In systemic vasculitis, interactions between antineutrophil cytoplasm autoantibodies (ANCAs) and neutrophils initiate endothelial and vascular injury. ANCAs directed against either myeloperoxiclase (MPO) or proteinase 3 (PR3) can activate cytokine-primed neutrophils by binding cell surface expressed MPO or PR3, with the concurrent engagement of Fc gamma receptors (Fc gammaR). Because roles for phospholipase D (PLD) and phosphatidylinositol 3 kinase (PI3K) have been demonstrated in Fc gammaR activation of neutrophils, this study investigated the hypothesis that ANCA stimulation of neutrophils involved a similar engagement of Fc gammaR and activation of PLD and PI3K. Pretreatment of tumor necrosis factor (TNF) alpha -primed neutrophils with antibodies against Fc gamma RII and Fc gamma RIII inhibited MPO-ANCA and PR3-ANCA induced superoxide generation, confirming that Fc gammaR ligation is involved in ANCA-mediated neutrophil activation. However, although stimulation of TNF-alpha -primed neutrophils by conventional Fc gammaR ligation, either using antibody-mediated cross-linking of Fc gammaR or aggregated IgG, induced PLD activation, ANCA stimulation did not. Moreover, although ANCA-induced neutrophil activation results in significant PI3K activation-as assessed by phosphatidylinositol 3,4,5-triphosphate generation-conventional Fc-IR ligation, but not ANCA, activates the p85/p110 PI3K subtype. Inhibition of ANCA-induced superoxide generation with pertussis toxin suggests that ANCAs activate the p101/p110 gamma PI3K isoform. In addition, the kinetics of activation of protein kinase B differs between conventional Fc gammaR ligation and ANCA stimulation of neutrophils. These results demonstrate that though ligation of Fc gamma RIIa and Fc gamma RIIIb may be necessary, it is likely that ANCAs require other membrane cofactors; for neutrophil activation. (C) 2001 by The American Society of Hematology.