The alterations in cytokine mRNA expressions and productions by fluoride in a murine macrophage cell line evaluated by real-time PCR and ELISA

摘要

We hypothesized that macrophages have a role in the alterations in bone metabolism induced by fluoride via cytokines. In this study, interleukin-10 (IL-10) and IL-12 in addition to tumor necrosis factor α (TNFα) and IL-1β were evaluated for their mRNA expressions in J774.1 cells by quantitative real-time PCR and the protein levels in the supernatant from the cell culture by ELISA at 0, 100, 300, and 1000 μM fluoride. At 18 hours after incubation, J774.1 cells were activated by lipopolysaccharide. At 6 hours after the activation, RNAs of the cells were sampled. The mRNA expressions for TNFα, IL-1β, IL-10, and IL-12p40 were analyzed by real-time PCR. The supernatant from the cell culture were sampled at 24 hours after the activation and determined for these cytokine levels by ELISA.The cell viability in the 1000 μM group at 6 hours after the activation was significantly lower than that in the control. The mRNA expressions of TNFα, IL-1β and IL-10 in the 1000 μM group were significantly higher than those in the control. Although it did not reach the significant level, the mRNA expression of IL-12p40 in the 1000 μM group was elevated more than that in the control. The mean values of concentrations of TNFα and IL-1β in the supernatant in the 1000 μM group were significantly lower than those in the control. While, there was no significant difference for the concentration of IL-10 in the supernatant among the groups. For IL-12p40, the mean value of the concentration in the 1000 μM group was significantly higher than that in the control. The effects of fluoride on the cytokines produced by macrophages were not common for all types of cytokines. In conclusion, the effects of fluoride on the immune system may vary via alterations in cytokine productions by macrophages, resulting in the alterations in bone metabolism.