title: Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration.
abstract: 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.
authors.affiliation: Epithelial Pathobiology Unit and Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA. achin@emory.edu
authors.forename: Alex C
authors.initials: AC
authors.lastname: Chin
authors.forename: Winston Y
authors.initials: WY
authors.lastname: Lee
authors.forename: Asma
authors.initials: A
authors.lastname: Nusrat
authors.forename: Nathalie
authors.initials: N
authors.lastname: Vergnolle
authors.forename: Charles A
authors.initials: CA
authors.lastname: Parkos
mesh_terms: D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology*; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000276:immunology / Q000378:metabolism; D018920:Coculture Techniques; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D007413:Intestinal Mucosa / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D053146:Myeloblastin / Q000494:pharmacology; D020556:Neutrophil Infiltration / Q000187:drug effects / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D010196:Pancreatic Elastase / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D044463:Receptor, PAR-1 / Q000276:immunology* / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D013379:Substrate Specificity; D019108:Tight Junctions / Q000276:immunology; D010975:Platelet Aggregation Inhibitors
chemical_list: D002630:Chemotactic Factors; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D053146:Myeloblastin; D010975:Platelet Aggregation Inhibitors