1 Rheumatology and Clinical Immunology and
2 Department of Immunology/Immunotherapy Laboratory, University Medical Center Utrecht, Utrecht, The Netherlands
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Abstract |
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Methods. Mononuclear cells from peripheral blood and synovial fluid were isolated and FcR expression levels on CD14-positive cells were analysed by flow cytometry. The effects of patient and disease characteristics and the use of DMARDs were assessed.
Results. A high expression level of FcRIIa and high percentages of Fc
RIIIa-expressing monocytes were found in RA patients with a high erythrocyte sedimentation rate. DMARD-naive early RA patients had higher Fc
RIIa expression levels but a similar amount of Fc
RIIIa-positive monocytes compared with RA patients using DMARDs. In synovial fluid, Fc
RIIa expression levels were lower than in RA peripheral blood, whereas the percentage of Fc
RIIIa-positive monocytic cells was higher in synovial fluid than in peripheral blood.
Conclusions. These data point to the involvement of FcRs, specifically Fc
RIIa and IIIa, in the immune response of RA and suggest that Fc
R expression levels are susceptible to modulation by DMARD therapy.
KEY WORDS: Fc Receptor, Rheumatoid arthritis, Monocytes/macrophages, DMARDs.
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Introduction |
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Circulating autoantibodies, such as rheumatoid factor (RF) [4], antiperinuclear factor and anti-keratin antibodies, have been characterized in RA. Although the pathological role of these autoantibodies is not fully understood, they contribute to disease by forming immune complexes [5]. Binding of immunoglobulin G (IgG)-containing immune complexes to receptors for the Fc domain of IgG (FcRs), expressed on the cell surface of different cell types, including monocytes and macrophages, leads to activation of these cells. This initiates a diversity of effector functions, such as phagocytosis, antigen presentation, antibody-dependent cellular cytotoxicity and the release of proinflammatory and tissue-destructive mediators [6, 7]. Therefore, Fc
R expression levels may play an important role in the activation and function of monocytes/macrophages and thus in the initiation and persistence of the immune response and tissue damage in RA.
Three classes of human IgG receptors (FcRI, II and III) have been described on human leucocytes. Fc
RI (CD64) is the high-affinity receptor, binding both monomeric and immune-complexed IgG. Fc
RII (CD32) exhibits low affinity for IgG and interacts only with polymeric or complexed IgG. Two subclasses are recognized, Fc
RIIa and IIb, with rather different biological roles. Fc
RIII (CD16) also exists as two isoforms, IIIa and IIIb, and binds IgG with intermediate affinity. The Fc
RIIIb isoform is expressed only on polymorphonuclear leucocytes. Fc
RI, IIa and IIIa are stimulatory receptors, characterized by an intracellular immunoreceptor tyrosine-based activation motif (ITAM), whereas Fc
RIIb is an inhibitory receptor, with an inhibitory motif (ITIM) [7, 8]. Fc
RI and IIa are constitutively expressed on the cell surface of monocytes and macrophages, whereas Fc
RIIIa is found only on a small fraction (<10%) of circulating monocytes [9]. Recently, Fc
RIIb has been identified in human blood monocytes by the reverse transcriptionpolymerase chain reaction and Western blotting [10].
FcR expression levels are known to be influenced by cytokines, which also play an important role in RA, and thereby cellular functions can be altered. Interferon
(IFN-
) and IL-10, both produced in significant amounts in RA patients, up-regulate Fc
RI and IIa, whereas IL-4 and IL-13 down-regulate expression levels of Fc
RI and IIa. Fc
RIIIa is selectively induced by transforming growth factor ß (TGF-ß) and down-regulated by TNF-
[1113].
In several inflammatory diseases, monocytes are activated and exhibit increased surface expression levels of FcRI, such as in Wegener's granulomatosis [14] and sepsis [15], and may exhibit more Fc
RIIIa-expressing monocytes, as in sepsis [16], HIV [17] and tuberculosis [18]. With regard to RA, few initial studies have been performed, none of which has described all three Fc
Rs [1921]. These studies were inconclusive regarding Fc
R expression levels and disease activity and did not address the use of disease-modifying anti-rheumatic drugs (DMARDs).
Because the activation of monocytes/macrophages may well be modulated by the expression levels of FcRs, we investigated the expression patterns of all three stimulatory Fc
Rs on peripheral blood monocytes of RA patients and compared expression levels with those of healthy controls and paired synovial fluid macrophages. We determined the relationship of Fc
R expression levels with patient and disease characteristics and the use of anti-rheumatic drugs.
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Patients and methods |
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Cell separation
Immediately after collection, peripheral blood and synovial fluid were diluted 1:1 with Dulbecco's Modified Eagle Medium (DMEM) (Gibco, New York, USA) supplemented with 1% penicillin, streptomycin sulphate and glutamine (PSG). Mononuclear cells were isolated by density centrifugation using FicollPaque (Pharmacia Biotech, Uppsala, Sweden) according to standard procedures.
Analysis of Fc receptor expression levels
Isolated mononuclear cells were washed in phosphate-buffered saline containing 1% bovine serum albumin and 0.1% sodium azide and incubated with 10 µl monoclonal antibodies for 30 min at 4°C. Double staining for monocytes and one of the three different FcRs was performed using phycoerythrin (PE)-conjugated CD14 (Clone Tük 4; Dako, Glostrup, Denmark) and either fluorescein isothiocyanate (FITC)-conjugated anti-Fc
RI (CD64; 32.2; Medarex, Annandale, NJ, USA); anti-Fc
RII (CD32; IV.3; Medarex) or anti-Fc
RIII (CD16; 3G8; Medarex). An isotype-matched control (FITC/PE) was obtained from Immunotech (Marseille, France). The isoforms Fc
RIIa (stimulatory) and Fc
RIIb (inhibitory) are extracellularly 92% homologous, but differ intracellularly with either an activation (ITAM) or inhibitory motif (ITIM), respectively. The CD32 monoclonal antibody IV.3 (Medarex) is preferentially directed against Fc
RIIa and does not stain Fc
RIIb. There are no antibodies available to detect solely Fc
RIIb on the cell surface [25, 26]. Anti-Fc
RIII (CD16; 3G8, Medarex) does not discriminate between Fc
RIIIa and Fc
RIIIb, but Fc
RIIIb is expressed only on polymorphonuclear cells, which are not present in the MNC fraction.
Flow cytometry was performed by fluorescence-activated cell-sorting using a FACScan (Becton Dickinson, Alphen a/d Rhijn, The Netherlands) and analysed with WinMDI software (Becton Dickinson). For the analysis of monocytes, a live gate was set for viable monocytes based on their forward/sideways scatter. Furthermore, cells were gated for CD14PE expression. Expression levels of FcRI, IIa and IIIa were determined as the geometrical mean fluorescence intensity (MFI) of CD64, CD32 and CD16 on CD14-positive cells. Because not all CD14-positive cells are CD16-positive, Fc
RIIIa was also expressed as the percentage of CD16-positive monocytes. The percentage of monocytes out of the total number of peripheral blood mononuclear cells (PBMC) was determined. A representative scattergram of a FACS analysis is shown in Fig. 1
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Incubation experiments
We tested whether the observed FcR expression levels were influenced or underestimated by interaction with immune complexes present in peripheral blood or synovial fluid from RA patients, either because of steric hindrance or internalization of Fc
Rs. For this purpose, mononuclear cells from healthy controls were incubated with serum from healthy controls, RA serum and RA synovial fluid for different periods.
PBMC obtained from healthy donors (n=8) were incubated in the presence of 50% (v/v) DMEM, 1% PSG and either 50% (v/v) human pooled adult male AB+ serum (Red Cross Blood Transfusion Center, Utrecht, The Netherlands), 50% (v/v) pooled RA serum or 50% (v/v) pooled RA synovial fluid. Serum and synovial fluid were obtained from five RF-positive RA patients with active disease, then pooled and heat-inactivated at 56°C for 1 h. Cells were incubated at a density of 5x106 cells/ml in 96-well plates.
An incubation time of 30 min at 4°C was used to assess the influence of steric hindrance by immune complexes at the binding site of the monoclonal antibodies used. An incubation time of 1 h at 37°C under culture conditions (5% CO2 in humid air) was used to analyse the internalization of FcR upon binding by immune complexes [25]. Following incubation, cells were washed and prepared for flow cytometry as described above.
Statistical analysis
Non-parametric tests were used for comparisons between RA patients and healthy controls (MannWhitney U-test and 2 test) and for comparisons between healthy controls and RA subpopulations (KruskalWallis test). The Wilcoxon signed ranks test was used to compare Fc
R expression levels of CD14-positive cells from paired samples of synovial fluid and peripheral blood and for the analysis of incubation experiments. Correlations were evaluated with Spearman correlation analyses. A P value of
0.05 was considered significant.
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Results |
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RA patients with high ESR (28 mm/h) and low ESR (<28 mm/h) were compared to determine whether ESR was associated with Fc
R expression levels (Fig. 2B
). Fc
RI expression levels were not statistically higher in patients with high ESR (mean MFI±S.D., 61.9±29) compared with patients with low ESR (50.9±27) and no differences with respect to control subjects (49.1±21) were found. The expression levels of Fc
RIIa were higher in patients with high ESR compared with RA patients with low ESR (+19%, P=0.03) and compared with healthy donors (+17%, P=0.04). Levels of Fc
RIIa in patients with low ESR were similar to those in healthy controls. The average expression level of Fc
RIIIa per cell was comparable for the high- and the low-ESR groups. However, RA patients with high ESR had more Fc
RIIIa-positive monocytes compared with patients with low ESR (+186%, P < 0.001) and compared with the control group (+300%, P=0.005). The percentage of Fc
RIIIa-positive monocytes was not significantly different between patients with low ESR and healthy individuals. For all RA patients, the expression level of Fc
RIIa (r=0.438, P=0.002) and the percentage of Fc
RIIIa-bearing monocytes (r=0.489, P=0.01) both correlated positively with ESR.
In addition, FcR expression levels were related to DMARD medication. Expression levels in DMARD-naive RA patients (n=9) were compared with those in RA patients receiving DMARD therapy (n=37) (Fig. 2C
). Fc
RI expression levels were not significantly higher in DMARD-naive RA patients (65.6±29.3) compared with RA patients using DMARDs (54.6±28.3) and compared with healthy subjects (49.1±21). Fc
RIIa expression levels were higher in DMARD-naive than in the DMARD-using RA patients (+20%, P=0.05) and higher than in healthy subjects (+26%, P=0.03); RA patients using DMARDs and healthy individuals had similar Fc
RIIa expression. The average intensity of Fc
RIIIa per cell was comparable for DMARD-naive and DMARD-using groups. The percentage of Fc
RIIIa-positive monocytes was equal in the two RA subpopulations. Both DMARD-naive patients (+277%, P=0.01) and RA patients using DMARDs (+232%, P<0.001) had more Fc
RIIIa-expressing monocytes compared with healthy controls.
FcR expression levels in peripheral blood vs synovial fluid
To investigate FcR expression levels on inflammatory synovial fluid macrophages, eight paired samples of peripheral blood and synovial fluid were analysed (Fig. 3
). No differences were observed for Fc
RI and Fc
RIIIa expression levels per cell (MFI). Fc
RIIa expression levels were lower (-51%, P=0.02) in synovial fluid than in peripheral blood. In contrast, 3.5 times more monocytic cells expressed Fc
RIIIa in synovial fluid than in peripheral blood (P=0.001).
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Incubation experiments
PBMC from healthy donors were incubated with 50% AB+ serum from healthy controls, RA serum or synovial fluid from RF-positive RA patients at 4°C for 30 min to analyse steric hindrance. The expression levels of FcRI, IIa and IIIa were similar for the three incubation conditions. Incubation at 37°C for 1 h to analyse the internalization of Fc
R also yielded similar data for the three incubation conditions. No statistically significant differences were observed, which indicates that immune complexes present in RA serum or synovial fluid do not interfere with the determination of Fc
R expression levels, either through steric hindrance or internalization (data not shown).
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Discussion |
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FcRIIa expression levels and the percentage of Fc
RIIIa-expressing monocytes were higher in RA patients with high ESR compared with RA patients with low ESR and healthy controls. Moreover, they correlated positively with the ESR. Although the same tendency was found for Fc
RI expression levels, this was not significant statistically. We have to keep in mind, however, that we analysed a relatively small, heterogeneous group of RA patients. The high percentage of circulating Fc
RIIIa-expressing monocytes has not been reported before in RA patients, but corresponds with the percentages found in infectious diseases [1618]. Higher expression levels of Fc
RI and Fc
RIIa in RA patients with active disease compared with healthy controls have been reported in previous studies [20, 21]. However, one of these studies only described patients with active disease, whereas we showed that patients with low ESR did not differ from controls. The other study showed no differences between RA patients with active disease and those in complete remission and suggested an intrinsic defect in Fc
R expression on monocytes of RA patients. Our results refute this suggestion because we found RA patients with a low ESR to be similar to healthy controls and different from those with a high ESR.
With respect to medication, our data showed higher expression levels of FcRIIa in DMARD-naive RA patients, while Fc
RIIa expression levels of DMARD-using RA patients were comparable to those of healthy controls. Fc
RI expression levels had the same tendency but this was also not statistically significant. The percentage of Fc
RIIIa-expressing monocytes, however, was high in all RA patients regardless of the use of DMARDs. It is important to note that, on average, the ESR values of DMARD-naive RA patients and DMARD-using RA patients were similar. The differences found cannot, therefore, be attributed to the ESR. Although the disease duration differed between the two groups, there was no association with disease duration and Fc
R expression levels. These data thus indicate a possible role for anti-rheumatic drugs in the modulation of Fc
RIIa (and perhaps Fc
RI). Methotrexate was the most frequently used drug in our RA population, but group numbers using the same therapy regimen in this heterogeneous population were too small; therefore no statements can be made about separate drugs. It has been demonstrated that high-dose glucocorticoid therapy in MS patients selectively depletes Fc
RIIIa-expressing blood monocytes [27]. Therefore, the effects of individual DMARDs on Fc
Rs deserve to be studied in greater detail.
We further investigated FcR expression on inflammatory RA synovial fluid macrophages compared with paired peripheral blood monocytes. In concordance with previous studies, expression levels of Fc
RI were similar in the two compartments [19, 20] and synovial fluid revealed 3.5 times as many Fc
RIIIa-expressing macrophages [28]. TGF-ß, abundantly present in synovial fluid, is able to induce Fc
RIIIa on monocytes and is held responsible for the high percentage of Fc
RIIIa-expressing macrophages in synovial fluid [28, 29]. Fc
RIIa expression on synovial fluid macrophages has not been studied previously. To our surprise we found lower Fc
RIIa expression levels in synovial fluid, whereas the level of Fc
RIIa on peripheral blood monocytes was related to disease activity. We excluded interference of immune complexes in synovial fluid with the staining procedures as a possible cause for the low expression. The decreased Fc
RIIa expression levels on synovial fluid monocytic cells may be attributed to the presence of IL-13 in RA synovial fluid, which has been shown to down-regulate the expression level of Fc
RIIa [30, 31].
The fact that the expression of FcRIIa and Fc
RIIIa is associated with a high ESR indicates a role for Fc
Rs in the disease process. Cytokines that are increased in RA, such as IFN-
and IL-10, can enhance the expression of Fc
Rs, and high Fc
R expression levels can lead to activation of monocytic cells and contribute to disease activity. This is supported by data from human and animal studies. Fc
RIIIa-positive blood monocytes, of which we found an increased percentage in RA peripheral blood, were found to exhibit features of tissue macrophages and were postulated to play a role in proinflammatory immune responses [32]. We reported that up-regulation of Fc
RI and Fc
RIIa expression levels on monocytes from RA patients, which were primed by IL-10, was associated with high cytokine production upon immune complex stimulation [33]. In mice, which naturally lack Fc
RIIa, induction of FcR
-chain deficiency blocks the function of Fc
RI and Fc
RIII and results in protection against antigen-induced-arthritis [34]. In experimental immune complex-mediated arthritis, Fc
R expression levels were related to the severity of synovial inflammation and cartilage destruction [35].
In summary, the above results show elevated expression of mainly the stimulatory receptors FcRIIa and IIIa on monocytes of RA patients, which was associated with active disease, on the basis of the high ESR levels. Binding of IgG-containing immune complexes to monocytes/macrophages leads to activation of these cells, and high expression levels may result in chronic inflammation and cartilage destruction. The fact that RA patients treated with anti-rheumatic drugs exhibited lower Fc
R expression levels suggests that Fc
R expression levels are susceptible to modulation by medication. Together, these data indicate that the expression levels of Fc
Rs are important factors in the persistent inflammatory response in RA.
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Acknowledgments |
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Notes |
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References |
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