Phosphatidylinositol 3-Kinase Controls Antineutrophil Cytoplasmic Antibodies—Induced Respiratory Burst in Human Neutrophils

ABSTRACT. Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor α (TNF-α) in vitro . Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-α–primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3- and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-α and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-α priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation. A total of 20 ± 3 nmol O 2 − /0.75 × 10 6 PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 μM) decreased this amount to 0.3 ± 2.6 nmol ( n = 10, P versus 1.6 ± 3.6 ( n = 10, P