Figure 1.
Functional TLRs were excessively expressed in RASF.
A, The expression of TLRs, including TLR2, TLR3, TLR4, and TLR9 in RASF was detected by RT-PCR analysis. GAPDH was used as the loading control. B, The expression of TLR2, TLR3, TLR4, and TLR9 in RASF (n = 5) and OASF (n = 5) was determined by realtime PCR (Wilcoxon signed-rank test, *P<0.05). RASF were stimulated with 25 µg/ml ploy(I:C) for 5 min, 15 min, and 30 min, respectively. Then the cells were harvested and lysised for western blot analysis of p-IκBα (C), p-ERK (D), p-p38 (E), and p-IRF3 (F). β-actin was used as the loading control. The results were representative of at least three experiments.
Figure 2.
TLR agonists promoted RASF to produce inflammatory cytokines.
RASF were stimulated with TLR2 agonist PGN (10 µg/ml), TLR3 agonist ploy(I:C) (25 µg/ml), TLR4 agonist LPS (100 ng/mL), or TLR9 agonist CpG (10 µg/ml). 4 h later, the cells were harvested for RT-PCR (A–C, a) and realtime PCR (A–C, b) analyses of IL-6, IL-8, and TNF-α; 24 h later, the cell culture supernatants were collected for ELISA analyses of IL-6, IL-8, and TNF-α (A–C, c). The results were presented as mean+SEM of five independent experiments (Wilcoxon signed-rank test, *P<0.05 vs. vehicle control).
Figure 3.
vLigation of TLRs stimulated MMPs and VEGF production in RASF.
RASF were stimulated with 10 µg/ml PGN, 25 µg/ml ploy(I:C), 100 ng/mL LPS, or 10 µg/ml CpG for 4 h. Then the cells were harvested for RT-PCR analysis of MMP-1 (A); realtime PCR analyses of MMP-1 (B), MMP-3 (C), MMP-9 (E), and VEGF (F). And also, after stimulation for 24 h, the cell culture supernatants were collected for MMP-3 ELISA (D). The results were presented as mean+SEM of five independent experiments (Wilcoxon signed-rank test, *P<0.05 vs. vehicle control).
Figure 4.
TLR activation enhanced RASF-mediated inflammatory Th1 and Th17 responses.
A, RASF were stimulated with 25 µg/ml ploy(I:C) or 100 ng/ml LPS for 24 h, then the expression levels of TSP-1, SDF-1, and IL-15 were determined by realtime PCR analyses (Wilcoxon signed-rank test, *P<0.05). B, After activation with 25 µg/ml ploy(I:C) or 100 ng/ml LPS for 24 h, RASF were co-cultured with allogeneic CD4+ RA patient T cells in the direct contact or the transwell systems for 5 days. Then the T cells were harvested for flow cytometry analysis of the percentage of Th1 and Th17 cells (Wilcoxon signed-rank test, *P<0.05). Accordingly, the cell culture supernatants were collected for ELISA analyses of IFN-γ and IL-17 (C) levels (Wilcoxon signed-rank test, *P<0.05 vs. unstimulated control). D, RASF/T cell co-culture was performed as described above under Th17 polarization conditions. 5 days later, the cell culture supernatants were collected for IL-17 ELISA (Wilcoxon signed-rank test, *P<0.05). The data were shown as mean+SEM of four separate assays.