Expression of osteoclast differentiation factor in rheumatoid arthritis

Objective. To analyze the expression pattern of osteoclast differentiation factor (ODF) and its contribution to osteoclastogenesis in rheumatoid arthr itis (RA), Methods. The expression of ODF was analyzed by reverse transcriptase-polyme rase chain reaction (RT-PCR) in RA synovial fibroblasts (RASF) isolated fro m 7 RA patients and in normal skin fibroblasts, Using RNA probes specific f or ODF, in situ hybridization was performed. Immunohistochemical double lab eling for CD68 was applied to characterize the ODF-expressing cells. ODF pr otein and messenger RNA (mRNA) expression by RASF with or without 1,25(OH)( 2)D-3 was studied by Western blot analysis and quantitative real-time PCR, In addition, we performed coculture experiments with RASF and normal periph eral blood mononuclear cells with or without 1,25(OH)(2)D-3. Results, 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 l abeling demonstrated ODF mRNA expression mainly in CD68-fibroblast-like syn oviocytes and CD68+ multinucleated osteoclast-like cells. By Western blotti ng, all RASF expressed ODF protein. However, different levels of ODF expres sion were found in the RASF from different patients. Interestingly, RASP ex pressing higher levels of ODF induced a larger number of osteoclast-like ce lls than did RASF expressing only low levels of ODF, Although 1,25(OH)(2)D- 3 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)(2)D-3, Conclusion. The present results suggest that activated RASF, by expressing ODF, play an important role in rheumatoid bone destruction. Moreover, the d ata provide evidence that RASF not only activate osteoclasts, but also cont ribute directly to osteoclastogenesis.