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.     

Chemokines act as phosphatidylserine-bound “find-me” signals in apoptotic cell clearance

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed “find-me” signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other “eat-me” signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein–coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.

Chemokines attract leukocytes by activating chemokine receptors, but many also bind anionic phospholipids. This study shows that phosphatidylserine-binding chemokines endow extracellular apoptotic bodies with “find-me” signals that trigger phagocyte migration for potential apoptotic cell clearance.     

Phosphatidylserine recognition by phagocytes: a view to a kill

The redistribution of phosphatidylserine (PS) to the external surface of the plasma membrane is a key element of apoptotic cell recognition and is a molecular cue that dying cells should be engulfed. Phagocytes interact with PS on apoptotic cells through either the PS receptor or secreted bridging proteins called opsonins. The study of two secreted PS opsonins, MFG-E8 and Gas6 and their receptors alphavbeta5 (and alphavbeta3) integrin and Mer tyrosine kinase, respectively, have provided insights into the temporal and spatial aspects of Rac1 activation following the recognition and internalization of apoptotic cells. Disruption of PS opsonins and their signaling pathways often manifest conditions of inflammation and autoimmune disease. Here, we review recent studies involving PS opsonins, their receptors and their role in the phagocytosis of apoptotic cells.     

Epidermal growth factor-like domain repeat of stabilin-2 recognizes phosphatidylserine during cell corpse clearance

Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor β. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.     

Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.     

Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.     

Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor

Rapid phagocytic clearance of apoptotic cells is crucial for the prevention of both inflammation and autoimmune responses. Phosphatidylserine (PS) at the external surface of the plasma membrane has been proposed to function as a general 'eat me' signal for apoptotic cells. Although several soluble bridging molecules have been suggested for the recognition of PS, the PS-specific membrane receptor that binds directly to the exposed PS and provides a tickling signal has yet to be definitively identified. In this study, we provide evidence that stabilin-2 is a novel PS receptor, which performs a key function in the rapid clearance of cell corpses. It recognizes PS on aged red blood cells and apoptotic cells, and mediates their engulfment. The downregulation of stabilin-2 expression in macrophages significantly inhibits phagocytosis, and anti-stabilin-2 monoclonal antibody provokes the release of the anti-inflammatory cytokine, transforming growth factor-beta. Furthermore, the results of time-lapse video analyses indicate that stabilin-2 performs a crucial function in the rapid clearance of aged and apoptotic cells. These data indicate that stabilin-2 is the first of the membrane PS receptors to provide tethering and tickling signals, and may also be involved in the resolution of inflammation and the prevention of autoimmunity.     

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.     

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 exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression

Phosphatidylserine (PS) exposure on the surface of cells has been considered a characteristic feature of apoptosis. However, we demonstrate herein that externalization of PS occurs in a cell-type-specific, albeit caspase-dependent, manner. Moreover, we could find no correlation in six different cell lines between the level of expression of the phospholipid (PL) scramblase and the capacity of these cells to externalize PS during apoptosis. Overexpression of PL scramblase in Raji cells, which exhibit low constitutive expression of this enzyme, by retroviral transduction of PL scramblase or treatment of the cells with interferon-alpha, failed to confer the capacity to expose PS in response to apoptotic stimuli. However, the lack of PS exposure in some cell types was not due to their inability to translocate PS molecules to the cell surface, since incubation with thiol reactive agents, such as N-ethylmaleimide, disulfiram and diamide, yielded rapid and pronounced PS exposure in all cell lines. These data suggest that plasma membrane PS exposure is not an obligatory component of the apoptotic phenotype, and that PL scramblase is not the sole determinant of PS externalization in apoptotic cells when this occurs.     

Exposure of Phosphatidylethanolamine on the Surface of Apoptotic Cells

In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells. The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells. The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells. The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a fluorescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V. This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.     

Surfactant protein A (SP-A) binds to phosphatidylserine and competes with annexin V binding on late apoptotic cells

The role of surfactant protein A (SP-A) in the recognition and clearance of apoptotic cells is well established, but to date, it is still not clear which surface molecules of apoptotic cells are involved in the process. Here we present evidence that phosphatidylserine (PS) is a relevant binding molecule for human SP-A. The binding is Ca²⁺-dependent and is not inhibited by mannose, suggesting that the sugar-binding site of the carbohydrate recognition domain (CRD) of SP-A is not involved. Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells, and this was consistent for Jurkat cells and neutrophils. Supporting these data, confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells. However, we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface, as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.     

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.     

Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine

The efficient phagocytosis of apoptotic cells by macrophages reduces the potential for an inflammatory response by ensuring that the dying cells are cleared before their intracellular contents are released. Early apoptotic cells are targeted for phagocytosis through the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. In this report, we show that the oxidant H(2)O(2) inhibits phagocytosis of apoptotic cells even though the cells express functional PS on their surface. Thus, B lymphoma cells induced to undergo apoptosis by the chemotherapy drug etoposide are efficiently phagocytosed by macrophages in a process that is mediated by PS (inhibitable by PS liposomes). Exposure of the apoptotic cells to H(2)O(2) inhibits phagocytosis even though the cells still express functional PS on their surface. In addition, Jurkat cells and thymocytes induced to undergo apoptosis by H(2)O(2) alone are poorly phagocytosed. Inhibition of phagocytosis by H(2)O(2) cannot be attributed to oxidative inactivation or redistribution of PS on the cell surface. The results indicate that PS externalization is necessary but is not sufficient to target apoptotic cells for phagocytosis. Another phagocytosis recognition factor must therefore exist to facilitate uptake of apoptotic cells, and this factor is sensitive to modification by H(2)O(2).     

Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes

When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.     

Tethering and tickling: a new role for the phosphatidylserine receptor

Several receptors are implicated in apoptotic cell (AC) uptake by phagocytic cells; however, their relative dominance in mammalian systems remains to be established. New studies shed light on the role of the phosphatidyl serine (PS) receptor (PSR). Ligation of PSR by PS on AC surfaces is considered essential for signaling uptake of ACs that are tethered to phagocytes via other receptors.     

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.     

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H₂O₂ was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA₂ was blocked by a selective inhibitor of Lp-PLA₂, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me'' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.     

TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of Pro‐Phagocytic Signals From Apoptotic to Viable Cells

The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. J. Cell. Physiol. 232: 2271–2279, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells.

One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.