M1 Macrophage-Derived Interleukin-6 Promotes the Osteogenic Differentiation of Ligamentum Flavum Cells

STUDY DESIGN: Basic experimental study. OBJECTIVE: The aim of this study was to clarify the role of macrophages (Mφs) in the osteogenic differentiation of ligamentum flavum (LF) cells. SUMMARY OF BACKGROUND DATA: Mφs and secreted factors are involved in the regulation of cell osteogenic differentiation, and play an important role in the process of heterotopic ossification. Whether Mφs are involved in the development of ossification of the ligamentum flavum (OLF) have not been reported. METHODS: The expression of CD68+ Mφs in ossified LF tissue was identified by immunohistochemical staining. THP-1 cells were polarized to M1 and M2, and identified by flow cytometry and immunofluorescence. The alkaline phosphatase activity and osteogenic differentiation-related gene expression in LF cells were evaluated following incubation with each Mφs conditioned medium (CM). Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines in the supernatants, and qPCR was used to detect the expression of the corresponding receptors in the LF cells after incubation with the CM. LF cells were induced with CM-M1 in the presence of neutralizing antibodies to further test whether cytokines secreted by M1 Mφs impacted their osteogenic differentiation. RESULTS: CD68+ Mφs were found on the OLF samples. THP-1 cells were polarized into M1 and M2, and both M1 and M2 Mφs promoted the osteogenic differentiation of LF cells. The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, and IL-6 in M1 Mφ supernatants were greater than those in M2, and greater levels of these cytokine receptors were observed in LF cells induced with CM-M1 than those with CM-M2. Osteogenic differentiation of LF cells induced by CM-M1 decreased after IL-6 was neutralized; however, not after IL-1β and TNF-α were neutralized. CONCLUSION: M1 Mφ-derived IL-6 promotes the osteogenic differentiation of LF cells, which may be a pathway in which Mφs regulate the osteogenic differentiation of LF cells.