AB0095 Attempt to develop a chimeric co-culture system to differentiate mouse osteoclasts by culturing mouse precursor cells with human synovial fibroblasts. (12th June 2018)

• Record Type: Journal Article
• Authors: Yazawa, H.; Yanagisawa, M.; Aizaki, Y.; Araki, Y.; Sato, K.; Mimura, T.
• Is Part Of: Annals of the rheumatic diseases. Volume 77(2018)Supplement 2
• Page Start: 1243
• Page End: 1243
• Publication Date: 2018-06-12
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2018-eular.3574
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : Background: In rheumatoid arthritis (RA), inflammatory synovial tissue called the pannus proliferates and erodes the articular cartilage and bone in the affected joints. Osteoclasts, multinucleated cells of monocyte/macrophage lineage, are implicated in the bone destruction in RA. Thus, osteoclasts are considered an important therapeutic target in the prevention of the joint destruction. Mouse bone marrow cells differentiate into osteoclasts when co-cultured with osteoblasts or stromal cells in the presence of reagents such as 1, 25-dihydroxyvitamin D3 (1, 25(OH)2 D3 ) and prostaglandin E2 (PGE2 ). There are no osteoblasts in the RA synovium, but there are fibroblasts and various inflammatory cells such as macrophages and lymphocytes. Thus, synovial fibroblasts may function as supporting cells for osteoclastogenesis in place of osteoblasts. Objectives: The aim of this study was to establish a chimeric co-culture system of osteoclast differentiation using human synovial fibroblasts and mouse monocyte/macrophage lineage cells. Methods: Synovial tissues were obtained from RA patients who underwent joint replacement surgery. Mouse osteoblasts were obtained from the calvariae of 2- or 3-day-old newborn C57BL/6 (B6) mice. Mouse bone marrow cells were prepared from femoral bones. Osteoclasts were visualised with tartrate-resistant acid phosphatase (TRAP) staining. The protein levels of RANKL and its decoy receptor, osteoprotegerin (OPG), in the culture supernatant were quantified using ELISA. Results: We confirmed that mouse osteoclasts could be differentiated in vitro by culturing bone marrow cells in the presence of human M-CSF and RANKL. We then cultured mouse osteoclast precursors with human synovial fibroblasts in the presence of 1, 25(OH)2 D3 and PGE2 . The murine cells seemed to disappear in the course of the co-culture, whereas they survived when exogenous human M-CSF was added to the system. Interestingly, however, they did not become TRAP-positive multinuclear cells, suggesting that synovial fibroblasts do not provide a sufficient amount of the osteoclast differentiation factor, RANKL. Following these results, we used ELISA to quantify the level of human RANKL and OPG in the culture supernatant of synovial fibroblasts. Predictably, the level of RANKL was below the detection limit with or without the presence of 1, 25(OH)2 D3 and/or PGE2 . In contrast, that of OPG was very high, irrespective of the reagents added. We also quantified the levels of mouse RANKL and OPG in the culture supernatant of mouse osteoblasts. As expected, RANKL was detectable in this case. Interestingly, the level of OPG was very high and comparable to that of human OPG produced from synovial fibroblasts. Conclusions: Synovial fibroblasts cannot be substituted for osteoblasts in a co-culture system of osteoclast differentiation. This is probably because synovial fibroblasts do not provide sufficient RANKL and M-CSF. Instead, they produce a large amount of OPG. This may be a mechanism by which ectopic osteoclastogenesis is inhibited. Thus, the osteoclasts observed in the pannus may be dependent on membrane-bound RANKL from other sources, like lymphocytes, or may be differentiated by stimulation with cytokines other than RANKL, such as TNF-α and IL-6. 1 2 References: [1] Yokota K, et al. Arthritis Rheumatol2014;66:121–9. [2] O'Brien W, et al. Arthritis Rheumatol2016;68:2889–2900. Disclosure of Interest: None declared