Sato T, Abe E, Jin C H, Hong M H, Katagiri T, Kinoshita T, Amizuka N, Ozawa H, Suda T
Research Laboratories 3, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan.
Endocrinology. 1993 Jul;133(1):397-404. doi: 10.1210/endo.133.1.8319587.
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.
我们之前报道过，1α,25 - 二羟基维生素D3[1α,25-(OH)2D3]在体外和体内均能特异性地刺激骨中补体第三成分（C3）的产生。在本研究中，我们使用针对C3和C3受体（Mac 1、8C12和7G6）的抗体以及纯化的小鼠C3蛋白，通过骨髓培养来研究C3在骨中的可能作用。由基质细胞响应1α,25-(OH)2D3优先产生的C3蛋白分布于巨噬细胞样单核细胞和少核小细胞中。在骨髓培养物中加入抗C3抗体和1α,25-(OH)2D3，不仅显著抑制了抗酒石酸酸性磷酸酶（TRAP）阳性单核细胞和多核细胞的出现，还抑制了巨噬细胞样单核细胞和基质细胞的生长。抗C3抗体对破骨细胞样细胞形成的抑制作用在6天培养期的第2 - 4天添加时最为显著，这对应于破骨细胞发育的增殖后期和分化早期。添加抗C3受体抗体也抑制了1α,25-(OH)2D3诱导的破骨细胞样细胞形成。当通过包被C3的绵羊红细胞结合或免疫染色检测C3受体时，C3受体阳性细胞的定位与C3蛋白阳性细胞的定位完全一致。C3受体主要在巨噬细胞样单核细胞、TRAP阳性单核细胞和少核TRAP阳性小细胞中表达。多核TRAP阳性大细胞对C3受体完全呈阴性。当将巨噬细胞集落刺激因子（M - CSF）、纯化的C3蛋白和1α,25-(OH)2D3单独或联合添加到骨髓甲基纤维素培养物中时，1α,25-(OH)2D3显著抑制了M - CSF依赖性集落形成，但同时添加C3可防止这种抑制。这些结果提供了额外的证据，表明破骨细胞祖细胞确实是单核细胞 - 巨噬细胞谱系的细胞。基质细胞响应1α,25-(OH)2D3产生的C3可能通过增强骨髓细胞的M - CSF依赖性增殖和诱导破骨细胞分化，以某种方式参与破骨细胞的发育。
