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* Epithelial Pathobiology Unit and Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322; and
Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada; INSERM U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; and Université Toulouse III Paul Sabatier, Toulouse, France
Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn’s disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by research fellowships from the Canadian Association of Gastroenterology, Canadian Institutes of Health Research, Axcan Pharma, and the Crohn’s and Colitis Foundation of America (to A.C.C.); by grants from the Alberta Heritage Foundation for Medical Research, and the Canadian Institutes of Health Research, INSERM-Avenir, and the Foundation Bettencourt-Schueller (to N.V.); National Institutes of Health Grants DK59888 (to A.N.), DK079392, and DK72564 (to C.A.P.), and a Digestive Diseases Minicenter Grant DK064399 (epithelial cell culture core and microscopy core support).
2 Address correspondence and reprint requests to Dr. Alex C. Chin or Dr. Charles Parkos, Department of Pathology and Laboratory Medicine, Emory University, 615 Michael Street, Atlanta, GA 30322. E-mail address: achin{at}emory.edu, cparkos{at}emory.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; PAR, protease-activated receptor; MLCK, myosin L chain kinase; AAPV, N-(methoxysuccinyl)alanylalanylprolylvalyl chloromethyl ketone; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; TER, transepithelial electrical resistance; siRNA, small interfering RNA.
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