Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-beta expression in the joint

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self-limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro- and anti-inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6] and anti-inflammatory cytokines [IL-10, IL-1ra, transforming growth factor (TGF)-beta1, TGF-beta2 and TGF-beta3] in joints was carried out over the course of the disease. Both pro- and anti-inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti-inflammatory cytokines, particularly TGF-beta1 and TGF-beta2, increased. Surprisingly, TNF-alpha continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF-alpha during the period of disease remission had no effect on TGF-beta expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro- and anti-inflammatory cytokine expression in the joints. Of the anti-inflammatory cytokines studied, TGF-beta1 and TGF-beta2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.     

Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids.

Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids.     

Experimental animal models for rheumatoid arthritis

Rheumatoid Arthritis (RA) is an autoimmune systemic disorder of unknown etiology and is characterized by chronic inflammation and synovial infiltration of immune cells. RA is associated with decreased life expectancy and quality of life. The research on RA is greatly simplified by animal models that help us to investigate the complex system involving inflammation, immunological tolerance and autoimmunity. The animal models of RA with a proven track record of predictability for efficacy in humans include: collagen type II induced arthritis in rats as well as mice, adjuvant induced arthritis in rats and antigen induced arthritis in several species. The development of novel treatments for RA requires the interplay between clinical observations and studies in animal models. However, each model features a different mechanism driving the disease expression; the benefits of each should be evaluated carefully in making the appropriate choice for the scientific problem to be investigated. In this review article, we focus on animal models of arthritis induced in various species along with the genetic models. The review also discussed the similarity and dissimilarities with respect to human RA.     

糖皮质激素对类风湿关节炎炎性因子改变及效果的观察

目的：分析糖皮质激素对类风湿关节炎炎性因子改变及效果。方法：选取我院2014年8月至2015年8月门诊及住院就诊的80例类风湿关节炎患者,依据抽签法分成对照组与观察组,每组40例,对照组采取常规治疗,观察组基于对照组联合糖皮质激素治疗,对比分析两组治疗后炎性因子、RF、ESR、晨僵、关节肿胀数及关节压痛数、DAS评分及VAS评分变化、治疗有效率与不良反应。结果：治疗后,观察组炎性因子TNF-α低于对照组[(31.23±8.74) vs.(36.46±9.12) pg/mL],IL-6低于对照组[(2.62±0.31) vs.(3.67±0.36) pg/L],CRP低于对照组[(15.26±1.03) vs.(17.38±1.10) mg/L],比较有显著差异(P<0.05);观察组RF低于对照组[(15.34±1.21) vs.(20.25±1.24) U/mL],ESR低于对照组[(24.36±1.57) vs.(28.95±1.60) mm/h],晨僵少于对照组[(16.73±1.28) vs.(18.97±1.34)],关节肿胀数低于对照组[(47.28±0.40) vs.(6.73±0.45)],关节压痛数低于对照组[(6.18±0.52) vs.(9.36±1.05) min],比较差异显著(P<0.05);观察组DAS28低于对照组[(1.93±0.20) vs.(2.18±0.26)分],VAS低于对照组[(2.23±0.25) vs.(3.07±0.12)分],比较差异明显(P<0.05);治疗有效率95.00%高于对照组80.00%(P<0.05),观察组总不良反应率25.00%高于对照组20.00%,但无统计学意义(P>0.05)。结论：类风湿关节炎运用糖皮质激素治疗安全性高,可有效降低炎性因子水平,改善患者临床症状,促进身心恢复。     

Reduction of monocyte-macrophage activation markers upon anti-CD4 treatment. Decreased levels of IL-1, IL-6, neopterin and soluble CD14 in patients with rheumatoid arthritis.

Anti-CD4 MoAbs have been successfully used in initial treatment trials of rheumatoid arthritis. One remarkable feature of this therapy was the early reduction of synovitis along with a decrease of the erythrocyte sedimentation rate (ESR) and the C-reactive protein (CRP). Since not only T helper cells, but also monocytes-macrophages bear the CD4 antigen, the question was raised whether the immediate effects observed may have been in part due to an influence on the mononuclear phagocyte system. Immediately after MoAb infusions, a significant reduction of the absolute peripheral blood monocyte count down to 30% (P < 0.001) was noted within the first hour of injection. In contrast to strikingly elevated levels of soluble CD4 after treatment which was indicative of T cell lysis, soluble CD14 levels did not rise, but rather decreased from previously elevated levels. Before treatment, activation of the monocyte-macrophage system had been signified by elevated serum levels of IL-1, IL-6, CRP and neopterin as well as a marked in vitro production of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6. Subsequent anti-CD4 treatment resulted in a rapid and significant reduction of monocyte-derived circulating cytokines and mediators concordant with a reduced capacity to produce IL-1, TNF-alpha, and IL-6 in those patients who demonstrated clinical benefits. Therefore, studies of monocyte activation markers may be useful in identifying subsequent responders to anti-CD4 therapy.     

Essential role of neutrophils in anti-type II collagen antibody and lipopolysaccharide-induced arthritis

In mice arthritis model induced by anti-type II collagen (CII) antibodies and lipopolysaccharide (LPS), most of cells that infiltrated into the joint space were neutrophils. To investigate the role of neutrophils in the pathogenesis of arthritis, we depleted the neutrophils in vivo by injection of the antibody against Gr-1 expressed mainly on neutrophils. The neutrophil depletion completely inhibited the arthritis development. Furthermore, neutrophil depletion in mice that had already developed arthritis ameliorated the disease. These results showed that neutrophils are indispensable not only for the development, but also for the maintenance of arthritis. Next, we tried to develop arthritis in C5-deficient mice to investigate the involvement of C5a, one of chemotactic factors for neutrophils. C5-deficient mice showed significant reduction in arthritis development in comparison with wild type mice. Injection of pertussis toxin (Ptx) into the mice, which inhibits the signals from the inhibitory G-protein coupled-receptors including the C5a receptor, suppressed the development of arthritis. Furthermore, Ptx also ameliorated the arthritis when injected into mice that had already developed the disease. These results suggest the important role of chemotactic factors involving C5a and inhibitory G-protein (Gi)-coupled receptors not only in the development, but also in the maintenance of arthritis.     

Retroviral gene therapy of collagen-induced arthritis by local delivery of IL-4

Rheumatoid arthritis (RA) is an autoimmune arthritis, for which treatment options remain limited. This study investigated the potential role of adoptive cellular gene therapy as a novel means for treating the RA animal model collagen-induced arthritis (CIA). Adoptive transfer of antigen-specific T-cell hybridomas retrovirally transduced to express IL-4 1 day before booster immunization significantly reduced the number of inflamed joints. Cell transfer after clinical onset of disease had no therapeutic effect. Bioluminescence imaging showed that the hybridomas migrated to the inflamed joints, thus delivering the regulatory protein locally at the site of inflammation. The homing was, at least in part, due to chemotaxis in response to proinflammatory chemokines that are expressed in inflamed joints. There were no significant changes in the cytokine milieu of the draining lymph nodes, nor in the systemic levels of anti-collagen antibodies in treated mice. We conclude that the beneficial clinical effects observed in our model were most likely based on the local action(s) of IL-4 in the inflamed joints and that the local delivery (and effects) of regulatory cytokines, like IL-4, constitutes a novel and effective method of preventing organ-specific autoimmune disease and of minimizing systemic adverse effects.     

Driving chronicity in rheumatoid arthritis: perpetuating role of myeloid cells

Acute inflammation is a complex and tightly regulated homeostatic process that includes leucocyte migration from the vasculature into tissues to eliminate the pathogen/injury, followed by a pro‐resolving response promoting tissue repair. However, if inflammation is uncontrolled as in chronic diseases such as rheumatoid arthritis (RA), it leads to tissue damage and disability. Synovial tissue inflammation in RA patients is maintained by sustained activation of multiple inflammatory positive‐feedback regulatory pathways in a variety of cells, including myeloid cells. In this review, we will highlight recent evidence uncovering biological mechanisms contributing to the aberrant activation of myeloid cells that contributes to perpetuation of inflammation in RA, and discuss emerging data on anti‐inflammatory mediators contributing to sustained remission that may inform a novel category of therapeutic targets.     

Arthritis induced by continuous infusion of hr-interleukin-1 alpha into the rabbit knee-joint

Continuous infusion of 200 ng/day hrIL-1 alpha for 14 days into knee-joints of rabbits leads to a severe arthritis of low aggressivity. This arthritis shows simultaneously characteristics of acute (serous and fibrinous exudation, polymorph infiltration, etc.) as well as chronic (synovial cell proliferation and fibrosis, pannus formation, cartilage and bone erosion, etc.) inflammation. The arthritis was associated with a distinct loss of metachromasia of the articular cartilage. These results indicate that IL-1 might play an important role in the induction and maintenance of arthritis.     

Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid.

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha and transforming growth factor-beta (TGF-beta) isoforms for varying time points up to 72 h at 37 degrees C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-beta isoforms and TNF-alpha and down-regulated by IL-4 and IL-10, with no effect observed with IL-1beta, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-alpha and not TGF-beta isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-alpha.     

类风湿性关节炎患者T细胞亚群失衡与炎性粘附分子的关系

目的：探讨类风湿性关节炎（RA）患者细胞免疫调节功能异常与参与介导免疫炎性损伤的细胞粘附分子之间的关系。方法：以ELISA方法检测40例RA患者外周血中IL－2、IL－10、 sICAM-1、sVCAM-1水平。结果：RA患者TH1，细胞因子IL－2水平明显低于健康对照组；TH2细胞因子IL－10及细胞粘附分子sICAM-1/sVCAM-1水平明显高于健康对照组，各组间比较均具有显著性差异（P＜0．01）。结论：RA患者高水平的 IL－10与低水平的IL－2提示TH2细胞功能亢进，进而TH1细胞被抑制，并导致细胞因子谱的偏移；高水平的sICAM-1、sVCAM-1是RA患者慢性炎症损伤的重要介质；异常升高的IL－10与细胞粘附分子的共同作用是导致RA患者病理损伤的重要原因。     

Blocking of YY1 reduce neutrophil infiltration by inhibiting IL-8 production via the PI3K-Akt-mTOR signaling pathway in rheumatoid arthritis

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder whose pathology involves multiple immune cell types, including B and T lymphocytes as well as myeloid cells. While it is clear that autoantibody-producing B cells, as well as CD4⁺ T cell help, are key contributors to disease, little is known regarding the role of innate lymphoid cells such as natural killer (NK) cells in the pathogenesis of SLE. We have characterized the phenotype of NK cells by multi-color flow cytometry in a large cohort of SLE patients. While the overall percentage of NK cells was similar or slightly decreased compared to healthy controls, a subset of patients displayed a high frequency of NK cells expressing the proliferation marker, Ki67, which was not found in healthy donors. Although expression of Ki67 on NK cells correlated with Ki67 on other immune cell subsets, the frequency of Ki67 on NK cells was considerably higher. Increased frequencies of Ki67⁺ NK cells correlated strongly with clinical severity and active nephritis and was also related to low NK cell numbers, but not overall leukopenia. Proteomic and functional data indicate that the cytokine interleukin-15 promotes the induction of Ki67 on NK cells. These results suggest a role for NK cells in regulating the immune-mediated pathology of SLE as well as reveal a possible target for therapeutic intervention.     

Chloroquine inhibits T cell proliferation by interfering with IL-2 production and responsiveness.

Chloroquine (Chl) is an anti-rheumatic drug that is widely used in the treatment of rheumatoid arthritis (RA). It seems that T cells are important in the pathogenesis of RA, but it is not known whether Chl acts via inhibition of T cell function. We here present evidence that Chl, just like cyclosporine A (CsA), inhibits T cell proliferation as induced with immobilized alpha CD3 MoAb in a concentration-dependent manner, at least partly through interfering with the production of IL-2 protein and the induction of IL-2 mRNA. Furthermore, Chl impedes the responsiveness of T cell clones to IL-2 since (1) the inhibition of alpha CD3 MoAb-induced proliferation by Chl could not be reversed by rIL-2 and (2) Chl directly blocks IL-2-driven proliferation of cloned T cells. Chl appeared to interfere with the internalization (50% inhibition) and degradation (total blockade) of rIL-2. Finally, the combination of Chl and CsA synergistically inhibited T cell proliferation. We conclude that Chl may inhibit functional properties of human T cells, although the drug is 100- to 1000-fold less potent than CsA in inhibiting T cell proliferation and IL-2 production, respectively. It is speculated that the in vitro effects of Chl might be relevant in explaining the anti-rheumatic effect of this drug in patients with RA.     

The role of T helper type 17 cells in inflammatory arthritis

While T cells have been implicated in the pathogenesis of inflammatory arthritis for more than three decades, the focus on the T helper type 17 (Th17) subset of CD4 T cells and their secreted cytokines, such as interleukin (IL)‐17, is much more recent. Proinflammatory actions of IL‐17 were first identified in the 1990s, but the delineation of a distinct Th17 subset in late 2005 has sparked great interest in the role of these cells in a broad range of immune‐mediated diseases. This review summarizes current understanding of the role of Th17 cells and their products in both animal models of inflammatory arthritis and human immune‐driven arthritides.     

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-α)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13

TNF-α is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-α transgene modified with the 3′ untranslated region of β-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-α-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4 weeks old. Control groups of transgenic mice were engrafted with β-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-α transgene, endogenous mouse TNF-α and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3 weeks of treatment (P < 0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-α transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.     

Selective accumulation of CCR5+ T lymphocytes into inflamed joints of rheumatoid arthritis

Chemokines and their receptors play critical roles in the selectiverecruitment of various subsets of leukocytes. Recent studieshave indicated that some chemokine receptors are differentiallyexpressed on T_h1 and T_h2 cells. However, available data concerningthe presence of T cells with a T_h1 or a T_h2 character and theexpression of chemokine receptors on infiltrating T cells inthe rheumatic joint are still limited. In this study, we investigatedthe expression of CC chemokine receptor 4 (CCR4) and CCR5, whichhave been shown to be preferentially expressed on T_h2 and T_h1respectively on T cells from rheumatoid arthritis (RA) patients.Although both CCR5⁺ and CCR4⁺ CD4⁺ T cell populations were observedin peripheral blood mononuclear cells from healthy controlsand osteoarthritis patients, these cell populations were decreasedin patients with active RA. In contrast, the vast majority ofsynovial fluid (SF) T cells from active RA patients expressedCCR5 but not CCR4. CCR5 ligands, MIP-1 and RANTES, were foundin RA SF at high levels. CCR5⁺ CD4⁺ T cells from SF mononuclearcells of RA patients produced IFN- but not IL-4 in responseto anti-CD3 stimulation in vitro. These results indicated thatdifferential expression of chemokine receptors plays a criticalrole for selective recruitment of pro-inflammatory T cells intothe joints of RA.     

Suppression of arthritis by forced expression of cyclin-dependent kinase inhibitor p21(Cip1) gene into the joints

Rheumatoid synovial fibroblasts (RSF) express cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(Cip1) when they are growth-inhibited in vitro. The induction of p16(INK4a) is characteristic of RSF and intra-articular p16(INK4a) gene therapy has been shown to suppress adjuvant arthritis (AA) of rats. The other inducible CDK inhibitor, p21(Cip1), has multiple functions depending on the cell type. They include inhibition of CDK as well as promotion of active CDK complex formation and induction of apoptosis. This study is to discern the biological effects of p21(Cip1) gene transfer into RSF and its therapeutic effects on AA. A recombinant adenovirus containing a human p21(Cip1) gene and control adenoviruses were prepared. RSF infected with these viruses were examined for their cell growth. Apoptotic cell death was evaluated by nuclear staining and DNA fragmentation analysis. In vivo gene therapy of rat AA was carried out by intra-articular injection of the viruses. Severity of the arthritis was clinically scored. The treated joints were examined histologically and proliferating cell nuclear antigens (PCNA) were detected immunohistochemically. The adenoviral p21(Cip1) gene transfer inhibited growth of RSF without inducing apoptosis. p21(Cip1) gene therapy suppressed AA clinically and histologically. The effects were comparable to p16(INK4a) gene therapy. PCNA expression was reduced in the p21(Cip1)-treated joints. The adenoviral gene transfer of p21(Cip1) ameliorated rat AA. The effect was attributable to inhibition of proliferation. Because p21(Cip1) is induced more easily by many chemicals than p16(INK4a), it also appears to be a feasible target in developing anti-rheumatic drugs.     

Activation of human fibroblast-like synoviocytes by uric acid crystals in rheumatoid arthritis

Hyperuricemia-mediated uric acid crystal formation may cause joint inflammation and provoke the destruction of joints through the activation of inflammasome-mediated innate immune responses. However, the immunopathological effects and underlying intracellular regulatory mechanisms of uric acid crystal-mediated activation of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) have not been elucidated. Therefore, we investigated the in vitro effects of monosodium urate crystals, alone or in combination with the inflammatory cytokines tumor-necrosis factor (TNF)-α or interleukin (IL)-1β, on the activation of human FLS from RA patients and normal control subjects and the underlying intracellular signaling mechanisms of treatment with these crystals. Monosodium urate crystals were able to significantly increase the release of the inflammatory cytokine IL-6, the chemokine CXCL8 and the matrix metalloproteinase (MMP)-1 from both normal and RA-FLS (all P<0.05). Moreover, the additive or synergistic effect on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS was observed following the combined treatment with monosodium urate crystals and TNF-α or IL-1β. Further experiments showed that the release of the measured inflammatory cytokine, chemokine and MMP-1 stimulated by monosodium urate crystals were differentially regulated by the intracellular activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways but not the p38 mitogen-activated protein kinase pathway. Our results therefore provide a new insight into the uric acid crystal-activated immunopathological mechanisms mediated by distinct intracellular signal transduction pathways leading to joint inflammation in RA.