Shigeyama Y, Pap T, Kunzler P, Simmen B R, Gay R E, Gay S
WHO Collaborating Center for Molecular Biology and Novel Therapeutic Strategies for Rheumatic Diseases, University Hospital, Zurich, Switzerland.
Arthritis Rheum. 2000 Nov;43(11):2523-30. doi: 10.1002/1529-0131(200011)43:11<2523::AID-ANR20>3.0.CO;2-Z.
To analyze the expression pattern of osteoclast differentiation factor (ODF) and its contribution to osteoclastogenesis in rheumatoid arthritis (RA).
The expression of ODF was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in RA synovial fibroblasts (RASF) isolated from 7 RA patients and in normal skin fibroblasts. Using RNA probes specific for ODF, in situ hybridization was performed. Immunohistochemical double labeling for CD68 was applied to characterize the ODF-expressing cells. ODF protein and messenger RNA (mRNA) expression by RASF with or without 1,25(OH)2D3 was studied by Western blot analysis and quantitative real-time PCR. In addition, we performed coculture experiments with RASF and normal peripheral blood mononuclear cells with or without 1,25(OH)2D3.
By RT-PCR, ODF mRNA expression was found in all RASF investigated, but not in normal skin fibroblasts. In situ hybridization revealed that in RA synovial tissues, ODF mRNA was expressed mainly in the lining layer and at sites where synovium was attached to bone. Immunohistochemical double labeling demonstrated ODF mRNA expression mainly in CD68-fibroblast-like synoviocytes and CD68+ multinucleated osteoclast-like cells. By Western blotting, all RASF expressed ODF protein. However, different levels of ODF expression were found in the RASF from different patients. Interestingly, RASF expressing higher levels of ODF induced a larger number of osteoclast-like cells than did RASF expressing only low levels of ODF. Although 1,25(OH)2D3 did not alter the levels of ODF expression in RASF on either Western blot or quantitative real-time PCR, osteoclastogenesis required the presence of 1,25(OH)2D3.
The present results suggest that activated RASF, by expressing ODF, play an important role in rheumatoid bone destruction. Moreover, the data provide evidence that RASF not only activate osteoclasts, but also contribute directly to osteoclastogenesis.
分析破骨细胞分化因子（ODF）的表达模式及其在类风湿关节炎（RA）破骨细胞形成中的作用。
采用逆转录聚合酶链反应（RT-PCR）分析从7例RA患者分离的RA滑膜成纤维细胞（RASF）及正常皮肤成纤维细胞中ODF的表达。使用ODF特异性RNA探针进行原位杂交。应用CD68免疫组化双重标记来鉴定表达ODF的细胞。通过蛋白质免疫印迹分析和定量实时PCR研究有或无1,25(OH)2D3时RASF中ODF蛋白和信使核糖核酸（mRNA）的表达。此外，我们进行了RASF与正常外周血单个核细胞在有或无1,25(OH)2D3情况下的共培养实验。
通过RT-PCR发现，在所研究的所有RASF中均有ODF mRNA表达，而正常皮肤成纤维细胞中未检测到。原位杂交显示，在RA滑膜组织中，ODF mRNA主要表达于衬里层以及滑膜与骨附着处。免疫组化双重标记表明ODF mRNA主要表达于CD68 - 成纤维样滑膜细胞和CD68 + 多核破骨样细胞。通过蛋白质免疫印迹分析，所有RASF均表达ODF蛋白。然而，不同患者的RASF中ODF表达水平不同。有趣的是，与仅低水平表达ODF的RASF相比，高水平表达ODF的RASF诱导产生的破骨样细胞数量更多。尽管1,25(OH)2D3在蛋白质免疫印迹分析或定量实时PCR中均未改变RASF中ODF的表达水平，但破骨细胞形成需要1,25(OH)2D3的存在。
目前的结果表明，活化的RASF通过表达ODF在类风湿性骨破坏中起重要作用。此外，数据表明RASF不仅激活破骨细胞，还直接促进破骨细胞形成。
