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The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b(558), the cytosol factors p47(phox), p67(phox), p40(phox), and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47(phox), as well as the recruitment of flavocytochrome b(558), p47(phox), and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b(558), p47(phox), or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67(phox) into detergent-resistant microdomains. Moreover, methyl-beta-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.

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.

Vitamin E (α-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. α-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O⨪2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of α-tocopherol on O⨪2 production by human monocytes. In activated monocytes O⨪2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, α-tocopherol (but not β-tocopherol) inhibited O⨪2production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that α-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47phox were reduced in monocytes preincubated with α-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47phox was similar to that of neutrophil p47phox, indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47phox was immunoprecipitated from α-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by α-tocopherol in a specific manner compared with that of β-tocopherol or Trolox®. Membrane translocation of PKC was not affected. These results show that α-tocopherol inhibits O⨪2production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47phox results from a decrease in PKC activity.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O·2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67 phox . In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67 phox . Using an anti-p67 phox antibody or an anti-p47 phox antibody, we showed that phosphorylated p67 phox and p47 phox form a complex. Phosphoamino acid analysis of the phosphorylated p67 phox revealed only32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67 phox is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67 phox in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67 phox was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67 phox is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.