Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis

Autophagy and apoptosis cooperate to modulate cell survival. Neutrophils are short-lived cells and apoptosis is considered to be the major mechanism of their death. In the present study, we addressed whether autophagy regulates neutrophil apoptosis and investigated the effects of autophagy inhibition on apoptosis of human neutrophils. We first showed that the established autophagy inhibitors 3-methyladenine (MA) and chloroquine (CQ) markedly accelerated spontaneous neutrophil apoptosis as was evidenced by phosphatidylserine exposure, DNA fragmentation and caspase-3 activation. Apoptosis induced by the autophagy inhibitors was completely abrogated by a pan-caspase inhibitor Q-VD-OPh. Unexpectedly, both MA and CQ significantly delayed neutrophil apoptosis induced by TNF-alpha, although the inhibitors did attenuate late pro-survival effect of the cytokine. The effect was specific for TNF-alpha because it was not observed in the presence of other inflammation-associated cytokines (IL-1 beta or IL-8). The autophagy inhibitors did not modulate surface expression of TNF-alpha receptors in the absence or presence of TNF-alpha. Both MA and CQ induced a marked down-regulation of a key anti-apoptotic protein Mcl-1 but did not affect significantly the levels of another anti-apoptotic protein Bcl-X-L. Finally, to confirm the effects of the pharmacological inhibition of autophagy by a genetic approach, we evaluated the consequences of siRNA-mediated autophagy suppression in neutrophil-like differentiated HL60 cells. Knockdown of ATG5 in the cells resulted in accelerated spontaneous apoptosis but attenuated TNF-alpha-induced apoptosis. Together, these data suggest that autophagy regulates neutrophil apoptosis in an inflammatory context-dependent manner and mediates the early pro-apoptotic effect of TNF-alpha in neutrophils.