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

J Biol Chem. 1999 Jul 16;274(29):20704-8. doi: 10.1074/jbc.274.29.20704.


Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the 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-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47(phox) than with GM-CSF alone and fMLP alone. GM-CSF-induced p47(phox) phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47(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) induced 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 to 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 priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47(phox) on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • Enzyme Inhibitors / pharmacology
  • Granulocyte-Macrophage Colony-Stimulating Factor / pharmacology*
  • Humans
  • N-Formylmethionine Leucyl-Phenylalanine / pharmacology
  • NADPH Oxidases
  • Neutrophils / drug effects*
  • Neutrophils / metabolism
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Respiratory Burst / drug effects*
  • Superoxides / metabolism


  • Enzyme Inhibitors
  • Phosphoproteins
  • neutrophil cytosol factor 67K
  • Superoxides
  • N-Formylmethionine Leucyl-Phenylalanine
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Protein Kinase C