The biological roles of the third component of complement in osteoclast formation

We previously reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] tissue-specifically stimulated the production of the third component of complement (C3) in bone in vitro and in vivo. In the present study, we examined the possible roles of C3 in bone using bone marrow cultures with antibodies against C3 and C3 receptors (Mac 1, 8C12, and 7G6) and the purified mouse C3 protein. The C3 protein produced preferentially by stromal cells in response to 1 alpha,25-(OH)2D3 was distributed in macrophage-like mononuclear cells and small cells with few nuclei. Adding anti-C3 antibody together with 1 alpha,25-(OH)2D3 to bone marrow cultures greatly inhibited not only the appearance of tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells, but also the growth of macrophage-like mononuclear cells and stromal cells. The inhibitory effect of anti-C3 antibody on osteoclast-like cell formation was most prominent when it was added between days 2-4 of the 6-day culture period, which corresponded to the late proliferative phase and the early differentiation phase of osteoclast development. Adding anti-C3 receptor antibodies also inhibited osteoclast-like cell formation induced by 1 alpha,25-(OH)2D3. When C3 receptors were detected by the binding of C3-coated sheep red blood cells or immunostaining, the localization of C3 receptor-positive cells coincided exactly with that of C3 protein-positive cells. C3 receptors were expressed mainly in macrophage-like mononuclear cells, TRAP-positive mononuclear cells, and TRAP-positive small cells with few nuclei. TRAP-positive large cells with many nuclei were totally negative for C3 receptors. When macrophage-colony-stimulating factor (M-CSF), the purified C3 protein, and 1 alpha,25-(OH)2D3 were added to bone marrow methylcellulose cultures, separately or in combination, M-CSF-dependent colony formation was strikingly inhibited by 1 alpha,25-(OH)2D3, but the inhibition was prevented by simultaneously adding C3. These results provide additional evidence that osteoclast progenitors are indeed cells of the monocyte-macrophage lineage. It is likely that the C3 produced by stromal cells in response to 1 alpha,25-(OH)2D3 is somehow involved in osteoclast development by potentiating M-CSF-dependent proliferation of bone marrow cells and induction of osteoclast differentiation.