Alan D. Pemberton

Tissue-specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut

Serine proteinases with trypsin‐like (tryptase) and chymotrypsin‐like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa‐specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa‐specific chymase, mouse mast cell proteinase‐1 (mMCP‐1), is regulated by transforming growth factor‐β1 (TGF‐β1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase‐activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin‐I to angiotensin‐II. Mucosa‐specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in innate responses to infection.

Mouse Mast Cell Tryptase mMCP-6 Is a Critical Link between Adaptive and Innate Immunity in the Chronic Phase of Trichinella spiralis Infection

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.

Innate BALB/c Enteric Epithelial Responses to Trichinella spiralis: Inducible Expression of a Novel Goblet Cell Lectin, Intelectin-2, and Its Natural Deletion in C57BL/10 Mice

Infection of mice with the nematode parasite Trichinella spiralis induces changes in the proteome of the jejunal epithelium, including substantial up-regulation of a novel variant of interlectin. In this study we sequence this novel lectin, termed intelectin-2, and compare expression levels during T. spiralis infection of resistant (BALB/c) with susceptible (C57BL/10) mouse strains. Intelectin-2 was cloned and sequenced from BALB/c mRNA extracted on day 14 of infection, and was found to have 91% amino acid identity with intelectin (within our study termed intelectin-1). Intelectin-2 transcripts were up-regulated early (day 3) during infection with T. spiralis in BALB/c mice, suggesting an innate response, and levels remained high through to day 14 (time of parasite rejection). Immunohistochemistry of jejunal sections with a rabbit polyclonal Ab to Xenopus laevis 35-kDa cortical granule lectin (XL35; 68% identity with intelectin-2) followed a similar pattern, with intense labeling of goblet and Paneth cells at day 14. However, intelectin-2 transcripts and protein were absent, and immunohistochemistry negative when C57BL/10 mice were infected with T. spiralis. Genomic PCR and Southern blotting confirmed that the intelectin-2 gene is absent from the C57BL/10 genome. The presence of intelectin-2 in resistant BALB/c mice, its absence from the susceptible C57BL/10 strain and the kinetics of its up-regulation during T. spiralis infection suggest that this novel lectin may serve a protective role in the innate immune response to parasite infection.

Innate immune response mechanisms in the intestinal epithelium: potential roles for mast cells and goblet cells in the expulsion of adult Trichinella spiralis

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.

Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis

ABSTRACT Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).

Proteomic identification of interactions between histones and plasma proteins: Implications for cytoprotection

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE‐K mouse small intestinal epithelial cells were protected from histone‐induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone‐interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone‐induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross‐linking properties. It was particularly notable that inter‐α‐trypsin inhibitor was prominent among the histone‐precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo.

Background Gastrointestinal nematode infection is associated with mucosal mast cell (MMC) hyperplasia. In the mouse, this is accompanied by the release of substantial quantities of the chymase mouse mast cell proteinase‐1 (mMCP‐1) into the gut lumen and peripheral bloodstream. Expression of mMCP‐1 is largely restricted to intraepithelial MMC and is thought to play a role in the regulation of epithelial permeability. MMCs also express mouse mast cell proteinase‐2 (mMCP‐2), but less is known about the expression or biological function of this proteinase.

Expression of Integrin-αE by Mucosal Mast Cells in the Intestinal Epithelium and Its Absence in Nematode-Infected Mice Lacking the Transforming Growth Factor-β1-Activating Integrin αvβ6

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-αEβ7 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-αEβ7 and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-β1. However, TGF-β1 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-αEβ7 in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-β6 subunit and, as consequence, do not express the epithelial integrin-αvβ6, integrin-αE 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-αvβ6 is to activate latent TGF-β1, these findings strongly support a role for TGF-β1 in both the recruitment and differentiation of murine MMCs during nematode infection.

Acute phase proteins in grass sickness (equine dysautonomia).

Four acute phase proteins were assayed in the serum of normal horses and those with acute, subacute and chronic grass sickness, colic and inflammatory conditions, in order to investigate their diagnostic value in grass sickness. The grass sickness and inflammation group had a significantly increased haptoglobin concentration (P less than 0.01-P less than 0.001). Orosomucoid was elevated in acute, subacute and chronic grass sickness and inflammation (P less than 0.001, P less than 0.001, P less than 0.05 and P less than 0.05, respectively). Highest concentrations of haptoglobin and orosomucoid were recorded in subacute grass sickness. Ceruloplasmin was significantly higher in acute grass sickness cases than all other groups except the colic group (P less than 0.05-P less than 0.01). alpha 2-macroglobulin was significantly higher in acute grass sickness than normal, colic and chronic grass sickness cases (P less than 0.01, P less than 0.05 and P less than 0.05). The time scale of changes suggests that the stimulus to haptoglobin and orosomucoid synthesis occurs at the onset of clinical signs whereas the increase in ceruloplasmin and alpha 2-macroglobulin is more likely to reflect haemoconcentration.

The Goblet Cell Is the Cellular Source of the Anti-Microbial Angiogenin 4 in the Large Intestine Post Trichuris muris Infection

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.