Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-ζ-dependent pathway

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O-2(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O-2(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O-2(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I-A PI3K, but not translocation of the p110gamma subunit of class I-B PI3K, to the neutrophil membrane. Inhibition of protein kinase C (PKCzeta inhibited MBP-stimulated O-2(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta(Thr(505).) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I-A PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I-A PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O-2(-) production in response to MBP stimulation.