Priming of human neutrophil respiratory burst by granulocyte macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47(phox)

Neutrophil superoxide production can be potentiated by prior exposure to "p riming" agents such as granulocyte/macrophage colony stimulating factor (GM -CSF), Because the mechanism underlying GM-CSF-dependent priming is not und erstood, we investigated the effects of GM-CSF on the phosphorylation of th e cytosolic NADPH oxidase components p47(phox) and p67(phox). Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47(phox ) but not that of p67(phox). Addition of formyl-methionyl-leucyl-phenylalan ine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosph orylation of p47(phox) than with GM-CSF alone and fMLP alone. GM CSF-induce d p47(phox) phosphorylation was time- and concentration-dependent and ran p arallel to the priming effect of GM-CSF on superoxide production. Two dimen sional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phos phorylation of one major peptide. fMLP alone induced phosphorylation of sev eral peptides, an effect enhanced by GM-CSF pretreatment, In contrast to fM LP and phorbol la-myristate 13-acetate, GM-CSF-induced phosphorylation of p 47(phox) was not inhibited by the protein kinase C inhibitor GF109203X, The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3 -kinase inhibitor wortmannin inhibited the phosphorylation of p47(phox) ind uced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13- acetate. GM-CSF alone did not induce p47(phox) Or P67(phox) translocation t o the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47(phox) and p67(phox). Taken together, these results show that the primin g action of GM-CSF on the neutrophil respiratory burst involves partial pho sphorylation of p47(phox) on specific serines and suggest the involvement o f a priming pathway regulated by protein-tyrosine kinase and phosphatidylin ositol 3-kinase.