Down-regulation of activating Fc{gamma} receptors on monocytes of patients with rheumatoid arthritis upon methotrexate treatment

S. Wijngaarden1, J. A. G. van Roon1,2, J. G. J. van de Winkel2,3, J. W. J. Bijlsma1 and F. P. J. G. Lafeber1

1 Rheumatology and Clinical Immunology, 2 Immunotherapy Laboratory, Department of Immunology, University Medical Centre Utrecht and 3 Genmab, P.O. Box 85500, 3508 GA Utrecht, The Netherlands.

Correspondence to: J. A. G. van Roon, Department of Rheumatology and Clinical Immunology, University Medical Centre Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: J.vanRoon{at}azu.nl


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. To determine the effect of methotrexate (MTX) on expression levels of activating receptors for IgG (Fc{gamma}Rs) on monocytes of rheumatoid arthritis (RA) patients in relation to changes in disease activity.

Methods. The effect of MTX on Fc{gamma}Rs on monocytes of RA patients was evaluated ex vivo as well as in vitro. Recently diagnosed, disease-modifying antirheumatic drug (DMARD)-naive RA patients were treated with low-dose MTX. At baseline and 16 weeks after the start of MTX treatment, changes in Fc{gamma}R expression levels on peripheral blood monocytes were evaluated by fluorescence-activated cell sorting analysis and were correlated to changes in disease parameters. To study the direct effects of MTX on monocytes, these cells were isolated from peripheral blood monocytes of healthy controls and cultured with MTX. Other monocyte surface molecules (CD40, CD80, CD86, MHC class II) were also determined to test the specificity of the effect on Fc{gamma}R expression levels.

Results. Eleven out of 15 patients improved clinically (mean disease activity score before 6.2 ± 0.8 vs 4.3 ± 1.7 after). Sixteen weeks after the start of MTX therapy, the expression levels of Fc{gamma}RI and IIa on monocytes were significantly decreased, whereas the decreases in Fc{gamma}RIIIa expression levels on monocytes were less marked. The percentage decrease in Fc{gamma}RI expression correlated with the percentage decrease in CRP and well-being. In vitro MTX selectively decreased Fc{gamma}RI and Fc{gamma}RIIa expression levels of isolated monocytes, in contrast to other surface molecules.

Conclusion. The disease-modifying effect of MTX in the treatment of RA is accompanied by down-regulation of activating Fc{gamma}RI and IIa on monocytes, which could be a direct effect of MTX on monocytes. This down-regulation represents a new mode of action of MTX which should be considered in RA patients, especially during conditions that could give rise to monocyte activation by IgG-containing immune complexes, e.g. during antibody-based therapy of RA.

KEY WORDS: Fc{gamma}, receptor, Methotrexate, Rheumatoid arthritis, Monocytes/macrophages


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Monocytes and macrophages are key players in the chronic inflammation of rheumatoid arthritis (RA). They produce proinflammatory mediators, such as interleukin-1ß (IL-1ß) and tumour necrosis factor {alpha} (TNF-{alpha}), which contribute to persistent synovial inflammation, finally resulting in cartilage and bone destruction [1, 2]. Immunoglobulin (Ig) G-containing immune complexes, e.g. rheumatoid factor (RF), abundantly present in RA serum and synovium, can cause powerful activation of monocytes and macrophages [3]. This activation is mediated by binding of immune complexes to IgG Fc receptors (Fc{gamma}Rs). Fc{gamma}Rs bind the Fc part of IgG, and thereby link the humoral and cellular branches of immunity. Triggering of Fc{gamma}Rs on monocytes initiates phagocytosis, antigen presentation, antibody-dependent cell-mediated cytotoxicity and the release of proinflammatory and tissue-destructive cytokines and enzymes, such as IL-1ß, TNF-{alpha} and matrix metalloproteinases. An important role of Fc{gamma}Rs in autoimmune diseases, including RA, has been indicated in numerous animal and human studies [4, 5].

Three classes of human leucocyte Fc{gamma}Rs, widely distributed on haematopoietic cells, have been identified: Fc{gamma}RI, II and III, with the isoforms IIa and IIb, and IIIa and IIIb. They differ in structure, cellular distribution, affinity for IgG and function. According to their function, Fc{gamma}Rs are divided into activating receptors (Fc{gamma}RI, IIa and IIIa) and inhibitory receptors (Fc{gamma}RIIb), with either an intracellular immunoreceptor tyrosine-based activation motif (ITAM) or inhibitory motif (ITIM) [4–6]. On the cell surface of the circulating blood monocytes in healthy controls Fc{gamma}RI and IIa are expressed constitutively, whereas Fc{gamma}RIIIb is absent and Fc{gamma}RIIIa is only present on a small percentage of monocytes (~10%) [7]. Due to the homology between Fc{gamma}RIIa and Fc{gamma}RIIb, antibodies recognizing only the extracellular part of Fc{gamma}RIIb have not been generated. Detection of Fc{gamma}RIIb on the cell surface of monocytes has not been documented. Alteration of expression levels of activating Fc{gamma}Rs alters immune complex-mediated effector functions, such as production of cytokines and catabolic enzymes [4–6]. Therefore studies on the regulation of these activating receptors in RA are important.

Previously, in a cross-sectional study in RA patients, we have shown that expression levels of activating Fc{gamma}Rs on peripheral blood monocytes of early, disease-modifying antirheumatic drug (DMARD)-naive RA patients are higher compared with RA patients with established RA using DMARDs. This suggested that expression levels of activating Fc{gamma}Rs are susceptible to modulation by DMARD therapy [8].

Low-dose methotrexate (MTX) is our first-choice DMARD in the treatment of RA [9]. Although its efficacy has been proven extensively, the exact mechanism of action is still unknown. Anti-inflammatory as well as immunomodulatory effects of MTX have been attributed to folate antagonism, resulting in impaired synthesis of DNA, RNA and proteins, inhibition of polyamine synthesis and increased release of adenosine [10, 11]. Several inhibitory effects of MTX on monocyte functions have been reported in vitro and in vivo. Inhibition of cytokine production by monocytes, such as IL-1ß, IL-6 and IL-8, and increased production of cytokine inhibitors, such as IL-1 receptor antagonist and soluble TNF receptor, have been demonstrated [11, 12].

Since regulation of expression levels of activating Fc{gamma}Rs may be essential in the regulation of monocyte/macrophage activity, we investigated whether expression levels of activating Fc{gamma}RI, IIa and IIIa on monocytes are modulated upon MTX treatment and whether this is related to clinical outcome. We therefore performed a longitudinal study examining recently diagnosed RA patients at baseline and 16 weeks after the initiation of MTX therapy. To gain insight into the mechanism of action of MTX, direct effects of MTX on the regulation of activating Fc{gamma}Rs on monocytes were studied in vitro.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Fifteen recently diagnosed DMARD- and corticosteroid-naive RA patients (11 females, four males) visiting our out-patient clinic were studied. RA was defined by the 1987 revised American College of Rheumatology (ACR) criteria [13]. Disease duration of all patients was less than 1 yr. The mean age (S.D.) was 58 ± 12) yr. Eight patients were RF-positive. All patients started oral treatment with low-dose MTX (7.5 mg weekly). If there was no clinical response, the MTX dose was increased monthly: by 7.5 mg after the first month and 5 mg in the months thereafter (mean 12.5 mg after 16 weeks). Other DMARDs were not allowed. The following clinical and laboratory parameters were assessed: tender and swollen joint scores, morning stiffness, well-being [visual analogue scale (VAS)], erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). At baseline and 16 weeks after MTX initiation venous blood samples were drawn for ex vivo analysis of Fc{gamma}R expression levels on monocytes. Clinical response was defined as 20% improvement in the Disease Activity Score (DAS) [14]. Approval to use the blood samples mentioned above was given by the UMC Utrecht Medical Ethical Committee. Each patient gave informed consent.

Isolation of cells
Venous blood of RA patients and healthy controls was diluted 1:1 with RPMI 1640 (Gibco Invitrogen, Life Technologies, UK) supplemented with 1% penicillin, streptomycin sulphate and glutamine. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Ficoll Paque (Pharmacia Biotech, Uppsala, Sweden).

For in vitro analysis of MTX, monocytes were isolated from PBMC of healthy donors by depletion of non-monocytes. In short, T cells, NK cells, B cells, dendritic cells and basophils were depleted using a monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing a cocktail of hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies. Anti-hapten magnetic microbeads were added and the magnetically labelled cells were depleted by retaining them on a MACS LS+ magnetic column using a MidiMACS magnet (Miltenyi). The negative effluent, containing untouched monocytes, was checked for cell purity by flow cytometry and was always over 95%.

Antibodies
For analysis of Fc{gamma}R expression levels on monocytes by fluorescence-activated cell sorting (FACS), the following fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies were used in combination with phycoerythrin (PE)-conjugated CD14 (Tük4; Dako, Denmark): anti-Fc{gamma}RI (CD64; 32.2), anti-Fc{gamma}RIIa (CD32; IV.3) and anti-Fc{gamma}RIII (CD16; 3G8) (all from Medarex, Annandale, NJ, USA). The isoforms Fc{gamma}RIIa and IIb are extracellularly 92% identical, but differ intracellularly [15]. The CD32 monoclonal antibody IV.3 is directed against Fc{gamma}RIIa and does not stain Fc{gamma}RIIb [8, 16]. Fc{gamma}RIIb has only been detected on mRNA and intracellular protein level but is not yet identified on the cell surface of monocytes. Discrimination between Fc{gamma}RIIIa and Fc{gamma}RIIIb is not possible using the CD16 monoclonal antibody 3G8. Fc{gamma}RIIIb, however, is solely expressed on polymorphonuclear cells, which are not present in the mononuclear cell fraction used in our study.

To check the purity of the isolated CD14+ cells after MACS isolation, the following antibodies were used to determine the percentage of positive cells (by flow cytometry): PE-conjugated CD8 (HIT8A), FITC-conjugated CD19 (HD37; Dako, Glostrup, Denmark), phycoerythrin-cyanin S.1 (PECY5)-conjugated CD4 (MT310; Dako), FITC-conjugated CD14 (RMO52) PE-conjugated CD45 (MMU19.2) (Immunotech, Marseille, France).

To measure expression of surface markers upon in vitro culture of monocytes, in addition to the antibodies mentioned above, the following monoclonal antibodies were used: CD40 (5C3), CD80 (L307.4), CD86 (IT2.2) (all from BD, Pharmingen, San Diego, CA, USA) and HLAII (Tü39; BD Pharmingen). For all of the above stainings, isotype-matched control pairs (FITC/PE; Immunotech, Marseille, France) were used.

FACS analysis
PBMC or monocytes were incubated for 30 min at 4°C with monoclonal antibodies, then washed in phosphate-buffered saline containing 0.1% sodium azide and suspended in the same buffer, now containing 2% paraformaldehyde (PFA). Flow cytometry was performed on a FACScan (Becton Dickinson) and analysed using CellQuest software. For analysis of monocytes, gates were set around viable monocytes, based on their forward/sideward light scatter pattern and CD14 expression. With respect to Fc{gamma}R expression levels, Fc{gamma}RI, IIa and IIIa were determined as geometrical mean fluorescence intensity (MFI) on CD14+ cells. Since not all CD14+ cells are Fc{gamma}RIIIa+, Fc{gamma}RIIIa was also determined as the percentage of Fc{gamma}RIIIa+ monocytes.

In vitro incubation of monocytes with methotrexate
To study direct effects of MTX on monocytes, freshly isolated monocytes of healthy controls (n = 6) were cultured in the absence or presence of 10–6 M Methotrexatum (Proderma, Hilversum, The Netherlands) for 4 days. This concentration of MTX, used in vitro, corresponds with the average concentration of MTX found in serum upon treatment [17]. Monocytes were cultured in flat-bottom 24-well microtitre plates at a concentration of 5 x 105 cells per well in a total volume of 1 ml RPMI 1640 (Gibco, UK) supplemented with 1% penicillin, streptomycin sulphate, glutamine and 10% human heat-inactivated pooled AB+ serum (Red Cross Blood Transfusion Center, Utrecht, The Netherlands). Following culture, monocytes were harvested on ice and prepared for flow cytometry as described above. Apart from staining for Fc{gamma}RI, IIa and IIIa, cells were also stained with the following monoclonal antibodies: CD40, CD80, CD86 and MHC class II.

Viability of monocytes after culture was verified with Trypan blue solution 0.4% (Sigma-Aldrich, Irvine, UK) and was always more than 95%. No differences in viability between cultures with or without MTX were observed.

Statistical analysis
We tested for normal distribution by skewness (not exceeding 1 or –1) and using the Shapiro–Wilk test (testing for normal distribution under 50 samples). According to this test, non-parametric tests were used for ex vivo data. The Wilcoxon signed ranks test was used to compare Fc{gamma}R expression levels and clinical parameters before and after the start of MTX therapy. The Mann–Whitney U-test was used for comparison of Fc{gamma}R expression levels and clinical parameters of responders vs non-responders. Correlations were evaluated with Spearman correlation analysis. A parametric test was used for in vitro data. The paired-samples t-test compared Fc{gamma}R expression levels of paired blood samples cultured with or without MTX. A value of P ≤ 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical responses
Table 1 shows the clinical features of RA patients before and 16 weeks after starting MTX therapy. Based on a 20% improvement of DAS score, 11 patients (73%) improved clinically (responders), whereas four patients (27%) were non-responders. There were no statistically significant differences between responders and non-responders regarding age, sex, RF positivity or baseline clinical parameters.


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TABLE 1. Disease characteristics and Fc{gamma}R expression levels before and 16 weeks after starting MTX therapy

 
Down-regulation of Fc{gamma}RI and IIa expression levels on PBMC upon MTX treatment
The average values of Fc{gamma}R expression levels of all patients are given in Table 1. Representative histograms of activating Fc{gamma}R expression levels on monocytes of one RA patient before and after MTX are shown in Fig. 1. Sixteen weeks after initiating MTX therapy, Fc{gamma}RI and IIa expression levels on PBMC were decreased compared with baseline levels (Table 1 and Fig. 2). The mean decrease in Fc{gamma}RI expression level was –17 (33)% (from median MFI 53.0 at baseline to MFI 50.5; P = 0.03) (Fig. 2A). Fc{gamma}RI expression levels decreased in 11 of 15 patients, whereas in four patients levels increased. On average, MTX treatment decreased Fc{gamma}RIIa expression levels by –26 (20)% (from MFI 161.2 at baseline to MFI 128.0 upon MTX treatment; P = 0.003). This decrease was observed in 14 out of 15 patients (Fig. 2B). The MFI of Fc{gamma}RIIIa was not significantly changed after 16 weeks (Fig. 2C). However, the percentage Fc{gamma}RIIIa+ monocytes in patients on average decreased by 29% (from median values of 18.2% at baseline to 17.2% upon MTX treatment; P = 0.057), significantly in those patients who had more than 20% Fc{gamma}RIIIa+ monocytes at baseline. The latter patients all had fewer circulating Fc{gamma}RIIIa+ monocytes after 16 weeks of treatment (n = 7; from 34.0% at baseline to 16.4% after MTX treatment), while the percentage of Fc{gamma}RIIIa+ monocytes in patients with baseline levels below 20% was not significantly altered (median at baseline 16.4 %; after 16 weeks 20.0%).



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FIG. 1. Representative histograms of ex vivo expression levels of activating Fc{gamma}Rs on PBMC at baseline and 16 weeks after starting MTX therapy. PBMC were stained for Fc{gamma}RI (CD64), Fc{gamma}RIIa (CD32) or Fc{gamma}RIIIa (CD16) together with CD14 (monocytes). The mean fluorescence intensities (MFI) of Fc{gamma}RI (A), IIa (B) and IIIa (C) on CD14+ cells are given. The percentages of Fc{gamma}RIIIa+CD14+ cells, indicated by the marker line M1, which was set on the basis of the isotype control, are also given (C). The filled histograms show levels at baseline, while the open histograms represent Fc{gamma}R expression levels after 16 weeks of MTX therapy. An isotype-matched control is depicted as a dotted line in all panels. The MFI is indicated for Fc{gamma}R expression levels before and after treatment with MTX.

 


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FIG. 2. Ex vivo expression levels of activating Fc{gamma}Rs on PBMC of individual RA patients at baseline and 16 weeks after starting MTX therapy. PBMC of RA patients (n = 15) were analysed by flow cytometry for Fc{gamma}RI, IIa and IIIa expression levels on CD14+ cells. The mean fluorescence intensities (MFI; median) are given for (A) Fc{gamma}RI (baseline 53.0; 16 weeks 50.5), (B) IIa (baseline 161.2; 16 weeks 128.0) and (C) IIIa (baseline 59.5; 16 weeks 67.6) on CD14+ cells, and (D) the percentage of Fc{gamma}RIIIa+CD14+ cells (baseline 18.2; 16 weeks 17.2). P values for differences between average Fc{gamma}R expression levels at baseline and 16 weeks, calculated using the Wilcoxon signed ranks test, are also given.

 
Changes in activating Fc{gamma}RI expression levels correlated with changes in clinical parameters. The percentage decrease in Fc{gamma}RI upon MTX therapy correlated strongly with the percentage decrease in CRP levels ({rho} = 0.802; P<0.001; Fig. 3, upper panel, n = 15) and the percentage decrease in well-being (VAS) ({rho} = 0.79; P = 0.001; Fig. 3, lower panel, n = 14, one missing value). Changes in Fc{gamma}RI expression levels did not correlate significantly with other disease parameters (DAS, ESR, swollen joints, tender joints and morning stiffness). Furthermore, in this relatively small group, no significant correlations of changes in Fc{gamma}RIIa and Fc{gamma}RIIIa with changes in disease parameters were found (data not shown). Clinical responders and non-responders had similar Fc{gamma}R expression levels at baseline.



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FIG. 3. The decrease in percentage change in Fc{gamma}RI expression levels (at week 16 compared with baseline) on monocytes from RA patients correlates with a decrease in percentage change in CRP levels (upper panel, n = 15) and well-being (n = 14, one missing value, lower panel) using a visual analogue scale (both at week 16 compared with baseline). The Spearman correlation coefficients ({rho}) and P values are given.

 
MTX down-regulates Fc{gamma}RI and IIa expression levels on monocytes in vitro
To determine whether the observed effects of MTX on monocytic Fc{gamma}R expression in vivo were mediated by a direct effect of MTX, isolated monocytes of healthy controls were cultured in the presence of MTX. In this way the influence on monocytes of cell–cell interactions with other inflammatory cells (e.g. T cells) and RA-specific inflammatory mediators were excluded. After culture for 4 days under control conditions, all Fc{gamma}Rs on monocytes are up-regulated and all monocytes become Fc{gamma}RIIIa-positive (data not shown). Monocytes cultured in the presence of MTX (10–6 M) displayed lower expression levels of Fc{gamma}RI and IIa, whereas Fc{gamma}RIIIa expression levels were not significantly decreased by MTX (Fig. 4). To investigate if these direct effects of MTX on Fc{gamma}Rs were distinct from other surface markers, several surface molecules on monocytes were determined as well (all n = 6). The expression levels of CD40 [MFI before 46.9 (19.3), after 45.1 (17.6)], CD80 [MFI before 45.6 (11.4), after 44.6 (10.6)], CD86 [MFI before 778.6 (206.5), after 904.1 (218.1)] and MHC class II [MFI before 2835 (669), after 2980 (781)] were not significantly changed by MTX.



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FIG. 4. MTX reduces Fc{gamma}RI, IIa and IIIa expression levels on monocytes in vitro. Monocytes of healthy controls (n = 6) were cultured for 4 days in the absence or presence of MTX (10–6 M). Fc{gamma}R expression levels were determined by flow cytometry. The relative mean fluorescence intensity (MFI) of Fc{gamma}RI (CD64), Fc{gamma}RIIa (CD32) and Fc{gamma}RIIIa (CD16) on monocytes cultured in the presence of MTX are given (shaded bars) compared with cultures without MTX (100%, black bar). Asterisks indicate statistically significant differences (P<0.05, calculated using a paired samples t-test) between cultures with and without MTX.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Treatment of RA patients with MTX is accompanied by distinct down-regulation of Fc{gamma}RI and IIa expression levels on PBMC compared with other monocytic surface molecules. Although on average Fc{gamma}RIIIa expression levels and the percentage of Fc{gamma}RIIIa+ monocytes were not changed statistically significantly upon MTX treatment, a tendency towards a decrease was observed. The present study strengthens recently published data from a cross-sectional study suggesting that DMARD therapy decreases Fc{gamma}RI and Fc{gamma}RIIa expression levels in RA patients [8]. Although in that cross-sectional study MTX was the most frequently used DMARD (68% of the patients), no reliable conclusions could be drawn with respect to the effects of MTX, due to co-medication and heterogeneity of patients.

In vivo it is difficult to test whether down-regulation of Fc{gamma}RI and IIa is indirectly caused by clinical improvement or by a direct effect of MTX on monocytes. Down-regulation of Fc{gamma}RI and IIa on monocytes by MTX might be the result of diminished disease activity or could contribute to clinical improvement, since previously these have been shown to correlate with parameters of disease activity [8]. Our in vitro studies showed lower Fc{gamma}RI and IIa expression levels (and to a lesser extent Fc{gamma}RIIIa) on isolated monocytes cultured with MTX, while other monocyte surface markers were not significantly changed by MTX. These data indicate a clear direct effect of MTX on the expression of activating Fc{gamma}Rs on monocytes. In vivo, down-regulation of activating Fc{gamma}Rs is therefore expected to be at least a partial direct effect of MTX on monocytes, although an indirect effect by other intermediates or as a result of clinical improvement cannot be ruled out.

An in vitro study by Seitz et al. demonstrated, as in our in vitro study, no statistically significant effect of MTX on Fc{gamma}RIIIa expression levels. However, in contrast to our results, also no effect of MTX on Fc{gamma}RI expression was observed [18]. In this latter study, PBMC were cultured with lower concentrations of MTX, while we used isolated monocytes. These methodological differences in culture conditions might account for the difference in results. In line with this assumption, at lower MTX concentrations (10–7 M) we observed no significant decreases in monocytic Fc{gamma}R expression levels (data not shown).

Seventy-three per cent of the RA patients responded to MTX treatment, based on 20% improvement in DAS scores. The percentage change in Fc{gamma}RI expression levels upon MTX treatment correlated strongly with the percentage change in disease parameters such as CRP and VAS well-being. The decreased levels of CRP could be the result of decreased monocyte activation by immune complexes present in RA patients due to down-regulated Fc{gamma}RI expression on monocytes. Decreased immune complex stimulation via Fc{gamma}RI can lead to reduced production of proinflammatory cytokines, such as IL1-ß, TNF-{alpha} and IL-6, which can induce hepatic production of CRP [6]. The relationship between Fc{gamma}RI expression on monocytes and CRP, which we and others have demonstrated previously, supports this notion [19]. Data showing that RA patients responding to MTX exhibit decreased serum levels of IL-6 are also in line with this suggestion [20, 21]. The lack of a significant correlation between the changes in (low-affinity) Fc{gamma}RIIa and the changes in clinical parameters may be due to the contribution of this Fc{gamma}R to macrophage activation and consequently disease activity. This may differ from Fc{gamma}RI, the high affinity Fc{gamma}R, the expression of which may lead to greater changes in macrophage activation and disease activity in response to immune complexes. In addition, there is substantial variation in Fc{gamma}R expression levels and disease activity as well as variation in responsiveness to MTX in individual patients, both in vitro and in vivo. In the relatively small population of patients that we have studied this could explain the lack of correlation between changes in Fc{gamma}RIIa and changes in disease activity, in contrast to Fc{gamma}RI. Considering the contribution of Fc{gamma}RIIa in triggering macrophages, we feel that suppression of Fc{gamma}RIIa (in our study ~30%) plays a role in prevention of macrophage activation.

Down-regulation of the activating Fc{gamma}RI and IIa upon MTX treatment is thus suggested to contribute to clinical improvement. We previously indicated that failure of human recombinant IL-10 treatment in RA patients, an otherwise anti-inflammatory cytokine, might be due to up-regulation of Fc{gamma}RI and IIa on monocytes. This was demonstrated ex vivo as well as in vitro. The in vitro up-regulation of Fc{gamma}RI and IIa by IL-10 resulted in higher immune-complex-induced TNF-{alpha} production [22]. This supports the assumption that a decrease in activating Fc{gamma}R expression levels upon treatment will result in decreased production of proinflammatory cytokines upon IgG-containing immune complex stimulation.

A dominant role of Fc{gamma}RIIIa in the pathogenesis of RA has been suggested, being up-regulated in active RA and present on approximately 60% of synovial fluid macrophages [8, 23]. Fc{gamma}RIIIa+ monocytes have been shown to possess features of proinflammatory tissue macrophages [24] and triggering of Fc{gamma}RIIIa with small immune complexes induced TNF-{alpha} and TNF-{alpha}-dependent IL-1 release by human monocytes [25]. The percentage Fc{gamma}RIIIa+ monocytes was not decreased statistically significantly upon MTX treatment. Although the percentage of Fc{gamma}RIIIa+ monocytes declined in RA patients who had high numbers (more than 20%) of circulating Fc{gamma}RIIIa+ monocytes at baseline, they never reached values seen in healthy controls (~10%) and the reduction was not related to changes in disease activity. On the contrary, another study revealed a decline in Fc{gamma}RIIIa+ monocytes correlating to a decrease in CRP levels, upon combination therapy of low-dose prednisolone in combination with MTX or sulphasalazine [26]. In addition, selective depletion of Fc{gamma}RIIIa+ monocytes has also been reported upon high-dose glucocorticoid therapy in patients treated for multiple sclerosis [27]. These data suggest that perhaps co-therapy using MTX with glucocorticoids or other drugs is required to normalize the percentage of Fc{gamma}RIIIa+ monocytes. Neither study allows a comparison of Fc{gamma}RIIIa expression levels relative to changes in other Fc{gamma}Rs since only Fc{gamma}RIIIa was studied.

The role of individual Fc{gamma}Rs in the initiation, perpetuation and joint destruction in RA needs to be further elucidated. It is postulated, however, that in these patients the balance of activating and inhibitory Fc{gamma}Rs is of importance. Co-ligation of immune complexes to inhibitory and activating Fc{gamma}Rs inhibits the activating signals. In this way a threshold is set for immune complex stimulation, determining the final effector cell response and consequently their state of activation [28, 29].

We demonstrated that MTX down-regulates activating Fc{gamma}RI and IIa expression levels, which may thus prevent activation of monocytes/macrophages via immune complexes. This differs from biological agents (e.g. anti-TNF-{alpha} and anti-IL-1) designed to block proinflammatory mediators produced by activated monocytes/macrophages. Therefore, our data underline the importance of Fc{gamma}Rs in the pathogenesis of RA and suggest down-regulation of activating Fc{gamma}Rs to be of additional value in the treatment of RA. This should be considered particularly in antibody-based treatments, where immune-complex-mediated activation of monocytes can become disadvantageous.


    Acknowledgments
 
This work was financially supported by the Dutch Arthritis Association (Nationaal Reumafonds).

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 13 February 2004; revised version accepted 26 January 2005.