Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice

In contrast to resident constitutive mast cells (CMCs), mucosal MCs (MMCs) appear in the lung and trachea of sensitized mice only following inhalation challenge. We monitored the influx and maturation of MCs by their expression of Kit, FcɛRI, β7-integrin and side scatter (SSC) by flow cytometry. Influx of MC progenitors (MCps) (FcɛRI^lo, Kit^int, β7^hi, and SSC^lo) peaks 1 day after challenges and subsides to baseline by day 7 after challenge. The mature MMCs appear as a distinct population on day 7 and peak at day 14 with higher SSC and FcɛRI expression, but lower β7 and Kit expression. A distinct transitional population is present between 1 and 7 days after challenge. Maturation occurs more rapidly in the trachea. The resident tracheal CMCs had higher SSC, FcɛRI, and Kit and lower β7-integrin expression than the MMCs. By histology, the MMCs follow similar kinetics to the flow cytometry-identified mature MMCs and are notably persistent for >42 days. Steroid treatment reduced inflammation and MCp influx but had no effect on established MMCs. Thus, changes in SSC, FcɛRI, and Kit together with the expression of αE/α4:β7-integrins characterizes the development of induced MMCs from MCps and distinguishes them from resident CMCs in the trachea and large airways.     

Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode-infected and normal BALB/c mice.

Soluble granule chymases in rodent intestinal mucosal mast cells (IMMCs) may play an important role in altering epithelial permeability during immediate hypersensitivity reactions. Using a monoclonal antibody against the chymase mouse mast cell protease-1 (mMCP-1), we have shown that it is constitutively expressed in < or = 20% of esterase-positive (esterase+) IMMCs but not in esterase+ gastric mucosal mast cells (GMMCs) in normal BALB/c mice. Intestinal infection with mouse- or rat-adapted strains of Nippostrongylus brasiliensis resulted in IMMC hyperplasia with 100% of esterase+ IMMCs expressing mMCP-1. In contrast, there was a variable response in terms of numbers of GMMCs and of the proportion expressing mMCP-1. Esterase+ mast cells in the gastric submucosa, muscularis, ear pinna, lung parenchyma, major airway submucosa, and peritoneal cavity did not express mMCP-1. The few airway esterase+ mast cells expressing mMCP-1 were, like the great majority of IMMCs and GMMCs, located intraepithelially. In conclusion, mMCP-1 is predominantly expressed by intraepithelial mucosal mast cells but not in all sites; the immunological stimulus associated with intestinal nematodiasis substantially up-regulates mMCP-1 expression by mast cells in the jejunum but not in the stomach; IMMCs and GMMCs in BALB/c mice are phenotypically and possibly functionally distinct.     

Expression of mast cell proteases in rat lung during helminth infection: mast cells express both rat mast cell protease II and tryptase in helminth infected lung

BACKGROUND: The phenotype of proliferated mast cells in Nippostrongylus brasiliensis-infected rat lung has been identified as mucosal mast cells (MMC) but not connective tissue mast cells (CTMC). However, a previous study of ours showed that the expression of rat mast cell tryptase (RMCT) mRNA, which has been reported to be confined to CTMC, significantly increased in rat lung 14 days after infection. METHODS: The expression of four mast cell proteases in rat lung during the course of infection with N. brasiliensis was examined by RNA blot analysis. Immunohistochemical analysis of RMCT and rat mast cell protease (RMCP) II, which has been reported to be confined to MMC was also performed. RESULTS: The number of lung mast cells did not change until 7 days after infection, then gradually increased until 21 days after infection. The expression of the RMCP II gene had increased 14 and 21 days after infection. In addition, the expression of the RMCP I and RMCT genes had also increased at the same time points, but RMCP III had not. By immunohistochemistry, most of the mast cells in infected lung were identified as RMCP II+/RMCT- (MMC), but both RMCP II+ and RMCT+ mast cells were also observed. CONCLUSIONS: The present results suggest that mast cell phenotype alteration or a distinct mast cell subset might be present in N. brasiliensis-infected rat lung, and therefore N. brasiliensis-infected rat lung may be a useful tool for studying the differentiation mechanism of mast cells.     

HtrA1 is a novel mast cell serine protease of mice and men

Mast cells are rich sources of proteases, such as tryptases and chymases that control many physiological and pathological processes, for example vascular permeability, smooth muscle cell proliferation or extracellular matrix remodeling. Murine mucosal mast cells mature under the influence of TGF-beta and play a role in asthmatic and anti-helminthic immune responses. In an attempt to identify novel genes that are highly upregulated during mucosal mast cell differentiation, we detected HtrA1 protease as a novel protein in mast cells by microarray experiments. HtrA1 level was much higher in murine mucosal than in connective tissue-type mast cells. Furthermore, HtrA1 is not localized in the secretory granules and is constitutively secreted by human mast cells. Although HtrA1 has been attributed a TGF-beta-inhibitory activity, it did not show any influence on TGF-beta-induced mucosal mast cell differentiation. As many extracellular target proteins have been suggested for HtrA1, this protease may participate in the mast cell-induced extracellular remodeling.     

IgE and mast cells in host defense against parasites and venoms

IgE-dependent mast cell activation is a major effector mechanism underlying the pathology associated with allergic disorders. The most dramatic of these IgE-associated disorders is the fatal anaphylaxis which can occur in some people who have developed IgE antibodies to otherwise innocuous antigens, such as those contained in certain foods and medicines. Why would such a highly “maladaptive” immune response develop in evolution and be retained to the present day? Host defense against parasites has long been considered the only beneficial function that might be conferred by IgE and mast cells. However, recent studies have provided evidence that, in addition to participating in host resistance to certain parasites, mast cells and IgE are critical components of innate (mast cells) and adaptive (mast cells and IgE) immune responses that can enhance host defense against the toxicity of certain arthropod and animal venoms, including enhancing the survival of mice injected with such venoms. Yet, in some people, developing IgE antibodies to insect or snake venoms puts them at risk for having a potentially fatal anaphylactic reaction upon subsequent exposure to such venoms. Delineating the mechanisms underlying beneficial versus detrimental innate and adaptive immune responses associated with mast cell activation and IgE is likely to enhance our ability to identify potential therapeutic targets in such settings, not only for reducing the pathology associated with allergic disorders but perhaps also for enhancing immune protection against pathogens and animal venoms.     

SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is highly expressed in normal airways, but is dramatically decreased in allergic and cigarette smoke exposure settings. We have previously demonstrated SPLUNC1 in vitro antibacterial property against Mycoplasma pneumoniae (Mp). However, its in vivo biological functions remain unclear. The objectives of this study were to determine the in vivo functions of SPLUNC1 following bacterial (eg, Mp) infection, and to examine the underlying mechanisms. We generated SPLUNC1-deficient mice and utilized transgenic mice overexpressing human SPLUNC1 exclusively within the airway epithelium. These mice were infected with Mp and, twenty-four hours post infection, their host defense responses were compared to littermate controls. Mp levels and inflammatory cells increased in the lungs of SPLUNC1(-/-) mice as compared to wild type controls. SPLUNC1 deficiency was shown to contribute to impaired neutrophil activation. In contrast, mice overexpressing hSPLUNC1 exclusively in airway epithelial cells demonstrated lower Mp levels. Furthermore, neutrophil elastase activity was significantly increased in mice overexpressing hSPLUNC1. Lastly, we demonstrated that SPLUNC1 enhanced Mp-induced human neutrophil elastase (HNE) activity, and HNE directly inhibited the growth of Mp. Our findings demonstrate a critical in vivo role of SPLUNC1 in host defense against bacterial infection, and likely provide a novel therapeutic approach to restore impaired lung innate immune responses to bacteria in patients with chronic lung diseases.     

Gut mucosal mast cells in Nippostrongylus-primed rats are the major source of secreted rat mast cell protease II following systemic anaphylaxis

The distribution of the predominant chymotrypsin-like enzyme of mucosal mast cells (rat mast cell protease II: RMCP II) was examined in naive and Nippostrongylus-primed rats both before and after the induction of systemic anaphylaxis. Anaphylactic secretion of RMCP II following i.v. challenge of primed rats with worm antigen was accompanied by significant depletion of this enzyme from the jejunal and gastric mucosae; the concentrations were not altered in the ileum and colon. Despite significant increases in the levels of RMCP II in lung and mesenteric lymph node following infection with N. brasiliensis there was no anaphylactic depletion of this enzyme from these sites. No RMCP II was detected in liver, spleen, kidney or bone marrow either before or after systemic anaphylaxis. Mucosal mast cells were depleted from the jejunal, gastric and colonic mucosae following antigen challenge of primed rats. These data provide further evidence that gastrointestinal mucosal mast cells are the major source of secreted RMCP II following systemic anaphylaxis in the rat.     

Mucosal vaccination increases endothelial expression of mucosal addressin cell adhesion molecule 1 in the human gastrointestinal tract

Homing of leukocytes to various tissues is dependent on the interaction between homing receptors on leukocytes and their ligands, addressins, on endothelial cells. Mucosal immunization results in homing of antigen-specific lymphocytes back to the mucosa where they first encountered the antigen. However, it is unknown whether this homing of antigen-specific cells is mediated by an altered endothelial addressin expression after vaccination. Using different immunization routes with an oral cholera vaccine, we show that the endothelial expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is increased in the gastric and upper small intestinal mucosae after immunization through various local routes in the upper gastrointestinal tract. In contrast, rectal immunization did not influence the levels of MAdCAM-1 in the gastric or duodenal mucosa. Furthermore, we show that MAdCAM-1 can be induced on human endothelial cells by tumor necrosis factor alpha (TNF-alpha) and gamma interferon. The vaccine component cholera toxin B subunit (CTB) increased MAdCAM-1 expression on endothelial cells in cultured human gastric explants, an effect that seemed to be mediated by TNF-alpha. In conclusion, MAdCAM-1 expression is increased in the upper gastrointestinal tract after local immunizations with a vaccine containing CTB. This strongly suggests the involvement of MAdCAM-1 in the preferential homing of mucosal lymphocytes to their original site of activation.     

Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection

Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis.     

Natural killer cells participate in the early defense against Leishmania major infection in mice

In this study the role of natural killer (NK) cells in the course of experimental Leishmania major infection was investigated. NK cells in genetically resistant C57BL/6 mice were depleted by in vivo administration of anti-asialo-GM1 or anti-NK1.1 antibodies. A marked exacerbation of the infection was found in the NK-depleted mice within the first two weeks of infection. Both the local tissue swelling and the number of parasites in the lesions were significantly higher than in normal animals. Lymph node cells taken from infected NK-depleted mice released less interferon-γ (IFN-γ) when cultured in vitro. As an alternate approach we have used poly I: C treatment in order to activate NK cell activity in vivo in BALB/c mice, which are genetically susceptible to L. major infection. Poly I: C treatment led to milder symptoms and to a significantly lower parasite burden in the early course of infection. Lymph node cells from infected and poly I: C-treated BALB/c mice released higher amount of IFN-γ in vitro than cells from control mice. These data show that NK cells are active participants in the non-specific phase of anti-leishmanial activity in the control of parasite multiplication early in the course of L. major infection in mice.     

Role of mucosal mast cells in intestinal graft-versus-host reaction in the mouse

Hyperplasia of mucosal mast cells (MMC) is found in many enteropathies which are caused by T lymphocytes, but their exact role is unknown. In this study we have investigated whether MMC play a part in the immunologically mediated enteropathy which occurs in mice with graft-versus-host reaction (GvHR). There were simultaneous increases in the numbers of jejunal MMC and in the concentrations of mouse intestinal mast cell proteinase both in serum and in the intestine in two separate models of GvHR. Although these changes developed in parallel with the evolving GvHR, there was no correlation between the degree of MMC activation and the severity of the intestinal pathology. In addition, mast cell deficient W/Wv mice developed systemic and intestinal GvHR as severe as their normal congenic littermates, despite a markedly deficient MMC response. We conclude that the role of MMC in enteropathy may be to regulate or repair T lymphocyte mediated immunopathology.     

Quantitative analysis of mucosal mast cell protease in the intestines of Nippostrongylus-infected rats.

Following infection with the intestinal nematode Nippostrongylus brasiliensis, mucosal mast cell (MMC) proliferated in the jejunum and the peak of this response was associated with a nine-fold increase in the level of mucosal mast cell protease (RMCPII) in the mucosa. At this stage the protease constituted 10%-15% of the soluble protein from gut homogenates. Concomitant immunoperoxidase studies showed that during the early proliferation of the MMC, only a proportion of the cells in lamina propria and none of the MMC within the epithelium contained detectable RMCPII. Fourteen and 20 days post infection and following a secondary challenge, staining for RMCPII in lamina propria MMC was much stronger and a few intraepithelial mast cells also contained RMCPII. With time after infection an increasing proportion of intestinal goblet cells were specifically labelled, indicating an accumulation either of RMCPII or of an antigenically similar enzyme within mucous glycoproteins. The significance of the high levels of protease in parasitized gut and of its apparent cellular distribution is discussed in relation to the protective response against the parasite.     

Neutrophils are critical for host defense against primary infection with the facultative intracellular bacterium Francisella tularensis in mice and participate in defense against reinfection.

It is generally believed that immunity to experimental infection with the facultative intracellular bacterium Francisella tularensis is an example of T-cell-mediated immunity that is expressed by activated macrophages and mediated by Francisella-specific T cells. According to the results presented herein, neutrophils are also essential for defense against primary infection with this organism. It is shown that mice depleted of neutrophils by treatment with the granulocyte-specific monoclonal antibody RB6-8C5 are rendered defenseless against otherwise sublethal doses of F. tularensis LVS inoculated intravenously or intradermally. In neutrophil-depleted mice, the organism grew progressively in the livers, spleens, and lungs to reach lethal numbers, whereas infection was resolved in normal mice. Although neutrophils were found to resistance to reinfection, their participation was less important. The results suggest that neutrophils are needed for defense against primary infection because they serve to restrict the growth of F. tularensis before it reaches numbers capable of overwhelming a developing specific immune response. The exact way that neutrophils achieve this is not clear at this time, although it is probable that they contribute in ways other than by ingesting and killing the bacterium.     

Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues

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.     

Aberrant mucosal mast cell protease expression in the enteric epithelium of nematode-infected mice lacking the integrin alphavbeta6, a transforming growth factor-beta1 activator

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.     

Characterization of the CD8+ T cell responses directed against respiratory syncytial virus during primary and secondary infection in C57BL/6 mice

The BALB/c mouse model for human respiratory syncytial virus infection has contributed significantly to our understanding of the relative role for CD4+ and CD8+ T cells to immune protection and pathogenic immune responses. To enable comparison of RSV-specific T cell responses in different mouse strains and allow dissection of immune mechanisms by using transgenic and knockout mice that are mostly available on a C57BL/6 background, we characterized the specificity, level and functional capabilities of CD8+ T cells during primary and secondary responses in lung parenchyma, airways and spleens of C57BL/6 mice. During the primary response, epitopes were recognized originating from the matrix, fusion, nucleo- and attachment proteins, whereas the secondary response focused predominantly on the matrix epitope. C57BL/6 mice are less permissive for hRSV infection than BALB/c mice, yet we found CD8+ T cell responses in the lungs and bronchoalveolar lavage, comparable to the responses described for BALB/c mice.     

The role of galanin in the differentiation of mucosal mast cells in mice

Mast cells are generally classified into two phenotypically distinct populations: mucosal-type mast cells (MMCs) and connective tissue-type mast cells (CTMCs). However, the molecular basis determining the different characteristics of the mast cell subclasses still remains unclear. Unfortunately, the number of mast cells that can be obtained from tissues is limited, which makes it difficult to study the function of each mast cell subclass. Here, we report the generation and characterization of MMCs and CTMCs derived from mouse BM mast cells (BMMCs). We found that the expression of galanin receptor 3 was elevated in MMCs when compared to the expression in CTMCs. Moreover, intraperitoneal injection of a galanin antagonist reduced MMCs and inhibited the inflammation of dextran sodium sulfate-induced colitis in mice. Therefore, these results suggest that galanin promotes MMC differentiation in vivo, and provide important insights into the molecular mechanisms underlying the differentiation of mast cell subclasses.     

Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse-high specificity indicates high substrate selectivity

Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.