Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells
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1Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Japan. oishi-k@net.nagasaki-u.ac.jp
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-1beta 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.
Insights
Pseudomonas aeruginosa infection triggers interleukin-8 (IL-8) production in airway cells. A specific protein, nitrite reductase, from P. aeruginosa was identified as the cause, contributing to airway inflammation.
Area of Science:
- Microbiology
- Immunology
- Pulmonology
Background:
- Persistent Pseudomonas aeruginosa infection is linked to elevated interleukin-8 (IL-8) and neutrophil infiltration in chronic airway diseases.
- The precise mechanisms by which P. aeruginosa infection induces IL-8 in airway cells remain incompletely understood.
Purpose of the Study:
- To investigate the role of P. aeruginosa components in stimulating IL-8 production from human bronchial epithelial cells.
- To identify the specific molecule within P. aeruginosa responsible for inducing IL-8.
Main Methods:
- Purification of a protein from P. aeruginosa sonicated supernatants using ion-exchange chromatography.
- N-terminal sequencing and immunoblotting to identify the purified protein.
- Assessing IL-8 production in human bronchial epithelial cells (BET-1A) stimulated with purified protein and immunoprecipitates.
Main Results:
- A 59-kDa heat-stable protein with IL-8-inducing activity was purified.
- The protein was identified as Pseudomonas nitrite reductase based on N-terminal sequencing and antibody binding.
- Nitrite reductase stimulated IL-8 mRNA expression in BET-1A cells within 1-3 hours, independent of new protein translation.
- Nitrite reductase induced IL-8 production in various respiratory cells, but TNF-alpha and IL-1beta primarily in macrophages.
Conclusions:
- Pseudomonas nitrite reductase is a key factor inducing IL-8 production in respiratory cells.
- This mechanism likely contributes to the pathogenesis of chronic airway diseases and persistent P. aeruginosa infections.