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Mast cells are key initiators of allergic, anaphylactic and inflammatory reactions, producing mediators that affect vascular permeability, angiogenesis and fibrosis. Glucocorticoid pharmacotherapy reduces mast cell number, maturation and activation but effects at physiological levels are unknown. Within cells, glucocorticoid concentration is modulated by the 11β-hydroxysteroid dehydrogenases (11β-HSDs). Here we show expression and activity of 11β-HSD1, but not 11β-HSD2, in mouse mast cells with 11β-HSD activity only in the keto-reductase direction, regenerating active glucocorticoids (cortisol, corticosterone) from inert substrates (cortisone, 11-dehydrocorticosterone). Mast cells from 11β-HSD1-deficient mice show ultrastructural evidence of increased activation, including piecemeal degranulation and have a reduced threshold for IgG immune complex-induced mast cell degranulation. Consistent with reduced intracellular glucocorticoid action in mast cells, levels of carboxypeptidase A3 mRNA, a glucocorticoid-inducible mast cell-specific transcript, are lower in peritoneal cells from 11β-HSD1-deficient than control mice. These findings suggest that 11β-HSD1-generated glucocorticoids may tonically restrain mast cell degranulation, potentially influencing allergic, anaphylactic and inflammatory responses.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

SUMMARYGastrointestinal infection with the nematode Trichinella spiralis is accompanied by a rapid and reversible expansion of the mucosal mast cell and goblet cell populations in the intestinal epithelium, which is associated with the release of their mediators into the gut lumen. Both goblet cell and mast cell hyperplasia are highly dependent on mucosal T-cells and augmented by the cytokines IL-4 and IL-13. However, the contribution of both mast and goblet cells, and the mediators they produce, to the expulsion of the adults of T. spiralis is only beginning to be elucidated through studies predominantly employing T. spiralis-mouse models. In the present article, we review the factors proposed to control T. spiralis-induced mucosal mast cell (MMC) and goblet cell differentiation in the small intestine, and focus on some key MMC and goblet cell effector molecules which may contribute to the expulsion of adult worms and/or inhibition of larval development.

Background: Mast cell degranulation is believed to act as a key event in initiating and maintaining airway response to allergen challenge in human asthma. It is hypothesized that the mast cell may play a similar role in equine heaves, which shares many similarities with occupational dust-induced asthma., Objective: The aim of this study was to quantify the mast cell proteinase tryptase in bronchoalveolar lavage fluid (BALF) from control and heaves-susceptible horses and to investigate tryptase mRNA and protein expression in pulmonary mast cells., Methods: Equine BALF tryptase concentrations were determined by ELISA from control and heaves-susceptible horses pre and post 24 h hay/straw challenge (HSC). Tryptase mRNA and protein expression were investigated by quantitative PCR and immunohistochemistry in bronchial and bronchiolar tissue samples of control and heaves-susceptible horses., Results: Both control and heaves-susceptible horses had significantly increased BALF tryptase concentrations following HSC (P=0.003 and 0.034, respectively). Increased numbers of tryptase-expressing intra-epithelial mast cells were demonstrated in heaves horses, but not controls, following challenge (P=0.02). Bronchiolar tissue from heaves horses removed from challenge contained significantly lower tryptase transcripts than that from control horses (P=0.02)., Conclusion: Mast cell degranulation and tryptase release into the airways occur following HSC of control and heaves-susceptible horses. The greater number of mast cells available in the bronchiolar epithelium of heaves horses may be clinically significant in the pulmonary inflammatory response of heaves.

Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.

Mast cells migrate to the mucosal epithelium during intestinal nematode infections in mice, where they express abundant mucosal mast cell-specific proteases, mouse mast cell protease-1 and -2 (MCPT1 and MCPT2). Expression of these proteases is strictly controlled by transforming growth factor-beta1 (TGF-beta1) in the epithelium. In vitro homologues of mucosal mast cells are generated by culturing bone marrow-derived mast cells (BMMC) in the presence of TGF-beta1. We examined the proteome of BMMC cultured either in the presence of TGF-beta1 (n = 5) or of a neutralising anti-TGF-beta1 antibody (n = 5). Cell extracts were examined by 2-DE, and changes in expression levels of protein spots were determined by densitometry. Spots of interest were identified by tryptic peptide mapping. In addition to the up-regulation of MCPT1 and MCPT2, which accounted for approximately 40% of all soluble protein in the TGF-beta1 treated cells, MCPT7 was modestly up-regulated by TGF-beta1, and calnexin was up-regulated fivefold. A 7.6-fold down-regulation of galectin-1 was verified by Western blotting and FACS analysis. Galectin-1 is located on the cell surface where it mediates cellular adhesion to basement membranes. Regulation of its expression by TGF-beta1 may be of relevance to mast cell adhesion within the epithelium.

Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit(+) population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B(4). Other activation products were inactive. LTB(4) was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB(4) receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB(4). Furthermore, LTB(4) was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB(4) was a potent chemoattractant for human cord blood-derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB(4) in regulating tissue mast cell numbers.

Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.

Upregulation of CGRP-immunoreactive (IR) primary afferent nerve fibers accompanied by mastocytosis is characteristic for the Schistosoma mansoni-infected murine ileum. These mucosal mast cells (MMC) and CGRP-IR fibers, which originate from dorsal root (DRG) and nodose ganglia, are found in close apposition. We examined interactions between primary cultured MMC and CGRP-IR DRG neurons in vitro by confocal recording of intracellular Ca(2+) concentration ([Ca(2+)](i)). The degranulatory EC(50) for the mast cell secretagogue compound 48/80 (C48/80; 10 microg/ml) and the neuropeptides CGRP (2.10(-8) M) and substance P (SP; 3.10(-8) M) were determined by measurement of extracellular release of the granule chymase, mouse mast cell protease-1. Application of C48/80 (10 microg/ml) and CGRP and SP (both 10(-7) M) to Fluo-4-loaded MMC induced a transient rise in [Ca(2+)](i) after a lag time, indicative of mast cell degranulation and/or secretion. The CGRP response could be completely blocked by pertussis toxin (2 microg/ml), indicating involvement of G(i) proteins. Application of MMC juice, obtained by C48/80 degranulation of MMC, to Fluo-4-loaded DRG neurons induced in all neurons a rise in [Ca(2+)](i), indicative of activation. Degranulation of MMC by C48/80 in culture dishes containing Fluo-4-loaded DRG neurons also caused activation of the DRG neurons. In conclusion, these results demonstrate a bidirectional cross-talk between cultured MMC and CGRP-IR DRG neurons in vitro. This indicates that such a communication may be the functional relevance for the close apposition between MMC and CGRP-IR nerve fibers in vivo.

Mucosal mast cells (MMC) play an important role in the immune response against selected species of intestinal nematode. The kinetics with which different strains of inbred mice resolve infection with Trichinella spiralis correlates with their ability to mount MMC responses in the intestinal mucosa. Homologues of MMC that express and constitutively secrete abundant amounts of the granule chymase, mouse mast cell protease-1 (mMCP-1), can be generated in vitro from bone marrow cultures supplemented with interleukins-3 and -9, stem cell factor and transforming growth factor-beta1. Using the enhanced growth characteristics of these MMC homologues, a novel limiting dilution assay for mast cell precursor (MCp) frequency has been developed. The assay is highly specific, in that cultures containing mast cells are identified with mMCP-1 specific antibody, and almost three-fold more sensitive than previously published systems. MCp frequencies were compared in BALB/c and C57/BL10 strains of mice that, respectively, respond rapidly and slowly to infection with T. spiralis. MCp frequency (1/378 bone marrow cells) was significantly greater in BALB/c than C57/BL10 mice (frequency: 1/751). Similarly the rate of growth of MMC homologues and the production of mMCP-1 was significantly greater in BALB/c than in C57/BL10 bone marrow cultures.

Background: The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated., Objective: To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2., Methods: MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA., Results: mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2., Conclusion: The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.

The immunoregulatory cytokine transforming growth factor (TGF)-beta(1) is secreted as a biologically inactive complex with latency-associated peptide, which must be modified by local factors to expose the functionally active cytokine. The epithelial integrin alpha(v)beta(6) mediates local activation of TGF-beta(1) in the lung and beta(6)(-/-) mice exhibit exaggerated pulmonary inflammation, but their response to inflammatory stimuli in the gut has not been investigated. We found that both beta(6) and TGF-beta(1) are constitutively expressed in the jejunal epithelial compartment in uninfected mice and during infection with the intestinal nematode Nippostrongylus brasiliensis. We also present data showing that beta(6)(-/-) mice are seriously compromised in their ability to mount a mucosal mast cell response after infection, and there is a significant reduction in the expression and systemic release of the granule chymase, mouse mast cell protease-1. Because in vitro expression of this chymase is regulated by TGF-beta(1), these data indicate that in the absence of alpha(v)beta(6) epithelially expressed TGF-beta(1) may not be activated, with a consequent absence of expression of mouse mast cell protease-1 and down-regulation of the mucosal mast cell response.

We previously reported that mast cell tryptase is a potent mitogen for cultured airway smooth-muscle cells, but the early intracellular signals mediating this response are not known. In many cells, proliferative effects are mediated by a mitogen-activated protein kinase signaling pathway involving Raf-1, MAP kinase kinases (MEKs), and extracellular signal-regulated protein kinases (ERKs) 1 and 2. Therefore, we tested for tryptase-induced activation of ERK1 and 2 in cultured dog tracheal smooth-muscle cells. Tryptase, in nanomolar concentrations which potently stimulated DNA synthesis, increased dual phosphorylation of ERKs in cellular lysates as well as ERK2 kinase activity in immunoprecipitates. Pretreatment of cells with the MEK inhibitor PD098059 abolished tryptase-induced increases in DNA synthesis and attenuated increases in ERK2 activity. Irreversible inhibition of tryptase's proteolytic activity, using p-amidino phenylmethanesulfonyl fluoride, attenuated tryptase-induced increases in DNA synthesis and dual phosphorylation of ERKs by 76% and 40 to 60%, respectively. Tryptase also increased c-fos transcription as quantified in polymerase chain reactions. In concentrations that caused similar increases in DNA synthesis, tryptase and platelet-derived growth factor (PDGF-BB) increased ERK activity (and c-fos transcription) with markedly different kinetics, the tryptase-induced responses being slower in onset and more sustained. We conclude that tryptase-induced mitogenesis in airway smooth-muscle cells requires activation of ERK1 and 2; that these responses depend partially, but not completely, upon tryptase's properties as a protease; and that they are slower in onset and more sustained than those induced by PDGF-BB.

Hyperplasia of airway smooth muscle cells is present in the airways of asthmatic patients and may contribute to the development of the bronchial hyperresponsiveness that occurs in these patients. Because tryptase is an abundant component of mast cell granules and has demonstrated growth-stimulatory effects in other mesenchymal cells (J. Clin. Invest. 1991; 88:493-499), the goal of our study was to determine whether tryptase is a mitogen for airway smooth muscle cells. The mitogenic effects of tryptase were tested in passages 1 through 5 of dog tracheal smooth muscle cells, either by counting smooth muscle cells or by monitoring uptake of bromodeoxyuridine (BrdU) into cellular DNA during S-phase. With respect to its efficacy, at a near maximal concentration (4 nM), tryptase increased cell numbers 2.1 +/- 0.2- or 2.8 +/- 0.6-fold above controls after 2 or 4 days, respectively, and these increases were approximately the same as those induced by platelet-derived growth factor (50 ng/ml) or 10% calf serum. With respect to potency, tryptase caused concentration-dependent increases in BrdU uptake, as detected in an enzyme-linked immunosorbent assay or by counting BrdU-labeled nuclei, with an EC50 of 2 nM. Pretreatment of tryptase with diisopropylfluorophosphate, to reduce markedly its catalytic as a activity as a proteinase, attenuated its growth-stimulated effects by 58 +/- 16%. Tryptase-induced mitogenesis was not a nonspecific effect of all serine proteinases, because thrombin, another proteinase with mitogenicity for fibroblasts, stimulated neither increases in cell counts nor BrdU uptake in our cells. We conclude that tryptase is a potent mitogen for airway smooth muscle cells in culture.

In 12 anesthetized dogs, we tested the effects of pharmacologic beta-adrenergic stimulation on the severity of tracheal mast cell reactions in vivo (Am Rev Respir Dis 1979; 119:62). In control dogs, tracheal arterial injections of compound 48/80 produced arteriovenous differences in histamine concentrations across the trachea (35 +/- 15 ng/ml), but not the lung; this finding suggested selective degranulation of tracheal mast cells. Tracheal arteriovenous histamine differences correlated (r = 0.70) with tracheal contractions (12 +/- 4 g/cm) induced by compound 48/80 in these animals. In other dogs, injections of isoproterenol (10 micrograms/kg) before compound 48/80 prevented the development of both tracheal arteriovenous differences and contractions. We concluded that pharmacologic beta-adrenergic stimulation inhibited degranulation of tracheal mast cells. The preparation appears suitable for examining the effects of endogenous stimuli on the severity of mast cell reactions in the central airways of anesthetized dogs.