Induction of Neutrophil Apoptosis by thePseudomonas aeruginosaExotoxin Pyocyanin: A Potential Mechanism of Persistent Infection

Abstract

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 μM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.

Selective Roles for Toll-Like Receptor (TLR)2 and TLR4 in the Regulation of Neutrophil Activation and Life Span

“…Dichlorodihydrofluorescein diacetate (DCF) is a cell-permeable compound, which becomes fluorescent upon oxidation by hydrogen peroxide and other reactive oxygen species and was used to measure neutrophil respiratory burst as described previously (25). Briefly, neutrophils were preincubated with 5 M DCF for 30 min at 37°C and 5% CO 2 , and then treated with pLPS, Pam 3 CSK 4 , or medium (RPMI 1640 plus 10% FCS plus penicillin and streptomycin) for 30 min, after which time fMLP or medium was added for an additional 15 min.…”

The Pro-apoptotic Protein Bim Is a Convergence Point for cAMP/Protein Kinase A- and Glucocorticoid-promoted Apoptosis of Lymphoid Cells

“…epithelial cells) and protects other cell types (e.g. neutrophils and eosinophils) from drug-and cytokine-promoted apoptosis (1)(2)(3). The greater susceptibility of certain tumor cells to cAMP-mediated growth arrest and/or apoptosis has spurred interest in developing cAMP analogs or related agents as anti-cancer drugs (4 -6), including those for glucocorticoid-resistant malignancies (4 -7).…”

Caspase-3-dependent Cell Death in B lymphocyte Caused by Pseudomonas aeruginosa Pyocyanin

“…The cytotoxicity of pyocyanin were also confirmed by previous studies on bronchial epithelium, pulmonary epithelium, and neutrophil. [6][7][8] Pyocyanin induced production of inflammatory mediators and caused cellular change on bronchial epithelium. Those mechanisms involved disturbance of intracellular antioxidant function that result in the inhibition of catalase activity.…”

“…This cytotoxin is responsible for the bacterial invasion, induction of inflammatory reaction, and cellular damage on respiratory epithelium cells, apoptotic cell death on neutrophil, and persistent infection caused by P. aeruginosa. [6][7][8] In adition, pyocyanin also cause apoptotic cell death on neutrophil. This ability facilitate the bacterium to excuse from the host protection mechanism and lead to persistent infection.…”

“…This ability facilitate the bacterium to excuse from the host protection mechanism and lead to persistent infection. 7,8 Apoptosis also known as programmed cell death. This occurs as cellular response to various stimulants including inflammation caused by bacterial infection.…”