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.
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 (KitW⁻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.
Regulation of intestinal epithelial barrier function is critical for preventing the passage of luminal contents, while concurrently maintaining vital nutrient uptake. Disruptions in the intestinal ...barrier are a consequence of numerous diseases and recently have been postulated to be an important factor in the development of diseases, such as food allergies and inflammatory bowel disease. Mast cells are highly granulated immune cells involved in innate and adaptive immunity that reside in the intestine and are classically known for their effector role in allergy and inflammation. They store and when activated, for example by allergens or pathogens, secrete a variety of preformed mediators. Additionally, mast cells undergo a constitutive, low-level “piecemeal” release of selected mediators. Mast cells and their mediators, including proteases, cytokines and lipidderived mediators, can alter intestinal barrier function during inflammation and disease. However, there is a lack of knowledge concerning regulation of the intestinal barrier under healthy, homeostatic conditions. The major objective of this dissertation was to define the role of mast cells in the regulation of intestinal epithelial barrier function, in particular, during homeostasis and in relation to food allergic reactions. We demonstrate that mice deficient in mast cells or the mast cell chymase Mcpt4 have alterations in their homeostatic intestinal barrier, including decreased intestinal permeability, decreased epithelial cell migration, altered intestinal morphology and decreased expression of the tight junction protein claudin-3. Bone marrow-derived mast cell reconstitution experiments confirmed that mast cells regulate the homeostatic intestinal barrier in a chymase/Mcpt4-dependent manner. We examined the mechanism by which chymase disrupts barrier function and show that chymase induces a biphasic change in intestinal epithelial cell barrier function. Phase I was associated with decreased expression of the tight junction protein claudin-5, followed by Phase II, which was characterized by increased protease activated receptor-2 activation and matrix metalloprotease-2 production and activation. Both phases require the chymotryptic activity of chymase, suggesting proteolytic cleavage to remove claudin-5 from the tight junction and to activate pro-matrix metalloprotease-2. In addition to regulating the intestinal barrier during homeostasis, our work defines a critical role for mast cells in altering barrier function both in the predisposition to and pathology of food allergies. Depletion or pharmacologic stabilization of mast cells demonstrates that mast cells regulate intestinal permeability in a murine model of food allergies. Transgenic intestinal overexpression of IL-9 induced an intestinal mastocytosis and mast cell-dependent increase in intestinal permeability. Notably, the mast cell-mediated increase in intestinal permeability was sufficient to facilitate oral sensitization to food antigens and the development of clinical symptoms of food allergies. Collectively, this work defines a critical homeostatic and pathogenic role for mast cells in the regulation of intestinal barrier function, dysregulation of which can lead to food antigen sensitization and the induction of food allergies.
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