The induction of OPN expression by cytokines and the blocking effect of anti-IL-10 antibody. (A) PBMCs selected from RA patients (n = 10, as described in the Figure 6 legend) were cultured in the presence and absence of the indicated cytokines at a final concentration of 25 ng/ml for 48 hours. T cells were purified by magnetic bead separation and were subject to real-time PCR analysis of OPN expression. The purity of the isolated T cell preparations was greater than 97%. (B) The same PBMC preparations were cultured in the presence and absence (Medium [control]) of recombinant IL-10 used at the indicated concentrations to determine the dose-response pattern and kinetics of OPN expression in T cells. The resulting cells were harvested at the indicated time points and T cells were purified prior to real-time PCR analysis for OPN expression. (C) In parallel experiments, the same PBMCs (10 untreated samples) were cultured in the presence and absence (Medium [control]) of SF at a dilution of 1:5. A purified mouse anti-human IL-10 antibody or a mouse control antibody of matched isotype (anti-lysozyme antibody), respectively, was added at a final concentration of 5 μg/ml. T cells were subsequently isolated and analyzed for OPN expression by real-time PCR. The purity of T cells was greater than 97%. Dash indicates absence of antibody. (D) CD45RA⁺ and CD45RO⁺CD45RA^– T cell preparations were purified (n = 10; cell purity >97%) using magnetic beads coupled with specific antibodies and analyzed, together with PBMC preparations, for the expression of OPN by real-time PCR. Relative expression of OPN gene was calculated as described in the Figure 1 legend. In all cases, asterisks indicate statistically significant differences between the groups (P < 0.05).