Materials and methods

Patients and samples
The total patient population consisted of 32 patients with RA (25 women and seven men; mean ± standard deviation age, 52.8 ± 12.4 years) diagnosed according to the revised 1987 criteria of the American College of Rheumatology (formally, the American Rheumatism Association) [25]. All patients were receiving prednisolone (≤ 7.5 mg/day) and disease-modifying antirheumatic drugs. Clinical parameters in the study patients were as follows (mean ± standard deviation): erythrocyte sedimentation rate, 55.9 ± 35.4 mm/hour; serum C-reactive protein (CRP) level, 32.0 ± 32.0 mg/l; and IgM class rheumatoid factor titer, 142 ± 158 U/ml. Patients were divided into two groups: 24 patients with active disease, who had multiple tender and/or swollen joints and elevated serum CRP level (≥ 10 mg/l); and eight patients with inactive disease, who satisfied the American College of Rheumatology preliminary criteria for clinical remission [26]. Sixteen healthy volunteers (11 women and five men; age, 45.8 ± 11.2 years) served as controls. ST samples were obtained from three RA patients undergoing total knee replacement. All patients gave informed consent.

Isolation of CD4+ T cells
Peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood samples by centrifugation over Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden). CD4+ T cells were purified from PBMC by positive selection using anti-CD4 mAb-coated magnetic beads (Miltenyi Biotec, Gladbach, Germany), according to the manufacturer's instructions. CD4+ T cells were isolated from ST samples, as previously described [27]. Briefly, fresh ST samples were fragmented and digested with collagenase and DNase for 1 hour at 37°C. After removing tissue debris, ST cell suspensions in culture medium (RPMI 1640 medium; Life Technologies, Gaithersburg, MD, USA) supplemented with 25 mM HEPES (2 mM L-glutamine, 2% nonessential amino acids, 100 IU/ml penicillin, and 100 mg/ml streptomycin; Life Technologies) with 10% heat-inactivated FCS (Life Technologies) were incubated at 37°C in six-well plates (Coster, Cambridge, MA, USA) for 45 min. Non-adherent cells were harvested and CD4+ T cells were purified by positive selection as already described.

Culture of CD4+ T cells
PB CD4+ T-cell populations were resuspended at a density of 1 × 106 cells/ml in culture medium with 10% FCS, and 0.5 ml cell suspensions were dispensed into the wells of 24-well microtiter plates (Coster) coated with 1 μg/ml anti-CD3 mAb (Immunotech, Marseille, France). The cells were incubated with 1 μg/ml anti-CD28 mAb (Immunotech) in the presence or absence of the indicated concentrations of IL-10 (Becton Dickinson, San Jose, CA, USA) at 37°C in a humidified atmosphere containing 5% CO2 [28]. Culture supernatants were collected 36 hours later and cell-free samples were stored at -30°C until cytokine assay.
To examine the effect of IL-6 on T-cell responsiveness to IL-10, CD4+ T cells from healthy controls were incubated in culture medium with 10% FCS in the presence or absence of 10 ng/ml IL-6 (Becton Dickinson) for 36 hours. Cells were then stimulated for 36 hours with anti-CD3 mAb and anti-CD28 mAb in the presence or absence of 1 ng/ml IL-10. Culture supernatants were measured for IFN-γ concentrations.

Flow cytometric analysis for IL-10R1 expression
A sample of 5 × 105 cells of PBMC was resuspended in PBS with 1% FCS. PBMC were incubated with saturating concentrations of anti-IL-10R1 mAb (IgG1; R&D systems, Minneapolis, MN, USA) or with isotype-matched control mAb (Immunotech), followed by incubation with FITC-conjugated goat anti-mouse IgG1 polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Cells were then incubated with phycoerythrin-conjugated anti-CD4 mAb (Becton Dickinson). Cells were washed well with 1% FCS/PBS between incubations. Analysis was performed on a FACScan flow cytometer (Becton Dickinson).

Immunoassay for IFN-γ and IL-2
Concentrations of IFN-γ and IL-2 in culture supernatants of CD4+ T cells were measured in duplicate by the quantitative sandwich ELISA using cytokine-specific capture with biotinylated detection mAb and recombinant cytokine proteins (all from Becton Dickinson), according to the manufacturer's protocol. The detection limits for IFN-γ and IL-2 were 15 pg/ml.

Isolation of mRNA and real-time PCR
Total cellular RNA was extracted from PB CD4+ T cells using an RNA isolation kit (RNeasy Mini kit; Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. cDNA was synthesized from total RNA with Molony murine leukemia virus reverse transcriptase (US Biochemical, Cleveland, OH, USA) and oligo-(dT)15 primers (Promega, Madison, WI, USA). Real-time PCR was performed with the LightCycler Instrument (Roche Diagnostics, Penzberg, Germany) in glass capillaries. The reaction mix containing Taq DNA polymerase and DNA double-strand-specific SYBR Green I dye (Lightcycler FastStart DNA Master SYBR Green I; Roche Diagnostics) and specific primers were added to cDNA dilutions.
The cDNA samples were denatured at 95° C for 10 min, and were then amplified for 40–50 cycles: at 95° C (10 s), at 65° C (15 s), and 72° C (22 s) for β-actin; at 95° C (10 s), at 62° C (15 s), and at 72° C (10 s) for SOCS1; and at 96° C (10 s), at 68° C (15 s), and at 72° C (15 s) for SOCS3. Amplification curves of the fluorescence values versus cycle number were obtained, and a melting curve analysis was then performed. The levels of SOCS1 and SOCS3 expression were determined by normalizing relative to β-actin expression. The forward and reverse primers were as follows: for β-actin, 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGATTTC-3' ; for SOCS1, 5'-AGACCCCTTCTCACCTCTTG-3' and 5'-GCACAGCAGAAAAATAAAGC-3' ; and for SOCS3, 5'-CCCGCCGGCACCTTTCTG-3' and 5'-AGGGGCCGGCTCAACACC-3'.

Western blot analysis
CD4+ T cells were stimulated for 20 min by the indicated concentrations of IL-10 and IL-6 at a density of 5 × 105 cells in 0.5 ml culture medium with 10% FCS. To examine the effect of serum IL-6 on STAT phosphorylation, normal CD4+ T cells were stimulated for 20 min with 30% active RA serum in culture medium with 40 μg/ml neutralizing goat anti-IL-6 polyclonal antibody (IgG; Techne, Princeton, NJ, USA) or control goat IgG (Techne). Whole cell lysates were prepared by placing cells in 100 μl SDS lysing buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue). Then 20 μl protein samples were fractionated on 10% SDS-polyacrylamide gels and were transferred to nitrocellulose membranes (Amersham, Buckinghamshire, UK), and the membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20.
Tyrosine phosphorylation of STAT1 and STAT3 was detected using commercial available kits (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer's instructions. Briefly, the membrane was incubated with the antibodies (rabbit IgG) anti-STAT1 antibody, anti-phosphorylated tyrosine 701 of STAT1 antibody, anti-STAT3 antibody, and anti-phosphorylated tyrosine 705 of STAT3 antibody, diluted as recommended at 1/2000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA. Antibody binding was detected by horseradish peroxidase-conjugated anti-rabbit IgG antibody diluted at 1/4000 with Tris-buffered saline with 0.1% Tween 20 with 5% BSA, and was revealed using the chemiluminescence system. Protein bands were quantified by densitometry using NIH-Image analysis, and STAT phosphorylation was compared with the total amount of STAT protein. IFN-γ-stimulated Hela cells were used as a positive control for STAT1 phosophorylation.

Statistical analysis
Data are expressed as the mean value ± standard error of the mean or box plots. The statistical significance of differences between two groups was determined by the Mann–Whitney U test or the Wilcoxon signed rank test. P < 0.05 was considered significant.