Figure 1.

Effects of Th1, Th2, and T reg cells on in vitro osteoclastogenesis. (A) Schematics of two culture systems for osteoclast differentiation and Th cell addition. In the RANKL–M-CSF system, mouse nonadherent BMCs were stimulated with M-CSF for 2 d and adherent cells were used as BMMs. After BMMs were stimulated with recombinant RANKL and M-CSF for 3 d, the formation of TRAP⁺ MNCs was analyzed. In the co-culture system, BMCs were co-cultured with osteoblasts stimulated with VitD₃ and PGE₂, and the formation of TRAP⁺ MNCs was observed 7 d after the addition of BMCs. (B) Inhibitory effects of Th1 and Th2 cells on TRAP⁺ MNC formation in the RANKL–M-CSF system. Th cells (4,000 or 20,000 cells/ml) were added at the same time as RANKL (day 0) with (black bars) or without (white bars) anti-CD3 mAb. n.d., not detected. (C) Inhibitory effects of Th1 and Th2 cells on TRAP⁺ MNC formation in the co-culture system. The same number of T cells as in B was added 2 d after BMC addition (day 2). (D) Microphotographs of the in vitro osteoclast formation systems in the presence of Th1 or Th2 cells (20,000 cells/ml) with anti-CD3 mAb (TRAP staining). (E) Cytokine profile of culture supernatants in the presence of Th cells and 1 μg/ml of soluble anti-CD3 mAb (the RANKL–M-CSF system on day 2). Without restimulation by anti-CD3 mAb, cytokine production was much less than this result and was difficult to detect after 2-d culture with osteoclast precursor cells (not depicted). (F) Effects of Th1 and Th2 cells (20,000 cells/ml plus anti-CD3 mAb) on WT or IFN-γ receptor–deficient (Ifngr1 ^−/−) osteoclast precursor cells. (G) Effects of isolated CD4⁺CD25⁺ T reg cells (4,000 or 20,000 cells/ml plus anti-CD3 mAb) on osteoclastogenesis in vitro. n.s., not significantly different. The survival of a considerable number of T reg cells after 3 d was confirmed by CFSE staining (not depicted).