Deciphering opsonized zymosan–induced phosphorylation of p47phox and NADPH oxidase activation in human neutrophils

ABSTRACT: Neutrophils play a key role in innate immunity by killing microbes through phagocytosis and superoxide anion production by the phagocyte reduced NAD phosphate (NADPH) oxidase. The signaling pathways regulating NADPH oxidase activation in neutrophils have been extensively studied using soluble agonists, but are less understood during phagocytosis, a fundamental function of neutrophils. The aim of this study was to investigate the phosphorylation of the cytosolic NADPH oxidase protein p47phox in human neutrophils stimulated by serum-opsonized zymosan (OZ), which induces phagocytosis, using antibodies against phosphorylated sites. The results show that OZ induced rapid phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328, followed by rapid dephosphorylation. Interestingly, despite the transient nature of p47phox phosphorylation, OZ-induced NADPH oxidase activity was sustained for a longer period in cells and in isolated membranes. OZ-induced p47phox phosphorylation was concentration dependent and preceded particle ingestion. Immunoglobulin G (IgG)- and complement protein fragment 3bi (C3bi)-opsonized zymosan similarly induced rapid phosphorylation and dephosphorylation of p47phox on Ser304 to Ser328, suggesting that IgG Fc-gamma receptors (FcγR) and complement receptor 3 (CR3) are involved in this process. Inhibitors of sarcome (Src) tyrosine kinase, spleen tyrosine kinase (Syk), phosphoinositide 3-kinase (PI3K), phospholipase C (PLC), phospholipase D (PLD), Ca2+, and protein kinase C beta 2 (PKCβ2) inhibited OZ-induced phosphorylation of p47phox. These results suggest that (1) OZ-induced p47phox phosphorylation on Ser304 to Ser328 is required for the initiation of NADPH oxidase activation but not for its maintenance during phagocytosis, (2 )the membrane receptors FcγR and CR3 mediate this phosphorylation, and (3) Src and Syk tyrosine kinases, PI3K, PLD, Ca2+, and PKCβ2 control the phosphorylation of p47phox during phagocytosis.