Fig. 3.
Fig. 3. Inhibition of TNF-α–induced osteoclastogenesis by GM-CSF. / The c-Fms+RANK− cells were cultured in the presence of 100 ng/mL M-CSF with various concentrations of TNF-α or 25 ng/mL sRANKL. After 6 days of culture, cells were subjected to a TRAP activity assay (A) or TRAP staining (bar = 25 μm) (B). Osteoclastogenesis reached a plateau at 25 ng/mL TNF-α (A), and multinuclear TRAP-positive cells were also formed (B). Expression of vitronectin receptors (integrin αv and β3) or a CTR were detected by RT-PCR in cells cultured with M-CSF and TNF-α for 6 days (C). NC indicates no template control. Osteoclast differentiation was induced by 2 cytokine combinations: M-CSF and sRANKL and, also, M-CSF and TNF-α. Differentiation induced by the former was completely inhibited by the addition of RANK−Fc in a dose-dependent manner, but differentiation induced by the latter was not. By contrast, GM-CSF inhibited both in a dose-dependent manner (D).

Inhibition of TNF-α–induced osteoclastogenesis by GM-CSF.

The c-Fms+RANK− cells were cultured in the presence of 100 ng/mL M-CSF with various concentrations of TNF-α or 25 ng/mL sRANKL. After 6 days of culture, cells were subjected to a TRAP activity assay (A) or TRAP staining (bar = 25 μm) (B). Osteoclastogenesis reached a plateau at 25 ng/mL TNF-α (A), and multinuclear TRAP-positive cells were also formed (B). Expression of vitronectin receptors (integrin αv and β3) or a CTR were detected by RT-PCR in cells cultured with M-CSF and TNF-α for 6 days (C). NC indicates no template control. Osteoclast differentiation was induced by 2 cytokine combinations: M-CSF and sRANKL and, also, M-CSF and TNF-α. Differentiation induced by the former was completely inhibited by the addition of RANK−Fc in a dose-dependent manner, but differentiation induced by the latter was not. By contrast, GM-CSF inhibited both in a dose-dependent manner (D).

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