Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells

Abstract

Persistent infection with Pseudomonas aeruginosa increases interleukin-8 (IL-8) levels and causes dense neutrophil infiltrations in the airway of patients with chronic airway diseases. To investigate the role of P. aeruginosa infection in IL-8 production in the airway of these patients, we examined whether cell lysates of P. aeruginosa could cause IL-8 production from human bronchial epithelial cells. Diluted sonicated supernatants of P. aeruginosa (SSPA) with a mucoid or nonmucoid phenotype stimulated human bronchial epithelial (BET-1A) cells to produce IL-8. In this study, we have purified a 59-kDa heat-stable protein with IL-8-inducing activity from the SSPA by sequential ion-exchange chromatography. The N-terminal sequence of this purified protein completely matched a sequence at the N-terminal part of the mature protein of nitrite reductase from P. aeruginosa. In addition, immunoblotting with a polyclonal immunoglobulin G (IgG) against recombinant Pseudomonas nitrite reductase demonstrated a specific binding to the purified protein. Furthermore, the immunoprecipitates of the SSPA with a polyclonal IgG against recombinant nitrite reductase induced a twofold-higher IL-8 production in the BET-1A cell culture than did the immunoprecipitates of the SSPA with a control IgG. These lines of evidence confirmed that Pseudomonas nitrite reductase was responsible for IL-8 production in the BET-1A cells. The purified nitrite reductase induced maximal expression of IL-8 mRNA in the BET-1A cells at 1 to 3 h after stimulation, and the IL-8 mRNA expression declined by 8 h after stimulation. New protein translation was not required for nitrite reductase-mediated IL-8 mRNA expression in the BET-1A cells. Nitrite reductase stimulated the BET-1A cells, as well as human alveolar macrophages, pulmonary fibroblasts, and neutrophils, to produce IL-8. In contrast, nitrite reductase induced significant levels of tumor necrosis factor alpha and IL-1␤ protein only in human alveolar macrophages. These data support the notion that nitrite reductase from P. aeruginosa induces the production of inflammatory cytokines by respiratory cells and may contribute to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infection.

Induction of Interleukin 8 (IL‐8) Production by Pseudomonas Nitrite Reductase in Human Alveolar Macrophages and Epithelial Cells

“…In addition, we previously confirmed PNRmediated IL-8 production in a human pulmonary fibroblast cell line (CCD-18 Lu) (18). The present study showed a similar PNR-mediated up-regulation of IL-8 mRNA expression…”

“…The sonicated supernatant of P. aeruginosa (SSPA) was obtained following ultracentrifugation at 18,000 X g for 60 min at 4 C and filtration through a 0.45 um filter. The PNR was purified as previously described (18). The purified PNR contained 30 ng of LPS per milliliter by a sensitive Toxicolor test (Seikagaku Kogyo Co., Tokyo) (16), and was stored at -80 C until use.…”

“…We recently reported that nitrite reductase from P . aeruginosa induced inflammatory cytokine such as IL -8 from bronchial epithelial cells in vitro (18) . Pseudomonas nitrite reductase (PNR) with a molecular weight of 60,204 Da is recognized as a periplasmic component active in energy generation (27).…”

Nitrite Reductase from Pseudomonas aeruginosa Released by Antimicrobial Agents and Complement Induces Interleukin-8 Production in Bronchial Epithelial Cells

“…with 25 g of amphotericin B per ml, 25 U of penicillin per ml, and 25 g of streptomycin per ml in a 24-well plate coated with fibronectin and collagen (24). The BET-1A cell line was employed for determination of nonlipopolysaccharide (LPS)-mediated IL-8 production because of its lack of responsiveness to LPS stimulation (27). After confluent cultures had been washed with HEPES-buffered saline, cells were incubated with purified PNR or the aliquots of bacterial culture with antimicrobial agents or absorbed normal human serum (AbsNHS), diluted with LHC-9 medium.…”

“…Immunoblotting. The purified PNR or aliquots of bacterial culture containing antimicrobial agents or complement were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (10% polyacrylamide), transferred to a polyvinylidene difluoride membrane, analyzed by immunoblotting with a polyclonal rabbit immunoglobulin G (IgG) against recombinant PNR at 1 g/ml (27) and horseradish peroxidase-conjugated streptavidin (Amersham, Little Chalfont, United Kingdom), and subjected to detection by enhanced chemiluminescence (Amersham).…”