Increase in expression of receptor activator of nuclear factor κB at sites of bone erosion correlates with progression of inflammation in evolving collagen‐induced arthritis

Objective

The receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) pathway is critical in osteoclastogenesis and bone resorption and has been implicated in the process of focal bone erosion in arthritis. This study was undertaken to identify in vivo the hitherto‐unknown origin and localization of RANK‐expressing osteoclast precursor cells at sites of bone erosion in arthritis.

Methods

DBA‐1 mice were immunized with bovine type II collagen/Freund's complete adjuvant and were given an intraperitoneal booster injection of type II collagen on day 21. Arthritis was monitored visually, and joint pathology was examined histologically. RANK and RANKL expression were analyzed using specific immunohistochemistry, and tartrate‐resistant acid phosphatase (TRAP) staining was performed. In addition, TRAP and cathepsin K messenger RNA expression were analyzed by in situ hybridization.

Results

A marked increase in the number of cells expressing RANK correlated with the progression of synovial inflammation and clinical disease severity in evolving collagen‐induced arthritis (CIA). Interestingly, RANK expression demonstrated a gradient pattern with increased numbers of RANK‐positive cells within the synovial infiltrate in areas closer to periosteum and cortical bone. Cells expressing RANK included cells in synovial tissue, bone lining cells on the surface of trabecular bone at sites of erosion, and cells in periosteal areas adjacent to synovial inflammation. In areas where RANK‐positive cells were abundant, TRAP‐positive, multinucleated osteoclast‐like cells were also present at sites of focal bone erosion, suggesting differentiation of synovially derived RANK‐positive osteoclast precursor cells into osteoclasts. In addition, TRAP– and cathepsin K–double‐positive osteoclast‐like cells were detected on the synovial side of cortical bone at sites of early and advanced cortical bone erosion. Sites of RANK expression also correlated well with sites of RANKL expression, and there was a close correlation of the temporal expression of the receptor–ligand pair.

Conclusion

Cells expressing RANK increased in abundance with the progression of arthritis in evolving CIA, and sites of RANK‐expressing cells correlated with sites of TRAP‐positive, multinucleated osteoclast‐like cells as well as with sites of RANKL expression. These data support the hypothesis that the RANK/RANKL pathway plays an important role in the process of bone erosion in CIA.

     

Expression of osteoclast differentiation factor at sites of bone erosion in collagen-induced arthritis

OBJECTIVE: To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA). METHODS: After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe. RESULTS: TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion. CONCLUSION: Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.     

The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation

OBJECTIVE: Spondylarthritis (SpA) and rheumatoid arthritis (RA) have different patterns of bone damage, with more pronounced bone erosions in RA. The RANK/RANKL/osteoprotegerin (OPG) system plays a central role in bone resorption by promoting the maturation and activation of osteoclasts. To assess the potential role of this system in the distinct bone phenotype, we studied the synovial expression of these mediators in SpA and RA peripheral synovitis. METHODS: Synovial biopsy specimens were obtained from the actively inflamed peripheral joints of 35 patients with SpA and 19 patients with RA. Paired synovial biopsy samples were obtained from 24 patients with SpA after tumor necrosis factor alpha (TNFalpha) blockade. Synovial tissue sections were immunostained for RANKL, OPG, RANK, and TRAP and assessed by semiquantitative scoring and digital image analysis. RESULTS: After extensive validation of the reactivity and specificity of the antibodies, we demonstrated the abundant expression of RANKL and OPG in SpA synovitis. RANKL was expressed by both fibroblast-like synoviocytes and sublining T lymphocytes. RANK-positive osteoclast precursors but no mature TRAP-positive osteoclasts were present in the inflamed tissue. The expression of these mediators was not different between patients with nonpsoriatic SpA, patients with psoriatic SpA, and patients with RA, was not related to the degree of systemic or local inflammation, and was not significantly modulated by highly effective treatment with TNFalpha blockers. Only the subset of patients with the best systemic response to TNFalpha blockade had decreased RANKL expression in the intimal lining layer. CONCLUSION: The relative protection against bone erosions in SpA cannot be explained by qualitative or quantitative differences in the synovial expression of RANKL, OPG, and RANK. The abundant expression of these factors in SpA peripheral synovitis is largely disconnected from systemic and local inflammation.     

RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis

OBJECTIVES: Receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been demonstrated to be critical regulators of osteoclast generation and activity. In addition, RANKL has been implicated as an important mediator of bone erosion in rheumatoid arthritis (RA). However, the expression of RANKL and OPG at sites of pannus invasion into bone has not been examined. The present study was undertaken to further elucidate the contribution of this cytokine system to osteoclastogenesis and subsequent bone erosion in RA by examining the pattern of protein expression for RANKL, OPG and the receptor activator of NF-kappaB (RANK) in RA at sites of articular bone erosion. METHODS: Tissues from 20 surgical procedures from 17 patients with RA were collected as discarded materials. Six samples contained only synovium or tenosynovium remote from bone, four samples contained pannus-bone interface with adjacent synovium and 10 samples contained both synovium remote from bone and pannus-bone interface with adjacent synovium. Immunohistochemistry was used to characterize the cellular pattern of RANKL, RANK and OPG protein expression immediately adjacent to and remote from sites of bone erosion. RESULTS: Cellular expression of RANKL protein was relatively restricted in the bone microenvironment; staining was focal and confined largely to sites of osteoclast-mediated erosion at the pannus-bone interface and at sites of subchondral bone erosion. RANK-expressing osteoclast precursor cells were also present in these sites. OPG protein expression was observed in numerous cells in synovium remote from bone but was more limited at sites of bone erosion, especially in regions associated with RANKL expression. CONCLUSIONS: The pattern of RANKL and OPG expression and the presence of RANK-expressing osteoclast precursor cells at sites of bone erosion in RA contributes to the generation of a local microenvironment that favours osteoclast differentiation and activity. These data provide further evidence implicating RANKL in the pathogenesis of arthritis-induced joint destruction.     

Enhanced osteoclastogenesis in patients with tophaceous gout: urate crystals promote osteoclast development through interactions with stromal cells

OBJECTIVE: To analyze cellular mechanisms of bone erosion in gout. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with gout were analyzed for the presence of osteoclast precursors. Fixed tophus and bone samples were analyzed by immunohistochemistry. Mechanisms of osteoclastogenesis were studied by culturing murine preosteoclast RAW 264.7 cells, bone marrow stromal ST2 cells, and human synovial fibroblasts with monosodium urate monohydrate (MSU) crystals. RESULTS: PBMCs from patients with severe erosive gout had the preferential ability to form osteoclast-like cells in culture with RANKL and monocyte colony-stimulating factor (M-CSF). The number of PBMC-derived tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells strongly correlated with the number of tophi (r = 0.6296, P = 0.630). Patients with severe erosive and tophaceous gout also had higher circulating concentrations of RANKL and M-CSF. Furthermore, greater numbers of TRAP-positive multinucleated cells were cultured from SFMCs derived from gouty knee effusions than from paired PBMCs (P = 0.004). Immunohistochemical analysis demonstrated numerous multinucleated cells expressing osteoclast markers within tophi and at the interface between soft tissue and bone. MSU crystals did not directly promote osteoclast formation from RAW 264.7 cells in vitro. However, MSU crystals inhibited osteoprotegerin gene and protein expression in ST2 cells and human synovial fibroblasts, without significantly altering RANKL gene expression. Conditioned medium from ST2 cells cultured with MSU crystals promoted osteoclast formation from RAW 264.7 cells in the presence of RANKL. CONCLUSION: Chronic tophaceous and erosive gout is characterized by enhanced osteoclast development. These data provide a rationale for the study of osteoclast-targeted therapies for the prevention of bone damage in chronic gout.     

OPG inhibits gene expression of RANK and CAII in mouse osteoclast-like cell

This study was designed to determine the effects of the osteoprotegerin (OPG) on the mRNA expression of carbonic anhydrase II (CAII) and the receptor activator of NF-??B (RANK) in mouse osteoclast-like cells. Marrow cells were harvested from femora and tibiae of mouse and cultured in 6-well chamber slides. After 1?day of incubation, the marrow cells were exposed to M-CSF (25?ng/ml), RANKL (50?ng/ml), and different concentrations of OPG (50, 75, and 100?ng/ml, respectively) for 3?days. Osteoclast-like cells were confirmed by both tartrate-resistant acid phosphatase (TRAP) stain and bone resorption assay. The expression of RANK and CAIImRNA was determined with real-time fluorescent quantitative polymerase chain reaction. The numbers of multinucleated, TRAP-positive osteoclast-like cells, and resorption pits formed were observed. Compared with the M-CSF?+?RANKL group, RANKmRNA expression was statistically decreased in the M-CSF and M-CSF?+?RANKL?+?OPG (100?ng/ml) groups (P?=?0.004, P?=?0.024, respectively); Compared with the M-CSF, M-CSF?+?RANKL, and M-CSF?+?RANKL?+?OPG (100?ng/ml) group, CAIImRNA expression in the M-CSF?+?RANKL?+?OPG (75?ng/ml) groups was statistically decreased (P?=?0.001, P?=?0.008, and P?=?0.036, respectively). These data suggest that OPG could regulate the expression of RANK and CA II mRNA in the marrow culture system.     

Role of ascorbic acid in the osteoclast formation: induction of osteoclast differentiation factor with formation of the extracellular collagen matrix

Osteoclasts are bone-resorbing multinucleated cells. Tartrate-resistant acid phosphatase-positive (TRAP-positive) mononuclear and multinucleated cells, which are osteoclast-like cells (OCLs), were formed as a result of the coculture of mouse bone marrow cells and clonal stromal ST2 cells in the presence of 1alpha,25-dihydroxy-vitamin D3. Removal of ascorbic acid from the culture medium prevented the formation of TRAP-positive OCLs. Addition of ascorbic acid to the medium formed TRAP-positive OCLs, and the effect of ascorbic acid was dose-dependent. When we examined the level of messenger RNA (mRNA) for osteoclast differentiation factor (RANKL/ODF) in ST2 cells, we found that ascorbic acid caused an approximately 5-fold increase in the level of this mRNA. The half-life of the mRNA was unaffected by ascorbic acid. To characterize the mechanism of action of ascorbic acid, we investigated the relationship between formation of TRAP-positive OCLs and formation of the collagen matrix. Inhibitors of the formation of collagen triple helices blocked both the formation of TRAP-positive OCLs and the expression of the mRNA for RANKL/ODF in response to ascorbic acid. Our findings suggest that ascorbic acid might be essential for osteoclastogenesis and might induce the formation of TRAP-positive OCLs via induction of the synthesis of RANKL/ODF that is somehow mediated by the extracellular matrix.     

Mechanisms of Disease: the link between RANKL and arthritic bone disease

Chronic inflammation and bone loss are closely linked pathophysiologic events. The most typical example of inflammatory bone loss is seen in patients with rheumatoid arthritis who develop systemic osteopenia as well as local breakdown of bone in the direct vicinity of inflamed joints. Understanding the mechanisms of arthritic bone degradation is crucial for designing therapies that can specifically protect joints from structural damage. Since osteoclast differentiation and activity are key events in arthritic bone damage, the signals that trigger osteoclastogenesis are potential therapeutic targets. Receptor activator of nuclear factor-kappaB (RANK) is activated by its ligand, RANKL, an essential molecule for osteoclast development: in the absence of RANKL or RANK, osteoclast differentiation from monocyte precursors does not occur. RANKL is expressed on T cells and fibroblasts within the synovial inflammatory tissue of patients with RA and its expression is regulated by proinflammatory cytokines. In animal models of arthritis, blockade of RANKL-RANK interactions, or a genetic absence of RANKL or RANK, protects against joint damage despite the presence of joint inflammation. Therefore, inhibition of RANKL is regarded as a promising future strategy for inhibiting inflammatory bone loss in patients with chronic inflammatory arthritis.     

Receptor activator of nuclear factor kappaB ligand is expressed in resident and inflammatory cells in aseptic and septic prosthesis loosening

OBJECTIVE: The pathogenesis of periprosthetic bone loss in aseptic and septic prosthesis loosening is unclear. There is considerable evidence that macrophages and osteoclasts play a key role in focal bone erosion and osteolysis around the prosthesis. RANKL (receptor activator of nuclear factor kappaB ligand) was shown to be a potent osteoclastogenic factor, and to be involved in bone destruction of myeloma and rheumatoid arthritis patients. Osteoprotegerin (OPG) is the natural RANKL inhibitor and may prevent periprosthetic bone loss. METHODS: The presence and distribution of RANKL, its receptor RANK and OPG in the periprosthetic interface of septically (n = 5) and aseptically (n = 6) loosened prostheses was examined by immunohistochemistry and immunoblotting. Additionally, the immunophenotype of the inflammatory infiltrate was determined [CD3, CD68, Ki-67, tartrate-resistant acid posphatase (TRAP)]. RESULTS: Aseptic and septic cases revealed a different histopathologic pattern. However, in all cases RANKL and RANK could be demonstrated in macrophages and giant cells. In addition, RANKL detected by immunoblot analysis proved to have the same molecular weight as a recombinant RANKL used as a control (31 kD and approximately 48 kD). OPG was detected in aseptic loosening, where macrophages showed a strong staining, but multinucleated giant cells were only weakly stained. A weak OPG staining was also observed in septic loosening. CONCLUSION: The pathogenesis of bone loss in septic loosening remains unclear, because the septic membrane bears few macrophages and giant cells, and half of them express OPG. In aseptic loosening, macrophages might not be stimulated by RANKL as a result of OPG expression. But multinucleated giant cells may be activated, as they hardly express OPG. They might be responsible for periprosthetic bone loss in aseptic loosening as a result of their RANKL and RANK expression.     

Minodronic acid influences receptor activator of nuclear factor kB ligand expression and suppresses bone resorption by osteoclasts in rats with collagen-induced arthritis

We investigated the inhibitory mechanism of bone resorption by minodronic acid in collagen-induced arthritis (CIA) in rats. Four groups of female Sprague–Dawley rats, aged 7 months, were studied: three groups of collagen-sensitized rats, including one placebo-administered group (CIA-P), and two minodronic acid-administered groups at 0.2 mg/kg/2 day (CIA-BIS) and 2.0 mg/kg/2 day (CIA-BIS10). These were studied with an additional untreated observation group (Cont group). Minodronic acid was administered orally a day after the initial sensitization. The femoral posteromedial condyle was analyzed histologically and immunohistologically 4 weeks after the initial sensitization. Western blotting was also performed to assess the receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) expression of the knee joints. In CIA-P rats, many tartrate-resistant acid phosphatase (TRAP)-positive cells were found at the pannus-lining layer and the epiphyseal medulla. The bone-lining cells in the epiphyseal medulla and the cells in the pannus strongly expressed RANK and RANKL. In the minodronic acid-administered group, the number of TRAP-positive cells and the severity of arthritis were reduced. The reduction in the CIA-BIS10 group was significant compared with the CIA-P group (P < 0.05). Dosage-dependent reduction of RANK and RANKL expression was confirmed by immunohistology and Western blotting. With or without minodronic acid administration, no apoptotic cells were found in any groups using the TdT-mediated dUTP-biotinnick end labeling (TUNEL) method. The expression of OPG was not clear in all groups. These results demonstrated that minodronic acid inhibited the differentiation and the activation of osteoclasts not by inducing apoptosis but by inhibiting the RANKL–RANK system, and thereby suppressing bone resorption.     

白细胞介素-23刺激的类风湿关节炎成纤维细胞对人破骨样细胞形成的研究

目的 探讨向细胞介素(IL)-23刺激的类风湿关节炎(RA)滑膜成纤维细胞(FLS)在人破骨样细胞形成中的作用.方法 用不同浓度(1、5和10 ng/ml)的IL-23刺激RA和骨关节炎(OA)患者滑膜FLS 72 h,实时荧光定量聚合酶链反应(real time-PCR)检测RA和OA滑膜FLS核因子κB受体激活剂配体(RANKL)mRNA表达.分离人外周血单核细胞(MN),与IL-23刺激的RA和OA滑膜FLS共培养,通过抗酒石酸酸性磷酸酶(TRAP)染色观察是否有破骨样细胞形成;同时,real time-PCR法检测破骨样细胞形成的分子标志.结果 不同浓度的IL-23均能上调RA滑膜FLS RANKL的表达;但几乎不诱导OA滑膜细胞RANKL的表达.在IL-23刺激的RA FLS和MN共培养体系中能观察到TRAP染色阳性的破骨样细胞形成;并且破骨样细胞形成的分子标志表达增高.而无IL-23刺激的RA FLS或IL-23刺激的OAFLS和MN共培养体系中未见TRAP染色阳性的破骨样细胞的形成.结论 IL-23通过诱导RA滑膜细胞RANKL的表达促进人MN分化衍生为破骨样细胞.     

Synovial fibroblasts promote osteoclast formation by RANKL in a novel model of spontaneous erosive arthritis

OBJECTIVE: Erosion of cartilage and bone is a hallmark of rheumatoid arthritis (RA). This study was undertaken to explore the roles of hyperproliferating synovial fibroblasts and macrophages in abnormal osteoclast formation, using the recently described BXD2 mouse model of RA. METHODS: Cell distribution in the joints was analyzed by immunohistochemistry, using tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclasts. To identify the defective cells in BXD2 mice, mouse synovial fibroblasts (MSFs) were cultured with bone marrow-derived macrophages. Osteoclast formation was assayed by TRAP staining and bone resorption pit assay, and the cytokine profiles of the MSFs and macrophages were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: In BXD2 mice, TRAP-positive osteoclasts were found at sites of active bone erosion, in close proximity to hyperproliferating synovial fibroblasts. On coculture, MSFs from BXD2 mice, but not C57BL/6 mice, produced high levels of RANKL messenger RNA, induced macrophages to form osteoclasts, and actively eroded bone slices, through a mechanism(s) that could be blocked by pretreatment with osteoprotegerin. Although macrophages from BXD2 mice expressed higher basal levels of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 than those from C57BL/6 mice, abnormal osteoclast formation was not due to enhanced sensitivity of the BXD2 mouse macrophages to RANKL. TNFalpha, produced by both BXD2 MSFs and BXD2 mouse macrophages, had a strong stimulatory effect on RANKL expression. CONCLUSION: BXD2 MSFs produce RANKL and induce the development of osteoclasts from macrophages. The enhanced production of RANKL is possibly due to autocrine stimulation, together with paracrine stimulation by factors produced by macrophages.     

Bone destruction in arthritis

Rheumatoid arthritis (RA) is characterised by the presence of an inflammatory synovitis accompanied by destruction of joint cartilage and bone. Destruction of cartilage matrix results predominantly from the action of connective tissue proteinases released by RA synovial tissues, chondrocytes, and pannus tissue. Several lines of evidence in RA and in animal models of arthritis support a role for osteoclasts in the pathogenesis of bone erosions. RA synovial tissues produce a variety of cytokines and growth factors that may increase osteoclast formation, activity, and/or survival. These include interleukin 1alpha (IL1alpha) and beta, tumour necrosis factor alpha (TNFalpha), IL11, IL17, and macrophage colony stimulating factor (M-CSF). Receptor activator of NFkappaB ligand (RANKL) is an essential factor for osteoclast differentiation and also functions to augment T cell-dendritic cell cooperative interactions. CD4+ T cells and synovial fibroblasts derived from RA synovium are sources of RANKL. Furthermore, in collagen induced arthritis (CIA), blockade with osteoprotegerin (OPG), a decoy receptor for RANKL, results in protection from bone destruction. To further evaluate the role of osteoclasts in focal bone erosion in arthritis, arthritis was generated in the RANKL knockout mouse using a serum transfer model. Despite ongoing inflammation, the degree of bone erosion in arthritic RANKL knockout mice, as assessed by microcomputed tomography and correlated histopathological analysis, was dramatically reduced compared with that seen in arthritic control mice. Cartilage damage was present in both the arthritic RANKL knockout mice and in arthritic control littermates, with a trend toward milder cartilage damage in the RANKL knockout mice. This study supports the hypothesis that osteoclasts play an important part in the pathogenesis of focal bone erosion in arthritis, and reveals distinct mechanisms of cartilage destruction and bone erosion in this animal model of arthritis. Future directions for research in this area include the further investigation of a possible direct role for the RANKL/RANK/OPG system in cartilage metabolism, and the possible role of other cell types and cytokines in bone erosion in arthritis.     

Tartrate-resistant acid phosphatase-positive cells in the synovial–cartilage junction and bone marrow during the progression of collagen-induced arthritis in adult rats

We investigated the time-course changes in bone destruction in rats with collagen-induced arthritis (CIA). The synovial–cartilage junction (SCJ) and epiphyseal bone marrow of the femoral posteromedial condyle in CIA rats were evaluated histologically and immunohistologically at 2, 3, 4, 6, and 8 weeks after sensitization. Two weeks after sensitization, tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells formed resorption lacunae on the lateral side of the cortical bone under the SCJ. No resorption lacunae were observed in bone marrow. Three weeks after sensitization, resorption lacunae on the lateral side of the cortical bone expanded, but no bone marrow invasion by pannus was observed. In bone marrow, many TRAP-positive multinuclear cells appeared and formed resorption lacunae in subchondral bone. Four weeks after sensitization, cortical bone was destroyed, and pannus had invaded the bone marrow. After six weeks, trabecular bone and subchondral bone plate were extensively resorbed by TRAP-positive cells. Bone destruction in CIA began with the appearance of TRAP-positive cells on the lateral side of the cortical bone under the SCJ, followed by the TRAP-positive multinuclear cells in bone marrow, which were morphologically unconnected to the SCJ lesions. These histological findings suggested that bone destruction in the early stage of arthritis occurred in two anatomically different regions.     

Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis

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.     

Osteoclast differentiation factor induces synovial macrophage–osteoclast differentiation in rheumatoid arthritis

The aim of this study was to clarify the role of osteoclast differentiation factor (ODF) and osteoprotegerin (OPG) in synovial macrophage–osteoclast differentiation. Synovial macrophages were cultured in the presence of macrophage-colony-stimulating factor (M-CSF) and/or ODF. OPG was added to cocultures of synovial macrophages and UMR106. The cultures on glass coverslips were stained with osteoclast-associated markers, tartrate-resistant acid phosphatase (TRAP), and vitronectin receptor (VNR), as well as macrophage-associated markers CD11b and CD14. Functional evidence of osteoclast formation was determined by a resorption pit assay. To investigate whether rheumatoid arthritis (RA) synovial cells expressed messenger RNA (mRNA) for ODF, OPG, and the receptor activator of NF-κB (RANK), we performed a polymerase chain reaction (PCR) analysis. The addition of M-CSF or ODF alone induced TRAP-positive multinucleated cell formation. Resorption pits were rarely detected with M-CSF alone. ODF was capable of inducing bone resorption and enhancing osteoclastogenesis, as well as bone resorption in the presence of M-CSF. In the coculture system, both osteoclast formation and bone resorption were inhibited by OPG in a dose-dependent manner. In all experiments, synovial cells, including macrophages and fibroblasts, expressed the mRNA for RANK, ODF, and OPG. Our findings suggest that ODF plays a role in regulating RA synovial macrophage–osteoclast differentiation, and that synovial cells might have the ability to produce ODF. OPG might be further developed as a new strategy for treating bone destruction in RA joints. Received: January 30, 2001 / Accepted: May 18, 2001