Type I cAMP-dependent protein kinase delays apoptosis in human neutrophils at a site upstream of caspase-3.

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX +/- 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had no effect on apoptosis in the absence of the analogue. In contrast, site-selective cAMP analogues that specifically activated the type I cAPK, but not type II cAPK, synergistically attenuated apoptosis. Exposure to 8-CPT-cAMP delayed, in parallel, the activity of caspase-3 (CPP-32beta), whereas mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD98059, had no effect on CHX-induced apoptosis +/- 8-CPT-cAMP. Together these results indicate that type I cAPK activation is necessary and sufficient to mediate cAMP-induced delay in human neutrophil apoptosis induced by several mechanisms and suggest that one of the major sites of cAPK action is upstream of caspase-3 (CPP-32beta) activation.