Enzyme histochemistry of tryptase in stomach mucosal mast cells of the mouse.

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.     

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.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

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.     

Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization.

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.     

Spatial distribution of mast cells in chronic venous leg ulcers

Chronic venous leg ulcers (CVUs) show chronic inflammation but different pathological changes occur in different parts of the ulcer. There is a lack of re-epithelialisation and defective matrix deposition in the ulcer base but epidermal hyperproliferation and increased matrix deposition in the surrounding skin. The role of mast cells in wound healing, inflammation, fibrosis and epidermal hyperproliferation has been extensively studied but less is known about their role in CVUs. In the present study, we investigated the distribution of mast cells in CVUs with specific consideration of the differences between the ulcer base and the skin surrounding the ulcer. Both histochemical and immunohistological methods were used to detect the mast cell marker tryptase in frozen sections of CVU biopsies. Mast cells were counted in the dermis of normal skin, in the ulcer base and in the skin surrounding the ulcer. Double immunofluorescence staining was used to study the location of mast cells in relation to blood vessels. In normal skin few mast cells were seen in the dermis but none in the epidermis. However in CVUs there was a significant increase in intact and degranulated mast cells in the surrounding skin and ulcer edge (184 per field, p<0.003) of CVUs and a significant reduction in the ulcer base (20.5 per field p<0.05) in comparison to normal skin (61 per field). In CVUs mast cells showed a characteristic location near the epithelial basement membrane whilst mast cell granules and phantom cells (mast cells devoid of granules) were predominantly seen in the epidermis. In the dermis, mast cells were seen associated with blood vessels. The marked increase in mast cells in the surrounding skin of CVUs and depletion of mast cells in the ulcer base could implicate mast cell mediators in the pathological changes in CVUs particularly in the epidermal and vascular changes occurring in the surrounding skin.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

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.     

Concomitant detection of mucosal mast cells and eosinophils in the intestines of normal and nippostrongylus-immune rats

Summary The granules of mucosal mast cells (MMC) in the rat and man apparently poorly fixed with formaldehyde and special fixation techniques are normally used to demonstrate MMC glycosaminoglycans (GAG) in these two species. However such techniques do not permit the study of MMC granule enzyme cytochemistry or the demonstration of eosinophils. We have, therefore, examined some histochemical and immunocytochemical properties of MMC and eosinophils in normal and parasitised rats following various fixation procedures. Immersion fixation of rat intestine in 4% paraformaldehyde for 6 h not only facilitated the demonstration of MMC glycosaminoglycans with basic dyes but also permitted the concomitant staining of cosinophils with acidophilic dyes. A MMC granule-associated serine esterase was also demonstrated by enzyme cytochemistry and rat mast cell protease II was detected within MMC granules by immunocytochemistry. This new methodology obviates the requirement for separate fixation procedures in the identification and characterisation of MMC/eosinophil interactions in normal and parasitised rat intestinal mucosa.     

Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.     

Distribution, density and heterogeneity of canine mast cells and influence of fixation techniques

 The present study was carried out to determine the physiological distribution of mast cell numbers and types in the dog according to tissue location, staining and fixation methods. Tissue samples from stomach, duodenum, lung, lymph node, skin and uterus were evaluated. Samples were fixed in formalin as well as in Carnoy’s fluid. The average number of mast cells was determined using a metachromatic staining method. Protease content of mast cells was examined with a double enzyme-immunohistochemical staining technique, using a histochemical reaction for chloroacetate esterase to detect chymase activity and an immunohistochemical staining method for the detection of tryptase. Canine mast cells can be subdivided into formalin-sensitive and -resistant mast cells. Three subtypes were identified according to their content of the mast cell-specific proteases tryptase (T) and chymase (C): T-, TC- and C-mast cells. Significant differences regarding the distribution of mast cell subtypes as well as the influence of the fixation method can be observed. This underlines the fact that data regarding mast cell heterogeneity from other species, obtained by different fixation methods, are not comparable. This fact has to be taken into consideration when evaluating mast cell subtypes under pathological conditions. Accepted: 29 January 1998     

Detection and partial characterization of a human mast cell carboxypeptidase

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.     

Evaluation of human peripheral blood leukocytes for mast cell tryptase

Murine monoclonal and goat polyclonal antibodies against tryptase, the dominant neutral protease and protein component in secretory granules of human mast cells, were used to assess the presence of tryptase in peripheral leukocytes. Carnoy's fluid-fixed cytocentrifuge preparations of enriched populations of lymphocytes, monocytes, eosinophils, and neutrophils showed no reactivity with anti-tryptase antibodies by a sensitive indirect immunoperoxidase procedure. Dispersed human lung mast cells showed strong granular cytoplasmic staining with both antibodies, whereas only approximately 50% of the peripheral blood basophils detectable with Wright's stain were detected with anti-tryptase antibodies, and these showed a staining pattern that was faint, granular, and cytoplasmic at high concentrations of antibody. At lower antibody concentrations mast cell staining was still intense, whereas basophils were not stained. Extracts of neutrophils and lymphocytes of up to 90% purity had undetectable amounts of tryptase by an ELISA sandwich immunoassay, as well as undetectable enzymatic activity with tosyl-L-gly-pro-lys-p-nitroanilide (a sensitive substrate for tryptase) in the presence of soybean trypsin inhibitor. Extracts of basophil-enriched (6 to 50% purity) preparations contained 0.046 +/- 0.013 pg of tryptase per basophil by the immunoassay along with 2 X 10(-9) +/- 0.8 X 10(-9) U of tryptase-like enzyme activity per basophil, compared with corresponding values of 12 pg, 480 X 10(-9) U of tryptase per human lung mast cell. Thus very small amounts of tryptase are present in human basophils (approximately 0.4% of that found in mast cells), but not in other peripheral leukocytes.     

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.     

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.     

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.     

Human mast cell carboxypeptidase. Selective localization to MCTC cells

Two murine mAb were prepared against human mast cell carboxypeptidase (HMC-CP) purified from human skin, and were termed CP1 and CP2, respectively. Double immunohistochemical labeling of Carnoy's-fixed sections of human skin, lung, and gastrointestinal tissue with CP1 and CP2, respectively, and with a murine monoclonal antitryptase antibody demonstrated that HMC-CP was selectively present in a subset of human mast cells. Double labeling experiments with CP1 and CP2, respectively, and a murine anti-chymase mAb demonstrated the presence of HMC-CP in the tryptase-positive, chymase-positive mast cell type (MCTC) only. Immunohistochemical labeling of peripheral blood leukocytes resulted in staining of monocytes with CP2 but not with CP1. In addition to chymase and a cathepsin-G like proteinase, HMC-CP is another neutral protease that is selectively present in the MCTC tryptase-positive, chymase-positive mast cells type of mast cell, thus extending the biochemical definition of human mast cell heterogeneity.     

Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.     

Number, fixation properties, dye-binding and protease expression of duodenal mast cells: comparisons between healthy subjects and patients with gastritis or Crohn’s disease

Summary There is an accumulation of evidence to suggest that mast cells may play a key role in gastrointestinal inflammation. We have investigated the numbers and heterogeneity in staining properties of mast cells in biopsies of the duodenum of normal subjects (n = 10), and of normal duodenum from patients with Crohn’s disease of the ileum and/or colon (n = 7) or with Helicobacter-associated gastritis of the antrum/corpus (n = 6). In normal donors, two subsets of mast cells, one located in the duodenal mucosa and the other in the submucosa, were clearly distinguished by their morphology and dye-binding properties. Whereas submucosal mast cells stained metachromatically with Toluidine Blue after neutral formalin fixation and emitted a yellow fluorescence after staining with Berberine sulphate, those in the mucosa were invisible using these stains. In patients with gastritis or Crohn’s disease, there were marked changes in the numbers of mucosal mast cells compared with control subjects, even though the duodenal biopsies were from apparently uninvolved tissue. Gastritis was associated with increased mucosal mast cell numbers (controls: 187 ± 23 cells mm−2; gastritis: 413 ± 139 cells mm−2; p = 0.0004), but mean mucosal mast cell counts in the uninvolved duodenum of Crohn’s patients were actually decreased (34 ± 30 cells mm−2, p = 0.0147). The clear differentiation between mucosal and submucosal mast cells on the basis of metachromasia with Toluidine Blue was not seen in biopsies from the patients with gastritis or Crohn’s disease. Previous studies which have suggested that there are no distinct mucosal and submucosal mast cell subsets in the human intestine may, therefore, have been affected by the use of tissue from diseased subjects. Heterogeneity in the expression of mast cell tryptase and chymase was seen by immunohistochemistry using specific antibodies, but the relative numbers of mast cell subsets were critically dependent on the methods used. Using a sensitive staining procedure, the majority of mucosal mast cells stained positively for chymase as well as for tryptase, an observation confirmed by immunoelectron microscopy and immunoabsorption studies. Our findings suggest that early stages in intestinal inflammation may be reflected in changes in mast cell numbers and in their staining properties, and call for a reappraisal of mast cell heterogeneity in the human intestinal tract This revised version was published online in November 2006 with corrections to the Cover Date.     

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.