Apoptosis: eating sensibly

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NATURE CELL BIOLOGY VOLUME 7 | NUMBER 12 | DECEMBER 2005 1161

this mechanism exists simply to downreg-ulate Notch activity or whether, similarly to mammalian β-arrestin, it also serves to mediate aspects of Notch signalling (Fig. 1b), requires futher investigation.

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Apoptosis: eating sensiblyChristopher D. Gregory and Simon B. Brown

Phagocytes may engulf both apoptotic and viable cells via calreticulin on the surface of the target cell, through its interaction with the phagocyte receptor, LRP. In reality, however, only apoptotic cells are engulfed, apparently because their surface ligand CD47 is prevented from activating the inhibitory phagocyte receptor SIRPα.

Over the past several years, a growing number of cell biologists have become fascinated with apoptosis, a form of programmed cell death, and it is now widely accepted that this physi-ological cell death is a fundamental feature of life in multicellular organisms. With the dawn-ing of the ‘age of apoptosis’, substantial progress has been made in understanding the molecu-lar cell biological mechanisms that underlie the initiation, execution and regulation of this cell death programme. However, less is known about the detailed mechanisms driving the fate of apoptotic cells, which, unless they are lost from tissue surfaces, are rapidly engulfed by phagocytes (either by neighbouring tissue cells or by macrophages, the professional scavengers of apoptotic cells)1,2. Phagocytosis of apoptotic cells protects the immediate neighbourhood from direct damage by degradative cellular components (for example, proteases3) leak-ing from dead cells. Furthermore, activation of phagocytes by apoptotic cells can suppress inflammatory responses and result in immu-nological tolerance1. Breakdown in certain clearance mechanisms that leads to persistence of apoptotic cells in situ may have pathologi-cal consequences, such as autoimmune reac-tions2. The importance of efficiently removing

apoptotic cells is emphasized by the evolution of molecular mechanisms to ensure effective recognition of apoptotic cells by phagocytes. Now, a study published by Gardai et al. in the journal Cell provides insights into one pathway that involves calreticulin and CD47 (ref. 4).

Interactions between apoptotic cells and phagocytes can be divided into five distinct phases: recognition, tethering, phagocyte signalling, engulfment and intracellular deg-radation (Fig. 1). Numerous glycoproteins and intercellular ‘bridging’ molecules on the phagocyte cell surface have been implicated in the various stages that lead to apoptotic cell engulfment — that is, assembly of a ‘phagocytic synapse’ (reviewed recently in ref. 2) — but a detailed knowledge of the underlying recep-tor–ligand interactions and downstream sig-nalling events is lacking. Little is known about the surface of an apoptotic cell and how it is recognized by phagocytes. One known signal for engulfment is a change in phospholipid composition, particularly the redistribution of phosphatidylserine (PS) from the inner- to the outer-plasma membrane leaflet5. However, although it may well be necessary, exposure to PS is not sufficient to drive apoptotic cell engulfment, and there are numerous instances of cells that are not phagocytosed despite exposure of PS2. Other apoptotic cell-surface changes that have been implicated in the clear-ance process include the presence of specific immunoglobulin superfamily members and

carbohydrates, but the underlying mechanisms also remain unclear1,2.

It is crucial that phagocytes can effectively discriminate viable cells from those that are dead or dying. But how is this achieved? The recent paper by Gardai et al.4 sheds new light on this process and proposes a two-step mecha-nism requiring calreticulin on the target cell. This pleiotropic protein, renowned for its ER localization, is up-regulated in stressed cells6 and commonly associates with the cell surface through as yet unknown mechanisms. In their previous work, this group had suggested that calreticulin associates with LRP/CD91 as part of a receptor complex on the engulfing cell that binds apoptotic cells7. In their new study, however, the group conclude that calreticulin functions not on the phagocyte, but rather on the apoptotic cell and binds in trans to phago-cyte LRP/CD91 to stimulate phagocytosis (Fig. 2). The authors show that apoptotic leu-kocytes and fibroblasts are required to expose or somehow bind calreticulin if they are to be phagocytosed efficiently. Furthermore, this mechanism appears to be important for both classes of phagocyte involved in apoptotic cell clearance in vivo (that is, neighbouring cells and scavenging macrophages).

Given its known ability to associate with LRP/CD91, it is perhaps not surprising that calreticulin seems to activate phagocyto-sis of apoptotic cells through this receptor. Calreticulin seems to stimulate membrane

Christopher D. Gregory and Simon Brown are in the University of Edinburgh/MRC Centre for Inflammation Research, Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK.e-mail: [email protected]

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1162 NATURE CELL BIOLOGY VOLUME 7 | NUMBER 12 | DECEMBER 2005

ruffling and macropinocytosis of macro-phages, as well as activate the Rac-1 GTPase that is crucial for the engulfment of dying cells8. A potentially confounding problem with this mechanism, as Gardai et al. show, is that calreticulin is not a specific surface feature of apoptotic cells, although after induction of apoptosis its levels are increased and its surface distribution is altered, becom-ing associated with PS. This is reminiscent of other molecules, including ICAM-3 and CD31 (which are present on both viable and apoptotic cell surfaces), or thrombospon-din (which, similarly to calreticulin, can associate with both types of cell surface) — all function at apoptotic cell surfaces to foster the selective engulfment of apoptotic cells9–11. Considering how calreticulin may promote selective phagocytosis of apoptotic cells, Gardai et al. propose that calreticulin-dependent engulfment requires an inhibi-tory pathway that constitutively suppresses phagocytosis of viable cells to be switched off. The concept that an inhibitory mechanism can be disabled by the apoptosis programme is not new and builds on the observation that the presence of inhibitory-receptor ligands, such as CD31 on leukocytes10 and CD47 on erythrocytes12, suppresses their phagocy-tosis. Indeed, Gardai et al. focus on CD47 and contend that it is this broadly expressed glycoprotein that determines whether a cell

is engulfed or not through the effects of the calreticulin–LRP interaction.

CD47 is the physiological ligand of SIRPα1 (intriguingly, also known as ‘macrophage fusion receptor’), a widely distributed inhibi-tory receptor that can recruit tyrosine kinases and phosphatases such as SHP-1 after trigger-ing by ligand-induced clustering13. Supported by observations indicating first, that erythro-cytes from CD47–/– mice are rapidly engulfed by macrophages when transferred to wild-type animals (though notably are not engulfed by macrophages from CD47–/– animals)12, and sec-ond, that the CD47/ SIRPα pathway can inhibit phagocytosis of IgG or complement-opsonised cells 14, Gardai et al. present data suggesting that when CD47 is present on viable cells it engages with SIRPα on phagocytes to inhibit activa-tion of engulfment mechanisms. They show that murine CD47–/– leukocytes and erythro-cytes are phagocytosed by macrophages in the absence of apoptosis. Furthermore, antibody blockade of either CD47 or SIRPα stimulated engulfment of apparently viable human neu-trophils. Thus, it seems that SIRPα signalling blocks engulfment of viable cells. In support of this, Gardai et al. also demonstrate phos-phorylation of macrophage SIRPα by viable, but not apoptotic, cells. They also show that a SHP-1 tyrosine phosphatase specific inhibitor can marginally promote engulfment of viable neutrophils. Taken together, these findings

suggest that the CD47–SIRPα interaction is key to protecting viable cells from phagocytosis.

This epic story offers two key insights: first, the potential of calreticulin and LRP to pro-mote engulfment of both viable and apoptotic cells; second, the suppression of this process in viable, but not apoptotic, cells by CD47 and SIRPα. So how do these mechanisms link together? Although the authors show that engulfment of CD47–/– erythrocytes depends on calreticulin and LRP, it is surprising that they do not show data on more relevant targets, such as CD47–/– non-erythroid cells; unlike erythrocytes, these cells have the potential to undergo apoptosis. Their contention is that CD47 on apoptotic cells is somehow prevented from interacting with SIRPα and this allows (perhaps by default) engulfment of the cell via calreticulin and LRP.

But what is different about CD47 on apop-totic cells? One obvious possibility is that CD47 is downregulated from the apoptotic cell surface. This seems to be the case for apoptotic neutrophils and fibroblasts, but not for apoptotic lymphocytes. It seems, however, that in all cases CD47 becomes qualitatively changed on apoptotic cell sur-faces, achieving a patchy distribution that may somehow modulate its ability to interact with SIRPα4. Definitive mechanisms under-lying the apparent qualitative changes in CD47 after induction of apoptosis need to

PhagocyteViable cell Apoptotic cell

Engulfmentmachinery

Engulfmentmachinery

Engulfmentmachinery

Engulfmentmachinery

Inhibition 1 Recognition

3 Signalling 4, 5 Engulfment and degradation

2 Tethering

Receptors Ligands

Inhibitory

Disabled inhibitoryTethering

Tethering & signalling

Signalling

Bridging

a b

Figure 1 Progressive molecular interactions activating phagocytosis of apoptotic cells. (a) Viable cells may activate inhibitory (‘Don’t eat me’) phagocyte signals that suppress intercellular adhesion and engulfment mechanisms. (b) Apoptotic cells fail to activate such inhibitory mechanisms and instead display ‘Eat me’ signals. Cell–cell interactions (1) are initiated

that may progress to strong intercellular adhesion (2) and culminate in the formation of a phagocytic synapse (3), a critical series of molecular interactions that may be required to activate the phagocyte beyond a threshold that leads to engulfment of the apoptotic cell and its subsequent degradation (4, 5).





NATURE CELL BIOLOGY VOLUME 7 | NUMBER 12 | DECEMBER 2005 1163

Macrophage

‘Disabled’ CD47

Apoptoticcell

SIRPα

αvβ3PS

LRP/CD91

MFG-E8

CD14 ACAMP

Calreticulin

Phagocyticsynapse

Figure 2 Formation of a phagocytic synapse between an apoptotic cell and a macrophage. Gardai et al.4 suggest that calreticulin functions as a ligand for macrophage LRP/CD91, clustering with exposed PS on the apoptotic cell remotely from CD47, which is no longer able to activate inhibitory signals through its phagocyte receptor, SIRPα. PS may interact with several phagocyte receptors. An indirect interaction with the vitronectin receptor integrin αvβ3 through the intermediate molecule MFG-E8 is shown. Also shown is CD14, the pattern recognition receptor that functions as a tethering receptor, interacting with the apoptotic cell surface through putative ACAMPs (apoptotic cell-associated molecular patterns) that may resemble microbial structures2. See refs 1, 2, 8 and 10 for examples of other molecules implicated in this process.

be substantiated and will be key to under-standing this model.

This paper makes an attractive proposition, pushing forward the notion10 that inhibitory signals may prevent default engulfment by neighbouring phagocytes and scavenging macrophages alike. Its apparent simplicity challenges the current molecular complex-ity that pervades this field2. How do other apoptotic cell-surface changes and receptors mediating phagocyte interaction relate to this model? For example, what is the relationship to

PS redistribution (the most renowned change in the apoptotic cell surface), and what are the roles of PS receptors? Intriguingly, calreticulin seems to be distributed in similar locations to PS on the apoptotic cell surface, whereas CD47 becomes patched elsewhere (Fig. 2) 4. As CD47 has been reported previously to function as a tethering receptor for apoptotic cells15, a func-tion that is not immediately reconcilable with that proposed by Gardai et al., the new model may be considered controversial. The relevance of the mechanism in vivo has not yet been

rigorously addressed, as the calreticulin knock-out mouse is an embryonic lethal; during embryonic development at least, macrophages seem dependent on calreticulin to effectively engulf apoptotic cells, as the latter are less efficiently phagocytosed in calreticulin-null embryos4. These initial in vivo investigations, however, do not discriminate between possible roles of calreticulin as a phagocyte receptor or apoptotic cell ligand. Thus, the relative impor-tance of this new model will become apparent when extensive in vivo studies are performed, as for other molecules at the phagocytic syn-apse2. Meanwhile, the proposed mechanisms will undoubtedly spark much discussion and new investigation.

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Gregory, C. D. J. Immunol. 162, 6800–6810 (1999).10. Brown, S. et al. Nature 418, 200–203 (2002).11. Savill, J., Hogg, N., Ren, Y. & Haslett, C. J. Clin. Invest.

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Apoptosis: eating sensibly