Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen‐induced arthritis: Possible involvement of osteopontin in bone destruction in arthritis

Objective

To investigate the involvement of osteopontin (OPN) in bone destruction in a murine experimental arthritis model of collagen‐induced arthritis (CIA).

Methods

The expression of OPN was examined at both the messenger RNA (mRNA) and protein levels in various arthritic lesions in mice with CIA by in situ hybridization and immunohistochemistry, respectively. In addition, the expression of αvβ3 integrin, a receptor for OPN, the ligation of which is thought to be essential for bone resorption by osteoclasts, was examined by immunohistochemistry. Plasma concentrations of OPN were measured at different time points in the course of CIA by enzyme‐linked immunosorbent assay.

Results

OPN mRNA was detected mainly at sites of bone erosion in arthritic lesions, where activated osteoclasts were present; OPN protein was also detected at sites of bone erosion. In the arthritic synovium, OPN was predominantly expressed in the synovial lining layer, but not in lymphoid aggregates. In addition, αvβ3 integrin was detected coincident with OPN at sites of bone erosion (bone–pannus junction). Plasma OPN levels were markedly elevated at the time points that corresponded to arthritis flares, and higher levels were maintained during the progression of arthritis.

Conclusion

OPN may mediate bone resorption by osteoclasts in arthritis through ligation with its receptor, αvβ3 integrin. OPN may be a useful therapeutic target molecule in the prevention of bone destruction in arthritis.

     

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.     

Evaluation of inflammatory change and bone erosion using a murine type II collagen-induced arthritis model

The exact mechanism of rheumatoid arthritis (RA) is unclear, but a combination of genetic, environmental and hormonal factors is thought to be involved. This study examined the progressive arthritic reaction of murine type II collagen-induced arthritis (CIA), a representative animal model of RA. Arthritic reactions, including inflammation and bone erosion were examined using an objective non-invasive method. Two scoring systems were used to evaluate changes in cutaneous inflammation and bone erosion during RA progression. The severity of inflammation was evaluated by visual scoring of erythema and edema, while the degree of bone erosion was quantified by macroradiographical erosion analysis of specific bones. A significant difference was observed in both visual (P = 0.0001, n = 7) and radiographic (P < 0.0001, n = 7) examinations for the RA group as compared to the control. The relationship between inflammatory change and erosive change in bone showed a significant positive correlation, r = 0.9550 (P < 0.0001, n = 7). The overall rate of asymmetry was 25.23% in both fore- and hindpaws. The results generated from these experiments show that murine CIA is a promising model for elucidating the mechanism of RA. In addition, the results of this study may be used for monitoring RA progression as well as screening therapy efficacy in the joint pathology.     

Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis

OBJECTIVE: To study the effect of zoledronic acid (ZA) on synovial inflammation, structural joint damage, and bone metabolism in rats during the effector phase of collagen-induced arthritis (CIA). METHODS: CIA was induced in female dark agouti rats. At the clinical onset of CIA, rats were assigned to treatment with vehicle or single subcutaneous doses of ZA (1.0, 10, 50, or 100 microg/kg). Clinical signs in all 4 paws were scored on a daily basis. After 2 weeks, the joints in the hind paws were assessed using plain radiographs, microfocal computed tomography (micro-CT), histologic scoring, and histomorphometry, and the serum levels of type I collagen crosslinks were measured by enzyme-linked immunosorbent assay. RESULTS: Although ZA mildly exacerbated synovitis, it effectively suppressed structural joint damage. At doses of >/=10 microg/kg, ZA significantly reduced radiographic bone erosions, Larsen scores, and juxtaarticular trabecular bone loss as quantified by micro-CT. ZA prevented increased type I collagen (bone) breakdown in CIA and diminished histologic scores of focal bone erosion by up to 80%. Increases in the percentage of eroded surface, osteoclast surface, and osteoclast numbers associated with CIA were prevented by ZA, even though synovitis scores were unchanged. CONCLUSION: Single doses (>/=10 microg/kg) of ZA strikingly reduced focal bone erosions and juxtaarticular trabecular bone loss, although synovitis was mildly exacerbated. Targeting osteoclasts with ZA may therefore be an effective strategy for preventing structural joint damage in rheumatoid arthritis.     

Role of major histocompatibility complex genes in murine collagen-induced arthritis: a model for human rheumatoid arthritis

Collagen-induced arthritis is an animal model for rheumatoid arthritis that shares a number of clinical, hematologic, serologic, and radiographic features with human disease. Predisposition to rheumatoid arthritis has been associated with major histocompatibility complex (MHC) class II genes, HLA-DRB*0401/DQB1*0302 and resistance to DRB1*0402 and DQ6 genes. Animal models allow one to study the genetics and immunologic processes of individual genes involved in the complex human diseases. To study the interactions between class II molecules and to define their role in arthritis, the authors generated HLA-DR and -DQ transgenic mice. HLA transgenes are expressed on cell surface and can positively select CD4 cells. A peripheral tolerance is maintained to the trans-genes even though an efficient T cell response to immunodominant antigens similar to human T cells is observed. Using HLA-DQ/DR double transgenic mice, the studies show that complementation between DQ and DR molecules contributes to predisposition to and severity of, or protection from, arthritis. Thus, these mice provide a powerful tool to understand the role of HLA molecules in the predisposition to and immunotherapy for human disease.     

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

OBJECTIVE: The receptor activator of nuclear factor kappaB (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.     

The destruction evaluation in different foot joints: new ideas in collagen-induced arthritis rat model

Collagen-induced arthritis (CIA) has been widely used as the animal model of rheumatoid arthritis since 1977, while till now, no paper has depicted the destruction characteristics in different foot joints. In this study, we observed the differences among the foot joint destruction process of CIA to elucidate further the pathological process of this model. CIA was induced in male Wistar rat immunized with bovine type II collagen and Freund’s incomplete adjuvant. Radiological studies were performed 1, 2, 4, 6, and 8 months after the second immunization to follow the development of disease. At last, all the animals were killed and histological research was performed. In the histological observation, three main types of joint destructions such as subchondral side erosion, external joint erosion and the cartilaginous fusion of articular cartilage were identified. All these destruction forms exist in one joint or several different joints. Furthermore, we found that tartrate-resistant acid phosphatase (TRAP) stain-positive cells participated in the destruction of articular cartilage. These new findings showed that in the disease process of the CIA model, different foot joints show different destruction characteristics and cartilaginous fusion of foot joints is another typical pathological characteristic. Our research was supported by Shanghai Key Laboratory of Orthopaedic Implant (08DZ2230300).     

Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation, cartilage destruction, and bone erosion

OBJECTIVE: Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium of rheumatoid arthritis (RA) patients. This T cell cytokine is implicated in the initiation phase of arthritis. However, the role of IL-17 during the effector phase of arthritis has still not been identified; this was the objective of the present study. METHODS: Mice with collagen-induced arthritis (CIA) were treated with polyclonal rabbit anti-murine IL-17 (anti-IL-17) antibody-positive serum or normal rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and treated with anti-IL-17 antibody or control serum. Arthritis was monitored visually, and joint pathology was examined radiologically and histologically. Systemic IL-6 levels were measured by enzyme-linked immunosorbent assay, and local synovial IL-1 and receptor activator of NF-kappaB ligand (RANKL) expression was analyzed using specific immunohistochemistry. RESULTS: Treatment with a neutralizing anti-IL-17 antibody after the onset of CIA significantly reduced the severity of CIA. Radiographic analysis revealed marked suppression of joint damage in the knee and ankle joints. Histologic analysis confirmed the suppression of joint inflammation and showed prevention of cartilage and bone destruction after anti-IL-17 antibody therapy. Systemic IL-6 levels were significantly reduced after anti-IL-17 antibody treatment. Moreover, fewer IL-1beta-positive and RANKL-positive cells were detected in the synovium after treatment with neutralizing IL-17. Interestingly, initiation of anti-IL-17 antibody therapy during a later stage of CIA, using mice with higher clinical arthritis scores, still significantly slowed the progression of the disease. CONCLUSION: IL-17 plays a role in early stages of arthritis, but also later during disease progression. Systemic IL-6 was reduced and fewer synovial IL-1-positive and RANKL-positive cells were detected after neutralizing endogenous IL-17 treatment, suggesting both IL-1-dependent and IL-1-independent mechanisms of action. Our data strongly indicate that IL-17 neutralization could provide an additional therapeutic strategy for RA, particularly in situations in which elevated IL-17 may attenuate the response to anti-tumor necrosis factor/anti-IL-1 therapy.     

Effects of pravastatin in murine collagen-induced arthritis

Here we evaluated whether 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have beneficial effects for collagen-induced arthritis (CIA). DBA/1 mice were immunized with bovine type-II collagen and administered 100 mg/kg of pravastatin interperitoneally. We measured the effects of pravastatin for CIA including infiltration of macrophages at the synovial membrane and production of anti-type-II collagen antibodies and cytokines. Adverse reactions of pravastatin were also measured. The pravastatin-treated mice had delayed onset of CIA compared with the controls. The involvement of inflammatory cells in the synovial membrane and the expression of monocyte chemotactic protein-1 (MCP-1) mRNA in the joint were reduced. Moreover, some cytokines (TNF-α, IL-6, IFN-γ) and MCP-1 levels in the supernatants of spleen cells cultured with pravastatin decreased. Meanwhile, adverse reactions of pravastatin, such as peritonitis, were not detected. Pravastatin may have good prospects for treating some anti-inflammatory effects on human rheumatoid arthritis.     

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.     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白在胶原诱导性关节炎大鼠中的疗效观察及其对骨桥蛋白的影响

目的 观察重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)在胶原诱导性关节炎(CIA)大鼠中对滑膜、关节软骨等的保护作用,并分析其对骨桥蛋白表达的影响.方法 建立CIA模型,第13天开始对治疗组采用rhTNFR:Fc腹腔注射治疗(10 mg/kg,隔日1次),每周测量体质量及踝关节的前后径,第36天处死大鼠,苏木素-伊红(HE)及甲苯胺蓝染色观察踝关节组织病理变化并进行病理评分;酶联免疫吸附试验(ELISA)检测血浆肿瘤坏死因子(TNF)-α和骨桥蛋白水平,免疫组织化学法检测踝关节骨桥蛋白的组织学表达.多组间均数比较采用单因素方差分析.结果 病理评分定量分析结果 显示造模组与治疗组比较,治疗组关节病理评分显著降低(分别为8.2±1.0与4.8±1.4,P<0.05);ELISA 结果 显示造模组平均血浆TNF-α和骨桥蛋白值分别为(713±146)pg/ml,(4.3±0.6)ng/ml,治疗组分别为(68±20)pg/ml,(4.2±0.6)ng/ml,2组间比较,血浆TNF-α值差异有统计学意义(P<0.05),而骨桥蛋白值差异无统计学意义(P=0.688);免疫组织化学显示骨桥蛋白主要表达在滑膜衬里层、软骨表面,造模组与治疗组间比较差异有统计学意义(P<0.05).结论 rhTNFR:Fc能显著减轻CIA大鼠关节及软骨的破坏,并能显著减少骨桥蛋白在关节滑膜的表达,延缓病情的进展.但不能减少骨桥蛋白在外周血浆中的表达,推测其原因可能为骨桥蛋白不直接参与炎症的发展过程,而主要参与骨质的破坏及吸收过程.

Abstract：

Objective To investigate the protection effects of recombinant human tumor necrosis factor-α receptor Ⅱ :IgG Fc fusion protein for injection (rhTNFR:Fc) on rats with collagen-induced arthritis (CIA) and analyze osteopontin (OPN) changes following therapy in order to understand its primary mechanism of action. Methods CIA was induced by bovine Ⅱ collagen (B Ⅱ C) injection. Rats were treated with rhTNFR:Fc from the 13th day after the first injection of B Ⅱ C till the 36th day. The anterior-posterior diameters of ankle joints and weight were measured weekly. The pathological score was evaluated by HE staining and toluidine blue staining. The blood plasma TNF-α and OPN levels were measured by ELISA and the histology expression was evaluated by immuno-histochemistry. Comparisons between groups were performed with one-way ANOVA. Results Quantitative analysis showed pathological score in the model group and treatment group was significantly reduced in joint pathology (8.2±1.0 vs 4.8±1.4, P<0.05). The mean plasma levels of TNF-α and OPN values were (713±146) pg/ml, (4.3±0.6) ng/ml respectively in the model group,but those of the treatment group were (68±20) pg/ml, (4.2±0.6) ng/ml. Serum TNF-α values were significantly different (P<0.05) between the two groups, while no significant difference was found in the value of plasma OPN (P=0.688) between the two groups. rhTNFR:Fc could reduce the cells OPN expression in the interface layer of the synovium and cartilage (P<0.05). Conclusion Pathology scores and ELISA results haveshown that rhTNFR:Fc has good therapeutic efficacy. It can significantly reduce the bone and cartilage damage of CIA mouse model, and can significantly reduce the expression of OPN in the sliding joints, thereby delay disease progression. However, it can not reduce the expression of OPN in the peripheral blood plasma.OPN may be involved in bone destruction and resorption rather than in inflammatory process.     

Alpha-lipoic acid suppresses the development of collagen-induced arthritis and protects against bone destruction in mice

Objective To test the ability of alpha-lipoic acid (LA) to attenuate the development of collagen-induced arthritis (CIA) in mice. Methods Mice were divided into three groups and treated with intraperitoneal administration of LA (10 or 100 mg/kg) or placebo. Clinical, histologic, and biochemical parameters were assessed. Human synovial fibroblasts and peripheral blood mononuclear cells were cocultured in various concentrations of LA to evaluate the effects on osteoclastogenesis. Results LA was associated with a dose-dependent reduction of CIA, as well as preventing bone erosion and destructive changes. Intracellular reactive oxygen species in lymphocytes obtained from inguinal lymph nodes, which was significantly higher in CIA than control mice, was significantly reduced in CIA by LA. The concentrations of TNF-α, IL-1β, and IL-6 in the paws, and synovial NF-κB binding, all of which were markedly higher in CIA than control mice, were reduced by treatment with LA. In addition, LA inhibited the formation of human osteoclasts in vitro. Conclusion Amelioration of joint disease by LA was associated with reduction in oxidative stress, as well as inhibition of inflammatory cytokine activation and NF-κB DNA binding activity. Moreover, LA inhibited bone destruction in vivo and osteoclastogenesis in vitro. Collectively, these results indicate that LA may be a new adjunctive therapy for rheumatoid arthritis.     

Fcgamma receptors directly mediate cartilage, but not bone, destruction in murine antigen-induced arthritis: uncoupling of cartilage damage from bone erosion and joint inflammation

OBJECTIVE: To determine the relationship between synovial inflammation and the concomitant occurrence of cartilage and bone erosion during conditions of variable inflammation using various Fcgamma receptor knockout (FcgammaR(-/-)) mice. METHODS: Antigen-induced arthritis (AIA) was introduced in the knee joints of various FcgammaR(-/-) mice and wild-type controls. Joint inflammation and cartilage and bone destruction levels were determined by histologic analysis. Cathepsin K, RANKL, and osteoprotegerin (OPG) levels were detected by immunolocalization. RESULTS: In FcgammaRIIb(-/-) mice, which lack the inhibiting Fcgamma receptor IIb, levels of joint inflammation and cartilage and bone destruction were significantly higher (infiltrate 93%, exudate 200%, cartilage 100%, bone 156%). AIA in mice lacking activating FcgammaR types I, III, and IV, but not FcgammaRIIb (FcR gamma-chain(-/-) mice), prevented cartilage destruction completely. In contrast, levels of bone erosion and joint inflammation were comparable with their wild-type controls. Of great interest, in arthritic mice lacking activating FcgammaR types I, II, and III, but not IV (FcgammaRI/II/III(-/-) mice), levels of joint inflammation were highly elevated (infiltrate and exudate, 100% and 188%, respectively). Cartilage destruction levels were decreased by 92%, whereas bone erosion was increased by 200%. Cathepsin K, a crucial mediator of osteoclasts, showed a strong correlation with the amount of inflammation but not with the amount of activating FcgammaR, which was low in osteoclasts. RANKL, but not OPG, levels were higher in the inflammatory cells of arthritic knee joints of FcgammaRI/II/III(-/-) mice versus wild-type mice. CONCLUSION: Activating FcgammaR are crucial in mediating cartilage destruction independently of joint inflammation. In contrast, FcgammaR are not directly involved in bone erosion. Indirectly, FcgammaR drive bone destruction by regulating joint inflammation.     

The effect of endostatin evaluated in an experimental animal model of collagen-induced arthritis

OBJECTIVE: To investigate the arthritis-inhibiting effect of endostatin, a potent angiogenesis inhibitor, on type II collagen-induced arthritis (CIA). METHODS: In an experimental system of prophylactic administration, endostatin was administered once daily at 1 mg/kg/day or 10 mg/kg/day for 2 weeks from before the onset of arthritis. In the experimental system of therapeutic administration, mice with an arthritis score of 1 to 3 were administered endostatin once daily at 10 mg/kg. In the experimental system of continuous administration, endostatin was administered using an osmotic pump capable of continuously administering a calculated dose of 1 mg/kg/day for 2 weeks. RESULTS: Arthritis scores were lower in a dose-dependent manner in the prophylactic administration group than in the control group. Arthritis scores were lower in the therapeutic administration group than in the control group. Compared with the once-daily dosage regimen, the administration of endostatin by an osmotic pump achieved a similar arthritis-inhibiting effect at one-tenth of the dose. CONCLUSION: Both prophylactic and therapeutic administration of endostatin inhibited type II CIA in mice. The administration method using an osmotic pump is useful.