Modulation of tryptase release from human tonsil mast cells by protease inhibitors

(1)

Modulation of tryptase release from human tonsil mast cells by protease inhibitors

Shaoheng He^1,2, Hua Xie¹

Allergy & Inflammation Research Institute, 22 Xin-Ling Road, Shantou University Medical College, Shantou 515031, China

Immunopharmacology Group, University of Southampton, Southampton, UK Correspondence:Shaoheng He, e-mail: shoahenghe@hotmail.com

Abstract:

AIM. To examine the influence of protease inhibitors on tryptase release, and as a comparison the influence of the inhibitors on histamine secretion was assessed.METHODS. Enzymatically dispersed cells from human tonsil were challenged with anti-IgE or calcium ionophore A23187 (CI) in the absence or presence of the tryptase and chymase inhibitors, and tryptase and histamine release was determined. RESULTS. IgE-dependent tryptase release from dispersed tonsil mast cells was inhibited by a maximum of approximately 35.5% and 35.7% by N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), respectively. The similar degree of inhibition of CI-induced tryptase release was observed also with these two inhibitors. Preincubation of TLCK or TPCK with cells at 37°C for 20 min before addition of the stimulus improved slightly their ability to inhibit anti-IgE and CI-induced tryptase release. Protamine showed dual action on tryptase release from tonsil mast cells.

The concentration-dependent inhibition of anti-IgE and CI-induced release of histamine from tonsil mast cells was also observed with TLCK, TPCK and protamine. The maximum inhibition of anti-IgE-induced histamine release was approximately 26.6%, 30.8% and 30.1% with TLCK, TPCK and protamine, respectively. At the concentrations tested, TLCK and TPCK by themselves did not stimulate tryptase and histamine release from tonsil mast cells.CONCLUSION. It was demonstrated that protease inhibitors were able to inhibit IgE-dependent tryptase release from human tonsil mast cells, which suggests strongly that they can be developed to a novel class of anti-inflammatory drugs to treat allergic conditions in man.

Key words:

tryptase, histamine, mast cell, anti-IgE, protease inhibitor

Abbreviations: TLCK – N-a-p-tosyl-L-lysine chloromethyl ketone, TPCK – N-tosyl-L-phenylalanyl chloromethyl ketone, CI – calcium ionophore A23187

Introduction

Mast cell degranulation is a key event in the pathogenesis of allergic disease. When allergens cross-link

their specific IgE on the surface of sensitized mast cells, degranulation occurs. Upon degranulation, a range of mediators are released from mast cells including histamine, tryptase, chymase, heparin and some cytokines [33].

Tryptase is an almost unique product of human mast cell granules and has been serving as a marker of mast cells for the last two decades. Increased levels of tryptase in biological fluids have been observed in a number of diseases such as in the serum from cases

Pharmacological Reports 2005, 57, 523530 ISSN 1734-1140

(2)

of patients with allergic rhinitis [19], in skin blister fluid from subjects with allergic contact dermatitis [4]

and in synovial fluid from patients with arthritis [6].

In recent years, increased tryptase levels were found also to be associated with asthma [3, 8], acute appendicitis [9], blood clotting [28], hay fever [21] and skin allergic reactions [23], indicating further that mast cell degranulation, tryptase release in particular, is likely to play a role in the pathogenesis of these diseases.

For more than four decades, histamine has been widely used as a marker of mast cell degranulationin vitro, and numerous anti-allergic drugs such as so- dium cromoglycate, lodoxamide, salbutamol, ketotifen, terfenadine and cetirizine [26, 27] and sal- meteral [7] were reported to be able to inhibit anti- IgE-induced histamine release from human mast cells.

In recent years, inhibitors of tryptase [14, 16] and chymase [13] were discovered to possess the ability to inhibit histamine release from human skin, tonsil and synovial mast cells [15], suggesting that these inhibitors are likely to be developed as a novel class of mast cell stabilizer. However, little is known of the actions of tryptase and chymase inhibitors on tryptase release from human mast cells. We, therefore, investigated the effects of these two types of inhibitors on IgE- dependent or independent tryptase release from human tonsil mast cells in the current study. For comparison, the actions of these inhibitors on histamine release were also investigated in the same experimental system.

Materials and Methods

Materials

The following compounds were purchased from Sigma (St. Louis, Mo., USA): TLCK, TPCK, protamine, calcium ionophore A23187 (CI), histamine di- hydrochloride, collagenase (type I), hyaluronidase (type I), bovine serum albumin (BSA, fraction V), penicillin and streptomycin, extravidin peroxidase, o-phenylene diamine (OPD). Minimum Essential Me- dium (MEM) containing 25 mmol/lN-(2-hydroxyethyl)- piperazine-N’-2-ethanesulfonic acid (HEPES) and FCS were from Gibco (Paisley, Renfrewshire, UK).

Andrew F. Walls (University of Southampton, UK).

Histamine plate was from RefLab (Copenhagen, Den- mark). HEPES and all other chemicals were of ana- lytical grade.

Dispersion of mast cells

Human tonsil tissue was obtained at tonsillectomy.

Only macroscopically normal tissue was used for the study. The mast cell dispersion procedure employed was the same one as that described previously with human tonsils [14]. Briefly, finely chopped tissue was incubated with 1.5 mg/ml of collagenase and 0.75 mg/ml of hyaluronidase in MEM containing 2%

fetal calf serum (1 g tonsil/10 ml buffer) for 60 min at 37°C. Dispersed cells were separated from undigested tissue by filtration through nylon gauze (pore size 100mm diameter), washed and maintained in MEM (containing 10% FCS, 200 U/ml of penicillin, 200mg/ml of streptomycin) on a roller overnight at room tem- perature. Mast cell purity, as determined by light mi- croscopy after staining by alcine blue, ranged from 0.5 to 1.1%.

Mast cell challenge

Dispersed cells were resuspended in HEPES-buffered salt solution (HBSS, pH 7.4) with CaCl₂and MgCl₂ (complete HBSS), and 100ml aliquots containing 4–6

× 10³mast cells were added to a 50ml of anti-IgE, CI, or inhibitor in complete HBSS and incubated for 15 min at 37°C. The reaction was terminated by the addition of 150ml of ice cold incomplete HBSS and the tubes were centrifuged immediately (500 × g, 10 min, 4°C). All experiments were performed in duplicate. For the measurement of total histamine concentration, in certain tubes the suspension was boiled for 6 min. Supernatants were stored at –20°C until histamine and tryptase concentrations were determined (in duplicate for each tube).

Inhibition of tryptase and histamine release

For certain experiments, protease inhibitor was preincubated with cells for 20 min before anti-IgE or CI were added. For other experiments, protease inhibitor was added to cells at the same time with anti-IgE or

(3)

CI (no preincubation period). Data were expressed as the percentage inhibition of tryptase and histamine release, taking into account the mediator release in the presence and absence of the inhibitor. As for our previous experiments, the optimal tryptase and histamine release from colon mast cells was induced by 10mg/ml of anti-IgE or 1 mg/ml of CI [17], and, therefore, they were chosen as standard concentrations throughout the study.

Histamine measurement

Histamine concentrations were determined using a glass fibre-based, fluorometric assay [14]. The procedure involves the binding of histamine to a glass- fiber matrix (RafLab, Copenhagen, Denmark) and its detection spectrophotometrically with Perkin-Elmer LS 2 Detector, (Denmark) following addition of OPD.

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.

Tryptase measurement

Tryptase concentrations were measured with a sand- wich ELISA procedure with a specific polyclonal antibody against human tryptase as the capture antibody and AA5 a monoclonal antibody specific for human tryptase as the detecting antibody [6].

Statistical analyses

Statistical analyses were performed by using SPSS software. Data were expressed as the mean ± SEM.

Where analysis of variance (ANOVA) indicated significant differences between groups, for the pre- planned comparisons of interest, Student’st-test was ap- plied. For all analyses, p < 0.05 was taken as significant.

Results

Effect of protease inhibitors, anti-IgE and CI on tryptase and histamine release from mast cells

At 15 min following incubation, anti-IgE at 10mg/ml and CI at 1 µg/ml were able to induce 7.2 ± 0.7 ng/ml

and 10.6 ± 0.9 ng/ml tryptase release from tonsil mast cells, respectively, whereas at the same time point spontaneous tryptase release (buffer alone) was 2.2 ± 0.6 ng/ml. The same concentrations of anti-IgE and CI were also able to provoke significant tryptase release from tonsil mast cells following 35 min incubation period. All protease inhibitors tested had no stimulatory effect on tonsil mast cells at both 15 min and 35 min incubation periods, except for protamine at 100 ng/ml, which was able to elicit 11.5 ± 2.5 ng/ml tryptase release from tonsil mast cells. TLCK, TPCK and protamine at the concentrations tested had no effect on histamine release from tonsil mast cells following both 15 min and 35 min incubation periods.

However, in the same experiments, anti-IgE and CI were able to induce up to 15.7% and 31.1% net histamine release, respectively.

Inhibition of tryptase release from mast cells by protease inhibitors

Both TLCK and TPCK were able to inhibit IgE- dependent tryptase release from tonsil mast cells in a concentration-dependent manner. Up to approximately 35.5% or 35.7% inhibition of anti-IgE-induced tryptase release were achieved with 100mg/ml TLCK or 80 mg/ml TPCK, respectively, when they were added to cells at the same time with anti-IgE (Fig.

1A). Similarly, TLCK and TPCK were also capable of inhibiting CI-induced tryptase release from tonsil mast cells in a concentration-dependent pattern with up to 32.3% or 34.4% inhibition, being achieved with 100mg/ml of TLCK or 80 mg/ml of TPCK, respectively (Fig. 1B). Preincubation of TLCK or TPCK with cells for 20 min at 37°C improved slightly their ability to inhibit anti-IgE-dependent and CI-induced tryptase release, but those improvements did not reach the levels of statistical significance (Fig. 1 A and 1B).

At the lower concentrations (0.1 and 1mg/ml), protamine had little effect on anti-IgE- or CI-induced tryptase release; but at the higher concentrations (10 or 100mg/ml), this tryptase inhibitor showed an additive stimulatory effect on anti-IgE- or CI-induced tryptase release. The amount of tryptase released from tonsil cells was up to 27.7 ± 6.6 and 23.7 ± 4.3 ng/ml, respectively when 100 mg/ml of protamine and 10mg/ml of anti-IgE or 1 mg/ml of CI were added to cells at same time. In comparison, 100mg/ml of protamine, 10 mg/ml of anti-IgE or 1 mg/ml of CI alone stimulated only 11.5 ± 2.5, 7.2 ± 0.7 and 10.6 ± 0.9 ng/ml

Modulation of tryptase release by protease inhibitors

Shaoheng He et al.

(4)

proximately 7.2 ± 0.7 and 10.6 ± 0.9 ng/ml induced by anti-IgE and CI, respectively

(5)

tryptase release, respectively in the same experiments.

However, preincubation of 10 mg/ml of protamine with cells for 20 min before addition of anti-IgE or CI did not induce significant tryptase release from tonsil mast cells.

tryptase release, respectively in the same experiments.

However, preincubation of 10 mg/ml of protamine with cells for 20 min before addition of anti-IgE or CI did not induce significant tryptase release from tonsil mast cells.

Inhibition of histamine release from mast cells by protease inhibitors

The concentration-dependent inhibition of anti-IgE- induced release of histamine from tonsil mast cells was observed when TLCK, TPCK or protamine were added to cells, respectively, with the stimulus at the same time. The maximum inhibition of histamine release induced by anti-IgE was approximately 26.6%, 30.8% and 30.1% with 100mg/ml of TLCK, 80 mg/ml of TPCK and 100 mg/ml of protamine, respectively.

Preincubation of various concentrations of TLCK, TPCK and protamine with cells at 37°C for 20 min before challenging them with anti-IgE did not signifi- cantly alter the inhibitory activities of these inhibitors (Fig. 2). TLCK, TPCK or protamine were also able to inhibit CI-induced histamine release from tonsil mast

cells with up to approximately 26%, 29.2% and 35.1% being observed with 100 mg/ml of TLCK, 80mg/ml of TPCK and 100 mg/ml of protamine, respectively. Preincubation of TLCK, TPCK or protamine with cells for 20 min at 37°C improved slightly their ability to inhibit CI-induced histamine release (Fig. 3).

Discussion

We found that inhibitors of tryptase and chymase were able to inhibit anti-IgE- and CI- induced tryptase release from dispersed human tonsil mast cells in the current study, which may indicate a potential of a novel therapy for the treatment of allergic or other mast cell-related diseases.

Up to approximately 35.5% inhibition of IgE- dependent tryptase release from tonsil mast cells was observed with the inhibitor of chymase, TPCK indicating that a chymase activity was involved in the process of IgE-dependent human mast cell degranula-

Shaoheng He et al.

%inhibitionofhistaminerelase

Fig. 2.Inhibition of anti-IgE (10 µg/ml)-induced histamine release from dispersed tonsil mast cells by the protease inhibitors TLCK, TPCK and protamine. The various concentrations of inhibitors were added to cells at the same time with anti-IgE (no preincubation) or preincubated with cells at 37°C for 20 min before addition of the stimulus. Data are presented as the mean ± SEM for six separate experiments performed in duplicate. * p < 0.05 compared with the responses with uninhibited controls. The unblocked release of histamine was approximately 13.3 ± 3.3 and 31± 6.8% induced by anti-IgE and CI, respectively

(6)

tion. This was consistent with our previous finding that chymase inhibitors were able to inhibit IgE- dependent histamine release [13], which indirectly proved that tryptase and histamine are likely to share a similar degranulation process. Similar to TPCK, a tryptase inhibitor, TLCK inhibited by up to some 35.7% anti-IgE-induced tryptase release from tonsil mast cells, which implicated that a tryptase activity is also likely to be involved in the process of mast cell degranulation. This result was consistent with our previous finding that tryptase inhibitors were able to inhibit IgE-dependent histamine release [14, 15].

Since a majority of these inhibitors at the concentrations used in the current study are able to inhibit by more than 95% tryptase or chymase activity in enzyme assays [12], the incomplete inhibition of tryptase release from mast cells may suggest that some pathways other than tryptase and chymase pathways were involved in the anti-IgE-induced degranulation of tonsil mast cells. In comparison, in the parallel experiments, TLCK and TPCK were also able to inhibit by a maximum of up to approximately 26.6% and 30.8% IgE-dependent histamine release from tonsil mast cells. The degree of inhibition was quite similar

to that for tryptase release, but much lower than that reported previously, which found that TPCK was able to completely inhibit FceRI- mediated histamine release from cultured human mast cells [34]. TPCK was reported previously also to be able to inhibit CI-induced histamine release from human lung mast cells [18].

CI is a calcium carrier that can elevate the calcium concentration in cytoplasms of mast cells [10], and, therefore, it is involved in the late stage of the process of mast cell degranulation. The inhibition of CI- induced tryptase release by the inhibitors of tryptase and chymase in the current study may suggest the involvement of tryptase and chymase activities in mast cell degranulation process at the late stage and most likely after influx of calcium ions into mast cells. The evidence that tryptase and chymase are sited in the granules of mast cells in their fully active form [25]

supported further the likelihood that these two mast cell serine proteases were involved in the activation of tonsil mast cells.

The result that tryptase levels were elevated when 10 and 100 mg/ml of protamine were added to cells alone or with anti-IgE or CI was unexpected. This was most likely due to the tetrameric structure of tryp-

%inhibitionofhist

Fig. 3.Inhibition of calcium ionophore (CI, 1µg/ml)-induced histamine release from dispersed tonsil mast cells by the protease inhibitors TLCK, TPCK and protamine. The various concentrations of inhibitors were added to cells at the same time with CI (no preincubation) or preincubated with cells at 37°C for 20 min before addition of the stimulus. Data are presented as the mean ± SEM for six separate experiments performed in duplicate. * p < 0.05 compared with the responses with uninhibited controls. The unblocked release of histamine was approximately 15.7 ± 4.5 and 31.1 ± 4.6% induced by anti-IgE and CI, respectively

(7)

tase being dissociated by protamine [11], thus more tryptase monomer existed in supernatants, being recognized by AA5 as an intact tryptase molecule. How- ever, it is difficult to exclude the possibility that protamine selectively stimulated tryptase release from human tonsil mast cells. Protamine by itself at the concentrations examined did not stimulate histamine release from tonsil mast cells. The reason for the difference between our finding and the previous reports, which showed that protamine could stimulate histamine release from guinea pig [1] and rat mast cells [2], was most likely due to the difference in mast cell sources.

The observation that preincubation of the inhibitors with the cells for 20 min before challenging them with anti-IgE had little impact on inhibition of IgE- dependent histamine release was unexpected, never- theless, it may suggest that the actions of these inhibitors are rather rapid and the involvement of tryptase and chymase activities in anti-IgE-induced tryptase and histamine release is likely to occur at the late stage of the degranulation process.

The inhibitory action of TPCK on anti-IgE- (34) and CI-(18) induced histamine release from human mast cells has been reported previously. However, the degree of inhibition achieved in the present study was by up to 40%, which was much lower than that reported previously (complete inhibition of anti-IgE induced histamine release by 3.52 mg/ml of TPCK) (34). While TLCK was reported to have no influence on CI-induced histamine release from human lung mast cells (18), it was shown to elicit potent inhibitory effect on compound 48/80-induced histamine release from rat mast cells (31). The current experimental result showed that TLCK did inhibit both anti-IgE- and CI-induced histamine release from human tonsil mast cells, which may reflect another mast cell het- erogeneity in man.

Over the years, many compounds including sodium cromoglycate, lodoxamide, salbutamol, ketotifen, terfenadine and cetirizine have been recognized as mast cell stabilizers or histamine receptor antagonists, and have been used as anti-allergic drugs in day-to-day clinical practice. However, only less than 40% inhibition of IgE-dependent mast cell degranulation can be achieved with these compounds, which is much less than that achieved with inhibitors of tryptase [14] and chymase in the similar experimental system [13].

Moreover, some of the latest reports on tryptase inhibitors highlighted the importance of this potential anti-inflammatory drugs. Inhaled APC366 was able to

attenuate allergen-induced late-phase airway obstruction in asthma [22], and APC2059 could improve the symptomatic scores of the patients with mildly to moderately active ulcerative colitis in an open-label pilot study [30]. Our findings in the current study may, at least partially, explain the mechanism by which tryptase inhibitors could treat these diseases.

The successful treatment of acute ulcerative colitis [20] and Crohn’s disease [24] with mast cell stabilizer drug ketotifen further strongly suggested that inhibitors of tryptase and chymase are likely to become a novel class of anti-inflammatory drugs with their anti- inflammatory actions and mast cell stabilizing proper- ties.

In conclusion, the inhibitors of both tryptase and chymase were able to inhibit IgE-dependent and CI- induced tryptase release from tonsil mast cells, indicating that they are likely to be developed to a novel class anti-inflammatory drugs to treat allergic or other mast cell-associated inflammatory conditions in man.

Acknowledgments:

This project was supported by the grants from the Li Ka Shing Foundation, Hong Kong, China (No. C0200001), the Major State Basic Research Program of China (973 Program) (No. 2001CB510009), the National Natural Science Foundation of China (No. 30471601), the Planned Science and Technology Project of Guang Dong Province, China (No. 2003B31502) and the National Natural Science Foundation of Guang Dong Province, China (No. 04106122).

References:

1. Augusto C, Lunardi LO, Vugman I: Non cytotoxic guinea- pig mesenteric mast cell stimulation by protamine. Agents Actions, 1987, 22, 185–188.

2. Augusto C, Lunardi LO, Vugman I: Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism. Cell Mol Biol, 1990, 36, 357–361.

3. Bettiol J, Radermecker M, Sele J, Henquet M, Cataldo D, Louis R: Airway mast-cell activation in asthmatics is associated with selective sputum eosinophilia. Allergy, 1999, 54, 1188–1193.

4. Brockow K, Abeck D, Hermann K, Ring J: Tryptase concentration in skin blister fluid from patients with bullous skin conditions. Arch Dermatol Res, 1996, 288, 771–773.

5. Broide DH, Gleich GJ, Cuomo AJ, Coburn DA, Feder- man EC, Schwartz LB, Wasserman SI: Evidence of on- going mast cell and eosinophil degranulation in symptomatic asthma airway. J Allergy Clin Immunol, 1991, 88, 637–648.

6. Buckley M, Walters C, Brander M, Wong WM, Cawley MI, Ren S, Schwartz LB et al.: Mast cell activation in ar-

Shaoheng He et al.

(8)

tent and long-acting inhibitor of inflammatory mediator release from human lung. Br J Pharmacol, 1991, 104, 672–676.

8. Carroll NG, Mutavdzic S, James AL: Increased mast cells and neutrophils in submucosal mucous glands and mucus plugging in patients with asthma. Thorax, 2002, 57, 677–682.

9. Coskun N, Sindel M, Elpek GO: Mast cell density, neu- ronal hypertrophy and nerve growth factor expression in patients with acute appendicitis. Folia Morphol, 2002, 61, 237–243.

10. Foreman JC, Mongar JL, Gomperts BD: Calcium iono- phores and movement of calcium ions following the physiological stimulus to a secretory process. Nature, 1973, 245, 249–251.

11. Hallgren J, Estrada S, Karlson U, Alving K, Pejler G:

Heparin antagonists are potent inhibitors of mast cell tryptase. Biochemistry, 2001, 40, 7342–7349.

12. He S, Chen P, Chen HQ: Modulation of enzymatic activity of human mast cell tryptase and chymase by proteinase inhibitors. Acta Pharmacol Sin, 2003, 24, 923–929.

13. He S, Gaça MDA, McEuen AR, Walls AF: Inhibitors of chymase as mast cell stabilising agents: the contribution of chymase in the activation of human mast cells. J Phar- macol Exp Ther, 1999, 291, 517–523.

14. He S, Gaça MDA, Walls AF: A role for tryptase in the activation of human mast cells: Modulation of histamine release by tryptase and inhibitors of tryptase. J Pharma- col Exp Ther, 1998, 286, 289–297.

15. He S, Gaça MD, Walls AF: The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors. Eur J Pharmacol, 2001, 412, 223–229.

16. He S, McEuen AR, Blewett SA, Li P, Buckley MG, Leufkens P, Walls AF: The inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin G. Bio- chem Pharmacol, 2003, 65, 1007–1015.

17. He S, Xie H, He YS: Induction of tryptase and histamine release from human colon mast cells by IgE-dependent or -independent mechanisms. World J Gastroenterol, 2004, 10, 319–322.

18. Hultsch T, Ennis M, Heidtmann HH: The effect of serine esterase inhibitors on ionophore-induced histamine release from human pulmonary mast cells. Agents Actions, 1988, 23, 198–200.

19. Jarjour NN, Calhoun WJ, Schwartz LB, Busse WW: Ele- vated bronchoalveolar lavage fluid histamine levels in allergic asthmatics are associated with increased airway obstruction. Am Rev Respir Dis, 1991, 144, 83–87.

20. Jones NL, Roifman CM, Griffiths AM, Sherman P: Ke- totifen therapy for acute ulcerative colitis in children:

a pilot study. Dig Dis Sci, 1998, 43, 609–615.

21. KleinJan A, McEuen AR, Dijkstra MD, Buckley MG, Walls AF, Fokkens WJ: Basophil and eosinophil accu- mulation and mast cell degranulation in the nasal mucosa

cell tryptase by inhaled APC366 attenuates allergen- induced late-phase airway obstruction in asthma.

J Allergy Clin Immunol, 2001, 107, 1039–1045.

23. Macfarlane AJ, Kon OM, Smith SJ, Zeibecoglou K, Khan LN, Barata LT, McEuen AR et al.: Basophils, eosi- nophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin.

J Allergy Clin Immunol, 2000, 105, 99–107.

24. Marshall JK, Irvine EJ: Ketotifen treatment of active colitis in patients with 5-aminosalicylate intolerance. Can J Gastroenterol, 1998, 12, 273–275.

25. McEuen AR, He S, Brander ML, Walls, AF: Guinea pig lung tryptase: localisation to mast cells and characterisa- tion of the partially purified enzyme. Biochem Pharma- col, 1996, 52, 331–340.

26. Okayama Y, Benyon RC, Lowman MA, Church MK: In vitro effects of H-antihistamine and PGD release from mast cells of human lung, tonsil, and skin. Allergy, 1994, 49, 246–253.

27. Okayama Y, Church MK: 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, 1992, 97, 216–222.

28. Samoszuk M, Corwin M, Hazen SL: Effects of human mast cell tryptase and eosinophil granule proteins on the kinetics of blood clotting. Am J Hematol, 2003, 73, 18–25.

29. Schwartz LB, Metcalfe DD, Miller JS, Earl H, Sullivan T: Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis. N Engl J Med, 1987, 316, 1622–1626.

30. Tremaine WJ, Brzezinski A, Katz JA, Wolf DC, Fleming TJ, Mordenti J, Strenkoski-Nix LC et al.: Treatment of mildly to moderately active ulcerative colitis with a tryptase inhibitor (APC 2059): an open-label pilot study. Ali- ment Pharmacol Ther, 2002, 16, 407–413.

31. Vassimon CS, Rothschild AM: Compound 48/80-induced secretion of histamine from rat peritoneal mast cells de- pends on a tryptase controlled step also leading to chymase activity. Agents Actions, 1990, 30, 150–152.

32. Walls AF, Bennett AR, Godfrey RC, Holgate ST, Church MK: Mast cell tryptase and histamine concentrations in bronchoalveolar lavage fluid from patients with intersti- tial lung disease. Clin Sci, 1991, 81, 183–188.

33. Walls AF, He S, Buckley MG, McEuen AR: Roles of the mast cell and basophil in asthma. Clin Exp Allergy Rev, 2001, 1, 68–72.

34. Yanagida M, Fukamachi H, Takei M, Uzumaki H, Saito TT, Iikura Y, Nakahata T: Effect of a chymotrypsin-like inhibitor, TPCK, on histamine release from cultured human mast cells. J Pharm Pharmacol, 1997, 49, 537–541.

Received:

November 4, 2004; in revised form: June 7, 2005.