) and increased secretion of tryptase in the airways (6, 21, 46). Tryptase has been demonstrated to possess a range of other inflammatory actions, acting as a stimulus for the degranulation of mast cells and eosinophils, for granulocyte accumulation, and for cytokine release from various cell types (reviewed in Ref. 43). A report that activation of PAR-2 can provide prostaglandin E₂-mediated bronchoprotection in the airways (11) contrasts with the observation that tryptase can induce hyperresponsiveness in vitro (2) and indicates that tryptase may be involved in a complex interplay of processes in vivo. Nevertheless, tryptase has the potential to be a key mediator of disease in bronchial asthma, and it is a promising target for therapeutic intervention.