Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Neutrophils aid host defense by killing invading microorganisms through production of highly reactive oxygen species (ROS) 1 generated by activation of the NADPH oxidase complex. When released inappropriately into the extracellular milieu, these ROS can cause persistent inflammation and considerable damage to the surrounding, healthy tissues. To prevent calamitous release of ROS, macrophages remove excess activated neutrophils from an inflammatory site in a regulated way, through processes that ensure swift resolution of inflammation yet make provision for neutrophils to fulfill their microbicidal function. Phagocytic cells carry out this clearance by recogniz-

No abstract

A common feature of the apoptotic program is phospholipid signaling aimed at the generation of "eat me" signals on the surface of the apoptotic cell that make it recognizable by phagocytes (1). This apoptotic signaling is mediated through the loss of plasma membrane phospholipid asymmetry and the concomitant externalization of phosphatidylserine (PS) 1 (2). PS-dependent signaling is coupled to the final common pathway of apoptosis, i.e. the caspase-driven dismantling of the cell, thus allowing for effective phagocytosis and clearance of cell corpses. The importance of phagocytosis of apoptotic cells for prevention of spillage of cellular contents and resultant tissue disruption and inflammation has been emphasized in numerous studies in recent years (3, 4); however, the specific mechanisms that govern PS externalization and recognition during apoptosis remain to be elucidated. We have recently shown that PS externalization during apoptosis is preceded by its selective oxidation, likely catalyzed by cytochrome c released from mitochondria (5, 6). We have also demonstrated that oxidized PS (PS-OX) may serve as an "eat me" signal for macrophage receptor(s), thus facilitating recognition and PS-dependent engulfment of apoptotic cells. (7,8) Based on these observations, we hypothesized that PS oxidation acts as an essential component of the signaling pathway that is required for PS externalization and the safe clearance of apoptotic cells by macrophages (5, 8, 9, 10). Our hypothesis predicts that lipid antioxidants capable of blocking PS oxidation will inhibit PS externalization and/or recognition of apoptotic cells by phagocytes. An important feature of such a lipid antioxidant, however, is that it should block phospholipid oxidation without affecting other redox-sensitive mechanisms.Our previous work has established that a phenolic antitumor drug, etoposide (a topoisomerase II inhibitor), acts as a powerful lipid antioxidant but does not protect thiols against oxidation because of a relatively high reactivity of etoposide phenoxyl radicals toward SH-groups (11). Etoposide has been reported to cause DNA damage and induce apoptosis accompanied by ROS generation (12, 13). It is conceivable that the fastidious lipid antioxidant traits of etoposide may dissociate PS-dependent signaling pathways of apoptosis from the final common pathway for apoptosis by inhibiting PS oxidation. However, etoposide effects on phospholipid peroxidation, as * This work was supported by National Institutes of Health Grants HL70755, CA90787, and GM64610 and the Swedish Society for Medical Research (to B. F.). J. C. Y. has a significant equity interest, as defined by the U. S. Public Health Service, in Bristol-Myers Squibb Co., a manufacturer of etoposide, used in this research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ ‡ To whom correspondence should be addre...