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Arthritis Res Ther. 2009; 11(1): R6. Published online 2009 January 14. doi: 10.1186/ar2590. | PMCID: PMC2688236 |
Copyright © 2009 Li et al.; licensee BioMed Central Ltd. Increased expression of FcγRI/CD64 on circulating monocytes parallels ongoing inflammation and nephritis in lupus Yi Li,  1 Pui Y Lee, 1,2 Eric S Sobel, 1 Sonali Narain, 1 Minoru Satoh, 1 Mark S Segal, 2 Westley H Reeves, 1 and Hanno B Richards 1,31Division of Rheumatology & Clinical Immunology, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA 2Division of Nephrology, Hypertension and Transplantation, Department of Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610-0221, USA 3Schering-Plough Corporation, Kenilworth, NJ 07033-0530, USA Received August 29, 2008; Revisions requested October 24, 2008; Revised November 21, 2008; Accepted January 14, 2009. This is an open access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The high-affinity receptor for IgG Fcγ/CD64 is critical for the development of lupus nephritis (LN). Cross-linking Fc receptor on recruited monocytes by IgG-containing immune complexes is a key step in immune-complex-mediated nephritis in systemic lupus erythematosus (SLE). The goal of this study was to determine whether expression of Fc receptor (FcγR) I on circulating monocytes is associated with systemic inflammation and renal disease in SLE patients. Methods We studied 205 SLE patients (132 with LN and 73 without LN) along with 74 healthy control individuals. Surface expression of CD14 (monocytes), FcγRI/CD64, FcγRII/CD32, and FcγRIII/CD16 was evaluated by flow cytometry. Monocyte function was assessed by determining the migratory capacity and the ability to produce CCL2 (monocyte chemotractic protein 1). High-sensitivity C-reactive protein, C3 and C4 were measured by nephelometry. Results There was little difference in the expression of FcγRIII/CD16 or FcγRIII/CD32 on circulating monocytes between patients with SLE and control individuals. In contrast, FcγRI/CD64 expression was significantly higher in SLE patients and even higher in patients with LN. FcγRI/CD64 expression was positively associated with serum creatinine and indicators of systemic inflammation. Monocytes from patients with high FcγRI/CD64 expression also exhibited increased chemotaxis and capacity to produce monocyte chemotractic protein 1. Conclusions Increased FcγRI/CD64 expression on circulating monocytes parallels systemic inflammation and renal disease in SLE patients. We propose that circulating monocytes activated by immune complexes and/or proinflammatory mediators upregulate surface expression of FcγRI/CD64 in SLE. The enhanced chemotactic and inflammatory potential of the activated monocytes may participate in a vicious cycle of immune cell recruitment and renal injury in SLE. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against a wide array of self-antigens [ 1]. Formation of immune complexes (ICs) between these autoantibodies and the target antigens has been linked to the development of lupus nephritis (LN) [ 2, 3]. Deposition of ICs in the kidneys activates monocyte/macrophages by interacting with Fc receptor (FcγR) I and FcγRIII, initiating an inflammatory cascade of cytokines and chemokines. The release of proinflammatory mediators such as monocyte chemotractic protein 1 (MCP-1 (CCL2)), macrophage inflammatory protein 1 (CCL3) and fractalkine (CX 3CL1) recruits monocyte/macrophages and other immune effector cells, culminating in tissue damage [ 4, 5]. Three classes of FcγRs are expressed on circulating human monocytes. FcγRI/CD64 is a high-affinity receptor constitutively expressed at substantial levels by monocytes [ 6]. Monocytes also express high levels of FcγRII/CD32, a low-affinity receptor for ICs with two functionally distinct isoforms. In contrast, FcγRIII/CD16, a receptor with moderate affinity for complexed IgG, is present on only about 10% to 15% of circulating monocytes [ 7]. FcγRI, FcγRIIa and FcγRIII are activating Fc receptors bearing intracytoplasmic tyrosine-based activation motifs that trigger monocyte activation upon receptor aggregation. FcγRIIb, on the other hand, contains an immunoreceptor tyrosine-based inhibitory motif and functions as an inhibitory Fc receptor upon interacting with ICs [ 8]. The balance of activating and inhibitory FcγR determines the magnitude of the cellular response in monocytes. Enhanced expression of activating FcγRs or decreased expression of the inhibitory FcγR can lower the activation threshold, leading to the production of inflammatory cytokines that may promote LN [ 9]. Conversely, NZB/W F1 mice deficient in FcγRI/III are protected from LN despite developing extensive IC deposits [ 10]. As in Wegener's granulomatosis [ 11] and rheumatoid arthritis [ 12], circulating monocytes in SLE are activated and exhibit increased surface expression of FcγRI/CD64 [ 13]. Whether this increase in activating FcγR on monocytes is related to development of LN, however, is unknown. To investigate the possible role of activating FcγR in human LN, we examined the expression of FcγRI/CD64, FcγRIII/CD16 and FcγRII/CD32 on circulating monocytes from SLE patients, and the relationship of FcγR expression levels to renal involvement and chemokine production. Study population 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. | Table 1Demographics, laboratory characteristics and clinical characteristics of participants |
Cell surface staining 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). Analysis of chemokine production 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. Chemotaxis assay 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. Measurement of C-reactive protein and complement 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]. In vitro stimulation of healthy donor peripheral blood mononuclear cells 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. Statistical analysis 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. We assessed the surface expression of FcγRs on monocytes from SLE patients with or without LN and from healthy control individuals. Demographics and clinical/laboratory data are summarized in Table 1. There was no difference in the percentage of circulating CD14 + monocytes between SLE patients with or without LN and normal control individuals (Table 2). Absolute monocyte counts, however, were significantly decreased in SLE patients with/without LN when compared with normal control individuals (268 ± 29 cells/μl and 254 ± 39 cells/μl, respectively, versus 357 ± 32 cells/μl; both P < 0.005, Student's t test). | Table 2Comparison of cell surface marker expression by CD14+ monocytes |
Increased FcγRI/CD64 expression on SLE monocytes 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 , top and Table 2). Although circulating monocytes from SLE patients also uniformly expressed FcγRI/CD64 (Figure , 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 and Table 2). In contrast, CD32 expression was similar on CD14 + monocytes from SLE patients versus normal healthy control individuals (Figure 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 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 ). In contrast, the expression of FcγRII and FcγRIII was similar among the different classes of LN (data not shown). Increased FcγRI/CD64 expression is associated with impaired renal function 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 , left) and blood urea nitrogen levels (r2 = 0.12, P = 0.001; Spearman's correlation) (Figure , middle), as well as with the degree of proteinuria (microalbumin/creatinine ratio, r2 = 0.10, P < 0.001; Spearman's correlation) (Figure , right). Increased levels of FcγRI/CD64 expression are associated with ongoing inflammation 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 ). FcγRI/CD64 expression showed an inverse relationship with serum C3 ( r2 = 0.07, P < 0.0001; Spearman's correlation) (Figure ) 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 ). 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. FcγRI/CD6hi monocytes have an activated phenotype Monocyte migration to the kidneys and the subsequent release of inflammatory mediators are thought to be critical steps initiating renal damage [ 18, 19]. 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 ). 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 ). Since the binding of CRP to FcγRI/CD64 and FcγRIIa/CD32a can lead to increased inflammatory cytokine production [ 21- 23], 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 and Figure ). 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 ). 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. Medication effects on FcγRI/CD64 expression 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 ). There also was no apparent effect of antimalarial, cytotoxic or statin therapy on the expression of FcγRs (Figure ). Stratifying patients based on the prednisone dose revealed that a daily dosage ≥ 40 mg was associated with decreased FcγRI/CD64 expression on monocytes (Figure ). 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 ). 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). Effect of cytokines on FcγRI/CD64 expression 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 ). 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 ), high concentrations of dexamethasone (≥ 10-4 M) inhibited the upregulation of FcγR1/CD64 expression induced by IFNγ, IFNα and IL-12 (Figure ). This effect was not seen with lower concentrations of dexamethasone. In mouse models of SLE, monocytes/macrophages bearing activating Fc receptors are pivotal to the development of IC-mediated glomerulonephritis [ 25, 26]. There is indirect evidence that the same may be true of human lupus [ 27, 28], although the relationship between activating FcγR expression and the pathogenesis of human LN is less clear than in the mouse. In the present study, we examined FcγR expression in more than 200 SLE patients. The levels of FcγRI/CD64 expression on circulating monocytes were significantly elevated in SLE patients, especially in those with LN. Increased monocyte FcγRI/CD64 expression also was associated with markers of impaired renal function impairment and with a greater ability to migrate and secrete the chemokine MCP-1. The proinflammatory role of activating FcγR in LN is evident in mice deficient in FcγRI/III, which are protected from the development of renal disease despite the presence of glomerular IC deposits [ 10]. A recent study showed that the expression of FcγRI/III by monocytes was both necessary and sufficient to trigger nephritis in NZB/W F1 mice [ 26]. In contrast, the inhibitory FcγRIIb suppresses inflammation and spontaneous activation of autoreactive lymphocytes and autoantibody production in mice [ 26, 29]. In human SLE, several groups have shown the abnormal upregulation of activating Fcγ receptors on monocytes [ 13, 30]. One relatively small study, however, found no significant difference in FcγRI/CD64 or FcγRIII/CD16 expression on SLE monocytes compared with healthy controls [ 31]. About two-thirds of the patients studied here had elevated levels of monocyte surface CD64 in the present study, a discrepancy that may be due to the relatively small number of subjects studied previously. Consistent with the observations of others [ 13, 28], our data show that the activating receptor FcγRIII/CD16 also is upregulated in SLE patients compared with healthy control individuals. In line with murine lupus data, our data support the idea that activating FcγRs play a crucial role in IC-mediated organ damage in SLE. Although NZB/W F1 mice deficient in activating FcγRs are protected from renal disease, the relative contributions of the individual activating FcγRs have not been studied further. Our data show that although both FcγRIII/CD16 and FcγRI/CD64 expression were elevated, increased FcγRIII/CD16 expression was not associated with LN, suggesting that activation via FcγRI/CD64 may be more significant to the pathogenesis of human LN. Moreover, we found no difference in the surface expression of FcγRII/CD32 on monocytes between the SLE patients and healthy control individuals, although interpretation of this finding is limited by the inability of the anti-CD32 antibody to distinguish the activating FcγRIIa and inhibitory FcγRIIb. Expression of the inhibitory FcγRIIb in peripheral blood mononuclear cells from SLE patients has been recently studied using specific antibodies [ 32]. While low expression levels were found on B-lymphocyte subsets, FcγRIIb/CD32b expression was not impaired on monocytes from SLE patients. The importance of FcγRs in the pathogenesis of SLE is further illustrated by extensive polymorphism studies involving FcγRII/CD32 and FcγRIII/CD16. Several of these polymorphisms – including FcγRIIa-131R, FcγRIIIa-176F, and FcγRIIIb-NA2 – have been associated with lupus susceptibility [ 33, 34]. Importantly, some of them cause functional alterations of the inhibitory receptor [ 35, 36] while others are associated with reduced surface expression of FcγRIIb on both memory and plasma B lymphocytes [ 37]. To our knowledge, however, polymorphisms involving FcγRI/CD64 have not been linked to SLE. The markedly elevated expression of FcγRI/CD64 among SLE patients with LN (Figure ) may serve as a surrogate marker of renal disease that correlates with both established measures of renal dysfunction (increased serum creatinine, blood urea nitrogen, and proteinuria) and inflammation (elevated serum CRP, C3 deficiency). Monocyte FcγRI/CD64 expression, however, did not correlate with overall disease activity as assessed by the Systemic Lupus Erythematosus Disease Activity Index (data not shown). This was not due to medication use, since FcγRI/CD64 levels on circulating monocytes were unaffected by treatment with prednisone at doses < 40 mg/day, or by antimalarials, cytotoxic agents, or statins. In contrast, higher doses of prednisone (≥ 40 mg/day) or dexamethasone treatment in vitro reduced FcγRI/CD64 expression, possibly due to direct effects on proinflammatory cytokine production [ 38, 39] or to the generation of a subset of anti-inflammatory monocytes that secrete IL-10 [ 40, 41]. Our in vitro data suggest that IL-12, IFNγ, and IFNα are potential inducers of FcγRI/CD64 expression in SLE. Interestingly, excess production of all three of these cytokines promotes LN in mice [ 42- 44]. In human LN, increased levels of IFNγ, IL-12 and IFNα/β are found in the kidney [ 45, 46]. Dysregulation of IFNα production is also associated with renal involvement [ 47]. Our data are consistent with the possibility that the overproduction of one or more of these cytokines promotes LN by enhancing the recruitment of proinflammatory (CD64 +) monocytes/macrophages to the renal glomerulus. Although there was a highly significant correlation between FcγRI/CD64 expression and several markers of renal involvement or inflammation (Figure ), the r2 values were in some cases relatively low. This indicated the existence of additional variables, at present undefined, affecting FcγRI/CD64 expression. Elucidating the variables that affect FcγRI/CD64 expression, perhaps including serum levels of the cytokines examined in our in vitro studies, will require further study. FcγRI/CD64 plays a role in phagocytosis, cytolysis, degranulation, and induction of inflammatory cytokines. Additionally, FcγRI-deficient mice display defective peritoneal monocyte infiltration in response to ICs [ 48]. Consistent with these studies, our data demonstrated that circulating human monocytes from patients with upregulated FcγRI/CD64 expression exhibited increased migratory capacity and MCP-1 production in response to LPS or CRP stimulation. Monocyte/macrophage infiltration is important in promoting mesangial hypercellularity and the development of glomerulosclerosis in both human and animal models [ 49, 50]. Additionally, the number of infiltrating monocytes/macrophages is associated with more severe renal injury and poor prognosis in LN [ 50, 51]. As seen in animal models [ 10, 26, 52], monocytes expressing FcγRI/CD64 may be important to the pathogenesis of IC-mediated nephritis in SLE. Elevated production of IFNα and IFNγ in SLE may induce the expression of FcγRI/CD64 monocytes and facilitate the infiltration of these cells to the sites of IC deposition in the kidney [ 48]. Since IFNα and IFNγ also stimulate the production of monocyte attractants such as MCP-1, the presence of these cytokines in the kidney also may promote the influx of monocytes. In turn, signal transduction downstream of FcγRI/CD64 leads to monocyte activation and further production of inflammatory cytokines and chemokines. These events could culminate in a vicious cycle of renal inflammation and monocyte infiltration, ultimately leading to permanent tissue damage. Our study demonstrates that elevated surface expression of FcγRI/CD64 is associated with ongoing systemic inflammation and renal disease in lupus patients. We propose that upregulation of FcγRI/CD64 expression on circulating monocytes may be a useful surrogate marker of monocyte activation in SLE. BSA: bovine serum albumin; CRP: C-reactive protein; DMEM: Dulbecco's modified Eagle's medium; FcγR: Fcγ receptor; IC: immune complex; IFN: interferon; IL: interleukin; LN: lupus nephritis; LPS: lipopolysaccharide; MCP-1: monocyte chemotractic protein 1; PBS: phosphate-buffered saline; SLE: systemic lupus erythematosus; TNF: tumor necrosis factor. The authors declare that they have no competing interests. WHR and HBR contributed equally to this work. YL carried out data analysis and interpretation, and the study design, and assisted in manuscript preparation. PYL participated in date analysis and interpretation, and assisted in manuscript preparation. ESS participated in acquisition of data and patient recruitment. SN participated in statistical analysis. MS assisted in data interpretation. MSS participated in acquisition of data and patient recruitment. WHR carried out the study design and data interpretation, and assisted in patient recruitment and preparation of the manuscript. HBR conceived of the study and coordinated patient recruitment, data analysis and preparation of the manuscript. All authors read and approved the final manuscript. The present work was supported by grants from the National Institutes of Health (K08 DK02890 and MO1 RR00082) and the Greater Florida Chapter of the Lupus Foundation of America and the Lupus Research Institute (New York, USA). PYL is an NIH T32 trainee (DK07518). The authors thank Marlene Sarmiento, Annie Chan, Frances Reeves, Ashley Armstrong, Emily Naglich, Kate Brunner and UF GCRC staff for clinical assistance; and Barbara Kolheffer, Christopher Kennedy and Ed Butfiloski for technical assistance. - Reeves WH, Narain S, Satoh M. Autoantibodies in systemic lupus erythematosus. In: Koopman WJ, Moreland LW, editor. Arthritis and Allied Conditions. Philadelphia, PA: Lippincott Williams & Wilkins; 2004. pp. 1497–1521.
- Lukacs K, Kavai M, Banyai A, Sonkoly I, Vegh E, Szabo G, Szegedi G. Effects of immune complexes from SLE patients on human monocyte locomotion and Fc receptor function. Ann Rheum Dis. 1984;43:729–733. doi: 10.1136/ard.43.5.729. [PubMed]
- McLigeyo SO. Pathogenesis of lupus nephritis: a review. East Afr Med J. 1998;75:628–631. [PubMed]
- Rovin BH, Song H, Birmingham DJ, Hebert LA, Yu CY, Nagaraja HN. Urine chemokines as biomarkers of human systemic lupus erythematosus activity. J Am Soc Nephrol. 2005;16:467–473. doi: 10.1681/ASN.2004080658. [PubMed]
- Li Y, Tucci M, Narain S, Barnes EV, Sobel ES, Segal MS, Richards HB. Urinary biomarkers in lupus nephritis. Autoimmun Rev. 2006;5:383–388. doi: 10.1016/j.autrev.2005.10.006. [PubMed]
- Salmon JE, Pricop L. Human receptors for immunoglobulin G: key elements in the pathogenesis of rheumatic disease. Arthritis Rheum. 2001;44:739–750. doi: 10.1002/1529-0131(200104)44:4<739::AID-ANR129>3.0.CO;2-O. [PubMed]
- Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol. 2007;81:584–592. doi: 10.1189/jlb.0806510. [PubMed]
- Pricop L, Redecha P, Teillaud JL, Frey J, Fridman WH, Sautes-Fridman C, Salmon JE. Differential modulation of stimulatory and inhibitory Fcγ receptors on human monocytes by Th1 and Th2 cytokines. J Immunol. 2001;166:531–537. [PubMed]
- Ravetch JV, Bolland S. IgG Fc receptors. Annu Rev Immunol. 2001;19:275–290. doi: 10.1146/annurev.immunol.19.1.275. [PubMed]
- Clynes R, Dumitru C, Ravetch JV. Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science. 1998;279:1052–1054. doi: 10.1126/science.279.5353.1052. [PubMed]
- Muller Kobold AC, Kallenberg CG, Tervaert JW. Monocyte activation in patients with Wegener's granulomatosis. Ann Rheum Dis. 1999;58:237–245. doi: 10.1136/ard.58.4.237. [PubMed]
- Wijngaarden S, van Roon JA, Bijlsma JW, Winkel JG van de, Lafeber FP. Fcγ receptor expression levels on monocytes are elevated in rheumatoid arthritis patients with high erythrocyte sedimentation rate who do not use anti-rheumatic drugs. Rheumatology (Oxford). 2003;42:681–688. doi: 10.1093/rheumatology/keg174. [PubMed]
- Fries LF, Mullins WW, Cho KR, Plotz PH, Frank MM. Monocyte receptors for the Fc portion of IgG are increased in systemic lupus erythematosus. J Immunol. 1984;132:695–700. [PubMed]
- Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG, Talal N, Winchester RJ. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982;25:1271–1277. doi: 10.1002/art.1780251101. [PubMed]
- 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;35:630–640. doi: 10.1002/art.1780350606. [PubMed]
- Barnes EV, Narain S, Naranjo A, Shuster J, Segal MS, Sobel ES, Armstrong AE, Santiago BE, Reeves WH, Richards HB. High sensitivity C-reactive protein in systemic lupus erythematosus: relation to disease activity, clinical presentation and implications for cardiovascular risk. Lupus. 2005;14:576–582. doi: 10.1191/0961203305lu2157oa. [PubMed]
- Sjowall C, Bengtsson AA, Sturfelt G, Skogh T. Serum levels of autoantibodies against monomeric C-reactive protein are correlated with disease activity in systemic lupus erythematosus. Arthritis Res Ther. 2004;6:R87–R94. doi: 10.1186/ar1032. [PubMed]
- Lloyd CM, Minto AW, Dorf ME, Proudfoot A, Wells TN, Salant DJ, Gutierrez-Ramos JC. RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis. J Exp Med. 1997;185:1371–1380. doi: 10.1084/jem.185.7.1371. [PubMed]
- Kelley VR. Leukocyte–renal epithelial cell interactions regulate lupus nephritis. Semin Nephrol. 2007;27:59–68. doi: 10.1016/j.semnephrol.2006.09.008. [PubMed]
- Tucci M, Barnes EV, Sobel ES, Croker BP, Segal MS, Reeves WH, Richards HB. Strong association of a functional polymorphism in the monocyte chemoattractant protein 1 promoter gene with lupus nephritis. Arthritis Rheum. 2004;50:1842–1849. doi: 10.1002/art.20266. [PubMed]
- Marnell L, Mold C, Du Clos TW. C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol. 2005;117:104–111. doi: 10.1016/j.clim.2005.08.004. [PubMed]
- Bang R, Marnell L, Mold C, Stein MP, Clos KT, Chivington-Buck C, Clos TW. Analysis of binding sites in human C-reactive protein for Fc{γ}RI, Fc{γ}RIIA, and C1q by site-directed mutagenesis. J Biol Chem. 2005;280:25095–25102. doi: 10.1074/jbc.M504782200. [PubMed]
- Tron K, Manolov DE, Rocker C, Kachele M, Torzewski J, Nienhaus GU. C-reactive protein specifically binds to Fcγ receptor type I on a macrophage-like cell line. Eur J Immunol. 2008;38:1414–1422. doi: 10.1002/eji.200738002. [PubMed]
- Sumegi A, Antal-Szalmas P, Aleksza M, Kovacs I, Sipka S, Zeher M, Kiss E, Szegedi G. Glucocorticosteroid therapy decreases CD14-expression and CD14-mediated LPS-binding and activation of monocytes in patients suffering from systemic lupus erythematosus. Clin Immunol. 2005;117:271–279. doi: 10.1016/j.clim.2005.09.002. [PubMed]
- Park SY, Ueda S, Ohno H, Hamano Y, Tanaka M, Shiratori T, Yamazaki T, Arase H, Arase N, Karasawa A, Sato S, Ledermann B, Kondo Y, Okumura K, Ra C, Saito T. Resistance of Fc receptor-deficient mice to fatal glomerulonephritis. J Clin Invest. 1998;102:1229–1238. doi: 10.1172/JCI3256. [PubMed]
- Bergtold A, Gavhane A, D'Agati V, Madaio M, Clynes R. FcR-bearing myeloid cells are responsible for triggering murine lupus nephritis. J Immunol. 2006;177:7287–7295. [PubMed]
- Parris TM, Kimberly RP, Inman RD, McDougal JS, Gibofsky A, Christian CL. Defective Fc receptor-mediated function of the mononuclear phagocyte system in lupus nephritis. Ann Intern Med. 1982;97:526–532. [PubMed]
- Salmon JE, Millard S, Schachter LA, Arnett FC, Ginzler EM, Gourley MF, Ramsey-Goldman R, Peterson MG, Kimberly RP. FcγRIIA alleles are heritable risk factors for lupus nephritis in African Americans. J Clin Invest. 1996;97:1348–1354. doi: 10.1172/JCI118552. [PubMed]
- Lin Q, Xiu Y, Jiang Y, Tsurui H, Nakamura K, Kodera S, Ohtsuji M, Ohtsuji N, Shiroiwa W, Tsukamoto K, Amano H, Amano E, Kinoshita K, Sudo K, Nishimura H, Izui S, Shirai T, Hirose S. Genetic dissection of the effects of stimulatory and inhibitory IgG Fc receptors on murine lupus. J Immunol. 2006;177:1646–1654. [PubMed]
- Salmon JE, Kimberly RP, Gibofsky A, Fotino M. Defective mononuclear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligand binding and internalization. J Immunol. 1984;133:2525–2531. [PubMed]
- Hepburn AL, Mason JC, Davies KA. Expression of Fcγ and complement receptors on peripheral blood monocytes in systemic lupus erythematosus and rheumatoid arthritis. Rheumatology (Oxford). 2004;43:547–554. doi: 10.1093/rheumatology/keh112. [PubMed]
- Mackay M, Stanevsky A, Wang T, Aranow C, Li M, Koenig S, Ravetch JV, Diamond B. Selective dysregulation of the FcγIIB receptor on memory B cells in SLE. J Exp Med. 2006;203:2157–2164. doi: 10.1084/jem.20051503. [PubMed]
- Dijstelbloem HM, Bijl M, Fijnheer R, Scheepers RH, Oost WW, Jansen MD, Sluiter WJ, Limburg PC, Derksen RH, Winkel JG van de, Kallenberg CG. Fcγ receptor polymorphisms in systemic lupus erythematosus: association with disease and in vivo clearance of immune complexes. Arthritis Rheum. 2000;43:2793–2800. doi: 10.1002/1529-0131(200012)43:12<2793::AID-ANR20>3.0.CO;2-6. [PubMed]
- Brown EE, Edberg JC, Kimberly RP. Fc receptor genes and the systemic lupus erythematosus diathesis. Autoimmunity. 2007;40:567–581. doi: 10.1080/08916930701763710. [PubMed]
- Li X, Wu J, Carter RH, Edberg JC, Su K, Cooper GS, Kimberly RP. A novel polymorphism in the Fcγ receptor IIB (CD32B) transmembrane region alters receptor signaling. Arthritis Rheum. 2003;48:3242–3252. doi: 10.1002/art.11313. [PubMed]
- Floto RA, Clatworthy MR, Heilbronn KR, Rosner DR, MacAry PA, Rankin A, Lehner PJ, Ouwehand WH, Allen JM, Watkins NA, Smith KG. Loss of function of a lupus-associated FcγRIIb polymorphism through exclusion from lipid rafts. Nat Med. 2005;11:1056–1058. doi: 10.1038/nm1288. [PubMed]
- Su K, Yang H, Li X, Gibson AW, Cafardi JM, Zhou T, Edberg JC, Kimberly RP. Expression profile of FcγRIIb on leukocytes and its dysregulation in systemic lupus erythematosus. J Immunol. 2007;178:3272–3280. [PubMed]
- Mozo L, Suarez A, Gutierrez C. Glucocorticoids up-regulate constitutive interleukin-10 production by human monocytes. Clin Exp Allergy. 2004;34:406–412. doi: 10.1111/j.1365-2222.2004.01824.x. [PubMed]
- Garrelds IM, van Hal PT, Haakmat RC, Hoogsteden HC, Saxena PR, Zijlstra FJ. Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids. Mediators Inflamm. 1999;8:229–235. doi: 10.1080/09629359990397. [PubMed]
- Llorente L, Richaud-Patin Y, Wijdenes J, Alcocer-Varela J, Maillot MC, Durand-Gasselin I, Fourrier BM, Galanaud P, Emilie D. Spontaneous production of interleukin-10 by B lymphocytes and monocytes in systemic lupus erythematosus. Eur Cytokine Netw. 1993;4:421–427. [PubMed]
- Xia CQ, Peng R, Beato F, Clare-Salzler MJ. Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity. Scand J Immunol. 2005;62:45–54. doi: 10.1111/j.1365-3083.2005.01640.x. [PubMed]
- Haas C, Ryffel B, Le Hir M. IFN-γ is essential for the development of autoimmune glomerulonephritis in MRL/Ipr mice. J Immunol. 1997;158:5484–5491. [PubMed]
- Calvani N, Satoh M, Croker BP, Reeves WH, Richards HB. Nephritogenic autoantibodies but absence of nephritis in Il-12p35-deficient mice with pristane-induced lupus. Kidney Int. 2003;64:897–905. doi: 10.1046/j.1523-1755.2003.00178.x. [PubMed]
- Nacionales DC, Kelly-Scumpia KM, Lee PY, Weinstein JS, Lyons R, Sobel E, Satoh M, Reeves WH. Deficiency of the type I interferon receptor protects mice from experimental lupus. Arthritis Rheum. 2007;56:3770–3783. doi: 10.1002/art.23023. [PubMed]
- Uhm WS, Na K, Song GW, Jung SS, Lee T, Park MH, Yoo DH. Cytokine balance in kidney tissue from lupus nephritis patients. Rheumatology (Oxford). 2003;42:935–938. doi: 10.1093/rheumatology/keg255. [PubMed]
- Peterson KS, Huang JF, Zhu J, D'Agati V, Liu X, Miller N, Erlander MG, Jackson MR, Winchester RJ. Characterization of heterogeneity in the molecular pathogenesis of lupus nephritis from transcriptional profiles of laser-captured glomeruli. J Clin Invest. 2004;113:1722–1733. [PubMed]
- Zhuang H, Narain S, Sobel E, Lee PY, Nacionales DC, Kelly KM, Richards HB, Segal M, Stewart C, Satoh M, Reeves WH. Association of anti-nucleoprotein autoantibodies with upregulation of type I interferon-inducible gene transcripts and dendritic cell maturation in systemic lupus erythematosus. Clin Immunol. 2005;117:238–250. doi: 10.1016/j.clim.2005.07.009. [PubMed]
- Heller T, Gessner JE, Schmidt RE, Klos A, Bautsch W, Kohl J. Cutting edge: Fc receptor type I for IgG on macrophages and complement mediate the inflammatory response in immune complex peritonitis. J Immunol. 1999;162:5657–5661. [PubMed]
- Saito T, Yusa A, Soma J, Ootaka T, Sato H, Ito S. Significance of leukocyte infiltration in membranous nephropathy with segmental glomerulosclerosis. Nephron. 1998;80:414–420. doi: 10.1159/000045213. [PubMed]
- Yang N, Isbel NM, Nikolic-Paterson DJ, Li Y, Ye R, Atkins RC, Lan HY. Local macrophage proliferation in human glomerulonephritis. Kidney Int. 1998;54:143–151. doi: 10.1046/j.1523-1755.1998.00978.x. [PubMed]
- Weidner S, Carl M, Riess R, Rupprecht HD. Histologic analysis of renal leukocyte infiltration in antineutrophil cytoplasmic antibody-associated vasculitis: importance of monocyte and neutrophil infiltration in tissue damage. Arthritis Rheum. 2004;50:3651–3657. doi: 10.1002/art.20607. [PubMed]
- Clynes R, Maizes JS, Guinamard R, Ono M, Takai T, Ravetch JV. Modulation of immune complex-induced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors. J Exp Med. 1999;189:179–185. doi: 10.1084/jem.189.1.179. [PubMed]
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