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.