Spontaneous Neutrophil Apoptosis Involves Caspase 3-mediated Activation of Protein Kinase C-δ

Abstract Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389–8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-β and -δ in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-δ was directly involved in the induction of apoptosis. PKC-δ can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-δ in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-δ fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-δ by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-α, -β, or -ζ had no significant effect. Rottlerin and LY379196 inhibit PKC-δ and PKC-β, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-δ but is MAP kinase-independent.