Combination of Tumor Necrosis Factor α and Interleukin-6 Induces Mouse Osteoclast-like Cells With Bone Resorption Activity Both In Vitro and In Vivo

Objective. To clarify the function of osteoclast-like multinuclear cells differentiated from bone marrow-derived macrophages (BMMs) by a combination of tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6), and to investigate the molecular mechanisms underlying the differentiation. Methods. BMMs were stimulated by TNF alpha and/or IL-6. The cells were then compared with conventional osteoclasts differentiated in vitro by RANKL. An in vitro pit formation assay on dentine slices and an in vivo resorption assay of calvarial bones were performed. We also evaluated the activities and expression levels of NF-kappa B, c-Fos, and NF-ATc1, which are essential to the differentiation of conventional osteoclasts. Small interfering RNA was used to knock down c-Fos. The effects of genetic ablation of STAT-3 and pharmacologic inhibitors of NF-AT, JAK, and ERK were also studied. Results. Osteoclast-like cell differentiation depended on TNF alpha and IL-6 and was not inhibited by osteoprotegerin. These differentiated cells were associated with both in vitro and in vivo bone resorption activity. TNF alpha and IL-6 had a synergistic effect on the activity and expression of c-Fos. Knockdown of c-Fos inhibited the expression of NF-ATc1 and the differentiation of osteoclast-like cells. All of these inhibitors blocked differentiation of the cells in vitro, but surprisingly, the conditional knockout of STAT-3 did not. Tofacitinib also inhibited the bone destruction caused by TNF alpha and IL-6 in vivo. Conclusion. Our results demonstrate that a combination of the inflammatory cytokines TNF alpha and IL-6 can induce osteoclast-like cells that have in vitro and in vivo bone-resorptive activity.