RELEASE OF THE MUCOSAL MAST-CELL GRANULE CHYMASE, RAT MAST-CELL PROTEASE-II, DURING ANAPHYLAXIS IS ASSOCIATED WITH THE RAPID DEVELOPMENT OFPARACELLULAR PERMEABILITY TO MACROMOLECULES IN RAT JEJUNUM

The soluble granule chymase, rat mast cell protease-II (RMCP-II), is a bundantly expressed in intestinal mucosal mast cells (MMC) but its fun ction is not known. One hypothesis is that RMCP-II degrades the epithe lial basement membrane and promotes the loss of enterocytes typically associated with type I hypersensitivity reactions in the rat. To test this hypothesis more directly, ex vivo perfusion of the cranial mesent eric artery and jejunal lumen was used to monitor the anaphylactic rel ease of RMCP-II and its effects on mucosal permeability and epithelial integrity. Within 2 min of intravascular challenge with soluble adult Nippostrongylus brasiliensis worm antigen there was a 1,000-fold (P<0 .02) increase in the concentration of RMCP-II in the vascular perfusat e from the jejunum of Nippostrongylus-sensitized rats but not the cont rols. Similarly, translocation of RMCP-II into the gut lumen increased 10-fold (P<0.02) after 2 min only in worm antigen-challenged immune r ats. Using an identical protocol, but incorporating Evans blue-labeled human serum albumin (EB-HSA) in the vascular perfusate, the timing of the release of RMCP-II into the two compartments was very similar to the first experiment and furthermore the translocation of EB-HSA incre ased 18-fold (P<0.05) after 4 min in sensitized rats challenged with w orm antigen. To examine the effects of RMCP-II more directly 1 mg of t he highly purified chymase was introduced into the cranial mesenteric artery in ex vivo perfused normal rats. A significant (P<0.05) 70-fold increase in concentration of RMCP-II in jejunal perfusate occurred af ter 6 min. In a repeat dose-response experiment, infusion of 0.375, 0. 75, or 1.5 mg of RMCP-II, together with EB-HSA, established that the c umulative amounts of RMCP-II and EB-HSA translocated from the vasculat ure to the gut lumen in each perfusion (during the 10-min period of RM CP-II infusion) were significantly correlated. Analysis of intestinal perfusates by SDS-PAGE and by Western blotting using monoclonal anti-R MCP-II antibody confirmed that there was a concomitant translocation o f both the protease and EB-HSA into the gut lumen. Histological evalua tion of the mucosa failed to reveal any significant morphological chan ge in any of the experiments. The rapid development of macromolecular leak, its association with the translocation of RMCP-II, and the absen ce of gross epithelial lesions, suggest for the first time that a mast cell granule chymase increases epithelial permeability via a paracell ular route and implies that the substrate may be a protein, or protein s, in the epithelial junctional complex.