THE RAT MUCOSAL MAST-CELL CHYMASE, RMCP-II, ALTERS EPITHELIAL-CELL MONOLAYER PERMEABILITY IN ASSOCIATION WITH ALTERED DISTRIBUTION OF THE TIGHT JUNCTION PROTEINS ZO-1 AND OCCLUDIN

Mucosal mast cells undergo hyperplasia in a variety of inflammatory bo wel diseases including nematode infection in man and animals. The intr a-epithelial localization of these cells make their soluble mediators prime candidates for modulators of epithelial function. In particular previous in vivo and ex vivo studies have established a link between t he release of the highly soluble mast cell granule chymases and increa sed mucosal permeability: The hypothesis that the rat mast cell protea se, RMCP-II, directly increases permeability to macromolecules via the paracellular route is tested in this study. Monolayers of epithelial cells (Madin-Darby canine kidney cell line) were exposed to varying co ncentrations of RR ICP-II in vitro, in the absence of other cell types or mediators, and the effect on permeability and tight junction assoc iated proteins was investigated. Basolateral, but not apical, exposure of polarized MDCK monolayers on porous supports to RMCP-II led to con centration- (>100 mu g/ml) and time-dependent increases in electrical conductance and permeability to mannitol (MW182) and inulin (MW5000), which was accompanied by decreases in the immunostaining of the tight junction-associated proteins occludin and ZO-1. Furthermore, prolonged exposure to RMCP-II (>12 hours) resulted in the formation of identifi able gaps separating adjacent epithelial cells, in the absence of evid ence of cytotoxicity Inhibition of RMCP-II with Soya bean trypsin inhi bitor completely abrogated the response, demonstrating that proteolysi s was required. These data provide direct evidence that the rat mast c ell chymase RMCP-II can, in the absence of other inflammatory mediator s, increase epithelial permeability via an effect on the paracellular route.