IL-23 Dependent and Independent Stages of Experimental Arthritis: No Clinical Effect of Therapeutic IL-23p19 Inhibition in Collagen-induced Arthritis

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.     

Eosinophils attenuate arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway

Rheumatoid arthritis (RA) represents a type of autoimmune disease that mainly affect the joints due to persistent synovitis. Eosinophils were Th2 effector cells that have been shown to have anti-inflammatory role recently. In this study, we aimed to investigate the effects of eosinophils transfer on arthritis and underlying mechanisms. DBA/1 mice were induced with collagen-induced arthritis (CIA) and treated with purified eosinophils at different time points. We showed that eosinophils transfer attenuated arthritis in CIA mice. Meanwhile, TNF-α, IL-6, IL-12 and iNOS levels were decreased whereas TGF-β, IL-10, IL-13 and Arg1 levels were increased after eosinophil transfer. In vitro stimulation of bone marrow-derived macrophage (BMDM) with LPS and IFN-γ induced high expression of CD68, iNOS, TNF-α, IL-6, and IL-12, while treatment with eosinophils downregulated their expression levels. Furthermore, high levels of p-IκB and p-P38 expression in BMDM induced by LPS and IFN-γ could be suppressed by eosinophil treatment, and a P38 or IκB inhibitor accelerated the effect of eosinophils on macrophage polarization. Our results demonstrate that eosinophils exert anti-inflammatory effects in arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway.     

Protection against collagen-induced arthritis by electrotransfer of an expression plasmid for the interleukin-4

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, leading to cartilage and bone destruction. We investigated whether the electrotransfer of IL-4 DNA could regulate the disease progress of murine collagen-induced arthritis (CIA). The maximum serum level of mIL-4 was measured by 340 pg/ml on day 1 following DNA transfer. The onset of severe CIA and the degree of synovitis and cartilage erosion were significantly reduced in mice treated with IL-4 DNA (P<0.05). The beneficial effect of IL-4 gene transfer lasted for at least 17 days subsequent to treatment. The expression of IL-1beta was considerably decreased in the paws by IL-4 DNA transfer (P<0.01). On the contrary, the ratio of TIMP2 to MMP2 significantly increased in the IL-4 DNA-treated group (P<0.01). These data demonstrated that electroporation-mediated gene transfer could provide a new approach as an IL-4 therapy for autoimmune arthritis.     

CXCR3 antagonist AMG487 suppresses rheumatoid arthritis pathogenesis and progression by shifting the Th17/Treg cell balance

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by uncontrolled joint inflammation and damage to bone and cartilage. Previous studies have shown that chemokine receptors have important roles in RA development, and that blocking these receptors effectively inhibits RA progression. Our study was undertaken to investigate the role of AMG487, a selective CXCR3 antagonist, in DBA/1J mice bearing collagen-induced arthritis (CIA). Following induction of CIA, animals were treated with 5 mg/kg AMG487 intraperitoneally every 48 h, starting from day 21 until day 41 and evaluated for clinical score, and histological hallmarks of arthritic inflammation. We further investigated the effect of AMG487 on Th1 (T-bet), Th17 (IL-17A, RORγt, STAT3), Th22 (IL-22), and T regulatory (Treg; Foxp3 and IL-10) cells in splenic CXCR3⁺ and CD4⁺ T cells using flow cytometry. We also assessed the effect of AMG487 on T-bet, RORγt, IL-17A, IL-22, Foxp3, and IL-10 at both mRNA and protein levels using RT-PCR and Western blot analyses of knee samples. The severity of clinical scores, and histological inflammatory damage decreased significantly in AMG487-treated compared with CIA control mice. Moreover, the percentage of Th1, Th17, and Th22 cells decreased significantly and that of Treg cells increased in AMG487-treated mice. We further observed that AMG487-treatment downregulated T-bet, IL-17A, RORγt, and IL-22, whereas it upregulated Foxp3 and IL-10 mRNA and protein levels. This study demonstrates the antiarthritic effects of AMG487 in CIA animal model and supports the development of CXCR3 antagonists as a novel strategy for the treatment of inflammatory and arthritic conditions.     

Acceleration and increased severity of collagen-induced arthritis in P-selectin mutant mice.

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14-91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.     

Decreased IgG production but increased MIP-1beta expression in collagen-induced arthritis in C-C chemokine receptor 5-deficient mice

Collagen-induced arthritis (CIA) is a widely used model of human rheumatoid arthritis (RA) characterized by chronic inflammation of the synovial joints. The pathogenesis of RA and CIA has not been completely defined, but both involve the recruitment of leukocytes and lymphocytes to the joints and Th1-type cell mediated autoimmune responses. The C-C chemokine receptor 5 (CCR5) is preferentially expressed on Th1 cells and has been strongly implicated in inflammatory process through trafficking of leukocytes and lymphocytes into the sites of inflammation. We investigated the role of the CCR5 in CIA using CCR5 knockout mice (CCR5-/-) in which we analyzed the consequences of CCR5 deficiency for the immune response and inflammation. We found that CCR5-/- mice showed a significant reduction in the incidence of CIA after collagen II (CII)-immunization as compared to wild-type (CCR5+/+) mice. The reduced incidence seen in CCR5-/- mice was associated with these animals having significantly lower IgG levels, especially IgG2a and IgG2b antibodies against CII, as well as an obviously augmented IL-10 production in splenocytes. Overproduction of MIP-1beta in CCR5-deficient mice after CII-immunization may contribute partially to the occurrence of arthritis.     

IL-10-deficient B10.Q mice develop more severe collagen-induced arthritis, but are protected from arthritis induced with anti-type II collagen antibodies.

IL-10 is a pleiotropic cytokine with stimulatory and inhibitory properties, and is thought to have a protective role in rheumatoid arthritis and collagen-induced arthritis (CIA). In this study, we investigated how IL-10 deficiency affects CIA and anti-collagen type II (CII) Ab-transferred arthritis in C57BL/10.Q (B10.Q) mice. The B10.Q.IL-10(-/-) mice had an 8-cM 129/Ola fragment around the IL-10 gene. The mice were treated with antibiotics, appeared healthy, and had no colitis. T cells from IL-10(-/-) mice expressed similar levels of IFN-gamma, IL-2, and IL-4 after mitogen stimulation; however, macrophages showed a reduced TNF-alpha production compared with IL-10(+/-) littermates. IL-10(-/-) mice had an increased incidence, and a more severe CIA disease than the IL-10(+/-) littermates. To study the role of IL-10 in T cell tolerance, IL-10(-/-) were crossed into mice carrying the immunodominant epitope, CII(256-270), in cartilage (MMC) or in skin (TSC). Both IL-10(-/-) and IL-10(+/-) MMC and TSC mice were completely tolerized against CIA, indicating that lack of IL-10 in this context did not break tolerance. To investigate whether IL-10 was important in the effector phase of CIA, arthritis was induced with anti-CII Abs. Surprisingly, IL-10(-/-) were less susceptible to Ab-transferred arthritis, as only 30% showed signs of disease compared with 90% of the littermates. Therefore, IL-10 seemed to have a protective role in CIA, but seemed to exacerbate the arthritogenicity of anti-CII Abs. These data emphasize the importance of studying IL-10 in a defined genetic context in vivo, to understand its role in a complex disease like arthritis.     

Interleukin-35 (IL-35) inhibits proliferation and promotes apoptosis of fibroblast-like synoviocytes isolated from mice with collagen-induced arthritis

Rheumatoid arthritis (RA) is an inflammatory disorder of the joints that affects 0.5–1 % of adults. Excessive growth of the fibroblast-like synoviocytes (FLS) promotes hyperplasia of synovial tissues and causes its invasion into the bone and cartilage, which eventually causes deformity and dysfunction of affected joints. Interleukin 35 (IL-35) was shown to suppress the inflammatory responses to collagen-induced arthritis (CIA) via upregulation of T regulatory cells and suppression of T helper type 17 cells in a mouse model. To study the effects of IL-35 on the proliferation and apoptosis frequency of cultured FLS isolated from mice with CIA as well as to examine the effects of IL-35 on CIA in vivo. Thirty DBA/1 J mice, which are used as an animal model for RA, were divided randomly (ten mice per group) to a CIA group (collagen treatment), a CIA + IL-35 group (collagen and IL-35 treatments), and a control group (no treatment). Starting on the 24th day after collagen administration, IL-35 was injected intraperitoneally into mice of the CIA + IL-35 group once per day for 10 days. An arthritis index was calculated, and pathological analysis of synovial tissue was performed. FLS isolated from CIA mice were treated with various concentrations of IL-35 (12.5–100 ng/ml). The MTT assay was used to examine FLS proliferation, and apoptosis frequency of FLS was detected by flow cytometry. On day 24, the CIA mice began to exhibit arthritis symptoms, and the symptoms rapidly progressed with time. Treatment with IL-35 significantly alleviated arthritis symptoms and reduced the synovial tissue inflammation. In addition, IL-35 treatment inhibited proliferation and promoted apoptosis in cultured FLS from CIA mice in a dose-dependent manner. IL-35 could ameliorate the symptoms of arthritis in the CIA mouse model in vivo and inhibited FLS proliferation while promoting FLS apoptosis in vitro, thereby exhibited the potential in inhibiting the progression of RA.     

Therapeutic potential of human umbilical cord mesenchymal stem cells in the treatment of rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is a T-cell-mediated systemic autoimmune disease, characterized by synovium inflammation and articular destruction. Bone marrow mesenchymal stem cells (MSCs) could be effective in the treatment of several autoimmune diseases. However, there has been thus far no report on umbilical cord (UC)-MSCs in the treatment of RA. Here, potential immunosuppressive effects of human UC-MSCs in RA were evaluated.

Methods

The effects of UC-MSCs on the responses of fibroblast-like synoviocytes (FLSs) and T cells in RA patients were explored. The possible molecular mechanism mediating this immunosuppressive effect of UC-MSCs was explored by addition of inhibitors to indoleamine 2,3-dioxygenase (IDO), Nitric oxide (NO), prostaglandin E2 (PGE2), transforming growth factor β1 (TGF-β1) and interleukin 10 (IL-10). The therapeutic effects of systemic infusion of human UC-MSCs on collagen-induced arthritis (CIA) in a mouse model were explored.

Results

In vitro, UC-MSCs were capable of inhibiting proliferation of FLSs from RA patients, via IL-10, IDO and TGF-β1. Furthermore, the invasive behavior and IL-6 secretion of FLSs were also significantly suppressed. On the other hand, UC-MSCs induced hyporesponsiveness of T cells mediated by PGE2, TGF-β1 and NO and UC-MSCs could promote the expansion of CD4⁺ Foxp3⁺ regulatory T cells from RA patients. More importantly, systemic infusion of human UC-MSCs reduced the severity of CIA in a mouse model. Consistently, there were reduced levels of proinflammatory cytokines and chemokines (TNF-α, IL-6 and monocyte chemoattractant protein-1) and increased levels of the anti-inflammatory/regulatory cytokine (IL-10) in sera of UC-MSCs treated mice. Moreover, such treatment shifted Th1/Th2 type responses and induced Tregs in CIA.

Conclusions

In conclusion, human UC-MSCs suppressed the various inflammatory effects of FLSs and T cells of RA in vitro, and attenuated the development of CIA in vivo, strongly suggesting that UC-MSCs might be a therapeutic strategy in RA. In addition, the immunosuppressive activitiy of UC-MSCs could be prolonged by the participation of Tregs.     

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

Introduction

Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.

Methods

CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b⁺ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.

Results

CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b⁺ splenocytes into the synovial tissues.

Conclusions

The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.     

Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.     

Retinoic acid attenuates rheumatoid inflammation in mice

Retinoic acid is the active vitamin A derivative and is well-known to have diverse immunomodulatory actions. In this study, we investigated the impact of all-trans retinoic acid (ATRA), a biologic key metabolite of vitamin A, on the development of arthritis and the pathophysiologic mechanisms by which ATRA might have antiarthritic effects in animal model of rheumatoid arthritis (RA; collagen-induced arthritis [CIA] in DBA/1J mice). We showed that treatment with ATRA markedly suppressed the clinical and histologic signs of arthritis in the CIA mice. It reduced the expression of IL-17 in the arthritic joints. Interestingly, Foxp3(+) regulatory T cells were markedly increased and IL-17-producing CD4(+) T cells (Th17 cells) were decreased in the spleens of ATRA-treated mice. In vitro treatment with ATRA induced the expression of Foxp3 and repressed the IL-17 expression in the CD4(+) T cells in mice. ATRA suppressed the production of total IgG and IgG2a in splenocytes that were stimulated by LPS. It also reduced serum levels of total IgG and IgG2 anti-collagen Abs and germinal center formation in CIA mice. In addition, the ATRA-treated mice showed decreased osteoclast formation in arthritic joints. Moreover, ATRA downregulated the expression of receptor activator of NF-κB ligand, the leading player of osteoclastogenesis, in the CD4(+) T cells and fibroblast-like synoviocytes from patients with RA. Furthermore, ATRA prevented both human monocytes and mice bone marrow-derived monocytes/macrophage cells from differentiating into osteoclasts. These data suggest ATRA might be an effective treatment modality for RA patients.     

Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen

Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-beta from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-gamma when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.     

Development of proteoglycan-induced arthritis is independent of IL-17

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-gamma are secreted in CIA and EAE, disease is exacerbated in IFN-gamma- or IFN-gamma receptor-deficient mice due to the ability of IFN-gamma to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-gamma. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17-/-) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17-/- mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17-/- mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17-/- mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-kappaB ligand) expression. There were only subtle differences between WT and IL-17-/- mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.     

Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune-modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen-induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen-specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of T_H1 and T_H17 cells in the spleens of CIA mice. WKYMVm attenuated T_H1 and T_H17 differentiation in a dendritic cell (DC)-dependent manner. WKYMVm-induced beneficial effects against CIA and WKYMVm-attenuated T_H1 and T_H17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL-10 production from lipopolysaccharide-stimulated DCs and WKYMVm failed to suppress T_H1 and T_H17 differentiation in the presence of anti-IL-10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of T_H1 and T_H17 cells via IL-10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.     

Systemic metabolism of tryptophan and its catabolites,kynurenine and 3-HAA,in mice with inflammatory arthritis

Tryptophan is an essential amino acid. The liver is primary organ involved the oxidative catabolism of tryptophan. However, in the immune system, tryptophan and its catabolites, kynurenine and 3-hydroxyanthranilic acid (3-HAA), play an anti-inflammatory role. Rheumatoid arthritis (RA) is an autoimmune disease. Collagen induced arthritis (CIA) is an animal model of RA. Therefore, it was of interest to measure concentration of tryptophan, kynurenine and 3-HAA in mice with CIA. Concentration of tryptophan and 3-HAA was measured with HPLC methods. Concentration of kynurenine was measured with colorimetric test. mRNA expression for the kynurenine pathway genes was assessed using qRT-PCR. It has been found that in sera from diseased mice concentration of tryptophan was not changed. Concentration of kynurenine and 3-HAA was decreased. Moreover, in the livers from mice with CIA, concentration of tryptophan and kynurenine was decreased. These observations coincided with decreased mRNA expression for Ido2 and Afm and increased mRNA expression for Kynureninase in the liver. It has been also shown that in CIA the concentration of 3-HAA was increased in the kidneys.     

小鼠胶原诱导性关节炎模型的建立及其免疫学变化研究

目的 胶原诱导性关节炎模型（collagen induced arthritis,CIA）是研究类风湿性关节炎发病机制和治疗药物筛选的理想模型,也是目前国际上公认的关节炎模型.但是,目前鲜见Ⅱ型胶原诱导CIA模型的系统免疫学变化的报道.因此,本研究采用DBA/1小鼠诱导了CIA模型,并对其免疫学改变进行了系统研究.方法 将牛Ⅱ型胶原与完全弗氏佐剂混和并充分乳化,于DBA/1小鼠尾根部皮内注射进行初次免疫,20 d后同样方法进行再次免疫.应用千分尺测量CIA模型小鼠的左右两侧足掌厚度,并进行关节炎评分.酶联免疫吸附试验测定小鼠血清Ⅱ型胶原特异性抗体,Luminex技术和αLISA技术测定血清及培养上清中的细胞因子水平.结果CIA小鼠于造模后23 d开始,陆续出现前肢、后肢的红肿、功能障碍,发病率高达100％,且随着时间的延长其关节肿胀程度呈进行性加重,关节炎评分增高.CIA小鼠脾脏指数较正常组明显升高,且Ⅱ型胶原刺激的特异性T细胞增殖明显增强.细胞因子检测结果表明,脾细胞培养上清中IFN-γ和1L-4含量及IFN-γ/IL-4比值明显升高,TNF-α和IL-1β水平亦显著升高.此外,CIA小鼠血清中存在高水平的Ⅱ型胶原特异性抗体.结论 Ⅱ型胶原诱导CIA模型发病率高,免疫学改变以Th1细胞因子升高为主,兼有细胞免疫功能及体液免疫功能损伤.     

Prophylactic effect of the oral administration of transgenic rice seeds containing altered peptide ligands of type II collagen on rheumatoid arthritis

Rheumatoid arthritis is an autoimmune disease associated with the recognition of self proteins secluded in arthritic joints. We generated transgenic rice seeds expressing three types of altered peptide ligands (APL) and the T cell epitope of type II collagen (CII256–271). When these transgenic rice and non-transgenic rice seeds were orally administrated to DBA/1?J mice once a day for 14?days, followed by immunization with CII, the clinical score of collagen-induced arthritis (CIA) was reduced and inflammation and erosion in the joints were prevented in mice fed APL7 transgenic rice only. IL-10 production against the CII antigen significantly increased in the splenocytes and iLN of CIA mice immunized with the CII antigen, whereas IFN-γ, IL-17, and IL-2 levels were not altered. These results suggest that IL-10-mediated immune suppression is involved in the prophylactic effects caused by transgenic rice expressing APL7.