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

Abstract

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 O2−/0.75 × 106PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 μM) decreased this amount to 0.3 ± 2.6 nmol (n= 10,P< 0.05); the MPO-ANCA values were 23 ± 3versus1.6 ± 3.6 (n= 10,P< 0.05). p38 MAPK inhibition with 10 μM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-α and to ANCA. However, SB202190 but not LY294002 abrogated TNF-α–mediated ANCA antigen surface translocation, demonstrating that superoxide generation and ANCA antigen translocation proceed by separate mechanisms. Akt, PAK1, and Rac1 existed as cytosolic complex in resting PMN. TNF-α stimulation increased association of PAK1 with Akt. An MPO monoclonal antibody did not alter the Akt signaling complex further. The data demonstrate the importance of PI3-K for the ANCA-induced PMN oxidant production.