Production of RANKL by Memory B Cells: A Link Between B Cells and Bone Erosion in Rheumatoid Arthritis Nida Meednu, PhD, Hengwei Zhang, PhD, Teresa Owen, BS, Wen Sun, PhD, Victor Wang, Christopher Cistrone, MS, Javier Rangel-Moreno, PhD, Lianping Xing, BMed, PhD, and Jennifer H. Anolik, MD, PhD University of Rochester Medical Center, Rochester, New York Abstract Objective—Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage. The mechanisms of bone damage in RA are complex, involving activation of bone- resorbing osteoclasts (OCs) by synoviocytes and Th17 cells. This study was undertaken to investigate whether B cells play a direct role in osteoclastogenesis through the production of RANKL, the essential cytokine for OC development. Methods—RANKL production by total B cells or sorted B cell subpopulations in the peripheral blood and synovial tissue from healthy donors or anti–cyclic citrullinated peptide– positive patients with RA was examined by flow cytometry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. To define direct effects on osteoclastogenesis, B cells were cocultured with CD14+ monocytes, and OCs were enumerated by tartrate-resistant acid phosphatase staining. Results—Healthy donor peripheral blood B cells were capable of expressing RANKL upon stimulation, with switched memory B cells (CD27+IgD−) having the highest propensity for RANKL production. Notably, switched memory B cells in the peripheral blood from RA patients expressed significantly more RANKL compared to healthy controls. In RA synovial fluid and tissue, memory B cells were enriched and spontaneously expressed RANKL, with some of these cells visualized adjacent to RANK+ OC precursors. Critically, B cells supported OC differentiation in vitro in a RANKL-dependent manner, and the number of OCs was higher in cultures with RA B cells than in those derived from healthy controls. Conclusion—These findings reveal the critical importance of B cells in bone homeostasis and their likely contribution to joint destruction in RA. Address correspondence to Jennifer H. Anolik, MD, PhD, Department of Medicine, Division of Allergy, Immunology and Rheumatology, University of Rochester, 601 Elmwood Avenue, Box 695, Rochester, NY 14642. Jennifer_anolik@urmc.rochester.edu. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Anolik had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Meednu, Xing, Anolik. Acquisition of data. Meednu, Zhang, Owen, Sun, Wang, Cistrone, Rangel-Moreno, Xing, Anolik. Analysis and interpretation of data. Meednu, Zhang, Sun, Rangel-Moreno, Xing, Anolik. HHS Public Access Author manuscript Arthritis Rheumatol. Author manuscript; available in PMC 2016 July 21. Published in final edited form as: Arthritis Rheumatol. 2016 April ; 68(4): 805–816. doi:10.1002/art.39489. Author Manuscript Author Manuscript Author Manuscript Author Manuscript