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Proteinase-Activated Receptor-2 and Human Lung Epithelial Cells

Disarming by Neutrophil Serine Proteinases

Unité de Défense Innée et Inflammation, Unité Associée IP/Inserm 485, Institut Pasteur, Paris, France; Departments of Surgery and Physiology, University of California, San Francisco, California; and Department of Pharmacology and Therapeutics and Department of Medicine, The University of Calgary, Calgary, Alberta, Canada

Address correspondence to: Michel Chignard, Unité de Défense Innée et Inflammation, Unité Associée IP/Inserm 485, Institut Pasteur, 25, rue du Dr. Roux, 75015 Paris, France. E-mail: chignard{at}pasteur.fr

Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH₂ leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH₂. Preincubation of a PAR-2–transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH₂–binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2–mediated lung inflammation.

Abbreviations: bovine serum albumin, BSA • PMSF-inactivated cathepsin G, CG-PMSF • Dulbecco's modified Eagle's medium, DMEM • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • human bronchial epithelial cells, HBEC • Hank's balanced salt solution, HBSS • PMSF-inactivated elastase, HLE-PMSF • interleukin, IL • Kirsten murine sarcoma virus-transformed rat kidney, KNRK • proteinase-activated receptor, PAR • polymerase chain reaction, PCR • prostaglandin, PG • soybean trypsin inhibitor, SBTI

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