Enzyme histochemistry of rat mast cell tryptase

Fixation and staining conditions for rat mast cell tryptase and its histochemical distribution in different rat tissues were investigated. Prostate, skin, lung, gut, stomach and salivary glands were fixed in either aldehyde or Carnoy fixatives and then frozen or embedded in paraffin wax. Preservation of tryptase enzymic activity against peptide substrates required aldehyde fixation and frozen sectioning. Of the peptide substrates examined, z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthylamide proved the most effective for the demonstration of tryptase. Double staining by enzyme cytochemistry followed by immunological detection of tryptase showed that, in all tryptase-containing mast cells, the enzyme is at least in part active. Conventional dye-binding histochemistry was used to confirm the identity of mast cells. Aldehyde-fixed mucosal mast cells required a much shorter staining time with Toluidine Blue if tissue sections were washed directly in t-butyl alcohol. Double staining by enzyme cytochemistry and dye binding showed that tryptase is absent from mucosal and subepidermal mast cells, which are also smaller in size and appear to contain fewer granules than connective tissue mast cells. This study demonstrates that rat mast cell tryptase, unlike tryptases in other species, is a soluble enzyme. It is stored in an active form and is absent from some mast cell subpopulations in mucosa, skin and lung. © 1998 Chapman & Hall     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

An antibody raised against in vitro-derived human mast cells identifies mature mast cells and a population of cells that are Fc epsilon RI(+), tryptase(-), and chymase(-) in a variety of human tissues.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.     

Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues

Proteases present in mast cell granules have been harnessed to demonstrate mast cells in human tissues. A number of substrate mixtures were tested. D-Val-Leu-Arg-4-methoxy-2-naphthylamide (MNA) plus Fast Blue B was the best for identifying human mast cells, yielding the most specific and complete staining. The procedure is simple and the results are permanent. Cryostat sections of aldehyde-fixed routine preparations or paraffin sections of Carnoy-fixed tissues give the most satisfactory results. Mast cells are stained a strong red color that stands out distinctly from the surrounding tissues, so that they can be easily identified by simple microscopy. A double-staining technique, first for protease and subsequently using Alcian Blue, showed that as progressive protease staining occurs, the alcianophilia of mast cells is lost. This procedure demonstrated that mast cells in the mucosa of human gut generally required longer incubations to develop protease staining than in other connective tissue sites. In post-mortem tissues, mast cells retain their protease activity well and so can be demonstrated in cryostat sections of aldehyde-fixed material, giving a more complete picture than with Alcian Blue. The synthetic substrate D-Val-Leu-Arg-MNA can be recommended for routine identification of mast cells in human tissues.     

Demonstration of non-neural tryptophan 5-mono-oxygenase in mouse intestinal mucosa.

Tryptophan 5-mono-oxygenase was demonstrated and its activity was measured in mucosal extracts of the mouse digestive tract by means of highly sensitive h.p.l.c. detection. The intestinal enzyme was activated by anaerobic incubation with dithiothreitol, as are the enzymes from mouse mastocytoma cells and bovine pineal gland. The dithiothreitol-enhanced activity was highest at the proximal portion of colon followed by that at the duodenum, where the unenhanced activity/enhanced activity ratio was highest. The enzymic and immunochemical properties of the intestinal tryptophan 5-mono-oxygenase were similar to those of the mastocytoma enzyme. In contrast, the intestinal enzyme was immunochemically different from brain tryptophan 5-mono-oxygenase. The possibility that connective tissue and/or mucosal mast cells are responsible for some of the enzyme activity of the duodenal mucosa was ruled out by the demonstration of the activity in extracts from a mast-cell-deficient mutant mouse (W/Wv). The enzyme in the duodenum was found to reside between the upper villus region and the bottom of the crypt, suggesting that it is mainly of enterochromaffin cell and not of submucosal nerve plexus origin.     

Elevated proteinase activities in mouse lung tumors quantitated by synthetic fluorogenic substrates.

Proteinase activities in malignant and normal lung tissues were measured using two synthetic substrates that consist of a fluorophor coupled to a peptide moiety. The hydrolysis of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide and BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide were studied in homogenates of two types of mouse lung tumors, the Lewis lung tumor of the C57 black mouse and the KHT tumor of the C3H mouse. The activity of CBZ-Val-Lys-Lys-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.3 and a Km of 2.1 x 10(-4) M, required a thiol activator, and was inhibited by leupeptin suggesting the activity of a cathepsin B-like enzyme. The activity of BZ-Gly-Gly-Arg-4-methoxy-2-naphthylamide hydrolysis had a pH optimum of 6.7 and a Km of 3 x 10(-5) M. Lung tumor homogenates contained higher hydrolytic activities for both substrates than normal lung homogenates.     

Heparin antagonists are potent inhibitors of mast cell tryptase

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.     

Dog mastocytoma tryptase: affinity purification, characterization, and amino-terminal sequence

A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.     

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.     

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.     

Thymosin beta 4 expression in normal skin,colon mucosa and in tumor infiltrating mast cells

Mast cells (MCs) are metachromatic cells that originate from multipotential hemopoietic stem cells in the bone marrow. Two distinct populations of MCs have been characterized: mucosal MCs are tryptase-positive while mast cells in skin contain tryptase and chymase. We now show that a sub-population of MCs is highly immunoreactive for thymosin β₄, as revealed by immunohistochemical analyses of normal skin, normal colon mucosa and salivary gland tumors. Four consecutive serial sections from each case were immunostained for thymosin β₄ (Tβ₄), chymase, tryptase and stained for toluidine blue. In skin biopsies, MCs showed a comparable immunoreactivity for Tβ₄, chymase and tryptase. In normal colon mucosa the vast majority of mucosal MCs expressed a strong cytoplasmic immunoreactivity for tryptase and for Tβ₄, in the absence of chymase reactivity. A robust expression of Tβ₄ was detected in tumor-infiltrating and peritumoral mast cells in salivary gland tumors and breast ductal infiltrating carcinomas. Tumorinfiltrating MCs also showed a strong immunoreactivity for chymase and tryptase. In this paper, we first demonstrate that normal dermal and mucosal mast cells exhibit strong expression of thymosin β₄, which could be considered a new marker for the identification of mast cells in skin biopsies as well as in human tumors. The possible relationship between the degree of Tβ₄ expression in tumor-infiltrating mast cells and tumor behaviour warrants further consideration in future investigations.Key words: mast cells, thymosin β₄, tryptase, chymase.     

Evidence for the activation of PAR-2 by the sperm protease, acrosin: expression of the receptor on oocytes

Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.     

Cloning of the cDNA and gene of mouse mast cell protease-6. Transcription by progenitor mast cells and mast cells of the connective tissue subclass

The cDNA and gene for mouse mast cell protease-6 (MMCP-6) have been sequenced and show MMCP-6 to be translated as a prepro-enzyme with a 21-amino acid hydrophobic leader peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The mature form of the enzyme has 73% amino acid sequence identity with human and dog mast cell tryptases. The MMCP-6 gene includes 6 exons, with a total span of 1.8 kilobases. A 208-base pair intron was defined which separates the 5'-untranslated sequence of MMCP-6 from the translation initiation codon, thereby presenting a gene organization which distinguishes tryptic serine proteases from chymotryptic serine proteases of the mast cell secretory granule. By RNA blot analysis with a gene-specific probe, MMCP-6 has a unique subclass distribution in being transcribed in mouse connective tissue mast cells but undetectable in mucosal mast cells. MMCP-6 is the first serine protease of any class to be shown to be significantly transcribed in progenitor, bone marrow-derived mast cells, which can reconstitute both mucosal mast cell and connective tissue mast cell populations in mast cell-deficient mice.     

Human Tryptase Cleaves Pro-Nerve Growth Factor (Pro-NGF): HINTS OF LOCAL,MAST CELL-DEPENDENT REGULATION OF NGF/PRO-NGF ACTION*

Several factors regulate nerve growth factor (NGF), which is formed from pro-NGF by intracellular and extracellular enzymatic cleavage. The close proximity between mast cells expressing the protease tryptase and NGF-producing smooth muscle-like peritubular cells in the testes of infertile patients led us to examine whether tryptase is among those factors. Human peritubular cells express functional tryptase receptors (PAR-2). Recombinant enzymatically active β-tryptase increased NGF levels in the culture medium of primary human peritubular cells, but the peptide agonist for PAR-2 (SLIGKV) did not. Neither tryptase nor the peptide increased NGF mRNA levels. To test whether the increase in NGF is due to enzymatic activity of tryptase acting on pro-NGF, supernatants of peritubular cells and synthetic pro-NGF were treated with tryptase. Results of Western blot studies indicate enzymatic cleavage of pro-NGF by active tryptase. Heat-inactivated tryptase or SLIGKV was not effective. Mass spectrometry analysis of in vitro cleavage products from recombinant tryptase and synthetic pro-NGF revealed multiple cleavage sites within the pro-NGF sequence. The results also indicate the generation of mature NGF and smaller NGF fragments as a result of tryptase action. Thus, tryptase-secreting mast cells in the vicinity of pro-NGF/NGF-secreting cells in any human tissue are likely able to alter the ratios of pro-NGF/NGF. As NGF and pro-NGF have different affinities for their receptors, this indicates a novel way by which mast cells, via tryptase, can modify the microenvironment in human tissues with regard to neurotrophin actions.     

Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs

Our previous work has demonstrated that weaning at 19 days of age has deleterious effects on mucosal barrier function in piglet intestine that are mediated through peripheral CRF receptor signaling pathways. The objectives of the present study were to assess the impact of piglet age on weaning-associated intestinal dysfunction and to determine the role that mast cells play in weaning-induced breakdown of mucosal barrier function. Nursing Yorkshire-cross piglets were either weaned at 19 days of age (early-weaned, n = 8) or 28 days of age (late-weaned, n = 8) and housed in nursery pens. Twenty-four hours postweaning, segments of midjejunum and ascending colon from piglets within each weaning age group were harvested and mounted on Ussing chambers for measurements of transepithelial electrical resistance and serosal-to-mucosal [(3)H]mannitol fluxes. Early weaning resulted in reductions in transepithelial electrical resistance and increases in mucosal permeability to [(3)H]mannitol in the jejunum and colon (P < 0.01). In contrast, postweaning reductions in intestinal barrier function were not observed in piglets weaned at 28 days of age. Early-weaned piglet intestinal mucosa had increased expression of CRF receptor 1 protein, increased mucosal mast cell tryptase levels, and evidence of enhanced mast cell degranulation compared with late-weaned intestinal mucosa. Pretreatment of piglets with the mast cell stabilizer drug cromolyn, injected intraperitoneally 30 min prior to weaning, abolished the early-weaning-induced intestinal barrier disturbances. Our results indicate that early-weaning stress induces mucosal dysfunction mediated by intestinal mast cell activation and can be prevented by delaying weaning.     

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.     

Human lung tryptase. Purification and characterization

Human lung tryptase, a mast cell-derived trypsin-like serine protease, has been isolated from whole human lung tissue obtained at autopsy. Increased yields from this purification process have allowed extensive characterization of the enzyme. One of the critical steps in the purification scheme is the use of a linear heparin gradient to elute active material from cellulose phosphate. Gel filtration studies in 1.0 M NaCl yielded an apparent Mr = 135,000, and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated the presence of two active species with apparent Mr = 30,900 and 31,600. Enzymatic activity was sensitive to NaCl concentrations above 0.05 M and was only 50% in 0.15 M NaCl, decreasing to 18% in 0.6 M NaCl. The effects of synthetic and natural inhibitors have also been studied, confirming the enzyme's trypsin-like characteristics and demonstrating that naturally occurring serum inhibitors are incapable of diminishing its activity. A complete amino acid analysis showed a high tryptophan content. Lastly, antisera to human lung tryptase have been generated, and the immunological identity of active fractions has been investigated as well as the localization of the enzyme to the mast cell granule by immunohistochemical staining.