Immune regulation by apoptotic cell clearance

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Clearance of Dying Cells in Healthy and Diseased Immune Systems

Immune regulation by apoptotic cell clearance

Masato Tanaka, Kenichi Asano, and Chun-Hong QiuLaboratory for Innate Cellular Immunity, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, Japan

Address for correspondence: Masato Tanaka, Laboratory for Innate Cellular Immunity, RIKEN Research Center for Allergy andImmunology, 1-7-22, Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan. [email protected]

Apoptotic cell clearance by phagocytes is essential for the maintenance of self-tolerance under physiological condi-tions. Consistent with this, the intravenous injection of apoptotic cells can induce cell-associated antigen-specificimmunosuppression or tolerance. The intravenous injection of apoptotic cells expressed a fragment of myelinoligodendrocyte glycoprotein (MOG)-induced MOG-specific T cell tolerance and suppressed the development ofexperimental autoimmune encephalomyelitis. However, the suppressive effects of the MOG-expressing apoptoticcells were largely eliminated by masking phosphatidylserine (PS) exposed on the apoptotic cells, suggesting that thePS-dependent engulfment of apoptotic cells is required for the tolerance induction. We found that this mechanismof tolerance induction requires the contribution of two cell populations in the splenic marginal zone (MZ). The MZcontains two types of macrophages: marginal metallophilic macrophages and MZ macrophages. These macrophagescontribute to the rapid clearance of cell corpses in blood flow. In addition, we also found that CD8α+, CD103+

dendritic cells localizing in the MZ selectively phagocytose blood-borne dead cells and subsequently present deadcell-associated antigens to induce antigen-specific immunosuppression or tolerance.

Keywords: cell death; phagocytosis; macrophage; dendritic cell; self-tolerance

Apoptotic cell clearance by phagocytes

Every type of cell has its own life span, and aged cellsundergo apoptosis for their turnover. The immunesystem eliminates harmful cells, including virus-infected cells or cancer cells, by inducing apoptosis.Although a substantial number of cells constantlyundergo apoptosis even in physiological conditions,it is very difficult to detect the apoptotic cells as theyare, primarily because the apoptotic cells are swiftlyphagocytosed by phagocytes such as macrophagesand dendritic cells (DCs).1 Phosphatidylserine (PS)is exposed on the apoptotic cell surface, and mostphagocytes recognize this lipid through specific re-ceptors or soluble molecules. The milk fat globule-epidermal growth factor 8 (MFG-E8) was identifiedas one such soluble protein that is involved in apop-totic cell phagocytosis by phagocytes.2 The MFG-E8 has two factor VIII-homologous domains in itsC-terminus that are responsible for binding to PSexposed on the apoptotic cell surface. On the otherhand, its N-terminus has epidermal growth factor(EGF)-like domains that can bind �v�3 integrin ex-

pressed in phagocytes via its Arg-Gly-Asp (RGD)motif. MFG-E8 acts as a bridging molecule betweenapoptotic cells and phagocytes to promote dead cellclearance.

In the germinal centers of spleen and lymphnodes, a large number of B cells undergo apoptosisduring B cell selection. Tingible body macrophages(TBMs) are located in the germinal centers andare responsible for the phagocytosis of apoptoticB cells. Immunohistochemical analysis has revealedthat these macrophages produce MFG-E8. In MFG-E8-deficient mice, a large number of apoptotic lym-phocytes associate with TBMs in the germinal cen-ters, but most of them are not engulfed by TBMs,indicating that MFG-E8 is involved in apoptotic cellclearance by TBMs.3 In addition, MFG-E8-deficientmice develop splenomegaly and spontaneously pro-duce autoantibodies such as antinuclear antibod-ies and anti-DNA antibodies in an age-dependentmanner. In association with autoantibody pro-duction, the mice develop systemic autoim-mune disorders including glomerulonephritis. Theautoimmune disorders can be observed in various

doi: 10.1111/j.1749-6632.2010.05746.xAnn. N.Y. Acad. Sci. 1209 (2010) 37–42 c© 2010 New York Academy of Sciences. 37

Tolerance and apoptotic cell clearance Tanaka et al.

gene-targeting mice deficient in molecules involvedin apoptotic cell clearance. In addition, the injectionof the MFG-E8 mutant protein D89E, which exerts apotent inhibitory effect on the phagocyotsis of apop-totic cells by masking PS, induces the productionof autoantibodies, including antiphospholipid anti-bodies and antinuclear antibodies, in mice.4 Theseresults indicate that apoptotic cell clearance plays acritical role in the maintenance of self-tolerance.

Mechanisms of self-tolerance by apoptoticcell clearance

It is speculated that the apoptotic cell clearance byphagocytes contributes to the maintenance of self-tolerance by means of two different mechanisms.First, the apoptotic cell clearance prevents the releaseof intracellular materials from dying cells. Whenthe apoptotic cell clearance is impaired, intracellu-lar endogenous adjuvants, such as uric acid, high-mobility group box 1, and spliceosome-associatedprotein 130, may be leaked to the environment;this consequently induces immune activation.5–7 Inthis sense, the rapid clearance of apoptotic cells in-hibits the exposure of immunogenic materials fromdying cells. In addition to the rapid clearance ofcell corpses, phagocytes actively contribute to self-tolerance by presenting self-antigen derived fromapoptotic cells to T cells. In order to eliminate self-reactive T cells in the periphery, self-antigens shouldbe presented by antigen-presenting cells. One ofthe major sources of cellular antigens is self-deadcells. Tissue-resident antigen-presenting cells con-stantly phagocytose apoptotic cells generated duringnormal tissue turnover and migrate to the drain-ing lymph nodes where they present the antigenstaken up from apoptotic cells.8 This presentationof self-antigens leads to the deletion or anergy ofself-reactive T cells, thereby maintaining T cell tol-erance to self-antigens. In support of this idea, theinjection of apoptotic cells loaded with ovalbumin(OVA) induces the suppression of OVA-specific Tcell responses.9 In summary, phagocytes not onlyremove potentially dangerous “garbage” from theenvironment but also recycle the garbage for themaintenance of self-tolerance.

Intravenous injection of apoptotic cellsinduces immune tolerance

Several groups tried to utilize the immunosuppres-sive effects of apoptotic cells and were able to suc-

cessfully suppress pathological immune responsesby intravenously injecting dying cells. The intra-venous injection of apoptotic splenocytes from adonor strain prevented the rejection of heart al-lografts.10 It is also reported that the transfusion ofapoptotic � cells induced immune tolerance and thesubsequent suppression of diabetes in NOD mice.11

We recently found that the induction of toler-ance to cell-associated antigens could be appliedto the prevention of autoimmune diseases in amouse model.12 The immunization of mice withmyelin oligodendrocyte glycoprotein (MOG) pep-tide emulsified with complete Freund’s adjuvant in-duced experimental autoimmune encephalomyeli-tis (EAE), a mouse model of human multiplesclerosis. We established transformants expressingthe MOG fragment, and the injection of apop-totic cells expressing the MOG fragment suppressedthe development of EAE. The intravenous injec-tion of apoptotic cells expressing an MOG frag-ment reduced MOG-specific T cell responses buthad no effect on T cell response to an irrelevant anti-gen, indicating that the tolerance to cell-associatedantigens was specifically induced by the apoptoticcell injection. For the tolerance induction to cell-associated antigens, the characteristics of early phaseof apoptotic cells were required. The suppressiveeffects of apoptotic W3/MOG-L cells were largely,but not completely, eliminated when the mice wereinjected with D89E, an MFG-E8 mutant protein.This result suggests that the PS-dependent engulf-ment of apoptotic cells is required, at least in part,for the induction of tolerance to cell-associatedantigens.

Cellular mechanisms of toleranceinduction by apoptotic cell phagocytosis

Intravenously injected apoptotic cells are initiallyaccumulated in the splenic marginal zone (MZ)(Fig. 1). This observation indicates that somephagocytes in the spleen phagocytose the injecteddead cell corpses and that these cells present anti-gens derived from the dead cell corpses for toleranceinduction. MZ is capable of constantly screeningblood for foreign particles and organisms, as well asaberrant molecular debris and dying cells. MZ con-tains two types of macrophages: MZ macrophages(MZMs) and marginal metallophilic macrophages(MMMs). MMMs are localized at the inner borderof the MZ close to the white pulp, whereas MZMs

38 Ann. N.Y. Acad. Sci. 1209 (2010) 37–42 c© 2010 New York Academy of Sciences.

Tanaka et al. Tolerance and apoptotic cell clearance

Figure 1. Intravenously injected apoptotic cells accumulate in the marginal zone of the spleen. Fluorescent-labeled apoptotic cells(green) were intravenously injected into wild-type mice. After a one or four hour injection, spleen sections were stained with F4/80,ER-TR9, or MOMA-1 antibodies.

are found at the outer rim, near the red pulp. It ismost likely that these macrophages act as sentinelsfor the screening of blood-borne materials. How-ever, the detailed functions of these macrophagesare largely unknown.

To reveal the role of macrophages in the MZin the induction of immune tolerance to injectedcell-associated antigens, we generated transgenicmice in which macrophages in the MZ could betransiently deleted by diphtheria toxin (DT) injec-tion. Recently, a method for conditional cell abla-tion by the transgene of the human DT receptor(DTR), named TRECK (Toxin REceptor-mediatedCell Knockout), has been used in various researchfields.13–18 Human, but not mouse, heparin-bindingEGF-like growth factor (HB-EGF) exhibits strongbinding activity to DT. Thus, mouse cells are moreresistant to DT than human cells. When the humanHB-EGF gene was transduced into mice under thecontrol of a cell-specific promoter, the target cellswere transiently depleted by DT administration invivo. Human HB-EGF complementary DNA was in-troduced into the CD169 gene locus in the mice.12

DT administration to the mice (CD169-DTR mice)

resulted in the transient deletion of MZMs andMMMs, whereas red pulp macrophages, TBMs, orDCs were not affected (Fig. 2). We found that toler-ance induction to cell-associated antigens was im-paired in the MZM-depleted mice, indicating thatmacrophages in the MZ are indispensable for toler-ance induction to cell-associated antigens.

In the mice, the clearance of injected apop-totic cells was severely delayed in the absence ofmacrophages in the MZ. Circulating dead cells andnon-self-materials, such as injected latex beads andbacteria, were first trapped and accumulated in theMZ of spleen and then rapidly cleared. Even in theabsence of macrophages in the MZ, injected apop-totic cells were still accumulated in the MZ, indi-cating that other types of cells localized in MZ areresponsible for trapping circulating dead cells inspleen. However, there was a dramatic delay in cellcorpse clearance in the absence of macrophages inthe MZ, indicating that these macrophages play acritical role in clearance by phagocytosis. It is pos-sible that this delayed clearance of injected deadcells critically affects tolerance induction to cell-associated antigens.

Ann. N.Y. Acad. Sci. 1209 (2010) 37–42 c© 2010 New York Academy of Sciences. 39


Figure 2. Selective deletion of macrophages located inmarginal zone of spleen in CD169-diphtheria toxin receptor(DTR) mice. Wild-type or CD169-DTR mice were treated withDT. Spleen sections were stained with CD169 antibody, or B220(green) and CD11c antibody (red).

A subset of CD8α+ DCs is involved intolerance induction by apoptotic cellinjectionAn analysis of CD169-DTR mice clearly demon-strated that macrophages in the MZ play a criti-cal role in rapid clearance of blood-borne apop-totic cells and subsequent induction of tolerance todead cell-associated antigens. However, it is still un-clear whether these macrophages are involved inpresentation of cell-associated antigens. As thereis no useful strategy to isolate these macrophagesfor ex vivo experiments, we could not elucidate theantigen-presentation activity of these macrophages.Beside macrophages, CD11c+ DCs, possessing po-tent antigen-presentation activity, exist in spleen.Among the DC subsets, CD8�+ DCs dominantlyphagocytose injected apoptotic cells and presentcell-associated antigens to T cells, suggesting thatthey are responsible for the tolerance induction tocell-associated antigens.19,20 However, as it is re-ported that CD8�+ DCs are predominantly local-ized in the T cell zone,21 it remains unclear how these

DCs phagocytose blood-borne apoptotic cells accu-mulated in the MZ. Some CD8�+ DCs were recentlyfound to be localized in MZ,22 but it is still unclearwhether there are any differences in the phenotypeand function between the CD8�+ DCs in the MZand those in the T cell zone.

To elucidate the characteristics of the splenicCD8�+ DCs that are capable of phagocytosingapoptotic cells, we injected mice with fluorescence-labeled apoptotic cells and then isolated phagocyticand nonphagocytic CD8�+DCs by cell sorting.23

We compared their gene expression profiles by DNAmicroarray analysis and found that CD8�+ DCsin spleen consist of two subpopulations: CD103+,CD207+ DCs, and CD103−, CD207− DCs andthat only CD8�+, CD103+, CD207+ DCs phago-cytosed blood-borne apoptotic cells. Immunohis-tochemical analysis with anti-CD207 antibody re-vealed that these cells were preferentially localized inthe MZ. Moreover, this DCs subset migrated fromthe MZ into the T cell zone after phagocytosis ofapoptotic cells (Fig. 3). Consistent with their abil-ity to phagocytose apoptotic cells in vivo, CD8�+,CD103+ DCs exhibited strong presentation activ-ity of cell-associated antigens to CD8 T cells. Liuet al. recently reported that intravenous injectionof cytochrome c induced apoptotic cell death in afraction of CD8�+ DCs.24 We carefully examinedthis phenomenon, and found that CD8�+, CD103+

DCs were selectively deleted in vivo by cytochromec treatment. Consistent with the reduced number ofCD8�+, CD103+ DCs, cytochrome c treatment im-paired the phagocytosis of injected apoptotic cellsby CD8�+ DCs. In cytochrome c-treated mice, tol-erance to cell-associated antigens was no longer in-duced by apoptotic cell injection, indicating thatCD8�+, CD103+ DCs play a critical role in tol-erance induction to cell-associated antigens by thephagocytosis of blood-borne apoptotic cells.

Stimulation of Toll-like receptors (TLRs) affectsthe function of antigen-presenting cells and usuallyrenders these cells immunogenic. We found that in-jection of a relatively small amount of lipopolysac-charide or polyinosinic–polycytidylic acid drasti-cally reduced the number of CD8�+, CD103+ DCs.This disappearance of CD8�+, CD103+ DCs maybe responsible for the interference with toleranceinduction by TLR stimulation in vivo.

In summary, two types of phagocytes are in-volved in tolerance induction to cell-associated


Tanaka et al. Tolerance and apoptotic cell clearance

Figure 3. Migration of CD8�+, CD103+, CD207+ dendritic cells (DCs) into the T cell zone after phagocyotsis of apoptotic cells.Apoptotic W3 cells were intravenously injected into wild-type mice. Localization of CD207+ DCs was examined at the indicatedtime after injection.

antigens. It is most likely that macrophages andthe DC subset play distinct roles in tolerance in-duction to dead cell-associated antigens. However,macrophages phagocytosing apoptotic cells may ex-ert some effects in the function of the DC subset.We are now trying to elucidate the coordinationbetween the macrophages and DCs, which is nec-essary to achieve the tolerance induction to deadcell-associated antigens.

Immunological property of dead cellsFrom the data described above, we concluded thatapoptotic cells and their clearance actively con-tribute to suppression of or tolerance to cell-associated antigens. However, dead cells are notalways tolerogenic.25–27 For example, secondarynecrotic cells or subcutaneously injected apoptoticcells did not suppress EAE progression in the ex-perimental model described above. Furthermore,in contrast to the tolerance induction, dead cellscan be utilized for the induction of antitumor im-munity. Immunological consequence of dead cellsdepends on the nature of dead cells as well as type ofphagocyte that engulfs them. We recently reportedthat nonapoptotic macrophage death enhancedchemically induced tumorigenesis.28 It would beof great interest to reveal the mechanisms ofthe immune regulation by dead cells and theirclearance.

Acknowledgments

We are grateful to all the members of our labo-ratory at RIKEN Research Center for Allergy andImmunology.

Conflict of interest

The authors declare no conflict of interest.

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Immune regulation by apoptotic cell clearance