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in ApoptosisNucleosomes Are Exposed at the Cell Surface

Marko Radic, Tony Marion and Marc Monestier

http://www.jimmunol.org/content/172/11/6692doi: 10.4049/jimmunol.172.11.6692

2004; 172:6692-6700; ;J Immunol 

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Nucleosomes Are Exposed at the Cell Surface in Apoptosis1

Marko Radic,2* Tony Marion,* and Marc Monestier †

Apoptotic cells are considered the source of DNA, histones, and nucleoprotein complexes that drive the production of autoanti-bodies in systemic lupus erythematosus. However, the role of apoptotic cells in the activation of the immune system is not clear.To explore interactions that may initiate or sustain the production of anti-nuclear autoantibodies, we characterized the bindingof a large panel of monoclonal autoantibodies to apoptotic cells. Autoantibodies to DNA, individual core histones, histone-DNAcomplexes, or the native nucleosome core particle revealed a consistent and specific binding pattern in confocal microscopy.Immunoreactive epitopes were detected in the cytoplasm and accumulated along the surface of the fragmenting nucleus in acaspase-dependent manner. Ag-Ab complexes on nuclear fragments that had emerged from the plasma membrane were accessibleto anti-isotype-reactive microparticles. Moreover, autoantibodies specific for the nucleosome core or its molecular componentsselectively precipitated a complex of core histones and DNA from the cytosol at 4 h after induction of apoptosis. These observationsidentify distinct steps in the release of nucleosomes from the nucleus and their exposure at the cell surface. Furthermore, theresults indicate a direct role for nucleosomes in the execution of apoptosis, clearance of apoptotic cells, and regulation of anti-nuclear autoantibody production. The Journal of Immunology, 2004, 172: 6692–6700.

T wo fundamental principles of lupus pathogenesis are wellestablished. 1) The autoimmune response is directedagainst nuclear Ags, most notably DNA, histones, and

their complexes (1). 2) B cells producing anti-nuclear autoantibod-ies (ANA)3 are activated by and selected for binding to these selfAgs (2). Because binding is directed toward nucleoprotein com-plexes, the precise conformation and arrangement of such com-plexes may be important in driving B cell activation. However, itis impossible to test this hypothesis without knowing more aboutthe initial encounter between B cells and nuclear Ags.

Accumulating evidence implicates the process of apoptosis inthe induction of autoimmune disease. Deficiencies in serum pro-teins that assist in the recognition and clearance of apoptotic cellsincrease the risk of systemic autoimmune disease (3, 4). Mice withmutations in the structurally related receptor tyrosine kinases Mer,Axl, and Tyro3 have impaired apoptotic cell clearance and in-creased incidence of autoimmunity (5, 6). Homozygous deletionsof Mer impede uptake of apoptotic cells and result in increasedautoantibody titers (5), whereas disruptions of two or all threekinase activities are additive and yield pathologies that are char-acteristic of diverse autoimmune diseases (6).

Impaired clearance of apoptotic cells may predispose to auto-immunity by increasing the availability of cells that have pro-gressed to more advanced stages of cell death. Apoptosis consists

of a sequential series of reactions in which cysteine proteases(caspases) and other enzymes that are active in apoptosis modifytheir specific substrates. In addition, morphological changes estab-lish new molecular associations and rearrange cellular contents. Asa result, cells in more advanced stages of apoptosis are likely toyield a greater diversity of altered self Ags.

This view is supported by the observation that, in apoptosis,many lupus autoantigens are modified (7), clustered, and redistrib-uted into surface protrusions called blebs (8). One particularly dra-matic structural transition is the condensation and fragmentation ofthe nucleus and the movement of nuclear fragments to the cellsurface (9). However, it is not known whether and how the mod-ification and redistribution of nuclear Ags contribute to the induc-tion of ANA.

Opportunities for interactions between apoptotic cells and lym-phocytes arise throughout development and maturation of the im-mune response: apoptosis helps to curtail the development of au-toreactive B and T cells, eliminate ineffective lymphocytes, andregulate the extent of an immune response (10). As B cells shareanatomic sites with apoptotic cells during development, Ig-medi-ated binding of immature B cells to apoptotic cells in the bonemarrow may offer a suitable opportunity to avert autoimmunityand induce tolerance (11).

Alternatively, encounter between lymphocytes and apoptoticcells in peripheral tissues may breach tolerance and account forAbs that arise in autoimmune disease. Clearance defects may con-tribute to this outcome by providing tissue-specific autoantigens tocirculating B cells. Positive selection of B cells for binding toproteolipid complexes that assemble at the cell surface during ap-optosis and contain serum proteins, such as �2GPI or prothrombin,suggests stimulation of autoreactive B cells by apoptotic cells (12).This idea is supported by the fact that administration of apoptoticcells to nonautoimmune mice can induce the transient expressionof anti-phospholipid autoantibodies (13, 14). Nonetheless, it is notclear where and in what form nuclear Ags become accessible to theimmune system. The B cell response to nuclear Ags may criticallydepend on whether the Ags spill from the apoptotic cell in a hap-hazard manner or whether they are displayed at the apoptotic cellsurface as a result of an organized redistribution of nuclearcontents.

*Department of Molecular Sciences, University of Tennessee Health Sciences Center,Memphis, TN 38163; and †Department of Microbiology and Immunology, TempleUniversity School of Medicine, Philadelphia, PA 19140

Received for publication January 8, 2004. Accepted for publication March 18, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the National Institute of Allergy and Infectious Dis-eases (AI054938), an institutional grant from the University of Tennessee HealthSciences Center, the Center of Excellence in Structural Biology, the Center of Ex-cellence for Diseases of Connective Tissue, and the University of Tennessee Rheu-matic Disease Research Core Center of the National Institutes of Health.2 Address correspondence and reprint requests to Dr. Marko Radic, Department ofMolecular Sciences, University of Tennessee Health Science Center, 858 MadisonAvenue, Memphis, TN 38163. E-mail address: mradic@utmem.edu3 Abbreviations used in this paper: ANA, anti-nuclear Ab; DFF, DNA fragmentationfactor; z-VAD-(OMe)-fmk, Z-Val-Ala-Asp(OMe)-fluormethylketone.

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Previously, we explored interactions between apoptotic cellsand variants of a murine lupus autoantibody, 3H9 (15, 16). Al-though it has been reported that 3H9 does not bind DNA (17), theoverwhelming evidence indicates 3H9 is a multireactive Ab thatbinds DNA, nucleosomes, and anionic phospholipids (18). As istypical of human and murine anti-DNA autoantibodies, 3H9 ac-quired cationic residues during V, D, and J rearrangement andretained somatic mutations that improve binding to DNA and nu-cleosomes (19, 20). The 3H9 therefore embodies characteristics ofAg-selected ANA. In this study, we present the results of experi-ments designed to identify apoptotic cell ligands that mediate thebinding of 3H9 itself, its variants, and additional murine lupusautoantibodies. We report that autoantibodies to the nucleosomecore particle or to its individual components share the ability torecognize nuclear fragments that emerge as blebs at the surface ofapoptotic cells. Nucleosomes thus become available for interac-tions with the immune system as part of an organized and tightlyregulated series of morphological changes that define programmedcell death.

Materials and MethodsAntibodies

The 3H9 hybridoma was grown and the 3H9 Ab was purified, as described(21). The 3H9 VDJ was isolated as a 4.4-kb genomic EcoRI DNA fragmentand subjected to site-directed mutagenesis (19). The clone containing re-placements of aspartate 56 and serine 76 to arginines was designated asD56R/S76R VDJ and used to construct the D56R/S76RC�2b expressionplasmid (19). To construct the 3H9C�s plasmid, the 3H9 VDJ was ligatedto a 9.8-kb fragment of BALB/c genomic DNA containing the C� C regionexons and the polyadenylation site for the C terminus of the secreted IgM.Both H chain expression vectors were electroporated into the J558L plas-macytoma line using conditions previously described (19). Stable trans-fectants were isolated, secreted Abs were characterized by ELISA, andproductive clones were grown in culture.

Anti-DNA Abs from NZB � NZW F1 mice used in this study have beendescribed before (22, 23). Their specificities are summarized in Table I.Additional autoantibodies from diverse strains of mice that react againstnucleosome core particles, subnucleosome complexes, or individual nu-cleosome components (24–30) were used and are listed in Table II. As acontrol, we also used FC3, a previously described anti-cardiolipin autoan-tibody (31). These Abs were purified by protein G-Sepharose chromatog-raphy according to established procedures.

Cell culture and induction of apoptosis

Jurkat cells were grown, as described (16), and apoptosis was induced forbetween 4 and 16 h by the addition of 2.0 �M camptothecin (Sigma-Aldrich, St. Louis, MO), or 200 ng/ml anti-Fas Ab (clone 7C11; Beckman

Coulter, Brea, CA). To inhibit apoptosis, cultures were preincubated for 2 hwith 20 �M z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk; En-zyme System Products, Livermore, CA) before addition of the apoptosisinducer. At the end of the incubation period, 5 � 105 cells were countedand used in each binding reaction.

Ab-binding assays

Cells were washed in HBSS (Mediatech, Herndon, VA), supplemented to3 mM CaCl2, and fixed for 15 min in ice-cold 6% paraformaldehyde (Elec-tron Microscopy Sciences, Ft. Washington, PA) that was freshly preparedin the same buffer. The fixation step was performed early in our procedurebecause we observed that apoptotic blebs are unstable and retract in phys-iological buffer over time. Fixed cells were diluted in HBSS and 3 mMCaCl2, pelleted at 1600 rpm for 5 min, and blocked by resuspension inwash buffer (HBSS containing 3 mM CaCl2, 3% FBS, and 0.02% azide) for5 min. Cells were pelleted again and resuspended in the appropriate dilu-tion of the primary Ab in wash buffer. The working dilution for purifiedAbs was 20 �g/ml. Following incubation with the primary Ab, cells werewashed in wash buffer, pelleted as above, and incubated in a mixture ofAlexa Fluor 647 rabbit anti-mouse IgG (H � L) antisera (1/100 dilution),SYTOX Orange DNA stain (1/10,000 dilution), and Alexa Fluor 488 an-nexin V (1/70 dilution). In experiments using the 3H9/�1 IgM Ab, wedetected binding by using Alexa Fluor 647 goat anti-mouse IgM (�-chain-specific) antisera (1/100 dilution). All secondary reagents and stains wereobtained from Molecular Probes (Eugene, OR). Following incubation onice for 20 min, cells were washed as above and resuspended in wash buffercontaining 50% glycerol before mounting on 24-well, Teflon-printed mi-croscope slides (Electron Microscopy Sciences).

In control experiments designed to test the effect of membrane perme-abilization on Ab binding, we introduced two additional steps into theprotocol outlined above. Immediately following paraformaldehyde fixa-tion, cells were centrifuged, resuspended, and incubated in HBSS contain-ing 0.1% Triton X-100 for 15 min on ice. Following this incubation, cellswere washed in plain HBSS and blocked in wash buffer before proceedingwith Ab binding, as described above.

Experiments to determine whether Abs bound to the surface of apoptoticcells used 3H9 or LG4-1 (Table II), followed by incubation in the mixtureof fluorescent reagents listed above to which we added biotinylated goatanti-mouse IgG (H � L) Abs (1/100 dilution; Southern BiotechnologyAssociates, Birmingham, AL). Subsequently, cells were washed, pelleted,and resuspended in binding buffer containing 1.8 � 107 Neutr-Avidin-labeled 1.0-�m, yellow-green fluorescent microspheres (Fluospheres; Mo-lecular Probes). Cells were incubated for 20 min on ice and washed beforeproceeding with microscopy.

Confocal microscopy

Samples were viewed on a Zeiss LSM 510 laser-scanning microscope (CarlZeiss, Thorwood, NY), by using a �40, Plan-Apochromat oil-immersionlens, and excitation at 488, 543, and 633 nm. Detection channels recordedfluorescence emission above 650 nm for Alexa Fluor 647 (displayed as redfor consistency with our previous experiments (16)), between 560 and 615nm for SYTOX Orange (displayed as blue), and between 505 and 530 nm

Table I. Autoantibodies from (NZB � NZW)F1 mice

Ab ssDNASpecificitya

dsDNA Nucleosome Isotypeb Reference

17s.86 ��� � ���� IgG2a 2217s.115 ��� � ��� IgG2b 2217s.161 ���� � � IgG2a 22111s.109 ��� �� ���� IgG2a 22163p.106 �� ��� ��� IgG2a 22163p.64 ��� �� ��� IgG2a 22163p.119 ���� ���� ���� IgG2a 22452p.132 ��� � ��� IgG2a 23452s.88 �� ��� ���� IgG2a 23

a All hybridomas were originally selected for mAb binding to solid-phase DNA. dsDNA was sheared calf-thymus DNAsubsequently treated with S1 nuclease. ssDNA was denatured by heat. Binding to ssDNA and dsDNA was determined bycompetitive ELISA. �, No binding to competitor; �, 50% inhibition of maximum binding to solid-phase DNA by �10 �g/mlcompetitor; ��,�2 �g/ml competitor; ���, �1 �g/ml competitor; and ����, �0.10 �g/ml competitor. The nucleosomeAg was isolated from the mouse P3 � 63-Ag8.653 cell line. Nucleosome binding was determined by direct ELISA. �, Nobinding; �, 50% of maximum binding to solid-phase Ag by �10 �g/ml mAb; ��, �1 �g/ml mAb; ���, �0.10 �g/ml mAb;and ����, �0.01 �g/ml mAb

b H chain isotype. All of the hybridomas produced �L chains.

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for Alexa Fluor 488 (displayed as green). Consecutive images were col-lected at intervals of between 0.4 and 0.8 �m to assemble complete three-dimensional representations of apoptotic cells.

SDS-PAGE and immunoprecipitation

Apoptosis was induced with camptothecin, as outlined above, and sampleswere withdrawn at various time points thereafter. Control cultures werepreincubated with z-VAD-fmk before addition of camptothecin. Cells wereharvested by centrifugation, washed in HBSS, and incubated at 108

cells/ml in hypotonic buffer containing 10 mM HEPES (pH 7.4), 10 mMNaCl, 1 mM EDTA, and 1 mM DTT for 15 min on ice. At that time,Nonidet P-40 was added to a final concentration of 0.5%, cells were vig-orously vortexed for 30 s, and the soluble cytoplasmic fraction was recov-ered as the supernatant following centrifugation for 5 min at 13,000 rpm.Proteins from the cytoplasmic fraction were analyzed by 15% denaturingPAGE, followed by Coomassie blue staining.

The cytoplasmic fraction prepared at 4 h after addition of camptothecinwas used for immunoprecipitations after adjustment of buffer to PBS andaddition of purified mAbs to 100 �g/ml final concentration. The bindingreactions were incubated on ice for 1 h. Separately, agarose beads conju-gated to goat anti-mouse IgG (Sigma-Aldrich) were incubated in PBSbuffer containing 1% BSA and washed in PBS containing 0.02% SDS and2.5 mM EDTA. The beads were added to the cytoplasmic extracts, and theimmune complexes were allowed to adsorb to the beads overnight at 4°C.The beads were briefly centrifuged to remove unbound proteins andwashed in PBS containing 0.01% SDS and 2.5 mM EDTA, and boundproteins were eluted in 2� gel-loading buffer. The eluates were analyzedon SDS-PAGE, as described above.

ResultsThe 3H9 and its variants bind to apoptotic blebs

As a first step toward identifying ligands that mediate autoantibodybinding to apoptotic cells, we asked whether differences in affinityor specificity among the many available variants of 3H9 correlatewith the binding to cells in apoptosis. Previously, we had used avariant of 3H9, the D56R/S76R single chain variable fragment, forbinding to apoptotic cells (15, 16). The D56R/S76R H chain differsfrom the 3H9 H chain by having arginines instead of the asparticacid at position 56 and the serine at position 76. As result of thesesubstitutions, D56R/S76R binds to DNA and phosphatidylserinewith higher relative affinity than 3H9 (15, 19). Binding of D56R/S76R was specific for cells in apoptosis and localized to surfaceblebs containing nuclear fragments (16).

To examine whether 3H9 itself binds apoptotic cells, apoptosiswas induced in Jurkat cells by treatment with camptothecin. The3H9 bound between 5 and 20% of all cells as early as 4 h afteraddition of camptothecin. Immunoreactive cells were mostly in theexecution stage of apoptosis, a stage characterized by condensationof chromatin and fragmentation of the nucleus (Fig. 1A). Previ-

ously, we had used flow cytometry, a technique that allows theanalysis of unfixed cells, and observed that a similar percentage ofcells bound our Ab (16). Therefore, we consider it unlikely thatfixation appreciably affects Ab binding. Confocal microscopy re-vealed that binding was specific for the periphery of nuclear frag-ments and preceded the complete fragmentation of the nucleus(Fig. 1A). In contrast, no binding of 3H9 to the interior of nuclearfragments was observed. Some nuclear fragments reacting with3H9 were at or near the cell surface, whereas others appeared wellwithin the perimeter of the cell. The location of the plasma mem-brane was determined by staining with fluorescent annexin V, aserum protein that binds phosphatidylserine after its exposure onthe cell surface in apoptosis. The plasma membrane appeared dis-organized in some areas of the cell surface, exhibiting gaps thatmay allow access of macromolecules to the cell interior (Fig. 1A).

We next examined whether different L chain V regions or Hchain isotypes are compatible with binding to apoptotic blebs. Forthat purpose, the 3H9 VH was expressed as part of an IgM H chainand the D56R/S76R VH as part of an IgG2b H chain. Both Hchains were combined with V�1, in place of the original 3H9 Lchain, V�4. Previous experiments had established that V�1 iscompatible with DNA binding when paired with either 3H9 orD56R/S76R IgG2b H chain (19). Like 3H9 itself, both H/L com-binations bound the surface of nuclear fragments (Fig. 1, B and C).Moreover, Ab binding was particularly strong along the peripheryof fragments protruding from the cell surface. In such cases, do-mains of nuclear fragments reacting with Abs abutted sharplyagainst the annexin V-labeled domains of the plasma membrane.In addition, a partially emerged nuclear fragment exhibited a con-striction near the base of the exposed portion of the bleb (Fig. 1B).These observations suggest that nuclear fragments are in tight ap-position with the plasma membrane as they emerge at the cellsurface.

To test whether the nuclear envelope restricts access to Agscontained in nuclear fragments, we conducted experiments thatincluded a Triton X-100 membrane permeabilization step. Strongand uniform binding to the inside and the periphery of nuclearfragments was observed only when detergent was used before theaddition of Ab (Fig. 1D). The observed binding was reminiscent ofthe homogeneous nuclear pattern seen with fixed and detergent-treated cells in assays used to identify ANA in lupus. The 3H9 isANA positive by this criterion (21).

The binding to the nuclear interior in detergent-treated cellssharply contrasted with the exclusive binding to the periphery of

Table II. Autoantibodies to individual histones, subnucleosome complexes, and the nucleosome coreparticle

mAb Specificity Strain Isotype Reference

PL9-11 DNA MRL/� IgG3 24BWA3 H2A and H4 B � W/F1 IgG1 25LG11-2 H2B MRL/lpr IgG2a 26LG2-2 H2B MRL/lpr IgG2a 26PR1-1 H2B MRL/lpr IgG2b 26LG2-1 H3 MRL/lpr IgG2a 25LG10-1 H3-H4-DNA MRL/lpr IgG2b 27PL2-6 H2A-H2B-DNA MRL/� IgG2b 28PL9-3 H2A-H2B-DNA MRL/� IgG2a 24LG8-1 H2A-H2B-DNA MRL/lpr IgG2b 27NZA2 NCPa NZB IgG2a 27LG4-1 NCP MRL/lpr IgG2a 29MGC23 NCP MRL/lpr IgG2b 27MRA12 H1 MRL/lpr IgG2a 30

a NCP, nucleosome core particle.

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nuclear fragments in cells that were maintained in physiologicalbuffer throughout the experiment (Fig. 1, A–C). This observationindicates that the surface of nuclear fragments is accessible to Abs,whereas binding to the nuclear interior requires detergent treat-ment. Alternatively, the packing or conformation of nucleosomesin the nucleus is refractory to Ab binding, unless altered by deter-gent. Controls confirmed that Ab binding to nuclear fragments wasspecific, as secondary anti-mouse Abs alone did not produce de-tectable binding (Fig. 1E). Moreover, Ab binding required caspaseactivity, as pretreatment of cells with the broad caspase inhibitorz-VAD-fmk prevented binding (Fig. 1F).

Monoclonal autoantibodies from NZB � NZW F1 mice bindapoptotic blebs

To examine whether binding to apoptotic cells is a general featureof murine ANA, we selected a diverse set of autoantibodies fromfemale animals derived by crossing NZB and NZW parentalstrains of mice. Like autoimmune MRL/lpr mice, the strain fromwhich 3H9 was derived, NZB � NZW F1 mice also exhibit anAg-driven, oligoclonal Ab response to DNA, nucleosomes, andhistones (22, 23). We selected nine previously described autoan-tibodies that showed widely different relative preference forssDNA vs dsDNA and variable binding to nucleosomes (Table I).For example, 163p.119 bound strongly to ssDNA and dsDNA aswell as nucleosomes, whereas 17s.161 exclusively bound ssDNA.Overall, all Abs bound ssDNA, four had little or no detectablebinding to dsDNA, and all but one bound nucleosomes. In addi-tion, the V gene used in these Abs was diverse, involving repre-sentatives from four VH families and five V� groups.

Despite the differences in structure and fine specificity amongthis group of ANA, all exhibited specific binding to the peripheryof nuclear fragments (Fig. 2, A–I). The Abs allowed us to visualizemorphologies corresponding to the different stages of apoptosis.For example, some images showed Abs bound to the periphery ofnuclear fragments that formed blebs at the cell surface (Fig. 2, A–Gand I), whereas others showed binding to fragments in the cellinterior (e.g., Fig. 2, H and I). Collectively, the images allowed usto identify successive stages in the process of nuclear fragmenta-tion, the migration of nuclear fragments to the cell surface, theprotrusion of fragments from the plasma membrane, and the sep-aration of apoptotic bodies from the remainder of the dying cell.

Because systemic autoimmunity in humans and mice manifestsin the production of a wide variety of autoreactive Abs, we com-pared ANA with autoantibodies directed against phospholipids. Asa representative anti-phospholipid autoantibody, we selected FC3,an anti-cardiolipin autoantibody (31). Previously, it had been re-ported that anti-cardiolipin Abs bind to the surface of apoptoticcells (32). The FC3 Ab bound the apoptotic cell surface in a punc-tate pattern that partially overlapped with the binding of annexinV, but showed little or no overlap with the distribution of nuclearfragments (Fig. 2J). In contrast, an IgG1/� Ab without self-spec-ificity gave no detectable binding to apoptotic cells in our assay(Fig. 2K).

Autoantibodies to nucleosomes bind nuclear fragments andapoptotic blebs

Autoantibodies in systemic lupus erythematosus and murine mod-els of this disease often recognize more than one nuclear Ag. The3H9, for example, binds DNA and nucleosomes. Careful fraction-ation of nuclear Ags allows screening for autoantibodies to nu-cleosomes, subnucleosomes, individual histones, or DNA (24–30).In this study, we used autoantibodies that preferentially bind DNAor the isolated histones H2B or H3, an Ab to a shared epitope inhistones H2A and H4, as well as Abs to the two subnucleosomecomplexes H2A/H2B/DNA and H3/H4/DNA (Table II). Remark-ably, each of these Abs bound the surface of nuclear fragments(Fig. 3, A–F). Most notably, autoantibodies to the complete nu-cleosome core particle also exhibited the same binding pattern(Fig. 3G). The observed binding was most intense along nuclearfragment surfaces that protruded from the cell surface (Fig. 3, A, F,and G).

A dramatic exception to the shared binding specificity of anti-histone autoantibodies was observed with the anti-H1 histone AbMRA12. This Ab bound in a diffuse, granular pattern throughoutthe nucleus at an early stage of apoptosis (Fig. 3H). In contrast, we

FIGURE 1. The 3H9 and its variants bind to nuclear fragments. Jurkatcells treated with camptothecin were fixed and incubated with 3H9 (A), orAbs composed of the D56R/S76R IgG2b H chain and the �1 L chain (B),or the 3H9 IgM H chain and the �1 L chain (C), before detection of boundAbs with anti-mouse Abs (displayed in red). Each of the Abs bound nearthe surface of nuclear fragments, and binding was most intense along theside facing the cell’s exterior. DNA, bound by Sytox Orange, is displayedin blue, and annexin V in green. Controls included cells treated with 0.1%Triton X-100 before binding of 3H9 (D), or cells incubated in the absenceof primary Abs (E). Ab binding required caspase activity, as cells pre-treated with z-VAD-fmk failed to bind Abs (F). In addition to the com-posite color image, corresponding separate color images are shown forA–D. These and the following images represent individual cross-sectionstaken from complete three-dimensional serial sections of each cell.

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could not detect MRA12 binding in exponentially growing cells,nor in cells that had initiated nuclear fragmentation. These results,in combination with the results of others (33), suggest that one ormore of the histone H1 subtypes may become accessible, modified,or mobilized in a transient manner at an early stage of apoptosis.The binding of MRA12 to the nuclear interior underscores theunique reactivity of the surface of nuclear fragments with autoan-tibodies to the nucleosome core particle. This result suggests aninteresting and testable possibility: nucleosomes that are arrayed atthe surface of nuclear fragments may expose structural epitopesthat are particularly favorable targets for binding of ANA.

Detection of nucleosomes in the cytoplasm and on the surface ofapoptotic cells

The earliest evidence of nucleosome core Ags outside of the ap-optotic cell nucleus was a diffuse granular pattern of 3H9 binding

throughout the cytoplasm of cells at 3 h following camptothecinaddition (Fig. 4A). Only certain segments of the nuclear peripheryreacted with the Ab at this time. Because of the brief time that hadelapsed since the induction of apoptosis, and the partial immuno-reactivity of the nuclear periphery, we infer that the diffuse cyto-plasmic staining corresponds to a distinct, initial stage of a pre-sumed pathway that transports nucleosomes from the apoptoticnucleus to the cell surface.

To confirm that nucleosome core particles exit the nucleus at thisstage of apoptosis, we examined cytoplasmic extracts by gel electro-phoresis and immunoprecipitation. Our analysis revealed the simul-taneous increase in the abundance of four proteins corresponding insize to the core histones (Fig. 4B). In fact, this was the most notablechange in the protein composition of the apoptotic cytoplasm thatoccurred between 2 and 4 h after addition of camptothecin. Previ-ously, Wu et al. (34) had observed that histones from apoptotic Jurkat

FIGURE 2. Autoantibodies from (NZB � NZW) F1 mice bind to the surface of nuclear fragments and to blebs formed by protruding nuclear fragments.Abs whose Ag preference was determined by ELISA (Table I) were used to examine specificity for molecular features of apoptotic cells. Results wereobtained using 17s.86 (A), 17s.115 (B), 17s.161 (C), 111s.109 (D), 163p.106 (E), 163p.64 (F), 163p.119 (G), 452p.132 (H), and 452s.88 (I). Most nuclearfragments (blue) are recognized along their surface by the different ANA (red), although some nuclear fragments remain unstained (B and F–I) and otherspredominantly show binding along the surfaces exposed to the exterior of the cell (A, C, and G). The almost complete separation of a nuclear fragmentfrom the remainder of the cell may represent a transition from a bleb to an apoptotic body (C). For comparison, FC3, an anti-cardiolipin autoantibody froman NZW � BSXB F1 mouse (J), or an isotype-matched nonautoreactive IgG (K) was used. FC3 bound to discrete domains of the plasma membrane thatdid not overlap with nuclear fragments. Binding of 163p.119 (G) is also shown in a set of three perpendicular sections (L).

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cells are more easily solubilized. The appearance of histones in thecytoplasm could be blocked by the broad caspase inhibitor, z-VAD-fmk (Fig. 4B).

To establish whether the cytoplasmic histones remained assem-bled into a particle resembling the nucleosome, we conducted im-munoprecipitations with three Abs that recognize differentepitopes of the core particle. Precipitates obtained with the anti-DNA Ab PA4, the anti-H2B Ab LG2-2, or the anti-nucleosome AbLG4-1 contained all four core histone proteins (Fig. 4C), suggest-ing that these individual components of the core particle are as-sembled in a complex that is structurally related to the nucleosome.Analysis of the DNA from the immunoprecipitate showed thepresence of fragments whose size was consistent with a nucleoso-mal ladder (data not shown).

To determine whether, at the cell surface, nuclear autoantigensremain shielded by a membrane, or exposed and available for di-rect macromolecular contacts, we used fluorescent microbeads asindicators for the ability of nucleosome Ags to act as ligands forcellular receptors. Following binding of either of two Abs, 3H9(Fig. 4D) or LG4-1 (Fig. 4E), to apoptotic cells, we added a mix-ture of fluorescent and biotinylated anti-mouse IgG Abs. The mix-ture of reagents allowed us to visualize the distribution of the pri-mary Abs while testing whether avidin-coated, fluorescentmicrospheres could access the bound Abs. The binding sites of3H9 and LG4-1 on the apoptotic cell surface were indeed acces-sible to the microspheres (Figs. 4, D and E). In contrast, no bindingwas observed in the absence of primary Abs (data not shown), orin the absence of biotinylated goat anti-mouse Abs (Fig. 4F).These results conclusively demonstrate that nucleosome core par-ticles are exposed at the surface of apoptotic blebs.

DiscussionMany scenarios have been envisioned for the interaction betweennucleosomal autoantigens and the immune system. That such in-teractions are relevant is implied by the fact that autoantibodies toDNA and histones are the defining characteristics of systemic lu-pus. That such interactions must occur is evident from the molec-ular characteristics of such autoantibodies that can only be ex-plained by positive Ag selection. To uncover the mechanism ofanti-nuclear autoantibody selection, we have relied on murine lu-pus autoantibodies. We find that cells in apoptosis release nucleo-somes into the cytoplasm and attach them to the outside of nuclearfragments. Subsequently, nuclear fragments migrate to the cell sur-face, break through the plasma membrane, and separate from theremainder of the dying cell. Nucleosomes thus become accessiblefor interactions with receptors, including B cell receptors, at thesurface of blebs and apoptotic bodies. The results point to anovel role for nucleosomes in apoptosis, clearance, and immuneregulation.

The pathway of nucleosome externalization at the cell surfacerequires a detailed integration with other events in apoptosis. Weshow that recovery of nucleosomes from the cytoplasm is pre-vented by inhibition of caspases (Fig. 4B), implying the require-ment for upstream enzymatic activity. Cleavage of apoptotic chro-matin by nucleases, such as DNA fragmentation factor 40 (DFF40)(35), may be the required event. Proteolysis of the DFF40-associ-ated chaperone DFF45 by caspase 3 allows the catalytic subunit todegrade linker DNA. Inhibition of DFF40 activity inhibits chro-matin condensation and nuclear fragmentation (35). Conceivably,cleavage of linker DNA contributes to the separation of a subset ofnucleosomes from chromatin and its exit from the nucleus.

Coincident with the appearance of cytoplasmic nucleosomes,the H1.2 linker histone is also released from the nucleus (36). Thecytoplasmic H1.2 is capable of signaling to the mitochondria to

FIGURE 3. Autoantibodies to individual core histones or components ofthe nucleosome core particle bind the surface of nuclear fragments and apo-ptotic blebs. Autoantibodies listed in Table II were used for binding to apo-ptotic Jurkat cells. Representative images obtained with Abs to DNA (PL9-11;A), a shared epitope on histones H2A and H4 (BWA3; B), core histones H2B(LG11-2; C) or H3 (LG2-1; D), the subnucleosome complexes H3-H4-DNA(LG10-1; E) and H2A-H2B-DNA (LG8-1; F), or the complete nucleosomecore particle (NZA2; G) are shown. Binding at the surface of nuclear frag-ments was observed, and protruding nuclear fragments bound Abs along thesurface facing the cell’s exterior (A, F, and G). In contrast, binding of MRA12,a mAb to the linker histone H1, was limited to an early stage of apoptosis,before nuclear condensation and fragmentation, and resulted in a diffuse andgrainy distribution of signal throughout the nucleus (H).

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induce cytochrome c release and mediate additional signals in thecaspase cascade (36). It is not known how histone H1.2 or a com-plete nucleosome exits the nucleus. Because the execution phase ofapoptosis coincides with the increase in the permeability of thenuclear pores (37), export of nucleosomes may occur by passivediffusion or active transport. In either case, the opportunity fornucleosomes to exit the nucleus may only be transient becausenuclear pores coalesce and are degraded concomitant with thefragmentation of the nucleus (38).

Upon exiting the nucleus, nucleosomes disperse throughout thecytoplasm, then attach to binding sites on the outer membrane ofnuclear fragments (Fig. 4A). Nucleosomes attach before nuclearfragmentation is complete (Figs. 1A and 3E) and remain associatedwith the nuclear periphery through the transition of nuclear frag-ments into apoptotic bodies, independent membrane-bound parti-cles that form by separation of a bleb from the remainder of thecell (Fig. 2C). Therefore, the role of nucleosomes may include themovement of nuclear fragments to the cell surface and interactionswith the plasma membrane. The transport of nuclear fragments tothe plasma membrane is an active process that is likely to requirethe participation of the cytoskeleton. Transport is, at least in part,regulated by the activity of p160ROCK/ROK� (ROCK) I kinase,as inhibition of ROCK I limits the formation of surface blebs (16).It will be important to test whether known histone modificationsthat occur in apoptosis (39) favor attachment of cytoplasmic nu-

cleosomes to the nuclear envelope and contribute to interactionswith the cytoskeleton.

Nuclear fragments that have reached the cell surface and formedsurface blebs (Figs. 1C, 2G, and 3A) afford an opportunity to eval-uate the interaction between nuclear fragments and the plasmamembrane. The protruding portions of nuclear fragments reactwith our Abs and exhibit a sharp boundary with domains of theplasma membrane that react with annexin V, indicating an unusualinteraction between the nuclear envelope and the plasma mem-brane. As confirmed by the binding of the fluorescent beads toblebs (Fig. 4, D and E), the plasma membrane ceases to surroundthe nuclear fragments as they emerge at the cell surface. In addi-tion, nuclear Ags do not appreciably disperse onto the adjacentannexin V-reactive membrane domains, indicating that the plasmamembrane and the nuclear envelope do not fuse. Therefore, wefavor the alternative that the polyionic nucleosome arrays on thenuclear envelope disrupt the structural integrity of the plasmamembrane and facilitate the emergence of nuclear fragments at thesurface of the apoptotic cell.

Once at the surface, nucleosomes may assist in the recognitionand clearance of apoptotic remains. Professional phagocytes ex-press a variety of receptors capable of recognizing apoptotic cells(40). Among the receptors identified to date, the phosphatidylser-ine receptor characterized by Fadok et al. (41) assumes an impor-tant role in directing the uptake of apoptotic cells by phagocytes.

FIGURE 4. Distinct steps of nucleosome exposure in apoptotic cells. The earliest evidence of nucleosome-like particles in the cytoplasm was a diffuse,granular cytoplasmic binding of 3H9, observed 3 h after addition of camptothecin (A). A time course of nucleosome release was established by analyzingthe protein content of cytoplasmic extracts prepared at the indicated times after addition of camptothecin (B). The m.w. of marker proteins and theapproximate positions of the four core histones are indicated. The changes are caspase dependent, as shown by preincubation of cells with z-VAD-fmk.Immunoprecipitations established that Abs to DNA, histone H2B, or the complete nucleosome sequester equivalent amounts of all four core histones,whereas agarose beads alone are ineffective (C). Nonapoptotic cytosols did not contain appreciable amounts of precipitable core histones (data not shown).Nucleosome Ags become accessible at the cell surface, as shown by incubation of apoptotic cells with 3H9 (D) or the anti-nucleosome autoantibody LG4-1(E), followed by addition of a mixture of fluorescently labeled and biotinylated anti-mouse Abs and, in a subsequent step, the addition of yellow-greenfluorescent microbeads conjugated to avidin. D and E, Projection views constructed by merging the individual optical sections, to illustrate the arrangementof beads relative to blebs. An individual section demonstrates that Abs bound to the nuclear periphery are accessible to the fluorescent beads (inset of D).Beads failed to bind blebs when the biotinylated Abs were omitted, and the few beads that remained were randomly distributed (F). The bright fluorescenceemitted by the beads precluded the simultaneous detection of the annexin V-associated signal.

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Exposure of phosphatidylserine is one of the earliest indicationsthat cells are committed to die (42). However, recognition of phos-phatidylserine is not sufficient for uptake of apoptotic cells. Toinduce uptake, signaling via the phosphatidylserine receptor mustbe accompanied by binding to additional phagocyte receptors (43).Additional receptors that bind to apoptotic cells include Mer andthe related tyrosine kinases (5, 6), CD14 (44), the ABC-1 trans-porter (45), and the CD91-calnexin complex (46).

Binding of phagocyte receptors to apoptotic cells takes advan-tage of two families of structurally related serum proteins, the de-fense collagens (collectins) and the pentraxins (40). These proteinsact as bridging molecules for efficient disposal of cells and cellularparticles produced during more advanced stages of cell death. In-terestingly, members of the collectin family, such as C1q (47) andmannose-binding lectin (46), and the pentraxin family, such asC-reactive protein (48), and serum amyloid P (49), bind to blebs atthe apoptotic cell surface. Our demonstration that nucleosome coreparticles are exposed on apoptotic blebs raises the possibility thatbinding to nucleosomes assists in the recognition and clearance ofapoptotic cells. Indeed, in vitro, both serum amyloid P (3) andC-reactive protein (50) recognize chromatin as well as purifiednucleosome core particles. Similarly, binding of IgM Abs to nu-clear Ags on the surface of apoptotic cells may induce C1q dep-osition and increase clearance efficiency.

Direct evidence for a role of chromatin cleavage products inclearance comes from studies in Caenorhabditis elegans, in whichinactivation of apoptotic nucleases decreases the efficiency of ap-optotic cell uptake (51). Additional evidence for the role of nu-cleosomes in the recognition and clearance of apoptotic cellscomes from the observation that an excess of nucleosomes par-tially inhibits clearance of apoptotic cells by macrophage (52). Thesimplest interpretation of this experimental result is that nucleo-somes interact with receptors that mediate clearance.

Our observation that nucleosomes are displayed on the surfaceof apoptotic cells also has implications for understanding toleranceand autoimmunity. An immune response to nuclear Ags, includinghistones, DNA, and their complexes, is difficult to induce. Never-theless, the nucleosome core particle is the primary target of au-toantibodies in systemic lupus erythematosus. Moreover, autoan-tibodies to DNA, histones, and nucleosomes exhibit clear evidenceof positive selection for binding to these nuclear Ags. Presently,neither the mechanisms establishing tolerance nor the circum-stances that induce anti-nuclear autoantibody production are wellunderstood. Our observations may provide a structural basis for theimportance of nucleosomes in the induction of autoimmunity inlupus.

The tethered exposure of nucleosomes on the surface of apo-ptotic blebs offers a rationale for the rigorous tolerance to chro-matin Ags. Interactions between immature B cells and surface-exposed nucleosomes on a neighboring apoptotic cell are expectedto trigger strong stimuli toward central deletion. This expectationis in line with the fact that B cells with Ig H and L chain transgenesencoding 3H9 do not mature beyond the pre-B cell stage (11),unless they succeed in editing the 3H9 specificity by revision oftheir Ig receptor genes (53). Similarly, mice expressing the D56R/S76R H chain develop very few Ig-positive B cells, if receptorediting is suppressed (54). Tight binding to apoptotic cells in thebone marrow may therefore abort further B cell development.

Nevertheless, our results also establish that B cells with Ig re-ceptors for apoptotic cells may reach the peripheral immune or-gans of nonautoimmune mice. B cells with receptors composed ofthe 3H9 H chain and the �1 L chain (analogous to the Ab shownin Fig. 1C) reach the spleen, yet are profoundly anergized and

short-lived (55). Circumstances may arise when encounter withapoptotic cells stimulates activation and proliferation of such cells.

A key feature of Ig receptor-mediated activation is that interac-tions between Ig receptors and membrane-bound or particulateAgs generate far more potent signals than interactions with solubleAgs (56, 57). Signaling elicited by binding of apoptotic cells tonucleosome-specific Ig receptors may be particularly consequen-tial because nucleosome-containing immune complexes inducenear-maximal B cell proliferation (58). DNA from such immunecomplexes may engage Toll-like receptor-9 and generate signalsthat synergize with the signals generated by the Ig receptor. Thus,binding of B cells to blebs or apoptotic bodies is predicted togenerate powerful signals that may reverse anergy, stimulate pro-liferation, and induce Ab secretion. In that case, B cells with re-ceptors for apoptotic cells may provide a starting point for auto-immune responses.

AcknowledgmentsWe acknowledge helpful criticisms and comments from Martin Weigert,Antony Rosen, Joyce Rauch, Jan Erikson, Dr. Brian Cocca, and Amy Clinethat shaped the final text of the manuscript. Jan Erikson’s help was essen-tial in recovering the 3H9 IgM/� transfectoma for the work shown. Thanksto Tim Higgins, senior illustrator, for careful professional attention tographic representations of our experimental results.

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