Shaoheng He

Human mast cell tryptase: a stimulus of microvascular leakage and mast cell activation

We have investigated the potential of tryptase to stimulate an increase in microvascular permeability following injection into the skin of guinea pigs. Tryptase was isolated from high salt extracts of human lung tissue by octyl-agarose and heparin-agarose chromatography. Injection of purified tryptase (2.5 ng-2.5 microg/site) into the skin of guinea pigs which had been injected intravenously with Evans blue dye provoked a dose-dependent increase in microvascular permeability. The skin reactions elicited by tryptase were apparent up to 80 min following injection, while histamine-induced microvascular leakage resolved completely by 40 min. Heat-inactivation of tryptase, or preincubating the proteinase with certain proteinase inhibitors, significantly reduced the extent of microvascular leakage, suggesting dependency on an intact catalytic site. No evidence was found for a synergistic or antagonistic interaction between tryptase (2.5 ng-2.5 microg/site) and histamine (1-10 microg/site) when these mast cell products were injected together. Addition of heparin to tryptase (10:1; w/w) prior to injection was without effect on tryptase-induced microvascular leakage. Pretreatment of guinea pigs with a combination of the histamine H1 receptor antagonist pyrilamine and the histamine H2 receptor antagonist cimetidine (both 10 mg/kg), partially abolished tryptase-induced microvascular leakage as well as attenuating the reaction to histamine. Reasoning that the microvascular leakage induced by tryptase is likely to involve the release of histamine, we investigated the ability of tryptase to stimulate histamine release from dispersed guinea-pig skin and lung cells in vitro. Tryptase was found to induce concentration-dependent histamine release from both sources of tissue. Mast cell activation stimulated by tryptase in vitro was inhibited by heat treating the enzyme or by addition of proteinase inhibitors, suggesting a requirement for an intact catalytic site. Histamine release was inhibited also by preincubating cells with the metabolic inhibitors antimycin A and 2-deoxy-D-glucose indicating that the mechanism was energy-requiring and non-cytotoxic. We conclude that human mast cell tryptase may be a potent stimulus of microvascular leakage. The activation of mast cells by this proteinase may represent an amplification process in allergic inflammation.

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

1 The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin‐agarose affinity chromatography and Sephacryl S‐200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. 2 Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea‐pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. 3 Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils, 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7×10−13 mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. 4 The chymase induced cell accumulation in both the guinea‐pig and mouse models was dependent on an intact catalytic site, being reduced by co‐injection of proteinase inhibitors or heat inactivation of the enzyme. 5 Co‐injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co‐injected into the mouse peritoneum. 6 Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.

The induction of a prolonged increase in microvascular permeability by human mast cell chymase.

Chymase is a major constituent of the secretory granules of human mast cells, but little is known of the contribution of this serine proteinase in acute allergic reactions. We have purified chymase from human skin tissue, and have investigated its potential to induce microvascular leakage in vivo. Injection of chymase into the skin of guinea pigs provoked an increase in microvascular leakage within 20 min. Although skin reactions were smaller than those elicited with similar quantities of histamine at this time point, they were much longer-lived, and were still apparent 120 min following injection. Chymase induced microvascular leakage was reduced in the presence of soybean trypsin inhibitor, and abolished by heat inactivating the enzyme, indicating dependence on an intact catalytic site. Little evidence was found for synergistic interactions between chymase and either histamine or tryptase. Antihistamine pretreatment of animals did not reduce the magnitude of skin reactions to chymase suggesting that they were not mediated by histamine release. Chymase could contribute to increases in microvascular permeability following mast cell degranulation in allergic disease.

Granulocyte Recruitment by Human Mast Cell Tryptase

Eosinophil Epithelial cells Mast cell Neutrophil Tryptase Correspondence to: Dr. Andrew F. Walls, University Medicine, Level F, Southampton General Hospital, Tremona Road, Southampton SO16 6YD (UK) Tryptase is a tetrameric serine protease which is stored almost exclusively in the secretory granules of mast cells. Substantial quantities of this enzyme are released as a consequence of mast cell activation in allergic disease. The pathobiological role of tryptase is not well understood, though this enzyme can cleave a number of potential substrates [reviewed in ref. 1]. Tryptase can efficiently degrade certain regulatory peptides including the bronchodilator, vasoactive intestinal peptide and the potent vasodilator cal-citonin-gene-related peptide; it may have a role in kinin generation, and by activating stromelysin, it may participate in processes of tissue degradation, and it can act as a growth factor for fibroblasts. Microvascular leakage has been observed in the skin of guinea pigs following injection of purified human mast cell tryptase. As we have found that the increase in vascular permeability can be inhibited by antihistamine pretreatment, and that tryptase can stimulate histamine release from guinea pig mast cells in vitro, it would seem likely that the effect is mediated by tryptaseinduced mast cell activation. Injection of tryptase into guinea pig skin or the mouse peritoneum results, within 6 h, in a neutrophil-rich inflammatory infiltrate. We have investigated potential mechanisms of human granulocyte recruitment by tryptase using in vitro models. In modified Boyden chambers with neutrophils purified from peripheral blood on Lymphoprep, we have found that tryptase can itself act as a chemoattractant even at concentrations of 6 mU/ml (where 1 mU is defined as that amount which can hydrolyse 1 nmol of the peptide substate N-αbenzoyl-DI-arginine /?-nitroanilide per minute at 25 °C). This action was inhibited by protease inhibitors including leupeptin, and by heat treatment, indicating dependence on an active catalytic site. Incubation of purified neutrophils with tryptase was associated with a shape change reaction within minutes, as revealed by scanning electron microscopy.

The inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin G

Inhibitors of mast cell tryptase and chymase can be effective as mast cell stabilising compounds. Lactoferrin has been reported to inhibit tryptase activity, but its actions on other serine proteases of mast cells and its potential to alter mast cell function are not known. We have examined the ability of lactoferrin to inhibit mast cell tryptase, chymase and cathepsin G, and investigated its potential to modulate the activation of human mast cells. Enzymatically dispersed cells from human skin, lung and tonsil were challenged with anti-IgE or calcium ionophore A23187, following incubation with recombinant human lactoferrin, and histamine release determined. IgE-dependent histamine release from skin mast cells was inhibited by up to 50% following incubation with lactoferrin (50 or 500 nM). Tonsil mast cells were also stabilised by lactoferrin, but not those from lung. Calcium ionophore A23187-induced histamine release was not altered by lactoferrin. A double-labelling immunocytochemical procedure revealed the presence of lactoferrin in 4-6% of mast cells, and this proportion increased to 40% following incubation with lactoferrin. Lactoferrin did not inhibit cleavage of synthetic substrates by tryptase and chymase directly, though it was able to diminish the ability of heparin to stabilise tryptase. Cathepsin G activity was inhibited by lactoferrin. The ability of lactoferrin to inhibit IgE-dependent activation of human mast cells and modulate protease activity suggests that the release of this neutrophil product may have a role in the downregulation of allergic inflammation.

Guinea pig lung tryptase: Localisation to mast cells and characterisation of the partially purified enzyme☆

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.

The activation of synovial mast cells: modulation of histamine release by tryptase and chymase and their inhibitors

Mast cells have been implicated as having pivotal roles in arthritis, but little is known of the processes leading to the activation of synovial mast cells or their potential for pharmacological control. We have investigated the ability of tryptase and chymase, and inhibitors of these major mast cell proteases to modulate the activation of mast cells from human synovial tissue. The tryptase inhibitor drug N-(1-hydroxy-2-naphthoyl)-L-arginyl-L-prolinamide hydrochloride (APC366) inhibited immunoglobulin E (IgE)-dependent histamine release in a dose-dependent manner, with about 70% inhibition being achieved at a dose of 300 microM. Histamine release stimulated by calcium ionophore A23187 was also inhibited by this compound. The chymase inhibitor chymostatin inhibited IgE-dependent histamine release by approximately 60% at 1 microg/ml. Tryptase at concentrations of 3.0 microg/ml and greater stimulated histamine release from synovial cells, which was dependent on catalytic activity, whereas chymase had little effect on these cells. The activation of mast cells by tryptase may represent an amplification process in the synovium. The mast cell stabilising properties of inhibitors of tryptase and chymase could be of therapeutic value in arthritis.

Experimental Activation of Mast Cells and Their Pharmacological Modulation

The activation of mast cells is of pivotal importance in the pathogenesis of allergic conditions. Mast cell activation can provoke rapid increases in microvascular permeability, induce bronchoconstriction after blood flow, stimulate the recruitment and activation of other inflammatory cells, and has come to be associated with the processes of tissue remodeling and fibrosis. Such changes may be mediated by the release of a range of potent mediators of inflammation: preformed in secretory granules, or newly generated, or both. There are major differences in the responsiveness to various stimuli and to pharmacological agents for mast cells from different body compartments. A method is presented here for the purification of mast cells from enzymatically dispersed human tissues. The methods described for the experimental activation of mast cells can be readily adapted to studies with cell lines or mast cells obtained through long-term culture.