【作者】 李甲振；

【导师】 沈文律； 孔抗美；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 背景类风湿关节炎(rheumatoid arthritis，RA)是一种慢性、自身免疫性、炎症性关节病，表现为对称性多关节炎，可侵犯多个大小关节，并伴有全身多系统损害。病变晚期受累的关节发生严重变形，使患者完全丧失劳动能力，给社会和家庭带来沉重的负担。关节滑膜炎是RA的基本病理改变，也是关节软骨和软骨下骨破坏的病变基础。RA的发病机制迄今不明了，近年来的研究表明RA的发病与致病因素引起免疫系统功能紊乱有密切关系。细胞免疫异常在RA的发病中起重要作用，在RA关节的滑膜、血管翳、软骨及软骨下骨中有大量表达激活标志的CD4~+ T细胞浸润，这些细胞可与滑膜中的抗原发生免疫应答，细胞因子在这一过程中发挥着重要作用。已知细胞因子肿瘤坏死因子α(tumor necrosis factor，TNF)、白细胞介素1(interleukin，IL)、IL-2、IL-6、IL-10、IL-12、IL-15、IL-18及干扰素γ(interferon，IFN)等参与了RA的病理过程，其中，以TNF-α和IL-1尤为重要。IL-21是近年来发现的一种细胞因子，属于I型细胞因子家族，主要是由CD4~+Th2细胞合成和分泌，为4个螺旋结构，与IL-2、IL-4和IL-15具有同源性。IL-21受体(IL-21R)与IL-2Rβ链、IL-4Rα链和IL-9R有同源性，其主要表达在淋巴组织的B细胞、T细胞和NK细胞上。IL-21的生物学功能主要是刺激T细胞增殖、NK细胞增殖和分化以及CD40特异性应答B细胞的增殖，IL-21功能的发挥依靠其受体的共用γ链，主要通过JAK-STAT途径传递信号。IL-21及其受体在人类疾病中的确切作用还不清楚，已有资料显示IL-21在人类自身免疫性疾病如克隆氏病、系统性硬皮病中有调节作用，提示IL-21在RA的发病中可能也有重要免疫调节作用。目的通过检测IL-21受体在RA患者的滑膜、关节液和外周血单个核淋巴细胞中的表达，以及对IL-21诱导RA患者T细胞激活、促炎症细胞因子分泌作用的研究，探讨IL-21在RA发病中的作用，以期为临床治疗RA提供一种新的有效途径。材料和方法收集14例RA、12例骨关节炎(osteoarthritis，OA)和8例膝关节损伤患者的滑膜组织，免疫组化分析各组病例中IL-21R在滑膜组织中的表达。收集38例RA、24例OA和26例健康体检者的外周血，12例RA、10例OA和7例正常对照的关节液，用流式细胞仪分析各组病例中IL-21R在外周血和关节液单个核淋巴细胞表面的表达。分离、纯化外周血或关节液中淋巴细胞，抗CD3单克隆抗体刺激，在培养不同时间点(8、12、24和48h)检测IL-21R在CD4~+T细胞中的表达。并且，用IL-21刺激激活T淋巴细胞，通过液闪技术、流式细胞分析仪和ELISA法检测分析淋巴细胞的增殖及其促炎细胞因子的分泌。结果1．IL-21R在RA患者的滑膜组织中呈高表达，RA患者滑膜内膜层和下层IL-21R阳性细胞数显著增加，而且总体细胞数目中IL-21R阳性细胞数目(12.3±2.4％)显著高于OA患者(2.1±0.6％)和正常对照组(1.2±0.3％)(P＜0.001)，OA和正常对照组之间的差异无统计学意义。2．用流式细胞分析检测在外周血和关节液单核细胞中IL-21R阳性的细胞数。结果显示RA患者的外周血和关节液中，CD4~+T、CD8~+T细胞、B细胞和NK细胞表达IL-21R的细胞数显著高于对照组(P＜0.05)。3．在正常对照组的外周血和关节液中CD4~+T细胞，在体外经抗CD3刺激后8hIL-21R迅速降低，其后持续较低的表达水平。RA患者中CD4~+T细胞IL-21R水平，在抗CD3刺激后降低缓慢，在培养8、12、24和48h均显著高于对照组。这些结果表明IL-21R主要在RA患者外周血和关节液的单核细胞中表达，在体外刺激中消失较慢，提示IL-21R可能与促炎反应免疫应答有关。4．在RA患者和正常对照组的外周血T细胞中，CD69无或较低表达。经抗CD3抗体刺激后，CD69在二组中表达均增加，但是培养8h时RA患者显著高于对照组(P＜0.05)。IL-21可上调CD69表达，而且RA患者CD69表达水平显著高于对照组(P＜0.05)。RA患者关节液中的T细胞CD69表达显著高于对照组(P＜0.01)。而在IL-21刺激后，二组关节液T细胞CD69表达均上调，在8h时达到高峰，以后降低；而且在RA患者关节液T细胞CD69表达水平在8、12、24、48h均显著高于对照组。提示IL-21可导致RA患者T细胞活性显著增加，以及诱导外周血T细胞的增殖。5．对T细胞诱导增殖效应进行分析，显示RA患者在抗CD3和IL-21刺激下外周血和关节液T细胞增殖指数显著高于对照组。提示IL-21是一个RA患者T细胞强有力的诱导因子，在炎症损伤中外周血和关节液T细胞活化以及对IL-21反应性更加敏感。6．在经抗CD3抗体和IL-21刺激的RA患者的外周血和关节液T细胞培养的上清液中，检测T细胞分泌的细胞因子(如：INF-γ、TNF-α、IL-2和IL-10)。显示RA患者的T细胞与对照组比较分泌较高水平的INF-γ和TNF-α(P＜0.05)。这些结果提示IL-21可显著激发RA患者T细胞的活性，诱导了促炎细胞因子的分泌，与RA的发生发展有密切关系。结论本实验首先观察了IL-21受体在类风湿关节炎患者的滑膜、关节液和外周血中单核淋巴细胞中的表达，在此基础上观察了IL-21受体在CD4~+ T细胞中的表达，进一步观察了抗CD3抗体刺激后IL-21受体在CD4~+ T细胞中的表达变化，最后检测了IL-21对CD4~+ T细胞的激活增殖效应以及促炎因子的分泌作用。结果证实IL-21受体在类风湿关节炎患者的滑膜、CD4~+ T细胞呈高表达，IL-21可诱导T细胞激活、增殖和增加促炎细胞因子的分泌。根据目前研究的结果，我们可得出以下结论：1．IL-21受体在类风湿关节炎患者滑膜中呈现高表达。2．IL-21是类风湿关节炎发病过程中重要的促炎细胞因子。3．阻断IL-21生物学活性对类风湿关节炎临床治疗可能是一有效的方法。更多还原

【Abstract】 BACKGROUDRheumatoid arthritis (RA) is a familiar chronic, systemic and inflammatoryautoimmune disease, in which main clinical features are chronic and symmetricpoly-synovium joints inflammation and extra-articular lesions. RA patients will loseall labor force at the late stage, characterized by the joints deformity. It increased theheavy burden for society and patient’s family.An inflamed synovium is central to the pathophysiology of RA, which is also keyfeature in the induction of joint destruction. The pathogenesis of RA is still elusive.Recent years, increasing evidences have proved that RA is closely associated withabnormal immunological response in the joints. Cellular immune dysregulation playsan important role in the pathogenesis of RA. There are large number of CD4~+ T cellinfiltrates in synovial tissues, these cells are activated and produce high levels ofproinflammatory cytokines, such as TNF-α, IL-1, IL-2, IL-6, IL-10, IL-12, IL-15,IL-18 and IFN-γ,. Importantly, TNF-αand IL-1 are major proinflammatory cytokinesin the induction of joint damage.Interleukin-21 (IL-21) is a recently discovered cytokine and is a novel classⅠcytokine. IL-21 was identified as a four-helix-bundle cytokine that is mosthomologous to IL-15and also has significant homology to IL-2 and IL-4. IL-21R hadthe highest homology to IL-2Rβand IL-4Ruand IL-9R. IL-21 is secreted by CD4~+T-helper cells and selectively modifies both humoral and cell-mediated immuneresponses through an interaction with various cell types including T, B, NK cells anddendritic cells. IL-21R is expressed in T, NK, B and dendritic cells and plays an important role in regulating these cell functional responses. Like IL-2, IL-21 exhibitsa broad capacity to regulate T cell functions. IL-21 is also critical for normal humoralimmunity, and synergizes with anti-CD40 mAb to stimulate B cell activation, clonalexpansion and maturation.It is not known the exact role of IL-21 and it’s receptor in the pathogenesis ofhuman diseases. Some data have shown that IL-21 is involved in the diseasedevelopment of Crohn’s disease and systemic scleroderma, suggesting that IL-21 mayalso play a role in the pathogenesis of RA.OBJECTIVETo investigate the role of IL-21 in the pathogenesis of RA, we analyzed theIL-21R expression in peripheral blood and synovial fluid mononuclear cells, as wellas its in situ expression in synovium and IL-21 induced peripheral blood T cell (PB-T)and synovial fluid T cell (SF-T) activation, proliferation and TNF-αand IFN-γ,secretion. This could help us determine the correlation of RA with IL-21, and find anew and effective approach to treat RA.METHODS14 cases of RA, 12 cases of osteoarthritis (OA) and 8 cases of acute injuriedpatients’ knee joint synovial tissue were obtained. They were immediately embeddedin OCT compound, frozen in liquid nitrogen, and stored at—80℃forimmunohistochemistry. Heparinized peripheral blood samples were obtained from 38patients with RA and 24 patients with OA. In addition, blood samples from 26 healthyvolunteers were also taken for comparison. Synovial fluid (SF) was also taken from12 patients with RA and 10 patients with OA. In addition, SF from 7 patients who hadno any visible and microscopic signs of inflammation after arthroscopic examinationwas used as controls. The diagnosis of RA and OA patients was described accordingto the advised criterion of American College Rheumatology (ACR). None receivedany immunosuppressants and target cytokine therapy. The frequency of IL-21 R+ cellsin PBMC and SFMC was determined on a flow cytometry. PB- and SF-CD4~+ T cellswere purified and stimulated with coated anti-CD3 mAb and IL-21R expression wasdetermined at various time intervals of 8, 12, 24 and 48 h of culture. After stimulated with coated anti-CD3 and IL-21, the role of IL-21 inducing PB-T and SF-T cellactivation, proliferation and proinflammatory cytokine secretion was measured byELISA, flow cytometry and a beta scintillation counter.RESULTS1. IL-21R was highly expressed in synovial tissues of RA patients. IL-21R~+ cellswere significantly increased in the synovial intimal lining and sublining layers in RApatients compared with those in controls. The number of IL-21R~+ cells among totalcell number was significantly higher in inflamed synovial tissue of RA patients (12.3±2.4％) compared with that from OA patients (2.1±0.6％) and healthy controls (1.2±0.3％) (P＜0.001). No difference was observed between OA patients and healthycontrols.2. After confirming in situ expression of IL-21R in synovial tissues of RApatients, we wanted to address the frequencies of IL-21R~+ cells in PBMC and SFMCusing a flow cytometry. CD4~+, CD8~+ T cells, B cells and NK cells in peripheral bloodand synovial fluid of RA patients expressed significantly higher levels of IL-21Rcompared with those in healthy controls (P＜0.05).3. IL-21R was rapidly decreased in healthy PB- and SF-CD4~+ T cells 8 h afteranti-CD3 in vitro stimulation, with a relatively low level of IL-21R expressionthereafter. However, the levels of IL-21R in RA CD4~+ T cells were found to bedecreased slowly after anti-CD3 stimulation, but still significantly higher comparedwith controls at 8, 12, 24 and 48 h of culture (P＜0.05). These results indicate thatIL-21R expression is mainly expressed in freshly isolated mononuclear cells fromperipheral blood and synovial fluid and disappear slowly after in vitro stimulation,suggesting that IL-21R may be involved in proinflammatory immune response insynovial membrane.4. Very low to absent levels of CD69 expression were detected on freshlyisolated PB-T cells from both RA and controls. After stimulation with coatedanti-CD3, CD69 expression was observed to be increased in both groups, but it wassignificantly higher in RA PB-T cells as compared to healthy controls (P＜0.05 at 8 hof culture). Interestingly, IL-21 further upregulated CD69 expression in PB-T cells, and the levels of CD69 were markedly higher in RA PB-T cells compared withcontrols (P＜0.05 at 8 and 12 h of culture). Moreover, CD69 expression was observedto be markedly increased in freshly isolated SF-T cells than in controls (P＜0.01).Interestingly, CD69 was upregulated in SF-T cells from both groups after IL-21stimulation, reached at the peak level 8 h, and decreased thereafter. However, itslevels were also significantly higher in RA SF-T cells than in healthy controls at 8, 12,24 and 48 h of culture (P＜0.05). Therefore, these data indicate that IL-21 stronglyinduces RAT cell activation.5. Since IL-21 could upregulate RA T cell activation, we further determined theproliferation-inducing effect of IL-2I on these T cells. PB- and SF-T cells from RAsynovial patients, when stimulated with coated anti-CD3 and IL-21, had asignificantly higher proliferative index than those from healthy controls. These resultsindicated that IL-21 is a strong inducer of RA T cells, suggesting that PB- and SF-Tcells are activated during inflammatory injury and become more responsive to IL-21.6. We explored whether IL-21 could contribute to proinflammatory cytokineproduction in the pathogenesis of RA. To this end, PB- or SF-T cells from RA patientswere stimulated with anti-CD3 and IL-21. T cell-derived cytokines (e.g., IFN-γ,,TNF-α, IL-2, IL-10) were measured by ELISA in culture supernatants. T ceils fromRA patients secreted significantly higher levels of TNF-αand IFN-γ,as. compared withthose from healthy controls (P＜0.05). These data demonstrated that IL-21 stronglytriggers RA T cell activation and induces proinflammatory cytokine secretion and maybe directly associated with the development of RA.CONCLUSION1. IL-21R is highly expressed in synovial tissue of RA patients.2. IL-21 may be an important proinflammatory cytokine in the pathogenesis.3. Targeted therapy directed blocking IL-21 may be helpful in the treatment ofRA.更多还原