ABSTRACT
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.
Subject(s)
Chymases/physiology , Intestinal Mucosa/enzymology , Intestinal Mucosa/physiology , Matrix Metalloproteinase 2/physiology , Receptor, PAR-2/physiology , Animals , Bone Marrow Cells/enzymology , Bone Marrow Cells/physiology , Caco-2 Cells , Chymases/antagonists & inhibitors , Chymases/genetics , Claudin-5/physiology , Fluorescent Antibody Technique , Humans , Lentivirus/genetics , Mast Cells/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Permeability , Tight Junctions/physiology , Transduction, GeneticABSTRACT
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.
Subject(s)
Chymases/physiology , Intestine, Small/physiology , Mast Cells/physiology , Serine Endopeptidases/physiology , Animals , Caco-2 Cells , Cell Movement/physiology , Chymases/deficiency , Chymases/genetics , Chymases/pharmacology , Claudin-3 , Epithelium/physiology , Homeostasis , Humans , In Vitro Techniques , Intestine, Small/cytology , Jejunum/cytology , Jejunum/physiology , Mast Cells/transplantation , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Permeability/drug effects , Recombinant Proteins/pharmacology , Serine Endopeptidases/deficiency , Serine Endopeptidases/geneticsABSTRACT
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.