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 alpha (TNF-alpha) 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-alpha-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-alpha 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-alpha 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 x 10(6) PMN/45 min was released after stimulation with PR3-ANCA. LY294002 (5 microM) decreased this amount to 0.3 +/- 2.6 nmol (n = 10, P < 0.05); the MPO-ANCA values were 23 +/- 3 versus 1.6 +/- 3.6 (n = 10, P < 0.05). p38 MAPK inhibition with 10 microM SB202190 that also decreased ANCA-induced superoxide generation prevented S473 phosphorylation of Akt in response to TNF-alpha and to ANCA. However, SB202190 but not LY294002 abrogated TNF-alpha-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-alpha 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.