Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus
© Li et al.; licensee BioMed Central Ltd. 2010
Received: 16 November 2009
Accepted: 18 May 2010
Published: 18 May 2010
More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.
Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.
Circulating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.
Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.
It has become increasingly clear that the autoantibody responses characteristic of systemic lupus erythematosus (SLE), such as anti-double-stranded (ds) DNA and anti-Sm, as well as certain clinical manifestations, notably lupus nephritis, are linked to the overproduction of type I interferon (IFN-I) [1–5]. The importance of IFN-I in autoimmunity is evident in the association between autoimmune manifestations and IFN-α treatment in some patients with hepatitis C infection, malignant carcinoid syndrome, or chronic myelogenous leukemia [6–8]. A positive fluorescent antinuclear antibody test can be found in up to 22% of patients treated with IFN-α , and the onset of SLE, autoimmune (Hashimoto) thyroiditis, autoimmune hemolytic anemia, rheumatoid arthritis, vasculitis, and other autoimmune diseases has been reported after IFN-α therapy [7, 9, 10].
More than half of SLE patients display abnormally high expression of a group of IFN-I-stimulated genes (ISGs), a feature associated with active disease, renal involvement, and the production of autoantibodies against DNA-protein and RNA-protein autoantigens [1–5]. Because of the inherent insensitivity and unreliability of measuring IFN-I protein levels in the blood, the level of ISG transcript expression in peripheral blood mononuclear cells (PBMCs) is frequently used as a measure of IFN-I activity [1–5]. However, these assays are costly and time consuming. Flow cytometry may afford a rapid and less expensive means of evaluating IFN-I levels than RNA-based methods. The objective of this study was to identify proteins encoded by ISGs expressed on the cell surface that can be used clinically to evaluate IFN-I levels in SLE. We show that CD64 (Fcγ receptor I) expression on monocytes can be used to assess IFN-I levels rapidly and reliably in clinical samples and may be well suited to monitoring disease activity and response to therapy.
Materials and methods
Patients and controls
Demographics, laboratory, and clinical characteristics of subjects
(n = 83)
(n = 108)
Mean age (years)
Disease duration (years)
12.1 ± 0.7
ACR criteria (mean)
6.2 ± 0.2
123.4 ± 5.7
95.4 ± 5.5
25.7 ± 3.5
19.7 ± 1.5
Medication use (%)
Mean dose (mg/day)
Isolation of RNA from PBMCs
Blood was collected in PAXgene tubes, and total RNA was isolated by using the PAXgene RNA kit (Qiagen, Valencia, CA, USA). RNA (1 to 2 μg per sample) was treated with DNase I (Invitrogen) to remove genomic DNA and reverse transcribed to cDNA by using Superscript II First-Strand Synthesis System (Invitrogen) for RT-PCR. RNA and cDNA samples were stored at -70°C until used.
Real-time quantitative PCR
Expression levels of three IFN-I-inducible genes, myxoma resistant gene-1 (MX1), interferon-inducible protein 44 (IFI44), and Ly6E, were determined in duplicate by real-time PCR (SYBR Green Core Reagent Kit, Applied Biosystems, Foster City, CA, USA). As demonstrated in previous studies, these ISGs are robust markers of IFN-I upregulation associated with SLE [3–5]. Gene expression was normalized to β-actin, and expression relative to the sample with the lowest expression was calculated by using the 2-ΔΔCt method . Amplification conditions were as follows: 95°C for 10 minutes, followed by 45 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 25 seconds, and elongation at 72°C for 25 seconds. After final extension at 72°C for 10 minutes, a melting-curve analysis was performed to ensure specificity of the products. For each ISG, a score was calculated based on the number of standard deviations above or below the mean expression of the designated control group . The ISG index was determined based on the average of individual ISG scores (that is, (MX1 + Ly6E + IFI44)/3) [3, 13]. Primers were as follows: β-actin forward 5'-TCC CTG GAG AAG AGC TAC GA-3'; reverse 5'-AGC ACT GTG TTG GCG TAC A-3'; MX1 forward 5'-CAC GAA GAG GCA GCG GGA TCG-3', reverse 5'-CCT TGC CTC TCC ACT TAT CTT C-3'; Ly6E forward 5'-AGG CTG CTT TGG TTT GTG AC-3', reverse 5'-AGC AGG AGA AGC ACA TCA GC-3'; and IFI44 forward 5'-CTG GGG CTG AGT GAG AAA GA-3', reverse 5'-AGC GAT GGG GAA TCA ATG TA-3'; CXCL9 forward 5'-TGC TGG TTC TGA TTG GAG TG3', reverse 5'-TCA ATT TTC TCG CAG GAA GG-3'; CD14 forward 5'-ATT TGG TGG CAG GAG ATC AA-3', reverse 5'-GCT TCC AGG CTT CAC ACT TG-3'; CD16 forward 5'-ACA GGT GCC AGA CAA ACC TC-3', reverse 5'-TTC CAG CTG TGA CAC CTC AG-3'; CD32 forward 5'-TTC AAG GCC AAC AAC AAT GA-3', reverse 5'-GGA GAA GGT GGG ATC CAA AT-3'; CD64 forward 5'-GTG TCA TGC GTG GAA GGA TA-3', reverse 5'-GCA CTG GAG CTG GAA ATA GC-3'; CCR2 forward 5'-ATC TCC GCC TTC ACT TTC TG-3', reverse 5'-AAT GCG TCC TTG TTC AAT CC-3'; CCL2 forward 5'-CTG CTC ATA GCA GCC ACC TT-3', reverse 5'-TCC TGA ACC CAC TTC TGC TT-3'; CX3CR1, forward 5'-GAC TGG CAG ATC CAG AGG TT-3', reverse 5'-ACC AAC AAA TTT CCC ACC AG-3'; CX3CL1, forward 5'-GGC TCC GAT ATC TCT GTC GT-3', reverse 5'-CTG CAC GTG ATG TTG CAT TT-3'.
Fluorescently tagged antibodies were from BD Bioscience (San Diego, CA, USA), unless otherwise indicated. Heparinized whole blood (100 μl) was stained with phycoerythrin (PE)-conjugated anti-CD64 (clone X54-5/7.1.1), PerCP-conjugated anti-CD14 (clone MΦP9), fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (clone 3G8), allophycocyanin (APC)-conjugated anti-CD32 (clone FLI8.26), PerCP- anti-HLA-II (clone L243), APC-conjugated anti-CD62L (clone DREG 56, eBioscience, San Diego, CA, USA), APC-conjugated anti-CCR2 (clone 48607, R&D Systems, Minneapolis, MN, USA), PE-anti-CX3CR1(clone 2A9-1, MBL International Corporation, Woburn, MA, USA), for 20 minutes in the dark. After erythrocyte lysis, cells were washed with PBS/1%BSA/0.01% NaN3 and fixed in 2% paraformaldehyde PBS. For dendritic cell characterization, cells were stained with Lin-FITC (a cocktail of anti-CD3, -CD14, -CD16, -CD19, -CD20, and -CD56), anti-CD123-PE (clone 9F5), anti-HLA-DR-PerCP, and anti-CD11c-APC. For T- and B-cell characterization, anti-CD3-FITC (clone UCHT1, eBioscience) and -CD19-PerCP (clone SJ25C1) were used. Cells (105) were analyzed by 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; lymphocyte gates were set based on forward/sideward light scatter. CD16, CD32, and CD64 expression levels were expressed as the geometric mean fluorescence intensity (MFI). Data were analyzed by using FCS Express 2.0 (De Novo Software, Ontario, Canada). Preliminary studies indicated that CD64 expression on monocytes is stable for at least 24 hours after blood collection (our unpublished observations). Intracellular protein expression of CCL2 was determined by using anti-human CCL2 (clone 5D3-F7; BD Pharmingen) as described previously .
Culture of PBMCs with cytokines or serum
Human PBMCs were isolated from healthy donor cells by Ficoll density-gradient centrifugation. PBMC were plated on 24-well plates (106 cells per well) in DMEM supplemented with 10% fetal bovine serum, 20 mmol/L L-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cytokines were from BD Bioscience, unless otherwise indicated. Cells were incubated for 19 hours at 37°C in medium containing 25% serum from either SLE patients (n = 65) or healthy controls (n = 44), or in the presence of recombinant human IFN-α (4 ng/ml; PBL Biomedical, Piscataway, NJ, USA), TLR4 agonist (ultrapure E. coli lipopolysaccharide (LPS), 1 μg/ml, Sigma-Aldrich), TLR7 agonist (R848, 1 μg/ml; Invivogen, San Diego, CA, USA), or TLR9 agonist (CpG-A ODN2216, 10 ng/ml; Invivogen). The concentration of TLR ligands used in these experiments was determined based on our preliminary studies using PBMCs from healthy controls. For each TLR ligand, the lowest concentration that induced maximal CD64 expression on control monocytes after 19 hours was selected (data not shown). In some experiments, the soluble viral IFN-I antagonist B18R (from vaccinia virus Western Reserve strain, 0.1 μg/ml; eBioscience, San Diego, CA, USA), anti-human IFN-γ (2 μg/ml), anti-human IL-12 (2 μg/ml), or isotype control mouse IgG1 (Biolegend, San Diego, CA, USA) was added 1 hour before stimulation with TLR agonists. Flow cytometry was performed immediately after incubation. For the analysis of serum-induced CD64 expression, ΔMFI was calculated by subtracting baseline CD64 MFI from the MFI of CD64 expression after incubation with serum from healthy controls (n = 44) or SLE patients (n = 65). A positive ΔMFI indicates an upregulation of CD64 expression compared with the baseline levels. All serum samples were stored at -80°C before these experiments. For real-time quantitative PCR studies, PBMCs (106 cells/well) were treated with PBS or recombinant IFN-α (4 ng/ml), and RNA isolation was performed after 6 hours. Average fold-differences in mRNA expression in PBMCs treated with PBS or IFN-α (n = 4 per group) were determined with real-time PCR, whereas changes in protein levels on monocytes were measured with flow cytometry. Positive values denote increased expression after IFN-α treatment compared with PBS treatment.
Differences between disease groups and normal controls were evaluated by using Student's two-tailed t test unless the data were not normally distributed, in which case the Mann-Whitney U test was used. Changes in CD64 and ISG expression levels after high-dose corticosteroid therapy were assessed by using the paired Student t test. Correlation coefficients were calculated by using Spearman's rank correlation. Data are presented as mean ± SEM. Analyses were performed by using Prism software, version 4.0 (GraphPad Software, San Diego, CA, USA). A P value of < 0.05 was considered significant.
CD64 expression on monocytes is upregulated in SLE patients
Comparisons of CD64 expression (mean fluorescence intensity) with disease manifestations and medication use
495.2 ± 15.9
455.5 ± 14.1
502.3 ± 16.0
446.9 ± 14.4
479.3 ± 13.2
456.4 ± 17.2
499.2 ± 14.4
448.7 ± 15.4
463.3 ± 24.8
475.1 ± 11.6
488.9 ± 18.9
462.6 ± 12.5
499.0 ± 23.3
463.1 ± 11.9
558.8 ± 26.8
450.7 ± 10.9
495.0 ± 15.3
446.2 ± 14.2
507.0 ± 13.8
439.5 ± 17.9
489.9 ± 12.0
399.7 ± 23.7
476.0 ± 15.7
476.2 ± 15.4
528.5 ± 20.4
512.9 ± 24.9
510.4 ± 18.1
556.0 ± 31.9
503.9 ± 16.7
557.9 ± 22.6
517.1 ± 37.9
483.9 ± 16.7
CD64 expression is IFN inducible and correlates with the interferon signature
Detailed analysis of CD64 with flow cytometry showed that the addition of IFN-α to monocytes from healthy donors stimulated its surface expression in a dose-dependent manner. This effect was blocked completely by pretreatment with the soluble vaccinia virus IFN-I antagonist B18R (Figure 2b). In contrast, surface expression of other FcγRs (CD16 and CD32) was unaffected by IFN-α treatment (Figure 2c and 2d).
Recent studies suggest that activation of Toll-like receptor (TLR) 7 and TLR9 may be upstream of the aberrant production of IFN-I in SLE [19–23]. Similar to direct stimulation with IFN-α, treatment with the TLR7 ligand R848 or the TLR9 ligand ODN2216 both induced monocyte surface expression of CD64, an effect that was abolished by pretreatment with B18R (Figure 2e). In contrast, the low level of CD64 upregulation in response to the TLR4 ligand LPS was unaffected by IFN-I blockade. These observations demonstrated that CD64 expression on monocytes is inducible by direct IFN-I stimulation or by TLR7/9 agonists, which elicit IFN-I production.
IFN-γ is also a potent inducer of CD64 expression [24, 25]. To address the potential involvement of IFN-γ, we compared CD64 expression with the transcript levels of CXCL9, a chemokine strongly induced by IFN-γ but only weakly by IFN-I . No correlation was found between CD64 staining and CXCL9 expression (Figure 3c). Taken together, these data suggest that surface CD64 expression on monocytes from SLE patients reflects primarily IFN-I exposure.
SLE serum induces surface expression of CD64 on monocytes
These data strongly suggest that one or more serum mediator(s) were responsible for the upregulation of CD64 expression on monocytes from SLE patients. Supporting this view, the ability of individual SLE sera to induce CD64 expression correlated with CD64 expression on monocytes from the serum donor (r = 0.36; P < 0.05, Spearman's rank correlation; Figure 4b). The effects of SLE sera were inhibited by the addition of B18R, but not neutralizing antibodies to IL-12 or IFN-γ, indicating that IFN-I was the major factor responsible for CD64 upregulation (Figure 4c and 4d). In contrast, the addition of SLE sera slightly decreased the expression of CD32 and had little effect on the expression of CD16 (Figure 4e). Taken together, these findings support the utility of CD64 as a biomarker of IFN-I dysregulation in SLE.
Changes in CD64 expression after therapy
Elevated serum IFN-α was first noted in SLE patients about two decades ago . More recently, high levels of ISG expression in lupus PBMCs have been widely reported [1–5]. This "interferon signature" can be identified by using microarrays or real-time PCR. These approaches have limitations, including the time and labor required to prepare and handle mRNA from clinical samples and their expense. Here, we evaluated the utility of CD64 as a marker for rapidly assessing IFN-I overproduction. Initial suggestions that CD64 is an ISG arose from microarray studies [2, 16], although it remained unknown whether the increased gene expression was associated with changes at the protein level. Our data show for the first time that CD64 surface-staining intensity on monocytes correlates with ISG expression and disease manifestations in lupus patients and illustrate the potential utility of CD64 measurements for quantifying IFN-I levels in serum or other biologic fluids. We also show that CD64 may be used to monitor the effect of therapy on IFN-I levels.
Compared with real-time PCR and microarrays, CD64 expression on circulating monocytes provides a quick and relatively inexpensive means of assessing a patient's interferon status. One-step staining of whole-blood samples and analysis by using a standard four-color flow cytometer can be completed within 30 to 45 minutes, allowing results to be generated during a patient's clinic visit. Moreover, because MFI from flow cytometry staining is an absolute value, this assay avoids the need to normalize the data to actin or other housekeeping genes, as in real-time PCR assays.
The specificity of CD64 fluorescence intensity for IFN-I levels is suggested by several lines of evidence. CD64 expression was enhanced in a dose-dependent manner by IFN-α and was blocked by the viral IFN-I inhibitor B18R (Figure 2a). Similarly, TLR7 and TLR9 ligands enhanced monocyte surface CD64 expression in an IFN-I-dependent manner (Figure 2d). The ability of SLE sera to induce CD64 expression also depended on the presence of IFN-I. CD64 seems unique among the Fc receptors in its regulation by IFN-I. Although IFN-γ can also induce CD64 expression , its contribution to the interferon signature in SLE may be limited, as genes specifically induced by IFN-γ (that is, CXCL9) are not known to be upregulated in lupus patients . In contrast to the lack of a correlation between CD64 fluorescence intensity and CXCL9 expression, surface CD64 expression correlated with the transcript levels of several ISGs (MX1, IFI44, and Ly6E) (Figure 3). In addition, upregulation of monocyte CD64 in the presence of lupus serum was inhibited by the poxviral B18R protein (Figure 4), strongly suggesting that IFN-I in SLE sera upregulates CD64 expression. This effect was not seen with blockade of IFN-γ or IL-12 with neutralizing antibodies, despite of the ability of these cytokines to induce CD64 expression .
Dysregulated CD64 expression may have functional consequences, as this activating FcγR plays important roles in phagocytosis, cytolysis, and induction of inflammatory cytokines . The balance of activating (CD16, CD32a, CD64) and inhibitory (CD32b) FcγRs on antigen-presenting cells, such as monocytes, determines the response to immune complexes, which are produced abundantly in SLE. In addition, CD64 also is involved in the inflammatory response induced by C-reactive protein . Elevated expression of CD64 in SLE, therefore, may fuel the chronic inflammation associated with the autoimmune disease. This view is supported by animal studies, as the deletion of Fc receptor γ-chain, a critical signaling component of the activating FcγRs, is sufficient to inhibit the development of glomerulonephritis lupus-prone mice . The presence of Fc receptor γ-chain on monocytes/macrophages is required for this effect . Besides monocytes, neutrophils, certain dendritic cell subsets (especially myeloid dendritic cells), and eosinophils also express surface CD64 . In particular, the fluorescence intensity of CD64 on myeloid dendritic cells from lupus patients was increased and correlated highly with that on monocytes (data not shown). However, because of the paucity of circulating DCs in most SLE patients , they are technically difficult to analyze.
Some limitations to the clinical application of CD64 expression merit consideration, notably in patients with infections. Neutrophil CD64 has been used as a marker for sepsis [32–35] and to distinguish between infections with dsDNA and ssRNA viruses [35, 36]. It has been suggested that neutrophil CD64 expression can be used to aid in the diagnosis of infections in patients with rheumatoid arthritis . Conversely, in the setting of sepsis, the existence of preexisting autoimmune disease, especially lupus, is a potential confounder. In addition, although CD64 appears to be a surrogate marker of IFN-I activity based on our cross-sectional analysis, longitudinal studies are needed to assess the utility of this marker in monitoring changes in serum IFN-I levels over time.
Recently it was reported that Siglec-1 (CD169) expression on monocytes can be used as a biomarker for IFN-I responses in systemic sclerosis and SLE [16, 38, 39]. Compared with the two-step flow-cytometry assay for CD169, measuring CD64 on circulating monocytes is simpler, requiring only a single step. Whether CD169 is suitable for bioassays using serum samples has also not been tested. However, similar to the desirability of measuring the expression of more than one ISG with real-time PCR, the use of both CD64 and CD169 staining may be warranted to optimize the reliability of the assay.
Our results indicate that the fluorescence intensity of CD64 on circulating monocytes can be used to evaluate IFN-I levels. This flow-cytometry assay is faster and less labor intensive than the measurement of ISG gene expression. As novel agents targeting IFN-I are already in clinical trials , flow-cytometry analysis of CD64 expression may be a convenient and rapid approach for estimating IFN-I levels in SLE patients.
American College of Rheumatology
C4: complement component 3 and complement component 4
Fc gamma receptor
high-sensitivity C-reactive protein
type I interferon
myeloid dendritic cells
mean fluorescence intensity
peripheral blood mononuclear cells
systemic lupus erythematosus
SLE disease activity index
This work was supported by research grants AR44731 and AR51766 from the U.S. Public Health Service, by the Lupus Foundation of America, and by generous gifts from Lupus Link, Inc. (Daytona Beach, FL) and Mr. Lewis M. Schott to the UF Center for Autoimmune Disease. We thank Marlene Sarmiento, Annie Chan, and UF GCRC staff for assistance with clinical data collection and Ed Butfiloski for technical assistance.
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