ABCA1 and the engulfment of apoptotic cells

Programmed cell death is one of the major devices controlling cellular homeostasis. However, the generation of cell debris that follows the execution phase of apoptosis has to be backed up by their efficient removal by phagocyte. This highly dynamic process requires the concerted action of a number of surface molecules able to recognize early signals of membrane modifications on the apoptotic prey. Among those, the loss of phospholipid asymmetry and exposure of phosphatidylserine on the prey to be is determinant to engage phagocyte receptors and trigger the removal of corpses. A loss of membrane lipid asymmetry occurs also on the phagocyte determining its efficiency as an undertaker. Here we will discuss how, in our mind, the ATP binding cassette transporter, ABCA1, by its action on the arrangement of lipids at the phagocyte membrane, may actively promote their competence to engulf.     

The disposal of dying cells in living tissues

Cells continuously die and disappear from the midst of living tissues. However, some of their constituents survive. DNA is horizontally transferred to phagocytic cells, and apoptotic cell antigens shape the immune repertoire. When massive apoptosis occurs, which overwhelms tissue scavenger cells, or when the function of phagocytes abates, dying cells escape clearance in vivo. Remnant dying cells come to phagocytes disguised: factors capable to envelop their membranes pervade the entire organism, or are generated in given tissues. Some are constitutively present, while other are generated during early or late phases of the inflammatory response, possibly to face the further burden of the dead inflammatory cells. This camouflage influences the disposal of the corpses: decoying molecules either bridge the corpse to the phagocyte or hide it. Furthermore, factors associated to the plasma membrane of the apoptotic cell shape the signals the phagocyte releases in situ. Finally, molecules contained or released by the dying cell alter the apprehension by the phagocyte of its prey, influencing its immunogenicity.     

Metabolic connections during apoptotic cell engulfment

Billions of cells die via apoptosis every day and are swiftly removed. When a phagocyte engulfs an apoptotic cell, it essentially doubles its cellular contents, raising the question of how a phagocyte may manage the excess metabolic load. This Minireview discusses phagocyte cellular metabolism, the digestion of the ingested apoptotic cell, and the impact of these processes on engulfment.     

Beta-2-glycoprotein 1-dependent macrophage uptake of apoptotic cells. Binding to lipoprotein receptor-related protein receptor family members

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein beta-2-glycoprotein 1 (beta2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of beta2GP1-phosphatidylserine and beta2GP1-apoptotic cell complexes. Using recombinant beta2GP1, we also show that beta2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.     

Plasma membrane phospholipid asymmetry precedes DNA fragmentation in different apoptotic cell models

 Biochemical alterations occurring in many cell types during apoptosis include the loss of plasma membrane phospholipid asymmetry and nuclear DNA fragmentation. Annexin V staining detects phosphatidylserine translocation into the outer plasma membrane layer occurring during cell death, while the in situ tailing (IST or TUNEL) reaction labels the DNA strand breaks typical of apoptosis. To compare the time course of these processes we investigated methylprednisolone-induced apoptosis of rat thymocytes, topoisomerase inhibitor-induced apoptosis in the human histiocytic lymphoma cell line U937, and serum deprivation-induced apoptosis in the rat pheochromocytoma cell line, PC12. At all time points, FACS analysis and quantitative fluorescence light microscopy showed a higher proportion of annexin V-positive than IST-positive cells, with significantly different time courses in the apoptotic cell models investigated (Anova test). Results were confirmed by confocal microscopy. Our data indicate that the exposure of phosphatidylserine, a potential phagocyte recognition signal on the cell surface of apoptotic cells in vivo, precedes DNA strand breaks during apoptosis in different cell types. Accepted: 29 June 1998     

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.     

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation.     

Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.     

Phosphatidylserine, a death knell

Virtually every cell in the body restricts phosphatidylserine (PS) to the inner leaflet of the plasma membrane by energy-dependent transport from the outer to the inner leaflet of the bilayer. Apoptotic cells of all types rapidly randomize the asymmetric distribution, bringing PS to the surface where it serves as a signal for phagocytosis. A myriad of phagocyte receptors have been implicated in the recognition of apoptotic cells, among them a PS receptor, yet few ligands other than PS have been identified on the apoptotic cell surface. Since apoptosis and the associated exposure of PS on the cell surface is probably over 600 million years old, it is not surprising that evolution has appropriated aspects of this process for specialized purposes such as blood coagulation, membrane fusion and erythrocyte differentiation. Failure to efficiently remove apoptotic cells may contribute to inflammatory responses and autoimmune diseases resulting from chronic, inappropriate exposure of PS.     

The role of phosphatidylserine in recognition and removal of erythrocytes.

During the time that erythrocytes (RBC) spend in the circulation, a series of progressive events take place that lead to their removal and determine their apparent aging and limited survival. In addition, a fraction of RBC precursors will be removed during erythropoiesis by apoptotic processes, often described as "ineffective erythropoiesis". Both will determine the survival of erythroid cells and play an important role in red cell pathology, including hemoglobinopathies and red cell membrane disorders. The loss of phospholipid asymmetry, and the exposure of phosphatidylserine (PS) on the surface of plasma membranes may be a general trigger by which cells, including aging RBC and apoptotic cells, are removed. Oxidant stress and inactivation of the system that maintains phospholipid asymmetry play a central role in the events that will lead to PS exposure, death and removal.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Human adipose cells as candidates in defense and tissue remodeling phenomena

Macrophages are part of the immunity defense mechanism via oxidative burst and phagocytosis. They are also involved in tissue remodeling via cytokine secretion and apoptotic body clearance. Previously, we demonstrated that adipose cells and macrophages share some of their features and functions. Our aim was to further test this hypothesis in humans. We first demonstrated that human preadipocytes exhibit phagocytosis of yeast, this effect being specific compared to another fibroblastic cell type, the skin fibroblast. Furthermore, as in rodents, human preadipocytes exhibit anti-microbial activity. Finally, for the first time, it was shown that these cells were able to phagocyte apoptotic lymphocytes. Altogether, these data suggest an active involvement of fat cells in host defense and tissue remodeling, which might play an important role at the level of the whole organism due to the large amount of adipose tissue. This gives support for some observations linking obesity or cachexia to immunological disorders.     

The opsonin MFG-E8 is a ligand for the alphavbeta5 integrin and triggers DOCK180-dependent Rac1 activation for the phagocytosis of apoptotic cells

Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.     

Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death.

Signal-induced apoptosis is a normal phenomenon in which cells respond to changes in their environment through a cascade of intracellular biochemical changes culminating in cell death. However, it is not clear at what point in this process the cell becomes committed to die. An early biochemical change characteristic of cells undergoing apoptosis is the loss of plasma membrane asymmetry, such that high levels of phosphatidylserine become exposed on the outside cell surface. These cells can be recognized by staining with Annexin V, which binds to phosphatidylserine with high affinity. To investigate the mechanisms controlling signal-induced apoptosis we have examined the response of a B cell lymphoma to crosslinking of the membrane immunoglobulin (mIg) receptor. We have found that many of the cells that stain positive for Annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the signal is removed. These results indicate that Annexin V staining, and thus loss of membrane asymmetry, precedes commitment to apoptotic death in this system.     

Dangerous attraction: phagocyte recruitment and danger signals of apoptotic and necrotic cells

Tissue homeostasis in metazoa requires the rapid and efficient clearance of dying cells by professional or semi-professional phagocytes. Impairment of this finely regulated, fundamental process has been implicated in the development of autoimmune diseases, such as systemic lupus erythematosus. Various studies have provided us a detailed understanding of the interaction between dying cells and phagocytes as well as the current concept that apoptotic cell removal leads to a non- or anti-inflammatory response, whereas necrotic cell removal stimulates a pro-inflammatory reaction. In contrast, our knowledge about the soluble factors released from dying cells is rather limited, although meanwhile it is generally accepted that not only the dying cell itself but also the substances liberated during cell death contribute to the process of corpse clearance and the subsequent immune response. This review article is intended as an up-to-date survey over attraction and danger signals of apoptotic, primary and secondary necrotic cells, their function as chemoattractants in phagocyte recruitment, additional effects on the immune system, and the receptors, which are engaged in this scenario.     

Regulation of transbilayer plasma membrane phospholipid asymmetry

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.     

Clearance of apoptotic cells: getting rid of the corpses

The efficient elimination of apoptotic cells is crucial for tissue homeostasis in multicellular organisms. Secreted "find-me," exposed "eat-me," and lacking "don't-eat-me" signals comprise the central elements of apoptotic cell removal, thus preventing the release of intracellular contents into the surrounding tissue. This is of special importance, as there is growing evidence that the onset of autoimmune disorders can be linked to the inefficient removal of apoptotic cells. This review focuses on the signals displayed by apoptotic cells, the bridging and receptor molecules on the phagocyte, and is intended to present a simplified model of the phagocytic synapse. Additionally, the recent discovery of lysophosphatidylcholine functioning as soluble attraction signal is discussed in the general context of apoptotic cell clearance.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.     

Phospholipid scramblase: An update

The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.