Figure 5.

Suppressive effect of interleukin-27 (IL-27) on human monocyte-derived osteoclasts. (a) Human osteoclast precursor cells were cultured in the presence of 25 ng/ml macrophage colony-stimulating factor (M-CSF), 30 ng/ml receptor activator of nuclear factor-κB ligand (RANKL), and various concentrations of IL-27 to generate osteoclasts. After 9 days, the cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (original magnification, × 100) and the number of TRAP⁺ multinucleated cells was determined by light microscopy. Data are representative of at least three independent experiments (mean and SD). **P < 0·01; ***P < 0·001, compared with the M-CSF plus RANKL culture conditions. (b) The mRNA expression levels of cathepsin K and calcitonin receptor were quantified by real-time PCR and normalized to that of β-actin. Data are representative of at least three independent experiments (mean and SD). *P < 0·05; **P < 0·01; ***P < 0·001, compared with the M-CSF plus RANKL culture conditions. (c) Monocytes from human peripheral blood mononuclear cells were cultured for 3 days with 100 ng/ml M-CSF to form macrophage-like osteoclast precursor cells. After 3 days, the cells were further cultured in the presence of 25 ng/ml M-CSF, 30 ng/ml RANKL, and 1 ng/ml IL-27. After 3 days, the cells were immunostained to detect IFN-γ (upper). The expression of IFN-γ mRNA was quantified by real-time PCR (lower). Data are representative of at least three independent experiments (mean and SD). **P < 0·01. (d) The expression of IL-27, interferon-γ (IFN-γ), low-density lipoprotein receptor-related protein 4 (LRP4), and matrix metalloproteinase 19 (MMP19) were determined in the synovium of patients with rheumatoid arthritis (RA; n = 4) or osteoarthritis (OA; n = 4) by immunohistochemical staining. Representative photographs are shown.