the phagocytosis of apoptotic cells

doi:10.1006/smim.2001.0333, available online at http://www.idealibrary.com onseminars in IMMUNOLOGY, Vol. 13, 2001: pp. 365–372

The phagocytosis of apoptotic cells

Valerie A. Fadok a and Giovanna Chimini b,∗

Apoptosis is a genetically controlled event taking care of cellturnover in healthy adult tissues and of focal eliminationof cells during embryonic development. The initial phaseof the program leads to corpse generation and is followedby the equally crucial removal by phagocytes. In fact,engulfment is not mere clearing of cell remnants, but ratherelicits phagocyte responses able to modulate inflammationand immune reactions. The combined investigation ofnematode and mammalian models has allowed, in recentyears, a fast progression in the field; however, effort isstill required to dissect thoroughly the molecular rulesorchestrating engulfment.

Key words: apoptosis / engulfment / inflammation /membrane changes / phosphatidylserine

c© 2001 Academic Press

Taking care of corpses generated by apoptosis isa challenging task with crucial implication for themaintenance of general cellular homeostasis.

Several features account for the unicity of apoptoticengulfment among the different forms of phagocy-tosis. The very nature of the prey to be, a self-cellundergoing major surface modifications, is the start-point. The doomed cell is then recognized by a sur-prisingly high number of surface receptors which inturn trigger specific intracellular machineries. Finally,the activation of these signalling cascades elicits com-plex phagocyte responses able to modulate local in-flammation and systemic immunological responses.

From the aNational Jewish Medical and Research Center, 1400 JacksonStreet, Denver, CO 80206, USA and bCentre d’Immunologie deMarseille-Luminy, INSERM-CNRS-Universite de la Mediterranee, Case906, Parc Scientifique de Luminy, 13288 Marseille Cedex 09, France.*Corresponding author. Centre d’Immunologie de Marseille Luminy,INSERM-CNRS-Universite de la Mediterranee, Case 906, Parc Scientifiquede Luminy, 13288 Marseille Cedex 09, France.E-mail: [email protected]

c©2000 Academic Press1044–5323/01/060365+ 08/$35.00/0

Surface modifications

Morphological changes accompany the accomplish-ment of the apoptotic program (Figure 1).1 Cellsround up and detach from their neighbours. Thencondensation of both nucleus and cytoplasm takesplace without major modification of intracellularorganelles. The nucleus however fragments andcharacteristic membrane protuberances (blebs)appear. Their segmentation leads to apoptotic bodieswith yet an intact membrane. Interestingly, freebodies are rarely seen under physiological conditionsand mostly appear inside the phagocytes, avidlyingesting the corpses as soon as they are generated.

These visually appreciated features have now beentranslated into biochemical mechanisms.2 However,whether and how any of them is crucial in turninga mere corpse into an highly appetizing meal isstill to be elucidated. The apoptotic disassembly ofthe cell factory is the consequence of a caspase-orchestrated massive cleavage of downstream targetseach taking part to the generation of a distinctphenotype. Among those potentially able to affect theinteractions between prey and phagocyte, membraneblebbing has long remained enigmatic.

Several caspase targets involved in cytoskeletalrearrangements have been identified,3,4 but only re-cently the demonstration that a caspase-3-dependentcascade is at the origin of membrane blebs has beenreported.5,6 The target is a serine/threonine kinaseROCK I, one of the effectors for Rho GTPases, asubgroup of the ras super-family of small GTP bindingproteins.7,8 Those regulate cell mobility by acting onthe actin cytoskeleton and are required to generatethe force for migration, spreading and phagocytosis.9

During apoptosis ROCK I is not activated by thenatural Rho-dependent pathway but transformedinto a constitutively active form, which controls theassembly of cortical actomyosin microfilaments viathe phosphorylation of myosin regulatory light chain(MLC). It is possible that other ROCK substratescooperate with MLC in the induction of blebbing.

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V. A. Fadok and G. Chimini

Figure 1. Diagram illustrating the morphological featuresof apoptosis; from the detachment of a cell doomed to dieto its final engulfment by phagocytes. A schematic view ofthe molecular mechanism leading to membrane blebbingis inserted.5,6 ROCK: Rho activated serine/threoninekinases, MLC: myosin regulatory light chain, PAK: p21activated kinase.

Interestingly the classical activation pathway doesnot lead to blebbing but to cell contraction andthe expression of an engineered cleaved ROCK Iis sufficient to induce blebbing in the absence ofapoptosis.5,6

In spite of these mechanistic evidences, whetherbleb formation has any impact on the clearance ofthe apoptotic particle remains to be elucidated. Therelationship between blebbing and the exposure ofthe best known ‘eat me’ signal, phosphatidylserine(PS) exposure, is in fact still unclear. WhereasROCK I manipulation does not seem to modulate theexposure of PS at the cell surface,5,6 it has not beeninvestigated whether the modifications of membranefluidity (that is the outward PS flip) are a prerequisitefor blebbing to occur.

The loss of the asymmetrical distribution of PSis the major signal triggering recognition andingestion of an apoptotic prey.10 Molecularlyspeaking the event requires the inhibition of theaminophospholipid translocase, to stop the restlessinward flipping of PS, and the concomitant activationof a phospholipid scramblase, to actively routeoutward the PS located in the inner leaflet.11,12

Both events can result from an increased cellular

concentration of calcium but it is not clear whetherthis happens during apoptosis.13 In addition, none ofthe candidate translocases or scramblases have beenreported as a direct caspase target. The evidenceindicates that the phenomenon is caspase dependent,though dissociable from other biochemical eventswhich follow caspase activation.14

Why are PS so appetizing for a phagocyte? PS expo-sure has long been recognized as a mediator of cellinteractions and is definitely able to trigger recogni-tion by professional phagocytes.15 Amateurs, such asdendritic cell or fibroblast, are also able to performengulfment albeit less efficiently.16 The suboptimalefficiency and the distinctive morphological featureswhich accompany engulfment by amateurs may ac-tually reflect the recognition pathway preferentiallyengaged. In fact amateurs, as opposed to profession-als, make priviliged use of an integrin-mediated path-way for recognition and uptake.

Exposed PS trigger their own recognition by astereo specific receptor (PSR).17 It is also importantto underline that PS exposure is only a limited readout of the complex perturbation of the biophysicalproperties of the membrane occurring duringapoptosis. This was exemplified by the work ofTepper and co-workers.18 They showed in fact thatexposing anionic phospholipids on the outer leafletgenerates ‘per se’ major imbalances in the distributionof other lipid moieties, e.g. sphingomyelin (SM)which is massively flipped to the inner leaflet. Inits turn the inward dislocation of SM affects thedistribution and mobility of membrane cholesterol.

The organization of membrane lipid, which is a sen-sor of the biophyiscal properties of the membrane,seems to be at play also on the side of the effectorphagocyte.

Macrophages are the exception to the rule ofstrict asymmetrical distribution of phospholipidsat the plasma membrane and possess, in contrast,a rather ‘loose’ asymmetry of membrane lipids,19

like neoplastic cells, myoblasts and megacaryoblasts.These domains of non-asymmetrical distributioncorrespond to sites of preferential endocytic recyclingand are also not neutral with respect to engulfment.In fact the pharmacological rigidification of themembrane or the physical saturation of the exposedPS residues on the phagocyte surface is able impairuptake of corpses.20,21 An interaction between PSexposing membranes on both the phagocyte andprey seems thus to be required; this is dispensablewhen the same phagocyte ingest non-PS exposingparticles.

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What exactly this underlies remains elusive.We may envision the requirement of homotypicinteractions between anionic phospholipids on thetwo opposing membranes to reinforce tethering ofthe prey. Alternatively this may reflect a mechanisticadvantage provided by the PS-rich patches. Inthis context it is interesting to note that, both inmammals and in c. elegans, experimental evidencesuggests that the ABC transporters (ABC1 andCED-7, respectively22,23) implicated in engulfmentcan modulate membrane fluidity. In mice, the lossof ABC1 function leads to impaired engulfment‘in vivo’ and alters concomitantly the mobility ofphospholipids and cholesterol across and fromthe bilayer.24 In c. elegans the question has notbeen addressed directly. It has nonetheless beenreported that the absence of CED-7 hampers thelateral redistribution and clustering on the phagocytemembrane of the CED-1 scavenger receptor, an eventnormally triggered by the contact with a dying cell.25

We may thus easily infer a facilitator role of the ABCtransporters during engulfment exerted by theircontrol on the transversal and lateral distribution ofmembrane lipids.

Receptors involved in the recognition of anapoptotic cell

Several receptors have been implicated in therecognition and engulfment of apoptotic cells inrecent years and they have been already extensivelyreviewed (Figure 2).10,15 Unfortunately in mostcases the molecular entity that they recognize on thesurface of the prey is poorly defined, as it is the exactnature of most the signals exposed by the apoptoticcell. The sole exceptions are the already-mentionedexofacial flip of PS and the documented changesin surface sugars, which are probably recognizedby lectins on the macrophage surface. Most of theengulfment receptors are well known proteins suchintegrins of the αvβ3 or αvβ5 classes and the lipidscavenger receptors of both A and B classes. TheLPS receptor CD14 has also been involved in thetethering of apoptotic lymphocytes via interactionwith ICAM3 moieties on the prey surface.26

Recently two new molecules able to engageingestion have been identified: a specific receptorfor the PS (PSR)27 and the tyrosine kinase receptorMER.28 The first acting alone or in concert withCD36 provides the required stereospecificity for PSrecognition, whereas the second could act as a recep-

Figure 2. Surface molecules involved in the recognitionof apoptotic preys on the phagocyte membrane.10,26 PSRreceptor for phosphatidylserine, SR-A and CD36: scavengerreceptors of the A and B classes. MER: tyrosine kinasereceptor MER.28 Integrins involved in the process areαvβ3 and αvβ5.The membrane localization of the ABC1transporter is also shown.

tor for gas-6 (the product of growth arrested specificgene 6) a soluble protein previously implicated as amediator of macrophage binding to PS.15

Phagocytic machineries

The first insights into the signalling cascades activatedby engulfment downstream to surface receptors camefrom the worm. From genetic analysis it has beendefined that two major pathways are at work.29

Recently Riedden and Horvitz30 thoroughly dissectedone of those and identified both the proteins involvedand their molecular interactions. These are calledCED-2 CED-5 and CED-10 and correspond on thebasis of sequence conservation to the mammalianCrk-II/Dock-180 and Rac-1.

The evolutionary parallel, however, goes furthersince in mammals the occupation of αvβ5 in dendriticcells and in non-professional phagocytes elicits thephosphorylation of p130 cas and the sequentialrecruitment of Crk-II and Dock-180 in a membranecomplex which in turn leads to the activation ofRac-1 (Figure 3).31,32 Their formal implicationin engulfment has been further demonstratedby the analysis of the effect of the reduced orincreased function on the phagocytic efficiency.31,32

This p130cas/CrkII/Dock180 collaborative action,previously associated with cell migration in vertebratesystems, perfectly reconstitutes the worm ced-2,ced-5 and ced-10 pathway. An additional piece ofinformation comes from the analysis of the eventstriggered in macrophages by the surface occupationof αvβ3 integrin.33 There pseudopod protrusion andthe Rac-induced formation of an actin cup has beenreported. In this cell system, the reconstitution of aCrk-II/Dock-180 pathway has not been attempted

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Figure 3. Molecular pathways of engulfment.The A panels illustrate the pathway triggered by surface occupation of integrins in mammals which correspond to thepathway controlled by ced-2, ced-5 and ced-10 in the nematode.30–32 The ligand for integrins on the surface of the apoptoticcell is unknown.10 Recruitment of tyrosine kinases (Tyr K) to the phagocytic cup occurs downstream to Rac activation.33

The involvement of PI3 kinases is also shown.33 Arrows indicate actin polymerization. The B panels illustrate a speculativemodel for the second pathway controlled by ced-1, ced-6 and ced-7 in the nematode.29 The ABC1 transporter (CED-7)facilitates engulfment by fine tuning the biophysical properties of the membrane (indicated by the arrow).24 These allowthe redistribution of the nematode CED-1 around the apoptotic body.25 A similar situation is envisioned for CD36 (SR)and the PSR in mammals. Activation of this pathway results in the dimerization of the mammalian CED-6 (h CED-6).35

Question marks illustrate missing links. Ps exposed on the apoptotic cells are also shown.

but Rac controls the local recruitment of tyrosinekinases and the downstream activation of one of theisoforms of the PI3-kinase.

If the ced-2, ced-5 and ced-10 pathway is maintainedin vertebrates as an integrin mediated cascade it ispossible that the pathway controlled by ced-1, ced-6and ced-7 corresponds to a PS governed recognitionsystem in mammals (Figure 3). The receptorswould be the scavenger CD36 and PS-receptors,possibly working in concert. The parallel is, however,highly speculative since CED-1 belongs to the familyof scavenger receptors but does not bear obvioushomology to the scavengers implicated in engulfmentin mammals.25,34 This recognition, whose efficiencyis fine tuned by the activity of the ABC transporters,will lead to the recruitment of the adaptor moleculehuman CED-6. There is no report both in the wormand vertebrates on molecular interactions betweenany of these partners, nor has it been investigated

whether any of the mammalian surface receptorsclusters around the prey after the initial contact.Moreover, although both the human and nematodeCED-6 are potentially able to interact with kinases orother effectors, the search for downstream targetshas failed so far.35,36

A number of crucial questions are still to beelucidated; among them it would be of interest toinvestigate whether cross-talks exist between thetwo pathways. Two observations suggests that theover-expression of the effector of one of the twopathways (namely CED-6) can overcome defectsof the furthermost effector of the parallel pathway(namely CED-10).36 Similarly it has been reportedthat phagocytes actively promote the death ofthe prey.37,38 How it happens is far from beingunderstood but this may well represent a backwardresponse triggered by the signalling machinery atwork during the ingestion of the prey.

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Functional consequences of the engulfment ofapoptotic cells by phagocytes

The engulfment occurs prior to lysis of the apoptoticcell, thereby preventing the release of potentiallyproinflammatory and pro-immunogenic intracellularcontents.39 Engulfment is believed to be a silent pro-cess, in that phagocytes are not stimulated to releaseproinflammatory cytokines or lipid mediators.40–42

Even neutrophils which have progressed to lateapoptosis have been found to be noninflammatory.43

In fact, engulfing macrophages become activelyanti-inflammatory and release suppressive mediatorssuch as TGFβ, IL10, PGE2, and possibly others.44–46

These mediators block endotoxin or zymosan-stimulated release of proinflammatory cytokines andchemokines by macrophages. In some cases, however,apoptotic cells can induce the transcription ofproinflammatory chemokines, and it has been shownthat in the presence of massive apoptosis, transientinfiltration of neutrophils is observed.47 There aretwo potential explanations for these discrepancies. Atleast in the case of some proinflammatory cytokines,the ability of apoptotic cells to suppress may occurpost-transcriptionally.45 Alternatively, it may bepossible to overwhelm the phagocyte clearancecapacity, resulting in secondary necrosis and leakageof intracellular contents from cells originally dyingby apoptosis. Determining the clearance capacity ofmacrophage populations in vivo has become a criticalgoal, and it is highly likely that there are differencesbetween various resident populations and thosemacrophages called into an inflammatory site.48

Recently there has been great interest inunderstanding how apoptotic cells affect the abilityof the dendritic cell (DC) to induce an immuneresponse. DC are able to phagocytose apoptotic cells,although not as efficiently as macrophages.49–52

The data on whether a DC can mature and presentantigens derived from apoptotic cells are conflicting.At least in vitro, several publications suggest thatDC can phagocytose apoptotic virally infected ortumor cells and present tumor-derived antigens viacross presentation to CD8+ cytotoxic T cells, andthat the activated CTL can kill the target cells, (e.g.References 50,51,53,54), at least in vitro. Rovere et al.have suggested that this outcome results fromdelayed clearance,51 which may reflect the relativeinefficiency of the DC to engulf apoptotic cellsand therefore an increased sensitivity to clearanceoverload. The observations that DC can cross-presentantigens to CTL form the basis for the generation

of DC-based vaccines for the treatment of severalcancers. However, examination of the methodologyused to induce apoptosis suggests that in some casesthe apoptotic cells consist of a mix of apoptoticand necrotic cells (e.g. Reference 54) or that theapoptotic cells were stressed by heat shock orinfected with virus, thus providing promaturationsignals.50,53,55,56 In the hands of some investigators,the binding/engulfment of purely apoptoticpopulations by apoptotic cell receptors on DCis non-stimulatory, whereas binding/engulfment ofnecrotic cells, particularly those derived from tumors,strongly stimulate the DC to mature and activate Tcells.50,56–59 A number of intracellular proteins,including heat shock proteins, have been reportedto activate DC, suggesting that cells must lyse beforethey can promote an immune response.55,56

So how then does the apoptotic cell blockmacrophage proinflammatory responses and, at leastin some cases, the ability of dendritic cells to producea productive immune response? One potentialclue lies with the observations that apoptoticcells loose phospholipid asymmetry and exposephosphatidylserine externally;60 several investigatorshave reported that phosphatidylserine must beexposed in order for engulfment to occur.61,62

In addition, the triggering of a newly describedphosphatidylserine receptor appears essentialfor engulfment and for the release of TGFβ bymacrophages and other phagocytes17 (Hoffmanet al. submitted). Thus, a reasonable hypothesis isthat triggering of the phosphatidylserine receptor(PSR) by exposed phosphatidylserine (PS) on theapoptotic cell inhibits inflammation. The inhibitoryeffects on dendritic cell function may result fromblocking inflammation or from direct effects of PSRstimulation.

This hypothesis was derived from several observa-tions. The direct stimulation of the PSR mimics theeffects of apoptotic cells in vivo and in vitro and in-duces the release of TGFβ, PGE2, IL10, and possi-bly other anti-inflammatory mediators44,46 (Huynh,Fadok, and Henson, unpublished data). The uptakeof purely apoptotic cells by dendritic cells fails to stim-ulate antigen presentation,51,54,56,58,63,64 whereas theexposure to necrotic cells, particularly those derivedfrom tumors and their lysates, stimulates it.58 Manypathogens which evade immune destruction eitherexpress PS on their membranes (e.g. cytomegalovirusand other herpes viruses, Leishmania) or induce apop-tosis and are engulfed with PS-expressing apoptoticcells (e.g. trypanosomes).10 Many malignant tumor

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cells either expose PS when viable65,66 or continu-ally shed intact PS exposing membrane vesicles67–69

(Fadok, Henson, unpublished data).This hypothesis still fits with the ability of the

immune system to mount an effective responseto pathogens, most of which do not express PSexternally, and therefore will not trigger thePSR. DC, binding microbial products via patternrecognition molecules will therefore mature andactivate T cells. In contrast, those DC binding orengulfing apoptotic cells or membrane vesicleswill receive a strong inhibitory signal, pre-emptingantigen presentation and T cell activation. Whatis not yet clear is how signaling through the PSRinteracts with signaling from pattern recognitionmolecules known to be involved in DC maturation.Does signaling through the PSR always overridesignals from proinflammatory receptors? Or is theoutcome determined by the balance between signalsfrom the anti- and pro-inflammatory receptors?We favour the latter hypothesis. We have someevidence to suggest that signaling through the FcR inmacrophages can override signaling via the PSR, butthat signaling from the PSR appears to overrides thatderived from TLR4 (via endotoxin stimulation).44

It will be important to determine the hierarchy ofresponses, as well as what receptors or combinationsof receptors must be engaged to overcome the PSRnegative signal. A potential role for other apoptoticcell receptors in the generation of the suppressiveresponse must also be examined.

Understanding the role of the PSR and otherapoptotic cell receptors in DC function and inantigen presentation is critical given the emphasis onusing DC which have engulfed apoptotic cells as thebasis for vaccines in the treatment of malignancies.It is clear that malignant melanoma cells expressthe PSR, can ingest apoptotic cells, and can secreteTGFβ in response to apoptotic cells or to triggeringPSR with the anti-receptor antibody (Geske, Fadok,unpublished data). It is also clear that melanomacells vesiculate continuously in culture and thatseveral tumors express small amounts of PS evenwhen viable.65,66 These observations indicate thattumors may use PS and the PSR as a weaponagainst an immune response. By the time thetumor has metastasized, the immune system hasbecome refractory to activation against tumor-specific antigens. This hypothesis may also explainwhy although tumor-specific T cells can be found inpatients with malignancies, they are not often activein vivo.

Concluding remarks

The number of investigations addressing thediverse aspects of engulfment of apoptotic cells byprofessional and amateur phagocytes have grownimpressively in recent years. The complexity of thephenomenon, which in mammals has been mostlystudied for in vitro systems, urges the developmentand analysis of in vivo models of delayed engulfment.Only those will allow one to address the impactof a suboptimal performance of the ‘sentinels’macrophages on the regulation and resolution ofinflammatory responses, on the onset of immuneresponses and in tissue remodelling.

Acknowledgements

GC wishes to thank C. Beziers La Fosse for help withdiagrams and ARC (Association pour la Recherchesur Le Cancer), LNCC (Ligue National Contre leCancer) and the Association Vaincre la Mucoviscidosefor support. The authors wish also to acknowledgethe contribution to the field of numerous scientists,whose work could not be quoted due to spacelimitations.

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