Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms

Caspase activation, exposure of phosphatidylserine (PS) on the outer surfac e of the plasma membrane, and rapid phagocytic removal of dying cells are k ey features of apoptosis, Nonapoptotic/necrotic modes of death occur indepe ndent of caspase activation, but the role of phagocytosis is largely unknow n. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were st imulated by several different methods that all caused caspase-independent d eath. First, we induced necrosis by combining toxins with ATP-depleting age nts. Under these conditions, neither PS was exposed nor were such cells pha gocytosed before their death. However, once the plasma membrane integrity w as lost, the dead cells were rapidly and efficiently engulfed by HMDM, Next , we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk, Under these conditions, death occurred by d elayed necrosis and without exposure of BS, Nevertheless, such lethally cha llenged cells were phagocytosed before the loss of membrane integrity. Fina lly, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neur ons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed startin g at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-m ediated PS exposure and phagocytosis. Essentially, similar phagocytosis dat a were obtained For all models with HMDM and microglia, We conclude that al so cells dying nonapoptotically and independent of caspase activation may b e recognized and removed before, or very quickly after, membrane lysis.