Bid truncation, bid/bax targeting to the mitochondria, and caspase activation associated with neutrophil apoptosis are inhibited by granulocyte colony-stimulating factor.

Neutrophil apoptosis constitutes a way of managing neutrophil-mediated reactions. It allows coping with infections, but avoiding overt bystander tissue damage. Using digitonin-based subcellular fractionation and Western blotting, we found that spontaneous apoptosis of human neutrophils (after approximately 20 h of culture) was associated with translocation of two proapoptotic Bcl-2 homologues, Bid and Bax, to the mitochondria and truncation of Bid, with subsequent release of Omi/HtrA2 and Smac/DIABLO into the cytosol. These events were accompanied by processing and increased enzymatic activity of caspase-8, -9, and -3. A G-CSF-mediated reduction in apoptosis coincided with inhibition of all these reactions. The G-CSF-induced effects were differentially dependent on newly synthesized mediators. Whereas inhibition of Bax targeting to the mitochondria and inhibition of caspase activation by G-CSF were dependent on protein synthesis, Bid truncation and redistribution were prevented by G-CSF regardless of the presence of the protein synthesis inhibitor cycloheximide. Apparently, the observed Bid changes were dispensable for neutrophil apoptosis. Although the regulators of the inhibitor of apoptosis proteins (IAPs), Omi/HtrA2 and Smac/DIABLO, were released into the cytosol during apoptosis, we did not observe cleavage of X-linked IAP, which suggests that another mechanism of IAP deactivation is involved. Together our results support an integrative role of the mitochondria in induction and/or amplification of caspase activity and show that G-CSF may act by blocking Bid/Bax redistribution and inhibiting caspase activation.