H. R. P. Miller

Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract

The cytokine interleukin (IL) 25 has been implicated in the initiation of type 2 immunity by driving the expression of type 2 cytokines such as IL-5 and IL-13, although its role in the regulation of immunity and infection-induced inflammation is unknown. Here, we identify a dual function for IL-25: first, in promoting type 2 cytokine-dependent immunity to gastrointestinal helminth infection and, second, in limiting proinflammatory cytokine production and chronic intestinal inflammation. Treatment of genetically susceptible mice with exogenous IL-25 promoted type 2 cytokine responses and immunity to Trichuris. IL-25 was constitutively expressed by CD4+ and CD8+ T cells in the gut of mouse strains that are resistant to Trichuris, and IL-25–deficient mice on a genetically resistant background failed to develop a type 2 immune response or eradicate infection. Furthermore, chronically infected IL-25−/− mice developed severe infection-induced intestinal inflammation associated with heightened expression of interferon-γ and IL-17, identifying a role for IL-25 in limiting pathologic inflammation at mucosal sites. Therefore, IL-25 is not only a critical mediator of type 2 immunity, but is also required for the regulation of inflammation in the gastrointestinal tract.

Mast cells disrupt epithelial barrier function during enteric nematode infection.

We have investigated the influence of mast cells on the barrier function of intestinal epithelium during nematode infection. Trichinella spiralis infection induces a strong type 2 cytokine-mediated inflammation, resulting in a critical mucosal mastocytosis that is known to mediate expulsion of the parasites from the intestine. The host response to infection is also characterized by an increase in mucosal leakiness. We show here that intestinal epithelial permeability is markedly elevated during infection, with kinetics that mirror the adaptive immune response to primary and secondary infection. Furthermore, we have identified degradation of the tight junction protein, occludin, thereby providing a mechanism for increased paracellular permeability during helminth infection. We further demonstrate by using anti-c-kit antibody and IL-9 transgenic mice that mast cells are directly responsible for increasing epithelial paracellular permeability and that mice deficient in a mast cell-specific protease fail to increase intestinal permeability and fail to expel their parasite burden. These results provide the mechanism whereby mucosal mast cells mediate parasite expulsion from the intestine.

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.

The IL-27 Receptor (WSX-1) Is an Inhibitor of Innate and Adaptive Elements of Type 2 Immunity

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1−/− mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.

IgE Enhances Parasite Clearance and Regulates Mast Cell Responses in Mice Infected with Trichinella spiralis

Trichinella spiralis infection elicits a vigorous IgE response and pronounced intestinal and splenic mastocytosis in mice. Since IgE both activates mast cells (MC) and promotes their survival in culture, we examined its role in MC responses and parasite elimination in T. spiralis-infected mice. During primary infection, wild-type but not IgE-deficient (IgE−/−) BALB/c mice mounted a strong IgE response peaking 14 days into infection. The splenic mastocytosis observed in BALB/c mice following infection with T. spiralis was significantly diminished in IgE−/− mice while eosinophil responses were not diminished in either the blood or jejunum. Similar levels of peripheral blood eosinophilia and jejunal mastocytosis occurred in wild-type and IgE-deficient animals. Despite the normal MC response in the small intestine, serum levels of mouse MC protease-1 also were lower in parasite-infected IgE−/− animals and these animals were slower to eliminate the adult worms from the small intestine. The number of T. spiralis larvae present in the skeletal muscle of IgE−/− mice 28 days after primary infection was about twice that in BALB/c controls, and the fraction of larvae that was necrotic was reduced in the IgE-deficient animals. An intense deposition of IgE in and around the muscle larvae was observed in wild-type but not in IgE null mice. We conclude that IgE promotes parasite expulsion from the gut following T. spiralis infection and participates in the response to larval stages of the parasite. Furthermore, our observations support a role for IgE in the regulation of MC homeostasis in vivo.

Mucosal mast cells and the allergic response against nematode parasites.

IgE-mediated Type-I allergic reactions at nematode-infected mucosal surfaces are considered to have a direct protective function. The contribution of mucosal mast cells (MMC) to these mucosal allergic responses is reviewed. In addition to the T helper 2 cell-mediated regulation of MMC hyperplasia during nematode infection the kit ligand, stem cell factor (SCF), plays a key role in the early development of the MMC response. Studies in the mouse suggest that MMC protect against certain nematodes which enter the mucosa but not against lumen dwelling nematodes. The protective roles of MMC in other species, including sheep, are less certain and there is some evidence that MMC might enhance parasite fecundity. The measurement of MMC-specific granule chymases released systemically, and into the gut lumen, permits precise monitoring of mast cell activation and suggests that the secreted chymases may target epithelial junctional complex proteins, thereby causing increased mucosal permeability. The abundant intraepithelial MMC found in parasitised mucosa may, therefore, serve as epithelial gatekeepers permitting the translocation of plasma proteins onto the mucosal surface.

Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors

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) B4. Other activation products were inactive. LTB4 was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB4 receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB4. Furthermore, LTB4 was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB4 was a potent chemoattractant for human cord blood–derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB4 in regulating tissue mast cell numbers.

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.

Systemic release of a mast cell proteinase following nematode infections in sheep.

Summary An enzyme‐linked immunosorbent assay (ELISA) for sheep mast cell proteinase (SMCP) has been developed. Concentrations of SMCP in homogenates of abomasal tissue from parasite‐immune sheep (341 μg SMCP/g tissues) were raised when compared to those in normal (non‐infected) abomasa (0±145 μg SMCP/g tissue). SMCP was not detected in sera from normal animals challenged with Haemonchus contortus but was present 1±4 ng SMCP/ml) was detected in lymph from 2/3 and 4/5 immune animals between 1 and 4 days post‐challenge with 50000 larvae, but not from normal animals. SMCP was not detected in lymph from immune animals following challenge with 1000 Ostertagia larvae. The relatively low concentrations of SMCP in blood and lymph reflect the presence of proteinase inhibitor(s) which interfered with the ELISA.

The isolation and characterization of globule leucocytes: their derivation from mucosal mast cells in parasitized sheep

Summary Ovine mucosal mast cells and globule leucocytes have been isolated from the abomasum of normal sheep, and from animals challenged with Ostertagia circumcincta. The ultrastructural, morphological and histochemical properties of these cells have been investigated. The granules of ovine mucosal immunoglobulin and a serine esterase. These cells also possess surface immunoglobulin. Cells morphologically intermediate between mucosal mast cells and bulin. Cells morphologically intermediate between mucosal mast cells and globule leucocytes have similar granule and surface properties. These observations, together with quantitative data, indicate that alterations in the granule structure of mucosal mast cells as a consequence of prolonged antigenic challenge give rise to mast cells in the epithelium which, in the past, have been commonly recognized as globule leucocytes.