Characterization and identification of subpopulations of mononuclear preosteoclasts induced by TNF-α in combination with TGF-ß in rats.

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-ß, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-ß specifically increased Kat1(+)c-fms(+) and Kat1(+)c-fms(-) cells but not Kat1(-)c-fms(+) cells. Kat1(-)c-fms(+) cells appeared in early stages of culture, but Kat1(+)c-fms(+) and Kat1(+)c-fms(-) cells increased later. Preosteoclasts induced by TNF-α, TGF-ß, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-ß, and M-CSF induced Kat1(+)CD11b(+) cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1(+)c-fms(+) cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-ß efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.

Infection of RANKL-primed RAW-D macrophages with Porphyromonas gingivalis promotes osteoclastogenesis in a TNF-α-independent manner.

Infection of macrophages with bacteria induces the production of pro-inflammatory cytokines including TNF-α. TNF-α directly stimulates osteoclast differentiation from bone marrow macrophages in vitro as well as indirectly via osteoblasts. Recently, it was reported that bacterial components such as LPS inhibited RANKL-induced osteoclastogenesis in early stages, but promoted osteoclast differentiation in late stages. However, the contribution to osteoclast differentiation of TNF-α produced by infected macrophages remains unclear. We show here that Porphyromonas gingivalis, one of the major pathogens in periodontitis, directly promotes osteoclastogenesis from RANKL-primed RAW-D (subclone of RAW264) mouse macrophages, and we show that TNF-α is not involved in the stimulatory effect on osteoclastogenesis. P. gingivalis infection of RANKL-primed RAW-D macrophages markedly stimulated osteoclastogenesis in a RANKL-independent manner. In the presence of the TLR4 inhibitor, polymyxin B, infection of RANKL-primed RAW-D cells with P. gingivalis also induced osteoclastogenesis, indicating that TLR4 is not involved. Infection of RAW-D cells with P. gingivalis stimulated the production of TNF-α, whereas the production of TNF-α by similarly infected RANKL-primed RAW-D cells was markedly down-regulated. In addition, infection of RANKL-primed macrophages with P. gingivalis induced osteoclastogenesis in the presence of neutralizing antibody against TNF-α. Inhibitors of NFATc1 and p38MAPK, but not of NF-κB signaling, significantly suppressed P. gingivalis-induced osteoclastogenesis from RANKL-primed macrophages. Moreover, re-treatment of RANKL-primed macrophages with RANKL stimulated osteoclastogenesis in the presence or absence of P. gingivalis infection, whereas re-treatment of RANKL-primed macrophages with TNF-α did not enhance osteoclastogenesis in the presence of live P. gingivalis. Thus, P. gingivalis infection of RANKL-primed macrophages promoted osteoclastogenesis in a TNF-α independent manner, and RANKL but not TNF-α was effective in inducing osteoclastogenesis from RANKL-primed RAW-D cells in the presence of P. gingivalis.