ARTHRITIS & RHEUMATISM Vol. 50, No. 1, January 2004, pp 265–276 DOI 10.1002/art.11419 © 2004, American College of Rheumatology Systemic Tumor Necrosis Factor  Mediates an Increase in Peripheral CD11b high Osteoclast Precursors in Tumor Necrosis Factor –Transgenic Mice Ping Li, Edward M. Schwarz, Regis J. O’Keefe, Lin Ma, R. John Looney, Christopher T. Ritchlin, Brendan F. Boyce, and Lianping Xing Objective. To investigate the mechanisms whereby tumor necrosis factor  (TNF) increases osteoclasto- genesis in vivo. Methods. TNF-transgenic (TNF-Tg) and wild- type mice injected with TNF were studied. In vitro osteoclastogenesis assays, monocyte colony-forming as- says, and fluorescence-activated cell sorting were per- formed using splenocytes, peripheral blood mononuclear cells (PBMCs), and bone marrow cells to quantify and characterize osteoclast precursors (OCPs). Etanercept, a TNF antagonist, was used to block TNF activity in vivo. The effects of TNF on proliferation, apoptosis, and differentiation of OCPs were assessed using 5-bromo-2- deoxyuridine labeling, annexin V staining, and reverse transcriptase–polymerase chain reaction. Results. OCP numbers were increased 4–7-fold in PBMCs and spleen, but not in bone marrow of TNF-Tg mice. The OCPs in spleen were in the CD11b high popu- lation and contained both c-Fms and c-Fms cells. The increased number of OCPs correlated with the initiation of detectable TNF in serum and the onset of inflammatory arthritis in TNF-Tg mice. Etanercept eliminated the increase in peripheral OCPs. TNF did not affect proliferation, survival, or differentiation of CD11b high splenocytes in vivo or in vitro, but caused a rapid increase in CD11b cells in blood within 4 hours of a single injection and an accumulation of CD11b high OCPs in spleen after 3 days of multiple injections. Conclusion. Systemic TNF induces a marked in- crease in circulating OCPs that is reversible by anti-TNF therapy and may result from their mobilization from bone marrow. Our findings provide a new mechanism whereby TNF stimulates osteoclastogenesis in patients with in- flammatory arthritis, suggesting that CD11b PBMCs could be used to evaluate a patient’s potential for erosive disease and the efficacy of anti-TNF therapy. Chronic inflammatory bone diseases, such as rheumatoid arthritis (RA), are accompanied by bone loss around affected joints due to increased osteoclastic resorption. This process is mediated largely by increased local production of proinflammatory cytokines (1,2). These cytokines may act directly on cells in the osteo- clast lineage or indirectly by affecting the production of the essential osteoclast differentiation factor, receptor activator of NF-B ligand (RANKL), and/or its soluble decoy receptor, osteoprotegerin, by osteoblast/stromal cells (3). TNF is a major mediator of inflammation; the importance of TNF in the pathogenesis of various forms of bone loss is supported by several lines of experimental and clinical evidence (4). However, TNF is not essential for osteoclastogenesis (5), erosive arthri- tis (6), or osteolysis (7), because these can occur in the absence of TNF. The critical question of how TNF increases osteoclastogenesis in vivo remains to be an- swered. Osteoclasts are multinucleated cells formed by fu- sion of mononuclear precursors in the monocyte/ macrophage lineage (colony-forming unit–macrophage [CFU-M]). Cell culture techniques (8) and studies of transgenic and knockout mice (9) have advanced our understanding of osteoclastogenesis and established that Supported by USPHS NIH research grants AR-45791, AR- 43510, AR-44220, and AR-48697. Ping Li, PhD, Edward M. Schwarz, PhD, Regis J. O’Keefe, MD, PhD, Lin Ma, MS, R. John Looney, MD, Christopher T. Ritchlin, MD, Brendan F. Boyce, MD, Lianping Xing, PhD: University of Rochester Medical Center, Rochester, New York. Address correspondence and reprint requests to Lianping Xing, PhD, Department of Pathology, University of Rochester Medi- cal Center, 601 Elmwood Avenue, Box 626, Rochester, NY 14642. E-mail: Lianping_Xing@URMC.Rochester.Edu. Submitted for publication March 18, 2003; accepted in revised form September 4, 2003. 265