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Ca2+ activation of cytosolic calpain induces the transition from apoptosis to necrosis in neutrophils with externalized phosphatidylserine

Physiologically, apoptotic neutrophils are ingested before they undergo necrosis. However, failure of ingestion will lead to necrosis of neutrophils and the unregulated release of neutrophil‐derived pathogenic molecules, such as protease and hydrolases. Understanding the mechanism of postapoptotic necrosis is thus clearly important. Here, we monitored the apoptotic‐to‐necrotic transition in individual‐aged human neutrophils in vitro by imaging fluorescent probes for externalized PS, cytosolic Ca2+, and membrane integrity. This showed that prenecrotic‐aged neutrophils with externalized PS had a significantly elevated cytosolic‐free Ca2+ level. A further unregulated Ca2+ influx into PS‐externalized neutrophils always preceded the necrotic transition. Ca2+ elevation was not simply a consequence of aging, as PS externalization was not uniform in similarly aged neutrophil populations. PS‐externalized neutrophils could be induced to undergo necrosis experimentally by simply elevating cytosolic Ca2+ further with ionomycin. This effect was observed only in neutrophils that had externalized PS, and was independent of the time after their isolation from blood (i.e., in vitro age). As pharmacological inhibition of calpain‐1 inhibition significantly reduced this CAIN, it was concluded that the apoptotic‐to‐necrotic transition was a consequence of uncontrolled calpain activation that resulted from Ca2+ overload in PS‐externalized neutrophils.