A Role for Tryptase in the Activation of Human Mast Cells: Modulation of Histamine Release by Tryptase and Inhibitors of Tryptase 1

Transcription

1 /98/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 1 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286: , 1998 A Role for Tryptase in the Activation of Human Mast Cells: Modulation of Histamine Release by Tryptase and Inhibitors of Tryptase 1 SHAOHENG HE, MARIANNA D. A. GAÇA and ANDREW F. WALLS Immunopharmacology Group, University of Southampton, Southampton General Hospital, Southampton, SO16 6YD, United Kingdom Accepted for publication March 4, 1998 This paper is available online at Tryptase is a tetrameric serine proteinase that constitutes approximately 20% of the total protein within human mast cells and is responsible for at least 95% of the trypsin-like activity in lysates of mast cells derived from lung and skin tissue (Schwartz, 1990). It is stored in the secretory granules in a catalytically active form (Glenner and Cohen, 1960) and is secreted along with histamine, heparin and other mast cell granule products on mast cell degranulation (Schwartz et al., 1981). Because it is stored almost exclusively in mast cells (Walls et al., 1990a), this proteinase has attracted particular attention as a marker for mast cells and for mast cell activation. Relatively high concentrations of tryptase have been detected in the serum from cases of systemic anaphylaxis (Schwartz et al., 1987a), in bronchoalveolar lavage fluid from patients with bronchial asthma (Broide et al., 1991) or interstitial lung disease (Walls et al., 1991), in nasal lavage fluid of patients with allergic rhinitis (Jarjour et al., 1991), in skin blister fluid from subjects with allergic contact dermatitis Received for publication November 7, This work was supported by grants from the Medical Research Council and the Wessex Medical Trust, UK. ABSTRACT Tryptase, the most abundant protein product of human mast cells is emerging as an important mediator and target for therapeutic intervention in allergic disease. We have investigated the potential of tryptase and inhibitors of tryptase to modulate histamine release from human mast cells. Addition of purified human tryptase in concentrations ranging from 1 to 100 mu/ml stimulated a concentration-dependent release of histamine from cells dispersed from tonsil, although not from skin tissue. The reaction depended on an intact catalytic site being inhibited by heat inactivation of the enzyme, or by preincubating with the tryptase inhibitors APC366 or leupeptin or the tryptic substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA). Tryptaseinduced histamine release took approximately 6 min to reach completion, appeared to require exogenous calcium and magnesium, and on the basis of inhibition by antimycin A and 2-deoxy-D-glucose, seemed to be a noncytotoxic process. Preincubation of cells with tryptase at concentrations that were suboptimal for histamine release had little effect on their responsiveness to anti-immunoglobulin (Ig) E or to calcium ionophore A23187, but at higher concentrations their subsequent activation was inhibited. APC366 significantly inhibited histamine release induced by anti-ige or calcium ionophore from both tonsil and skin cells, with up to 90% inhibition being observed at a concentration of 100 M with skin. IgE-dependent histamine release was inhibited also by leupeptin, benzamidine and BAPNA. Tryptase may act as an amplification signal for mast cell activation, and this could account at least partly for the potent mast cell stabilizing properties of tryptase inhibitors. (Brockow et al., 1996) and in synovial fluid from patients with arthritis (Buckley et al., 1997). Evidence is emerging that this major secretory product of the human mast cell may be a key mediator of allergic inflammation and a promising target for therapeutic intervention (Walls, 1995). In seeking to determine the contribution of tryptase in acute inflammatory responses it is particularly important to consider the early cellular events. Studies of human tryptase function in animal models have suggested that certain of the effects noted may depend on the ability of tryptase to activate mast cells. The increase in microvascular permeability induced by tryptase in guinea pig skin, but not that elicited by bradykinin or tissue kallikrein, can be abrogated by pretreatment of guinea pigs with histamine H 1 and H 2 receptor antagonists (He and Walls, 1997). Moreover, addition of tryptase to dispersed guinea pig lung and skin tissue can provoke the release of histamine by a noncytotoxic mechanism in vitro. Although not investigated directly, the finding that both skin microvascular leakage (Molinari et al., 1995) and bronchoconstriction (Molinari et al., 1996) stimulated by tryptase in sheep also could be blocked by a histamine an- ABBREVIATIONS: BAPNA N-benzoyl-DL-arginine-p-nitroanilide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NA, nitroanilide; MES, 2-(N-morpholino)ethanesulfonic acid; MEM, minimal essential medium; BSA, bovine serum albumin; HEPES, N-2-hydroxyethylpiperzine-N -2-ethanesulfonic acid; Ig, immunoglobulin; FCS, fetal calf serum; HBSS, Hanks balanced salt solution. 289

2 290 He et al. Vol. 286 tagonist provides evidence that this human proteinase may induce the activation of mast cells in this species also. Several proteinases have been characterized as secretagogues for mast cells. Thus the degranulation of rodent mast cells may be stimulated by bovine pancreatic trypsin (Lagunoff et al., 1975), thrombin (Razin and Marx, 1984), -chymotrypsin or rat mast cell chymase (Schick et al., 1984). In addition, certain broad spectrum proteinase inhibitors can reduce IgE-dependent histamine release from chopped guinea pig (Austen and Brocklehurst, 1961) or human lung tissue (Kaliner and Austen, 1973), and inhibitors of chymase reduce IgE-dependent histamine release from human tonsil mast cells (Dietze et al., 1990). However, the actions of human tryptase on human mast cells have not been investigated, and the potential of inhibitors and substrates of tryptase to act as mast cell stabilizers is not known. In the present study, we investigated the ability of human tryptase to stimulate histamine release from dispersed human skin and tonsil cells and to modulate histamine release by other stimuli. Our findings suggest that tryptase released from degranulated mast cells has the capacity to activate certain mast cell populations. This could provide the basis of an amplification system in allergic disease, which could be susceptible to control by tryptase inhibitors. Materials and Methods Reagents. The following compounds were purchased from Sigma (Poole, Dorset, UK): leupeptin, benzamidine, NA substrates (BAPNA, N-succinyl-L-Ala- L-Ala- L-Ala-p-NA, N-succinyl- L-Ala- L- Ala-L-Pro-L-Phe-p-NA), porcine heparin glycosaminoglycan, histamine dihydrochloride, collagenase (type I), hyaluronidase (type I), BSA (fraction V), penicillin and streptomycin, MEM containing 25 mm HEPES, heparin agarose, calcium ionophore A23187, dimethyl sulfoxide, tris-base, MES, antimycin A, 2-deoxy-D-glucose. Goat antihuman IgE (inactivated) was purchased from Serotec (Kidlington, Oxford, UK); HEPES and all other chemicals were of analytical grade from BDH (Poole, Dorset, UK); CNBr-activated Sepharose 4B from Pharmacia (Milton Keynes, UK); FCS from Gibco (Paisley, Renfrewshire, UK); o-phthaldialdehyde from Fluka (Gillingham, Dorset, UK); Coomassie protein assay reagent from Pierce (Rockford, IL); silver staining kit from Bio-Rad (Hemel Hempstead, UK); the Toxicolor System for the assay of endotoxin from Seikagaku (Tokyo, Japan). The tryptase inhibitor APC366 [N-(1-hydroxy-2-naphthoyl)- L-arginyl-L-prolinamide hydrochloride] was kindly provided by Axys Pharmaceutical Corporation (South San Francisco, CA). Purification and characterization of tryptase. Tryptase was purified from human lung tissue by high salt extraction, heparin agarose chromatography and an immunoaffinity chromatography procedure with tryptase-specific monoclonal antibody AA5 coupled to agarose as described previously (He et al., 1997). Fractions of purified tryptase were concentrated in C-30 Centricon centrifugal concentrators (Amicon, Stonehouse, Gloucestershire, UK) and stored at 80 C until use. Enzymatic activity was determined by measuring spectrophotometrically at 410 nm the hydrolysis of 20 mm BAPNA in 0.1 M Tris-HCl, 1 M glycerol, ph 8.0, containing 1 mg/ml BSA, at 25 C. Protein concentrations were determined by the Coomassie blue dye binding procedure with a BSA standard (according to the manufacturer s protocol). The specific activity of tryptase was 1.8 U/mg, where one unit of enzyme was taken as the amount that catalyzed the cleavage of 1 mol of BAPNA per minute at 25 C. Analysis by SDS-PAGE with silver staining revealed a single diffuse band with an apparent molecular weight of approximately 32 kda and the identity as tryptase was confirmed by Western blotting with monoclonal antibody AA5 (Walls et al., 1990b). Levels of contaminating endotoxin in the final preparations of tryptase were very low. Using the Toxicolor System according to the instructions provided by the manufacturer, endotoxin was found to be present at less than 22 pg/ml in a stock solution, which indicates contamination at the level of less than 40 pg/mg tryptase. No contamination with chymase was detected by use of the substrate 0.7 mm N-succinyl- L-Ala- L-Ala-L-Pro-L-Phe-p-NA in 1.5 M NaCl, 0.3 M Tris, ph 8.0, and 5% ethanol, or by use of elastase with 1.4 mm N-succinyl-L-Ala- L-Ala-L-Ala -p-na in the same buffer as used with BAPNA. Preparation of compounds. Because tryptase is enzymatically unstable in physiological solutions, considerable care was taken in its preparation and storage. Purified tryptase was maintained in a high salt buffer (10 mm MES, 1 M NaCl, ph 6.8) in the presence or absence of heparin. Immediately before addition to cells, tryptase was diluted first with sterile distilled water, adjusting the NaCl concentration to 0.15 M, and then with normal saline to obtain the required tryptase concentration. Where added, proteinase inhibitors or the buffer alone were incubated with tryptase for 30 min on ice before adding to the cells. Mast cell dispersion and challenge. Human tonsil and skin tissues were freshly obtained at tonsillectomy or circumcision operations in children. Cells in the macroscopically normal tissue were dispersed enzymatically by a procedure similar to that used previously with human tonsil (Okayama et al., 1994) or skin tissues (Benyon et al., 1987). Tissue was chopped finely with scissors into fragments of 0.5 to 2.0 mm 3, washed twice with MEM containing 2% FCS (500 g, 8 min, 25 C), and incubated at 37 C with 1.5 mg/ml collagenase and 0.75 mg/ml hyaluronidase in the same buffer, but containing 200 U/ml penicillin, 200 g/ml streptomycin (1 g tonsil/10 ml buffer; 1 g skin/15 ml buffer) for 60 min with tonsil tissue and 75 min with skin tissue. Dispersed cells were separated from undigested tissue by filtration through nylon gauze (pore size, 100 m diameter) and washed twice with MEM containing 2% FCS. Mast cell numbers were determined by light microscopy after staining with the Kimura staining solution. Dispersed cells were maintained in MEM (containing 10% FCS, 200 U/ml penicillin, 200 g/ml streptomycin) on a roller overnight at room temperature. After washing twice with HBSS, ph 7.4, without added calcium or magnesium (500 g, 10 min, 25 C), the cells were resuspended in HBSS with 1.8 mm CaCl 2 and 0.5 mm MgCl 2 (complete HBSS), and warmed at 37 C for 5 min. Aliquots of 100 l containing 4 to mast cells were added to a 50- l aliquot of purified tryptase, control secretagogue or inhibitor in complete HBSS and incubated for 15 min at 37 C. The reaction was terminated by the addition of 150 l ice-cold HBSS and the tubes centrifuged immediately (500 g, 10 min, 4 C). All experiments were performed in duplicate. For the measurement of total histamine concentration in some tubes, the suspension was boiled for 6 min. Supernatants were stored at 20 C until histamine concentrations were determined. Histamine measurement. A glass fiber-based, fluorometric assay for histamine was used as described previously (Nolte et al., 1987). Sample, control buffer or histamine standard (50 ng/ml) was pipetted into the well of a 96-well microtiter plate coated with a glass-fiber matrix (Lundbeck Diagnostics, Copenhagen, Denmark) that selectively binds histamine, 50 l of sample volume per well. After 1 h incubation at 37 C, the plate was rinsed with distilled water, and 150 l of 0.4% SDS solution was added to each well. After 30 min incubation at 37 C, the plate was rinsed again with distilled water, and 75 l of coupling reagent [containing 0.5 mg/ml o-phthaldialdehyde, 5% (v/v) methanol and 2 mg/ml NaOH] was added to each well. The plate was then kept in the dark for 10 min at room temperature, and finally 75 l of 58 mm HClO 4 was added to stop the histamine-o-phthaldialdehyde reaction. The histamine concentration was measured on a spectrophotofluorometer (Perkin-Elmer LS 2, Denmark). Histamine release was expressed as a percentage of total cellular histamine levels and corrected for the spontaneous release measured in tubes in which cells had been incubated with the HBSS diluent alone (i.e., percentage net histamine release [hista-

3 1998 Role of Tryptase in Mast Cell Activation 291 mine release with stimulus spontaneous histamine release]/total histamine content 100). The lower limit of detection of the assay was 1 ng/ml, and interassay variability was less than 3%. Statistics. All statistical analyses were performed with StatView software (Version 4.02, Abacus Concepts, Berkeley, CA). Data are shown as the mean S.E. for the number of experiments indicated. Where analysis of variance indicated significant differences between groups, for the preplanned comparisons of interest, the paired Student s t test was applied. For all analyses, P.05 was taken as significant. Results Tryptase-induced histamine release. Incubation of mast cells dispersed from tonsil tissue with purified tryptase throughout the concentration range 0.3 to 100 mu/ml stimulated a dose-dependent release of histamine (fig. 1A). Under similar conditions, in preliminary experiments carried out with a range of concentrations of anti-ige or calcium ionophore A23187, maximal noncytotoxic histamine release for both tonsil and skin tissues was observed with 1% anti-ige or with 1 M calcium ionophore (data not shown), and both 1% anti-ige and 1 M calcium ionophore were included as positive controls in all experiments (fig. 1B). The amount of histamine release induced from tonsil cells by tryptase represented two thirds of that which could be elicited with anti-ige. However, in similar experiments performed with dispersed skin mast cells, tryptase throughout the concentration range 0 to 30 mu/ml failed to stimulate histamine release regardless of whether or not heparin was present (fig. 1A), although the mean net release with anti- IgE was only % in these experiments (fig. 1B). Basal levels of tryptic activity (assayed by BAPNA cleavage) in culture media of dispersed tonsil or skin cells were and mu/ml, respectively. There was no significant correlation between the extent of histamine release induced by tryptase (30 mu/ml) and by either anti-ige (1%) or calcium ionophore (1 M), when data were analyzed separately for tonsil or skin cells, or with all data considered together. The time course of histamine release stimulated by tryptase was relatively slow and appeared to be biphasic, with maximal release not being achieved until approximately 6 min after addition of the enzyme (fig. 2). In comparison, maximum histamine release in response to 1% anti-ige was observed by 3 min and in response to 1 M calcium ionophore by 5 min (data not shown). Because tryptase is enzymatically unstable, but may be stabilized by heparin in vitro (Schwartz and Bradford, 1986), heparin was added to tryptase (1 g heparin per mu tryptase) before incubating with cells in certain experiments. However, heparin appeared to reduce histamine release induced by lower concentrations ( 100 mu/ml) of tryptase from tonsil mast cells (fig. 1A). It was confirmed that addition of heparin significantly reduced the spontaneous loss of tryptase activity in cell supernatants which occurred after incubation with tonsil or skin cells, as well as reducing the loss of activity which occurred in the absence of cells (table 1). To further investigate the effects of heparin on the activation of tonsil mast cells, the cells were preincubated with 3 or 30 g/ml of heparin for 0, 5 and 30 min before challenge with anti-ige or calcium ionophore. At all time points, heparin was without effect on the extent of anti-ige or calcium iono- Fig. 1. (A) Histamine release from tonsil or skin cells incubated with tryptase ( ), tryptase with heparin ( E ) or heat-treated tryptase ( ). The quantity of tryptase is expressed in terms of enzyme activity, except in the heat-treated preparation, for which activity before heat inactivation is indicated. Data are presented as mean S.E. for 6 to 12 experiments. (B) Mean net histamine release ( S.E.) with anti-ige and calcium ionophore A23187 (12 experiments with tonsil cells and 11 with skin cells) is shown. Mean net spontaneous histamine release ( S.E.) was % or % with tonsil or skin cells, respectively. * P.05 and ** P.005 compared with the base-line values, P.05 compared with the response with tryptase alone. Fig. 2. Time course of tryptase-induced release of histamine from dispersed tonsil cells. Data shown are the mean S.E. of the percentage of maximum histamine release induced by tryptase in three separate experiments. * P.05 compared with the base-line response.

4 292 He et al. Vol. 286 TABLE 1 Loss of enzymatic activity after incubation of tryptase (30 mu/ml) with dispersed tonsil or skin cells or with medium alone for 15 min at 37 C Tryptase was added in the presence or absence of 30 g/ml heparin, and BAPNA cleaving activity was determined in supernatants after centrifugation at 4 C for 10 min. Values are mean S.E. for three separate experiments. Cell preparation % Loss in Tryptase Activity Without heparin With heparin Tonsil * Skin * Medium alone * *P.05 in comparison with the loss in activity which occurred without heparin. P.05 in comparison with the tryptase activity added. phore induced histamine release. Incubation of heparin alone at concentrations either 3 or 30 g/ml with cells for 15, 20 or 45 min did not alter the extent of histamine release from these cells (data not shown). Inhibition of tryptase-induced histamine release. Heating tryptase at 56 C for 60 min abolished its ability to stimulate histamine release from tonsil cells (fig. 1A), as well as its ability to cleave the chromogenic substrate, BAPNA. The proteinase inhibitor leupeptin and the tryptase inhibitor drug APC366 were investigated for their ability to inhibit histamine release from tonsil cells stimulated by tryptase. Tryptase was preincubated for 30 min on ice with these inhibitors at concentrations which were sufficient to inhibit the cleavage of BAPNA by tryptase but which did not themselves stimulate histamine release. Both leupeptin and APC366 proved effective at reducing tryptase-induced histamine release (table 2). Addition of BAPNA to tryptase before incubation with cells also inhibited histamine release. When cells were incubated with the metabolic inhibitors 2-deoxy-D-glucose (10 mm) and antimycin A (1 M) for 40 min at 37 C before challenge with tryptase, no significant release of histamine was induced (table 3). Consistent with the idea that this is a noncytotoxic reaction was the observation that the presence of calcium and magnesium in the medium may be necessary for histamine release, at least with tryptase concentrations up to 10 mu/ml (fig. 3). Interaction of tryptase with other stimuli. Dispersed tonsil mast cells were preincubated with concentrations of tryptase ranging from 0.1 to 10 mu/ml in the presence or absence of heparin for 0, 5 or 30 min at 37 C before challenge with the standard doses of anti-ige (1%) or calcium ionophore (1 M). Concentrations of tryptase smaller than those that could induce histamine release TABLE 2 The effects of proteinase inhibitors and the substrate BAPNA on histamine release induced from dispersed tonsil cells by tryptase (10 mu/ml) Values shown are mean S.E. for three separate experiments with BAPNA and five to seven experiments with cells. Tryptase was preincubated with either proteinase inhibitor or BAPNA for 30 min on ice before adding to the cells or BAPNA. Inhibitor or Substrate BAPNA cleavage Inhibition of Net histamine release % % Leupeptin 10 g/ml * 64 17* APC M * 79 17* BAPNA 100 g/ml ND a 60 16* *P.05 compared with response with the uninhibited controls. a ND, not done. TABLE 3 The effect of metabolic inhibitors (MI) on histamine release induced from dispersed tonsil cells by tryptase, tryptase with heparin, anti-ige or calcium ionophore Values shown are mean S.E. for four separate experiments performed in duplicate. The cells were preincubated with metabolic inhibitors for 40 min at 37 C before addition of stimulus. Stimulus Net Histamine Release With MI Without MI % % Tryptase 10 mu/ml heparin * mu/ml heparin * mu/ml (no heparin) * Anti-IgE, 1% * 29 7 Calcium ionophore, 1 M * *P.05 compared with response with the uninhibited controls. Fig. 3. Histamine release from tonsil cells induced by tryptase ( ) or tryptase with heparin (E) in the presence (- - -) or absence ( ) of exogenous calcium and magnesium ions. The mean S.E. are shown for five separate experiments. * P.05 compared with the response with calcium and magnesium in the buffer. themselves, neither primed the cells nor inhibited subsequent activation by the other stimuli (data not shown). However, preincubating cells with tryptase at concentrations capable of stimulating histamine release did significantly reduce the degree of histamine release elicited after challenge with either anti-ige or calcium ionophore (fig. 4). Similar findings were observed when more limited experiments were performed with preparations of skin cells (n 2 or 3; data not shown) Inhibition of histamine release by tryptase inhibitors or substrate. Preincubation of dispersed tonsil or skin cells with various doses of APC366 for periods of 0, 5 or 30 min before challenge with either anti-ige or calcium ionophore resulted in a dose-dependent inhibition of histamine release (fig. 5). The inhibitory actions of APC366 were particularly potent with skin mast cells, and significant inhibition of histamine release was achieved with a concentration as low as 1 M after 30 min incubation. With shorter incubation periods, however, higher doses of APC366 were required to achieve significant inhibition of histamine release. Ninety percent inhibition of both anti-ige and calcium ionophore-induced histamine release from skin cells was

5 1998 Role of Tryptase in Mast Cell Activation 293 Fig. 4. The effects of tryptase (10 mu/ ml) in the presence of heparin on (A) anti- IgE (1%) and (B) calcium ionophore A23187 (1 M) induced histamine release from dispersed human tonsil cells. The cells were preincubated with tryptase ( ) or buffer alone ( E ) for 0, 5 and 30 min at 37 C before challenge with stimulus. Mean S.E. are shown for six separate experiments. * P.05 compared with the responses with anti-ige or calcium ionophore alone. Fig. 5. Inhibitory actions of APC366 on (A) anti-ige (1%) and (B) calcium ionophore A23187 (1 M) induced histamine release from dispersed tonsil or skin mast cells. The cells were preincubated with various concentrations of APC366 for 0 ( ),5( ) and 30 ( E ) min, respectively, at 37 C before challenge. Data are presented as mean S.E. for 6 to 10 separate experiments. * P.05 compared with the responses with uninhibited controls. achieved with 100 M APC 366 after 30 min preincubation; IC 50 values of about 15 M were found with both stimuli. For purposes of comparison, more limited studies were performed with either 10 g/ml or 100 g/ml leupeptin or benzamidine hydrochloride, concentrations which effectively inhibited the catalytic activity of tryptase (as determined by measuring the cleavage of BAPNA) but which did not themselves cause apparent cell toxicity or liberation of histamine from tonsil mast cells. Both leupeptin and benzamidine at a concentration of 100 g/ml inhibited IgE-induced histamine release with the tonsil cells (fig. 6). Leupeptin also inhibited IgE-dependent histamine release from skin cells, although benzamidine had little effect at these concentrations with this source of tissue (data not shown). Neither leupeptin- nor benzamidineinhibited histamine release from calcium ionophore challenged tonsil or skin cells in parallel experiments (data not shown). Addition of the substrate BAPNA to tonsil cells before challenge with anti-ige elicited a dose-dependent inhibition of histamine release (fig. 7A). The degree of inhibition was less with calcium ionophore as the stimulus, but there was nevertheless significant inhibition of mast cell activation at the highest dose (fig. 7B).

6 294 He et al. Vol. 286 Fig. 6. Inhibitory actions of the proteinase inhibitors leupeptin and benzamidine (both at 10 and100 g/ml) on anti-ige induced histamine release from dispersed human tonsil cells. The cells were preincubated with a concentration of either leupeptin or benzamidine for 0 ( ),5(o) and 30 (s) min at 37 C before challenge. Mean S.E. are shown for five separate experiments. * P.05 compared with the responses with the uninhibited controls. Discussion With two sources of human mast cells we have established that inhibitors and substrates of tryptase may have potent mast cell-stabilizing properties. Our observation that tryptase is a stimulus for histamine release, at least from tonsil tissue, suggests that this major secretory product of human mast cells may itself have a key role in processes of mast cell activation. Moreover, the release of tryptase from activated mast cells may stimulate secretion from neighboring mast cells and thus provide an amplification signal in allergic disease. The proportion of the total cellular histamine released from dispersed tonsil cells by tryptase at concentrations of 30 or 100 mu/ml was of an order similar to the maximum histamine release elicited from these cells by antibody against IgE. Human tonsillar mast cells therefore would seem to resemble mast cells of the guinea pig lung and skin in being responsive to human tryptase (He and Walls, 1997). In contrast, we found that dispersed human foreskin mast cells released negligible quantities of histamine after incubation with tryptase. The possibility that the skin mast cells may have already been activated maximally in response to the release of endogenous tryptase during tissue processing cannot be excluded, although basal levels of tryptase activity measured in the culture supernatants were low. The differences in responsiveness to tryptase are more likely to reflect functional heterogeneity between mast cell populations at different anatomical sites in humans, and between mast cells at comparable sites in different mammalian species. This concept has been established with other secretagogues including neuropeptides and basic compounds (Church et al., 1997). As found in previous studies with human tonsil and skin tissues (Lowman et al., 1988a), the proportion of histamine released from skin cells with anti-ige and calcium ionophore was substantially less than that from tonsil cells. However, no direct relationship was found in our studies between the degree of releasability to tryptase and that to anti-ige or to calcium ionophore. Histamine release induced by tryptase and that by anti-ige are thus likely to be mediated by different processes. Although detailed information is now available on the mechanism of IgE-dependent mast cell activation (Kennerly and Duffy, 1993), much less is known of the other means by which human mast cells may be activated. One of the best studied non-ige-dependent processes in human mast cells is that stimulated by the neuropeptide substance P which is able to elicit histamine release from human skin mast cells, but not from tonsil, gut or lung mast cells (Lowman et al., 1988a; Church et al., 1991). Although mast cells which have degranulated in response to substance P appear similar at the ultrastructural level to those which have been activated by anti-ige (Caulfield et al., 1990), they seem to involve different receptors (Lowman et al., 1988b) and exhibit quite different kinetics of histamine release. Whereas substance P-induced histamine release is complete within 15 to 20 s (Benyon et al., 1987; Lowman et al., 1988b), IgE-dependent histamine release requires some 3 to 6 min to reach completion. Moreover, histamine release stimulated by anti-ige depends on the presence of calcium in the cell culture medium, whereas that induced by the nonimmunological stimulus is not (Benyon et al., 1987; Lowman et al., 1988b). The activation of human mast cells by other basic compounds, and by complement peptides C3a and C5a seems to share these characteristics (El Lati et al., 1994). The mechanism of tryptase-induced histamine release must be quite different from that of substance P and related secretagogues. Not only were tonsil mast cells responsive to tryptase, and skin mast cells unresponsive, but the time required to activate maximum histamine release was at 6 min more similar to that found for stimulation with anti-ige than that with substance P. In addition, we found that the presence of exogenous calcium and magnesium ions may be required for mast cell activation induced by tryptase. The activation of human mast cells by tryptase seems to involve a novel mechanism quite different from that characterized for other secretagogues. The observation that heat inactivation abrogated its secretagogue properties suggests that an intact catalytic site is required. Moreover, inhibiting tryptase activity with the tryptase inhibitor APC366 markedly reduced its potential to stimulate histamine release, as did the broad spectrum inhibitor leupeptin and the tryptic substrate BAPNA. The possibility cannot be excluded that the addition of these different inhibitors or substrates could have effects on mast cell responsiveness other than those attributable to the inhibition of exogenous tryptase. However, taken together our findings strongly suggest that a proteolytic process is involved in tryptase-induced activation of mast cells. The ability of heparin to stabilize tryptase activity (Schwartz and Bradford, 1986) was confirmed, even in the presence of the cell preparations. Although the ability of heparin to enhance tryptase-induced cleavage of defined substrates in vitro has been demonstrated (Alter et al., 1987),

7 1998 Role of Tryptase in Mast Cell Activation 295 Fig. 7. Inhibitory actions of the substrate BAPNA on (A) anti-ige (1%) and (B) calcium ionophore A23187 (1 M) induced histamine release from dispersed human tonsil cells. Cells were preincubated with various concentrations of BAPNA for 0 ( ),5( ) and 30 ( E ) min at 37 C before challenge. Values shown are the mean S.E. for seven separate experiments. * P.05 compared with the responses with the uninhibited controls. this effect has not been seen consistently in investigations of the actions of tryptase on cells (Cairns and Walls, 1996) or tissues (He and Walls, 1997; He et al., 1997), possibly because cell surface or tissue proteoglycans themselves can bind and stabilize tryptase. The finding of that the addition of heparin to tryptase actually inhibited tryptase-induced histamine release was surprising, but may be related to a quite separate effect of heparin on mast cells. Although in the present studies the addition of heparin did not alter the extent of either IgE-dependent or calcium ionophore-induced histamine release, Ahmed and colleagues (1993) previously reported that heparin can inhibit IgE-dependent histamine release from human uterine and rat peritoneal mast cells in vitro, as well as reduce allergen-induced airway and cutaneous responses in sheep. The modulating effects of heparin on mast cells seem to differ between mast cell populations and between stimuli of activation. Although the actions of tryptase on human mast cells have not been investigated previously, studies with several other proteinases have highlighted the potential involvement of proteolytic processes in mast cell activation. Most investigations reported to date have used rodent peritoneal cell models, preventing direct comparison with our findings with human cells, but observations that proteases as diverse as pancreatic trypsin (Lagunoff et al., 1975), -chymotrypsin (Schick et al., 1984), thrombin (Razin and Marx, 1984), rat mast cell chymase (Schick et al., 1984) and a variety of other proteases (Machado et al., 1996) can all stimulate histamine release, suggest that there could be various proteolytic mechanisms whereby these cells may become activated. The cleavage by tryptase of a substrate whether in the membrane or in the extracellular fluid could provide a signal for mast cell activation either directly or by generating an activating peptide. These possibilities have been raised for chymase-induced mast cell activation by Schick (1990) who found that rat chymase appeared to cleave a 90 kda membrane component of rat mast cells, and by Cochrane and colleagues (Cochrane et al., 1993) who reported that this proteinase could cleave albumin to release a histamine-releasing peptide. Recently a series of receptors for tryptic proteinases have been characterized, termed proteinase-activated receptor 1 (thrombin receptor) (Vu et al., 1991), 2 (Nystedt et al., 1994) and 3 (Ishihara et al., 1997), and in certain experimental systems tryptase is capable of activating the first two of these (Molino et al., 1997). The extent to which these receptors may be expressed on mast cells remains to be determined, but such a mechanism deserves consideration as a means whereby tryptase can activate human mast cells. When cells were preincubated for up to 30 min with tryptase at concentrations which were suboptimal for histamine release, before challenging with other stimuli, the response induced by anti-ige or calcium ionophore was barely affected. Only with concentrations of tryptase that were themselves able to stimulate histamine release was there an apparent reduction in histamine release in response to these other stimuli. Reports that preincubation of rat peritoneal mast cells with trypsin can prevent the subsequent release of histamine in response to chymotrypsin or chymase (Lagunoff et al., 1975; Schick et al., 1984) provide a parallel with our study. It is possible that tryptase may cleave IgE or the Fc R1 receptor. Alternatively, the reduced responsiveness may simply be a consequence of mast cell activation. Repeated antigen challenge at 30-min intervals has been found to inhibit the responsiveness of cultured rat mast cells to antigen (Shalit and Levi-Schaffer, 1995). Unlike many of the proteases demonstrated to have the capacity to stimulate mast cell activation, tryptase is a physiologically relevant stimulus. It has been estimated that there may be up to 35 pg tryptase per cell stored within secretory granules (Schwartz et al., 1987b), which suggests that high concentrations of this proteinase will be achieved in the immediate vicinity of degranulating mast cells. The concentrations of tryptase that elicited histamine release from tonsil mast cells are likely to be achieved in vivo (He and Walls, 1997). Moreover, the noncytotoxic nature of tryptaseinduced cell activation was indicated by the ability of the metabolic inhibitors antimycin A and 2-deoxy-D-glucose to abrogate the response. By acting as a stimulus of mast cell degranulation, tryptase could play a key role in allergic disease by amplifying the responses of mast cells to allergen and other stimuli. Consistent with this idea is the observation that the tryptase inhibitor drug APC366 in a dose-dependent manner inhibited both IgE-dependent and calcium ionophore-induced histamine release. The degree of inhibition achieved with APC366 for both tonsil and skin mast cells is high when

8 296 He et al. Vol. 286 compared with that reported for other drugs with mast cell stabilizing properties. With tonsillar cells, APC366 could be considered to have potency similar to the beta-2 adrenoceptor agonist salbutamol, which at a concentration of 1 M has been found to inhibit IgE-dependent histamine release by about 25% (Okayama and Church, 1992). In the same study, the antiallergic drug sodium cromoglycate reduced IgE-dependent histamine release from tonsil cells by only about 12% at a concentration of 1000 M. For skin mast cells, the potency of APC366 was quite remarkable, and at a concentration of 100 M inhibited histamine release by about 90%. By comparison, salbutamol has been reported to inhibit IgEdependent histamine release from skin cells by 20%, whereas sodium cromoglycate is without any inhibitory effects on histamine release from this source of mast cells (Okayama and Church, 1992). Although the addition of exogenous tryptase to skin cells did not elicit histamine release at the concentrations tested, the ability of APC366 to inhibit histamine release from these cells in response to anti-ige and calcium ionophore does suggest that endogenous tryptase could be involved in cell activation, nevertheless. Tryptase, which is stored in mast cell granules in an active form (Glenner and Cohen, 1960), may interact with substrates or receptors exposed during the degranulation process. When skin or tonsil cells were preincubated with APC366 for 30 min, the inhibitory actions tended to be greater than when this tryptase inhibitor was added at the same time as the stimulus. This may indicate that APC366 can interact with cells, possibly binding irreversibly to tryptase itself in the time-dependent manner described for this inhibitor (McEuen et al., 1996). Leupeptin and benzamidine which are relatively broad spectrum inhibitors of tryptic proteinases, as well as the substrate BAPNA, also inhibited histamine release from tonsil and skin cells. This supports the idea that the actions of APC366 were related to its properties as a proteinase inhibitor. Both leupeptin and benzamidine were less potent than APC366, and in contrast to APC366 and BAPNA, did not inhibit histamine release induced by calcium ionophore. It is possible that the quantities of these inhibitors added was not sufficient for inhibition, but we were constrained by their cytotoxicity at higher concentrations. The ability of APC366 to inhibit the activation of human mast cells in vitro is in keeping with its actions found in vivo in sheep models. Clark and colleagues (1995) reported that prophylactic administration of this compound can inhibit antigen-induced early as well as late increases in specific lung resistance, airway hyperresponsiveness to inhaled carbachol, microvascular leakage and lung tissue eosinophilia. Moreover, APC366 can inhibit immediate cutaneous responses to Ascaris suum in naturally sensitized sheep (Molinari et al., 1995). Although developed as an inhibitor of tryptase, APC366 is not selective for this proteinase, but it also can inhibit trypsin and thrombin to a certain degree (Molinari et al., 1995). It is possible therefore that the mast cell stabilizing actions of this drug may depend in part on the inhibition of another protease which is yet to be identified in addition to tryptase. The development of selective and potent inhibitors of mast cell tryptase should assist in elucidating some of the mechanisms of action of tryptase, as well as providing a powerful new class of antiallergic drugs which could be effective as mast cell stabilizers. References Ahmed T, Syriste T, Lucio J, Abraham W, Robinson M and D Brot J (1993) Inhibition of antigen-induced airway and cutaneous responses by heparin: A pharmacodynamic study. J Appl Physiol 74: Alter SC, Metcalfe DD, Bradford TR and Schwartz LB (1987) Regulation of human mast cell tryptase. Biochem J 248: Austen KF and Brocklehursr WE (1961) Anaphylaxis in chopped guinea pig lung: I. Effect of peptidase substrates and inhibitors. J Exp Med 113: Benyon RC, Lowman MA and Church MK (1987) Human skin mast cells: their dispersion, purification and secretory characterization. J Immunol 138: Brockow K, Abeck D, Hermann K and Ring J (1996) Tryptase concentration in skin blister fluid from patients with bullous skin conditions. Arch Dermatol Res 288: Broide DH, Gleich GJ, Cuomo AJ, Coburn DA, Federman EC, Schwartz LB and Wasserman SI (1991) Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J Allergy Clin Immunol 88: Buckley M, Walters C, Brander M, Wong WM, Cawley MID, Ren S, Schwartz LB and Walls AF (1997) Mast cell activation in arthritis: Detection of and tryptase, histamine and eosinophil cationic protein in synovial fluid. Clin Sci 93: Cairns JA and Walls AF (1996) Mast cell tryptase is a mitogen for epithelial cells: Stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J Immunol 156: Caulfield JP, El-Lati S, Thomas G and Church MK (1990) Dissociated human foreskin mast cells degranulate in response to anti-ige and substance P. Lab Invest 63: Church MK, El-Lati S and Caulfield JP (1991) Neuropeptide-induced secretion from human skin mast cells. Int Arch Allergy Appl Immunol 94: Church MK, Bradding P, Walls AF and Okayama Y (1997) Human mast cells and basophils, in Allergy and Allergic Diseases (Kay, AB, ed) pp , Blackwell, Oxford. Clark JM, Abraham WM, Fishman CE, Forteza R, Ahmed A, Cortes A, Warne RL, Moore WR and Tanaka RD (1995) Tryptase inhibitors block allergen-induced airway and inflammatory responses in allergic sheep. Am J Respir Crit Care Med 152: Cochrane DE, Carraway RE, Feldberg RS, Boucher W and Gelfand JM (1993) Stimulated rat mast cells generate histamine- releasing peptide from albumin. Peptides 14: Dietze SC, Sommerhoff CP and Fritz H (1990) Inhibition of histamine release from human mast cells ex vivo by natural and synthetic chymase inhibitors. Biol Chem Hoppe-Seyler 371: EL-Lati SG, Dahinden CA and Church MK (1994) Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells. J Invest Dermatol 102: Glenner GG and Cohen LA (1960) Histochemical demonstration of a species specific trypsin-like enzyme in mast cells. Nature 185: He S and Walls AF (1997) Human mast cell tryptase: A stimulus of microvascular leakage and mast cell activation. Eur J Pharmacol 328: He S, Peng Q and Walls AF (1997) Potent induction of a neutrophil- and eosinophilrich infiltrate in vivo by human mast cell tryptase: Selective enhancement of eosinophil recruitment by histamine. J Immunol 159: Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng YW, Timmons C, Tram T and Coughlin SR (1997) Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386: Jarjour NN, Calhoun WJ, Schwartz LB and Busse WW (1991) Elevated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Respir Dis 144: Kaliner M and Austen KF (1973) A sequence of biochemical events in the antigeninduced release of chemical mediators from sensitized human lung tissue. J Exp Med 138: Kennerly DA and Duffy PA (1993) Activation mechanisms of mast cells and basophils, in Allergy (Holgate ST and Church MK, eds) pp , Gower Medical Publishing, London. Lagunoff D, Chi EY and Wan H (1975) Effects of chymotrypsin and trypsin on rat peritoneal mast cells. Biochem Pharmacol 24: Lowman MA, Rees PH, Benyon RC and Church MK (1988a) Human mast cell heterogeneity: histamine release from mas cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli. J Allergy Clin Immunol 81: Lowman MA, Benyon RC and Church MK (1988b) Characterisation of neuropeptideinduced histamine release from human dispersed skin mast cells. Br J Pharmacol 95: Machado DC, Horton D, Harrop R, Peachell PT and Helm BA (1996) Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen- specific IgE. Eur J Immunol 26: McEuen AR, He S, Brander ML and Walls AF (1996) Guinea pig lung tryptase: Localisation to mast cells and characterisation of the partially purified enzyme. Biochem Pharmacol 52: Molinari JF, Moore WR, Clark J, Tanaka R, Butterfield JH and Abraham WM (1995) Role of tryptase in immediate cutaneous responses in allergic sheep. J Appl Physiol 79: Molinari JF, Scuri M, Moore WR, Clark J, Tanaka R and Abraham WM (1996) Inhaled tryptase causes bronchoconstriction in sheep via histamine release. Am J Respir Crit Care Med 154: Molino M, Barnathan ES, Numerof R, Clark J, Dreyer M, Cumashi A, Hoxie JA, Schechter N, Woolkalis M and Brass LF (1997) Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 272: Nolte H, Schiotz PO and Skov PS (1987) A new glass fibre based histamine analysis

9 1998 Role of Tryptase in Mast Cell Activation 297 for allergy testing in children: results compared with conventional leukocyte histamine release assay, skin prick test, bronchial provocation test, and RAST. Allergy 42: Nystedt S, Emilsson K, Wahlestedt C and Sundelin J (1994) Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91: Okayama Y and Church MK (1992) Comparison of the modulatory effect of ketotifen, sodium cromoglycate, procaterol and salbutamol in human skin, lung and tonsil mast cells. Int Arch Allergy Appl Immunol 97: Okayama Y, Benyon RC, Lowman MA and Church MK (1994) In vitro effects of H1-antihistamine and PGD2 release from mast cells of human lung, tonsil, and skin. Allergy 49: Razin E and Marx,. G (1984) Thrombin-induced degranulation of cultured bone marrow-derived mast cells. J Immunol 133: Schick B, Austen KF and Schwartz LB (1984) Activation of rat serosal mast cells by chymase, an endogenous secretory granule protease. J Immunol 132: Schick B (1990) Cleavage of a rat serosal mast cell membrane component during degranulation mediated by chymase, a secretory granule protease. Immunology 69: Schwartz LB, Lewis RA, Seldin D and Austen KF (1981) Acid hydrolases and tryptase from secretory granules of dispersed human lung mast cells. J Immunol 126: Schwartz LB and Bradford TR (1986) Regulation of tryptase from human lung mast cells by heparin: Stabilization of the active tetramer. J Biol Chem 261: Schwartz LB, Metcalfe DD, Miller JS, Earl H and Sullivan T (1987a) Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis. N Engl J Med 316: Schwartz LB, Irani AM, Roller K, Castells MC and Schechter NM (1987b) Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells. J Immunol 138: Schwartz LB (1990) Tryptase from human mast cells: Biochemistry, biology and clinical utility, in Neutral Proteases of Mast Cells (Schwartz LB, ed) pp , Monogr Allergy, Basel, Karger. Shalit M and Levi-Schaffer F (1995) Challenge of mast cells with increasing amounts of antigen induces desensitization. Clin Exp Allergy 25: Vu T-KH, Hung DT, Wheaton VI and Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: Walls AF, Jones DB, Williams JH, Church MK and Holgate ST (1990a) Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase. J Pathol 162: Walls AF, Bennett AM, McBride HM, Glennie MJ, Holgate ST and Church MK (1990b) Production and characterization of monoclonal antibodies specific for human mast cell tryptase. Clin Exp Allergy 20: Walls AF, Bennett AR, Godfrey RC, Holgate ST and Church MK (1991) Mast cell tryptase and histamine concentrations in bronchoalveolar lavage fluid from patients with interstitial lung disease. Clin Sci 81: Walls AF (1995) The roles of neutral proteases in asthma and rhinitis, in Asthma and Rhinitis (Busse WW and Holgate ST, eds) pp , Blackwell, Boston. Send reprint requests to: Dr. Andrew F. Walls, Immunopharmacology Group, Centre Block (Mail Point 825), Southampton General Hospital, Southampton SO16 6YD, United Kingdom.