FIG 2.

Effects of M1 macrophages on osteoclastogenesis induced in BM macrophages. After BM cells were cultured with M-CSF for 6 days, RANKL was applied to the BM culture (day 0). M0, M1, or M2 macrophages were generated ex vivo by incubating BM cells with M-CSF for 6 days, which was followed by incubation using M-CSF with or without LPS, IFN-γ, or IL-4 for an additional 24 h. The harvested M1, M2, and M0 macrophages from ex vivo culture were applied to osteoclasts on day 3 or day 6 (6,000 macrophages/well). On day 7, TRAP staining was performed. (A) Images of TRAP staining for RANKL-stimulated BM cells cocultured with or without polarized macrophages are shown. Bars indicate 100 μm. (B) The number of TRAP⁺ multinuclear osteoclasts was calculated. Data are means ± SD (n = 5). *, significant difference by Tukey's test (P < 0.05). (C) BM cells were cultured for 6 days with M-CSF only, and on day 6, media were changed to M-CSF and RANKL. Three days after RANKL addition, M0, M1, or M2 macrophages were applied to BM culture. After coculture of BM cells and macrophages for an additional 4 days, RNA was extracted and real-time PCR performed. The readout of each sample was converted to a relative expression level (REL) value using beta-actin as the internal control. Data in the graph represent means of REL values ± SD (n = 3). *, significant difference by one-way ANOVA with post hoc Tukey's test (P < 0.05).