Osteoclasts are bone-eroding cells that develop from monocytic precursor cells in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Osteoclasts are essential for physiological bone remodeling, but localized excessive osteoclast activity is responsible for the periarticular bone destruction that characteristically occurs in patients with rheumatoid arthritis (RA). The origin of osteoclasts at sites of bone erosion in RA is unknown. Natural killer (NK) cells, as well as monocytes, are abundant in the inflamed joints of patients with RA. We show here that such NK cells express both RANKL and M-CSF and are frequently associated with CD14 + monocytes in the RA synovium. Moreover, when synovial NK cells are cocultured with monocytes in vitro, they trigger their differentiation into osteoclasts, a process dependent on RANKL and M-CSF. As in RA, NK cells in the joints of mice with collagen-induced arthritis (CIA) express RANKL. Depletion of NK cells from mice before the induction of CIA reduces the severity of subsequent arthritis and almost completely prevents bone erosion. These results suggest that NK cells may play an important role in the destruction of bone associated with inflammatory arthritis.

IntroductionDendritic cells (DCs) are extremely potent antigen-presenting cells (APCs), capable of initiating primary T-cell responses by presenting antigenic peptides in association with major histocompatibility complex (MHC) class I and II molecules on the cell surface (reviewed in Banchereau and Steinman 1 ). Circulating DCs are rare, necessitating the use of in vitro cultivation protocols to generate sufficient DCs from bone marrow or monocytes for in vitro studies or therapeutic use. [2][3][4][5] One such protocol, culturing peripheral blood (PB) derived CD14 ϩ monocytes in the presence of exogenous granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7-10 days, 2 has become the standard for production of human monocyte-derived DCs (moDCs). These immature moDC can be further matured by exposing the cells to toll-like receptor agonists or molecules such as tumor necrosis factor ␣ (TNF␣), interferon-␥ (IFN␥), and CD40 ligand (CD154). 1,6 Although these in vitro methods have been shown to generate moDCs that can induce immune responses in vitro and in vivo, it is unknown whether such cells correspond to any DC subset present in vivo.DCs are believed to contribute to the pathophysiology of inflammatory diseases such as rheumatoid arthritis (RA), which is characterized by chronically inflamed joints and other characteristic findings. 7-9 A variety of leukocytes accumulate in the RA synovium and synovial fluid, including large numbers of monocytes derived from the circulation. 10 DCs are also present in these tissues, although the physiologic processes that guide their differentiation from monocytes are not known.Like monocytes, circulating natural killer (NK) cells are recruited to inflamed tissues, including the RA joint, [11][12][13][14][15] and they coexist in various lymphoid organs with monocytes and DCs. [16][17][18][19][20][21] NK cells were initially defined on the basis of their cytotoxic activity 22 ; this activity, together with their production of both proinflammatory and anti-inflammatory cytokines, is believed to be important in the defense against infectious agents, especially viruses (reviewed in Lanier 23 ). Other studies have suggested that NK cells can regulate myelopoiesis [24][25][26] and induce maturation of GM-CSF and IL-4 derived moDCs in vitro, 27-29 but whether NK cells affect early stages of monocyte differentiation into moDCs is not known. On this basis, and given the colocalization of NK cells with monocytes and DCs in the inflamed RA joint, we reasoned that NK cells might play a role in the differentiation of monocytes into moDCs. Materials and methods Cell isolation and culturePeripheral blood mononuclear cells (PBMCs) from healthy volunteer blood donations at the Stanford Blood Center and synovial fluid mononuclear cells (SFMCs) from patients with immune-mediated arthritis were isolated by Ficoll density gradient centrifugation. CD14 ϩ monocytes and CD56 ϩ NK cells were purified from PBMCs or SFMCs using anti-CD14 and anti-CD56-coated microbeads (...

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