Figure 5.

Effect of interleukin-34 (IL-34) on human peripheral blood mononuclear cells (PBMCs) migration and subsequent osteoclast (OC) formation. (A) Involvement of IL-34 in the migration of human PBMCs. Recombinant IL-34 (100 ng/ml) or CM (conditioned media) from rheumatoid arthritis (RA) fibroblast-like synovial cell (FLS) cultures with mouse immunoglobulin G (IgG) (50 ng/ml) or IL-34 antibody (Ab) (0, 1, 10, and 50 ng/ml) was added to the lower compartments of a transwell system, and PBMCs (1.2 × 10⁶) were added to the upper chamber of each transwell, Cells were allowed to migrate for six hours, and the number of PBMCs in the lower chamber was counted. Bars indicate the mean and standard deviation (SD) of the fold-change of PBMC migration relative to that of rIL-34 treatment (*P < 0.05, N.S., not significant, compared to PMBCs treated with rIL-34). (B) and (C) Involvement of IL-34 in osteoclast (OC) differentiation and activation. Isolated human PBMCs were cultured with macrophage colony-stimulating factor (M-CSF) (100 ng/ml) or IL-34 (100 ng/ml) plus receptor activator of nuclear factor kappa-B ligand (RANKL) (100 ng/ml) as a positive control or CM plus RANKL (100 ng/ml) in the presence of mouse IgG (50 ng/ml) or IL-34 Ab (50 ng/ml) for 12 to 14 days. Human osteoclasts showing tartrate resistant acid phosphatase (TRAP) positive (TRAP⁺) multinucleated cells (MNCs) (> 3 nuclei) on the same surface area were identified by TRAP staining (B) and counted under a light microscope (C). Scale bars = 50 μm. Bars indicate the mean and standard deviation (SD) of triplicate samples (*P < 0.05, N.S., not significant, compared to PBMCs cultured with M + R). rIL-34, recombinant IL-34; M, M-CSF; R, RANKL; none, no treatment; CM, conditioned media; IgG, isotype control; IL-34 Ab, anti-IL-34 blocking antibody.