Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

No abstract

No abstract

No abstract

No abstract

No abstract

NADPH oxidase Cytochrome b−245 Phosphorylation Protein kinase Neutrophil activation Respiratory burst

We have previously shown that in neutrophils classical transmembrane signaling consisting of increased [Ca2+]i and hydrolysis of phospholipids was not essential for phagocytosis mediated by more than one receptor (yeast‐IgG, yeast‐C3b/bi, yeast‐Con A). This work deals with the role of this transmembrane signaling in phagocytosis of erythrocyte (E) IgG, which is mediated only by receptors for IgG (FcγRs). The ingestion of E‐IgG was associated with an increase in [Ca+]i and production of inositol phosphates, phosphatidic acid, diacylglycerol, and arachidonic acid, via activation of phospholipases G, D and A2. Related to the same number of particles ingested, the respiratory burst and the transmembrane signaling during phagocytosis of E‐IgG were much smaller than during phagocytosis of yeast‐IgG. In Ca2+‐depleted neutrophils, where the increase in [Ca2+]i and hydrolysis of phospholipids were lacking, the phagocytosis of E‐IgG was depressed by about 60%; the respiratory burst was also depressed due to the decrease of ingestion and of stimulation of NADPH oxidase by residual phagocytosis. Pertussis toxin (PT) did not inhibit the phagocytosis of E‐ IgG but depressed by about 40% the stimulation of li‐ pidic transmembrane signaling and the respiratory burst in normal neutrophils. In Ca2+‐depleted neutrophils the toxin was without effect on ingestion and respiratory burst. Staurosporine did not inhibit the ingestion of E‐ IgG in normal and Ca2+‐depleted neutrophils but depressed by 30‐40% the respiratory burst in normal and not in Ca2+‐depleted neutrophils. Genistein, an inhibitor of tyrosine kinase, did not inhibit the ingestion of E‐IgG but depressed by 30‐40% the respiratory burst both in normal and Ca2+‐depleted neutrophils. These results demonstrate the following findings in human neutrophils. (1) Contrary to the phagocytosis mediated by more than one receptor (yeast‐IgG, yeast‐Con A, yeast‐C3b/bi), the transmembrane signaling involving increase in [Ca2+]i and hydrolysis of phospholipids plays a role in the phagocytosis and respiratory burst mediated by FcγRs alone. Thus, different signal transduction pathways can be involved in phagocytosis and associated respiratory burst depending on the receptor or combination of receptors activated. (2) FcγRs alone promote phagocytosis with two signaling pathways independent of and dependent on [Ca2+]i changes and phospholipid hydrolysis and insensitive to PT, staurosporine, and genistein. (3) The signaling pathways promoting phagocytosis triggered by FcγRs alone are in some way, or at some step, different from those that activate the respiratory burst.