The present study was approved by the University of Florida Institutional Review Board, and all subjects provided written informed consent prior to participation. SLE patients met at least four of the revised 1982 American College of Rheumatology criteria 14. Peripheral blood was collected from 205 patients and 74 healthy control individuals. In the patient group, 132 participants had either biopsy-proven or laboratory-proven LN and 73 had no evidence of LN. At each visit a medication history and key laboratory parameters were collected. Disease activity was assessed using the Systemic Lupus Erythematosus Disease Activity Index 15. Detailed demographics, clinical characteristics, medication usage and laboratory measurements for all groups are presented in Table 1.

Antibodies were obtained from BD Pharmingen (San Diego, CA, USA) unless indicated otherwise. Heparinized whole blood (100 μl) was stained with PerCP-conjugated anti-CD14 (clone MΦ P9), fluorescein isothiocyanate-conjugated anti-CD16 (clone 3G8), allophycocyanin-conjugated anti-CD32 (clone FLI8.26), anti-HLA-DR (clone LN3), anti-CD62L (clone DREG56; eBioscience, San Diego, CA, USA), phycoerythrin-conjugated anti-CD64 (clone X54-5/7.1.1), and anti-CD16 (clone 3G8) for 30 minutes in the dark. Following lysis of erythrocytes, cells were washed with PBS/1% BSA/0.01% NaN₃ and were fixed in 2% paraformaldehyde PBS. Cells (10⁵) were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA, USA).

Gates were set around monocytes based on their forward/sideward light scatter pattern and CD14 expression. Surface marker expression levels were expressed as the geometrical mean fluorescence intensity on monocytes. Since not all CD14 monocytes express CD16, CD32, CD62L and HLA-DR, expression was also expressed as the percentage of positive monocytes. Data analysis was performed using FCS Express 2.0 (De Novo Software, Thornhill, ON, Canada).

Heparinized whole blood was diluted 1:1 with DMEM (Mediatech, Inc., Herndon, VA, USA) containing 10% fetal bovine serum (Mediatech, Inc.), and was stimulated with lipopolysaccharide (LPS) (500 ng/ml, from Escherichia coli; Sigma Chemical Company, St Louis, MO, USA) or C-reactive protein (CRP) (50 ng/ml, purified from human serum, endotoxin-free; Calbiochem, La Jolla, CA, USA) in the presence of the protein transport inhibitor GolgiStop™ (BD Pharmingen). In all cases, cells were incubated at 37°C in a 5% CO₂ atmosphere for 4 hours. The dose of LPS and CRP and the length of incubation were optimized for chemokine production in preliminary experiments. Immediately after incubation, 100 μl aliquots of cells were stained with appropriate combinations of monoclonal antibodies for 30 minutes at 22°C in the dark. After incubation, 2 ml PharMlyse (BD Pharmingen) was added to lyse erythrocytes. After washing, cells were fixed and permeabilized with 200 μl Cytofix/Cytoperm solution (BD Pharmingen) for 20 minutes at 4°C. After two washes with Perm/Wash solution (BD Pharmingen), cells were resuspended in 100 μl Perm/Wash solution containing 1.5 μg/μl phycoerythrin-conjugated anti-MCP-1 clone (5D3-F7; BD Pharmingen) or the same concentration of phycoerythrin-conjugated mouse IgG1 as an isotype control. After incubating at 4°C for 30 minutes in the dark, cells were washed and analyzed by flow cytometry.

Peripheral blood mononuclear cells isolated from SLE patients and from healthy control individuals using Ficoll-Hypaque density gradient centrifugation were washed once and resuspended in DMEM containing 0.5% fetal bovine serum at a concentration of 10⁷ cells/ml. Medium containing MCP-1 (25 ng/ml; Research Diagnostics Inc., Flanders, NJ, USA) or medium alone as a control were added to the lower chambers of a 24-well Costar Transwell plate (Corning Inc. Corning, NY, USA). The cell suspension (100 μl) was added to the upper chamber, which was separated from the lower chamber by a polycarbonate membrane (8.0 μm pores). After incubation for 3 hours at 37°C, cells in the lower chamber were collected, stained with anti-CD14, anti-CD16, and anti-HLA-DR, and analyzed by flow cytometry. Results are presented as a migration index calculated by dividing the number of cells that migrated toward MCP-1 by the number of cells that migrated to medium alone.

High-sensitivity C-reactive protein, C3 and C4 assays were performed using a BN ProSpec^® Nephelometer (Dade Behring, Deerfield, IL, USA) as described elsewhere 16.

Peripheral blood mononuclear cells from healthy control individuals were plated on 24-well plates (10⁶ cells/well) in complete medium (DMEM supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin). All cytokines were from BD Bioscience unless indicated otherwise. Cells were incubated for 19 hours at 37°C in the presence of recombinant human IFNα (4 ng/ml; PBL Biomedical, Piscataway, NJ, USA), IFNγ (2 ng/ml), IL-4 (4 ng/ml), IL-6 (4 ng/ml), IL-8 (4 ng/ml), IL-12 (4 ng/ml), or CRP (50 ng/ml; Calbiochem). Flow cytometry was performed immediately after incubation. In some experiments, dexamethasone (10^-5 to 10^-3 M) was added to the culture 3 hours prior to the addition of cytokines.

Differences between disease groups and normal control individuals were evaluated using Student's two-tailed t test unless the data were not normally distributed, in which case the Mann–Whitney U test was used. Correlation coefficients were calculated using Spearman's rank correlation. Data are presented as the mean ± standard error of the mean. Analyses were performed using Prism software, version 4.0 (GraphPad Software, San Diego, CA, USA). For all analyses, P < 0.05 was considered significant.

In healthy control individuals, nearly all peripheral blood monocytes displayed surface expression of FcγRI/CD64 and FcγRII/CD32. Only 9.3 ± 0.7% of circulating monocytes, however, expressed FcγRIII/CD16 (Figure 1a, top and Table 2). Although circulating monocytes from SLE patients also uniformly expressed FcγRI/CD64 (Figure 1a, bottom), quantification of FcγRI/CD64 expression showed a significantly higher mean fluorescence intensity in SLE patients compared with healthy control individuals (521 ± 21 versus 319 ± 22; P < 0.001, Student's t test). The expression was even higher in patients with nephritis compared with those without nephritis (567 ± 28 versus 449 ± 31; P < 0.001, Student's t test) (Figure 1b and Table 2).

In contrast, CD32 expression was similar on CD14⁺ monocytes from SLE patients versus normal healthy control individuals (Figure 1c and Table 2). While the frequencies and absolute numbers of CD16⁺CD14⁺ monocytes were similar between SLE patients and control individuals, the intensity of CD16 staining was increased slightly in SLE patients with or without LN (12 ± 0.4 and 13 ± 0.6, respectively, versus control individuals 10 ± 0.6; both P < 0.01, Student's t test) (Figure 1d and Table 2). We also assessed the expression of HLA-DR and CD62L, markers related to monocyte activation, but found no significant differences between the groups (data not shown).

To further evaluate the relationship between FcγR expression and LN, we analyzed the expression of FcγRs on monocytes in 79 patients who had undergone renal biopsy (class II, n = 7; class III/IV, n = 43; and class V, n = 29). The presence of class III/IV or class V LN, but not of class II LN, was associated with increased expression of FcγRI/CD64 compared with SLE patients who did not have LN (Figure 1e). In contrast, the expression of FcγRII and FcγRIII was similar among the different classes of LN (data not shown).

Since FcγRI/CD64 expression on monocytes was greater in SLE patients with LN compared with SLE patients without LN, we investigated its relationship with individual markers of renal involvement. Increased expression of FcγRI/CD64 on monocytes correlated positively with elevated creatinine (r² = 0.27, P < 0.001; Spearman's correlation) (Figure 2a, left) and blood urea nitrogen levels (r² = 0.12, P = 0.001; Spearman's correlation) (Figure 2a, middle), as well as with the degree of proteinuria (microalbumin/creatinine ratio, r² = 0.10, P < 0.001; Spearman's correlation) (Figure 2a, right).

We next examined the relationship of FcγRI/CD64 expression with measures of systemic inflammation such as high-sensitivity CRP and complement C3 17. In patients with SLE, the expression of FcγRI/CD64 on monocytes was positively correlated with elevated serum levels of high-sensitivity CRP (r² = 0.14, P < 0.0001; Spearman's correlation) (Figure 2b). FcγRI/CD64 expression showed an inverse relationship with serum C3 (r² = 0.07, P < 0.0001; Spearman's correlation) (Figure 2c) but not with C4 (r² = 0.01, P = 0.14) (data not shown). Increased FcγRI/CD64 expression was also associated with anti-dsDNA autoantibodies (534.1 ± 21.4 versus 426.5 ± 21.0 mean fluorescence intensity units, P = 0.0005) (Figure 2d). Increased FcγRI/CD64 surface expression on monocytes was therefore associated with impaired renal function, anti-dsDNA autoantibody production, C3 consumption, and ongoing inflammation in SLE patients.

Monocyte migration to the kidneys and the subsequent release of inflammatory mediators are thought to be critical steps initiating renal damage 1819. We evaluated the migratory capacity of circulating monocytes from SLE patients using an in vitro transwell assay, and found that monocytes with elevated FcγRI/CD64 expression exhibited increased migration toward the chemokine MCP-1 (r² = 0.09, P = 0.005; Spearman's correlation) (Figure 3a).

As monocyte-derived proinflammatory cytokines and chemokines such as MCP-1 regulate immune cell infiltration and play an important role in organ damage in SLE 20, we examined the ability of CD64⁺ monocytes to produce MCP-1. After LPS stimulation, monocytes with high FcγRI/CD64 expression produced higher levels of the chemokine than CD64^- monocytes, as measured by intracellular staining (r² = 0.09, P < 0.001; Spearman's correlation) (Figure 3b).

Since the binding of CRP to FcγRI/CD64 and FcγRIIa/CD32a can lead to increased inflammatory cytokine production 212223, we stimulated monocytes from SLE patients with CRP (50 ng/ml) and analyzed the MCP-1 production. CRP and LPS elicited similar levels of intracellular MCP-1 staining (compare Figure 3b and Figure 3c). Consistent with the results with LPS stimulation, high FcγRI/CD64 surface expression was associated with increased intracellular MCP-1 production in response to CRP (r² = 0.26, P = 0.03; Spearman's correlation) (Figure 3c). Monocytes with elevated surface expression of FcγRI/CD64 therefore displayed a more activated phenotype in terms of migratory properties and MCP-1 production in response to either LPS or CRP.

Corticosteroids potently downmodulate certain inflammatory markers on circulating monocytes 24. Since about one-half of our SLE patients were treated with corticosteroids (Table 1), we asked whether the levels of FcγRI/CD64 expression by monocytes were affected by treatment. When analyzed as a group, patients treated with conventional doses of prednisone (< 40 mg/day) showed no difference in FcγRI/CD64 expression compared with those patients not treated with corticosteroids (Figure 4a). There also was no apparent effect of antimalarial, cytotoxic or statin therapy on the expression of FcγRs (Figure 4a). Stratifying patients based on the prednisone dose revealed that a daily dosage ≥ 40 mg was associated with decreased FcγRI/CD64 expression on monocytes (Figure 4b). A similar trend (not statistically significant) was seen at a dose of 20 to 30 mg/day. This effect was not seen at lower dosages (Figure 4b). Patients treated with ≥ 40 mg/day prednisone tended to display lower serum levels of C3 (67.6 ± 8.5 versus 93.7 ± 3.4 mg/dl; P < 0.05) and higher levels of blood urea nitrogen (36.9 ± 9.0 versus 15.6 ± 0.8 ng/dl; P < 0.05) compared with their counterparts given lower doses, consistent with higher disease activity (data not shown). There was no difference in Systemic Lupus Erythematosus Disease Activity Index scores, American College of Rheumatology criteria counts, serum creatinine, high-sensitivity CRP levels, or microalbumin/creatinine ratios between the groups (data not shown).

Several studies have shown that the expression of FcγRI/CD64 can be influenced by different cytokines in pathogenic circumstances. Dysregulation of proinflammatory cytokine production has also been well documented in SLE. To examine potential inducers of FcγRI/CD64 upregulation, we stimulated peripheral blood mononuclear cells from healthy control individuals with a panel of cytokines. Overnight incubation with IFNα, IFNγ, and IL-12 significantly increased FcγRI/CD64 expression on monocytes, whereas IL-6, IL-8, IL-10, TNFα, and CRP treatment did not (Figure 5a). Similar results were obtained when the experiment was performed using cultured THP-1 cells (data not shown).

Curiously, while the addition of dexamethasone to whole blood did not alter the steady-state levels of FcγRI/CD64 expression on monocytes in vitro (Figure 5b), high concentrations of dexamethasone (≥ 10^-4 M) inhibited the upregulation of FcγR1/CD64 expression induced by IFNγ, IFNα and IL-12 (Figure 5c). This effect was not seen with lower concentrations of dexamethasone.