Fig. 5.

TLR4 is involved in Hsp70-induced IL-10 production in FLSs. a Total RNA from RA FLSs or RAW264.7 macrophages was extracted and analyzed by RT-PCR for TLR2 and TLR4. GAPDH was used as a loading control. b RA FLSs or RAW264.7 macrophages were lysed and the protein levels of TLR2 and TLR4 expression in the cells were determined by Western blotting using specific mAb against TLR2 and TLR4. GAPDH was used as a loading control. c RA FLSs were pre-treated with anti-TLR2, anti-TLR4, or isotype-matched mAb (10 μg/ml) for 30 min. Then, the cells were incubated with Hsp70 (1 μg/ml) for 24 h and IL-10 concentrations were determined with ELISA. Data are shown as means ± SEM of three independent experiments. Asterisk, p < 0.05 versus Hsp70 alone group. d RA FLSs were pre-treated with monoclonal anti-TLR2 and anti-TLR4 (10 μg/ml) for 30 min. Then, the cells were incubated with PGN or LPS (100 ng/ml) for 24 h and IL-10 concentrations were determined with ELISA. Data are shown as means ± SEM of three independent experiments. Asterisk, p < 0.05 versus LPS alone group. Double asterisk, p < 0.05 versus PGN alone group