Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 10, October 2003, pp 2888–2897 DOI 10.1002/art.11237 © 2003, American College of Rheumatology Increased Apoptotic Neutrophils and Macrophages and Impaired Macrophage Phagocytic Clearance of Apoptotic Neutrophils in Systemic Lupus Erythematosus Yi Ren,1 Jinling Tang,2 M. Y. Mok,1 Albert W. K. Chan,1 Adrian Wu,1 and C. S. Lau1 Objective. To evaluate whether patients with systemic lupus erythematosus (SLE) have a higher rate of apoptosis in and secondary necrosis of polymorphonuclear neutrophils (PMNs) and macrophages compared with controls; to compare the rate of macrophage phagocytic clearance of apoptotic PMNs in patients with SLE and healthy controls; to evaluate whether in vitro PMN and macrophage apoptosis and secondary necrosis, and the ability of macrophages to phagocytose apoptotic bodies, are correlated with lupus disease activity; and to determine whether macrophage clearance of apoptotic bodies in patients with SLE and normal controls is related to certain serum factors. Methods. Thirty-six patients with SLE and 18 healthy, nonsmoking volunteers were studied. PMNs and monocytes were isolated from fresh blood and cultured in the presence of different sources of serum. Apoptotic PMNs and macrophages were examined by annexin V binding and morphology on May–Giemsa– stained cytopreparations, at different time points. The presence of secondary necrotic PMNs and macrophages was verified by staining with trypan blue. Macrophage phagocytosis of apoptotic PMNs was measured using a coded, observer-blinded, microscopically quantified phagocytosis assay. Cells were cultured in the presence of serum obtained from healthy subjects or from patients with SLE. Results. At 5 and 24 hours, the percentage of apoptotic PMNs from patients with SLE was significantly higher than that of PMNs from healthy subjects. At 24 and 48 hours, the percentage of secondary necrotic PMNs from patients with SLE was also significantly higher than the percentage of necrotic PMNs from controls. Serum from patients with SLE accelerated the rate of apoptosis in and secondary necrosis of PMNs from healthy subjects. Macrophages from SLE patients were less capable of phagocytosing apoptotic PMNs compared with macrophages obtained from controls. Macrophages from patients with active SLE were less capable of phagocytosing apoptotic PMNs than were macrophages from patients with inactive SLE, but the difference was not statistically significant. The percentage of phagocytosis of apoptotic PMNs by macrophages from SLE patients correlated negatively with the SLE Disease Activity Index, serum levels of anti–doublestranded DNA, and the erythrocyte sedimentation rate, and correlated positively with serum levels of C3, C4, and albumin, the hemoglobin level, and the leukocyte count. Serum from SLE patients not only significantly increased macrophage apoptosis in cells from healthy subjects but also remarkably down-regulated the clearance of apoptotic PMNs by macrophages from healthy subjects. In contrast, serum from healthy subjects significantly increased phagocytosis of apoptotic PMNs by macrophages from SLE patients. Conclusion. The observed increase of apoptotic PMNs and macrophages and the poor ability of macrophages from patients with SLE to phagocytose apoptotic bodies may indicate an impaired clearance mechanism, which may be mediated by factors in a patient’s serum. Supported by a grant from the Committee on Research and Conference, The University of Hong Kong, and a Research Grants Council Competitive Earmarked Research grant from the Hong Kong University Grants Council. 1 Yi Ren, MD, M. Y. Mok, MD, Albert W. K. Chan, BSc, Adrian Wu, MD, C. S. Lau, MD: Queen Mary Hospital, University of Hong Kong, Hong Kong; 2Jinling Tang, MD: Chinese University of Hong Kong, Hong Kong. Address correspondence and reprint requests to C. S. Lau, MD, University Department of Medicine, Room 807 Administration Block, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong. E-mail: email@example.com. Submitted for publication October 16, 2002; accepted in revised form June 6, 2003. Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with unknown etiology. 2888 APOPTOTIC CELL CLEARANCE IN SLE B cell hyperactivity with production of multiple autoantibodies is the hallmark of SLE. It is now known that such polyclonal B cell activation is T cell–dependent and autoantigen-driven (1). An excessive supply of autoantigens has been suggested to be secondary to increased apoptosis in SLE (2). Apoptosis is an energy-dependent process that leads to the programmed destruction of cells. It is tightly regulated by the expression of cell surface molecules such as Fas and intracellular protooncogenes including Bcl-2 and the Bax family (3). Fas ligation leads to the induction of apoptosis, while Bcl-2 and the Bax family molecules provide a positive signal for survival. A possible association between SLE and Fas-mediated apoptosis and the failure of cell tolerance was suggested when Chu et al (4) reported a defect in the expression of Fas messenger RNA in MRL/lpr mice. This hypothesis was later supported by the identification of the gld gene (which was phenotypically similar to lpr) as a nonfunctional Fas ligand gene (5). In human SLE, increased spontaneous in vitro apoptosis in cultured peripheral blood mononuclear cells (PBMCs) has been described (6). Nucleosomes, which are selectively produced during apoptosis, have been shown to be a major antigen for autoreactive T cells in SLE (7). The morphologic features of apoptosis include cytoplasmic blebbing, fragmentation and condensation of intracellular chromatin, internucleosomal digestion of DNA, and late membrane failure (8). In the late phase of apoptosis, anionic phospholipids (phosphatidylserine [PS] in particular), which are initially confined to the intracellular portion of the membrane of viable cells, are translocated to the outer surface (9). The exposure of anionic phospholipid moieties on the cell surface of apoptotic cells triggers the immediate engulfment of these cells by scavenger phagocytes. It is prudent that apoptotic bodies are effectively cleared to avoid release of intracellular contents that may be immunogenic, which may induce T and B cell autoreactivity (10,11). Impairment, if any, in phagocytic cell clearance of apoptotic bodies may therefore have a pathogenic role in SLE. The objectives of this study were, therefore, 1) to study the rate of apoptosis in and secondary necrosis of polymorphonuclear neutrophils (PMNs) and macrophages in patients with SLE and controls; 2) to measure the ability of phagocytic macrophages to clear apoptotic bodies in patients with SLE; 3) to evaluate whether the in vitro rate of PMN and macrophage apoptosis and secondary necrosis and changes in macrophage phagocytosis of apoptotic bodies are correlated with lupus disease activity; and 4) to 2889 determine whether factors in serum from patients with active SLE affect PMN and macrophage apoptosis and secondary necrosis and macrophage phagocytic clearance of apoptotic bodies. PATIENTS AND METHODS Tissue culture materials. All chemicals were purchased from Sigma (St. Louis, MO), unless indicated otherwise. Culture medium (Iscove’s modified Dulbecco’s medium [IMDM] and Dulbecco’s modified Eagle’s medium) and supplements (100 units/ml penicillin, 100 g/ml streptomycin, 2 mM glutamine, and 10% fetal calf serum) were obtained from Gibco (Grand Island, NY), and sterile tissue culture plasticware was obtained from Falcon Plastics (Cockeysville, MD). Subjects. Study subjects included 36 patients with SLE (4 men and 32 women, mean age 32.3 years [range 18–48 years]), of whom 11 had inactive disease and 25 had active disease at the time of study. All patients met the American College of Rheumatology classification criteria for SLE (12). SLE disease activity was assessed using the SLE Disease Activity Index (SLEDAI) (13). Patients were classified as having inactive disease if the SLEDAI score was persistently ⱕ4 for at least 4 months prior to blood sampling. Patients with active disease had a SLEDAI score of ⱖ5 at the time of study. Five patients, of whom 2 were newly diagnosed as having SLE, were receiving no treatment at the time of the study. Seventeen patients (the nonimmunosuppressive therapy group) were receiving low-dose steroids (prednisolone, ⱕ10 mg/day) and/or hydroxychloroquine, and 14 patients (the immunosuppressive therapy group) were receiving prednisolone at a dosage of ⬎10 mg/day and/or immunosuppressive agents. The immunosuppressive agents taken by patients with SLE included azathioprine (n ⫽ 11), methotrexate (n ⫽ 1), cyclosporin A (n ⫽ 1), and mycophenolate mofetil (n ⫽ 1). Table 1 shows the clinical characteristics and drug treatment of the 36 patients studied. Eighteen healthy, nonsmoking volunteers (6 men and 12 women, mean age 34 years [range 30–40 years]) were included as controls. This study was approved by The University of Hong Kong Faculty of Medicine Ethics Committee. Written informed consent was obtained from all subjects. Leukocytes. A 20-ml sample of venous blood was obtained from each subject. PMNs were isolated by dextran sedimentation and plasma–Percoll discontinuous densitygradient centrifugation and were “aged” in tissue culture in IMDM with 10% platelet-rich plasma-derived serum in order to undergo apoptosis, exactly as described previously (14). Monocytes were prepared from adherent PBMCs, using standard methods, and were cultured for 5 days in IMDM with 10% autologous serum to promote maturation into macrophages. Assessment of PMN morphology, and flow cytometric analysis. Apoptotic PMNs were assessed by microscopic examination of cytocentrifuge preparations fixed in methanol and stained with May–Giemsa (14). Typically, 500 counts were made from duplicate slides of the same experiment. Binding of fluorescein isothiocyanate–conjugated annexin V (Immunotech, Marseilles, France) was used to assess the exposure of PS. Propidium iodide and trypan blue were used to assess plasma 2890 REN ET AL Table 1. Clinical characteristics and drug treatment of 36 patients with SLE* Characteristic Value Sex, no. men/no. women Age, mean (range) years Disease duration, mean (range) years Patients with inactive disease/patients with active disease SLE drug treatment at time of study Patients with inactive disease No treatment Prednisolone, ⱕ10 mg/day and/or hydroxychloroquine Prednisolone, ⬎10 mg/day and/or immunosuppressive agents Patients with active disease No treatment Prednisolone, ⱕ10 mg/day and/or hydroxychloroquine Prednisolone, ⬎10 mg/day and/or immunosuppressive agents 4/32 32.3 (18–48) 6.2 (0–12) 11/25 3 5 3 2 12 11 * Except where indicated otherwise, values are the number of patients. Systemic lupus erythematosus (SLE) disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Inactive disease ⫽ SLEDAI score ⱕ4 for at least 4 months prior to blood sampling; active disease ⫽ SLEDAI score ⱖ5 at the time of study. Immunosuppressive agents included azathioprine (taken by 3 patients with inactive disease and 8 patients with active disease), methotrexate (1 patient with active disease), cyclosporin A (1 patient with active disease), and mycophenolate mofetil (1 patient with active disease). membrane permeability. Labeled cells were applied to an EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA), which automatically and simultaneously measured the fluorescence of individual cells identified by their size-dependent light-scattering properties. Interaction assay. A coded, observer-blinded, microscopically quantified phagocytosis assay of macrophage ingestion of apoptotic PMNs, which has been extensively described, illustrated, and validated (14), was used in this study. Apoptotic PMNs prepared as described above were washed once in phosphate buffered saline, suspended in IMDM and 0.5 ⫻ 106 PMNs in 50 l of medium, and added to each washed well of macrophages cultured in 96-well plates. After interaction for 30 minutes at 37°C in 5% CO2, the wells were washed in cold (4°C) 0.9% saline to remove noningested PMNs. The macrophage monolayer was then fixed in 2% glutaraldehyde in saline for 2 minutes, stained with myeloperoxidase, and the proportion of macrophages ingesting PMNs was counted by inverted light microscopy. The percentage of macrophages that had taken up apoptotic PMNs was regarded as the overall macrophage phagocytosis index. Additionally, to better assess macrophage phagocytosis activity, the percentage of macrophages that had ingested different numbers of apoptotic PMNs was evaluated. Effects of serum. To determine whether serum factors have an effect on PMNs and monocytes undergoing apoptosis, freshly isolated PMNs and monocytes from healthy subjects and patients with SLE were cultured and cross-cultured with IMDM in 10% untreated serum obtained from healthy sub- jects or patients with SLE. The rate of PMN and monocyte apoptosis was measured at different time points. To determine whether serum factors have an effect on phagocytosis of PMNs undergoing apoptosis by monocyte-derived macrophages, freshly isolated monocytes from healthy subjects and patients with SLE were cultured and cross-cultured with IMDM in 10% untreated serum from healthy subjects or patients with SLE for 5 days. Apoptotic PMNs were allowed to interact with day 5 macrophages for 30 minutes, and the percentage of macrophages taking up apoptotic PMNs was counted. Statistical analysis. The Mann-Whitney U test was used to compare the percentage of apoptotic and necrotic PMNs and macrophages, and macrophage phagocytosis of apoptotic PMNs, in patients with SLE (those with active or inactive disease and those who were or were not receiving immunosuppressive treatment) and controls. Spearman’s rank correlation test was used to examine the relationship between phagocytosis capacity and various characteristics of lupus disease activity, expressed as continuous variables. All analyses were conducted using SAS version 6.1 software (SAS Institute, Cary, NC). RESULTS Apoptotic PMNs isolated from patients with SLE and healthy subjects. PMNs from SLE patients and controls were isolated and cultured for different periods of time (0, 5, and 24 hours). At each time point, cells were harvested, and apoptosis was measured. Figure 1 shows the percentage of cells cultured with autologous serum that underwent spontaneous apoptosis at 0, 5, and Figure 1. Spontaneous apoptosis of polymorphonuclear neutrophils (PMNs) during culture of purified PMNs at 0, 5, and 24 hours. The percentage of cells undergoing apoptosis was determined after morphologic examination of May–Giemsa–stained cytospin preparations. PMNs were derived from 11 patients with inactive systemic lupus erythematosus (SLE), 25 patients with active SLE, and 18 healthy controls. Results are expressed as the percentage of apoptotic cells per total PMNs. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus controls. APOPTOTIC CELL CLEARANCE IN SLE Figure 2. Percentage of secondary necrotic PMNs detected by trypan blue assay after 24 and 48 hours of culture. PMNs were derived from 11 patients with inactive SLE, 25 patients with active SLE, and 18 healthy controls. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus controls. See Figure 1 for definitions. 24 hours. At time 0, there were no significant differences in spontaneous apoptosis between PMNs from patients with inactive (0.96 ⫾ 0.50%) or active (0.32 ⫾ 0.19%) SLE, and PMNs from healthy controls (0.10 ⫾ 0.10%). After 5 hours of culture, higher percentages of apoptotic PMNs from patients with active SLE (13.93 ⫾ 2.73%) and those with inactive SLE (8.64 ⫾ 1.54%) were detected when compared with controls (3.25 ⫾ 1.2%). Interestingly, the difference between the percentage of apoptotic PMNs from patients with active SLE and those with inactive SLE was significant (P ⬍ 0.05). The percentage of apoptotic PMNs at 24 hours was also determined. PMNs from patients with inactive SLE and patients with active SLE displayed accelerated apoptosis (71.67 ⫾ 5.46% and 71.9 ⫾ 4.04%, respectively) when compared with healthy subjects (58.04 ⫾ 3.94%) (P ⬍ 0.05). At 24 hours, there were no differences in apoptosis between PMNs from patients with inactive or active SLE and PMNs from patients who were or were not receiving immunosuppressive drugs. A negative correlation between the percentage of apoptotic PMNs and the peripheral neutrophil count (r ⫽ ⫺0.595, P ⬍ 0.02) was observed in patients with SLE. Secondary necrotic PMNs isolated from patients with SLE and healthy subjects. PMNs isolated from patients with SLE and healthy subjects were cultured for 24 and 48 hours, and necrotic PMNs were detected by trypan blue assay. At 24 hours and 48 hours, the percentage of necrotic PMNs from patients with inactive or active SLE was significantly higher than the percentage of necrotic PMNs from healthy subjects (Figure 2). At both 24 and 48 hours, there were no differences in 2891 PMN necrosis between patients with active SLE and patients with inactive SLE, and patients who were or were not receiving immunosuppressive drugs. Effects of serum on PMN apoptosis and necrosis. To study whether serum from patients with SLE affected apoptosis in and necrosis of healthy PMNs, PMNs from healthy subjects were cultured with serum from patients with active SLE, and apoptosis was detected (Figure 3A). Serum from patients with active SLE not only accelerated apoptosis of PMNs from healthy subjects at 24 hours (from 58.03 ⫾ 3.94% to 72.07 ⫾ 5.17%; P ⬍ 0.05), but also increased the percentage of necrotic PMNs at both 24 and 48 hours (Figure 3B). Serum from healthy subjects did not rescue SLE PMNs from undergoing apoptosis and necrosis (data not shown). Apoptosis in macrophages from SLE patients. Monocytes isolated from patients with SLE and healthy Figure 3. A, Modulation of apoptosis of control PMNs by serum from patients with SLE. PMNs from 11 healthy subjects were cultured with serum from patients with active SLE, and apoptosis was detected at 5 hours and 24 hours. B, Modulation of necrosis in control PMNs by serum from patients with SLE. PMNs from 11 healthy subjects were cultured with serum from patients with active SLE, and necrosis was detected at 24 hours and 48 hours. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus controls. See Figure 1 for definitions. 2892 REN ET AL from patients with SLE (Figure 5A) and healthy subjects (Figure 5B). The percentage of SLE macrophages ingesting apoptotic PMNs was 23.48 ⫾ 7.0%, while the percentage of healthy macrophages taking up apoptotic PMNs was 38.5 ⫾ 4.0% (P ⬍ 0.001) (Figure 6A). Data presented here were obtained from unselected patients. Furthermore, the percentage of phagocytosis of apopto- Figure 4. Apoptotic macrophages detected by flow cytometry. Monocytes isolated from 10 healthy control subjects (A) and 11 patients with active SLE (B) were cultured with autologous serum for 5 days. The percentage of apoptotic macrophages was determined by annexin V binding and propidium iodide (PI) staining. The mean ⫾ SEM percentage of annexin V binding in SLE and control macrophages was 23.25 ⫾ 3.6 and 2.02 ⫾ 0.8, respectively. The mean ⫾ SEM percentage of PI binding in SLE and control macrophages was 14.0 ⫾ 1.6 and 2.43 ⫹ 0.5, respectively. FITC ⫽ fluorescein isothiocyanate (see Figure 1 for other definitions). subjects were cultured for 5 days, and then apoptosis was detected. Macrophages undergoing apoptosis were also detected by flow cytometry (Figure 4). Macrophages from patients with active SLE had more annexin V binding compared with macrophages from healthy subjects. Serum from patients with active SLE accelerated apoptosis of macrophages from healthy subjects (data not shown). Macrophage phagocytosis of PMNs undergoing apoptosis. Monocytes from patients with SLE and healthy subjects were cultured for 5 days with serum obtained from either SLE patients or controls. Adherent cultures of day 5 macrophages were washed and interacted with apoptotic PMNs in medium with no added serum for 30 minutes. Figure 5 shows the results of macrophage phagocytosis of apoptotic PMNs obtained Figure 5. Decreased phagocytosis of apoptotic polymorphonuclear neutrophils (PMNs) by monocyte-derived macrophages from patients with systemic lupus erythematosus (SLE). PMNs within macrophages are readily identifiable by their brown staining for myeloperoxidase. A, Macrophages from patients with active SLE ingesting apoptotic PMNs (day 5). B, Macrophages from healthy subjects ingesting apoptotic PMNs (day 5). APOPTOTIC CELL CLEARANCE IN SLE 2893 Figure 7. Ingestion of apoptotic PMNs by macrophages from healthy subjects and patients with SLE. Day 5 macrophages from 8 SLE patients and 8 healthy subjects were allowed to interact with apoptotic PMNs for 30 minutes. The number of macrophages that had ingested different numbers of apoptotic PMNs was counted. Bars show the mean and SEM. See Figure 5 for definitions. Figure 6. Macrophage ingestion of apoptotic PMNs from healthy subjects and patients with SLE. Day 5 macrophages from SLE patients and healthy subjects (n ⫽ 12) were allowed to interact with apoptotic PMNs for 30 minutes. The number of macrophages ingesting apoptotic PMNs was then counted. A, Phagocytosis of apoptotic PMNs by macrophages from unselected SLE patients (n ⫽ 36). ⴱ ⫽ P ⬍ 0.001 versus controls. B, Phagocytosis of apoptotic PMNs by macrophages from patients with active (n ⫽ 25) or inactive (n ⫽ 11) SLE and healthy control subjects. ⴱ ⫽ P ⬍ 0.05 versus controls; ⴱⴱ ⫽ P ⬍ 0.001 versus controls. Bars show the mean and SEM. See Figure 5 for definitions. tic PMNs by macrophages from patients with inactive or active SLE was 29.2 ⫾ 5.2% and 17.8 ⫾ 3.2%, respectively (Figure 6B); this difference was not statistically significant (P ⫽ 0.081). The use of immunosuppressive agents did not appear to influence macrophage phagocytosis of apoptotic PMNs. There were no significant differences in phagocytosis between healthy subjects and patients with inactive SLE. Not only did patients with SLE have fewer macrophages that were capable of phagocytosing apoptotic PMNs, but also the capacity of the phagocytosiscompetent macrophages to ingest apoptotic bodies was lower in SLE patients compared with controls. Figure 7 shows the percentage of macrophages that ingested different numbers of apoptotic PMNs from patients with SLE and controls. Figure 8 shows the number of apo- ptotic PMNs within 100 phagocytosis-competent macrophages, as well as within a total of 100 macrophages (both phagocytosis-competent and phagocytosisnoncompetent) from patients with SLE and controls. Correlation between the percentage of phagocytosis of apoptotic PMNs by SLE macrophages and lupus disease activity parameters. The percentage of phagocytosis of apoptotic PMNs by macrophages from patients with SLE correlated negatively with the SLEDAI score, the serum level of anti–double-stranded DNA Figure 8. Impaired phagocytic capacity of SLE macrophages. The number of apoptotic PMNs (from patients with SLE [n ⫽ 8] and controls [n ⫽ 8]) within 100 phagocytically competent macrophages, and within a total of 100 macrophages (both phagocytically competent and phagocytically noncompetent) was counted. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus controls; ⴱⴱ ⫽ P ⬍ 0.001 versus controls. See Figure 5 for definitions. 2894 REN ET AL Table 2. Correlation between macrophage phagocytosis of apoptotic neutrophils and various parameters of SLE disease activity* Parameter r P SLEDAI Anti-dsDNA C3 C4 Serum albumin Hemoglobin Leukocyte count Erythrocyte sedimentation rate ⫺0.5169 ⫺0.7184 0.5011 0.5556 0.6058 0.5330 0.5181 ⫺0.5168 0.0336 0.0017 0.0480 0.0254 0.0129 0.0335 0.0335 0.0404 * SLE ⫽ systemic lupus erythematosus; SLEDAI ⫽ SLE Disease Activity Index; anti-dsDNA ⫽ anti–double-stranded DNA. increased in SLE (19). In that study, there was a positive correlation with anti-dsDNA antibodies and lupus disease activity, as measured by the Systemic Lupus Activity Measure score (20). It is interesting to note from our experiments that serum from patients with active SLE accelerated in vitro apoptosis in and secondary necrosis of PMNs, while serum from control subjects failed to prevent cells from SLE patients from undergoing apoptosis and secondary necrosis. These results suggest that both genetic and serum factors that regulate PMN apoptosis may be abnormal in patients with SLE. Apoptosis in PMNs is mediated by Fas, and Fas ligation is associated with widespread PS expression on the cell surface (21). (anti-dsDNA), and the erythrocyte sedimentation rate, and positively with the serum levels of C3, C4, and albumin, the hemoglobin level, and the leukocyte count (Table 2). Effects of serum on phagocytosis of apoptotic PMNs. We also studied whether serum from healthy subjects would rescue the impaired phagocytic capacity of macrophages obtained from patients with active SLE. Monocytes from patients with SLE were cultured for 5 days with serum obtained from healthy subjects and then interacted with apoptotic PMNs for 30 minutes. Healthy serum increased the percentage of phagocytosis of apoptotic PMNs by macrophages from patients with active SLE, from 23.48 ⫾ 7.0% to 52.92 ⫾ 6.9% (P ⬍ 0.05) (Figure 9A). In contrast, serum obtained from patients with active SLE decreased the percentage of macrophages ingesting apoptotic PMNs, from 41.4 ⫾ 5.3% to 14.0 ⫾ 2.3% (P ⬍ 0.05) (Figure 9B). DISCUSSION In this study, we observed that the rate of in vitro apoptosis of PMNs and macrophages was increased in patients with SLE when compared with controls. Patients with active disease had higher rates of in vitro apoptosis than did patients with inactive disease. Additionally, the in vitro rate of secondary necrosis of PMNs was increased in patients with SLE when compared with controls. Our findings are in accordance with previous studies, in which it was reported that increased apoptosis of peripheral blood cells has a pathogenic role in SLE (6,15–19). Most of these studies evaluated the increased spontaneous cell death of PBMCs in SLE; the rate of lymphocyte apoptosis correlated significantly with disease activity (6,15,17). Recently, neutrophil apoptosis, as assessed by flow cytometry using annexin V binding and fluorescence-labeled anti-Fas, was also shown to be Figure 9. Effects of serum on phagocytosis of apoptotic polymorphonuclear neutrophils (PMNs). A, Monocytes isolated from patients with active systemic lupus erythematosus (SLE) (n ⫽ 6) were cultured with autologous serum and serum from healthy subjects. The number of macrophages ingesting apoptotic PMNs was counted. B, Monocytes isolated from healthy subjects (n ⫽ 6) were cultured with autologous serum and serum from patients with active SLE. Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05. APOPTOTIC CELL CLEARANCE IN SLE Significantly increased PMN Fas expression in SLE has been reported (19). Besides being mediated by Fas expression, PMN apoptosis is regulated by a number of serum-soluble factors, primarily cytokines, including interleukin-1␤ (IL-1␤), IL-4, IL-6, granulocyte–macrophage colonystimulating factor (GM-CSF), and granulocyte colonystimulating factor, which delay apoptosis, and tumor necrosis factor ␣ (TNF␣), which induces apoptosis (22,23). Cytokine abnormalities have been reported in SLE (24). It is possible that the presence, or the lack, of some of these PMN apoptosis regulatory cytokines in the serum of patients with active SLE accelerates control of PMN apoptosis and secondary necrosis. This possibility should be further investigated. We previously examined the question of whether allospecific antibodies may have an effect on PMN apoptosis and necrosis, by performing cross-feeding experiments in which normal cells were incubated with sera from different healthy individuals. No differences in the rate of PMN apoptosis and necrosis were observed (Ren Y, et al: unpublished observations). Whether the use of steroids and other immunosuppressive drugs in the treatment of SLE may account for these observations was not demonstrated in the current study, because only a few patients were not receiving any medication at the time of the study. This is an inherent problem with most human SLE studies, because few patients are treatment naive. We did, however, evaluate whether the use of high-dose steroids (prednisolone, ⬎10 mg/day) and/or immunosuppressive drugs may influence our results. There were no significant differences in the rate of apoptosis and macrophage handling of apoptotic cells between patients in the immunosuppressive therapy group and those in the nonimmunosuppressive therapy group. This is probably related to heterogeneity of the clinical characteristics, disease activity, steroid dose, and use of different immunosuppressive drugs within the 2 treatment groups, which may confound the analysis. In SLE, an increased number of circulating PMNs is probably more important than is PBMC apoptosis, because PMNs are numerically greater and have a shorter lifespan compared with PBMCs. Increased apoptotic PMNs may contribute to an excess of autoantigens, including dsDNA and nucleosomes. Nucleosome release has recently been shown to be important in both the induction of SLE and the initiation of renal lesions by targeting autoantibodies to the glomerular basement membrane (25). The relationship between increased apoptosis, nucleosome release, and the formation of 2895 nucleosome–immunoglobulin complexes was confirmed by Licht et al (26), using lipopolysaccharide (LPS) administration to induce apoptosis in the MRL lupusprone model. A fast and efficient way to remove cells undergoing apoptosis and secondary necrosis is important for preventing the release of unwanted proteins that may accumulate within the lymphatic system and ultimately trigger antinuclear immune responses (11). In vivo, cells undergoing apoptosis are usually recognized and swiftly ingested by macrophages or neighboring cells acting as semiprofessional phagocytes. The phagocytes then actively down-regulate inflammatory and immune responses, further ensuring the safe clearance of apoptotic cells in vivo. The reduced phagocytic activity of PMNs, monocytes, and macrophages in patients with SLE is well established (27–29), and aberrations in the clearance of apoptotic materials by phagocytes have been hypothesized to play a role in the etiology of lupus (19,30). Another important finding of this study was that macrophages from patients with SLE were less capable of ingesting apoptotic PMNs when compared with macrophages from control subjects. This is in concordance with the finding by Herrmann et al (31) of decreased noninflammatory engulfment phagocytosis of apoptotic PBMCs. We went one step further, because our study is the first to show that the ability of macrophages to phagocytose apoptotic cells is inversely correlated with biochemical and serologic markers of lupus disease activity and the overall SLEDAI score. Additionally, these findings confirmed that impaired macrophage phagocytosis has a contributory role in the pathogenesis of SLE. The underlying mechanism of defective macrophage phagocytosis in SLE has not been fully evaluated. In cross-feeding experiments, Herrmann et al (31) showed that reduced clearance of apoptotic cells in SLE was not related to an “abnormal execution of apoptosis,” but rather to intrinsic phagocyte dysfunction in patients with SLE. In our study, serum obtained from patients with active SLE impaired the phagocytic ability of healthy control macrophages. Conversely, serum from control subjects restored the phagocytic ability of macrophages from patients with SLE. These data suggest that in patients with SLE, the impaired ability of macrophages to phagocytose apoptotic PMNs is secondary to the presence or absence of certain serum factors rather than being attributable to an intrinsic defect of the macrophage or its binding to apoptotic PMNs. The identity of the serum factor(s) that influ- 2896 ences macrophage phagocytosis of apoptotic cells is not known. Several recognition molecules or phagocyte receptors have been identified. These include lectins, ␣v␤3 (vitronectin receptor), thrombospondin (TSP), CD36 (TSP receptor), PS receptors, scavenger receptors, CD14 (the LPS receptor), ATP-binding cassette protein 1, and complement factors (11). One or more of these molecules may be lacking or may meet with interference in SLE. Cairns et al (32) also recently demonstrated reduced expression of CD44 on PMNs, which may result in poor recognition and clearance of these cells by monocyte-derived macrophages. A deficiency of complement and complement receptors confers an increased risk of SLE (33). We previously reported that insufficiency of mannosebinding lectin (MBL), a newly derived complementactivating factor, is associated with the development of SLE in Chinese individuals of southern origin (34,35). C1q and other complement factors have been shown to bind to surface blebs of apoptotic cells and facilitate phagocytic removal (36,37). Recently, MBL was shown to have similar properties (38). Thus, impairment in serum complement and/or MBL may render reduced clearance of apoptotic PMNs. It should be noted that in our study, the macrophage phagocytosis index correlated positively and significantly with serum levels of C3 and C4; serum MBL levels were not measured. Antiphospholipid antibodies (aPL), which are present in 20–40% of patients with SLE, may also disturb the safe clearance of apoptotic cells by macrophages that do not bear opsonic immunoglobulin (11). Alternatively, aPL may opsonize apoptotic cells so that macrophages incite inflammation (e.g., release of TNF␣) or autoimmunity (39). In our study, we evaluated the relationship between macrophage phagocytosis ability and serum levels of IgG and IgM anticardiolipin antibodies. However, no significant correlation was found (data not shown). The effects of other autoantibodies on macrophage apoptotic body phagocytosis have not been thoroughly studied, but it is tempting to speculate that anti-dsDNA antibodies, which cross-react with antinucleosome antibodies, may have a role. Indeed, nucleosomes have been shown to elicit a strong dosedependent inhibition of phagocytosis of apoptotic thymocytes by young macrophages from MRL mice (40). Antineutrophil cytoplasmic antibodies (ANCAs) have also been shown to induce the generation of reactive oxygen species and cause dysregulation of primed PMN apoptosis and clearance by macrophages (41). However, ANCAs are not commonly found in patients with SLE. REN ET AL Finally, it is possible that the presence of allospecific antibodies in a patient’s serum may interfere with the ability of macrophages to remove apoptotic cells. However, this possibility has not been studied previously. Proinflammatory cytokines have been shown to potentiate TSP-mediated phagocytosis of PMNs undergoing apoptosis (10). For example GM-CSF, IL-1␤, TNF␣, interferon-␣, and transforming growth factor ␤1 enhance phagocytosis, while IL-4 and IL-6 have no effects. As was mentioned above, SLE is associated with widespread changes in cytokine levels (24). It is possible that lowered levels of some of these proinflammatory cytokines may explain the results of our study. In conclusion, results of our study confirm previous findings of increased peripheral blood cell apoptosis that correlated with increased disease activity in SLE. To prevent induction of autoimmunity, it is prudent that apoptotic cells are effectively removed, and the most important mechanism for such removal is nonphlogistic phagocytic clearance by macrophages. Our data confirm that this mechanism is defective in SLE. Additionally, impairment in macrophage apoptotic cell clearance correlated with SLE disease activity. This further supports an important role of such a defect in the etiology of SLE. The reason why macrophages from patients with SLE are ineffective in engulfing apoptotic cells is not fully understood, but our results suggest that this is likely due to the presence or the lack of a specific serum factor(s) in these patients. Further studies in this area are required. ACKNOWLEDGEMENTS We thank Ms Y. Lo for her assistance in the collection of SLE clinical data and Ms K. Larm for secretarial help. REFERENCES 1. Mohan C, Adams S, Stanik V, Datta SK. Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus. J Exp Med 1993;177:1367–81. 2. Salmon M, Gordon C. The role of apoptosis in systemic lupus erythematosus. Rheumatology (Oxford) 1999;38:1177–83. 3. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–19. 4. Chu JL, Drappa J, Parnassa A, Elkon KB. The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn. J Exp Med 1993;178:723–30. 5. Takahashi T, Tanaka M, Brannan CI, Jenkins NA, Copeland NG, Suda T, et al. Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand. Cell 1994;76:969–76. 6. Emlen W, Niebur J, Kadera R. Accelerated in vitro apoptosis of lymphocytes from patients with systemic lupus erythematosus. J Immunol 1994;152:3685–92. APOPTOTIC CELL CLEARANCE IN SLE 7. Amoura Z, Chabre H, Koutouzov S, Lotton C, Cabrespines A, Bach JF, et al. Nucleosome-restricted antibodies are detected before anti-dsDNA and/or antihistone antibodies in serum of MRL-Mp lpr/lpr and ⫹/⫹ mice, and are present in kidney eluates of lupus mice with proteinuria. Arthritis Rheum 1994; 37:1684–8. 8. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239–57. 9. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995;182:1545–56. 10. Ren Y, Savill J. Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. J Immunol 1995;154:2366–74. 11. Ren Y, Savill J. Apoptosis: the importance of being eaten. Cell Death Differ 1998;5:563–8. 12. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271–7. 13. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI: a disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992;35:630–40. 14. Savill J, Henson PM, Haslett C. Phagocytosis of aged human neutrophils by macrophages is mediated by a novel “chargesensitive” recognition mechanism. J Clin Invest 1989;84:1518–27. 15. Richardson BC, Yung RL, Johnson KJ, Rowse PE, Lalwani ND. Monocyte apoptosis in patients with active lupus. Arthritis Rheum 1996;39:1432–4. 16. Lorenz HM, Grunke M, Hieronymus T, Herrmann M, Kuhnel A, Manger B, et al. In vitro apoptosis and expression of apoptosisrelated molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases. Arthritis Rheum 1997;40:306–17. 17. Georgescu L, Vakkalanka RK, Elkon KB, Crow MK. Interleukin-10 promotes activation-induced cell death of SLE lymphocytes mediated by Fas ligand. J Clin Invest 1997;100:2622–33. 18. Perniok A, Wedekind F, Herrmann M. Specker C, Schneider M. High levels of circulating early apoptotic peripheral blood mononuclear cells in systemic lupus erythematosus. Lupus 1998;7:113–8. 19. Courtney PA, Crockard AD, Williamson K, Irvine AE, Kennedy RJ, Bell AL. Increased apoptotic peripheral blood neutrophils in systemic lupus erythematosus: relations with disease activity, antibodies to double stranded DNA, and neutropenia. Ann Rheum Dis 1999;58:309–14. 20. Liang MH, Socher SA, Larson MG, Schur PH. Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus. Arthritis Rheum 1989;32: 1107–18. 21. Martin SJ, Finucane DM, Amarante-Mendes GP, O’Brien GA, Green DR. Phosphatidylserine externalization during CD95-induced apoptosis of cells and cytoplasts requires ICE/CED-3 protease activity. J Biol Chem 1996;271:28753–6. 22. Simon HU. Regulation of eosinophil and neutrophil apoptosis: similarities and differences. Immunol Rev 2001;179:156–62. 23. Akgul C, Moulding DA, Edwards SW. Molecular control of neutrophil apoptosis. FEBS Lett 2001;487:318–22. 24. Tsokos GC. Lymphocytes, cytokines, inflammation, and immune trafficking. Curr Opin Rheumatol 1995;7:376–83. 25. Kramers C, Hylkema MN, van-Bruggen MC, van de Lagemaat R, 2897 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. Dijkman HB, Assmann KJ, et al. Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo. J Clin Invest 1994;94:568–77. Licht R, van Bruggen MC, Oppers-Walgreen B, Rijke TP, Berden JH. Plasma levels of nucleosomes and nucleosome–autoantibody complexes in murine lupus: effects of disease progression and lipopolysaccharide administration. Arthritis Rheum 2001;44: 1320–30. Brandt L, Hedberg H. Impaired phagocytosis by peripheral blood granulocytes in systemic lupus erythematosus. Scand J Haematol 1969;6:348–53. Svensson BO. Serum factors causing impaired macrophage function in systemic lupus erythematosus. Scand J Immunol 1975;4: 145–50. Vazquez-Doval J, Sanchez-Ibarrola A. Defective mononuclear phagocyte function in systemic lupus erythematosus: relationship of FcRII (CD32) with intermediate cytoskeletal filaments. J Investig Allergol Clin Immunol 1993;3:86–91. Herrmann M, Zoller OM, Hagenhofer M, Voll R, Kalden JR. What triggers anti-dsDNA antibodies? Mol Biol Rep 1996;23: 265–7. Herrmann M, Voll RE, Zoller OM, Hagenhofer M, Ponner BB, Kalden JR. Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus. Arthritis Rheum 1998;41:1241–50. Cairns AP, Crockard AD, McConnell JR, Courtney PA, Bell AL. Reduced expression of CD44 on monocytes and neutrophils in systemic lupus erythematosus: relations with apoptotic neutrophils and disease activity. Ann Rheum Dis 2001;60:950–5. Hartung K, Baur MP, Coldewey R, Fricke M, Kalden JR, Lakomek HJ, et al. Major histocompatibility complex haplotypes and complement C4 alleles in systemic lupus erythematosus: results of a multicenter study. J Clin Invest 1992;90:1346–51. Lau YL, Lau CS, Chan SY, Karlberg J, Turner MW. Mannosebinding protein in Chinese patients with systemic lupus erythematosus. Arthritis Rheum 1996;39:706–8. Ip WK, Chan SY, Lau CS, Lau YL. Association of systemic lupus erythematosus with promoter polymorphisms of the mannosebinding lectin gene. Arthritis Rheum 1998;41:1663–8. Korb LC, Ahearn JM. C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited. J Immunol 1997;158:4525–8. Taylor PR, Carugati A, Fadok VA, Cook HT, Andrews M, Carroll MC, et al. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo. J Exp Med 2000;192:359–66. Ogden CA, deCathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA, et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 2001;194: 781–95. Manfredi AA, Rovere P, Galati G, Heltai S, Bozzolo E, Soldini L, et al. Apoptotic cell clearance in systemic lupus erythematosus. I. Opsonization by antiphospholipid antibodies. Arthritis Rheum 1998;41:205–14. Laderach D, Bach JF, Koutouzov S. Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL⫹/⫹ lupus-prone mice. J Leukoc Biol 1998;64:774–80. Harper L, Ren Y, Savill J, Adu D, Savage CO. Antineutrophil cytoplasmic antibodies induce reactive oxygen-dependent dysregulation of primed neutrophil apoptosis and clearance by macrophages. Am J Pathol 2000;157:211–20.