ARTHRITIS & RHEUMATISM Vol. 56, No. 10, October 2007, pp 3399–3411 DOI 10.1002/art.22947 © 2007, American College of Rheumatology
Opsonization of Late Apoptotic Cells by Systemic Lupus Erythematosus Autoantibodies Inhibits Their Uptake via an Fc␥ Receptor–Dependent Mechanism Esther Reefman, Gerda Horst, Marije T. Nijk, Pieter C. Limburg, Cees G. M. Kallenberg, and Marc Bijl SSA/Ro and IgG isolated from 5 antinuclear antibody (ANA)–positive patients with SS were also able to elicit these effects, whereas IgG from 5 ANA-negative patients with RA did not. The inhibitory effect of patient IgG was abolished by blocking either the Fc␥ receptors (Fc␥R) or the constant region of IgG, using a specific Fcblocking peptide. Conclusion. Autoantibodies from SLE patients are able to opsonize apoptotic cells and inhibit their uptake by macrophages via an Fc␥R-dependent mechanism.
Objective. Decreased clearance of apoptotic cells is suggested to be a major pathogenic factor in systemic lupus erythematosus (SLE). The aim of this study was to investigate whether the binding of SLE autoantibodies to apoptotic cells influences the phagocytosis of these cells by macrophages. Methods. Apoptosis was induced in a human T cell line (Jurkat) and a keratinocyte cell line (HaCaT) by ultraviolet B irradiation. Binding of purified IgG from 26 SLE patients and 15 healthy controls to apoptotic cells was assessed by flow cytometry and Western blotting. Phagocytosis of IgG-opsonized apoptotic cells by monocyte-derived macrophages was assessed by light microscopy. Similar experiments were performed with a monoclonal antibody against SSA/Ro and IgG fractions from 5 patients with Sjo ¨gren’s syndrome (SS) and 5 patients with rheumatoid arthritis (RA). Results. IgG fractions from all 26 SLE patients bound to late apoptotic, but not early apoptotic, cells. IgG fractions isolated from SLE patients with different autoantibody profiles showed comparable levels of binding. IgG fractions from healthy controls did not bind. Opsonization of apoptotic cells with IgG fractions from SLE patients resulted in a significant inhibition of phagocytosis as compared with healthy control IgG fractions. A monoclonal antibody directed against
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the presence of autoantibodies directed against nuclear and cytoplasmic antigens, in combination with diverse clinical disease manifestations. Several studies have indicated an important role of apoptotic cells in the induction of autoimmunity in SLE. Injection of large numbers of apoptotic cells into animals used as experimental models induced autoimmunity, suggesting that self tolerance can be broken by the accumulation of apoptotic cells (1,2). In addition, studies have indicated that phagocytosis of apoptotic cells is impaired in SLE (3,4). During the apoptotic process, intracellular autoantigens may become exposed, in altered or unaltered form, at the outer surface of apoptotic cells (5–8). Decreased clearance of apoptotic cells results in prolonged exposure of these cell surface–expressed autoantigens to the immune system in SLE, which might explain why autoantibodies against these intracellular antigens develop. Disturbance in the clearance of apoptotic cells, therefore, is considered one of the pathophysiologic mechanisms underlying the breakdown of tolerance and, subsequently, the induction of SLE (9). Breakdown of tolerance and production of auto-
Supported by a grant from the Dutch Kidney Foundation. Esther Reefman, PhD, Gerda Horst, BSc, Marije T. Nijk, BSc, Pieter C. Limburg, PhD, Cees G. M. Kallenberg, MD, PhD, Marc Bijl, MD, PhD: University Medical Center Groningen, and University of Groningen, Groningen, The Netherlands. Address correspondence and reprint requests to Esther Reefman, PhD, or Cees G. M. Kallenberg, MD, PhD, Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. E-mail:
[email protected] or
[email protected]. Submitted for publication June 19, 2006; accepted in revised form June 29, 2007. 3399
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Table 1. Autoantibody reactivity to various antigens in sera from 26 patients with systemic lupus erythematosus* Patient
SSA, 52 kd
SSA, 60 kd
SSB
nRNP
Sm
Histones
dsDNA, WHO units
Cardiolipin, GPL units
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 S R D
– – ⫹ – – – – ⫹ – ⫹ – – ⫹ ⫹ – – – ⫹ – – ⫹ – – ⫹ – –
– – ⫹ – – – ⫹ ⫹ ⫹ ⫹ – – ⫹ ⫹ – ⫹ ⫹ ⫹ – – ⫹ – – ⫹ – –
– – ⫹ – – – – – – – – – – ⫹ – – – ⫹ – – – – – – – –
– – – – – – – – – ⫹ ⫹ ⫹ – – – – – – – ⫹ ⫹ ⫹ ⫹ – ⫹ –
– – – – – – ⫹ – – – ⫹ – – – – – – – – – – – – – – ⫹
– – ⫹ – – ⫹ ⫹ ⫹ – ⫹ – ⫹ – – – – – – – – – – – – – –
⬍10 19 ⬍10 ⬍10 ⬍10 624 608 230 440 370 161 86 ⬍10 ⬍10 25 ⬍10 ⬍10 359 ⬍10 ⬍10 13 113 ⬍10 ⬍10 ⬍10 284
0 0 0 50 0 0 0 26 0 55 0 0 0 0 0 0 0 18 0 0 52 0 0 0 0 10
* Patients S, R, and D were selected for the presence of autoantibodies to SSA (52 and 60 kd), nuclear RNP (nRNP), and double-stranded DNA (dsDNA) in combination with Sm, respectively, without the concomitant presence of other autoantibodies. Anti-dsDNA antibodies are expressed in World Health Organization (WHO) units (14), as detected by the Farr assay (positive ⬎10). Anticardiolipin antibodies are expressed in IgG phospholipid (GPL) units.
antibodies generally precedes the onset of clinical manifestations in SLE (10). Little is known about the role of apoptotic cells during the course of the disease. In particular, their significance for boosting the autoimmune response and inducing lesions has not been studied. Also, the mechanisms involved in the reduced clearance of apoptotic cells have not yet been unraveled. Whether macrophages display intrinsic defects in the clearance of apoptotic cells in SLE is still debated (4,11–13). Alternatively, reduced clearance might result from serum factors. We recently showed that reduced levels of complement, as are present in the serum of patients with active disease, correlate with decreased apoptotic cell clearance in vitro (13). Furthermore, the presence of autoantigens on the apoptotic cell surface could, in theory, represent a binding site for autoantibodies. Binding of autoantibodies to apoptotic cells will enable their interaction with Fc␥ receptors (Fc␥R) on macrophages and thereby alter the route of phagocytosis as compared with non–IgG-opsonized apoptotic cells. In this study, we investigated whether autoantibodies derived from SLE patients bind to apoptotic cells.
In addition, we explored whether the binding of autoantibodies to apoptotic cells affects their uptake by monocyte-derived macrophages. PATIENTS AND METHODS Patient selection. Patients eligible for this study fulfilled at least 4 of the American College of Rheumatology (ACR) criteria for SLE (14). The Local Ethics Committee of the University Medical Center Groningen approved the use of patient sera for the study. IgG fractions were isolated from the serum of 26 consecutive SLE patients (mean ⫾ SD age 48.6 ⫾ 12.2 years) who were positive for antinuclear antibodies (ANAs), as determined by indirect immunofluorescence, and for various autoantigenic specificities as shown in Table 1. From this group, 3 patients were selected who had distinct autoantibody profiles. These patients tested positive only for autoantibodies directed against SSA/Ro (patient S), nuclear RNP (nRNP) (patient R), or double-stranded DNA (dsDNA) in combination with Sm (patient D) (Table 1). Autoantibodies to dsDNA were assessed by the Farr assay (Diagnostic Products, Los Angeles, CA), anticardiolipin IgG was tested using a Varelisa cardiolipin IgG enzyme immunoassay kit (Sweden Diagnostics, Freiburg, Germany), and reac-
OPSONIZATION OF APOPTOTIC CELLS BY SLE AUTOANTIBODIES
tivity to other antigens was tested by the Inno-LIA blot system (Innogenetics, Zwijndrecht, Belgium). In addition, sera from 5 patients with rheumatoid arthritis (RA; mean ⫾ SD age 49.2 ⫾ 8.7 years ) and 5 patients with primary Sjo ¨gren’s syndrome (SS; mean ⫾ SD age 50.4 ⫾ 18.6 years) were selected for study, and IgG fractions were isolated as described above. IgG fractions from the RA patients tested negative for ANAs and for reactivity against any of the antigens shown in Table 1. IgG fractions from the SS patients all tested positive for reactivity against SSA and 2 of the 5 tested positive for reactivity against SSB but did not react with the other antigens shown in Table 1. Furthermore, IgG was isolated from the sera of 15 age-matched healthy control subjects (mean ⫾ SD age 50.3 ⫾ 14.5 years). Chemicals and antibodies. Bovine serum albumin (BSA) and Lymphoprep were obtained from Fresenius Kabi (Oslo, Norway), and natrium citrate was from Merck (Darmstadt, Germany). Mouse monoclonal antibody directed against -actin was obtained from ICN (Aurora, OH), and fluorescein isothiocyanate (FITC)–labeled polyclonal rabbit anti-human IgG F(ab⬘)2 fragments and horseradish peroxidase (HRP)– labeled polyclonal rabbit anti-human Ig antibodies were from Dako (Glostrup, Denmark). Phycoerythrin (PE)–labeled polyclonal goat anti-mouse Ig (heavy plus light chains) IgG was obtained from Southern Biotechnology (Birmingham, AL). Antibody directed against nuclear matrix protein p84 was obtained from Novus Biologicals (Littleton, CO). Fc␥Rblocking antibodies, mouse anti-CD16 IgG clone AT10, and anti-CD64 IgG clone 10.1 were from BD PharMingen (San Diego, CA), and mouse anti-CD32 clone 3G8 was from Serotec (Oxford, UK). IgG-blocking peptide TG19320 (15) was kindly provided by Dr. G. Fassina (Xeptagen, Pozzuoli, Italy). Apoptosis induction. Jurkat cells (a human T cell line) were irradiated with 8.3 mJ/cm2 of ultraviolet B (UVB) using a TL12 broad-band UVB lamp (Philips, Best, The Netherlands) to induce apoptosis. Subconfluent monolayers of HaCaT cells (human keratinocyte cell line) were irradiated with 19.8 mJ/cm2 of UVB. Induction of apoptosis of freshly isolated neutrophils was achieved either by aging for 72 hours or by UV irradiation, as described elsewhere (16). We also used a general caspase inhibitor to inhibit apoptosis. Cells were incubated for 1 hour before induction of apoptosis with 2 concentrations of ZVAD (10 M and 20 M; Sigma, St. Louis, MO) in culture medium. Cells were subsequently tested for apoptosis induction using the methods described below. Apoptosis detection. Progression of apoptosis was assessed after incubating cells for 4–48 hours at 37°C in an atmosphere of 5% CO2, followed by staining with FITClabeled annexin V and assessment of propidium iodide (PI) uptake as described elsewhere (17). Cell DNA content was determined as described previously (18,19). Early apoptotic cells were defined as cells that bound annexin V but did not take up PI. Late apoptotic cells were defined as cells that both bound annexin V and took up PI. IgG purification and binding assay. IgG fractions were isolated using a HiTrap protein G column (Amersham, Uppsala, Sweden) and checked for endotoxin contamination. Apoptotic cells were washed with 1% BSA/phosphate buffered saline (PBS), resuspended in 1% BSA/PBS (2 ⫻ 106 cells/ml),
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and incubated with 10 g/ml of purified IgG for 30 minutes at 37°C in an atmosphere of 5% CO2. After washing, cells were incubated with 5 l of FITC-labeled rabbit anti-human IgG for 30 minutes at room temperature in the dark and were subsequently analyzed by flow cytometry. A combination of FITClabeled anti-human IgG and PE-labeled annexin V (BD PharMingen) was used to assess the apoptotic status of cells that bound IgG. To test the permeability of late apoptotic Jurkat cells for IgG molecules, cytoskeletal -actin was used as an intracellular marker. IgG-permeable cells were defined as cells that bound PE-labeled mouse anti–-actin. Late apoptotic Jurkat cells were double stained with anti–-actin (diluted 1:200 in 1% BSA/PBS) combined with IgG (10 g/ml) isolated from healthy controls or from SLE patient S, R, or D. After washing, cells were incubated with a combination of FITC-labeled rabbit anti-human IgG F(ab⬘)2 (5 l; undiluted on the pellet) and PE-labeled rabbit anti-mouse IgG (5 l; 1:10 dilution on the pellet) for 30 minutes at room temperature in the dark, and then analyzed by flow cytometry. Plasma membrane extraction and Western blotting. Nonirradiated, early apoptotic, and late apoptotic Jurkat cells (30 ⫻ 106 cells/ml) were resuspended in buffer containing 10 mM Tris HCl (pH 7.3), 1.5 mM MgCl2, 10 mM NaCl, and protease inhibitors (Complete; Roche, Indianapolis, IN) and incubated for 1 hour on ice. Cell membranes were disrupted by douncing, and subsequently, intact cells and nuclei were pelleted by centrifugation (1,200 revolutions per minute for 5 minutes). The supernatant was centrifuged in a high-speed centrifuge (12,000 rpm for 30 minutes), and pelleted plasma membranes were resuspended in 10 mM Tris HCl (pH 7.3). Plasma membrane fractions (1.8 g of protein per lane) were analyzed using standard Western blotting techniques. Briefly, after transfer to a nitrocellulose membrane, the membrane was blocked using 5% milk suspension, washed, and incubated for 1 hour at room temperature with 15 g/ml of IgG derived from controls or SLE patients. After washing, the membrane was incubated for 30 minutes at room temperature with a secondary antibody (HRP-labeled rabbit anti-human IgG antibody diluted 1:2,500 in blocking buffer) and analyzed using Lumi-Light Plus (Roche). Nuclear fractions were isolated using the NE-PER nuclear and cytoplasmic protein extraction reagent kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. Culture of monocyte-derived macrophages. Peripheral blood mononuclear cells were isolated from healthy controls as described previously (13). Phagocytosis assay. The phagocytosis assay was performed as described previously (17). Briefly, UVB-irradiated apoptotic cells were incubated with 10 g/ml of the IgG fractions (see above), and the cells were washed twice to remove nonbinding IgG. Subsequently, cells (2 ⫻ 106 Jurkat cells/well and 1 ⫻ 106 HaCaT cells/well) were incubated with monocyte-derived macrophages for 30 minutes at 37°C in an atmosphere containing 5% CO2, in the presence of 30% normal human serum. Staining procedures were subsequently performed, and the preparations scored for phagocytosis of apoptotic cells, as described previously (17). All experiments were performed in duplicate and scoring was blinded. Phagocytosis was expressed as either a phagocytosis index, which is the number of apoptotic cells internalized by 100 monocyte-
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Figure 1. Apoptosis induction by ultraviolet B (UVB) irradiation of Jurkat and HaCaT cell lines. A, Annexin V/propidium iodide (PI) staining (top) and DNA content (bottom; log of the PI value) in Jurkat cells before (viable) and at 4 and 24 hours after UVB irradiation. The lower right compartment represents early apoptotic cells, and the top right compartment represents late apoptotic cells. Percentages are as follows: for viable cells, 8.6% early and 3.8% late; at 4 hours after irradiation, 48.2% early and 10.9% late; and at 24 hours after irradiation, 21.1% early and 40.5% late. B, Annexin V/PI staining (top) and DNA content (bottom; log of the PI value) in HaCaT cells before (viable) and at 8 and 24 hours after UVB irradiation. The lower right compartment represents early apoptotic cells, and the top right compartment represents late apoptotic cells. Percentages are as follows: for viable cells, 3.9% early and 6.0% late; at 4 hours after irradiation, 11.2% early and 20.4% late; and at 24 hours after irradiation, 7.8% early and 58.4% late. C, Inhibition of apoptosis of Jurkat and HaCaT cells by various concentrations of ZVAD, a caspase inhibitor.
derived macrophages, or as the percentage inhibition as compared with the phagocytosis of nonopsonized apoptotic cells. Monoclonal antibody directed against SSA/Ro clone 2G10 (kindly provided by Prof. L. A. Aarden, Sanquin Research, Amsterdam, The Netherlands) was used for opsonization at a concentration of 10 g/ml as described for the IgG fractions. In all experiments comparing the effects of patient
IgG with control IgG, monocyte-derived macrophages from the same culture and serum from the same donor were used in order to prevent differences resulting from the culture or serum conditions. Blocking of Fc␥R-mediated uptake of opsonized apoptotic cells. Apoptotic cells were opsonized with SLE and control IgG as described above. Subsequently, cells were
OPSONIZATION OF APOPTOTIC CELLS BY SLE AUTOANTIBODIES
washed and incubated for 30 minutes at room temperature with various concentrations of TG19320 (0–100 g/ml) in 1% BSA/PBS. This allowed the binding of TG19320 to the Fc portion of IgG, which is supposed to block the subsequent binding to Fc␥R. After washing, cells were used in the phagocytosis assay as described above. Blocking antibodies were also used to block the various subclasses of Fc␥R on monocyte-derived macrophages (CD16, CD32, and CD64). The monocyte-derived macrophages were preincubated for 30 minutes at 4°C with 5 g/ml of blocking antibody diluted in 1% BSA/PBS. For double blocking of both CD32 and CD64, 5 g/ml of each antibody was used. Unbound antibody was removed by washing, and monocyte-derived macrophages were subsequently allowed to take up opsonized apoptotic cells as described above. Statistical analysis. Statistical significance was calculated using GraphPad Prism software (version 4.0; GraphPad Software, San Diego, CA). The Mann-Whitney U test was used for comparisons between groups. Correlations were calculated using Spearman’s rank correlation test. P values less than 0.05 were considered significant.
RESULTS Induction of apoptosis of Jurkat and HaCaT cells by UVB irradiation. Apoptosis was induced in Jurkat cells, a human T cell line, by UVB irradiation. Since photosensitivity is one of the manifestations of SLE, we also studied UVB-induced apoptosis in HaCaT cells, a keratinocyte cell line. After UVB irradiation and 4 hours of culture, the majority (mean ⫾ SD 54.0 ⫾ 5.1%; n ⫽ 3 experiments) of Jurkat cells were early apoptotic (annexin V positive/PI negative), whereas low levels (8.5 ⫾ 2.5%) of late apoptotic cells (annexin V positive/PI positive) were present (Figure 1A). At that time point, the DNA content was decreased in 35.5 ⫾ 3.1% (mean ⫾ SD) of Jurkat cells. At 24 hours after UVB irradiation, 47.3 ⫾ 2.7% of Jurkat cells were late apoptotic, with 60.6 ⫾ 6.3% of cells having a subdiploid DNA content. HaCaT cells were more resistant to UVB irradiation and needed twice the UVB dose that was used for the Jurkat cells to induce apoptosis. After 8 hours, only 11.2 ⫾ 2.3% and 20.4 ⫾ 4.1% of HaCaT cells were early apoptotic and late apoptotic, respectively (mean ⫾ SD; n ⫽ 3 experiments). DNA content was decreased in 16.5 ⫾ 2.1% of HaCaT cells at 8 hours (Figure 1B). After 24 hours 58.4 ⫾ 4.5% of HaCaT cells were late apoptotic, while ⬍10% of the cells were early apoptotic. According to the DNA content, 55.1% of HaCaT cells were apoptotic after 24 hours (Figure 1B), which further increased to 76.3% at 48 hours (data not shown). Treatment with the caspase inhibitor ZVAD blocked the induction of apoptosis of both cell lines to
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⬃10% of the level in the nonirradiated cell cultures (Figure 1C). Binding of SLE antibodies to apoptotic cells. IgG was isolated from the sera of 26 SLE patients with various autoantibody profiles (Table 1) and from the sera of 15 healthy control subjects. Binding of IgG to Jurkat cells that had been irradiated and then incubated for 4 hours (early apoptotic) and 24 hours (late apoptotic) was tested by flow cytometry. Since only a small proportion of HaCaT cells were apoptotic after 8 hours, we tested IgG binding to HaCaT cells that had been incubated for 24 hours after UVB irradiation. Also, since only a portion of the cells were apoptotic, we used both the total mean fluorescence intensity (MFI) and the binding index (BI) as indicators of binding. The BI is calculated by multiplying the percentages of cells binding IgG by the MFI of the positive peak. No significant binding of patient or healthy control IgG to viable or early apoptotic Jurkat cells could be detected (data not shown). IgG from all SLE patients showed increased binding to both late apoptotic Jurkat cells (Figure 2A) and late apoptotic HaCaT cells (data not shown) as compared with control IgG (P ⬍ 0.001). In both SLE patient and control IgG, binding to late apoptotic Jurkat cells correlated highly (r ⫽ 0.89, P ⬍ 0.0001) with the binding to late apoptotic HaCaT cells. To study possible differences in binding capacity between patients with distinct autoantibody profiles, IgG fractions from 3 of the SLE patients were selected for analysis. These patients were known to have autoantibodies directed against SSA/Ro (patient S), nRNP (patient R), or dsDNA in combination with Sm (patient D), without reactivity against the other nuclear and/or cytoplasmic antigens tested (Table 1). Autoantibodies from patients S, R, and D showed increased binding to late apoptotic Jurkat cells (Figure 2B) and late apoptotic HaCaT cells (data not shown) as compared with control IgG. Significantly more IgG from patient S (mean ⫾ SD BI 7.2 ⫾ 3.1 [⫻1,000]; P ⫽ 0.032), patient R (BI 11.7 ⫾ 4.1 [⫻1,000]; P ⫽ 0.029), and patient D (BI 11.3 ⫾ 3.7 [⫻1,000]; P ⫽ 0.029) bound to late apoptotic cells as compared with control IgG (BI 2.1 ⫾ 3.1 [⫻1,000]). The percentages of late apoptotic cells that bound IgG from these patients varied between 21% and 23% for the Jurkat cells and between 40% and 60% for the HaCaT cells (data not shown). Double staining using annexin V and SLE patient IgG showed that the SLE patient IgG bound only to apoptotic Jurkat cells (data not shown). Since late apoptotic cells are permeable to PI, it
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Figure 2. Binding of systemic lupus erythematosus (SLE) autoantibodies to apoptotic Jurkat cells and HaCaT cells. A, Binding of IgG fractions from 26 SLE patients with autoantibodies to multiple intracellular antigens and from 15 healthy controls to late apoptotic Jurkat cells. Binding is expressed as both the binding index (BI; % positive cells ⫻ mean fluorescence intensity [MFI] of the positive peak) (left) and the total MFI (right). Data are shown as box and whisker plots, where the boxes represent the 25th and 75th percentiles, the lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. ⴱⴱⴱ ⫽ P ⬍ 0.0001. B, Binding of IgG fractions from SLE patient S (anti-SSA/Ro antibodies), patient R (anti–nuclear RNP antibodies), and patient D (anti–double-stranded DNA and anti-Sm antibodies) and from a healthy control subject to late apoptotic Jurkat cells. Binding is expressed as both the BI (left) and the total MFI (right). Values are the mean and SD of 4 independent experiments. ⴱ ⫽ P ⬍ 0.05. C, Double staining of late apoptotic Jurkat cells with a monoclonal antibody to -actin as an intracellular marker of IgG permeability and with IgG fractions from SLE patients S, R, and D and from a healthy control subject. Values shown in each compartment are the percentage of apoptotic cells. D, Western blot analysis of the binding of IgG fractions from SLE patients S, R, and D and from a healthy control subject to membrane fractions derived from viable (V), early apoptotic (EA), and late apoptotic (LA) Jurkat cells (1.8 g/lane). Molecular sizes are shown at the left.
is possible that IgG from SLE patients could bind to autoantigens localized intracellularly in late apoptotic cells. To evaluate whether IgG bound intracellularly,
extracellularly, or both, we used -actin, an antibody directed against a strongly expressed cytoskeleton protein. Late apoptotic Jurkat cells were double-stained
OPSONIZATION OF APOPTOTIC CELLS BY SLE AUTOANTIBODIES
with IgG from healthy controls or with IgG from SLE patients S, R, and D in combination with the anti–actin antibody. As expected, viable cells stained positive for -actin only after cell membrane permeabilization (data not shown), which makes this a suitable antibody with which to study the IgG permeability of late apoptotic cells. Double staining of late apoptotic Jurkat cells revealed that ⬃40% of these cells were IgG-permeable (defined as anti–-actin positive) (Figure 2C). IgG derived from healthy controls did not stain late apoptotic cells as described above. Using IgG from patients S, R, and D, we found that ⬃40% of the cells stained double positive. Nevertheless, ⬃15% of late apoptotic cells were positive only for the SLE patient IgG, indicating that in these cells, the IgG bound solely to membraneexpressed antigens. The MFIs of the double-positive and single-positive populations of Jurkat cells were comparable. In addition, we studied the ability of IgG fractions from SLE patients S, R, and D to react with plasma membrane fractions isolated from viable, early apoptotic, and late apoptotic Jurkat cells. No contamination of nuclei in the plasma membrane fractions was detected by Western blotting, using a nuclear matrix protein (p84) as a nuclear marker (data not shown). Control IgG was only reactive with a protein of ⬃60 kd in the viable plasma membrane fraction. This reaction was nonspecific, since it was also detected using only the secondary antibody. Compared with control IgG, patient IgG fractions recognized several proteins in plasma membrane fractions of late apoptotic cells, occasionally 1 protein in early apoptotic Jurkat cells (patient S), and no proteins in viable cells (Figure 2D). Proteins of similar size could also be detected in nuclear extracts when they were run side by side (data not shown). The expected 52-kd and 60-kd SSA/Ro bands were detected in IgG from patient S, although many more proteins were recognized. IgG from patient R mainly recognized a protein of ⬃70 kd, which was expected to be nRNP, although other proteins were also weakly detected. Proteins of 52, 60, and 70 kd were not recognized by the IgG from patient D, which is consistent with the results of diagnostic testing for autoantibodies. However, the IgG from patient D also recognized multiple other proteins in the membranes of apoptotic cells. Inhibition of macrophage uptake of apoptotic cells by SLE antibody opsonization. Monocyte-derived macrophages were used to assess the uptake of late apoptotic Jurkat and HaCaT cells opsonized with IgG
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fractions from SLE patients, in the presence of normal human serum. Preincubation of late apoptotic HaCaT cells with control IgG fractions resulted in a phagocytosis index comparable to that of cells preincubated with PBS alone (mean ⫾ SD 69.0 ⫾ 1.9 versus 78.0 ⫾ 0.0). Opsonization of late apoptotic HaCaT cells with IgG fractions from SLE patients decreased the phagocytosis index to 38.0 ⫾ 3.4 (data not shown). Phagocytosis was inhibited by 51.3 ⫾ 4.3% after preincubation with IgG fractions from SLE patients, compared with 11.9 ⫾ 2.7% after preincubation with IgG fractions from healthy controls (data not shown). Similar results were obtained when Jurkat cells were used as a source of apoptotic cells (Figure 3A). Similar levels of inhibition were obtained when late apoptotic neutrophils, either aged or UVB irradiated, were opsonized with IgG isolated from SLE patients (Figure 3B). The level of inhibition of phagocytosis of opsonized late apoptotic cells correlated between the Jurkat cells and the HaCaT cells (r ⫽ 0.56, P ⫽ 0.0009). No correlation between the presence and levels of anticardiolipin antibodies, one of the known phospholipid autoantibodies, and levels of phagocytosis was found (data not shown). However, levels of inhibition of phagocytosis correlated with the BI of the IgG fractions for both the late apoptotic Jurkat cells (r ⫽ 0.42, P ⫽ 0.02) and the late apoptotic HaCaT cells (r ⫽ 0.52, P ⫽ 0.0007). To test whether the inhibitory effect was specific for IgG isolated from SLE patients, we also isolated IgG from 5 ANA-negative RA patients and 5 anti–SSApositive primary SS patients and performed the phagocytosis assays as described above. IgG isolated from SS patients was able to inhibit the uptake of apoptotic cells to the same extent as IgG isolated from SLE patients (mean ⫾ SD 39.7 ⫾ 3.9 versus 39.8 ⫾ 3.8%) (Figure 3C). IgG obtained from RA patients did not bind or inhibit the uptake of late apoptotic cells (Figure 3C). We confirmed that apoptotic cells were internalized and not merely bound to monocyte-derived macrophages by incubating monocyte-derived macrophages and late apoptotic Jurkat cells at 4°C and at 37°C (Figure 3D). At 4°C, late apoptotic Jurkat cells either did not bind or were localized to the side near the monocytederived macrophages, and internalization was rarely observed (phagocytosis index 1.0 ⫾ 1.0). At 37°C, internalization of late apoptotic Jurkat cells was clearly detectable (phagocytosis index 51.3 ⫾ 3.2), and apoptotic cells were localized in a vesicular structure in monocyte-derived macrophages. Next, we studied the uptake of late apoptotic Jurkat cells and HaCaT cells opsonized with IgG frac-
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Figure 3. Phagocytosis of apoptotic cells opsonized with IgG fractions from systemic lupus erythematosus (SLE) patients and healthy controls. A, Phagocytosis of monocyte-derived macrophages incubated with late apoptotic Jurkat cells opsonized with IgG fractions from 26 SLE patients and 15 healthy controls as compared with phosphate buffered saline (PBS)–treated cells (n ⫽ 4). Phagocytosis is expressed as both the phagocytosis index (PhI) (left) and the percentage inhibition of phagocytosis (right). Data are shown as box and whisker plots, where the boxes represent the 25th and 75th percentiles, the lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. ⴱⴱⴱ ⫽ P ⬍ 0.0001. B, Percentage inhibition of phagocytosis of apoptotic neutrophils after opsonization with IgG from 3 SLE patients and 3 healthy controls as compared with PBS-treated cells. Apoptosis was induced by ultraviolet B irradiation (UV) or by aging for 72 hours. Values are the mean ⫾ SD. PMN ⫽ polymorphonuclear cells. C, Percentage inhibition of phagocytosis of late apoptotic Jurkat cells after opsonization with IgG from 4 SLE patients, 5 rheumatoid arthritis (RA) patients, 5 Sjo ¨gren’s syndrome patients, and 4 healthy controls as compared with PBS-treated cells. Data are shown as box and whisker plots, where the boxes represent the 25th and 75th percentiles, the lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. ⴱ ⫽ P ⬍ 0.05. D, Phagocytosis and binding of late apoptotic Jurkat cells after incubation at 4°C or 37°C. The photomicrographs show that at 37°C, there is internalization of the late apoptotic Jurkat cells (original magnification ⫻ 200).
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Figure 4. Phagocytosis of late apoptotic cells opsonized with IgG fractions from systemic lupus erythematosus (SLE) patients whose sera were primarily reactive against SSA/Ro (patient S), nuclear RNP (patient R), and double-stranded DNA (patient D). A, Phagocytosis index (PhI) of late apoptotic Jurkat cells opsonized with IgG from SLE patients S, R, and D and from healthy controls as compared with phosphate buffered saline (PBS)–pretreated cells. B, Percentage inhibition of phagocytosis of late apoptotic Jurkat cells opsonized with IgG fractions from SLE patients S, R, and D and from healthy controls as compared with phagocytosis of PBS-treated cells. C, Percentage inhibition of phagocytosis of late apoptotic (LA) Jurkat and HaCaT cells opsonized with control IgG or with a monoclonal antibody (MoAb) directed against SSA/Ro as compared with phagocytosis of PBS-treated cells. Values are the mean and SEM of 4 independent experiments. ⴱ ⫽ P ⬍ 0.05.
tions from SLE patients S (SSA/Ro), R (nRNP), and D (dsDNA/Sm). Compared with late apoptotic Jurkat cells preincubated with PBS (54.8 ⫾ 3.2; n ⫽ 4) or control IgG (50.5 ⫾ 8.5; n ⫽ 4), the phagocytosis index was decreased when late apoptotic cells were preincubated with IgG fractions from patient S (30 ⫾ 8.1; n ⫽ 4), R (26.8 ⫾ 7.6; n ⫽ 4), or D (34.3 ⫾ 10.2; n ⫽ 4) (Figure 4A). Compared with control IgG, phagocytosis was inhibited by 40–50%, depending on patient IgG and the nature of the apoptotic cells (Figure 4B). To assess whether binding of an autoantibody with only 1 specificity was sufficient to induce the inhibitory effect, we used a monoclonal antibody di-
rected against SSA/Ro for opsonization of both apoptotic Jurkat and HaCaT cells. In phagocytosis assays, this monoclonal antibody also inhibited the uptake of late apoptotic cells by macrophages to a similar level (median ⫾ SEM 39.2 ⫾ 17.8%, as compared with 8.6 ⫾ 9.9% of controls) as patient IgG (Figure 4C). Dependence of apoptotic cell phagocytosis on serum concentration, IgG concentration, and IgG Fc and Fc␥R interactions. We investigated the effect of normal human serum on the phagocytosis of apoptotic cells by varying the serum concentrations from 50% to 10%. The phagocytosis index of late apoptotic Jurkat cells incubated with IgG fractions from healthy controls
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Figure 5. Dependence of phagocytosis of late apoptotic cells opsonized with IgG fractions from systemic lupus erythematosus (SLE) patients on the IgG concentration and interactions between Fc and Fc␥ receptors (Fc␥R). A, Effect of decreasing concentrations of IgG used for opsonizing late apoptotic Jurkat cells on the phagocytosis index (PhI). IgG fractions from SLE patients D and 8 and a healthy control subject were used at the concentrations shown. B, Effect of blocking the interaction of Fc and Fc␥R on SLE patient IgG–mediated inhibition of phagocytosis. Opsonized late apoptotic Jurkat cells were preincubated with different concentrations of the blocking peptide TG19320 and incubated with monocyte-derived macrophages. C, Effect of blocking CD16 (Fc␥RIII), CD32 (Fc␥RII), and CD64 (Fc␥RI) on the phagocytosis of late apoptotic Jurkat cells opsonized with SLE patient IgG. The percentage inhibition of monocyte-derived macrophage phagocytosis of apoptotic cells preincubated with either phosphate buffered saline (PBS) or IgG from healthy controls (C4 and C5) or 5 different SLE patients was examined in the absence of blocking (PBS) and after blocking the different Fc␥R (CD16, CD32, and CD64) or after blocking the IgG Fc with TG19320 peptide. D, Effect of blocking both CD32 and CD64 on the phagocytosis of late apoptotic (LA) Jurkat cells opsonized with IgG from SLE patients D and 5. Apoptotic cells were opsonized with IgG from both patient D (CD32 mediated) and patient 5 (CD64 mediated) and were examined in the absence of Fc␥R blockade (PBS), in the presence of anti-CD32 or anti-CD64 alone, after blocking with both anti-CD32 and anti-CD64, and after blocking with the IgG Fc–blocking peptide TG19320. Values are the mean and SEM of 2 independent experiments, except for D, which represents a single experiment.
or SLE patients did not change when the serum concentration was reduced from 50% to 30% (data not shown). Lower serum concentrations reduced the phagocytosis
index for late apoptotic cells opsonized with IgG fractions from controls and with IgG fractions from SLE patients. However, the inhibitory effect of opsonizing
OPSONIZATION OF APOPTOTIC CELLS BY SLE AUTOANTIBODIES
IgG fractions from SLE patients was still detectable at a serum concentration of 20%. At a serum concentration of 10%, late apoptotic cells opsonized with either healthy control IgG or patient IgG were taken up at a comparable, but low, rate (data not shown). Subsequently, we assessed the influence of the IgG concentration, as used for opsonizing late apoptotic Jurkat cells, on the level of phagocytosis. As expected, the concentration of IgG from healthy controls did not influence the phagocytosis index (Figure 5A). However, diluting IgG fractions from SLE patients increased the phagocytosis index to levels comparable with those of control IgG. To evaluate whether the inhibition of phagocytosis by IgG from SLE patients was dependent on interactions between Fc and Fc␥R, we blocked the Fc portion of IgG using the blocking peptide TG19320. Preincubation of SLE patient IgG–opsonized late apoptotic Jurkat cells with increasing concentrations of the Fc-blocking peptide increased the phagocytosis of these cells to levels similar to those of control IgG–opsonized late apoptotic cells (Figure 5B). In addition, we blocked the various Fc␥ receptors on monocyte-derived macrophages with Fc␥R-blocking antibodies and performed the phagocytosis assay. Phagocytosis of late apoptotic Jurkat cells pretreated with PBS or with IgG fractions from healthy controls was not influenced by Fc␥R blockade (data not shown). The inhibitory effect of opsonization with IgG fractions from SLE patients was strongly decreased by Fc␥R blockade, with a phagocytosis index comparable to that of late apoptotic cells blocked with the IgG Fc–blocking peptide (Figure 5C). Blocking of CD32 resulted in complete resolution of the inhibitory effect in 2 of the 5 patient IgG fractions tested. In 2 other patient IgG fractions, this occurred after blockade of CD64. The remaining patient IgG we tested required blockade of both CD32 and CD64 to abolish its inhibitory effect. Thus, the IgG fractions interacted differentially with Fc␥R. Blockade of both CD32 and CD64 was performed to test the independent contribution of these receptors to the inhibitory effect. Late apoptotic Jurkat cells were opsonized with a mixture of IgG from patient D (CD32 mediated) and patient 5 (CD64 mediated). Inhibition of phagocytosis mediated by these 2 SLE IgG could only be completely abolished by blocking both CD32 and CD64 (Figure 5D). This confirms the independent involvement of both CD32 and CD64 in the inhibition of phagocytosis of SLE IgG–opsonized late apoptotic cells.
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DISCUSSION Although SLE autoantigens are known to be exposed on the surface of apoptotic cells, little is known about the binding of IgG from SLE patients to these surface-exposed autoantigens and its subsequent effect on apoptotic cell clearance. In this study, we showed that IgG derived from SLE patients not only opsonized late apoptotic cells, but also inhibited their uptake by monocyte-derived macrophages. Thus far, binding of human autoantibodies to nuclear and/or cytoplasmic antigens expressed on apoptotic cells has only been investigated in a limited number of sera (8). In the present study, we used flow cytometric techniques to show that IgG isolated from SLE patients binds to late apoptotic cells. Because flow cytometry was used to analyze the binding, it could be argued that cell permeability, as demonstrated by PI staining, of late apoptotic cells could potentially allow the binding of IgG molecules to autoantigens localized intracellularly. Using an antibody to the cytoskeletal protein -actin, we showed that ⬃15% of late apoptotic cells were negative for -actin staining and were thus not permeable to IgG. This strongly suggests that patient IgG binds to the outer leaflet of the apoptotic cell membrane. Furthermore, IgG fractions from SLE patients with 3 distinct autoantibody profiles all recognized proteins in plasma membrane fractions derived from late apoptotic Jurkat cells and hardly any proteins in membrane fractions from early apoptotic Jurkat cells, findings that were consistent with the flow cytometry results. The comparable inhibitory behavior of the IgG fractions isolated from patients with these distinct autoantibody profiles indicates that opsonization of late apoptotic cells and its subsequent effect on phagocytosis is not restricted to one particular autoantibody profile. However, as illustrated by the experiment using a monoclonal antibody directed against SSA, binding of a single specificity is sufficient to reduce the phagocytosis index. Separate analysis of other antibody specificities involved in this inhibitory effect will be the subject of further studies. One could argue that the use of transformed cell lines as apoptotic targets may not reflect the physiologic situation. Therefore, we also performed these experiments using late apoptotic neutrophils. Phagocytosis was similarly inhibited in these primary cells by SLE IgG, indicating that these effects can be extrapolated to apoptotic nontransformed cells present in humans. Antibodies directed against nuclear antigens are
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not restricted to SLE patients. In patients with primary SS, antibodies directed against SSA/Ro are often found. To establish whether the inhibition of apoptotic cell clearance by opsonizing autoantibodies is SLE specific, we also tested IgG fractions isolated from anti-SSA/Ropositive primary SS patients and ANA-positive RA patients for their inhibitory effects on phagocytosis. Phagocytosis of late apoptotic cells was inhibited to a similar extent by IgG fractions isolated from the SS patients as by IgG fractions from the SLE patients, but not by IgG from the RA patients, who lacked autoantibodies directed against nuclear or cytoplasmic antigens. Therefore, inhibition of phagocytosis seems to be dependent on the presence of antibodies that recognize surface molecules expressed on apoptotic cells and is not specific for SLE autoantibodies. Facilitation of apoptotic cell uptake by antiphospholipid antibodies has been suggested by Manfredi et al (20). In our study, 3 of the 26 SLE patient IgG fractions lacked inhibitory capacity and even facilitated, to some extent, the uptake of late apoptotic Jurkat cells. However, we found no correlation between the presence and levels of IgG anticardiolipin antibodies, one of the known antiphospholipid autoantibodies, and the levels of phagocytosis (data not shown). The phagocytosis assay as applied by Manfredi et al used radioactively labeled apoptotic cells and detected levels of the radioactive label in monocyte-derived macrophages after a 1-hour contact between the cells. Despite washing the monocyte-derived macrophage monolayers, apoptotic cells that bind to monocyte-derived macrophages but are not phagocytosed will not be removed, and such cells were detected as well in the assay used by Manfredi. In the current study, we were able to discriminate, by microscopy, the uptake from the binding of apoptotic cells, which might explain differences in results. Recently, Clancy et al (21) also reported an inhibitory effect of anti-SSA/Ro and anti-SSB/La monoclonal antibodies on the uptake of apoptotic myocardiocytes. That study, as well as our study, strongly indicates that the binding of antibodies to apoptotic cells can have a negative effect on their uptake by macrophages. Some speculation about the mechanisms underlying the reduced uptake of apoptotic cells opsonized by SLE IgG is relevant. As suggested by Clancy et al (21), the antigens on apoptotic cells bound by autoantibodies may themselves be involved in the normal engulfment of apoptotic cells. SLE IgG might block molecules on the apoptotic cells that are necessary for recognition and facilitation of their uptake by macrophages, such as phosphatidylserine (PS) or C1q (22–25). Otherwise, we
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showed in our study that preincubation with a peptide that blocks the IgG Fc region or blockade of Fc␥ receptors on the macrophage abrogated the inhibitory effect of the SLE autoantibodies. This finding clearly indicates that Fc␥R engagement by the antibodies is necessary for the inhibitory effect. Additionally, the involvement of both CD64 and CD32 rules out the possibility that engagement of the inhibitory Fc␥RIIb was solely responsible for the inhibitory effect seen. Signaling via the Fc␥R may influence the capacity of monocyte-derived macrophages to internalize additional apoptotic cells (26). Triggering of Fc␥ receptors influences the signals provided by other receptors that are involved in uptake. This may lead to altered kinetics of uptake compared with uptake of non–IgG-opsonized apoptotic cells, as supported by a recent study showing that phagosome maturation is slower when cells are opsonized with IgG (27). The role of the PS receptor (PSR) in apoptotic cell uptake has been questioned (28) but cannot be excluded (25,29). Nevertheless, both Fc␥R and PSR are thought to use Rho and Rac to elicit actin polymerization (30). The simultaneous signaling of both receptors may result in a disorganized actin reorganization, resulting in decreased uptake. In contrast, PSR signaling also results in the release of antiinflammatory mediators, while Fc␥R engagement is known to trigger proinflammatory mediators. How these 2 pathways are involved in the actual phagocytic process is not known, but they may counteract each other and hamper the uptake of apoptotic cells. Further elucidation of the underlying mechanism should focus on the study of signaling changes in macrophages during interaction with IgG-opsonized apoptotic cells. This subject is currently being pursued in our laboratory. The role of autoantibodies in the clearance of apoptotic cells has, until now, not been explored. Here, we show that autoantibodies inhibit apoptotic cell clearance, which might further increase the apoptotic cell load in SLE patients. ACKNOWLEDGMENTS The authors thank Dr. G. Fassina (Xeptagen, Pozzuoli, Italy) for providing the Fc-blocking peptide TG19320 and Prof. L. A. Aarden (Sanquin Research, Amsterdam, The Netherlands) for providing the monoclonal antibodies directed against SSA/Ro. AUTHOR CONTRIBUTIONS Dr. Reefman had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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Study design. Reefman, Limburg, Kallenberg, Bijl. Acquisition of data. Reefman, Horst, Nijk. Analysis and interpretation of data. Reefman, Limburg, Bijl. Manuscript preparation. Reefman, Limburg, Kallenberg, Bijl. Statistical analysis. Reefman.
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