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As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (150 µm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.

Abstract Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair. (Funding; DEFRA/SHEFC (VT0102) and the BBSRC (BB/E01867X/1)).

Abstract Natural killer (NK) cells are well recognized as playing a key role in innate immune defence through cytokine production and cytotoxic activity; additionally recent studies have identified several novel NK cell functions. The ability to study NK cells in the sheep has been restricted due to a lack of specific reagents. We report the generation of a monoclonal antibody specific for ovine NKp46, a receptor which in a number of mammals is expressed exclusively in NK cells. Ovine NKp46+ cells represent a population that is distinct from CD4+ and γδ+ T-cells, B-cells and cells of the monocytic lineage. The NKp46+ cells are heterogenous with respect to expression of CD2 and CD8 and most, but not all, express CD16 - characteristics consistent with NK cell populations in other species. We demonstrate that in addition to populations in peripheral blood and secondary lymphoid organs, ovine NKp46+ populations are also situated at the mucosal surfaces of the lung, gastro-intestinal tract and non-gravid uterus. Furthermore, we show that purified ovine NKp46+ populations cultured in IL-2 and IL-15 have cytotoxic activity that could be enhanced by ligation of NKp46 in re-directed lysis assays. Therefore we conclude that ovine NKp46+ cells represent a population that by phenotype, tissue distribution and function correspond to NK cells and that NKp46 is an activating receptor in sheep as in other species.

Abstract Background C57BL/6J mice possess a single intelectin (Itln) gene on chromosome 1. The function of intelectins is not well understood, but roles have been postulated in insulin sensitivity, bacterial recognition, intestinal lactoferrin uptake and response to parasites and allergens. In contrast to C57BL/6J mice, there is evidence for expansion of the Itln locus in other strains and at least one additional mouse Itln gene product has been described. The aim of this study was to sequence and characterise the Itln locus in the 129S7 strain, to determine the nature of the chromosomal expansion and to inform possible future gene deletion strategies. Results Six 129S7 BAC clones were sequenced and assembled to generate 600 kbp of chromosomal sequence, including the entire Itln locus of approximately 500 kbp. The locus contained six distinct Itln genes, two CD244 genes and several Itln- and CD244-related pseudogenes. It was approximately 433 kbp larger than the corresponding C57BL/6J locus. The expansion of the Itln locus appears to have occurred through multiple duplications of a segment consisting of a full-length Itln gene, a CD244 (pseudo)gene and an Itln pseudogene fragment. Strong evidence for tissue-specific distribution of Itln variants was found, indicating that Itln duplication contributes more than a simple gene dosage effect. Conclusions We have characterised the Itln locus in 129S7 mice to reveal six Itln genes with distinct sequence and expression characteristics. Since C57BL/6J mice possess only a single Itln gene, this is likely to contribute to functional differences between C57BL/6J and other mouse strains.

Abstract Background Trichuris muris in the mouse is an invaluable model for infection of man with the gastrointestinal nematode Trichuris trichiura. Three T. muris isolates have been studied, the Edinburgh (E), the Japan (J) and the Sobreda (S) isolates. The S isolate survives to chronicity within the C57BL/6 host whereas E and J are expelled prior to reaching fecundity. How the S isolate survives so successfully in its host is unclear. Results Microarray analysis was used as a tool to identify genes whose expression could determine the differences in expulsion kinetics between the E and S T. muris isolates. Clear differences in gene expression profiles were evident as early as day 7 post-infection (p.i.). 43 probe sets associated with immune and defence responses were up-regulated in gut tissue from an E isolate-infected C57BL/6 mouse compared to tissue from an S isolate infection, including the message for the anti-microbial protein, angiogenin 4 (Ang4). This led to the identification of distinct differences in the goblet cell phenotype post-infection with the two isolates. Conclusion Differences in gene expression levels identified between the S and E-infected mice early during infection have furthered our knowledge of how the S isolate persists for longer than the E isolate in the C57BL/6 mouse. Potential new targets for manipulation in order to aid expulsion have been identified. Further we provide evidence for a potential new marker involving the acidity of the mucins within the goblet cell which may predict outcome of infection within days of parasite exposure.

Background Chronic and frequent alcohol (ethanol [EtOH]) intake has been associated with an increased incidence of several types of cancers including breast, mouth, throat, esophageal, stomach, and colorectal ( CRC). The underlying mechanism of this deleterious carcinogenic effect of alcohol has not been clearly established but inflammation may be 1 unifying feature of these cancers. We have recently shown that intestinal mast cells play a central role in intestinal carcinogenesis. In this study, we tested our hypothesis that mast cell-mediated inflammation is 1 underlying mechanism by which chronic alcohol promotes intestinal tumorigenesis. Methods APCΔ468 mice were fed either an alcohol-containing Nanji liquid diet or isocaloric dextrose-containing Nanji diet for 10 weeks and then sacrificed to collect small and large intestine samples. Assessments of tumor number and size as well as mast cell number and mast cell activity and histology score for invasion were compared between Control (dextrose-fed) and alcohol-fed APC∆468 mice. The effect of alcohol on mast cell-mediated tumor migration was also assessed using an in vitro migration assay. Results Alcohol feeding increased both polyp number and size within both the small and the large intestines of APC∆468 mice. Only alcohol-fed mice showed evidence of tumor invasion. Chronic alcohol feeding also resulted in an increased mast cell number and activity in tumor stroma and invading borders. In vitro migration assay showed that alcohol significantly increases mast cell-mediated tumor migration in vitro. Conclusions Our data show that chronic alcohol intake promotes: (i) intestinal tumorigenesis and tumor invasion in genetically susceptible mice; (ii) increases in polyp-associated mast cells; and (iii) mast cell-mediated tumor migration in vitro. Both our in vivo and in vitro studies suggest that mast cell-mediated inflammation could be 1 mechanism by which alcohol promotes carcinogenesis. [ABSTRACT FROM AUTHOR]

Background: Mouse angiogenin 4 (Ang4) has previously been described as a Paneth cell--derived antimicrobial peptide important in epithelial host defence in the small intestine. However, a source for Ang4 in the large intestine, which is devoid of Paneth cells, has not been defined. Methodology/Principal Findings: Analysis was performed on Ang4 expression in colonic tissue by qPCR and immunohistochemistry following infection with the large intestine dwelling helminth parasite Trichuris muris. This demonstrated an increase in expression of the peptide following infection of resistant BALB/c mice. Further, histological analysis of colonic tissue revealed the cellular source of this Ang4 to be goblet cells. To elucidate the mechanism of Ang4 expression immunohistochemistry and qPCR for Ang4 was performed on colonic tissue from T. muris infected mouse mutants. Experiments comparing C3H/HeN and C3H/HeJ mice, which have a natural inactivating mutation of TLR4, revealed that Ang4 expression is TLR4 independent. Subsequent experiments with IL-13 and IL-4 receptor alpha deficient mice demonstrated that goblet cell expression of Ang4 is controlled either directly or indirectly by IL-13. Conclusions:The cellular source of mouse Ang4 in the colon following T. muris infection is the goblet cell and expression is under the control of IL-13. [ABSTRACT FROM AUTHOR]

Background: Gastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection. Methodology/Principal Findings: The use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals. Conclusions/Significance: This study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals. [ABSTRACT FROM AUTHOR]

Background: Nuclear histones have previously been shown to aggregate LDL in vitro, suggestive of a possible proatherogenic role. Recent studies indicate that histones are released during acute inflammation, and therefore might interact with circulating lipoproteins in vivo. In view of the associative link between inflammation and cardiovascular disease, the behaviour of histones was investigated using in vitro models of LDL retention and foam cell formation. Methodology/Principal Findings: Heparin agarose beads were used as a model of a matrix rich in sulphated glycosaminoglycans, to which histones bind strongly. Histone-modified beads were observed to pull down more LDL from solution than untreated beads, indicating that histones can function as bridging molecules, enhancing LDL retention. Furthermore, addition of heparin inhibited histone-induced aggregation of LDL. To model foam cell formation, murine RAW 264.7 macrophages were incubated for 24 h in the presence of LDL, histones, LDL plus histones or vehicle control. Cells incubated with LDL in the presence of histones accumulated significantly more intracellular lipid than with LDL or histone alone. Conclusions/Significance: These results are consistent with a potential pro-atherogenic role for extracellular histones, which should be investigated further. [ABSTRACT FROM AUTHOR]

Background: Trichuris muris in the mouse is an invaluable model for infection of man with the gastrointestinal nematode Trichuris trichiura. Three T. muris isolates have been studied, the Edinburgh (E), the Japan (J) and the Sobreda (S) isolates. The S isolate survives to chronicity within the C57BL/6 host whereas E and J are expelled prior to reaching fecundity. How the S isolate survives so successfully in its host is unclear. Results: Microarray analysis was used as a tool to identify genes whose expression could determine the differences in expulsion kinetics between the E and S T. muris isolates. Clear differences in gene expression profiles were evident as early as day 7 post-infection (p.i.). 43 probe sets associated with immune and defence responses were up-regulated in gut tissue from an E isolate-infected C57BL/6 mouse compared to tissue from an S isolate infection, including the message for the anti-microbial protein, angiogenin 4 (Ang4). This led to the identification of distinct differences in the goblet cell phenotype post-infection with the two isolates. Conclusion: Differences in gene expression levels identified between the S and E-infected mice early during infection have furthered our knowledge of how the S isolate persists for longer than the E isolate in the C57BL/6 mouse. Potential new targets for manipulation in order to aid expulsion have been identified. Further we provide evidence for a potential new marker involving the acidity of the mucins within the goblet cell which may predict outcome of infection within days of parasite exposure. [ABSTRACT FROM AUTHOR]

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases. [Copyright &y& Elsevier]

Abstract We have previously shown that both atopic and normal dogs generate an IgG response to antigens of Malassezia pachydermatis . The aim of this study was to compare IgE responses to separated proteins of M. pachydermatis in 28 atopic dogs with Malassezia dermatitis and 22 clinically normal dogs using Western immunoblotting. Six different detection systems were evaluated in order to assess sensitivity and eliminate nonspecific binding and cross-reactivity. The protocol yielding the best results utilized a monoclonal mouse antidog IgE, an alkaline phosphatase conjugated goat antimouse IgG which had been passed through a canine IgG column 3 times, a chemiluminescent substrate and a digital imaging system. Proteins of 45, 52, 56 and 63 kDa were recognized by more than 50% of the atopic dog sera and thus represented major allergens. Only a minority of normal dogs showed faint IgE binding to these proteins. The results indicate that the majority of atopic dogs with Malassezia dermatitis have a greater IgE response than normal dogs, suggesting an IgE-mediated immune response may be clinically important in the pathogenesis of the disease. [ABSTRACT FROM AUTHOR]

Abstract: Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes. [Copyright &y& Elsevier]

There is established evidence that cytotoxic CD8 + T cells are important mediators of immunity against the bovine intracellular protozoan parasite Theileria parva However, the mechanism by which the specific CD8 + T cells kill parasitized cells is not understood. Although the predominant pathway used by human and murine CD8 + T cells to kill pathogen-infected cells is granule exocytosis, involving the release of perforin and granzyme B, there is to date a lack of published information on the biological activities of bovine granzyme B. The present study set out to define the functional activities of bovine granzyme B and determine its role in mediating the killing of T. parva -parasitized cells. DNA constructs encoding functional and nonfunctional forms of bovine granzyme B were produced, and the proteins expressed in Cos-7 cells were used to establish an enzymatic assay to detect and quantify the expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parva -specific CD8 + T cell clones were found to be significantly correlated with the levels of granzyme B protein but not the levels of mRNA transcript expression. Experiments using inhibitors specific for perforin and granzyme B confirmed that CD8 + T cell killing of parasitized cells is dependent on granule exocytosis and, specifically, granzyme B. Further studies showed that the granzyme B-mediated death of parasitized cells is independent of caspases and that granzyme B activates the proapoptotic molecule Bid., (Copyright © 2018 Yang et al.)

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Chronic and frequent alcohol (ethanol [EtOH]) intake has been associated with an increased incidence of several types of cancers including breast, mouth, throat, esophageal, stomach, and colorectal (CRC). The underlying mechanism of this deleterious carcinogenic effect of alcohol has not been clearly established but inflammation may be 1 unifying feature of these cancers. We have recently shown that intestinal mast cells play a central role in intestinal carcinogenesis. In this study, we tested our hypothesis that mast cell-mediated inflammation is 1 underlying mechanism by which chronic alcohol promotes intestinal tumorigenesis., Methods: APC(Δ468) mice were fed either an alcohol-containing Nanji liquid diet or isocaloric dextrose-containing Nanji diet for 10 weeks and then sacrificed to collect small and large intestine samples. Assessments of tumor number and size as well as mast cell number and mast cell activity and histology score for invasion were compared between Control (dextrose-fed) and alcohol-fed APC(∆468) mice. The effect of alcohol on mast cell-mediated tumor migration was also assessed using an in vitro migration assay., Results: Alcohol feeding increased both polyp number and size within both the small and the large intestines of APC(∆468) mice. Only alcohol-fed mice showed evidence of tumor invasion. Chronic alcohol feeding also resulted in an increased mast cell number and activity in tumor stroma and invading borders. In vitro migration assay showed that alcohol significantly increases mast cell-mediated tumor migration in vitro., Conclusions: Our data show that chronic alcohol intake promotes: (i) intestinal tumorigenesis and tumor invasion in genetically susceptible mice; (ii) increases in polyp-associated mast cells; and (iii) mast cell-mediated tumor migration in vitro. Both our in vivo and in vitro studies suggest that mast cell-mediated inflammation could be 1 mechanism by which alcohol promotes carcinogenesis., (Copyright © 2012 by the Research Society on Alcoholism.)

Sheep intelectin1 and sheep intelectin3 (sITLN1 and sITLN3) were cloned and sequenced. The amino acid sequences of sITLN1 and sITLN3 shared 86% and 91% homology with the previously cloned sheep intelectin2 (sITLN2), respectively. Expression of sITLN1 and sITLN3 transcript was demonstrated in abomasum, lung, colon and gastric lymph node, terminal rectum, skin, jejunum, mesenteric lymph node, ileal peyer's patches, brain, kidney, liver, spleen, skin, ear pinna, heart and ovary in normal sheep tissues. sITLN2 transcript expression was restricted to the abomasal mucosa in normal sheep tissues. Using a non selective chicken anti-intelectin antibody, tissue intelectin protein was demonstrated in mucus neck cells in the abomasum, mucus cells in the colon, free mucus in ileum, goblet cells in the lung, small intestinal epithelium and brush border, epidermal layer of the skin and skin sebaceous glands. The expression of the three sITLN transcripts was examined in two nematode infections in sheep known to induce a Th2 response; a Teladorsagia circumcincta challenge infection model and a Dictyocaulus filaria natural infection. The three sITLN were absent in unchallenged naïve lambs and present in the abomasal mucosa of both naïve and immune lambs following T. circumcincta challenge infection. Upregulation of sITLN2 and sITLN3 was shown in sheep lung following D. filaria natural infection. Intelectins may play an important role in the mucosal response to nematode infections in ruminants.

In the present study we have employed an in vitro organ challenge model to study the post-challenge responses in parasite naïve and immune gastric tissue of sheep, in an attempt to identify the host derived factors involved in immune exclusion of Teladorsagia circumcincta larvae. Proteins present in the epithelial cells and mucus from ovine abomasa following parasite challenge in previously naïve and immune animals were analysed through Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof)-MS and shotgun proteomics. MALDI-ToF analysis of epithelial cell lysates revealed that a number of proteins identified were differentially expressed in naïve and immune cells. These included intelectin and lysozymes, which were present at higher levels in epithelial cell lysates derived from immune samples. A large number of proteins were identified in the mucosal wash from immune tissue which were not present in the mucosal wash of the naïve tissue. Some of these proteins were present in washes of immune tissue prior to the parasite challenge including immunoglobulin A, galectin 14 and 15 and sheep mast cell protease 1. However, other proteins, such as calcium activated chloride channel and intelectin were only detected in the washings from the challenged tissue. The latter may be related to an enhanced mucus release, which may result in entrapment of infective larvae and thus reduced establishment in tissue that has been previously challenged with the parasite. In conclusion, several proteins have been identified which may be involved, either directly or indirectly, in the exclusion and immune elimination of incoming infective larvae. In the present study, the usefulness of the in vitro model has been confirmed, and the global proteomic approach has identified proteins that had not previously been associated with parasite exclusion from abomasal mucosa, such as the calcium activated chloride channel.

Although the innate immune function of mast cells in the acute phase of parasitic and bacterial infections is well established, their participation in chronic immune responses to indolent infection remains incompletely understood. In parasitic infection with Trichinella spiralis, the immune response incorporates both lymphocyte and mast cell-dependent effector functions for pathogen eradication. Among the mechanistic insights still unresolved in the reaction to T. spiralis are the means by which mast cells respond to parasites and the mast cell effector functions that contribute to the immunologic response to this pathogen. We hypothesized that mast cell elaboration of tryptase may comprise an important effector component in this response. Indeed, we find that mice deficient in the tryptase mouse mast cell protease-6 (mMCP-6) display a significant difference in their response to T. spiralis larvae in chronically infected skeletal muscle tissue. Mechanistically, this is associated with a profound inability to recruit eosinophils to larvae in mMCP-6-deficient mice. Analysis of IgE-deficient mice demonstrates an identical defect in eosinophil recruitment. These findings establish that mast cell secretion of the tryptase mMCP-6, a function directed by the activity of the adaptive immune system, contributes to eosinophil recruitment to the site of larval infection, thereby comprising an integral link in the chronic immune response to parasitic infection.

The granule-derived mouse mast cell proteases-1 and -2 (mMCP-1 and -2) colocalize in similar quantities in mucosal mast cells but micrograms of mMCP-1 compared with nanograms of mMCP-2 are detected in peripheral blood during intestinal nematode infection. This differential systemic response was investigated both in vitro and in vivo. Bone marrow-derived mucosal mast cell homologs released similar quantities of mMCP-1 and-2 concomitantly with beta-hexosaminidase in response to calcium ionophore ( approximately 60% release) or IgE/DNP (25% release). In contrast, serum from mice sensitized by infection with Nippostrongylus brasiliensis 10 days earlier contained >1500-fold more mMCP-1 (10,130 +/- 1,609 ng/ml) than mMCP-2 (6.4 +/- 1 ng/ml), but, in gut lumen, the difference was approximately 8-fold. After OVA sensitization, >600-fold more mMCP-1 (7,861 +/- 2,209 ng/ml) than mMCP-2 (12.8 +/- 4.7 ng/ml) was present in blood 1 h after challenge, but, in gut lumen, there were relatively comparable levels of mMCP-1 and -2. To estimate the rates of systemic accumulation and clearance, 10 microg of mMCP-1 or -2 was injected i.p. Plasma levels of injected mMCP-2 peaked (1%) at 15 min then declined, whereas levels of mMCP-1 were maximal (approximately 25%) at 3 h. Inactivation of mMCP-1 with PMSF before injection resulted in mMCP-2-like kinetics, but inhibition of mMCP-1 by serum gave kinetics similar to that of native mMCP-1. mMCP-1 isolated from serum is complexed with serpins and we conclude that both the accumulation and the longevity of mMCP-1 in blood is due to complex formation, protecting it from a pathway that rapidly clears mMCP-2, which is unable to form complexes with serpins.

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.