Figure 4

TLR3 expression was induced in macrophages with pristane stimulation. In splenic macrophages, TLR3 protein expression was detected by FACS at 0, 6 and 26 days after pristane injection. The proportion and mean fluorescence intensity (MFI) of cell surface and intracellular TLR3 were detected on His36⁺ (anti-rat macrophage) gated TLR3⁺ cells from splenocytes, and representative histograms (a) are showed in the course of arthritis. Data were collected after correction for isotype-matched IgG control. NR8383 cells were stimulated with a series of concentrations of pristane (0.1 μ, 1 μ, 10 μ, 100 μ, 1 m, 10 mM as theoretical pristane concentration), and TLR3 and TLR4 mRNA expression was detected at 48 hours after stimulation (b). The expression of TLR3 increased with a raised dosage of pristane (r² = 0.78) by Pearson correlation analysis. With 100 μM pristane stimulation, TLR3 (c) and IFN-β, TNF-α (d) mRNA expression was measured at 0, 8, 24, 48 and 72 hours. (e) NR8383 pretreated with anti-TLR3 or isotype control antibodies were stimulated by 100 μM pristane or 10 μg/ml polyI:C, and IFN-β, TNF-α mRNA expression was measured at 24 hours. mRNA expression level was detected by Real-time quantitative PCR, and data are presented as means ± SEM of four replicated determinations from three independent experiments. * represents the comparison with the control group (Blank in B and E, and 0 hour in C and D), and levels of significance were calculated by using Student's T test (* P < 0.05, ** P < 0.01, *** P < 0.001).