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The Journal of Immunology, 2004, 172: 6692-6700.
Copyright © 2004 by The American Association of Immunologists

Nucleosomes Are Exposed at the Cell Surface in Apoptosis1

Marko Radic2,*, Tony Marion* and Marc Monestier{dagger}

* Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, TN 38163; and {dagger} Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apoptotic cells are considered the source of DNA, histones, and nucleoprotein complexes that drive the production of autoantibodies 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 binding of a large panel of monoclonal autoantibodies to apoptotic cells. Autoantibodies to DNA, individual core histones, histone-DNA complexes, 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 a caspase-dependent manner. Ag-Ab complexes on nuclear fragments that had emerged from the plasma membrane were accessible to anti-isotype-reactive microparticles. Moreover, autoantibodies specific for the nucleosome core or its molecular components selectively precipitated a complex of core histones and DNA from the cytosol at 4 h after induction of apoptosis. These observations identify distinct steps in the release of nucleosomes from the nucleus and their exposure at the cell surface. Furthermore, the results indicate a direct role for nucleosomes in the execution of apoptosis, clearance of apoptotic cells, and regulation of anti-nuclear autoantibody production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Two fundamental principles of lupus pathogenesis are well established. 1) The autoimmune response is directed against nuclear Ags, most notably DNA, histones, and their complexes (1). 2) B cells producing anti-nuclear autoantibodies (ANA)3 are activated by and selected for binding to these self Ags (2). Because binding is directed toward nucleoprotein complexes, the precise conformation and arrangement of such complexes may be important in driving B cell activation. However, it is impossible to test this hypothesis without knowing more about the initial encounter between B cells and nuclear Ags.

Accumulating evidence implicates the process of apoptosis in the induction of autoimmune disease. Deficiencies in serum proteins that assist in the recognition and clearance of apoptotic cells increase the risk of systemic autoimmune disease (3, 4). Mice with mutations in the structurally related receptor tyrosine kinases Mer, Axl, and Tyro3 have impaired apoptotic cell clearance and increased incidence of autoimmunity (5, 6). Homozygous deletions of Mer impede uptake of apoptotic cells and result in increased autoantibody titers (5), whereas disruptions of two or all three kinase activities are additive and yield pathologies that are characteristic of diverse autoimmune diseases (6).

Impaired clearance of apoptotic cells may predispose to autoimmunity by increasing the availability of cells that have progressed 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 modify their specific substrates. In addition, morphological changes establish new molecular associations and rearrange cellular contents. As a result, cells in more advanced stages of apoptosis are likely to yield 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 redistributed into surface protrusions called blebs (8). One particularly dramatic structural transition is the condensation and fragmentation of the nucleus and the movement of nuclear fragments to the cell surface (9). However, it is not known whether and how the modification and redistribution of nuclear Ags contribute to the induction of ANA.

Opportunities for interactions between apoptotic cells and lymphocytes arise throughout development and maturation of the immune response: apoptosis helps to curtail the development of autoreactive B and T cells, eliminate ineffective lymphocytes, and regulate the extent of an immune response (10). As B cells share anatomic sites with apoptotic cells during development, Ig-mediated binding of immature B cells to apoptotic cells in the bone marrow may offer a suitable opportunity to avert autoimmunity and induce tolerance (11).

Alternatively, encounter between lymphocytes and apoptotic cells in peripheral tissues may breach tolerance and account for Abs that arise in autoimmune disease. Clearance defects may contribute to this outcome by providing tissue-specific autoantigens to circulating B cells. Positive selection of B cells for binding to proteolipid complexes that assemble at the cell surface during apoptosis and contain serum proteins, such as {beta}2GPI or prothrombin, suggests stimulation of autoreactive B cells by apoptotic cells (12). This idea is supported by the fact that administration of apoptotic cells to nonautoimmune mice can induce the transient expression of anti-phospholipid autoantibodies (13, 14). Nonetheless, it is not clear where and in what form nuclear Ags become accessible to the immune system. The B cell response to nuclear Ags may critically depend on whether the Ags spill from the apoptotic cell in a haphazard manner or whether they are displayed at the apoptotic cell surface as a result of an organized redistribution of nuclear contents.

Previously, we explored interactions between apoptotic cells and variants of a murine lupus autoantibody, 3H9 (15, 16). Although it has been reported that 3H9 does not bind DNA (17), the overwhelming evidence indicates 3H9 is a multireactive Ab that binds DNA, nucleosomes, and anionic phospholipids (18). As is typical of human and murine anti-DNA autoantibodies, 3H9 acquired cationic residues during V, D, and J rearrangement and retained somatic mutations that improve binding to DNA and nucleosomes (19, 20). The 3H9 therefore embodies characteristics of Ag-selected ANA. In this study, we present the results of experiments designed to identify apoptotic cell ligands that mediate the binding of 3H9 itself, its variants, and additional murine lupus autoantibodies. We report that autoantibodies to the nucleosome core particle or to its individual components share the ability to recognize nuclear fragments that emerge as blebs at the surface of apoptotic cells. Nucleosomes thus become available for interactions with the immune system as part of an organized and tightly regulated series of morphological changes that define programmed cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

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 fragment and subjected to site-directed mutagenesis (19). The clone containing replacements of aspartate 56 and serine 76 to arginines was designated as D56R/S76R VDJ and used to construct the D56R/S76RC{gamma}2b expression plasmid (19). To construct the 3H9Cµs plasmid, the 3H9 VDJ was ligated to a 9.8-kb fragment of BALB/c genomic DNA containing the Cµ C region exons and the polyadenylation site for the C terminus of the secreted IgM. Both H chain expression vectors were electroporated into the J558L plasmacytoma line using conditions previously described (19). Stable transfectants were isolated, secreted Abs were characterized by ELISA, and productive clones were grown in culture.

Anti-DNA Abs from NZB x NZW F1 mice used in this study have been described before (22, 23). Their specificities are summarized in Table I. Additional autoantibodies from diverse strains of mice that react against nucleosome core particles, subnucleosome complexes, or individual nucleosome components (24, 25, 26, 27, 28, 29, 30) were used and are listed in Table II. As a control, we also used FC3, a previously described anti-cardiolipin autoantibody (31). These Abs were purified by protein G-Sepharose chromatography according to established procedures.


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

 

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

 
Cell culture and induction of apoptosis

Jurkat cells were grown, as described (16), and apoptosis was induced for between 4 and 16 h by the addition of 2.0 µM 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 h with 20 µM z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk; Enzyme System Products, Livermore, CA) before addition of the apoptosis inducer. At the end of the incubation period, 5 x 105 cells were counted and used in each binding reaction.

Ab-binding assays

Cells were washed in HBSS (Mediatech, Herndon, VA), supplemented to 3 mM CaCl2, and fixed for 15 min in ice-cold 6% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) that was freshly prepared in the same buffer. The fixation step was performed early in our procedure because we observed that apoptotic blebs are unstable and retract in physiological buffer over time. Fixed cells were diluted in HBSS and 3 mM CaCl2, pelleted at 1600 rpm for 5 min, and blocked by resuspension in wash buffer (HBSS containing 3 mM CaCl2, 3% FBS, and 0.02% azide) for 5 min. Cells were pelleted again and resuspended in the appropriate dilution of the primary Ab in wash buffer. The working dilution for purified Abs was 20 µg/ml. Following incubation with the primary Ab, cells were washed in wash buffer, pelleted as above, and incubated in a mixture of Alexa Fluor 647 rabbit anti-mouse IgG (H + L) antisera (1/100 dilution), SYTOX Orange DNA stain (1/10,000 dilution), and Alexa Fluor 488 annexin V (1/70 dilution). In experiments using the 3H9/{lambda}1 IgM Ab, we detected binding by using Alexa Fluor 647 goat anti-mouse IgM (µ-chain-specific) antisera (1/100 dilution). All secondary reagents and stains were obtained from Molecular Probes (Eugene, OR). Following incubation on ice for 20 min, cells were washed as above and resuspended in wash buffer containing 50% glycerol before mounting on 24-well, Teflon-printed microscope slides (Electron Microscopy Sciences).

In control experiments designed to test the effect of membrane permeabilization on Ab binding, we introduced two additional steps into the protocol outlined above. Immediately following paraformaldehyde fixation, cells were centrifuged, resuspended, and incubated in HBSS containing 0.1% Triton X-100 for 15 min on ice. Following this incubation, cells were washed in plain HBSS and blocked in wash buffer before proceeding with Ab binding, as described above.

Experiments to determine whether Abs bound to the surface of apoptotic cells used 3H9 or LG4-1 (Table II), followed by incubation in the mixture of fluorescent reagents listed above to which we added biotinylated goat anti-mouse IgG (H + L) Abs (1/100 dilution; Southern Biotechnology Associates, Birmingham, AL). Subsequently, cells were washed, pelleted, and resuspended in binding buffer containing 1.8 x 107 Neutr-Avidin-labeled 1.0-µm, yellow-green fluorescent microspheres (Fluospheres; Molecular Probes). Cells were incubated for 20 min on ice and washed before proceeding with microscopy.

Confocal microscopy

Samples were viewed on a Zeiss LSM 510 laser-scanning microscope (Carl Zeiss, Thorwood, NY), by using a x40, Plan-Apochromat oil-immersion lens, and excitation at 488, 543, and 633 nm. Detection channels recorded fluorescence emission above 650 nm for Alexa Fluor 647 (displayed as red for consistency with our previous experiments (16)), between 560 and 615 nm for SYTOX Orange (displayed as blue), and between 505 and 530 nm for Alexa Fluor 488 (displayed as green). Consecutive images were collected at intervals of between 0.4 and 0.8 µm to assemble complete three-dimensional representations of apoptotic cells.

SDS-PAGE and immunoprecipitation

Apoptosis was induced with camptothecin, as outlined above, and samples were withdrawn at various time points thereafter. Control cultures were preincubated with z-VAD-fmk before addition of camptothecin. Cells were harvested by centrifugation, washed in HBSS, and incubated at 108 cells/ml in hypotonic buffer containing 10 mM HEPES (pH 7.4), 10 mM NaCl, 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 vigorously vortexed for 30 s, and the soluble cytoplasmic fraction was recovered as the supernatant following centrifugation for 5 min at 13,000 rpm. Proteins from the cytoplasmic fraction were analyzed by 15% denaturing PAGE, followed by Coomassie blue staining.

The cytoplasmic fraction prepared at 4 h after addition of camptothecin was used for immunoprecipitations after adjustment of buffer to PBS and addition of purified mAbs to 100 µg/ml final concentration. The binding reactions were incubated on ice for 1 h. Separately, agarose beads conjugated to goat anti-mouse IgG (Sigma-Aldrich) were incubated in PBS buffer containing 1% BSA and washed in PBS containing 0.02% SDS and 2.5 mM EDTA. The beads were added to the cytoplasmic extracts, and the immune complexes were allowed to adsorb to the beads overnight at 4°C. The beads were briefly centrifuged to remove unbound proteins and washed in PBS containing 0.01% SDS and 2.5 mM EDTA, and bound proteins were eluted in 2x gel-loading buffer. The eluates were analyzed on SDS-PAGE, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The 3H9 and its variants bind to apoptotic blebs

As a first step toward identifying ligands that mediate autoantibody binding to apoptotic cells, we asked whether differences in affinity or specificity among the many available variants of 3H9 correlate with the binding to cells in apoptosis. Previously, we had used a variant of 3H9, the D56R/S76R single chain variable fragment, for binding to apoptotic cells (15, 16). The D56R/S76R H chain differs from the 3H9 H chain by having arginines instead of the aspartic acid at position 56 and the serine at position 76. As result of these substitutions, D56R/S76R binds to DNA and phosphatidylserine with higher relative affinity than 3H9 (15, 19). Binding of D56R/S76R was specific for cells in apoptosis and localized to surface blebs containing nuclear fragments (16).

To examine whether 3H9 itself binds apoptotic cells, apoptosis was induced in Jurkat cells by treatment with camptothecin. The 3H9 bound between 5 and 20% of all cells as early as 4 h after addition of camptothecin. Immunoreactive cells were mostly in the execution stage of apoptosis, a stage characterized by condensation of chromatin and fragmentation of the nucleus (Fig. 1A). Previously, we had used flow cytometry, a technique that allows the analysis of unfixed cells, and observed that a similar percentage of cells bound our Ab (16). Therefore, we consider it unlikely that fixation appreciably affects Ab binding. Confocal microscopy revealed that binding was specific for the periphery of nuclear fragments and preceded the complete fragmentation of the nucleus (Fig. 1A). In contrast, no binding of 3H9 to the interior of nuclear fragments was observed. Some nuclear fragments reacting with 3H9 were at or near the cell surface, whereas others appeared well within the perimeter of the cell. The location of the plasma membrane was determined by staining with fluorescent annexin V, a serum protein that binds phosphatidylserine after its exposure on the cell surface in apoptosis. The plasma membrane appeared disorganized in some areas of the cell surface, exhibiting gaps that may allow access of macromolecules to the cell interior (Fig. 1A).



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  		FIGURE 1. The 3H9 and its variants bind to nuclear fragments. Jurkat cells treated with camptothecin were fixed and incubated with 3H9 (A), or Abs composed of the D56R/S76R IgG2b H chain and the {lambda}1 L chain (B), or the 3H9 IgM H chain and the {lambda}1 L chain (C), before detection of bound Abs with anti-mouse Abs (displayed in red). Each of the Abs bound near the surface of nuclear fragments, and binding was most intense along the side facing the cell’s exterior. DNA, bound by Sytox Orange, is displayed in 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 absence of primary Abs (E). Ab binding required caspase activity, as cells pretreated with z-VAD-fmk failed to bind Abs (F). In addition to the composite color image, corresponding separate color images are shown for A–D. These and the following images represent individual cross-sections taken from complete three-dimensional serial sections of each cell.

 
We next examined whether different L chain V regions or H chain isotypes are compatible with binding to apoptotic blebs. For that purpose, the 3H9 VH was expressed as part of an IgM H chain and the D56R/S76R VH as part of an IgG2b H chain. Both H chains were combined with V{lambda}1, in place of the original 3H9 L chain, V{kappa}4. Previous experiments had established that V{lambda}1 is compatible with DNA binding when paired with either 3H9 or D56R/S76R IgG2b H chain (19). Like 3H9 itself, both H/L combinations bound the surface of nuclear fragments (Fig. 1, B and C). Moreover, Ab binding was particularly strong along the periphery of fragments protruding from the cell surface. In such cases, domains of nuclear fragments reacting with Abs abutted sharply against the annexin V-labeled domains of the plasma membrane. In addition, a partially emerged nuclear fragment exhibited a constriction near the base of the exposed portion of the bleb (Fig. 1B). These observations suggest that nuclear fragments are in tight apposition with the plasma membrane as they emerge at the cell surface.

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

The binding to the nuclear interior in detergent-treated cells sharply contrasted with the exclusive binding to the periphery of nuclear fragments in cells that were maintained in physiological buffer throughout the experiment (Fig. 1, A–C). This observation indicates that the surface of nuclear fragments is accessible to Abs, whereas binding to the nuclear interior requires detergent treatment. Alternatively, the packing or conformation of nucleosomes in the nucleus is refractory to Ab binding, unless altered by detergent. Controls confirmed that Ab binding to nuclear fragments was specific, as secondary anti-mouse Abs alone did not produce detectable binding (Fig. 1E). Moreover, Ab binding required caspase activity, as pretreatment of cells with the broad caspase inhibitor z-VAD-fmk prevented binding (Fig. 1F).

Monoclonal autoantibodies from NZB x NZW F1 mice bind apoptotic blebs

To examine whether binding to apoptotic cells is a general feature of murine ANA, we selected a diverse set of autoantibodies from female animals derived by crossing NZB and NZW parental strains of mice. Like autoimmune MRL/lpr mice, the strain from which 3H9 was derived, NZB x NZW F1 mice also exhibit an Ag-driven, oligoclonal Ab response to DNA, nucleosomes, and histones (22, 23). We selected nine previously described autoantibodies that showed widely different relative preference for ssDNA vs dsDNA and variable binding to nucleosomes (Table I). For example, 163p.119 bound strongly to ssDNA and dsDNA as well as nucleosomes, whereas 17s.161 exclusively bound ssDNA. Overall, all Abs bound ssDNA, four had little or no detectable binding to dsDNA, and all but one bound nucleosomes. In addition, the V gene used in these Abs was diverse, involving representatives from four VH families and five V{kappa} groups.

Despite the differences in structure and fine specificity among this group of ANA, all exhibited specific binding to the periphery of nuclear fragments (Fig. 2, A–I). The Abs allowed us to visualize morphologies corresponding to the different stages of apoptosis. For example, some images showed Abs bound to the periphery of nuclear fragments that formed blebs at the cell surface (Fig. 2, A–G and I), whereas others showed binding to fragments in the cell interior (e.g., Fig. 2, H and I). Collectively, the images allowed us to identify successive stages in the process of nuclear fragmentation, the migration of nuclear fragments to the cell surface, the protrusion of fragments from the plasma membrane, and the separation of apoptotic bodies from the remainder of the dying cell.



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  		FIGURE 2. Autoantibodies from (NZB x 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 were obtained 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 nuclear fragments (blue) are recognized along their surface by the different ANA (red), although some nuclear fragments remain unstained (B and F–I) and others predominantly show binding along the surfaces exposed to the exterior of the cell (A, C, and G). The almost complete separation of a nuclear fragment from the remainder of the cell may represent a transition from a bleb to an apoptotic body (C). For comparison, FC3, an anti-cardiolipin autoantibody from an NZW x BSXB F1 mouse (J), or an isotype-matched nonautoreactive IgG (K) was used. FC3 bound to discrete domains of the plasma membrane that did not overlap with nuclear fragments. Binding of 163p.119 (G) is also shown in a set of three perpendicular sections (L).

 
Because systemic autoimmunity in humans and mice manifests in the production of a wide variety of autoreactive Abs, we compared ANA with autoantibodies directed against phospholipids. As a representative anti-phospholipid autoantibody, we selected FC3, an anti-cardiolipin autoantibody (31). Previously, it had been reported that anti-cardiolipin Abs bind to the surface of apoptotic cells (32). The FC3 Ab bound the apoptotic cell surface in a punctate pattern that partially overlapped with the binding of annexin V, but showed little or no overlap with the distribution of nuclear fragments (Fig. 2J). In contrast, an IgG1/{kappa} Ab without self-specificity gave no detectable binding to apoptotic cells in our assay (Fig. 2K).

Autoantibodies to nucleosomes bind nuclear fragments and apoptotic blebs

Autoantibodies in systemic lupus erythematosus and murine models of this disease often recognize more than one nuclear Ag. The 3H9, for example, binds DNA and nucleosomes. Careful fractionation of nuclear Ags allows screening for autoantibodies to nucleosomes, subnucleosomes, individual histones, or DNA (24, 25, 26, 27, 28, 29, 30). In this study, we used autoantibodies that preferentially bind DNA or the isolated histones H2B or H3, an Ab to a shared epitope in histones H2A and H4, as well as Abs to the two subnucleosome complexes H2A/H2B/DNA and H3/H4/DNA (Table II). Remarkably, each of these Abs bound the surface of nuclear fragments (Fig. 3, A–F). Most notably, autoantibodies to the complete nucleosome core particle also exhibited the same binding pattern (Fig. 3G). The observed binding was most intense along nuclear fragment surfaces that protruded from the cell surface (Fig. 3, A, F, and G).



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  		FIGURE 3. Autoantibodies to individual core histones or components of the nucleosome core particle bind the surface of nuclear fragments and apoptotic blebs. Autoantibodies listed in Table II were used for binding to apoptotic 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 nucleosome core particle (NZA2; G) are shown. Binding at the surface of nuclear fragments was observed, and protruding nuclear fragments bound Abs along the surface 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 and grainy distribution of signal throughout the nucleus (H).

 
A dramatic exception to the shared binding specificity of anti-histone autoantibodies was observed with the anti-H1 histone Ab MRA12. This Ab bound in a diffuse, granular pattern throughout the nucleus at an early stage of apoptosis (Fig. 3H). In contrast, we 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 or more 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 the unique reactivity of the surface of nuclear fragments with autoantibodies to the nucleosome core particle. This result suggests an interesting and testable possibility: nucleosomes that are arrayed at the surface of nuclear fragments may expose structural epitopes that are particularly favorable targets for binding of ANA.

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

The earliest evidence of nucleosome core Ags outside of the apoptotic cell nucleus was a diffuse granular pattern of 3H9 binding throughout the cytoplasm of cells at 3 h following camptothecin addition (Fig. 4A). Only certain segments of the nuclear periphery reacted with the Ab at this time. Because of the brief time that had elapsed since the induction of apoptosis, and the partial immunoreactivity of the nuclear periphery, we infer that the diffuse cytoplasmic staining corresponds to a distinct, initial stage of a presumed pathway that transports nucleosomes from the apoptotic nucleus to the cell surface.



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  		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 analyzing the protein content of cytoplasmic extracts prepared at the indicated times after addition of camptothecin (B). The m.w. of marker proteins and the approximate 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-green fluorescent microbeads conjugated to avidin. D and E, Projection views constructed by merging the individual optical sections, to illustrate the arrangement of 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 fluorescence emitted by the beads precluded the simultaneous detection of the annexin V-associated signal.

 
To confirm that nucleosome core particles exit the nucleus at this stage of apoptosis, we examined cytoplasmic extracts by gel electrophoresis and immunoprecipitation. Our analysis revealed the simultaneous increase in the abundance of four proteins corresponding in size to the core histones (Fig. 4B). In fact, this was the most notable change in the protein composition of the apoptotic cytoplasm that occurred between 2 and 4 h after addition of camptothecin. Previously, Wu et al. (34) had observed that histones from apoptotic Jurkat cells are more easily solubilized. The appearance of histones in the cytoplasm could be blocked by the broad caspase inhibitor, z-VAD-fmk (Fig. 4B).

To establish whether the cytoplasmic histones remained assembled into a particle resembling the nucleosome, we conducted immunoprecipitations with three Abs that recognize different epitopes of the core particle. Precipitates obtained with the anti-DNA Ab PA4, the anti-H2B Ab LG2-2, or the anti-nucleosome Ab LG4-1 contained all four core histone proteins (Fig. 4C), suggesting that these individual components of the core particle are assembled in a complex that is structurally related to the nucleosome. Analysis of the DNA from the immunoprecipitate showed the presence of fragments whose size was consistent with a nucleosomal ladder (data not shown).

To determine whether, at the cell surface, nuclear autoantigens remain shielded by a membrane, or exposed and available for direct macromolecular contacts, we used fluorescent microbeads as indicators for the ability of nucleosome Ags to act as ligands for cellular receptors. Following binding of either of two Abs, 3H9 (Fig. 4D) or LG4-1 (Fig. 4E), to apoptotic cells, we added a mixture of fluorescent and biotinylated anti-mouse IgG Abs. The mixture of reagents allowed us to visualize the distribution of the primary Abs while testing whether avidin-coated, fluorescent microspheres could access the bound Abs. The binding sites of 3H9 and LG4-1 on the apoptotic cell surface were indeed accessible to the microspheres (Figs. 4, D and E). In contrast, no binding was observed in the absence of primary Abs (data not shown), or in the absence of biotinylated goat anti-mouse Abs (Fig. 4F). These results conclusively demonstrate that nucleosome core particles are exposed at the surface of apoptotic blebs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many scenarios have been envisioned for the interaction between nucleosomal autoantigens and the immune system. That such interactions are relevant is implied by the fact that autoantibodies to DNA and histones are the defining characteristics of systemic lupus. That such interactions must occur is evident from the molecular characteristics of such autoantibodies that can only be explained by positive Ag selection. To uncover the mechanism of anti-nuclear autoantibody selection, we have relied on murine lupus autoantibodies. We find that cells in apoptosis release nucleosomes into the cytoplasm and attach them to the outside of nuclear fragments. Subsequently, nuclear fragments migrate to the cell surface, break through the plasma membrane, and separate from the remainder of the dying cell. Nucleosomes thus become accessible for interactions with receptors, including B cell receptors, at the surface of blebs and apoptotic bodies. The results point to a novel role for nucleosomes in apoptosis, clearance, and immune regulation.

The pathway of nucleosome externalization at the cell surface requires a detailed integration with other events in apoptosis. We show that recovery of nucleosomes from the cytoplasm is prevented by inhibition of caspases (Fig. 4B), implying the requirement for upstream enzymatic activity. Cleavage of apoptotic chromatin by nucleases, such as DNA fragmentation factor 40 (DFF40) (35), may be the required event. Proteolysis of the DFF40-associated chaperone DFF45 by caspase 3 allows the catalytic subunit to degrade linker DNA. Inhibition of DFF40 activity inhibits chromatin condensation and nuclear fragmentation (35). Conceivably, cleavage of linker DNA contributes to the separation of a subset of nucleosomes 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). The cytoplasmic H1.2 is capable of signaling to the mitochondria to induce cytochrome c release and mediate additional signals in the caspase cascade (36). It is not known how histone H1.2 or a complete nucleosome exits the nucleus. Because the execution phase of apoptosis coincides with the increase in the permeability of the nuclear pores (37), export of nucleosomes may occur by passive diffusion or active transport. In either case, the opportunity for nucleosomes to exit the nucleus may only be transient because nuclear pores coalesce and are degraded concomitant with the fragmentation of the nucleus (38).

Upon exiting the nucleus, nucleosomes disperse throughout the cytoplasm, then attach to binding sites on the outer membrane of nuclear fragments (Fig. 4A). Nucleosomes attach before nuclear fragmentation is complete (Figs. 1A and 3E) and remain associated with the nuclear periphery through the transition of nuclear fragments into apoptotic bodies, independent membrane-bound particles that form by separation of a bleb from the remainder of the cell (Fig. 2C). Therefore, the role of nucleosomes may include the movement of nuclear fragments to the cell surface and interactions with the plasma membrane. The transport of nuclear fragments to the plasma membrane is an active process that is likely to require the participation of the cytoskeleton. Transport is, at least in part, regulated by the activity of p160ROCK/ROK{beta} (ROCK) I kinase, as inhibition of ROCK I limits the formation of surface blebs (16). It will be important to test whether known histone modifications that occur in apoptosis (39) favor attachment of cytoplasmic nucleosomes to the nuclear envelope and contribute to interactions with the cytoskeleton.

Nuclear fragments that have reached the cell surface and formed surface blebs (Figs. 1C, 2G, and 3A) afford an opportunity to evaluate the interaction between nuclear fragments and the plasma membrane. The protruding portions of nuclear fragments react with our Abs and exhibit a sharp boundary with domains of the plasma membrane that react with annexin V, indicating an unusual interaction between the nuclear envelope and the plasma membrane. As confirmed by the binding of the fluorescent beads to blebs (Fig. 4, D and E), the plasma membrane ceases to surround the nuclear fragments as they emerge at the cell surface. In addition, nuclear Ags do not appreciably disperse onto the adjacent annexin V-reactive membrane domains, indicating that the plasma membrane and the nuclear envelope do not fuse. Therefore, we favor the alternative that the polyionic nucleosome arrays on the nuclear envelope disrupt the structural integrity of the plasma membrane and facilitate the emergence of nuclear fragments at the surface of the apoptotic cell.

Once at the surface, nucleosomes may assist in the recognition and clearance of apoptotic remains. Professional phagocytes express a variety of receptors capable of recognizing apoptotic cells (40). Among the receptors identified to date, the phosphatidylserine receptor characterized by Fadok et al. (41) assumes an important role in directing the uptake of apoptotic cells by phagocytes. Exposure of phosphatidylserine is one of the earliest indications that cells are committed to die (42). However, recognition of phosphatidylserine is not sufficient for uptake of apoptotic cells. To induce uptake, signaling via the phosphatidylserine receptor must be accompanied by binding to additional phagocyte receptors (43). Additional receptors that bind to apoptotic cells include Mer and the related tyrosine kinases (5, 6), CD14 (44), the ABC-1 transporter (45), and the CD91-calnexin complex (46).

Binding of phagocyte receptors to apoptotic cells takes advantage of two families of structurally related serum proteins, the defense collagens (collectins) and the pentraxins (40). These proteins act as bridging molecules for efficient disposal of cells and cellular particles produced during more advanced stages of cell death. Interestingly, members of the collectin family, such as C1q (47) and mannose-binding lectin (46), and the pentraxin family, such as C-reactive protein (48), and serum amyloid P (49), bind to blebs at the apoptotic cell surface. Our demonstration that nucleosome core particles are exposed on apoptotic blebs raises the possibility that binding to nucleosomes assists in the recognition and clearance of apoptotic cells. Indeed, in vitro, both serum amyloid P (3) and C-reactive protein (50) recognize chromatin as well as purified nucleosome core particles. Similarly, binding of IgM Abs to nuclear Ags on the surface of apoptotic cells may induce C1q deposition and increase clearance efficiency.

Direct evidence for a role of chromatin cleavage products in clearance comes from studies in Caenorhabditis elegans, in which inactivation of apoptotic nucleases decreases the efficiency of apoptotic cell uptake (51). Additional evidence for the role of nucleosomes in the recognition and clearance of apoptotic cells comes from the observation that an excess of nucleosomes partially inhibits clearance of apoptotic cells by macrophage (52). The simplest interpretation of this experimental result is that nucleosomes interact with receptors that mediate clearance.

Our observation that nucleosomes are displayed on the surface of apoptotic cells also has implications for understanding tolerance and autoimmunity. An immune response to nuclear Ags, including histones, DNA, and their complexes, is difficult to induce. Nevertheless, the nucleosome core particle is the primary target of autoantibodies in systemic lupus erythematosus. Moreover, autoantibodies to DNA, histones, and nucleosomes exhibit clear evidence of positive selection for binding to these nuclear Ags. Presently, neither the mechanisms establishing tolerance nor the circumstances that induce anti-nuclear autoantibody production are well understood. Our observations may provide a structural basis for the importance of nucleosomes in the induction of autoimmunity in lupus.

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

Nevertheless, our results also establish that B cells with Ig receptors for apoptotic cells may reach the peripheral immune organs of nonautoimmune mice. B cells with receptors composed of the 3H9 H chain and the {lambda}1 L chain (analogous to the Ab shown in Fig. 1C) reach the spleen, yet are profoundly anergized and short-lived (55). Circumstances may arise when encounter with apoptotic cells stimulates activation and proliferation of such cells.

A key feature of Ig receptor-mediated activation is that interactions between Ig receptors and membrane-bound or particulate Ags generate far more potent signals than interactions with soluble Ags (56, 57). Signaling elicited by binding of apoptotic cells to nucleosome-specific Ig receptors may be particularly consequential because nucleosome-containing immune complexes induce near-maximal B cell proliferation (58). DNA from such immune complexes may engage Toll-like receptor-9 and generate signals that synergize with the signals generated by the Ig receptor. Thus, binding of B cells to blebs or apoptotic bodies is predicted to generate powerful signals that may reverse anergy, stimulate proliferation, and induce Ab secretion. In that case, B cells with receptors for apoptotic cells may provide a starting point for autoimmune responses.


   Acknowledgments
 
We acknowledge helpful criticisms and comments from Martin Weigert, Antony Rosen, Joyce Rauch, Jan Erikson, Dr. Brian Cocca, and Amy Cline that shaped the final text of the manuscript. Jan Erikson’s help was essential in recovering the 3H9 IgM/{lambda} transfectoma for the work shown. Thanks to Tim Higgins, senior illustrator, for careful professional attention to graphic representations of our experimental results.


   Footnotes
 
1 This work was supported by the National Institute of Allergy and Infectious Diseases (AI054938), an institutional grant from the University of Tennessee Health Sciences Center, the Center of Excellence in Structural Biology, the Center of Excellence for Diseases of Connective Tissue, and the University of Tennessee Rheumatic Disease Research Core Center of the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Marko Radic, Department of Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163. E-mail address: mradic{at}utmem.edu Back

3 Abbreviations used in this paper: ANA, anti-nuclear Ab; DFF, DNA fragmentation factor; z-VAD-(OMe)-fmk, Z-Val-Ala-Asp(OMe)-fluormethylketone. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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