Figure 2

The effect of MIF on the expression of RANKL in RA human synovial fibroblasts. (a) Isolated RA synovial fibroblasts were incubated with rhMIF (0.1 to 10 ng/mL) for 72 hours, and mRNA was extracted and measured using real-time PCR. (b) Isolated RA synovial fibroblasts were incubated with rhMIF (0.1 to 10 ng/mL) for 72 hours, and protein was extracted and measured using western blot analysis. (c) RA synovial fibroblasts were cultured with 0.1 to 10 ng/mL of rhMIF for 72 hours and stained with an anti-RANKL antibody (red) (original magnification 400×). (d) Effect of neutralizing agents for known osteoclastogenic factors on MIF-induced RANKL expression. RA synovial fibroblasts were treated with rhMIF 5 ng/mL for 72 hours in the presence or absence of anti-IL-1β, anti-TNF-α, or anti-IL-6. RANKL mRNA expression was quantified using real-time PCR. (e) Isolated RA synovial fibroblasts were incubated with rhMIF (0.1 to 10 ng/mL) for 72 hours, and MIF-induced IL-1β mRNA expression was measured using RT-PCR. The data represent the mean and standard deviation of three separate experiments. *P < 0.05 and **P < 0.005. MIF, macrophage migration inhibitory factor; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-B ligand.