1 Department of Medicine, Division of Nephrology and Rheumatology, Klinikum Benjamin Franklin, Free University, Berlin and
2 Deutsches Rheuma Forschungszentrum Berlin, Berlin, Germany
Correspondence to:
J. Sieper, Department of Medicine, Rheumatology, Klinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany.
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Abstract |
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Methods.Eighteen patients with RA and 17 with ReA were studied. For intracellular staining of cytokines, SF mononuclear cells (MNC) from seven patients with RA, in comparison to eight patients with ReA, were triple stained with anti-IFN-, IL-4 and anti-CD4 or anti-CD8 monoclonal antibodies (mAb) and analysed by flow cytometry. Furthermore, in 13 patients with RA, immunohistology of SM was performed and compared with seven ReA patients. In addition, in six of the RA patients, synovial T cells were grown over 3 weeks in the presence of various cytokines and intracellular cytokine staining analysed by flow cytometry weekly.
Results.In SF, the mean percentage of IFN-+/CD4+ T cells in RA was almost 4-fold higher than the number of IL-4+/CD4+ T cells (11.3±5 vs 3.02±1.04; P=0.0012), while the ratio of IFN-
/IL-4+ CD4+ T cells was only 1.59 in ReA (P=0.047 for the ratio difference). A similar result was obtained for SM: the ratio of IFN-
/IL-4+ cells in RA was 4.3 (P<0.0001 for the IFN-
/IL-4 difference), but only 1.2 for ReA (P=0.02 for the ratio difference). Of the CD3+ cells in SM, 2.8% were positive for IFN-
and 0.4% for IL-4 in three RA patients. A decrease in the number of IFN-
-positive SF T cells and an increase in the number of IL-4-positive SF T cells could be achieved in vitro through IL-4, but not by IL-10 or transforming growth factor beta.
Conclusions.The Th1 pattern in the joint of RA patients demonstrated at the single-cell level may be important for the pathogenesis of RA and may provide a target for future immunotherapy. Our data suggest a therapeutic role for IL-4.
KEY WORDS: Th1 cytokines, Rheumatoid arthritis, Intracellular cytokines, Cytokine manipulation
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Introduction |
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The balance of T-helper type (Th) 1 and Th2 subsets is implicated in the regulation of many immune responses [3]. The Th1-type cytokines interferon gamma (IFN-) and TNF-
are required for response against intracellular infections, while Th2 cells (secreting IL-4, IL-5 and IL-10) are responsible for protection against extracellular ones. Th1 cytokines have also been implicated in the pathogenesis of autoimmune diseases and/or their animal models, such as multiple sclerosis [4], diabetes mellitus [5] and RA [6] in which a T-cell response against an unknown self-antigen may play a role. In contrast, the Th2-like cytokines IL-4 and IL-10 downregulate inflammation in these models [7].
Previously, we have demonstrated that in the synovium of RA patients IL-4-positive cells could be detected in fewer patients than in reactive arthritis (ReA), suggesting a Th1-like pattern in RA synovium and a Th0-like pattern in ReA synovium [8]. Provided that Th1 cells play an important role in the pathogenesis of RA, a change of this pattern would be a therapeutic option. However, although the Th1 pattern has been successfully manipulated in animal models [9], no such manipulations have been tried in RA patients.
In the present study, we have quantitatively analysed the cytokine pattern at the single-cell level in synovial fluid (SF) T cells by intracellular cytokine staining and flow cytometry, and in synovial membrane (SM) by immunohistology. We present further evidence that in the affected tissue and fluid of RA patients a high ratio of IFN-/IL-4-positive cells is present. We went on to investigate the possibility of manipulating the pattern in vitro through treatment with cytokines.
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Patients and methods |
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Cell cultures
SF MNC were separated by Ficoll-paque (Pharmacia, Uppsala, Sweden) density centrifugation as previously described and subsequently cultured with medium RPMI 1640 (GIBCOBRL, Life Technologies, Paisley, UK) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Biochrom KG, Berlin, Germany), 2 mM L-glutamine (Biochrom KG, Berlin, Germany) and 10% heat-inactivated fetal calf serum (FCS) (GIBCOBRL, Life Technologies, Paisley, UK) [12].
Intracellular cytokine staining and analysis by flow cytometry
The staining method for intracellular cytokines has been developed with modification from a technique described in the literature [13]. SF MNC (1x106 /ml) were stimulated with plate-bound anti-CD3 (UCTH, Pharmingen, San Diego, CA, USA; 10 µg/ml) plus soluble anti-CD28 (Immunotech, Hamburg, Germany; 1 µg/ml). After 4 h of activation, 2.5 µM monensin (Sigma, St Louis, MO, USA) was added and the cells were cultured for an additional 2 h. After these 6 h of activation, cells were then washed in phosphate-buffered saline (PBS), fixed for 20 min with PBS containing 4% formaldehyde (Merck, Darmstadt, Germany), washed twice with PBS, and permeabilized by incubation in PBS containing 0.5% saponin (Sigma, St Louis, MO, USA), 1% inactivated FCS and 0.1% sodium azide (permeabilizing buffer) for 10 min. All incubation steps were carried out at room temperature. For triple staining, cells were first stained with cy-chrome-labelled anti-CD4 or anti-CD8 monoclonal antibodies (mAb) (Pharmingen, San Diego, CA, USA) in PBS containing 1% inactivated FCS and 0.1% sodium azide (staining buffer) at room temperature for 20 min. After washing twice with staining buffer, cells were then fixed and permeabilized as described above. Permeabilized cells were incubated with phycoerythrin (PE)-labelled anti-human IL-4 mAb and fluorescein isothiocyanate (FITC)-labelled anti-human IFN- mAb (Pharmingen) in permeabilizing buffer. PE- or FITC-labelled isotype-matched mAb (Pharmingen) were used as a negative control. After 20 min incubation at room temperature, cells were washed twice with the permeabilizing buffer and further washed with staining buffer twice, resuspended in the staining buffer and analysed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). After gating either on all lymphocytes, or the CD4+ or the CD8+ populations, data were analysed using CELLQuest software and displayed as dot plots of FITC (x-axis) and PE (y-axis) fluorescence (four decade log scales). Quadrant markers were positioned to include >99% of control Ig staining cells in the lower left quadrant. The percentage of cytokine-producing cells within the gated population was measured in the upper left for IL-4 and in the lower right quadrants for IFN-
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Manipulation of synovial fluid T-cell cytokine pattern in vitro
SF MNC from RA patients were stimulated with coated anti-CD3 and soluble anti-CD28 as described above for short-term stimulation, in the absence or presence of the following recombinant human cytokines: IL-4, IL-10, IL-4+ IL-10, IL-12, transforming growth factor beta (TGF-ß). IL-2 (100 U/ml) was added to the culture at day 3. The stimulation was repeated weekly under the same condition at least for 3 weeks consecutively. To measure intracellular cytokine production, cells taken from these cell lines were stimulated at the end of each week with PMA (5 ng/ml) and ionomycin (1 ng/ml) for 4 h at 37°C, and monensin was added for the last 2 h of culture. The cells were fixed and stained for CD4 and CD8 surface markers and for intracellular cytokines using FITC- or PE-conjugated mAb specific for IFN-, IL-4, IL-10 and TNF-
as described above. The concentration was 5 ng/ml for IL-12 and 50 ng/ml for the other cytokines in all experiments.
Immunohistology: single staining for CD3, IFN- and IL-4
Immunohistology was performed on sections from SM of 12 patients with RA and six patients with ReA as described [14]. The following primary mAb were used: murine anti-human CD3 (UCHT1, IgG1, Dako, Glostrup, Denmark), murine anti-human IFN- (B-B1, IgG1, Biosource-International, Camarillo, CA, USA) and murine anti-human IL-4 (IgG1, Genzyme Diagnostics, Cambridge, MA, USA). For the staining, the primary antibody was detected with affinity-purified goat anti-mouse IgG antibodies (Dako) followed by APAAP complex (Dako), and visualized with the chromogen New Fuchsin (Merck, Darmstadt, Germany).
Immunohistology: double staining for CD3/IFN- and CD3/IL-4
Double staining for CD3/IFN- and CD3/IL-4 was performed in three RA patients (patients 9, 11 and 13) using immunofluorescence. Fixed sections were incubated with mouse anti-human IFN-
(dilution of 1:50; IgG1, Pharmingen) or with rat anti-human IL-4 (dilution 1:50; IgG1, Pharmingen), subsequently incubated with digoxigenin-N-hydroxysuccinimidester-conjugated sheep IgG [F(ab)2 fragment] against mouse Ig for IFN-
detection or with a biotin-SP-conjugated F(ab)2 fragment donkey anti-rat IgG (H+L) (Dianova, Hamburg, Germany) for IL-4 detection. Sections were then incubated with an anti-digoxigenin-fluorochrome-antibody (CyTM3-conjugated IgG fraction of mouse anti-digoxin) (Dianova) for IFN-
detection and with CyTM2-conjugated streptavidin (Dianova) for IL-4. For CD3/IFN-
double labelling, cells were then incubated with rabbit anti-human CD3 (dilution 1:100; Dako, Glostrup, Denmark) followed by incubation with a biotin-SP-conjugated donkey anti-rabbit IgG (H+L) (Dianova), followed by incubation with Cy2-conjugated streptavidin (dilution 1:200) (Dianova). For CD3/IL-4 double labelling, sections were incubated with mouse anti-human CD3 (dilution 1:100; Dako), followed by incubation with sheep IgG anti-mouse Ig conjugated to digoxigenin-N-hydroxysuccinimidester (Boehringer Mannheim, Germany), followed by incubation with CyTM3-conjugated IgG mouse anti-digoxin (Dianova, Denmark). All incubations were performed for 60 min at room temperature and for the fluochrome-conjugated antibodies in the dark. Between and after incubations, slides were washed twice with PBS for 5 min. All dilutions were made in 3% bovine serum albumin (BSA)/PBS.
Finally, cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) (Sigma, St Louis, MO, USA) for 10 min at room temperature in the dark and the sections embedded in UV-inert medium (Fuoromount-G; Bioproducts, Boehringer Ingelheim, Germany) for microscopy.
Counting of positively stained cells in synovial membrane
Cytokine-positive cells were counted in the lining and the sublining layers of synovium as described [15]. All sections were examined under x400 magnification with a 1 mm graticule. For each analysis, 13 tissue samples from each patient were examined. The mean number of positive cells in the lining and sublining layer per high-power field over the entire area of three sections in each patient was recorded. The mean count per square millimetre was calculated using a conversion factor (x0.0625-1), and the number of IFN-- and IL-4- positive cells for each patient and, in the case of double labelling, the numbers of CD3+/cytokine+ double-positive and CD3-/cytokine+ single-positive cells are presented. CD3- and cytokine-positive cells were counted first using different filters. Double-positive cells were identified subsequently by using a `combined' filter.
Statistics
Student's t-test was used to compare the mean (±S.D.) between related numbers. Differences between means were considered significant if the two-tailed P value was <0.05. All data were analysed by the program Instat.2.1 for Macintosh.
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Results |
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High ratio of IFN-/IL-4-producing synovial fluid CD4+ T cells in patients with RA compared to ReA
The clearest difference was found when only CD4+ T cells were analysed (Fig. 1). In RA, the mean percentage of IFN-
+ cells out of the CD4+ T-cell population (11.3±5) was almost 4-fold higher than for IL-4- positive cells (3.02±1.04; P = 0.0012). In contrast, in ReA, the frequency of IFN-
-and IL-4-positive CD4+ T cells was not different (5.35±4.8 and 4.34±1.72, respectively; P=0.79), resulting in a ratio of IFN-
/ IL-4+ T cells of 1.6. This ratio was 2.5-fold higher in RA than in ReA (P=0.047). Examples of the cytometry results after short-term stimulation with anti-CD3/anti-CD28 from two patients with RA (nos 2 and 5) and two patients with ReA (nos 6 and 8) are shown in Fig. 1A
. The numbers of cytokine-positive cells for all patients are shown in Fig. 1B
. The cytokine staining could be completely inhibited by pre-incubation of the anti-cytokine antibody with saturated concentrations of recombinant cytokines, indicating the specificity of the cytokine staining. The mean percentage of CD4+ T cells in RA patients was 52±9 and of CD8+ T cells 33±9; the corresponding data in ReA patients were 51±17 and 35±14.
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When CD8+ T cells were gated, five out of seven patients with RA and five out of eight patients with ReA showed a T cytotoxic 1- pattern. No significant differences between the ratios of IFN--positive and IL-4-positive cells were found in the two arthritides (9.26±4.7 for IFN-
vs 6.15±3.48 for IL-4 in RA, P=0.18, and 3.46±2.29 for IFN-
vs 2.22±1.09 for IL-4 in ReA, P=0.64). For total lymphocytes, the mean percentage of IFN-
-positive cells (10.23±4.9) was >2-fold higher than that of IL-4-positive cells in RA (4.8±0.9; P=0.014 for the difference), whereas the percentage of IFN-
- (4.3±3.4) and IL-4-positive cells (3.8±0.9) was similar in ReA.
To quantify further the level of cytokine secretion per cell, the mean fluorescence intensity (MFI) of cytokine-positive cells was compared between ReA and RA patients. The MFI of cytokine-positive cells was divided by the MFI of cytokine-negative cells (the lower left of the dot plot) in order to normalize the level of MFI for cytokine secretion. No clear difference in the intensity of fluorescence staining was found between these two groups of patients (data not shown).
High ratio of IFN-/IL-4-positive cells in synovial membrane of patients with RA compared to ReA
The mean ratio of IFN-/IL-4+ cells in RA was 4.3 (18.5±6.1/mm2 for IFN-
and 4.2±2.9/mm2 for IL-4; P<0.0001), but only 1.2 in ReA [12.3±9.7/mm2 (x3.5) for IFN-
and 9.9±8.6/mm2 (x3.5) for IL-4; difference not significant] (P=0.02 for the ratio difference). The number of CD3-positive cells in SM was ~3.5-fold higher in ReA (1607±1049/mm2 ) than in RA (458±163/mm2 ). Therefore, to correct the number of cytokine-positive cells for the same number of T cells, the number of cytokine-positive cells was divided by 3.5 for ReA patients (in Fig. 2
). The linear regression shown for RA and ReA patients in Fig. 2
indicates that the individual IFN-
/IL-4 ratio remains about the same for each patient group, while the number of cytokine-positive cells differs.
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In vitro stimulation in the presence of IL-10.
In general, the number of IL-10-secreting T cells was low. IL-10 alone had no significant effect on the number of IFN--, IL-4-or IL-10-positive T cells over 3 weeks (Fig. 4
).
In vitro stimulation in the presence of TGF-ß.
TGF-ß induced a small but partly significant reduction of IL-4-producing cells (IL-4 single positive: P=0.08, P=0.04, P=0.08 at week 1, 2 and 3, respectively, for CD4+ T cells, no significant reduction for CD8+ T cells; IL-4/IFN--double-positive cells: P=0.005, P =0.04, P=0.02 at week 1, 2 and 3, respectively, for CD4+ T cells, no significant reduction for CD8+ T cells). However, the number of IFN-
-positive cells remained unchanged.
In vitro stimulation in the presence of IL-12.
IL-12 had a small enhancing effect on IFN- secretion, which was not significant, while the number of IL-4-secreting cells was significantly and most effectively reduced, an effect which was stronger than that induced by TGF-ß (data not shown).
The number of TNF--positive T cells.
The number of TNF--positive T cells was not significantly influenced by any of the cytokines tested (data not shown).
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Discussion |
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The role of T cells in the pathogenesis of RA is still a matter of controversy, especially in long-standing disease [2, 16, 17]. The abundance of macrophage-derived cytokines such as TNF- and IL-1 in SF and SM [1], and the therapeutic benefit of antagonizing these cytokines [18], point to an important role for macrophages in the pathogenesis of RA. However, such treatment has not yet led to long-lasting remission. The evidence that a Th1-like response might be deleterious and a Th2-like response beneficial in autoimmune disease came first from animal models [47]. Early studies in man failed to detect T-cell cytokines in the rheumatoid SM [16]. However, there are now a growing number of reports on the presence of T-cell cytokines in the joint of RA patients [8, 1926]. Most of these studies report a Th1-type pattern with more IFN-
than IL-4, using the techniques of polymerase chain reaction (PCR), semi-quantitative PCR, in situ hybridization, immunohistology, measurement of soluble or intracellular cytokines in the joint or peripheral blood [27]. Despite these studies, there is still a controversy about the role of Th1 cytokines in RA [1, 2, 17, 23].
This study is the first to quantify the number of IFN-- and IL-4-positive cells at the single-cell level in SF and SM, and to compare the number of cytokine-positive cells with those found in another form of inflammatory arthritis. Most interestingly, a similar pattern was found in both SF and SM (Figs 1 and 2
): the number of IFN-
-positive cells was about four times higher than that of IL-4-positive cells in RA. In a recent investigation analysing SM-derived CD4+ T cells by flow cytometry, an even higher ratio was reported which was clearly higher than in PB [26]. For comparison, in ReA the number of IFN-
-positive cells was about the same as that of IL-4-positive cells. The presence of both T-cell cytokines in two different forms of arthritis stresses the importance of always analysing a control disease for comparison, to quantify the cytokine-positive cells and to compare ratios instead of absolute numbers. Indeed, the balance of these two T-cell cytokines seems to be crucial for the outcome of an immune response [3]. Previous studies trying to quantify T-cell cytokines at the single-cell level in RA did this only in SF [25, 28] or in SM [26, 29], and did not calculate a ratio of IFN-
/IL-4-positive cells because IL-4 was not detected or not looked for [25, 29].
Synovial fluid T cells were analysed by using the technique of intracellular cytokine staining and the number of cytokine-positive T cells was quantified by flow cytometry [13, 30]. We and others [20, 25, 26, 30] have shown that the frequency of cytokine-positive T cells, especially IL-4-positive ones, is very low and therefore normally not detectable by flow cytometry or ELISA without in vitro stimulation. Therefore, we used short-term mitogenic in vitro stimulation.
However, it was possible to quantify the number of cytokine-positive cells in SM (without stimulation) probably because of a higher frequency compared to SF. By double staining RA SM for CD3 and either IFN- or IL-4, we could show that only a minority of T cells (<3%) secrete cytokines (Fig. 3
). A similar result has been reported before for IFN-
, but not for IL-4 [29]. Cytokine-positive regulatory T cells in the range of 12% seem indeed to be sufficient to orchestrate a whole immune response, as suggested by animal models [31]. We could also show for the first time in RA synovium that the vast majority of IFN-
-positive cells were T cells (99%), while as many as 49% of IL-4-positive cells were non-T cells. The most likely non-T-cell source for IFN-
would be NK cells and, for IL-4 mast cells, basophils or NK1+ T cells.
The mean disease duration was clearly longer in patients with RA than with ReA. However, two recent studies demonstrated that the cell infiltrates in SM [32] and the amount of IFN- in RA SM [21] are similar in early and late RA, indicating that `early' RA may already have a chronic phenotype. In contrast, a distinct Th1/Th2 cytokine pattern was found in new-onset synovitis vs chronic RA by other investigators [33]. Nonetheless, the Th1-type cytokine pattern in the joints of patients with chronic RA demonstrated in our study could be a target of immunointervention independently of whether such a pattern is also present in early RA. There was no difference in the cytokine pattern in patients treated with second-line drugs compared to those not treated, indicating that activity might be more important than treatment.
ReA was chosen in this study for comparison because of the Th0/Th2-like pattern present in the joint [8, 34], which makes it a good control disease for RA. The finding of a Th1 pattern in the joints of some patients with ReA in this study suggests a more heterogeneous spectrum which needs further clarification. As can be seen from Table 2, there is no simple correlation of the cytokine pattern with disease duration, HLA-B27 status or the triggering bacterium. Using a nested PCR technique for the detection of cytokine mRNA, one recent study could not find a difference between RA and ReA membrane in IFN-
expression [23], while another report found less IFN-
in ReA than in RA [24]. According to our results, the IFN-
/IL-4 ratio is more relevant than the absolute numbers for IFN-
alone. Investigating the cytokine pattern in SF and SM by PCR, a recent study detected a higher IL-4/IFN-
ratio in patients with pauciarticular juvenile rheumatoid arthritis and with juvenile spondyloarthropathy compared to RA [35]. This would be in line with our results because of some similarity between these forms of juvenile arthritis and ReA [36].
Therefore, based on the evidence that both IFN-- and IL-4-positive cells are present in the joint of RA patients, but that the IFN-
-positive cells clearly outnumber the IL-4-positive ones, an obvious question is whether such an established Th1 pattern could be changed. Current approaches to the modulation of a Th1 pattern are discussed elsewhere in more detail [37]. Among them, IL-4 and IL-10 are so far the best candidates as agents of intervention. IL-4 has been reported to downregulate secretion of pro-inflammatory cytokines of cells derived from RA joints [38], to suppress IFN-
production of T cells in vitro [39] and to improve animal models of arthritis [7, 40]. In this study, we show that IL-4 is able to change an established Th1 pattern in vitro of T cells derived from RA joints by suppressing IFN-
-positive cells and by expanding IL-4-positive ones, both IL-4 single-positive and IL-4/ IFN-
-double-positive cells. It is possible that these effects are more pronounced if T cells (or patients) are treated for longer than 3 weeks. From our experiments, we cannot answer the question whether this was due to a true reversion from Th1 to Th2 cells. However, because there is good experimental evidence that a polarized Th1 or Th2 pattern is irreversible [39], it is more likely that IL-4 worked as an inhibitor of IFN-
secretion and that the new IL-4-positive cells were derived from expansion of IL-4+ T cells and/or from uncommitted precursor cells of the Th0 type by differentiation and clonal outgrowth [39, 40].
In contrast, the presence of IL-10 in our T-cell lines over 3 weeks did not change the number of T cells secreting IFN-γ, IL-10 or IL-4 significantly. Furthermore, IL-10 added to IL-4 proved no more effective than IL-4 alone. Thus, IL-10 neither suppressed IFN- secretion nor did it induce an expansion of IL-10+ T cells. These results are of special interest because recent evidence suggests that IL-10 may be a good candidate for the treatment of RA: IL-10 suppresses inflammatory cytokines of RA MNC in vitro [19, 41] and is an effective treatment of arthritis in animal models of the disease [7, 42]. Furthermore, a new T-cell type has been described, characterized by high IL-10 and low IL-4 secretion, which has the capacity to suppress ongoing Th1 responses [43]. However, our data suggest that manipulation of T cells, potentially capable of mediating long-lasting suppression [43], seems to be more difficult after disease onset.
It has also been reported that TGF-ß might be a potent suppressor of Th1 responses [4], but the only effect in our study was seen in a reduction of IL-4-positive T cells. IL-12 did not expand the population of IFN--positive T cells significantly during the 3 weeks, probably reflecting an already maximally polarized Th1 pattern. Anti-IL-12 could also be a therapeutic option for the downregulation of Th1 responses [44], although it was not tested in this study.
In summary, by quantifying IFN-- and IL-4-positive single cells in SF and SM, we demonstrated a shift towards a high IFN-
/IL-4 ratio (Th1 like) in the joints of RA patients in comparison to patients with ReA. Only IL-4 induced a suppression of Th1 cytokines (IFN-
) and an expansion of Th2 cytokines (IL-4) in vitro, while IL-10 was not effective. These results are of special interest with regard to the first clinical studies treating RA patients with IL-10 [45] or IL-4 (P. Miossec, personal communication). Although IL-10 might be effective if monocytes [41] and not T cells are the targets, new approaches in the treatment of RA should rather aim for immunomodulation of the T cell response [46].
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Acknowledgments |
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References |
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