Gastrointestinal (GI) nematode infections are an important public health and economic concern. Experimental studies have shown that resistance to infection requires CD4 ؉ T helper type 2 (Th2) cytokine responses characterized by the production of IL-4 and IL-13. However, despite >30 years of research, it is unclear how the immune system mediates the expulsion of worms from the GI tract. Here, we demonstrate that a recently described intestinal goblet cell-specific protein, RELM␤͞FIZZ2, is induced after exposure to three phylogenetically distinct GI nematode pathogens. Maximal expression of RELM␤ was coincident with the production of Th2 cytokines and host protective immunity, whereas production of the Th1 cytokine, IFN-␥, inhibited RELM␤ expression and led to chronic infection. Furthermore, whereas induction of RELM␤ was equivalent in nematode-infected wild-type and IL-4-deficient mice, IL-4 receptor-deficient mice showed minimal RELM␤ induction and developed persistent infections, demonstrating a direct role for IL-13 in optimal expression of RELM␤. Finally, we show that RELM␤ binds to components of the nematode chemosensory apparatus and inhibits chemotaxic function of a parasitic nematode in vitro. Together, these results suggest that intestinal goblet cell-derived RELM␤ may be a novel Th2 cytokine-induced immune-effector molecule in resistance to GI 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 cytokinemediated 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.T he adult stage of the nematode Trichinella spiralis resides within enterocytes of the jejunum. During parasite infection characteristic changes occur in the small intestine (1). It has long been known that the gut becomes edematous and inflamed, with these responses peaking at the time of parasite expulsion from the host, but the precise mechanisms involved have remained obscure. Infection induces leakiness in the intestinal epithelium that is considered to be a host defense mechanism against the parasite (the leak-lesion hypothesis) (2).We hypothesize that an increase in epithelial paracellular permeability resulting in the loss of parasites is a direct consequence of adaptive immunity. T. spiralis elicits a strong T helper 2 response resulting in intestinal goblet cell hyperplasia, eosinophilia, and a profound mucosal mastocytosis (3-5). Efficient parasite expulsion depends on CD4 ϩ T cells through control of the critical mast cell response (6). In the absence of intestinal mast cells the loss of parasites is markedly delayed (7). The mechanism by which mast cells induce parasite expulsion is unknown and is the focus of this study.Changes in epithelial paracellular permeability during the course of T. spiralis infection in mice and the role that the mast cell may play in inducing these changes were investigated. By depleting mast cells with anti-c-kit antibodies or by using IL-9 transgenic mice that overexpress mast cells (8), we present compelling evidence that mast cells are the key mediators of increased mucosal permeability. To understand further the action of mast cells on intestinal epithelium, we have infected mice deficient in mouse mast cell protease-1 (mMCP-1) that had been shown previously to delay parasite expulsion (9) and investigated whether this mast cell-specific proteinase is involved in increased epithelial perm...

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The β-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific β-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1−/− BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.

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.

This is the first report to demonstrate that canine atopic dermatitis is associated with over-production of IL-4. Clinical tolerance in healthy individuals appears to be associated with TGF-beta, although it is unclear if this reflects an active mechanism or simply non-responsiveness of the immune system. Th1 cytokines may be induced by subsequent self-trauma and secondary infections in atopic skin. We believe that these results better characterize spontaneously occurring canine atopic dermatitis. We further propose that this should be investigated as a possible animal model of human atopic dermatitis.

Intestinal mucosal mast cells (IMMC) express granule neutral proteases that are regulated by T-cell–derived cytokines, including interleukin-3 (IL-3) and IL-9, and by stem cell factor (SCF). The IMMC-specific chymase, mouse mast cell protease-1 (mMCP-1), is released in substantial quantities into the blood stream during gastrointestinal allergic responses. We used cultured bone marrow-derived mast cells (mBMMC) to identify cytokines that regulate the expression and extracellular release of mMCP-1. When grown in IL-3–rich WEHI (15% vol/vol) and 50 ng/mL recombinant rat SCF (rrSCF) bone marrow cells supplemented with IL-9 (5 ng/mL) differentiated into mBMMC that expressed a maximum of less than 250 ng mMCP-1/106 cells and 189 ng mMCP-1/mL of culture supernatant. Supplementation of the same three cytokines with transforming growth factor-β1(TGF-β1; 1 ng/mL) resulted in substantially enhanced expression (6 μg/106 mBMMC) and extracellular release (2 μg/mL of culture supernatant) of mMCP-1. The response to TGF-β1 was dose-dependent, with maximal effect at 1 ng/mL, and was associated with immunohistochemical and ultrastructural changes in the secretory granules. IL-9–induced expression of mMCP-1 may be due to endogenously expressed TGF-β1, because it was blocked by anti–TGF-β antibodies. In conclusion, the expression and extracellular release of the IMMC-specific chymase, mMCP-1, is strictly regulated by TGF-β1.

The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.

The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.