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 (O2-) 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 micrograms/mL pertussis toxin did not inhibit MBP-stimulated O2- production, and MBP did not stimulate an increase in diradylglycerol levels. MBP did stimulate a low 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 O2- 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 O2- production. In agreement with the pharmacologic sensitivity, MBP did not stimulate any 51Cr release. These data indicate that tyrosine kinase and calmodulin-dependent steps are involved in the noncytotoxic stimulation of neutrophil O2- production by MBP.