Hydrogen peroxide (H2O2) is produced during a variety of cellular procedures. In this paper, the regulatory role of H2O2, in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihythorhodamine (DHR) in order to study H2O2 synthesis and E. coli-FITC to study phagocytosis. Flow cytometcy revealed increased synthesis of H2O2 in polymorphonuclears which incorporated E coli-FITC. The blocking of H2O2 synthesis by specific inhibitors, N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. col( phagocytosis, as well. Immunoblot analysis of white blood cell protein extracts revealed that the blocking of NADPH oxidase and SOD decreased ERK-1/2 phosphorylation, while it had no effect on INK and p38. Confocal microscopy showed that phosphorylation of MAPKs and phagocytosis solely occur in the polymorphonuclear and not in mononuclear cells. The use of specific MAPKs inhibitors showed that all of them are necessary for phagocytosis, but only phospho-p38 affects H2O2 synthesis. The blocking of JNK phosphorylation, in the presence of E colt, evoked a further decrease of cytoplasmic p47 thus increasing its translocation onto the plasma membrane for the assembly of NADPH oxidase. It appears that newly synthesised H2O2 invigorates the phosphorylation and action of ERK-1/2 in E. ail( phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H2O2 production. (C) 2015 The Authors. Published by Elsevier B.V.
