Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.

Intestinal mast cell levels control severity of oral antigen-induced anaphylaxis in mice.

Food-triggered anaphylaxis can encompass a variety of symptoms that affect multiple organ systems and can be life threatening. The molecular distinction between non-life-threatening and life-threatening modes of such anaphylaxis has not yet been delineated. In this study, we sought to identify the specific immune functions that regulate the severity of oral antigen-induced anaphylaxis. We thus developed an experimental mouse model in which repeated oral challenge of ovalbumin-primed mice induced an FcεRI- and IgE-dependent oral antigen-triggered anaphylaxis that involved multiple organ systems. Strikingly, the severity of the systemic symptoms of anaphylaxis (eg, hypothermia) positively correlated with the levels of intestinal mast cells (r = -0.53; P < 0.009). In addition, transgenic mice with both increased intestinal and normal systemic levels of mast cells showed increased severity of both intestinal and extra-intestinal symptoms of IgE-mediated passive as well as oral antigen- and IgE-triggered anaphylaxis. In conclusion, these observations indicate that the density of intestinal mast cells controls the severity of oral antigen-induced anaphylaxis. Thus, an awareness of intestinal mast cell levels in patients with food allergies may aid in determining their susceptibility to life-threatening anaphylaxis and may eventually aid in the treatment of food-triggered anaphylaxis.

Interleukin-13 (IL-13)/IL-13 receptor alpha1 (IL-13Ralpha1) signaling regulates intestinal epithelial cystic fibrosis transmembrane conductance regulator channel-dependent Cl- secretion.

Interleukin-13 (IL-13) has been linked to the pathogenesis of inflammatory diseases of the gastrointestinal tract. It is postulated that IL-13 drives inflammatory lesions through the modulation of both hematopoietic and nonhematopoietic cell function in the intestine. To delineate the relevant contribution of elevated levels of intestinal IL-13 to intestinal structure and function, we generated an intestinal IL-13 transgenic mouse (iIL-13Tg). We show that constitutive overexpression of IL-13 in the small bowel induces modification of intestinal epithelial architecture (villus blunting, goblet cell hyperplasia, and increased epithelial proliferation) and epithelial function (altered basolateral → apical Cl(-) ion conductance). Pharmacological analyses in vitro and in vivo determined that elevated Cl(-) conductance is mediated by altered cystic fibrosis transmembrane conductance regulator expression and activity. Generation of iIL-13Tg/Il13rα1(-/-), iIL-13Tg/Il13rα2(-/-), and iIL-13Tg/Stat6(-/-) mice revealed that IL-13-mediated dysregulation of epithelial architecture and Cl(-) conductance is dependent on IL-13Rα1 and STAT-6. These observations demonstrate a central role for the IL-13/IL-13Rα1 pathway in the regulation of intestinal epithelial cell Cl(-) secretion via up-regulation of cystic fibrosis transmembrane conductance regulator, suggesting an important role for this pathway in secretory diarrhea.

Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury.

BACKGROUND: Administration of granulocyte-macrophage colony stimulating factor (GM-CSF) relieves symptoms in Crohn's disease (CD). It has been reported that reduced GM-CSF bioactivity is associated with more aggressive ileal behaviour and that GM-CSF-null mice exhibit ileal barrier dysfunction and develop a transmural ileitis following exposure to non-steroidal anti-inflammatory drugs (NSAIDs). STAT5 signalling is central to GM-CSF action. It was therefore hypothesised that GM-CSF signalling in non-haematopoietic cells is required for ileal homeostasis. METHODS: Bone marrow (BM) chimeras were generated by reconstituting irradiated GM-CSF receptor (gm-csfr) beta chain or GM-CSF (gm-csf) deficient mice with wild type BM (WTBM-->GMRKO and WTBM-->GMKO). Intestinal barrier function and the response to NSAID-induced ileal injury were examined. Expression of gm-csf, gm-csfr or stat5 in Caco-2 and HT-29 intestinal epithelial cell (IEC) lines was knocked down and the effect of GM-CSF signalling on IEC survival and proliferation was determined. RESULTS: Elevated levels of GM-CSF autoantibodies in ileal CD were found to be associated with dysregulation of IEC survival and proliferation. GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras exhibited ileal hyperpermeability. NSAID exposure induced a transmural ileitis in GM-CSF receptor-deficient mice and WTBM-->GMRKO chimeras. Transplantation of wild type BM into GM-CSF-deficient mice prevented NSAID ileal injury and restored ileal barrier function. Ileal crypt IEC proliferation was reduced in WTBM-->GMRKO chimeras, while STAT5 activation in ileal IEC following NSAID exposure was abrogated in WTBM-->GMRKO chimeras. Following knock down of gm-csf, gm-csfr alpha or beta chain or stat5a/b expression in Caco-2 cells, basal proliferation was suppressed. GM-CSF normalised proliferation of Caco-2 cells exposed to NSAID, which was blocked by stat5a/b RNA interference. CONCLUSIONS: Loss of GM-CSF signalling in non-haematopoietic cells increases NSAID ileal injury; furthermore, GM-CSF signalling in non-haematopoietic cells regulates ileal epithelial homeostasis via the STAT5 pathway. The therapeutic use of GM-CSF may therefore be beneficial in chronic ileitis associated with CD.

Paired immunoglobulin-like receptor B (PIR-B) negatively regulates macrophage activation in experimental colitis.

BACKGROUND & AIMS: Innate and adaptive immune responses are regulated by cross talk between activation and inhibitory signals. Dysregulation of the inhibitory signal can lead to aberrant chronic inflammatory diseases such as the inflammatory bowel diseases (IBD). Little is known about negative regulation of innate intestinal immune activation. We examined the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of macrophage function in innate intestinal immunity. METHODS: We examined the susceptibility of Pirb-/- and wild-type (WT) mice to dextran sodium sulfate (DSS)-induced colitis. We assessed proinflammatory cytokine release and mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) activation in Pirb-/- and WT macrophages following Escherichia coli stimulation. Macrophage transfer experiments were performed to define the role of PIR-B in the negative regulation of macrophage function in DSS-induced colitis. We also assessed expression of PIR-B human homologues (immunoglobulin-like transcript [ILT]-2 and ILT-3) in colon biopsy samples from healthy individuals (controls) and patients with IBD. RESULTS: Pirb-/- mice had increased susceptibility to DSS-induced colitis. In vitro analysis showed increased production of proinflammatory cytokines (interleukin-6, interleukin-1beta, and tumor necrosis factor alpha) and activation of MAPK and NF-kappaB in Pirb-/- macrophages following bacterial activation. Adoptive transfer of bone marrow-derived Pirb-/- macrophages into WT mice was sufficient to increase disease susceptibility. ILT-2 and ILT-3 were expressed on CD68+ and CD68- mononuclear cells and intestinal epithelium in colon biopsy samples from patients and controls. CONCLUSIONS: PIR-B negatively regulates macrophage functions in response to pathogenic bacteria and chronic intestinal inflammatory responses. Inhibitory receptors such as PIR-B might be used as therapeutic targets for treatment of patients with IBD.

Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism.

Altered intestinal barrier function is postulated to be a central predisposing factor to intestinal diseases, including inflammatory bowel diseases and food allergies. However, the mechanisms involved in maintaining homeostatic intestinal barrier integrity remain undefined. In this study, we demonstrate that mice deficient in mast cells (Kit(W-sh/W-sh) [Wsh]) or mast cell chymase (Mcpt4(-/-)) have significantly decreased basal small intestinal permeability compared with wild-type (WT) mice. Altered intestinal barrier function was linked to decreased intestinal epithelial cell migration along the villus/crypt axis, altered intestinal morphology, and dysregulated claudin-3 crypt expression. Remarkably, engraftment of Wsh mice with WT but not Mcpt4(-/-) mast cells restored intestinal epithelial cell migration, morphology, and intestinal epithelial barrier function. Collectively, these findings identify a mechanism by which mast cells regulate homeostatic intestinal epithelial migration and barrier function.

Intestinal barrier function: molecular regulation and disease pathogenesis.

The intestinal epithelium is a single-cell layer that constitutes the largest and most important barrier against the external environment. It acts as a selectively permeable barrier, permitting the absorption of nutrients, electrolytes, and water while maintaining an effective defense against intraluminal toxins, antigens, and enteric flora. The epithelium maintains its selective barrier function through the formation of complex protein-protein networks that mechanically link adjacent cells and seal the intercellular space. The protein networks connecting epithelial cells form 3 adhesive complexes: desmosomes, adherens junctions, and tight junctions. These complexes consist of transmembrane proteins that interact extracellularly with adjacent cells and intracellularly with adaptor proteins that link to the cytoskeleton. Over the past decade, there has been increasing recognition of an association between disrupted intestinal barrier function and the development of autoimmune and inflammatory diseases. In this review we summarize the evolving understanding of the molecular composition and regulation of intestinal barrier function. We discuss the interactions between innate and adaptive immunity and intestinal epithelial barrier function, as well as the effect of exogenous factors on intestinal barrier function. Finally, we summarize clinical and experimental evidence demonstrating intestinal epithelial barrier dysfunction as a major factor contributing to the predisposition to inflammatory diseases, including food allergy, inflammatory bowel diseases, and celiac disease.

IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity.

Previous mouse and clinical studies demonstrate a link between Th2 intestinal inflammation and induction of the effector phase of food allergy. However, the mechanism by which sensitization and mast cell responses occurs is largely unknown. We demonstrate that interleukin (IL)-9 has an important role in this process. IL-9-deficient mice fail to develop experimental oral antigen-induced intestinal anaphylaxis, and intestinal IL-9 overexpression induces an intestinal anaphylaxis phenotype (intestinal mastocytosis, intestinal permeability, and intravascular leakage). In addition, intestinal IL-9 overexpression predisposes to oral antigen sensitization, which requires mast cells and increased intestinal permeability. These observations demonstrate a central role for IL-9 and mast cells in experimental intestinal permeability in oral antigen sensitization and suggest that IL-9-mediated mast cell responses have an important role in food allergy.

Resistin-like molecule beta regulates innate colonic function: barrier integrity and inflammation susceptibility.

BACKGROUND: Resistin-like molecule (RELM) beta is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function primarily remains an enigma. OBJECTIVE: We sought to elucidate the function of RELM-beta in the gastrointestinal tract. METHODS: We generated RELM-beta gene-targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation. RESULTS: We show that RELM-beta is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-beta. Using acute colonic inflammatory models, we demonstrate that RELM-beta has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-beta regulates expression of type III regenerating gene (REG) (REG3beta and gamma), molecules known to influence nuclear factor kappaB signaling. CONCLUSIONS: These data define a critical role for RELM-beta in the maintenance of colonic barrier function and gastrointestinal innate immunity. CLINICAL IMPLICATIONS: These findings identify RELM-beta as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies.