Differences in expression pattern of molecules related to osteoclastogenesis between CD16+ and CD16- monocyte subsets. (a) Total RNA was extracted from freshly isolated CD16+ and CD16- monocytes or from the cells incubated in either macrophage colony-stimulating factor (M-CSF) (25 ng/ml) alone or M-CSF (25 ng/ml) + receptor activator of NF-κB ligand (RANKL) (40 ng/ml) for 3 days, and semiquantitative RT-PCR analysis was performed. Representative results from three independent experiments are shown. (b) The expression of receptor activator of NF-κB (RANK) and c-fms in unstimulated or stimulated monocytes was analyzed by western blotting. (c) Cell surface expression of c-fms on unstimulated CD16+ and CD16- monocytes was examined by three-color flow cytometry. Gates were set either for CD14+ CD16+ (left panel) or CD14+ CD16+ (right panel) monocytes. Histograms show the stained cells with anti-c-fms mAb (solid lines) and isotype-matched control (dotted lines). (d) Purified monocytes were allowed to adhere on plates overnight (unstimulated) or the cells treated with M-CSF (25 ng/ml) + RANKL (40 ng/ml) were examined for the expression of the αvβ3 heterodimer by immunofluorescent staining. Solid arrows indicate mononuclear αvβ3-positive cells. Dotted arrows indicate multinucleated αvβ3-positive cells. Original magnification, ×100. Results are representative of two independent experiments.