ANALYSIS OF SIGNALING EVENTS ASSOCIATED WITH ACTIVATION OF NEUTROPHIL SUPEROXIDE ANION PRODUCTION BY EOSINOPHIL GRANULE MAJOR BASIC-PROTEIN

This study was undertaken to identify the signaling events involved in activation of neutrophil superoxide anion (O-2(-)) production by eosinophil granule major basic protein (MBP). MBP did not produce an immediate increase in the cytosolic free calcium concentration ([Ca2+]i), characteristic of phospholipase C activation, but did cause a gradual increase in [Ca2+]i in cytochalasin B-treated cells. Preincubation with 0.01 to 3 mu g/mL pertussis toxin did not inhibit MBP-stimulated O-2(-) production, and MBP did not stimulate an increase in diradylglycerol levels. MBP did stimulate a tow level of phospholipase D activity, as measured by a time-dependent increase in phosphatidic acid and, in the presence of 0.5% ethanol, phosphatidylethanol. Inhibition of MBP-stimulated O-2(-) production by genistein and Western blot analysis using an antiphosphotyrosine antibody showed tyrosine kinase activation by MBP. Calmodulin antagonists (calmidazolium and W-7) caused up to 80% inhibition of MBP-stimulated O-2(-) production. In agreement with the pharmacologic sensitivity, MBP did not stimulate any Cr-51 release. These data indicate that tyrosine kinase and calmodulin-dependent steps are involved in the noncytotoxic stimulation of neutrophil O-2(-) production by MBP. (C) 1995 by The American Society of Hematology.