Novel pathways of F-actin polymerization in the human neutrophil

Recently we demonstrated the existence of a phosphatidylinositol 3-kinase (Pl3K)independent F-actin polymerization during neutrophil pseudopod extension. Here we examine the use of the Pl3K-dependent and Pl3K-independent pathways of activation by the N-formyl peptide receptor and the chemokine receptors, and the priming of the 2 pathways by granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin. The inhibition of Pl3K activity with wortmannin showed that rate of pseudopod extension stimulated with N-formyl-Met-Leu-Phe (fMLP was mostly dependent on Pl3K, while the rate of interleukin-8 (IL-8)-stimulated pseudopod extension was less dependent on Pl3K. The incubation of cells with either GM-CSF or insulin increased the rate of pseudopod extension by 50% when the cells were stimulated with IL-8 but not with fMLP. The stimulation with IL-8 phosphorylated the Pl3K regulatory subunit. This phosphorylation was enhanced by GM-CSF, which increased Pl3K activity and total phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) production. The effect of GM-CSF was blocked with wortmannin. In contrast, insulin did not increase p85 phosphorylation and did not enhance Pl3K activity or PtdIns(3,4,5)P-3 production. The effect of insulin was insensitive to wortmannin; however, it was blocked by an Src homology 2 (SH2)-binding peptide. These data indicate that priming of IL-8 activation with GM-CSF was mediated via the Pl3Ks of class I-A, while priming with insulin used a Pl3K-independent pathway. (C) 2003 by The American Society of Hematology.