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Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Maintenance of membrane lipid asymmetry is a dynamic process that influences many events over the lifespan of the cell. With few exceptions, most cells restrict the bulk of the aminophospholipids to the inner membrane leaflet by means of specific transporters. Working in concert with each other, these proteins correct for sporadic incursions of the aminophospholipids to the outer membrane leaflet as a result of bilayer imbalances created by various cellular events. A shift in the relative contribution in each of these activities can result in sustained exposure of the aminophospholipids at the cell surface, which allows capture of the cells by phagocytes before the integrity of the plasma membrane is compromised. The absence of an efficient recognition and elimination mechanism can result in uncontrolled and persistent presentation of self-antigens to the immune system, with development of autoimmune syndromes. To prevent this, phagocytes have developed a diverse array of distinct and redundant receptor systems that drive the postphagocytic events along pathways that facilitate cross-talk between the homeostatic and the immune systems. In this work, we review the basis for the proposed mechanism(s) by which apoptotic ligands appear on the target cell surface and the phagocyte receptors that recognize these moieties.

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The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca 2؉ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.Apoptosis, senescence, and necrosis are distinct cell death mechanisms in normal physiology and during pathological duress. Irrespective of mechanism, and with very few exceptions (1, 2), dying cells trigger downstream events that result in the appearance of phosphatidylserine (PS) 2 at the cell surface. This serves as a specific recruitment signal for phagocyte docking and subsequent engulfment and degradation of the apoptotic cell (2-4). PS externalization is critical to this process, since its absence results in impaired recognition and clearance of apoptotic debris that can lead to inflammatory and autoimmune responses (5, 6).Mammalian cells have an asymmetric transbilayer distribution of phospholipids such that most of the PS is localized at the inner membrane leaflet. This asymmetry is maintained by the activity of lipid-specific transporters that regulate the distribution of PS between bilayer leaflets, a process that is Ca 2ϩ -dependent (7, 8). Interestingly, lipid transport studies in (organelle-free) erythrocytes have shown that sulfhydryl-modifying reagents do not induce the outward movement of PS (9). These results are in sharp contrast to more recent observations demonstrating that nucleated cells externalize PS upon sulfhydryl modification (10, 11). This suggests that the appearance of PS at the cell surface is regulated by specific intracellular signaling events that are absent in erythrocytes.It is generally accepted that PS externalization during apoptosis occurs downstream to cytochrome c (cyt c) release. There is, however, no direct evidence indicating that cyt c release, caspase activation, or DNA fragmentation is related to, or responsible for PS exposure. In this study, we show that PS externalization can be specifically induced and reversed by a mechanism that is distinct and separable from other hallmarks of apoptosis and operates through a Ca 2ϩ -dependent mechanism that is inhibited by calcium channel blockers, suggesting involvement of L-type Ca 2ϩ channels. EXPERIMENTAL PROCEDURES Cells and Cell L...

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS-e.g., PS exposure in the outer leaflet ofthe cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells-serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival ofpathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7-to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.Macrophages play an important role as effector cells in host defense against cancer metastasis (1) and viral diseases (2). When appropriately activated, macrophages are able to recognize and destroy a variety of tumorigenic and virusinfected cells, including cells resistant to other host defenses such as T cells and natural killer cells (1), while leaving normal cells unharmed. Macrophage-mediated tumor cell killing has been shown to be independent of such cell characteristics as surface receptors, drug resistance, cell cycle, and metastatic potential (1,3).The mechanism responsible for the ability of mononuclear phagocytes to discriminate between normal and pathologic cells is not known. The broad spectrum of target cells susceptible to macrophage-mediated cytolysis might suggest, however, that a uniform surface moiety could be involved in target cell recognition.An interesting feature of some cell membranes is the asymmetric distribution of membrane phospholipids between the two leaflets of the bilayer (4). In red blood cells (RBC), for example, most membrane phospholipids show some preference for either leaflet, whereas phosphatidylserine (PS) is the only phospholipid that adopts a totally asymmetric distribution, being localized exclusively in the cell's inner leaflet (5-7). Although the mechanisms responsible for maintaining an asymmetric distribution of PS are still unclear, recent evidence suggests that the preservation of PS in the cell's inner leaflet is of central importance in cellular physiology. For example, the exposure of PS that occurs in activated platelets (8) and in sickled RBC (9, 10) regulates hemostasis by serving as a potent procoagulant surface (11, 12) and as a signal for triggering the recognition of these cells by macrophages (13). Related experiments have shown that artificially generated phospholipid vesicles (14,15) a...

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