Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu−/− mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

Clusterin (Clu), also known as apolipoprotein J, is a soluble 80-kDa disulfide-linked heterodimeric glycoprotein which is highly conserved during evolution and among mammals. 1 Clu is abundant in physiological fluids (concentrations ranging from 100 to 300 μg/ml in human serum) [2][3][4] and is induced in response to a wide variety of tissue injuries. Clu has chaperone activity and is a functional homolog to small heatshock proteins. 5,6 It binds hydrophobic domains of numerous non-native proteins and targets them for receptor-mediated internalization and lysosomal degradation. Clu also interacts with a broad spectrum of molecules (such as lipids, components of the complement system, amyloid-forming proteins, immunoglobulins) 7,8 and has been suggested to regulate several functions, such as complement activity, cellcell and cell-substratum interactions, and cell proliferation/ survival. 1 In various diseases, an accumulation of Clu has been reported in the injured organs. 9,10 Clu also interacts with different immune molecules; however, its potential role in immune responses remains unclear. Clu binds to some bacteria (Staphylococcus aureus and some Staphylococcus epidermidis strains) and bacterial proteins (such as the Streptococcus pyogenes extracellular protein SIC), [11][12][13] suggesting that it may modulate antimicrobial responses. Moreover, Clu limits the severity of induced autoimmune myocarditis 14 and pancreatitis. 15 Finally, the levels of circulating Clu in systemic lupus erythematosus, 16 as well as the expression of Clu mRNA in the synovium of rheumatoid arthritis patients, are decreased. 17 Phagocytosis of dying cells, a process called efferocytosis, is a complex mechanism that involves (i) exposure at the apoptotic cell surface of phosphatidylserine (PS) and membrane molecules that are altered during the apoptotic process, 18 and (ii) endocytic receptors expressed by phagocytes, such as members of the scavenger receptor family, 19 vitronectin receptors, 20 Fc receptors, 21 MER, 22 TIM-1 and TIM-4, 23 and CD91. 24 Soluble receptors (also called opsonins), such as C1q, mannose-binding lectin (MBL), 25 and milk fat globule-EGF factor 8 (MFG-E8), 26 bind to apoptotic cells and act as bridging molecules to favor their internalization by phagocytes. In a non-inflammatory environment, a rapid and efficient clearance of apoptotic cells maintains immune homeostasis and avoids the initiation of autoimmune responses. 27,28 In contrast, a dysfunction in the clearance of apoptotic cells may result in the release of danger molecules (referred to as danger-associated molecular patterns) that may favor the initiation of autoimmune responses. 27,28 The pivotal role played by soluble molecules in apoptotic cell clearance and, consequently the outcome of immune responses to apoptotic cell antigens, has been clearly evidenced in opsonin-deficient mice. For example, mice deficient in MFG-E8, C1q, or serum amyloid P component (SAP) show impaired clearance of apoptotic cells and develop a lupus-like disease characterized by elevated levels of autoantibodies and glomerulonephritis. [29][30][31] Some soluble innate immunity receptors involved in microbial recognition have also been implicated in apoptotic cell clearance. 19 As Clu binds to microbial moieties, [9][10][11] we investigated whether Clu might also mediate apoptotic cell clearance by phagocytes.

Results
Clu binds to late apoptotic cells. We first evaluated the ability of recombinant human Clu to bind to spontaneously dying human neutrophils. As previously described, 32 four populations can be distinguished by flow cytometry, based on annexin V (Ann V) and propidium iodide (PI) staining ( Figure 1a, left panel): viable (Ann V − PI − , corresponding to R1), early apoptotic (Ann V + PI − ; R2), late apoptotic (Ann V + PI + ; R3), and secondary necrotic cells (Ann V +/ − PI high ; R4). Results showed that Oregon Green 488 (OG)-labeled Clu (OG-Clu) binds to late apoptotic (R3) and, to a low extent, to secondary necrotic neutrophils (R4), but not to viable (R1) and early apoptotic (R2) neutrophils (Figure 1a, middle panels). A low binding of OG-Clu was also detected to heat-induced necrotic neutrophils (Figure 1a, right panel). Similar binding profiles were obtained using purified and recombinant Clu revealed by a FITC-labeled anti-Clu mAb ( Figure 1b). As control, 25,33 OG-C1q binds preferentially to late apoptotic and secondary necrotic cells (Figure 1b). No binding of the control protein OG-HSA (human serum albumin) was observed on dying cells (Figure 1b).
We next investigated whether Clu present in human serum also binds to late apoptotic cells. Dying neutrophils were incubated with human serum and bound Clu was detected using a FITC-labeled anti-Clu mAb. Results showed that Clu present in human serum binds to late apoptotic cells and that the level of binding was dependent on the concentration of serum used ( Figure 1c); no binding of the anti-Clu mAb on late apoptotic cells was observed in the absence of serum (Figure 1c), demonstrating that intracellular Clu does not translocate to the surface of dying cells during the apoptotic process. Finally, we observed that Clu also binds to (i) late apoptotic Jurkat cells (induced by etoposide or an anti-Fas mAb) (Figure 1d), (ii) apoptotic murine thymocytes (Supplementary Figure S1A), and (iii) tumor cells either irradiated or treated with etoposide (Supplementary Figure S1B), demonstrating that the binding of Clu to late apoptotic cells is not dependent on the cell type or on the apoptosis-inducing signal.
Clu binds to histones expressed at the surface of late apoptotic cells. The binding of OG-Clu to late apoptotic neutrophils is dose dependent, partly saturable (Figure 2a), and is inhibited in a dose-dependent manner, by unlabeled Clu, but not by HSA (Figure 2b). Fluorescence microscopy revealed an intense staining on bleb-like structures (Figure 2c). This binding was reduced on apoptotic neutrophils (Figure 2d), which were left to die in the presence of Y-27632, an inhibitor of membrane blebbing. 34 These observations suggested the presence of Clu-binding elements at the surface of late apoptotic cells.
The apoptotic process is accompanied by cell surface alterations, such as PS externalization, membrane relocalization of intracellular components, and oxidation of membrane molecules; 35,36 these motifs act as 'eat-me' molecules. In an attempt to characterize the nature of the Clu-binding motif(s), we evaluated whether a treatment with DNase, glycosidases, or pronase may modulate the binding of Clu to late apoptotic cells. DNase strongly upregulated the binding of Clu to apoptotic Jurkat cells (increase of 362 ± 51%; mean ± S.E.M., n = 4; Figures 3a and b); as a control for DNase efficiency, the staining with PI and the binding of an anti-dsDNA mAb was lower on DNase-treated than on non-treated apoptotic cells (Figures 3a and b). In contrast, pronase or glycosidases did not modulate the binding of Clu to apoptotic cells ( Figure 3b); as enzyme controls, pronase and glycosidases decreased the binding on late apoptotic cells of anti-CD45RA mAb 37 Figure S2B).
The fact that DNAse increased the binding of Clu to late apoptotic cells suggested that DNA may sterically mask Clubinding elements. As genomic DNA is associated with histones, we suspected that histones might represent Clu-binding elements. A solid-phase binding assay showed that Clu binds to immobilized H2A, H2B, H3, and H4, and, to a lower extent, to the linker subunit H1 ( Figure 3c). As control, 40,41 C-reactive protein (CRP) and SAP, but not HSA, also bind to histones (Figure 3c). No binding of Clu to immobilized dsDNA was observed ( Figure 3c); as a control, immobilized dsDNA was recognized by anti-dsDNA Abs (data not shown).
We then confirmed that histones, translocated to the surface of apoptotic cells, represent Clu-binding elements. As reported, 36 histones can be detected at the surface of late apoptotic neutrophils but not on viable, early apoptotic, and necrotic neutrophils (Figure 3d). Interestingly, DNAse increased the binding of an anti-histone Ab (Supplementary Figure S2C), confirming that DNA may mask Clu-binding motifs on histones at the surface of late apoptotic cells. Finally, confocal microscopy revealed a partial colocalization between OG-Clu and histones at the surface of apoptotic cells (Figure 3e).

Clu is involved in the clearance of apoptotic cells.
Opsonins act as bridging molecules to favor apoptotic cell clearance. We therefore examined whether Clu may be involved in apoptotic cell clearance using a FACS-based in vitro apoptotic cell engulfment assay. 42,43 Macrophages (Mφ) were fed with PKH67-labeled early or late apoptotic neutrophils, previously incubated or not with Clu, MBL, or HSA. Compared with the control protein HSA, Clu enhanced the phagocytosis of apoptotic cells (69 ± 12% increase; mean ± S.E.M., n = 6) in a similar manner to MBL (67 ± 14% increase) (Figure 4a) used as a positive control. 25 In agreement with the absence of binding to early apoptotic cells, Clu did not modulate the efferocytosis of AnnV + PI − cells (Figure 4a). In order to investigate the role of seric Clu, late apoptotic neutrophils were incubated with heatinactivated human serum, either depleted or not in Clu. Depletion of Clu reduced the phagocytosis of apoptotic cells (26 ± 6% decrease; mean ± S.E.M., n = 5); this inhibition was partially reversed by supplementing Clu-depleted serum with exogenous Clu (Figure 4b).
Prior to analyzing the role of Clu in the in vivo clearance of apoptotic cells, we confirmed the ability of Clu to promote the in vitro phagocytosis of apoptotic murine cells. Results showed that (i) late apoptotic thymocytes opsonized with Clu are more efficiently internalized by Mφ than apoptotic cells incubated with HSA (46 ± 7% increase; mean ± S.E.M., n = 5; Figure 4c), and (ii) that apoptotic thymocytes incubated with serum from Clu − / − mice were less efficiently engulfed by Mφ than apoptotic cells incubated with serum from wild-type (WT) mice (12 ± 2% decrease; mean ± S.E.M., n = 6; Figure 4d). The in vivo role of Clu in apoptotic cell clearance was investigated using Clu − / − mice. In a first set of experiments, we compared, in WT and Clu − / − mice, the clearance of dying thymocytes in which apoptosis was induced by dexamethasone sodium phosphate (Dex). 22,44 Remarkably, the thymus of Dex-injected Clu − / − mice contained approximately twofold more remnant apoptotic cells than Dex-injected WT mice (15 ± 3% versus 8 ± 2%; mean ± S.E.M., n = 5; Figure 4e); we excluded that this observation may result from an increased sensitivity of thymocytes from Clu − / − mice to Dex-induced apoptosis (Supplementary Figure S3A). In contrast, no difference was observed in Clu − / − and WT mice injected with PBS ( Figure 4e). In a second set of experiments, we analyzed the splenic clearance of PKH67-labeled apoptotic thymocytes injected intravenously in Clu − / − and WT mice. Two hours after injection, the spleens from Clu − / − mice contained more apoptotic cells than WT mice (0.56 ± 0.06% versus 0.44 ± 0.03%; mean ± S.E.M., n = 4; Figure 4f); this defect was maintained 6 h after apoptotic cell injection (data not shown). Importantly, Mφ from Clu − / − mice do not exhibit any defect in apoptotic cell phagocytosis (Supplementary Figure S3B).
Clu enhances CD4 + and CD8 + T-cell responses to an apoptotic cell-associated antigen. The engulfment of apoptotic cells by phagocytes leads to the presentation of apoptotic cell-derived antigens, a process contributing to the maintenance of peripheral tolerance. [45][46][47] We therefore analyzed whether Clu might promote apoptotic cell antigen presentation to CD4 + and CD8 + T cells. Murine thymocytes were loaded with ovalbumin (Ova) prior to apoptosis induction (Ova-Apopt). In a first set of experiments, dendritic cells (DCs) were incubated with Ova-Apopt previously incubated with Clu or HSA, before culture with Ova-specific OT1 CD8 + or OT2 CD4 + T cells. Results showed that the opsonization of Ova-Ova-Apopt with Clu enhanced the production of IL-2 by OT1 and OT2 T cells, compared with Ova-Ova-Apopt incubated with HSA (200 ± 64% and  Figure 5a). In a second set of experiments, DCs were incubated with Ova-Ova-Apopt previously incubated with 10% serum from Clu − / − or WT mice before culture with Ovaspecific T cells. The levels of IL-2 produced by OT1 and OT2 T cells were lower with Ova-Ova-Apopt incubated with serum from Clu − / − mice versus serum from WT mice (32 ± 8% and 34 ± 9% decrease, respectively; mean ± S.E.M., n = 4; Figure 5b).
Clu-deficient mice are sensitive to apoptotic cell-induced autoimmunity. A defect in apoptotic cell clearance may trigger an autoimmune response. 27,44,48 We thereby postulated that the absence of Clu might predispose mice to apoptotic cell-induced autoimmunity. We compared, in Clu − / − and WT mice, the appearance of signs of autoimmunity in a model of mild autoimmune response induced by repeated injections of apoptotic cells. 47,49 Results showed that, 2 weeks after the first injection of apoptotic cells, the levels of IgG anti-dsDNA Ab were increased in Clu − / − mice compared with WT mice (2638 U/ml ± 282 versus 1823 U/ml ± 450, respectively; Figure 6a). In contrast to WT mice, which only developed a slight and transient upregulation 6 weeks after the first injection of apoptotic cells, the levels of IgG anti-dsDNA Abs were significantly higher and maintained elevated in Clu − / − mice, 10 weeks after the first injection of apoptotic cells (Figure 6a). The basal levels of anti-dsDNA Abs remained stable in non-injected Clu − / − mice and were equivalent to the ones in WT mice, although a slight increase was observed as the animal aged (Figure 6a). Interestingly, no difference in the kinetics and amplitude of IgG anti-Ova Ab titers was observed between Clu − / − and WT mice immunized with Ova (Supplementary Figure S4A), suggesting that the induction of anti-dsDNA Abs in Clu − / − mice did not result from an abnormal capacity to mount a humoral response. Interestingly, glomerular IgG and complement component C4 deposits were observed in Clu − / − but not in WT mice, 10 weeks after injection of apoptotic cells ( Figure 6b); no deposit was observed in Clu − / − and WT mice injected with PBS (Supplementary Figure S4B). Upon injection of apoptotic cells, the spleen weight of Clu − / − mice was slightly but significantly increased, compared with WT mice (increase of 38 ± 10%; mean ± SEM, n = 4), 8 weeks after the first injection of apoptotic cells (Figure 6c). Moreover, 10 weeks after the first injection of apoptotic cells, relative to WT mice, the liver expression of SAP mRNA was enhanced in Clu − / − mice (Figure 6d).
The generation of class-switched IgG autoantibodies in Clu − / − mice suggested the role of T cells. We first analyzed the frequency of naive (CD44 − CD62L high ), central memory (CD44 + CD62L high ), and effector memory (CD44 + CD62L low ) 50 CD4 + and CD8 + T cells. Results showed an increase in the frequency of CD44 + CD62L low cells within both CD4 + and CD8 + T-cell subsets, 10 weeks after the first injection of apoptotic cells (Figure 7a). Moreover, the ratio of CD44 + CD62L low and CD44 + CD62L high cells among CD4 + and CD8 + T cells were significantly increased in the lymph nodes of Clu − / − (1.10 ± 0.07 and 0.28 ± 0.07, respectively; mean ± S.E.M., n = 5) compared with WT mice (0.70 ± 0.14 and 0.14 ± 0.03, respectively) ( Figure 7b). The total numbers of CD4 + and CD8 + T cells, B cells, Mφ, and DCs (Supplementary Figure S5A), as well as the frequency of regulatory, memory, naive, and activated T cells, and of activated B cells (Supplementary Figure S5B), were equivalent in the lymph nodes and spleens of non-treated 12-weekold WT and Clu − / − mice. We next determined whether the increased percentage of CD44 + CD62L low CD8 + and CD4 + T cells in apoptotic cell-injected Clu − / − mice had functional implications. Upon stimulation with phorbol myristic acetate (PMA) plus ionomycin, lymph node CD8 + and CD4 + T cells from Clu − / − mice produced significantly more IL-2 than cells from WT mice (Figure 7c), while only CD8 + T cells from Clu − / − mice produced significantly more IFN-γ than cells from WT mice (Figure 7d).

Discussion
Even though suspected, the potential role of Clu in immune homeostasis remains largely unexplored. We report here that Clu promotes the clearance of late apoptotic cells via its unique capacity to bind to histones translocated to the surface of apoptotic cells. Accordingly, Clu − / − mice develop signs of autoimmunity in a model of apoptotic cell-induced autoimmunity. These results identify Clu as a new bridging After apoptotic cell engulfment, professional antigenpresenting cells (APCs) activate tolerogenic pathways that prevent local inflammatory reactions. 44,48 They produce immunoregulatory cytokines (TGFβ, IL-10) and low or no proinflammatory cytokines and chemokines. 51 In this immunoregulatory environment, the presentation and crosspresentation of apoptotic cell antigens by APCs maintain peripheral T-cell tolerance. 47,52 In contrast, in the absence of prompt clearance, apoptotic cells may evolve into immunologically harmful secondary necrotic cells which release danger signals that may favor the initiation of an autoimmune response. 27,53 Necrotic cells also trigger the production of inflammatory mediators by APCs. 54 A rapid and efficient efferocytosis is thus required to maintain immune tolerance. In this study, we demonstrate that Clu, via its unique property to potentiate efferocytosis, prevents the in vivo generation of necrotic cells and thereby contributes to maintain selftolerance.
Despite the loss of billions of cells each day, the incidence of histologically detectable apoptotic cells is rare in normal tissues because of the efficiency of efferocytosis. 22,44 We showed that Clu binds specifically to late apoptotic cells, suggesting that, under normal physiological conditions, Clu will have only a minor role in apoptotic cell clearance. Accordingly, Clu − / − mice do not exhibit spontaneous signs of autoimmunity, as observed for most opsonin-deficient mice. Aged MBL-deficient mice do not develop autoimmunity even on a lupus-prone genetic background 129 × C57BL/6. 55 Moreover, SAP − / − and C1q − / − mice only develop autoimmunity on the mixed 129 × C57BL/6 or MRL/Mp background. 30,31 To our knowledge, MFG-E8 − / − mice are the only opsonin-deficient model that spontaneously develop an autoimmune phenotype with aging. 29 The role of Clu in efferocytosis suggests the existence of endocytic receptor(s) for Clu. Megalin was described as a receptor for Clu involved in the uptake of Clu-associated misfolded proteins at the cerebral vascular endothelium and choroid epithelium 56 and in the endocytosis of cellular debris by epithelial cells. 57 In humans, however, the expression of megalin is restricted to the proximal tubule of the kidneys, the choroid plexus epithelium, and ependymal cells lining the brain ventricules, 58 making it unlikely to represent a major endocytic receptor for Clu-mediated efferocytosis by phagocytes. Accordingly, we failed to detect megalin by human Mφ (unpublished observations). Recent studies have reported that Clu binds to some scavenger receptors 5 and DC-SIGN, 59 suggesting that these endocytic receptors may be involved in the internalization of Clu-opsonized apoptotic cells. Experiments are in progress to identify Clu-binding elements involved in the capture of late apoptotic cells by phagocytes.
We have observed that Clu binds specifically to blebs on late apoptotic cells, as reported for other opsonins, such as CRP and SAP. 60 The fact that the binding of Clu is not dependent on the cell type nor on the apoptosis-inducing method suggested that the Clu-binding motifs are conserved molecules. We demonstrate here that Clu binds to histones. Previous studies have shown that core histone subunits rapidly accumulate in the cytoplasm of early apoptotic cells 61,62 before accumulation on blebs. 36,63 Accordingly, we observed that histones are expressed by late but not early apoptotic cells, explaining the lack of binding of Clu to early apoptotic cells. These results confirm the role of histones as 'eat-me' molecules at the surface of late apoptotic cells.
Defects in apoptotic cell clearance and/or an excess of apoptotic cells make mice and humans susceptible to autoimmunity. 28 Although efferocytosis is mediated by multiple and partly redundant mechanisms to avoid the initiation of an autoimmune response, it has been reported that repeated injections of apoptotic cells may induce signs of autoimmunity (without clinical signs). 49 Considering the role of Clu in efferocytosis, we hypothesized that an excess of apoptotic cells could be less efficiently cleared in Clu − / − mice, leading to a more intense immune response. Indeed, we observed that Clu − / − mice are more sensitive to apoptotic cell-induced autoimmunity. They develop signs of autoimmunity, such as immunoglobulin and complement component C4 deposition within kidneys, autoantibody production, and splenomegaly. In parallel, we observed an activation of effector memory apoptotic cell antigen-specific T cells in Clu − / − mice. In line with these results, previous studies reported that, in models of autoimmune pancreatitis and myocarditis, Clu − / − mice develop more severe inflammatory lesions than WT mice. 14,15 However, the mechanism(s) involved in this protective role of Clu was not investigated. Considering our results, it is likely that, in these models associated with a massive cell death, the absence of Clu may have contributed to initiate apoptotic cell-driven autoimmunity.
In conclusion, we show that Clu is a non-redundant opsonin critically involved in the efferocytosis of late apoptotic cells and the maintenance of immune homeostasis. In vivo, Clu deficiency leads to a striking autoimmunity induced by the injection of apoptotic cells, a model that mimics a massive cell death that can occur during severe tissue injuries. These results also suggest that Clu may have a protective role against the establishment of chronic sterile inflammatory disorders. This study opens new insights into how to induce tolerance to self-antigens in autoimmune diseases and to optimize immunogenic cell death in antitumor immunotherapies.

Materials and Methods
Proteins and antibodies. Human Clu was purified from plasma, as previously described. 7 Recombinant human MBL and human and murine Clu were from Biotechne (Lille, France). HSA, C1q, and FITC-labeled WGA were from Sigma-Aldrich  Table 1.
Isolation and generation of murine leukocytes. C57BL/6 mice and Ova-specific T-cell receptor transgenic mice OT1 and OT2 (C57BL/6 background) were from Charles River Laboratories (L'Arbresle, France). Clu − / − mice (C57BL/6 background) were from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were bred and housed in a pathogen-free environment. Experiments were conducted according to institutional guidelines and were approved by the institutional ethics committee of Région des Pays de la Loire (agreement 2009.18).
Murine CD8 + and CD4 + T-cell purification: CD8 + T cells from OT1 mice and CD4 + T cells from OT2 mice were isolated from the spleen and lymph nodes using the CD8 + T-Cell Isolation Kit II and the CD4 + T-Cell Isolation Kit II, respectively, following the manufacturer's instructions (Miltenyi Biotech). Cell purity, determined by staining for CD3, CD4, CD8, and CD11c expression, was 499% (data not shown).
Induction of cell death. Spontaneous human neutrophil and murine thymocyte apoptosis was induced by incubating cells in RPMI 1640 medium containing 1% FCS. Staining with allophycocyanin (APC)-labeled Ann V (BD Pharmingen, San Diego, CA, USA) and PI (Sigma-Aldrich) allowed to distinguish four cell populations by flow cytometry, corresponding to viable (Ann V − PI − ), early apoptotic (Ann V + PI − ), late apoptotic (Ann V + PI + ) and secondary necrotic cells (Ann V +/ − PI high ). 32 In some experiments, cell necrosis was induced by incubating cells at 56°C for 30 min. Apoptosis of the human T-cell line Jurkat (ATCC, Manassas, VA, USA) was induced by a 24-h incubation with 20 μg/ml etoposide (Sigma-Aldrich) or 20 ng/ml anti-FAS mAb (clone CH-11; MBL International, Woburn, MA, USA). Before each experiment, the Ann V/PI staining was assessed to confirm cell apoptosis. In vivo apoptosis of cortical thymocytes was induced by injecting mice intraperitoneally with 0.2 mg Dex (Calbiochem) per 25 g body weight. 22,44 After 24 h, the level of thymocyte apoptosis was evaluated by flow cytometry, as described above.
Binding assays. Apoptotic or necrotic cells (1 × 10 5 cells/well) were resuspended in PBS containing 1% BSA (w/v) and incubated or not with 1 μM OG-labeled human Clu, recombinant Clu (rClu), C1q, or HSA for 20 min at room temperature. The binding of unlabeled Clu was detected with an anti-Clu mAb (Biotechne); mouse IgG1 antibody (R&D Systems) was used as a control. Bound antibodies were detected with FITC-labeled anti-mouse Ig Ab (BD Pharmingen). Fluorescence was analyzed by flow cytometry. In some experiments, apoptotic Jurkat T cells (containing at least 60% Ann V + PI + cells) were treated for 1 h with 500 μg/ml DNase or for 30 min with 100 μg/ml pronase (both from Roche, Mannheim, Germany) or for 4 h with protein deglycosylation mix (New England Biolabs) prior to the binding assay. In other experiments, binding of Clu to 'flip-flopped' erythrocytes 64 was measured. The binding of Clu, SAP, CRP, and HSA to histone subunits and dsDNA was measured by a solidphase binding assay. Briefly, 1 μg/ml of each histone subunit was coated on 96-well plates in 50 mM NaHCO 3 /Na 2 CO 3 buffer, pH = 9.6, for 12 h at 4°C. dsDNA was coated as previously described. 44 After blocking the nonspecific sites with PBS containing 5% BSA, plates were incubated for 2 h with 1 μg/ml of biotinylated Clu, SAP, CRP, and HSA and then with HRP-labeled streptavidin (Diaclone, Besançon, France) diluted 1 : 500 for 1 h. The coating of dsDNA was verified using an anti-dsDNA Ab (Immunotools) revealed by HRP-conjugated anti-mouse IgG Ab (Life Technologies, Saint Aubin, France). Optical density was read at λ = 492 nm.

Phagocytosis assays
Phagocytosis assay with human cells: Freshly isolated neutrophils were labeled with the green fluorescent dye PKH67 using the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), according to the manufacturer's instructions. Apoptosis was induced as described above. Mφ (2 × 10 5 cells/well) were cultured in 48-well plates for 12 h before the assay. PKH67-labeled early (corresponding to a cell population containing no late apoptotic cells) or late apoptotic neutrophils (corresponding to a cell population containing at least 80% late apoptotic cells) were incubated for 30 min in RPMI 1640 medium, containing or not 1 μM Clu, MBL, or HSA. After washing, 1 × 10 6 neutrophils were added to Mφ and incubated for 40 min at 37°C in RPMI 1640 medium. Non-internalized apoptotic cells were removed by washing Mφ with ice-cold PBS. 32 Cells were then incubated with an APC-labeled anti-HLA-DR mAb. Phagocytosis was analyzed by flow cytometry, as previously described. 32 In some experiments, apoptotic neutrophils were incubated for 30 min in RPMI 1640 medium containing 30% human serum, depleted or not in Clu, prior to the phagocytosis assay. Clu-depleted serums and control serums were prepared by passing human serums over a column of anti-Clu mAb or isotype control mAb covalently linked to agarose beads (co-IP columns; Pierce). Depletion was verified by quantifying Clu by ELISA (Biotechne); results showed that 89 ± 2% (mean ± S.E.M., n = 4) of Clu was removed from the serum (not shown).
Phagocytosis assay with murine cells: Freshly isolated thymocytes from C57BL/6 mice were labeled with PKH67, prior to apoptosis induction. BMDM (2 × 10 5 cells/well) were cultured in 48-well plates for 12 h before the assay. Opsonization of apoptotic thymocytes (80% late apoptotic cells) was performed as described above. After washing with ice-cold PBS, BMDM were incubated with phycoerythrin (PE)-labeled anti-F4/80 mAb. In some experiments, apoptotic thymocytes were incubated with 1% (v:v) serum from WT or Clu − / − mice, prior to incubation with BMDM. Results are expressed as a percentage of phagocytosis (percentage of PKH67 + events among the HLA-DR + or F4/80 + populations) or as a phagocytic index determined as follows: (percentage of phagocytosis × MFI of double-positive events)/100. 63 In vivo phagocytosis assay: Thymocytes from WT mice were labeled with PKH67 and apoptosis was induced as described above. Apoptotic thymocytes (1 × 10 8 cells containing at least 80% late apoptotic cells) were injected intravenously into Clu − / − and WT mice. The frequencies of PKH67-labelled cells among splenocytes were analyzed by flow cytometry 2 h after injection.
Laser confocal scanning microscopy. Human Mφ were cultured on glass slides in 48-well plates, 24 h prior to the phagocytosis assay. After phagocytosis, Mφ were washed with ice-cold PBS, fixed in 2% (w/v) paraformaldehyde (PFA) in PBS and stained with a PE-labeled anti-CD14 mAb. Slides were then mounted with ProlonGold reagent (Invitrogen). In some experiments, apoptotic neutrophils were incubated with OG-Clu as described above and fixed with 2% PFA in PBS. In others, apoptotic neutrophils were stained with anti-H2/H3/H4 mAb; bound mAb were detected with a PE-labeled anti-mouse Ig Ab (Dako, Glostrup, Denmark). Cells were then mounted on glass slides with ProlonGold reagent after nucleus staining with DAPI (Invitrogen). Staining was analyzed with a confocal laser scanning system (A1Rsi, Nikon, Tokyo, Japan).