Materials and methods

Patients
Informed consent was obtained from 24 patients (5 men and 19 women) with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) [17]. The age of the patients with RA was 50 ± 8 (mean ± SEM) years (range 23–71 years). All medications were stopped 48 hours before entry to the study. Comparisons were made with 14 patients with OA (3 men and 11 women) and with 14 healthy controls (3 men and 11 women) who had no rheumatic diseases. The mean ages of the patients with OA and the healthy controls were 50 ± 8 years (range 34–68 years) and 30 ± 6 years (range 24–57 years). Informed consent was obtained, and the protocol was approved by the Catholic University of Korea Human Research Ethics Committee.

Reagents
Recombinant IL-17, IL-18, IL-15, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-6 and IL-8 were purchased from R & D systems (Minneapolis, MN, USA). Recombinant transforming growth factor (TGF)-β was purchased from Peprotech (London, UK). Recombinant TNF-α and IL-1 were purchased from Endogen Inc. (Cambridge, MA, USA). Cyclosporin A was provided by Sandos Ltd. (Basel, Switzerland). Phytohemagglutinin (PHA), pyrrolidine dithiocarbamate (PDTC), rapamycin, dexamethasone and curcumin were all obtained from the Sigma Chemical Co. (St Louis, MA, USA). Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were obtained from BD Biosciences (San Diego, CA, USA). LY294002, SB203580, FK506, wortmannin and PD98059 were obtained from Calbiochem (Schwalbach, Germany).

Production of IL-17 by T cell receptor activation, cytokines or chemokines
PBMC were prepared from heparinized blood by Ficoll-Hypaque (SG1077) density-gradient centrifugation. Cell cultures were performed as described previously [18]. In brief, the cell suspensions were adjusted to a concentration of 106/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine. Cell suspension (1 ml) was dispensed into 24-well multi-well plates (Nunc, Roskilde, Denmark), and incubated for 24 hours at 37°C in 5% CO2. Subsequently, various concentrations of cyclosporin A (10–500 ng/ml) were added to the medium and cells were incubated for 24 hours. To each well was added FK506, rapamycin, curcumin, PDTC, LY294002, SB203580, PD98059, dexamethasone or wortmannin. After incubation for 24 hours (unless otherwise stated), cell-free media were collected and stored at -20°C until assayed. All cultures were set up in triplicate, and results are expressed as means ± SEM.

CD4+ T-cell isolation by MACS
Anti-CD4 microbeads were used essentially as recommended by the manufacturer (Miltenyi) [19]. PBMC were resuspended in 80 μl of FBS staining buffer. Anti-CD4 microbeads (20 μl) were added and incubated for 15 min at 6–12°C. Saturating amounts of fluorochrome-conjugated antibodies were added for a further 10 min. Cells were diluted in 2.5 ml of FBS staining buffer, pelleted, resuspended in 500 μl and magnetically separated, usually on an AutoMACS magnet fitted with a MACS MS column. Flow-through and two 1 ml washes were collected as the negative fraction. Enriched cells were collected in two 0.5 ml aliquots from the column after removal from the magnet. Alternatively, cells stained with anti-CD4–phycoerythrin were washed, magnetically labeled with anti-phycoerythrin microbeads (20 μl added to 80 μl of cell suspension; 15 min, 6–12°C), and magnetically separated as described above. The purity of cells was assessed by flow cytometric analysis of stained cells on a FACS Vantage sorter. Most (more than 97%) of the isolated cells had the CD4 T cell marker.

Enzyme-linked immunosorbent assay of IL-17
IL-17 in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay as described previously [20]. In brief, a 96-well plate (Nunc) was coated with 4 μg/ml monoclonal antibodies against IL-17 (R & D Systems) at 4°C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 hours at room temperature (22–25°C), test samples and the standard recombinant IL-17 (R & D Systems) were added to the 96-well plate and incubated at room temperature for 2 hours. Plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-17 (R & D Systems) for 2 hours at room temperature. After washing, streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma) was incubated for 2 hours, then washed again and incubated with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 M NaOH and the optical density of each well was read at 405 nm. The lower limit of IL-17 detection was 10 pg/ml. Recombinant human IL-17 diluted in culture medium was used as a calibration standard, ranging from 10 to 2000 pg/ml. A standard curve was drawn by plotting optical density against the log of the concentration of recombinant cytokines, and used for determination of IL-17 in test samples.

Quantification of IL-17 mRNA by semiquantitative reverse transcription–polymerase chain reaction
PBMC were incubated with various concentrations of anti-CD3 in the presence or absence of inhibitors (LY294002, PDTC). After 16 hours of incubation, mRNA was extracted with RNAzol B (Biotex Laboratories, Houston, TX, USA) in accordance with the manufacturer's instructions. Reverse transcription of 2 μg of total mRNA was performed at 42°C using the Superscript™ reverse transcription system (Takara, Shiga, Japan). PCR amplification of cDNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 U of Taq DNA polymerase (Takara) and 0.25 μM of sense and antisense primers. The reaction was performed in PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3) in a total volume of 25 μl. The following sense and antisense primers for each molecules were used: IL-17 sense, 5'-ATG ACT CCT GGG AAG ACC TCA TTG-3'; IL-17 antisense, 5'-TTA GGC CAC ATG GTG GAC AAT CGG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-CGA TGC TGG GCG TGA GTA C-3'; GAPDH antisense, 5'-CGT TCA GCT CAG GGA TGA CC-3'. Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) through cycles for 30 s of denaturation at 94°C, 1 min of annealing at 56°C for GAPDH and IL-17, followed by 1 min of elongation at 72°C. PCR rounds were repeated for 25 cycles each for both GAPDH and IL-17; this was determined as falling within the exponential phase of amplification for each molecule. The level of mRNA expression was presented as a ratio of IL-17 PCR product over GAPDH product.

Western blot analysis of Akt, phosphorylated Akt and IκB-α
PBMC were incubated with anti-CD3 (10 μg/ml) in the presence or absence of LY294002 (20 μM). After incubation for 1 hour, whole cell lysates were prepared from about 107 cells by homogenization in the lysis buffer, and centrifuged at 14,000 r.p.m. (19,000 g) for 15 min. Protein concentrations in the supernatants were determined with the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, membrane was preincubated with 0.1% skimmed milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 hours, then primary antibodies against Akt, phosphorylated Akt and IκB-α (Cell Signaling Technology Inc., Beverly, MA, USA), diluted 1:1000 in 5% BSA/TBS-T, were added and incubated overnight at 4°C. After washing four times with TBS-T, horseradish peroxidase-conjugated secondary antibodies were added and allowed to incubate for 1 hour at room temperature. After TBS-T washing, hybridized bands were detected with the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).

Gel mobility-shift assay of NF-κB binding site
Nuclear proteins were extracted from about 5 × 106 PBMC. Oligonucleotide probes encompassing the NF-κB binding site of the human IL-17 promoter (5'-ATG ACC TGG AAA TAC CCA AAA TTC-3') were generated by 5'-end labeling of the sense strand with [γ-32P]dATP (Amersham Pharmacia) and T4 polynucleotide kinase (TaKaRa). Unincorporated nucleotides were removed by NucTrap probe purification columns (Stratagene, La Jolla, CA, USA). Nuclear extracts (2 μg of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 μl of binding buffer consisting of 20 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 5% glycerol, 1 mg/ml BSA, 0.2% Nonidet P40 and 50 ng/μl poly(dI-dC). Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 × Tris-borate-EDTA buffer (pH 8.0) at 100 V. Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70°C with intensifying screens. Rabbit polyclonal antibodies against NF-κB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell viability (Trypan blue dye exclusion assay)
For cell viability assays, the trypan blue dye exclusion method was used to evaluate the potential of direct cytotoxic effect of inhibitors on cells. After incubation for 24 hours, the cells were harvested and the percentage cell viability was calculated with the formula 100 × (number of viable cells/number of both viable and dead cells) [21].

Statistical analysis
Data are expressed as means ± SEM. Statistical analysis was performed with Student's t-test for matched pairs. P values less than 0.05 were considered significant.