Hydrogen peroxide signals E. coli phagocytosis by human polymorphonuclear cells; up-stream and down-stream pathway

Hydrogen peroxide (Η(2)Ο(2)) is produced during a variety of cellular procedures. In this paper, the regulatory role of Η(2)Ο(2), in Escherichia coli phagocytosis by the human polymorphonuclears, was investigated. White blood cells were incubated with dihydrorhodamine (DHR) in order to study H(2)O(2) synthesis and E. coli-FITC to study phagocytosis. Flow cytometry revealed increased synthesis of H(2)O(2) in polymorphonuclears which incorporated E. coli-FITC. The blocking of H(2)O(2) synthesis by specific inhibitors, N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for superoxide dismutase (SOD), decreased E. coli 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 JNK 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 H(2)O(2) synthesis. The blocking of JNK phosphorylation, in the presence of E. coli, 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 H(2)O(2) invigorates the phosphorylation and action of ERK-1/2 in E. coli phagocytosis, while phospho-JNK and phospho-p38 appear to regulate H(2)O(2) production.