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Arthritis Rheum. Author manuscript; available in PMC Aug 27, 2009.
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PMCID: PMC2733836
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Association of Reactive Oxygen and Nitrogen Intermediate and Complement Levels With Apoptosis of Peripheral Blood Mononuclear Cells in Lupus Patients

Abstract

Objective

Both increased production of reactive oxygen and nitrogen intermediates (RONI) and reduced levels of complement may play a role in the increased apoptosis and reduced clearance of apoptotic cells in systemic lupus erythematosus (SLE). The objective of this study was to evaluate both processes in a parallel, prospective, longitudinal manner.

Methods

Sixty-seven SLE patients were evaluated during multiple visits, and 31 healthy control subjects were evaluated once or twice. Clinical and laboratory features of SLE disease activity were determined, and blood was collected for measurement of serum nitrate plus nitrite (NOx) levels and for isolation of peripheral blood mononuclear cells (PBMCs). PBMCs were cultured with a nitric oxide (NO) donor and SLE or control plasma, with or without heat inactivation, cobra venom factor (CVF), or lipopolysaccharide plus interferon-γ treatment. Cells were analyzed for apoptotic index (AI), cellular subsets, and RONI production.

Results

The PBMC AI was associated with SLE and was inversely associated with complement levels over time. Changes in the AI with addition of a NO donor was longitudinally associated with serum NOx levels, and stimulation of SLE PBMCs led to parallel increases in RONI production and apoptosis. Addition of SLE plasma resulted in a greater PBMC AI, an effect that was increased with heat inactivation and was corrected with CVF treatment.

Conclusion

These data suggest that the greater AI observed in SLE PBMCs relates to increased PBMC RONI production and reduced complement levels. The longitudinal nature of these parallel associations within individuals suggests that these processes are dynamic and additive.

Increasing attention is being focused on the role of apoptosis or reduced clearance of apoptotic/necrotic bodies in the pathogenesis of systemic lupus erythematosus (SLE) (1-3). Apoptosis is the process of programmed cell death that maintains immune tolerance and homeostasis of lymphocyte populations. Increased apoptosis of circulating immune cells and reduced clearance of apoptotic cells have been described in SLE (4-11). Either one or a combination of these abnormalities can lead to increased circulating nucleosome-containing apoptotic bodies (5,12) that can serve as autoantigens and mitogens to stimulate autoantibody production (13-15). The mechanism of this increase in apoptosis of peripheral leukocytes or reduction in clearance of apoptotic bodies in SLE is likely multifactorial and is still a matter of investigation. However, there is precedence for implicating enhanced reactive nitrogen intermediate (RNI) production and reduced complement-mediated clearance of apoptotic cells in this process.

Nitric oxide (NO) is a short-lived, membrane-permeable effector RNI. In the setting of inflammatory stimuli, NO is produced by inducible nitric oxide synthase (iNOS), which synthesizes logarithmically higher levels of NO and, under some circumstances, superoxide (O2). NO itself can have antiapoptotic effects, while peroxynitrite (ONOO), the product of NO and O2, can induce apoptosis (16).

We have reported increased markers of systemic production of RNI, such as NO, in SLE patients with increased disease activity and increased expression of iNOS protein in proliferative glomerulonephritis (17,18). Inducible NO synthase activity also appears to increase systemic production of reactive oxygen intermediates (ROIs) in murine lupus, a condition that can lead to increased peroxynitrite production (19,20). Investigators in our laboratory have observed that inhibition of iNOS activity leads to reduced apoptosis of splenocytes in the same murine model of lupus (21).

Another general mechanism for increased numbers of circulating or tissue apoptotic cells is reduced clearance of these cells. Several groups of investigators have described a reduced phagocytic capacity of SLE monocytes, macrophages, granulocytes, and CD34+ stem cell–derived phagocytic cells that appears to be intrinsic (1). However, deficiency of plasma factors, likely C1q, C4, and C3 complement, has been described in SLE, which leads to reduced phagocytosis of apoptotic cells by monocyte-derived macrophages from both healthy and SLE donors (22). Complement-dependent NO production has been reported in 2 models of immune complex–mediated glomerulonephritis, and complement receptor–mediated activation of nitric oxide synthase has been observed in macrophages (23-25). These observations suggest that increased production of reactive oxygen and nitrogen intermediate (RONI) in SLE may be related to complement activation.

To link these 2 pathogenic pathways, we determined longitudinal associations between in vitro apoptosis of SLE and control peripheral blood mononuclear cells (PBMCs), clinical measures of disease activity, and levels of serum nitrate plus nitrite (NOx; a surrogate for systemic NO production). We determined the effect of exogenous NO on PBMC apoptosis and observed associations between enhanced ROI and RNI production and apoptosis in resting and activated SLE PBMCs. Finally, we observed that elements of SLE plasma, likely complement-related, lead to an increased PBMC apoptotic index (AI) in vitro. Our results suggest that both complement deficiency and enhanced RONI production lead to increased levels of apoptotic PBMCs in SLE patients over time.

PATIENTS AND METHODS

Study subjects

Sixty-seven SLE patients and 31 healthy control subjects were enrolled in the study. All subjects provided written consent to participate in this study, and the study was approved by the institutional review board of the Medical University of South Carolina. The demographics of the subjects involved in the individual experiments were reflective of the group as a whole.

All SLE patients met at least 4 of the American College of Rheumatology criteria for SLE (26). Control subjects were free of autoimmune disease. Exclusion criteria were pregnancy, infection, or cancer (27). Subjects who smoked cigarettes were asked not to smoke for 24 hours prior to each visit (28). All subjects consumed a low nitrate/nitrite diet for 24 hours prior to each visit (27). Several SLE patients and controls were seen more than once (range 1–6 visits for a total of 135 SLE patient visits and 1–2 visits for a total of 39 control subject visits). All subjects were seen in our General Clinical Research Center, where a history was obtained (including smoking status and use of prednisone, immunosuppressive agents, nonsteroidal antiinflammatory drugs [NSAIDs], and/or hydroxychloroquine), and physical examination, phlebotomy, and urine collection were performed. Scores on the SLE Disease Activity Index (SLEDAI) were determined at each visit (29-31) by physicians (JCO and GSG) who were trained both internally and as part of multicenter trials that use this instrument.

Routine laboratory measures and sample processing

All laboratory evaluations used to determine SLEDAI scores were performed in the clinical laboratory at the Medical University of South Carolina using standard quality control measures. These laboratory measures included serum levels of C3, C4, creatinine, and anti–double-stranded DNA (anti-dsDNA) antibodies (by Crithidia luciliae assay or by enzyme-linked immunosorbent assay), a complete blood cell count, an erythrocyte sedimentation rate, urinary levels of protein and creatinine, and microscopic analysis of urine. Serum samples for measurement of NOx were centrifuged and frozen in aliquots at −80°C. Anticoagulated blood for separation of PBMCs was transferred within 30 minutes to the laboratory for PBMC isolation at room temperature. As a post hoc analysis, antiphospholipid antibody status was recorded in SLE patients in whom testing was performed as part of their routine medical care.

Isolation and culture of PBMCs

PBMCs used in all experiments were isolated from 60 ml of whole blood in citrate-containing tubes using a Ficoll-Paque Plus density gradient (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s recommendations. Plasma from the gradient was frozen at −80°C for future experiments. PBMCs were resuspended at 106 cells/ml in RPMI 1640 with 10% heat-inactivated fetal bovine serum, 63 mg/500 ml of l-arginine (catalog no. A8094; Sigma, St. Louis, MO), and penicillin/streptomycin/glutamine (penicillin G 10 units/ml, streptomycin 10 μg/ml [catalog no. 10378-016]; l-glutamine 2 mM [catalog no. 25030081]; all from Invitrogen, Carlsbad, CA). Then, 0.2 ml of this suspension was transferred to 96-well flat-bottomed culture plates (catalog no. 3595; Costar, Corning, NY).

Apoptosis of SLE and control PBMCs with exogenous NO in vitro

To each of the wells described above, a final concentration of either 0, 100, 250, 500, or 1,000 μM diethylenetriamine NONOate (DETA/NO; a slow-releasing NO donor) (catalog no. 430-014-M025; Alexis, San Diego, CA) was added from a sterile 10× stock in 0.01M NaOH (Sigma). Each culture condition was performed in duplicate. Camptothecin (catalog no. C9911; Sigma) was added to 1 well to act as a positive apoptosis control. Cells were cultured overnight at 37°C in an atmosphere of 5% CO2 and harvested for analysis of apoptosis as described below.

Measurement of apoptosis by propidium iodide (PI) staining and flow cytometry

PBMCs (5 × 105) from each sample were fixed and stained with PI as described elsewhere (32,33) and then analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA), with pulse processing and gating to exclude doublets, cell fragments, and apoptotic bodies using fluorescence channel 2 (FL2) width versus FL2 amplitude. Gated cells were analyzed using an FL2 histogram. Apoptotic cells appeared as a sub-G1 peak (32,33). The AI was calculated by determining the percentage of nuclei in the sub-G1 peak relative to all gated nuclei.

Measurement of serum NOx levels

Samples were filtered using a 20K cutoff centrifugal filter (catalog no. 42407; Millipore, Bedford, MA) and reduced in vanadium chloride (Sigma) at 95°C prior to analysis. Serum samples were analyzed for NOx levels by chemiluminescence detection using a Sievers NOA 280 analyzer (Sievers, Boulder, CO) according to the manufacturer’s instructions.

Culture of SLE and control PBMCs in human plasma

PBMCs isolated from 43 of the SLE patients and 31 control subjects to be cultured in human plasma were isolated by Ficoll gradient and cultured with 20 ng/ml of recombinant human macrophage colony-stimulating factor (catalog no. RDI-3025; Research Diagnostics, Concord, MA) in RPMI 1640 at 106 cells/ml. Cells were cultured in duplicate under each of the following 6 plasma conditions: 1) 25% fresh autologous plasma, 2) 25% frozen plasma from a random subject of the opposite study group (i.e., SLE PBMCs cultured with control plasma and vice versa), 3) 25% heat-inactivated autologous plasma (heated to 55°C for 30 minutes prior to addition to culture), 4) 25% heat-inactivated plasma from a subject of the opposite study group, 5) 25% autologous plasma treated with cobra venom factor (CVF) (catalog no. A600; Quidel, San Diego, CA) (diluted 1 μg/ml and incubated at 37°C for 90 minutes prior to addition to culture), and 6) 25% plasma from a subject of the opposite study group treated with CVF.

Control PBMCs were also cultured with 25% pooled normal human plasma (catalog no. ES1019P; Biomeda, Foster City, CA) to determine the effects of a plasma freeze–thaw cycle on PBMC apoptosis. For 2 control PBMC experiments, SLE plasma was also mixed 1:1 with control plasma. Cells for these experiments were cultured for 5 days to allow monocytes to differentiate and adhere. Both SLE and control PBMC cultures contained adherent cells, as determined by microscopy. Nonadherent cells were harvested after 5 days and analyzed for apoptosis by PI staining of DNA as described above. In some experiments, PBMCs were cultured in serum-free medium (X-vivo 10; Lonza, Basel, Switzerland), and adherent cells were analyzed as well.

Determination of stimulated ROI and RNI production relative to cell type and rates of apoptosis

SLE PBMCs were isolated as above and cultured overnight in 25% RPMI 1640 with autologous plasma, with either no treatment or treatment with 100 units/ml of interferon-γ (IFNγ) (catalog no. 285-IF; R&D Systems, Minneapolis, MN) plus 1 μg/ml of lipopolysaccharide (LPS) (catalog no. L4391; Sigma). Cells incubated with either 0.3% hydrogen peroxide (catalog no. H1009; Sigma) immediately prior to analysis or 250 μM S-nitroso-N-acetylpenicillamine (catalog no. N7927; Molecular Probes, Eugene, OR) for 4 hours served as ROI+ and RNI+ controls, respectively.

Cells were stained for assessment of RNI or ROI production by incubation in the dark at 37°C in an atmosphere containing 5% CO2 with 1 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (catalog no. D23844; Molecular Probes) in phosphate buffered saline (PBS) for 30 minutes or 0.1 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (catalog no. C6827; Molecular Probes) in PBS for 15 minutes, respectively. After washing, cells were stained with phycoerythrin (PE)–conjugated annexin V (catalog no. NXPE 1; R&D Systems, Minneapolis, MN), PE-conjugated anti-CD3, anti-CD19, or anti-CD14 antibodies (all from BD PharMingen, San Jose, CA), or with appropriate isotype controls.

Annexin V staining and PI (1 μg/ml) staining to exclude dead cells were performed according to the manufacturer’s protocol. PBMCs (106 cells/well) were washed in PBS and stained in 20 μl of cell-surface antibody on ice for 30 minutes. The cells were washed in PBS/0.1% bovine serum albumin (BSA). The last wash also contained 1 μg/ml of PI. The cells were resuspended in 200 μl of PBS with 0.1% BSA and analyzed on a FACSCalibur flow cytometer. The AI was calculated as the percentage of annexin V+/PI− cells of the total number of gated PI− cells.

Statistical analysis

Comparisons of demographic variables between SLE patients and control subjects were made using Pearson’s chi-square test. For continuous variables, Student’s t-test was used for normally distributed data; otherwise, the Mann-Whitney U test was used. For in vitro assays in which several culture conditions were used across multiple experiments, data were factor-corrected to reduce the effect of interexperiment variations and to maintain the original units measured (34). When data from different experiments represented multiple patient visits, bivariate correlations were determined between the input variables and the AI to select variables for multivariate analysis. For categorical variables, Pearson’s chi-square test was used for selection of variables for multivariate analysis. Selected variables (those with significant associations in these analyses) were then used as input variables in a type 3 generalized estimating equation analysis. This technique creates models that account for within-group and within-individual associations in longitudinal data. All data were expressed as the mean ± SEM. P values less than or equal to 0.05 were considered significant.

RESULTS

Characteristics of the study subjects

Sixty-seven SLE patients and 31 healthy control subjects were enrolled in the study. Their demographic and clinical characteristics are outlined in Table 1. Each experiment below involved a subset of this total cohort.

Table 1
Demographic and clinical characteristics of the SLE patients and control subjects*

Apoptosis of cultured SLE and control PBMCs

PBMCs from 33 SLE patients and 11 control subjects from the total cohort were cultured overnight. SLE PBMCs had significantly greater AIs (mean ± SEM 7.6 ± 0.9%) than the controls (3.5 ± 0.8%; P = 0.004) (Figure 1). SLE and control PBMCs were cultured in various concentrations of DETA/NO to determine the effects of NO on the PBMC AIs in these groups. SLE PBMCs cultured in 0, 100, 250, 500, and 1,000 μM DETA/NO had significantly greater AIs than did control PBMCs cultured under the same conditions. Among the SLE patients and control subjects, PBMCs cultured in 250, 500, and 1,000 μM DETA/NO had significantly greater AIs than did those cultured in 0 μM DETA/NO (Figure 2).

Figure 1
Apoptotic index (AI) of cultured peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and healthy control subjects. PBMCs from 33 SLE patients and 11 control subjects were cultured overnight, and nonadherent cells ...
Figure 2
Effect of exogenous nitric oxide (NO) on the apoptotic index (AI) of cultured peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and healthy control subjects. PBMCs from 33 SLE patients and 11 control subjects ...

Longitudinal associations between clinical variables and PBMC AIs

To explore mechanisms related to apoptosis of PBMCs in SLE patients, the 33 SLE patients and 11 control subjects from the above experiment were followed up in a prospective, longitudinal manner. Data were collected from 33 first visits, 21 second visits, 13 third visits, 6 fourth visits, 4 fifth visits, and 1 sixth visit of the SLE patients as well as from 11 first and/or second visits of the controls. During these quarterly study visits, SLEDAI elements were determined, and serum NOx levels were determined. Using generalized estimating equation modeling, serum C3 levels (C4 levels could be substituted with the same results), serum NOx levels, and the diagnosis of SLE were used as input variables after preselection of individual laboratory values from the SLEDAI elements with bivariate correlation. The AIs of cultured PBMCs from both study groups were significantly associated with C3 levels (negative association; P = 0.02) and with the diagnosis of SLE (positive association; P = 0.03).

Because the diagnosis of SLE was significantly associated with the PBMC AI, a separate analysis was performed among study visits from only the SLE patients and using serum C3 and NOx levels, the SLEDAI score, the prednisone dose, and peripheral white blood cell (WBC) counts as input variables. Among these variables, the C3 level and WBC count had a significant inverse association (P = 0.03 for each association) with apoptosis of unstimulated PBMCs. A separate analysis of the individual SLEDAI elements as categorical variables revealed no significant association between the PBMC AI and the individual elements of the SLEDAI.

Effect of exogenous NO on apoptosis of SLE and control PBMCs in vitro

Because DETA/NO significantly increased the level of in vitro apoptosis of SLE PBMCs at a single time point, measures of in vivo activation of the innate immune system (serum C3, C4, dsDNA antibody, and NOx levels) were compared with the change in levels of apoptosis with culture in exogenous NO (ΔAI; reported as the log[AI] of PBMCs cultured with 1,000 μM DETA/NO divided by the AI of cells cultured in 0 μM DETA/NO). From this analysis, serum C3/C4, dsDNA antibody, and serum NOx levels were used as input variables. The ΔAI was the output variable in the generalized estimating equation analysis. Serum NOx levels ex vivo were significantly associated (P = 0.03) with the ΔAI in vitro. Unlike the association between C3 levels and baseline levels of apoptosis, serum levels of C3 were not associated with the ΔAI (P = 0.3).

RONI production in activated SLE PBMC subsets

To explore the observed association between in vitro apoptosis with an exogenous NO donor and in vivo NO levels as a possible intrinsic or paracrine signal for apoptosis, the next experiments were designed to determine whether cellular RONI production was increased in concert with apoptosis in cultured, stimulated SLE PBMCs. PBMCs from 13 SLE patients from the total cohort were cultured with and without stimulation with LPS plus IFNγ. Nonadherent cells were costained for cellular subset surface markers and intracellular markers of RONI production. CD3+ and CD19+ cells demonstrated a significant increase in RONI production with stimulation, while CD14+ cells demonstrated a significant reduction in RONI production (Figure 3A).

Figure 3
Reactive oxygen and nitrogen intermediate (RONI) production by subsets of peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients. Adherent cells are compared with nonadherent cells. A, PBMCs from 13 SLE patients were ...

To study the effect of plasma on adherent CD14+ cells, a subsequent experiment was performed in which PBMCs from 4 SLE patients were cultured overnight in 25% autologous serum. Both adherent and nonadherent cells were analyzed by 4-color flow cytometry for PI, annexin V, cell surface markers, and RONI production. CD14+ cells were more frequently positive for RONI and apoptosis stains, suggesting that adherent CD14+ cells were more likely to produce RONI (Figure 3B). Relatively lower levels of apoptosis among CD3+ and CD19+ cells paralleled decreased production of RNI, but not ROI, in these cells.

Association between RONI production and apoptosis in SLE PBMCs

To determine the association between RONI production and apoptosis among SLE PBMCs, stimulated and unstimulated cells from some of the above cultures (n = 7 SLE patients) were stained for a marker of early apoptosis (PE-conjugated annexin V) instead of for individual cell subset markers, and apoptotic cells were analyzed for RONI production. RNI+ apoptotic cells outnumbered RNI− ones in both the stimulated and unstimulated states. The percentage of ROI+ apoptotic cells significantly outnumbered the percentage of ROI− ones only in the stimulated state (Figure 4). These data suggest that RNI production is associated with baseline apoptosis, whereas ROI production is increased in apoptotic cells only upon stimulation, as one might observe during SLE disease activity (17,18).

Figure 4
Effect of treatment with lipopolysaccharide (LPS) plus interferon-γ (IFNγ) on reactive oxygen and nitrogen intermediate (RONI) production by apoptotic peripheral blood mononuclear cells (PBMCs). PBMCs from 7 systemic lupus erythematosus ...

Plasma factors associated with apoptosis of PBMCs

Because in vitro apoptosis was associated with serum complement levels, we explored plasma factors in SLE patients and control subjects that might affect the PBMC AIs in vitro. PBMCs from 43 SLE patients and 31 control subjects were cultured in fresh autologous or frozen donor plasma from the opposite study group (SLE versus control). The PBMC AI was determined after 4–5 days in culture. When both SLE and control PBMCs were cultured in SLE plasma (Figure 5), the AI was increased relative to that seen in the same donor PBMCs cultured in control plasma.

Figure 5
Effect of systemic lupus erythematosus (SLE) plasma on the apoptotic index (AI) of cultured peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy control subjects. PBMCs from 43 SLE patients and 37 control subjects were cultured for ...

The same SLE and control PBMCs were cultured in heat-inactivated SLE and control plasma samples. The increase in AI with heat inactivation was significant for all conditions except for control PBMCs cultured in SLE plasma. When plasma was treated with CVF immediately before culture, both SLE and control PBMCs contained significantly lower AIs in all groups except for SLE PBMCs in SLE plasma (Figure 5). The increases in control PBMC AIs seen with the addition of SLE plasma were restored to control plasma levels when SLE plasma was mixed 1:1 with fresh control plasma (mean ± SEM AI 9.0 ± 0.2 with control:SLE plasma versus 7.0 ± 0.7 with control plasma and 16.3 ± 1.8 with SLE plasma; n = 2 subjects).

These experiments suggest that SLE plasma increases apoptosis or reduces phagocytosis of apoptotic cells in both SLE and control PBMCs in a manner similar to that of heat inactivation. The addition of CVF to plasma negated this effect, suggesting that activation of complement reduces apoptosis or increases phagocytosis.

The AIs of control PBMCs cultured with either frozen pooled or fresh autologous plasma were similar. PBMCs from 4 SLE patients were cultured overnight in 25% autologous plasma, control plasma, or serum-free medium, and viable adherent and nonadherent cells were analyzed by 4-color flow cytometry for annexin V, cell surface markers, and RONI production. The presence or type of plasma had no effect on this marker of early apoptosis (data not shown). This suggests that plasma affects the number of late, but not early, apoptotic cells. Similarly, the presence or type of plasma in culture had no effect on RONI production (data not shown).

DISCUSSION

This is the first study to show parallel intrinsic RONI-related and extrinsic complement-related in vivo processes that associate with an increased PBMC AI in SLE patients. The first of these processes is intrinsic and is likely related to the increased RONI production seen with disease activity (17,18). The second of these processes is extrinsic to PBMCs and is plasma, most likely complement, related. Both intrinsic and extrinsic factors have been implicated in dysregulated apoptotic processes in SLE, but both processes have not been observed in a longitudinal, parallel manner.

Among the intrinsic factors implicated in increasing levels of apoptosis in SLE is an increase in mitochondrial-mediated apoptosis (35). NO plays an antithetic role in signaling for apoptosis. At low levels, NO nitrosates procaspases 3 and 9 and prevents cleavage to their active enzymes, thus inhibiting the terminal phases of apoptosis. When produced in the presence of ROIs, NO combines with O2 to form peroxynitrite, which damages ATP synthase and causes cytochrome c release. This release results in activation of caspase 3 (16).

Nagy et al (36) have reported increased levels of T cell activation in lupus patients, with persistent membrane hyperpolarization and increased RNI production. Our study supports these observations by demonstrating that apoptotic lupus T cells are more likely to have increased RONI production and that activation of CD3+ and CD19+ cells increases the production of RONI in parallel with increased levels of apoptosis. Our findings differ from those reported by Nagy in that NO production by CD14+ cells was reduced in nonadherent CD14+ cells. This may be due to the fact that we analyzed nonadherent CD14+ cells that were cocultured with lymphocytes for RONI, whereas Nagy analyzed RNI production by isolated adherent CD14+ cells. When our experiments included adherent cells, CD14+ cells exhibited elevated baseline production of RNI in parallel with an increase in the AI. This suggests that increased RNI production occurs more readily among the more differentiated adherent phenotype of CD14+ cells. The increased AI among SLE PBMCs cultured with exogenous NO may have been due to an already increased baseline intrinsic ROI production in activated SLE PBMCs. Intrinsic O2 could thus have combined with exogenous NO to form peroxynitrite, which is known to induce mitochondrial-dependent apoptosis (16).

The second process associated with in vitro apoptosis of SLE PBMCs was dependent on SLE plasma elements. In our study, serum complement was inversely associated with unstimulated PBMC apoptosis over time in vitro. Experiments in which a marker of early apoptosis (annexin V staining) was not affected by SLE compared with control or no plasma suggests that the effect of SLE plasma occurs only in late apoptosis. This is consistent with previous studies in which low complement levels were associated at a single time point with increased apoptosis of cultured cells (37) or with a reduced phagocytosis capacity of macrophages cultured in sera from SLE patients deficient in complement (22). The observation that heat inactivation reverses the relative antiapoptotic effect of normal plasma suggests that complement is important in preventing in vitro apoptosis but does not fully support this as an exclusive mechanism. It is interesting that control PBMCs had numerically greater AIs in frozen SLE plasma than did SLE PBMCs in fresh autologous plasma. It is possible that a freeze–thaw cycle reduced already low complement levels in SLE plasma to a level low enough to affect opsonization.

We demonstrated that CVF pretreatment of plasma completely reversed any increase in the AI observed with SLE plasma. CVF is a stable form of C3b (38) that depletes complement substrate to form more C3b (39). C3b and inactivated C3b can act as opsonins by binding to CR2, CR3, and CR4 on phagocytes or red blood cells (2). This experiment demonstrated that addition of C3b is adequate to reverse the elevated AI seen in PBMCs cultured in SLE plasma. Whether this condition was adequate to overcome other SLE-related deficiencies of opsonins such as mannose-binding lectin, C1q, and C-reactive protein in SLE plasma is not known (22,40-42). Any number of these factors could be important in this population, and abnormalities in the clearance of apoptotic cells in SLE are likely heterogeneous (1). The fact that in a limited number of experiments, culture with equal amounts of lupus and control plasma reversed the increased AI seen with lupus plasma alone suggests that the abnormal elements of SLE plasma are not antibody related in these patients.

The observation that low complement levels were associated with an increased AI in SLE PBMCs is intriguing because in this experiment, apoptosis occurred in culture of heat-inactivated fetal bovine serum and not SLE plasma. There are several possible explanations for this observation that were not directly addressed by this study. First, cultured apoptotic cells may have already been opsonized by complement split products from native complement at the time of isolation. This possibility is less likely given that the percentage of apoptotic SLE PBMCs ex vivo is much lower than that observed after overnight culture (Oates JC, et al: unpublished observations), and the process of apoptosis and phagocytosis of any 1 cell would have been complete after overnight culture (43). Second, there is precedent for a direct effect of complement activation on RONI production. In 2 studies of Thy-1 nephritis, a model of immune complex nephritis, RONI synthesis was increased, an effect that was inhibited by treatment with anti–C5b–9 complex antibodies and soluble CR1 (23,24). While these studies suggest a direct role of activated complement in the production of RONI, in our experiments SLE plasma had no effect on RONI production in vitro. Finally, low C3 levels may simply coexist with activated T cells that occur during times of SLE disease activity. In support of this notion is 1 study demonstrating that CD3/CD28 costimulation of human peripheral blood lymphocytes led to significant increases in endothelial NOS and neuronal NOS protein expression (44). Both enzymes produce not only NO, but also, under the right conditions, O2 (45,46). Thus, low complement levels may be a marker of lymphocyte activation that results in increased RONI production, which, in turn, could trigger intrinsic and/or paracrine signaling for apoptosis.

In summary, this is the first study to demonstrate both in vivo and in vitro that parallel intrinsic RONI production and extrinsic complement activation lead to an increased AI in SLE PBMCs over time. These processes are temporally related in this cohort and may be directly related, given the known role of complement in the induction of iNOS protein expression. This raises the possibility that therapies designed to modulate autoantibody production could affect both processes. However, such therapies do not have an immediate effect due to the half-life of immunoglobulin. Thus, early intervention in SLE disease flares could target RONI production and thus reduce the burden of autoantigen presentation from apoptotic cells.

Acknowledgments

Special thanks to Haiquin Zheng for assistance with the flow cytometry assays and to the Flow Cytometry facility of the Medical University of South Carolina for use of its equipment.

Drs. Oates and Gilkeson’s work was supported by a research award from the Arthritis Foundation, a University Research Committee grant from the Medical University of South Carolina, a General Clinical Research Center grant to the Medical University of South Carolina (NIH grant 5M01-RR-01070), NIH grants (K08-AR-002193, AI-047469, AR-045476, and AR-04745), and Career Development, Research Enhancement Award Program, and Merit Review grants from the Medical Research Service, Ralph H. Johnson VA Medical Center, Charleston, SC.

Footnotes

Dr. Oates 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.

Study design. Oates, Gilkeson.

Acquisition of data. Oates, Farrelly, Hofbauer.

Analysis and interpretation of data. Oates, Farrelly, Hofbauer, Gilkeson.

Manuscript preparation. Oates.

Statistical analysis. Wang, Gilkeson.

Dr. Oates has received consulting fees (less than $10,000) from Genentech.

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