In vitro investigation of the roles of the proinflammatory cytokines tumor necrosis factor-α and interleukin-1 in murine osteoclastogenesis.

Whereas the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-кB ligand (RANKL) are essential and sufficient for osteoclastogenesis, a number of other cytokines including two proinflammatory cytokines, tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1), can exert profound effects on the osteoclastogenic process. However, the precise mode of action of TNF-α and IL-1 in osteoclastogenesis remains controversial. While some groups demonstrated that these two cytokines can promote murine osteoclastogenesis in vitro in the presence of M-CSF only, we and others showed that TNF-α-/IL-1-mediated osteoclastogenesis requires permissive levels of RANKL. This chapter describes the method that we have used to investigate the effects of TNF-α and IL-1 on osteoclast formation in in vitro osteoclastogenesis assays using primary murine bone marrow macrophages (BMMs). Detailed experimental conditions are provided and critical points are discussed to help the reader use the method to independently evaluate the roles of TNF-α and IL-1 in osteoclastogenesis in vitro. Moreover, this method can be used to further elucidate the signaling mechanisms by which these two cytokines act in concert with RANKL or with each other to modulate osteoclastogenesis.