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.     

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.     

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.     

Distribution and enzyme histochemical characterisation of mast cells in cats

Mast cells from 15 different cat organs were examined in terms of distribution and protease activity. The number of mast cells in each site was found to vary when visualised by metachromatic staining using Alcian Blue. Enzyme histochemical analysis revealed the existence of two subtypes of mast cells. These were categorised based on protease content, i.e. whether the mast cells contained chymase or tryptase. Tryptase-positive mast cells were clearly identifiable in every organ examined, whereas chymase-containing mast cells were predominantly observed in the ear (skin), tongue, spleen, and submucosa of the stomach and rectum. The chymase-reactive cells were not detected in the heart, or in the muscularis or serosa of the duodenum, jejunum, ileum or rectum. In addition, we suggest the existence of another subtype of mast cell containing both chymase and tryptase and localised within the ear (skin), tongue, spleen and submucosa of the rectum.     

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     

用免疫荧光标记对人组织中肥大细胞亚型的分选与形态观察

方法:利用中性蛋白酶成分、特征性酶抗体的免疫荧光染色和流式细胞仪确定分选肥大细胞亚型,以激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒。结果:三种免疫表型被确定:肥大细胞的类胰蛋白酶阳性(MCT)、类糜蛋白酶阴性;类糜蛋白酶阳性(MCC)、类胰蛋白酶阴性和类胰蛋白酶阳性、类糜蛋白酶阳性(MCTC)。肥大细胞内分泌颗粒分散或聚集存在,分泌颗粒突起分泌或以分散的方式释放。分泌颗粒大范围释放后,肥大细胞的形态结构发生了改变。结论:利用肥大细胞的特征性酶抗体、免疫荧光标记和流式细胞仪可将人组织中的肥大细胞分选纯化为三种亚型;以共聚焦显微镜显示肥大细胞含有丰富的分泌颗粒,它说明肥大细胞具备了为人体I型变态反应提供快速反应的物质基础。     

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.     

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca²⁺] in cells loaded with the fluorescent Ca²⁺ probe Fura-2. Tryptase produced transient cytosolic Ca²⁺ mobilization in keratinocytes, but not in fibroblasts. Ca²⁺ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca²⁺ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca²⁺ mobilization, nor did it affect Ca²⁺ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells. J. Cell. Physiol. 176:365–373, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.     

Chymotrypsin- and trypsin-type serine proteases in rat mast cells: properties and functions

Two of the major enzymes present in and released from rat mast cells are chymotrypsin-type serine protease (chymase) and trypsin-type serine protease (tryptase), and these have been postulated to be important in the inflammatory reactions. There have been no clear data regarding the trypsin-type protease in rat mast cells. Tryptase was recently purified from rat peritoneal mast cells with an associated protein (trypstatin) that inhibited the protease activity above pH 7.5. Chymase was also purified from rat peritoneal cells by employing a one-step method involving hydrophobic chromatography on octyl-Sepharose 4B or arginine-Sepharose 4B. The properties of chymase and tryptase were described in relation to substrate specificity and their relative sensitivity to inhibitors. It was found that proteolytic activities of these enzymes were modulated by naturally occurring substances, such as phosphoglycerides, long-chain fatty acids, and trypstatin. There is as yet little evidence for the physiological roles of these enzymes in the inflammatory reaction. It has been found that the specific, low-molecular-weight inhibitor of chymase, chymostatin, and that of tryptase, leupeptin, inhibit histamine release induced by addition of anti-rat IgE to mast cells. However, the inhibitors with molecular weights of more than 6000 were found to have no effect in this process. The data suggest that chymase and tryptase in mast cell granules play a crucial or significant role in the process of degranulation.     

Immunohistochemical Localization of Nerve Fibers in the Pseudocapsule of Fibroids

The pseudocapsule surrounding fibroids consists of compressed myometrium containing nerves and blood vessels that continue into adjacent myometrium. Oxytocin (OXT) is thought to affect wound healing after myomectomy. We determined the presence of OXT and protein gene product 9.5 (PGP9.5) immunoreactive nerve fibers in pseudocapsule compared to adjacent myometrium. Samples (N=106) of pseudocapsule and adjacent myometrium were collected from 57 women with uterine fibroids undergoing myomectomy, and stained with anti-OXT and PGP 9.5 antibodies to demonstrate the presence of nerve fibers. Nerve fibers in the pseudocapsule stained positively with OXT (89/106, 84.0%) and PGP 9.5 (94/106, 88.7%). The densities of nerve fibers staining with PGP 9.5 and OXT in the pseudocapsule were highest in the isthmus (23.68±22.45/mm² and 43.35±40.74/mm², respectively). There were no significant differences in the density of nerve fibers, stained with either OXT or PGP 9.5, between the pseudocapsule, and adjacent normal myometrium regardless of the fibroid location in the uterus (P>0.05). These results suggest that the pseudocapsule should avoid to be damaged during the myomectomy procedure.Key words: fibroid pseudocapsule, nerve fibers, oxytocin, myomectomy, protein gene product 9.5, immunohistochemistry     

Mast cell chymase. A potent secretagogue for airway gland serous cells

Submucosal glands are the major sources of airway secretions in most mammals. Mast cells are abundant in the environment of airway submucosal glands and are rich sources of secreted proteases. To investigate the hypothesis that mast cell proteases stimulate airway gland secretion, we studied the ability of the two major mast cell granule proteases, chymase and tryptase, to cause secretion of 35S-labeled macromolecules from a line of cultured bovine airway gland serous cells. Mast cell chymase and tryptase were purified from dog mastocytoma cells. Chymase markedly stimulated serous cell secretion in a concentration-dependent fashion with a threshold of 10(-10) M, whereas tryptase had no effect. The response to 10(-8) M chymase (1530 +/- 80% over base line) was approximately 10-fold higher than that evoked by other agonists such as histamine and isoproterenol. The predominant 35S-labeled macromolecule released by chymase was chondroitin sulfate proteoglycan, the glycoconjugate present in serous cell secretory granules. The response to chymase was non-cytotoxic and was blocked by active site inhibitors of chymase (soybean trypsin inhibitor and chymostatin) and by inhibitors of cellular energy metabolism (azide,2,4-dinitrophenol, dicumarol). Supernatant obtained by degranulation of mastocytoma cells caused a secretory response of comparable magnitude to that caused by chymase. These findings demonstrate that chymase, but not tryptase, is a potent secretagogue for airway gland serous cells, and they suggest a possible role for chymase-containing mast cells in the pathogenesis of airway hypersecretion.     

IL-16 regulation of human mast cells/basophils and their susceptibility to HIV-1

AIDS patients often contain HIV-1-infected mast cells (MCs)/basophils in their peripheral blood, and in vivo-differentiated MCs/basophils have been isolated from the blood of asthma patients that are HIV-1 susceptible ex vivo due to their surface expression of CD4 and varied chemokine receptors. Because IL-16 is a ligand for CD4 and/or an undefined CD4-associated protein, the ability of this multifunctional cytokine to regulate the development of human MCs/basophils from nongranulated progenitors residing in cord or peripheral blood was evaluated. After 3 wk of culture in the presence of c-kit ligand, IL-16 induced the progenitors residing in the blood of normal individuals to increase their expression of chymase and tryptase about 20-fold. As assessed immunohistochemically, >80% of these tryptase(+) and/or chymase(+) cells expressed CD4. The resulting cells responded to IL-16 in an in vitro chemotaxis assay, and this biologic response could be blocked by anti-IL-16 and anti-CD4 Abs as well as by a competitive peptide inhibitor corresponding to a sequence in the C-terminal domain of IL-16. The additional finding that IL-16 induces calcium mobilization in the HMC-1 cell line indicates that IL-16 acts directly on MCs and their committed progenitors. IL-16-treated MCs/basophils also are less susceptible to infection by an M/R5-tropic strain of HIV-1. Thus, IL-16 regulates MCs/basophils at a number of levels, including their vulnerability to retroviral infection.     

Rat mast cell tryptase

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells

Summary An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MC_T) or chymase together with tryptase (MC_TC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MC_T were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MC_T of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7–67%, average 30%), while this was not the case in the small intestinal mucosa (5–26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MC_T therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.     

Bovine mast cells: distribution, density, heterogeneity, and influence of fixation techniques

Mast cells can be distinguished according to various characteristics: rodent mast cells have been subtyped by histochemical criteria, whereas canine and human mast cells have been classified according to their proteases. Comparisons of mast cells from different species have therefore resulted in contradictory and confusing opinions on mast cell heterogeneity. Thus, it is essential to obtain species-specific data on mast cell density and heterogeneity. The present study was carried out to determine the physiological distribution of mast cell numbers and types in bovines according to tissue location, staining, and fixation methods. Samples were fixed in formalin or Carnoy’s fluid. The average number of mast cells was determined by using a metachromatic staining method. Protease content of mast cells was examined with a double-enzyme-immunohistochemical staining technique. Three mast cell subtypes were distinguished: T-, TC-, and C-mast cells. The T-mast cell was the predominant subtype in nearly all investigated organs and tissue locations. Only tryptase-positive mast cells could be demonstrated in bovine skin and uterus. No chymase activity was found in these organs, regardless of the fixation type. A larger number of mast cells was observed after fixation in Carnoy’s fluid. The three different mast cell subtypes were only demonstrated in formalin-fixed tissue; chymase-positive mast cells were not found after fixation in Carnoy’s fluid. Increasing experimental data suggest that mast cell subtypes have different functions in promoting and modulating inflammation and in remodeling the extracellular matrix. Since mast cell tryptase and chymase have different functional properties, these results may clarify the different reaction patterns observed in various organs and species.     

CD1d expressed in mast cell surface enhances IgE production in B cells by up-regulating CD40L expression and mediator release in allergic asthma in mice

Mast cells play important roles via FcεRI-mediated activation in allergic asthma. A nonpolymorphic MHC I-like molecule CD1d, which is mainly expressed in APCs, presents glycolipid Ag to iTCR on iNKT cells and modulates allergic responses. This study aimed to investigate the role of CD1d on IgE production and mast cell activation related to allergic asthma. Bone marrow-derived mast cells (BMMCs) from C57BL/6 Wild type (WT) or KO (CD1d^−/−) mice were activated with Ag/Ab (refer to WT-act-BMMCs and KO-act-BMMCs, respectively) or α-Galactosylceramide (WT-αGal-BMMCs, KO-αGal-BMMCs) in the presence of iNKT cells. WT, KO or BMMC-transferred KO mice were sensitized and/or challenged by OVA or α-Gal to induce asthma. KO-act-BMMCs reduced intracellular Ca^2 + levels, expression of signaling molecules (Ras, Rac1/2, PLA₂, COX-2, NF-κB/AP-1), mediator release (histamines, leukotrienes and cytokines/chemokines), and total IgE levels versus the corresponding WT-BMMCs. KO mice reduced total and OVA-specific serum IgE levels, number of mast cells, recruiting molecules (CCR2/CCL2, VCAM-1, PECAM-1), expression of tryptase, c-kit, CD40L and cytokine mRNA, co-localization of c-kit and CD1d or iNKT cells in BAL cells or lung tissues, and PCA responses, compared with the corresponding WT mice. BMMC-transferred KO-both mice showed the restoration of all allergic responses versus KO-both mice (Ag/Ab reaction plus α-Gal). KO-αGal-BMMCs or KO-αGal mice did not show any responses. Our data suggest that CD1d-expressed mast cells may function as APC cells for iNKT cells and exacerbate airway inflammation and remodeling through up-regulating IgE production via B cell Ig class switching and mediator release in mast cells of OVA-challenged mice.     

CD117-positive Cells of the Heart: Progenitor Cells or Mast Cells?

Human cardiac stem/progenitor cells and their potential for repair of heart injury are a current hot topic of research. CD117 has been used frequently as a marker for identification of stem/progenitor cells in the heart. However, cardiac mast cells, which are also CD117⁺, have not been excluded by credible means when selecting putative cardiac progenitors by using CD117 as a marker. We evaluated the relationship between CD117⁺ cells and mast cells in the left ventricle of human hearts (n=5 patients, ages 1 week–75 years) with the well-established mast cell markers tryptase, toluidine blue, and thionine. A large number (85–100%) of CD117⁺ cells in the human heart were specifically identified as mast cells. In addition, mast cells showed weak or moderate CD45 immunostaining signals. These results indicate that the majority of CD117⁺ cells in the heart are mast cells and that these cells are distinctly positive for CD45, although staining was weak or moderate. These results strongly suggest that the newly reported CD117⁺/CD45^dim/moderate putative cardiac progenitor cells are mast cells. The significance of this observation in stem cell research of the heart is discussed. (J Histochem Cytochem 58:309–316, 2010)