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072 NEUTROPHIL-EPITHELIAL INTERACTIONS ARE REGULATED BY PROTEASE-ACTIVATED RECEPTOR-1 AC Chin1, N Vergnolle2, A Nusrat1, CA Parkos1 Neutrophil (PMN) infiltration into the intestinal mucosa is a hallmark of active inflammatory bowel disease and contributes to epithelial pathophysiology. Previous studies have shown that PMN contact with basolateral surfaces of epithelial cells increases permeability and induces epithelial signaling events prior to transmigration. Activated PMNs mobilize proteases to the cell surface which in turn may disrupt epithelial barrier function through activation of epithelial protease-activated receptors (PARs).
1Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia; 2Mucosal Inflammation Research Group and Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta
AIM: To determine whether the increase in epithelial permeability induced by PMN contact is dependent on PAR signaling and whether this event regulates PMN transepithelial migration.
METHODS: Inverted T84 intestinal monolayers cultured on the underside of 0.4µm (transepithelial permeability assay) or 5.0µm (transepithelial migration assay) pore-sized transwell supports were incubated with or without peripheral blood PMNs in the presence or absence of an f-met-leu-phe (fMLP) gradient. PMNs or monolayers were pre-treated with or without 1mM AEBSF, 5µM SCH79797 (PAR-1 inhibitor) or P4pal-10 (PAR-4 inhibitor). In separate experiments, monolayers were incubated with: 1) 10µg/ml human PMN elastase (HNE), proteinase-3 (PR3), cathepsin G (CatG), 2) 50µM TFLLR (PAR-1 agonist), AYPGKF (PAR-4 agonist), or 3) 50µM RLLFT, YAPGKF (reverse peptides). Paracellular permeability was determined by measuring transepithelial resistance (TER) while PMN transmigration was determined by measuring myeloperoxidase activity.
RESULTS: In the presence of a transepithelial fMLP gradient, PMN contact with T84 monolayers enhanced paracellular permeability. PMNs pre-treated with AEBSF were unable to decrease TER and exhibited marked inhibition in transmigration. Pre-treatment of monolayers with SCH79797, but not P4pal-10, prevented the decrease in TER induced by PMN contact in the presence of an fMLP gradient and also decreased the rate of PMN transepithelial migration. Basolateral, but not apical, activation of PAR-1 with TFLLR decreased TER. Incubation with HNE and PR3, but not CatG, decreased TER. PMNs pre-treated with SCH79797 and/or P4pal-10 still decreased TER, but exhibited impaired transmigration. We postulate that regulation of PMN transepithelial migration is mediated, in part, by activation of epithelial PAR signaling events induced by proteases expressed on migrating PMNs.