PROTEINASE-3, THE MAJOR AUTOANTIGEN OF WEGENERS GRANULOMATOSIS, ENHANCES IL-8 PRODUCTION BY ENDOTHELIAL-CELLS IN-VITRO

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the ozurophilic granules of polymorphonuclear neutrophils, the domina nt cell type in vascular lesions during the early stages of systemic v asculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the c hemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefo re, human umbilical vein endothelial cells in culture were incubated w ith varying concentrations of highly purified proteinase 3, human neut rophil elastase, and cathepsin G for different time periods, The super natants were subsequently assessed for IL-8 antigen by using a sandwic h ELISA, The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion, Cathepsin G did not influence IL-8 production. Although the addition of an ai-proteinase inhibitor c ompletely abrogated elastase-mediated IL-8 production, ii did not sign ificantly influence the effect of proteinase 3, Both proteinase 3- and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the Finding of inc reased IL-8 mRNA levels in proteinase 3-treated human umbilical vein e ndothelial cells by using Northern blot analysis, Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastas e may contribute to a self-perpetuating process of neutrophil recruitm ent in acute inflammation by increasing de novo synthesis of IL-8 by e ndothelial cells, The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indica ting a hitherto unknown mode of action on endothelial cells.