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.     

The role of fibroblasts and fibroblast-derived factors in periprosthetic osteolysis

OBJECTIVE: This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption. METHODS: Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme-linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate-resistant acid phosphatase staining was used to identify multinucleated osteoclast-like cells. RESULTS: The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein 1 (MCP-1), interleukin-1beta (IL-1beta), IL-6, IL-8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up-regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP-1, IL-1beta, IL-6, IL-8, cyclooxygenase 1 (COX-1), COX-2, leukemia inhibitory factor 1, transforming growth factor beta1 (TGFbeta1), and TGFbeta receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFalpha, or IL-1beta. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony-stimulating factor induced osteoclastogenesis. CONCLUSION: Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.     

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.     

Expression of toll-like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin-12 and interleukin-18 via interferon-gamma

OBJECTIVE: To study the expression of Toll-like receptor 2 (TLR-2) and TLR-4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals. METHODS: Synovial tissue specimens from 29 RA patients were stained for TLR-2, TLR-4, and proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-12, IL-17, IL-18, and tumor necrosis factor alpha [TNFalpha]). The expression of TLR-2, TLR-4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL-12 and IL-18, and TLR expression was assessed using fluorescence-activated cell sorter analysis. Production of TNFalpha and IL-6 was measured using Luminex bead array technology. RESULTS: In RA synovial tissue, the expression of TLR-2 was slightly higher than that of TLR-4. Interestingly, both TLR-2 and TLR-4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL-12 and IL-18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL-12 and IL-18 resulted in increased expression of both TLR-2 and TLR-4; this could be blocked with anti-interferon-gamma (anti-IFNgamma) antibodies, suggesting a role for IFNgamma. Lipopolysaccharide- or lipoteichoic acid-mediated triggering of PBMCs incubated with IL-12/IL-18 or IFNgamma led to an increased production of both TNFalpha and IL-6, indicating the functionality of TLR-2 and TLR-4. CONCLUSION: TLR-2 and TLR-4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL-12 and IL-18. The synergistic effect of IL-12 and IL-18 on T cell IFNgamma production seems to regulate expression of TLR-2 and TLR-4 in the synovial tissue of RA patients.     

Imbalanced expression of RANKL and osteoprotegerin mRNA in pannus tissue of rheumatoid arthritis

OBJECTIVE: To test if the pannus tissue is characterized by a high receptor activator of nuclear factor kappaB ligand to osteoprotegerin (RANKL:OPG) ratio, which could explain local osteoclastogenesis and formation of bony erosions. METHODS: Messenger RNA and protein expressions of RANKL and OPG in rheumatoid and osteoarthritic tissue samples were measured using quantitative real-time RT-PCR and Western blot/densitometry. Pannus and synovitis fibroblasts explanted from tissue samples were cultured in vitro without and with TNF-alpha, IL-1Beta or IL-17 and analyzed quantitatively for RANKL expression. The ability of pannus fibroblasts to induce formation of multinuclear osteoclast-like cells from human monocytes, with macrophage-colony stimulating factor (M-CSF) but without RANKL added, was tested. Histochemical staining was used to assess the eventual presence of RANKL and tartrate resistant acid phosphatase positive osteoclast-like cells at the pannus-bone interface. RESULTS: RANKL:OPG ratios of messenger RNA (p<0.05) and protein level were high in pannus (2.06+/-0.73 and 2.2+/-0.65) compared to rheumatoid (0.62+/-0.13 and 1.31+/-0.69) and osteoarthritis (0.62+/-0.32 and 0.52+/-0.16) synovial membranes. Resting and stimulated (p dependent on the cytokine used) pannus fibroblasts produced RANKL in excess (p=0.0005) and unstimulated pannus fibroblasts also effectively induced osteoclast-like cell formation from monocytes in vitro without any exogenous RANKL added. Compatible with these findings, multinuclear osteoclasts-like cells were frequent in the fibroblast- and macrophage-rich pannus tissue at the soft tissue-to-bone interface. CONCLUSION: The high RANKL:OPG ratio, together with close fibroblast-to-monocyte contacts in pannus tissue, probably favor local generation of bone resorbing osteoclasts at the site of erosion in rheumatoid arthritis.     

Dysregulation of interleukin-10-dependent gene expression in rheumatoid arthritis synovial macrophages

OBJECTIVE: The inflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1) are produced by activated macrophages, are key mediators of pathogenesis, and are validated therapeutic targets in rheumatoid arthritis (RA) and seronegative spondylarthritis (SpA). IL-10 is a potent antiinflammatory cytokine that suppresses macrophage TNFalpha and IL-1 production, yet is not effective in suppressing inflammatory arthritis. To gain insight into IL-10 responses in inflammatory arthritis, we used microarray analysis to determine the patterns of IL-10-inducible gene expression in freshly isolated RA and seronegative SpA synovial macrophages. METHODS: Macrophages from the synovial fluid of 5 patients with RA and 3 with seronegative SpA (2 with psoriatic arthritis and 1 with ankylosing spondylitis) were isolated by positive selection and stimulated ex vivo with IL-10 or interferon-gamma (IFNgamma). Gene expression was analyzed using Affymetrix microarrays and protocols. Real-time polymerase chain reaction was used to confirm changes in gene expression. RESULTS: The number of genes induced by IL-10 in arthritic macrophages was markedly smaller than that induced in control macrophages, and the strength of induction was lower in arthritic macrophages for most genes. The residual response of arthritic macrophages to IL-10 stimulation was qualitatively altered, such that IL-10 preferentially increased expression of IFNgamma-inducible genes. In contrast, arthritic macrophages expressed many IFNgamma-inducible genes prior to stimulation, and their response to IFNgamma remained mostly intact. CONCLUSION: These results demonstrate that IL-10 responses are dysregulated in RA synovial macrophages. An altered biologic response to IL-10, with attenuation of its antiinflammatory function and a concomitant retention of IFNgamma-like activating functions, provides a basis for the lack of efficacy of IL-10 in suppressing inflammatory arthritis.     

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.     

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.     

Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.     

Cadherin-11 regulates fibroblast inflammation

Fibroblasts are important participants in inflammation. Although not leukocytes, their capacity to produce cytokines, chemokines, and other inflammatory factors locally in tissues suggests that they can contribute to inflammatory diseases. For example, fibroblasts in a rheumatoid arthritis (RA) joint are a dominant source of IL-6 and RANKL in the synovium, both of which are therapeutic targets for inflammation and bone erosion. Previously, we found that fibroblasts can be targeted by mAb directed against cadherin-11 (cad-11), a mesenchymal cadherin that fibroblasts selectively express. Targeting cad-11 significantly reduced inflammation as assessed by joint swelling and clinical inflammation scores. However, the mechanism by which anti-cad-11 reduced inflammation was not known. Here, we show that cad-11 engagement induces synovial fibroblasts to secret proinflammatory cytokines including IL-6. Cad-11 engagement strongly synergized with TNF-α and IL-1β in the induction of IL-6. Importantly, cad-11 activated MAP kinases and NF-κB for IL-6 induction. IL-6 levels in ankles of inflamed joints were reduced in cad-11 mutant mice compared to wild-type mice with inflammatory arthritis. Thus, we suggest that cad-11 modulates synovial fibroblasts to evoke inflammatory factors that may contribute to the inflammatory process in RA.     

Single and combined inhibition of tumor necrosis factor, interleukin-1, and RANKL pathways in tumor necrosis factor-induced arthritis: effects on synovial inflammation, bone erosion, and cartilage destruction

OBJECTIVE: To investigate the efficacy of single and combined blockade of tumor necrosis factor (TNF), interleukin-1 (IL-1), and RANKL pathways on synovial inflammation, bone erosion, and cartilage destruction in a TNF-driven arthritis model. METHODS: Human TNF-transgenic (hTNFtg) mice were treated with anti-TNF (infliximab), IL-1 receptor antagonist (IL-1Ra; anakinra), or osteoprotegerin (OPG; an OPG-Fc fusion protein), either alone or in combinations of 2 agents or all 3 agents. Synovial inflammation, bone erosion, and cartilage damage were evaluated histologically. RESULTS: Synovial inflammation was inhibited by anti-TNF (-51%), but not by IL-1Ra or OPG monotherapy. The combination of anti-TNF with either IL-1Ra (-91%) or OPG (-81%) was additive and almost completely blocked inflammation. Bone erosion was effectively blocked by anti-TNF (-79%) and OPG (-60%), but not by IL-1Ra monotherapy. The combination of anti-TNF with IL-1Ra, however, completely blocked bone erosion (-98%). Inhibition of bone erosion was accompanied by a reduction of osteoclast numbers in synovial tissue. Cartilage destruction was inhibited by anti-TNF (-43%) and was weakly, but not significantly, inhibited by IL-1Ra, but was not inhibited by OPG monotherapy. The combination of anti-TNF with IL-1Ra was the most effective double combination therapy in preventing cartilage destruction (-80%). In all analyses, the triple combination of anti-TNF, IL-1Ra, and OPG was not superior to the double combination of anti-TNF and IL-1Ra. CONCLUSION: Articular changes caused by chronic overexpression of TNF are not completely blockable by monotherapies that target TNF, IL-1, or RANKL. However, combined approaches, especially the combined blockade of TNF and IL-1 and, to a lesser extent, TNF and RANKL, lead to almost complete remission of disease. Differences in abilities to block synovial inflammation, bone erosion, and cartilage destruction further strengthen the rationale for using combined blockade of more than one proinflammatory pathway.     

类风湿关节炎炎性关节中高水平脂联素促进白细胞介素-6单核细胞趋化因子-1和核因子-κB受体活化因子配体表达

目的 研究类风湿关节炎(RA)炎性关节中高水平脂联素(AD)表达的意义.方法 酶联免疫吸附试验(ELISA)测定关节液中AD及白细胞介素(IL)-1β、IL-6、IL-8、肿瘤坏死因子(TNF)-α、单核细胞趋化因子(MCP)-1和基质金属蛋白酶(MMP)-9等促炎因子/趋化因子的含量,并分析AD与其相关性.双重免疫标记分析RA滑膜中AD在滑膜成纤维细胞(SFLs)、T淋巴细胞、单核细胞、中性粒细胞和肥大细胞的表达.ELISA测定重组AD因子刺激SFLs后培养上清液中细胞因子含量变化.实时荧光定量反转录-聚合酶链反应(real-time PCR)分析AD和IL-6共刺激对人滑膜细胞系MH7A中核因子-κB受体活化因子配体(RANKL)mRNA表达影响.3组间采用多变量方差分析,2组比较采用Student's t检验.Mann-Whitney U检验,相关性分析采用Spearman检验.结果 RA关节液中AD与IL-6含量呈正相关(r=0.27,P=0.03).AD与成纤维细胞双重免疫标记呈阳性反应.以重组AD因子30g/ml刺激SFLs 24 h后,培养上清液中IL-6和MCP-1分泌量与刺激前相比分别增加了11.4倍和8倍(P<0.05).AD和IL-6共刺激后,MH7A中RANKL mRNA表达显著增加(82±26).结论 炎性关节中高水平AD可促进IL-6、MCP-1和RANKL表达,其可能在RA慢性炎症和骨侵蚀过程中发挥重要作用.     

Peripheral blood T lymphocytes from patients with early rheumatoid arthritis express RANKL and interleukin-15 on the cell surface and promote osteoclastogenesis in autologous monocytes

OBJECTIVE: To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process. METHODS: T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate-resistant acid phosphatase staining and calcified matrix resorption activity. RESULTS: Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin-15 (IL-15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL-15, IL-17, tumor necrosis factor alpha (TNFalpha), and IL-1beta. OPG, anti-TNFalpha, and anti-IL-1beta demonstrated a cooperative inhibitory effect. At 1-year followup, surface RANKL and IL-15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment. CONCLUSION: T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.