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
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Abstract |
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Methods. The effect of MTX on FcRs 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
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
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 FcRI and IIa on monocytes were significantly decreased, whereas the decreases in Fc
RIIIa expression levels on monocytes were less marked. The percentage decrease in Fc
RI expression correlated with the percentage decrease in CRP and well-being. In vitro MTX selectively decreased Fc
RI and Fc
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 FcRI 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, receptor, Methotrexate, Rheumatoid arthritis, Monocytes/macrophages
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Introduction |
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Three classes of human leucocyte FcRs, widely distributed on haematopoietic cells, have been identified: Fc
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
Rs are divided into activating receptors (Fc
RI, IIa and IIIa) and inhibitory receptors (Fc
RIIb), with either an intracellular immunoreceptor tyrosine-based activation motif (ITAM) or inhibitory motif (ITIM) [46]. On the cell surface of the circulating blood monocytes in healthy controls Fc
RI and IIa are expressed constitutively, whereas Fc
RIIIb is absent and Fc
RIIIa is only present on a small percentage of monocytes (
10%) [7]. Due to the homology between Fc
RIIa and Fc
RIIb, antibodies recognizing only the extracellular part of Fc
RIIb have not been generated. Detection of Fc
RIIb on the cell surface of monocytes has not been documented. Alteration of expression levels of activating Fc
Rs alters immune complex-mediated effector functions, such as production of cytokines and catabolic enzymes [46]. 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 FcRs 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
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 FcRs may be essential in the regulation of monocyte/macrophage activity, we investigated whether expression levels of activating Fc
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
Rs on monocytes were studied in vitro.
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Patients and methods |
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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 FcR 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
RI (CD64; 32.2), anti-Fc
RIIa (CD32; IV.3) and anti-Fc
RIII (CD16; 3G8) (all from Medarex, Annandale, NJ, USA). The isoforms Fc
RIIa and IIb are extracellularly 92% identical, but differ intracellularly [15]. The CD32 monoclonal antibody IV.3 is directed against Fc
RIIa and does not stain Fc
RIIb [8, 16]. Fc
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
RIIIa and Fc
RIIIb is not possible using the CD16 monoclonal antibody 3G8. Fc
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 FcR expression levels, Fc
RI, IIa and IIIa were determined as geometrical mean fluorescence intensity (MFI) on CD14+ cells. Since not all CD14+ cells are Fc
RIIIa+, Fc
RIIIa was also determined as the percentage of Fc
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 106 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 FcRI, 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 ShapiroWilk 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 FcR expression levels and clinical parameters before and after the start of MTX therapy. The MannWhitney U-test was used for comparison of Fc
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
R expression levels of paired blood samples cultured with or without MTX. A value of P
0.05 was considered statistically significant.
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Results |
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Discussion |
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In vivo it is difficult to test whether down-regulation of FcRI and IIa is indirectly caused by clinical improvement or by a direct effect of MTX on monocytes. Down-regulation of Fc
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
RI and IIa expression levels (and to a lesser extent Fc
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
Rs on monocytes. In vivo, down-regulation of activating Fc
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 FcRIIIa expression levels. However, in contrast to our results, also no effect of MTX on Fc
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 (107 M) we observed no significant decreases in monocytic Fc
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 FcRI 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
RI expression on monocytes. Decreased immune complex stimulation via Fc
RI can lead to reduced production of proinflammatory cytokines, such as IL1-ß, TNF-
and IL-6, which can induce hepatic production of CRP [6]. The relationship between Fc
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
RIIa and the changes in clinical parameters may be due to the contribution of this Fc
R to macrophage activation and consequently disease activity. This may differ from Fc
RI, the high affinity Fc
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
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
RIIa and changes in disease activity, in contrast to Fc
RI. Considering the contribution of Fc
RIIa in triggering macrophages, we feel that suppression of Fc
RIIa (in our study
30%) plays a role in prevention of macrophage activation.
Down-regulation of the activating FcRI 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
RI and IIa on monocytes. This was demonstrated ex vivo as well as in vitro. The in vitro up-regulation of Fc
RI and IIa by IL-10 resulted in higher immune-complex-induced TNF-
production [22]. This supports the assumption that a decrease in activating Fc
R expression levels upon treatment will result in decreased production of proinflammatory cytokines upon IgG-containing immune complex stimulation.
A dominant role of FcRIIIa 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
RIIIa+ monocytes have been shown to possess features of proinflammatory tissue macrophages [24] and triggering of Fc
RIIIa with small immune complexes induced TNF-
and TNF-
-dependent IL-1 release by human monocytes [25]. The percentage Fc
RIIIa+ monocytes was not decreased statistically significantly upon MTX treatment. Although the percentage of Fc
RIIIa+ monocytes declined in RA patients who had high numbers (more than 20%) of circulating Fc
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
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
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
RIIIa+ monocytes. Neither study allows a comparison of Fc
RIIIa expression levels relative to changes in other Fc
Rs since only Fc
RIIIa was studied.
The role of individual FcRs 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
Rs is of importance. Co-ligation of immune complexes to inhibitory and activating Fc
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 FcRI and IIa expression levels, which may thus prevent activation of monocytes/macrophages via immune complexes. This differs from biological agents (e.g. anti-TNF-
and anti-IL-1) designed to block proinflammatory mediators produced by activated monocytes/macrophages. Therefore, our data underline the importance of Fc
Rs in the pathogenesis of RA and suggest down-regulation of activating Fc
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.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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