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Review R795 Apoptotic cell removal Peter M. Henson, Donna L. Bratton and Valerie A. Fadok Ingestion by professional or amateur phagocytes is the fate of most cells that undergo apoptosis. Studies in both Caenorhabditis elegans and mammals are now converging to reveal some of the key mechanisms and consequences of this removal process. At least seven corpse removal genes in nematodes have mammalian equivalents, and represent elements of signaling pathways involved in uptake. In mammals, a wide variety of apoptotic cell recognition receptors has been implicated and appears to be divided into two categories, involved in tethering the apoptotic cell or triggering an uptake mechanism related to macropinocytosis. Apoptotic cell removal is normally efficient and non-inflammatory. By contrast, the process may become subverted by parasites to yield a more favorable growth environment, or in other cases lead to fibrosis. Removal may also clinch the apoptotic process itself in cells not yet completely committed to death. Address: Program in Cell Biology and Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA. Correspondence: Peter M. Henson E-mail:[email protected] Introduction Cell recognition, engulfment and digestion are the final stages in the process of apoptosis. The outcome of these actions is the efficient deletion of unwanted or damaged cells, often considered a counterpoint to mitosis. That apoptosis is accompanied by rapid removal of the affected cells in situ has been recognized since the phenomenon was first characterized [1,2]. As discussed recently by Jack Cohen [3], a newer view of biology suggests significant overproduction, at the level of the organism, sperm, egg and embryo, of individual cells during development. In each case, excess material is removed and thus removal is an important element of overall control. Even in the adult organism, significant cell turnover seems to be the rule rather than the exception, including systems such as the brain that were long thought not to exhibit such plasticity. Turnover of cells in metazoa means apoptosis and, critically, apoptotic cell removal — often called corpse removal. However the mechanisms of apoptotic cell recognition and removal are only now becoming apparent, an understanding that represents a fascinating investigative interplay between genetic approaches in the nematode, Caenorhabditis elegans, and analyses of uptake mechanisms in mammalian systems (Figure 1). Current Biology 2001, 11:R795–R805 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. Combining studies in mammals and the worm Seven genes have recently been identified in C. elegans that appear to play a role in apoptotic corpse removal during larval development or in corpse digestion after engulfment [4–8]. One of the many strengths of the C. elegans system goes back to the original cell mapping studies that led to the identification of 131 specific somatic cells, as well as some 300 germ cells, that are destined for deletion (see [4,7]). Until recently, the genes involved in removal each appeared to correspond to steps in the signal pathways leading to engulfment, rather than to cell recognition steps per se. In contrast, studies in mammalian systems generated a host of potential recognition receptors (see [9–13]) but made little progress towards identifying uptake signaling molecules. Recent reports provide a crucial synthesis between these two approaches and highlight a number of important points, not the least of which is the broad commonality in mechanisms employed for apoptotic cell removal. It seems reasonable to suggest that potential redundancy among the recognition elements would make their identification by the genetic screening approaches used for C. elegans less likely. Such redundancy is certainly implicated in the studies of mammalian systems in vitro. On the other hand, the involvement of limited, and presumptively unique, signaling pathways for uptake could readily explain the R796 Current Biology Vol 11 No 19 Figure 1 A comparison of elements implicated in apoptotic cell uptake in (a) mammals and (b) C. elegans. The electron micrograph of uptake in the nematode (b) is taken with permission from [7]. 1. Surface changes on the apoptotic cells Mammal PS Oxidized phospholipid ABC-1 Altered carbohydrates ICAM3 Nematode PS? ced-7 2/3. Tethering and/or stimulation receptors (a) Macrophage Corpse PSR PSR? SR-A CD36/SR-B CD36? SREC? ced-1 CD68 αvβ3/αvβ5 integrins β1 integrins CD14 CR3 β2GP receptor Lectin(s) Collectin receptors (calreticulin/CD91) 4. Signaling molecules CrkII DOCK180 rac-1 ced-6 ABC-1 ELMO (b) Sheath cell Corpse ced-2 ced-5 ced-10 ced-6 ced-7 ced-12 5. Digestion Acid DNAase? nuc-1 Current Biology identification of genes within such pathways in the nematodes. These mechanisms for cell removal may also extend to necrotic cells and cell debris, even if the consequences of such clearance may sometimes be different [5,14,15]. An example of this synergism is the recent, elegant paper by Zhou et al. [7]. In this work, the last but one of the seven C. elegans genes to be characterized, ced-1, does seem to encode a potential recognition and signaling molecule that may initiate cell uptake. ced-1 was originally identified in the genetic screens for persistent cell corpses and encodes a protein that plays a critical role in engulfment. The protein is expressed in the membranes of phagocytosing cells and clusters around corpses during removal. Deletion of potential signaling domains within the intracellular portion of the molecule leads to defective apoptotic cell clearance, even in the face of unaltered clustering. These data suggest that CED-1 both binds to the target apoptotic cell and initiates signaling for uptake. While the specific ligand that might be recognized by CED-1 remains unknown, clustering, which is taken to be equivalent to ligand engagement, appears to be associated with the presence of the product of ced-7. This latter gene encodes a protein similar to ABC-1 in mammals [16], a member of the ATP binding cassette transporter family [17]. Defects in ABC-1 in man are associated with Tangier disease [18–20] and the molecule appears to be involved in cholesterol or phospholipid transport. Zhou et al. [7] suggest the possibility that CED-7 changes phospholipid distribution on the apoptotic cell (see below) in C. elegans and that this is what CED-1 recognizes. It seems reasonable to consider the apoptotic cell removal process to comprise five steps, namely: surface alterations and ligand expression on the apoptotic cell; recognition of these ligands by tethering receptors on the phagocyte; initiation of the appropriate signaling pathways in the phagocyte, which might be mediated by the tethering interaction but may also involve separate or additional uptake receptors; the process of engulfment; and finally, phagosome–endosome interactions leading to digestion. It is also important to emphasize that uptake is not limited to professional phagocytes, such as macrophages or immature dendritic cells in mammals, but may be performed by most cell types. An example of this is seen in the normal developmental clearance of cells from the interdigital webs in mice wherein removal occurs even in animals that do not contain macrophages — PU-1–/– mice — but at a slower rate [21]. Surface changes on apoptotic cells leading to recognition and removal Despite all the effort spent characterizing the mechanisms of apoptosis, comparatively little has been focused on surface changes on the apoptotic cell. A very clearly defined change is the exposure of phosphatidylserine on the apoptotic cell surface, shown first on lymphocytes [22] and now known to occur with essentially all apoptotic cells in mammals. Phosphatidylserine also appears to be involved in apoptotic cell removal in amphibians [23] and is seen Review during apoptosis in Drosophila [24]. Whether apoptotic cells in C. elegans express phosphatidylserine on their surface has still to be shown definitively. While the mechanism of phosphatidylserine exposure is still poorly understood at the molecular level, it appears to require activation of a phospholipid ‘scramblase’ that enhances bidirectional phospholipid flip-flop across the membrane bilayer, and inactivation of an aminophospholipid translocase that would normally return the phosphatidylserine to the inner membrane leaflet. Effects on both these systems can be seen during normal cell function but are reversible; the key difference in apoptosis may be the permanence of the effect. Candidate regulatory genes for these activities have been identified [25–28] although their exact role, identity or mode of action is by no means clear. A search of the C. elegans gene database reveals a molecule with considerable homology to the mammalian scramblase. And a putative aminophospholipid translocase — a member of the P-type ATPase family — is widely distributed, from plants to man [25,29–31]. Although the validity of this candidate has been challenged, more recent data in Arabidopsis support a role for these P-type ATPases as aminophospholipid translocases [29]. Another class of surface changes that has been implicated in apoptotic cell removal is alteration of membrane carbohydrates. The original observation of apoptotic cell recognition by macrophages suggested a lectin-like activity [32,33]. Alterations to surface charge on apoptotic cells were also attributed to carbohydrate moieties [32,34] although specific sugars on the apoptotic cell surface were not identified in either of these studies. On the other hand there is some evidence for increased expression of mannose on apoptotic cells leading to their removal within the liver [35,36]; this is an area that needs much more detailed investigation. A number of mechanisms for such changes to membrane carbohydrates can be envisioned. New or altered glycosylated molecules may be supplied to the cell-surface from internal membranous structures, possibly by direct fusion of elements from the endoplasmic reticulum with the plasma membrane. In addition, the normal turnover of membrane glycoproteins is likely to be significantly altered and decreased in apoptosis leading to gradual depletion of key molecules or their normal glycosylation. Any enzymatic alteration of surface oligosaccharides would require either the presence of enzymes in or on the membrane surface — so-called ectoenzymes — or in the extracellular medium. This issue represents a potential problem in all studies of apoptosis carried out in vitro, as some necrosis and cytolysis is bound to occur and could contribute intracellular enzymes to the system that might not be present during the efficient removal usually seen in vivo. R797 A completely different possibility is that appropriate glycoproteins become laterally redistributed or clustered in the membrane of apoptotic cells. This could be a critical determinant of recognition. For example, members of the family of defense collagens [37] that includes the C-type lectins known as Collectins as well as the first component of the classical complement pathway, C1q, have been shown to bind to highly localized sites on apoptotic cells [38–40]. Mannose binding lectin is one of these proteins and not surprisingly its attachment to apoptotic cells is blocked by high concentrations of free mannose. It is noteworthy that preliminary evidence shows that phosphatidylserine expression and membrane phospholipid redistribution can often be patchy (our unpublished observations) and highly localized. The relationship between phospholipid redistribution and other putative membrane changes to proteins or their carbohydrate substituents is poorly described. What is also not clear is the linkage between the two broad signaling pathways leading to nuclear apoptotic changes — those that involve mitochondria and those that do not — and surface changes. How the aminophospholipid scramblase is inactivated is not known. As far as phospholipid redistribution is concerned, cleavage of PKCδ by caspase 3 induces its unregulated activation, which, we suggest, leads in turn to a more persistent induction of the phospholipid scramblase [41]. What is not clear is how the actual phospholipid scrambling effect is mediated, which is where questions about the involvement of the ABC transporters might come in. These are known to transport phospholipids across cell membranes and it is certainly possible that ABC1 serves such a function in the apoptotic cell. However this suggestion, which has been raised by a number of groups (see [42,43]) is at present merely an interesting speculation. If ABC transporters are involved in phospholipid scrambling, the generality of phosphatidylserine expression during apoptosis dictates the participation of more than one member of this family as ABC1 is not generally distributed among different cell types. The phospholipid scramblase appears to require calcium and phosphorylation [41,44] for its activity, self aggregates and is known to scramble phospholipids in artificial membrane systems [45], albeit with low efficiency. It might therefore alter the membrane structure by itself to mediate transbilayer movement. However, although potentially present, it was not identified in the genetic screen for abnormal corpse removal in C. elegans. Another element that must be put into the equation is the suggestion that, in both mammals and the nematode, phosphatidylserine is expressed on both the apoptotic cell and the phagocyte during removal [7,43,46]. At this point, therefore, the role of ABC1 is difficult to unravel. In the mouse it seems to play a role in the phagocyte as R798 Current Biology Vol 11 No 19 macrophages from knockout mice show defects in apoptotic cell uptake [43]. The problem here is that most cell types have the capacity to recognize and ingest apoptotic cells — presumably a very primitive function — once again in the face of a restricted distribution of ABC1. would require a receptor on the phagocyte and an important recent paper [57] shows that Mer, a member of the Axl, Mer, Tyro3 family of receptor tyrosine kinases, may play such a role. Critically, mer–/– mice show a marked defect in removal of cells in the thymus. Recognition of apoptotic cells Whether the putative receptor, Mer, acts via Gas-6 and exposed phosphatidylserine in vivo is not yet known — it could also act along with some of the other recognition molecules as a signaling agent. It is important to note in this section that while many receptors have been suggested to participate in recognition of apoptotic cells in vitro and even in reconstituted transfected cells, far fewer candidates have been shown to play this role in vivo. In part this is likely to reflect the significant redundancy, and by implication, importance, of the removal processes. Two possible issues of importance may be gleaned from the above observations regarding surface changes on apoptotic cells. First, the classes of molecules that are altered, carbohydrates and phospholipids, are recognized in vivo by so-called pattern recognition molecules, which have recently been associated with the innate immune system. Secondly, aggregation of glycoproteins and phospholipids in the membrane of the apoptotic cell further fits this concept in that many of these recognition processes are thought to be low affinity, high avidity systems whose effectiveness is markedly enhanced by local aggregation. In mammals, phosphatidylserine expression on the apoptotic cell membrane appears to be a critical element for recognition or uptake, despite the abundance of potential recognition receptors [11–13,47,48]. In the absence of phosphatidylserine expression, uptake is minimal and deliberate replenishment of outer leaflet phosphatidylserine restores uptake [48]. The selectivity and stereospecificity of this restoration system strongly implicates the newly cloned phosphatidylserine receptor [12] in such uptake. A simple model suggests a variety of tethering ligands with a critical involvement of the phosphatidylserine receptor for initiation of the engulfment process. While a C. elegans homologue for the phosphatidylserine receptor was identified from the gene database [12], it too never appeared in the genetic screen for corpse removal. To this point, its possible role in apoptotic cell removal in nematodes is unclear (although it has been implicated in amphibians [23]). Careful examination of the C. elegans phosphatidylserine receptor candidate gene does reveal a deletion in a portion of the putative intracellular domain that might contain a tyrosine phosphorylation site. Whether this suggests that in the nematode the receptor serves more a tethering than signaling function remains to be determined. In mammals, the receptor also appears to play a key role in suppressing the inflammatory response during apoptotic cell removal [12,48,49]. The phosphatidylserine receptor in the phagocyte membrane would be expected to interact directly with phosphatidylserine on the apoptotic cell. Other phosphatidylserine-binding membrane proteins include the scavenger receptors (see below and [50–54]). Gas-6 is a soluble protein that can recognize and bind phosphatidylserine on apoptotic cells [55,56] and could act as a bridging molecule. Its ability to participate in uptake As noted above, the first study of apoptotic cell recognition suggested the involvement of a lectin [33]. More recently, the complement protein C1q was reported to bind to blebs on apoptotic keratinocytes [38,39] and to localized sites on other apoptotic cell types [40]. The same was seen for mannose binding lectin and surfactant proteins A and D (Vandivier W and Ogden CA, unpublished observations). Moreover, each of these molecules has been observed to enhance uptake of apoptotic cells into macrophages in vitro [40,58] and, for C1q [59] and surfactant protein D, in vivo as well (Vandivier W and Ogden CA, unpublished observations). These molecules appear to bind apoptotic cells through their globular head groups — the pattern recognition regions — and signal for uptake through their conserved collagen-like tails. Because of the potential involvement of mannose on the apoptotic cell, the mannose receptor may be an additional candidate, but there is as yet no evidence to support its involvement in mediating apoptotic cell recognition. On the other hand, scavenger receptors in the A and B group have been suggested to recognize apoptotic cells, particularly SR-A [9,60] and CD36 [61–65] or SR-B1 [51–53,66]. In Drosophila, a member of the SR-B family, Croquemort, has been shown to participate in corpse removal [67,68]. The ligands involved on the apoptotic cells are not known. Closely associated with CD36 activity are the αv integrins: αvβ3 on macrophages [65] and αvβ5 on dendritic cells [63] and retinal pigment epithelial cells [69]. Bridging by thrombospondin has been suggested in some of these CD36-mediated systems although, once again, the apoptotic cell ligands are unknown. As CD36 can directly bind anionic phospholipids [50,54] the potential interactions in this system are likely to be complex. ced-1, like the other six genes involved in C. elegans apoptotic cell removal, is also similar to a mammalian molecule, in this case, the scavenger receptor from endothelial cells, Review SREC [70]. SREC binds and internalizes acetylated lowdensity lipoproteins. However, it is not widely distributed among mammalian cell types and so cannot be solely responsible for apoptotic cell recognition. Nor does it exhibit the specificity for phosphatidylserine recognition, in contrast to the phosphatidylserine receptor, that characterizes this phospholipid’s involvement in apoptotic cell clearance. SREC has yet to be shown capable of mediating apoptotic cell uptake, even in endothelial cells. Intriguingly, although their sequences appear unrelated, SREC and the phosphatidylserine receptor are both found on the tip of human chromosome 17; the biological significance of this association is unknown. CD14 represents an interesting candidate for apoptotic cell recognition. This soluble or glycophosphoinositidelinked protein is involved in the appropriate interaction of lipopolysaccharides with Toll-like receptor 4. It has been implicated in recognition of apoptotic cells, particularly apoptotic lymphocytes. Most of the recognition processes do not seem to show much specificity for apoptotic cell type so this selectivity may either be a special case or may merely represent lack of detailed investigation in other systems. The counter-ligand on the apoptotic cell has been suggested to be ICAM3 [71] which is upregulated or exposed on apoptotic lymphocytes through an unknown mechanism. This again brings in something new, namely a protein ligand. While it seems likely that CD14 represents a tethering rather than signaling ligand as it does not exhibit a transmembrane domain, it may prove important in immune system development and function. These observations also raise the possibility that there will be other cell specific protein–protein interactions involved in apoptotic cell removal. Finally, one cannot ignore the possibility that apoptotic cells in mammals might be rendered more susceptible to phagocytosis in vivo by more conventional antibody–antigen interactions, the process of opsonization. For example, a number of studies have suggested that apoptotic cells initiate attachment of complement C3bi followed by deposition of IgG, and subsequent uptake through complement receptor 3, CR3 [72–74]. Once again, however, the structures on the apoptotic cells that might initiate this opsonization are not fully defined. R799 engulfment. CED-12 has recently been shown to participate in the CED-2, CED-5, CED-10 pathway in C. elegans. Two mammalian homologues of CED-12, ELMO1 and 2, seem to form a trimolecular complex with CrkII and DOCK180 that results in activation of Rac-1 (Ravichandran K, personal communication). However, mutation of any one of these four genes in C. elegans diminishes, but does not eliminate, corpse removal. CED-1, CED-6 and CED-7 seem to participate in an alternative path for uptake: double mutants of any of these three genes with one of the former group results in much more profound abnormalities in apoptotic cell clearance than the single mutation. CED-1 has already been discussed above and may represent a C. elegans recognition or signaling receptor. CED-7 is an ABC transporter that may be involved in both apoptotic cell and phagocyte. As discussed above, its activity, while very poorly understood, is most likely to be involved in membrane alterations. CED6 resembles an adaptor protein with a phosphotyrosine binding domain, and its mammalian homolog is implicated in phagocytes during apoptotic cell engulfment [77,78]. How CED-6 interacts with putative surface receptors or downstream effector molecules is not yet clear. In contrast, evidence for two separate uptake pathways in mammals is lacking. Possibly one or more of the different recognition molecules could initiate uptake by using different signal pathways but, at least to us, this seems unlikely. It has been suggested that uptake into professional phagocytes such as macrophages could be different from ingestion by tissue cells [81]. However, the effect is largely one of ingestion rate and efficiency and in our hands does not show differences in receptor usage or uptake mechanism. Even in C. elegans it is not clear how the proposed pathways are related. Uptake signaling pathways As actin dependent processes are presumably involved, Rho family GTPases are expected in both pathways. Rac-1 is the key candidate in the CED-2, 5, 10 cell uptake path and studies in murine macrophages suggest the participation of Rac-1 and Cdc42 [80]. A key problem with many of these investigations is the complexity of trying to sort out signaling processes with multiple, or even unknown, recognition molecules and receptors, and initial signal transduction molecules. The ability to address the processes initiated by an individual receptor would be of great value. Gene complementation studies in C. elegans suggest two related pathways for internalization of the apoptotic cells by phagocytes [7,75,76]. CED-2, CED-5 and CED-10 represent homologs of mammalian CrkII, DOCK180 and Rac1 and constitute one such pathway. Studies in mammalian systems also show these molecules are involved in apoptotic cell removal (see[43,77–80]). They are presumed to act at the point of cytoskeletal reorganization during It is clear that a critical issue in understanding apoptotic cell removal is whether the uptake processes and signaling pathways are unique. They are clearly different from Fc receptor driven phagocytosis. Stimulated macropinocytosis driven by growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and macrophage colony-stimulating factor (M-CSF) signaling through R800 Current Biology Vol 11 No 19 Figure 2 Phagocytosis of cells by the zipper mechanism or by stimulated macropinocytosis. It is suggested that most forms of apoptotic cell ingestion result from the macropinocytotic process. Zipper mechansim Stimulated macropinocytosis Membrane ruffles Current Biology their receptor tyrosine kinases may be a closer model. However, very preliminary evidence obtained in collaboration with Dr Kodimangalam Ravichandran suggests to us that upstream signaling elements resulting from growth factor stimulation may be different from those seen in apoptotic cell uptake. On the other hand, downstream molecules such as Rac-1 are common. The nematode studies seem to support this implication. From the point that Rho family GTPases become involved, the signaling for cytoskeletal reorganisation, ruffling, membrane extension and eventual fusion, along with other elements of internalization, may not be specific for apoptotic cells or their recognition receptors. However, key issues are the relationship between Cdc42 and Rac; how and when Rac-1 becomes localized to the site of uptake; whether the interaction of an apoptotic cell initiates activation and membrane alterations at only the local site of contact and stimulation; and any potentially unique processes involved in subsequent phagosome disposition, pH changes, lysosome and vesicle fusion. Because of the overall importance of the apoptotic cell removal process and its consequences, these are critical questions for future investigation. Mechanisms of uptake Ingestion of particles into cells occurs by a number of different mechanisms reflecting different receptors, signaling pathways and physical uptake processes. Immunoglobulin Fc receptor stimulation induces phagocytosis by a process that involves the sequential ligation of the receptors by Ig molecules on the particle as in a zipper [82–85]; this results in a phagosome with membranes in close contact with the ingested particle. Uptake through CR3–αmβ2 integrin involves the particle appearing to sink into the phagocytic cell, without the development of an obvious phagocytic cup. We have recently suggested that uptake of apoptotic cells through the types of receptors discussed above occurs by a process more akin to macropinocytosis (Figure 2). In stimulated macropinocytosis, induced, localized membrane ruffling is associated with enclosure of extracellular fluid followed by internalization within a spacious phagosome. Particles attached to the surface are taken in at the same time. In this system the receptors for macropinocytosis initiation could be on either the particle or the apoptotic cell, or could interact independently if the apoptotic cell were already tethered to the phagocyte membrane. We have called this a ‘tether and tickle’ mechanism ([86], Hoffmann P and de Cathelineau A, unpublished observations) and thereby imply a two-step uptake process with a requirement for apoptotic cell attachment and signaling that, in many cases, may be mediated by different receptors (Figure 3). The tether and tickle mechanism resembles that accompanying uptake of pathogenic Salmonella into epithelial cells. Here the virulence factor SopE [87,88] is injected into the cell by the bacterial Type III secretion system where it activates members of the Rho family of GTPases and causes membrane ruffling and macropinocytosis [89,90]. Salmonella lacking the virulence factor may still attach to the cell surface but are not ingested unless ruffling and Review macropinocytosis are induced by alternative stimuli, such as growth factors [91]. Ligation of some apoptotic cell recognition receptors such as CD36 or αv integrins induces tethering of cells to macrophages but without uptake. Inclusion of phosphatidylserine on the target cell surface in this circumstance initiates uptake. By contrast, phosphatidylserine on the target cell membrane without the tethering ligand is ineffective, we would suggest, because the affinity/avidity is too low to initiate the process. Independent stimulation of the phosphatidylserine receptor in the presence of previously tethered cells also initiates their uptake — a bystander mechanism akin to that outlined above for Salmonella. This dual process may provide significant checks and balances in the removal process, minimizing for example, uptake of cells that may transiently express phosphatidylserine during activation or of cells that may become transiently attached to potential phagocytes in the absence of the signaling receptor ligation, the tickle part of the process. Uptake of apoptotic cells by the tether and tickle mechanism would initially involve spacious phagosomes and the concurrent ingestion of extracellular fluid. This was found to be the case ([40], Hoffmann et al. unpublished observations), and was in marked contrast with uptake of cells, even apoptotic cells, via the Fc receptor after opsonization; here there is no evidence for fluid uptake into the phagosome. It should be noted that while macropinocytosis initially exhibits a spacious phagosome, this may soon be followed by condensation of the phagosome to yield more close apposition of the membranes to the ingested particles [89]. Although not studied to any degree, one might expect differences in subsequent disposition of the phagosomes, in digestion of the apoptotic cells and final processing of the contents. The significant body of literature showing macropinocytosis to be a key uptake mechanism in antigen processing and presentation supports this, as does the genetic evidence for specific genes involved in corpse digestion in C. elegans [75,92]. Points of regulation One aspect of regulation in the removal of apoptotic corpses seen in mammalian systems is the stimulation of macrophages to exhibit enhanced uptake. It is intriguing to note that in most investigators’ hands, only a proportion of macrophages, primary isolates or even cell lines, actually engulf apoptotic cells in vitro. Whether this reflects subtypes of cells, varying abilities to undergo the putative macropinocytotic process, cell–cell contact issues or heterogeneity in receptor expression, remains to be determined. Resident macrophages from lung or peritoneum are markedly inefficient at apoptotic cell clearance as are circulating monocytes. However, macrophages that have R801 Figure 3 Apoptotic cell Signaling ligands Signaling receptors Phagocyte Tethering ligands Tethering receptors Signaling for membrane ruffling and macropinocytosis Current Biology A proposed two component uptake mechanism for apoptotic cells involving a combination of tethering and signaling receptors — a ‘tether and tickle’ process. emigrated to, and matured within inflammatory sites are very effective [93,94]. As monocytes mature into macrophages in vitro they too develop an increased ability to recognize and ingest apoptotic cells. Part of this maturation may reflect stimulated increase in expression of the phosphatidylserine receptor [95,96]. It may also suggest levels of regulation that are only beginning to be explored. For example, stimulation of macrophage CD44 [97] and exposure to corticosteroids [98] or to lipoxins [99] have each been shown to enhance the ability of macrophages to recognize and engulf apoptotic cells. While the mechanisms for enhancement have not been clarified, these processes do seem to be selective for apoptotic cells. Maturation of monocytes into immature dendritic cells also increases their ability to ingest apoptotic cells. Immature dendritic cells show increased constitutive macropinocytosis so it may be that uptake in these cells is regulated more by the nature of the tethering interaction than specific stimulation of macropinocytotic signaling. Regulation of uptake efficiency is expected in resident tissue cells as well but has not yet been investigated to any significant extent. It would also seem likely that other combinations of stimuli and environmental factors yield circumstances that block or delay apoptotic cell recognition and uptake. For example, defective clearance of apoptotic inflammatory cells in cystic fibrosis has recently been attributed in part to cleavage of the recognition receptors by elastase (Vandivier et al., unpublished observations). R802 Current Biology Vol 11 No 19 Another area of increased interest is the likelihood that cells, including apoptotic cells, may under certain circumstances express ‘don’t eat me’ signals. The demonstration that CD47 expression by erythrocytes blocks their phagocytosis, through an interaction with the signal regulatory protein SIRPα [100,101], lends credence to such a concept. Whether this system, or others, operates in the context of apoptotic cell uptake remains to be firmly established. Consequences of recognition and removal Apoptotic cell clearance is efficient and non-inflammatory and may often be mediated in vivo by neighboring cells. Great efforts are currently being expended on understanding the sequelae of apoptotic cell deletion, largely because of its active anti-inflammatory and suggested anti-immunogenic effects. Such effects have been discussed in some detail (see [9–11,79,102–104]). Following initial observations that uptake does not induce the release of inflammatory mediators, it soon became apparent that inflammation is actively suppressed, both in vitro and in vivo, through the production of inhibitory factors such as transforming growth factor (TGF) β, prostaglandin E2 (PGE2) and interleukin-10 (IL-10). We, and others, have implicated the phosphatidylserine receptor and phosphatidylserine expression on the apoptotic cells as a major player in this suppression and have recently suggested that its effect may be extended to explain the inhibition of dendritic cell maturation and antigen presentation in the adaptive immune response [79,105]. This is in keeping with the suggested distinction between the interaction with dendritic cells of apoptotic cells — which inhibits maturation and antigen presentation — and necrotic cells [14,15,102,106–108]. It is worth noting in this regard that induced apoptosis and cell deletion in normal tissues often leads to replenishment and repair, in contrast to the removal of excess cells seen during development. It seems likely to us that growth and differentiation factors other than TGFβ may be produced in response to apoptotic cell recognition that could contribute to this return to normal structure and function. The obverse of this overall beneficial and essentially normal process are the circumstances where suppression of inflammation or immunity is essentially harmful. A clear example of this was shown in tissue responses to Trypanosoma cruzi, the cause of Chagas disease. Here, apoptotic cells enhance the survival of the parasites in macrophages through their induction of TGFβ and PGE2 [109]. Similarly there is increasing evidence that apoptosis in tumor cells might also have an overall negative effect on the host as a consequence of local suppression of inflammation and immunity. TGFβ itself has long been associated with the development of fibrotic reactions in tissues — a process that may lead to an important restoration of structural integrity in a skin wound, but long-term impairment in function in the lung or liver. These types of responses emphasize the need for balance in the response to apoptotic cell removal in normal cell turnover. In situations where cells are produced to excess, for example myofibroblasts in fibrotic wound healing, even here the processes of apoptosis and cell clearance appear critical to their resolution. A number of studies have addressed the ability of macrophages not only to engulf apoptotic cells but also to induce apoptosis in appropriate targets, especially in the context of tissue reorganization during development or after inflammatory or fibrotic changes [110–112]. Although not explicitly investigated in most of these experimental systems, a coordinated induction of apoptosis and cell removal seems likely to occur. Potential mechanisms include the production of oxidants, nitric oxide (NO), tumor necrosis factor (TNF) α or Fas ligand (FasL), and attachment of the macrophage to the target cell could readily enhance both apoptosis and subsequent removal. The genetic approach already discussed in C. elegans has recently made an intriguing contribution to our understanding of the combined apoptosis and engulfment process [113,114]. Apoptosis in worms is blocked in the absence of CED-3 or CED-4. However, in partial ced-3 mutants, showing only reduced numbers of apoptotic cells, the combined mutation of engulfment genes blocks apoptosis. The implication is that engulfment can contribute to the induction of cell death initiated by less than optimal activation of caspases. Once again the mechanisms are not yet defined although Green and Beere [115] have suggested a possible early discharge of lysosomal hydrolases akin to processes described many years ago in mammalian phagocytes [116–118] and colloquially called ‘frustrated phagocytosis’ or ‘regurgitation while feeding’. The two C. elegans studies also suggest that the terminal, caspase-dependent ‘execution’ step in apoptosis may not be as irreversible as is often implied — a concept that is perhaps more readily understood in the apparently more complex regulation processes seen during apoptosis in mammalian cells [119]. The take-home message for our understanding of apoptotic cell clearance is that the complexities of apoptotic cell engulfment are beginning to succumb to investigation, are likely to be unique and primitive, and appear to exhibit remarkable similarities across disparate members of the animal kingdom. References 1. 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