NITRITE REDUCTASE FROM PSEUDOMONAS-AERUGINOSA INDUCES INFLAMMATORY CYTOKINES IN CULTURED RESPIRATORY CELLS

Persistent infection with Pseudomonas aeruginosa increases interleukin -8 (IL-8) levels and causes dense neutrophil infiltrations in the airw ay of patients with chronic airway diseases. To investigate the role o f. aeruginosa infection in IL-8 production in the airway of these pati ents, eve examined whether cell lysates of P. aeruginosa could cause I L-S production from human bronchial epithelial cells. Diluted sonicate d supernatants of P. aeruginosa (SSPA) vith a mucoid or nonmucoid phen otype stimulated human bronchial epithelial (BET-IA) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein wit h IL-8-inducing activity from the SSPA by sequential ion-exchange chro matography. The N-terminal sequence of this purified protein completel y matched a sequence at the N-terminal part of the mature protein of n itrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas ni trile reductase demonstrated a specific binding to the purified protei n. Furthermore, the immunoprecipitates of the SSPA with a polyclonal I gG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BHT-IA cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed tha t Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. Tie purified nitrite reductase induced maximal expr ession of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-IS mRNA expression declined by 8 h after stimulation. New protein translation was not required far nitrite reductase-mediated IL -S mRNA expression in the BET-1A cells. Nitrite reductase stimulated t he BET-1A cells, as well as human alveolar macrophages, pulmonary fibr oblasts, and neutrophils, to produce IL-8. In contrast, nitrite reduct ase induced significant levels of tumor necrosis factor alpha and IL-1 beta protein only in human alveolar macrophages. These data support t he notion that nitrite reductase from P. aeruginosa induces the produc tion of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeru ginosa infection.