FIG 9.

Induction of caspase-3 in RANKL-stimulated BM cells by recombinant IL-12. Following 6 days of pretreatment of BM cells with M-CSF alone, the resulting BM cells were stimulated with RANKL in the presence or absence of recombinant IL-12 (50 ng/ml) for 48, 72, or 96 h and harvested for the following experiments. (A) Caspase-3 mRNA expression in the harvested cells was analyzed by real-time PCR. Asterisks indicate significant differences (P < 0.05) by t test between two groups at the same time point. Data are means of REL and SD (n = 3). (B and C) Representative images of annexin V staining (B) and percentages of annexin V-positive apoptotic cells (C) at 96 h after the stimulation with RANKL with or without IL-12 in the presence or absence of anti-IL-12 neutralizing Ab are shown. For determinations of the percentages of apoptotic cells, the number of apoptotic cells among all cells in the microscopic field (area, 100 μm by 100 μm) was counted and converted to a percentage by the following formula: [(number of apoptotic cells)/(number of all the cells) × 100]. The asterisks show significant differences (P < 0.05) by Tukey's test. Data are means of REL and SD (n = 3). The scale bar indicates 10 μm. (D and E) The effects of conditioned medium isolated from M1 and M2 of IFN-γ KO mice on the induction of annexin V expression by RANKL-stimulated BM cells were examined. Annexin V expression (D) and percentages of annexin V-positive apoptotic cells (E) are shown. After prestimulation with RANKL alone for 3 days, BM-derived macrophages were further incubated with the culture supernatant of macrophage (M0 or M1) derived from IFN-γ KO mice and RANKL in the presence or absence of anti-IL-12 neutralizing Ab for 96 h and subjected to annexin V staining. Measurement of percentages of annexin V⁺ apoptotic cells was carried out following the protocol shown in panel C. The asterisks indicate significant differences (P < 0.05) by Tukey's test. Data are means of REL and SD (n = 3). Scale bar, 10 μm.