Figure 4

Cocultures of monocytes and MIF-activated RA human synovial fibroblasts showed significantly more TRAP-positive multinucleated cells. (a) Schematics for cocultures of isolated human monocytes and RA synovial fibroblasts prestimulated with rhMIF for OC differentiation. RA synovial fibroblasts were pretreated with rhMIF 5 ng/mL for three days, monocytes were added to each well, and cocultures were maintained in α-MEM containing 10% horse serum and 25 ng/mL M-CSF for 21 days. TRAP-positive multinucleated cells were counted. Results are presented as the mean and standard deviation (SD) of four separate experiments. #P < 0.05 and **P < 0.005. (b) Schematics of cocultures of isolated human monocytes and RA synovial fibroblasts prestimulated by rhMIF with or without signal inhibitors for OC differentiation. RA synovial fibroblasts were pretreated with rhMIF 5 ng/mL for three days, monocytes were added to each well with the different signal inhibitors, and cocultures were maintained in α-MEM containing 10% horse serum and 25 ng/mL M-CSF for 21 days. TRAP-positive multinucleated cells were counted. Data represent the mean and SD of four separate experiments. *P < 0.05 and **P < 0.005. (c) The expression of TRAP, RANK, cathepsin K, CTR, MMP-9, and β-actin mRNA from differentiated OCs that were cocultured with MIF-stimulated RA synovial fibroblasts was measured using quantitative real-time PCR. (d) Differentiation into TRAP-positive multinucleated cells was decreased after blocking of IL-1β. **P < 0.005 versus monocyte + M-CSF + unstimulated RA FLS and P < 0.005 versus monocyte + M-CSF + MIF stimulated RA FLS. The data represent the mean and SD of four separate experiments. CTR, calcitonin receptor; FLS, fibroblast-like synoviocytes; MIF, macrophage migration inhibitory factor; MMP, matrix metalloproteinases; OC, osteoclast; RA, rheumatoid arthritis; TRAP, tartrate resistant acid phosphatase.