实验性自身免疫性葡萄膜视网膜炎大鼠T细胞受体Vβ8.3基因的表达

目的 探讨实验性自身免疫性葡萄膜视网膜炎(EAU)大鼠CD4⁺T细胞抗原受体(TCR)Vβ8.3基因的表达。 方法 Lewis大鼠18只分为EAU、Freund完全佐剂及空白对照组。用Fmoc 化学合成法合成光感受器间维生素A类结合蛋白（IRBP）R16多肽片段，以诱导EAU动物模型。利用磁吸附细胞分选法(MACS)分离大鼠脾脏CD4⁺T细胞，流式细胞术检测MACS分选前后CD4⁺T细胞比例，监测细胞分选效果。通过荧光定量-聚合酶链反应法检测大鼠脾脏CD4⁺T细胞的TCR Vβ8.3基因片段的表达。 结果 IRBP R16 免疫Lewis大鼠稳定地诱导出EAU动物模型；MACS分选CD4⁺T细胞的纯度较分选前明显增高 （P＜0.001）；IRBP R16 诱导的EAU大鼠CD4⁺T细胞受体Vβ8.3基因的表达显著高于空白对照组（P＜0.05）。 结论 在IRBP R16 诱导的EAU中存在着抗原特异性T细胞受体Vβ8.3基因的优势利用，为EAU的免疫治疗提供了新的思路。 （中华眼底病杂志，2004，20：167-167）     

实验性自身免疫性葡萄膜视网膜炎中浸润细胞的表型及其凋亡的研究

目的探讨实验性自身免疫性葡萄膜视网膜炎(experimental autoimmune uveoretinitis,EAU)中参与的细胞表型及其凋亡。方法用光感受器间维生素A类结合蛋白(interphotoreceptor retinoid-binding protein,IRBP)免疫16只Lewis鼠后，于眼组织切片和平片上进行免疫组织化学染色和原位 凋亡染色，所用抗体为抗单核细胞、巨噬细胞(EDI)、MHC-II类抗原(OX6)、T淋巴细胞(R73)的单克隆抗体，所用原位凋亡试剂盒为TACS1 Klenow。结果用IRBP免疫Lewis鼠后，16只鼠中12只发生了临床可见的葡萄膜炎，炎症平均得分为1.29±0.7级；免疫组织化学染色发现葡萄膜和视网膜中有大量的单核细胞、淋巴细胞及MHC-II⁺细胞浸润，这些组织中均可见浸润细胞的凋亡，虹膜睫状体中凋亡细胞明显多于脉络膜和视网膜中的凋亡细胞。结论单核巨噬细胞、淋巴细胞和MHC-II⁺细胞均参与了EAU的形成，在EAU早期即有浸润细胞发生凋亡，此可能是导致这种炎症迅速消退的重要机制。（中华眼底病杂志，2000，16：1-70）     

IRBP免疫鼠血清特异性抗体的测定及动态变化

我们首次在国内提纯了光感受器间维生素 A类结合蛋白(interphotoreceptor retinoid-binding protein,IRBP),将其免疫 Lewis 大鼠后动态测定了鼠血清抗IRBP 抗体和抗视网膜 S 抗原抗体.发现抗 IRBP 抗体于免疫后第7天出现,以后逐渐上升,于第26天达高峰,未测出抗 S 抗原抗体.根据特异性抗体与实验性自身免疫性葡萄膜视网膜炎(experimental autoimmune uveoretinitis,EAU)之间的关系,讨论了特异性体液免疫反应在 EAU发生中的作用.     

大鼠自身免疫性葡萄膜视网膜炎眼部细胞因子信号抑制因子的表达

目的:大鼠自身免疫性葡萄膜视网膜炎(experimentalautoimmune uveoretinitis,EAU)大鼠模型眼部研究细胞因子信号抑制因子(suppressor of cytokine signaling,SOCS)的表达。方法:用光感受器间维生素A类结合蛋白(interphotoreceptorretinoid-binding protein,IRBP)免疫Lewis鼠160只后,在眼组织切片上应用SOCS多克隆抗体进行免疫组织化学染色,观察其在眼组织中的表达并与正常大鼠40只做对照。结果:用IRBP免疫Lewis鼠后,免疫组织化学染色发现在虹膜、睫状体和视网膜部位可见SOCS-1和SOCS-5蛋白表达;而在正常Lewis鼠未见SOCS蛋白表达。统计学结果显示,SOCS-1和SOCS-5蛋白表达与EAU严重程度呈正相关(r1=0.954,r2=0.963,P<0.01)。结论:自身免疫性葡萄膜视网膜炎Lewis鼠出现SOCS阳性表达细胞在EAU的发病过程中起了一定的作用。     

大鼠的实验性自身免疫性葡萄膜视网膜炎的免疫致病机理

为了了解实验性自身免疫性葡萄膜视网膜炎(EAU)免疫致病机理,用Lewis鼠研究T淋巴细胞和肥大细胞的作用。用致敏的淋巴细胞特别是辅助性/诱异性T细胞亚群成功地转移给首次用来作实验的同系大鼠。接受了对S抗原致敏了的辅助性/诱导性T细胞的实验性自身免疫性葡萄膜视网膜炎(EAU)的大鼠充分表现了对此抗原的迟发型皮肤超敏反应但极轻微的Arthus反应。分析了环孢霉素、环磷酰胺,地塞米松等免疫抑制剂药物对S抗原免疫的鼠产生EAU的作用。通过选择性抑制对S抗原的迟发型皮肤超敏反应,说明仅有环孢霉素能完全抑制EAU的发生。这些资料说明T淋巴细胞在EAU致病机理中起主要的作用。根据在疾病发作以前,脉络膜的肥大细胞发生脱颗粒作用,说明除了淋巴细胞参与以外,脉络膜的肥大细胞也起了附带作用。     

调节性T细胞在实验性自身免疫性葡萄膜炎中的作用

目的探讨CD4^＋调节性T细胞（Treg）在实验性自身免疫性葡萄膜炎（EAU）病程进展中的作用。方法通过IRBP161-180和完全弗氏佐剂皮下免疫,在B10RⅢ小鼠诱导产生EAU,对眼内炎症行临床检查并对其严重程度予以评分。通过免疫磁珠分选EAU恢复期小鼠脾脏CD4^＋T细胞、CD8^＋T细胞、CD4^＋CD25^＋T细胞和CD4^＋CD25^-T细胞。通过流式细胞术分析脾脏CD4^＋CD25^＋Foxp3^＋T细胞的比例。通过体内过继转移评价Treg的抑制效应。结果小鼠诱导EAU后产生眼内炎症反应。将EAU恢复期小鼠脾细胞、脾CD4＋T细胞或脾CD4^＋CD25^＋T细胞过继转移入正常小鼠继而皮下免疫诱导EAU,眼底检查显示受者小鼠眼内炎症程度明显降低。相反,正常小鼠脾细胞、EAU恢复期脾CD8^＋T细胞或CD4^＋CD25^-T细胞过继转移无此效应。流式细胞分析显示随着EAU的发展,脾内CD4^＋CD25^＋T细胞比例和CD4^＋CD25^＋Foxp3^＋T细胞比例逐步升高,并在恢复期维持较高水平。结论 CD4^＋Treg随着EAU的病程进展数量增多,它们可能参与EAU的自发缓解。     

实验性自身免疫性葡萄膜炎小鼠T细胞变化的动态研究

目的：了解实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis, EAU)不同进展阶段小鼠T细胞动态变化,对葡萄膜炎治疗方案的优化及疗效评价提供指导。

方法：用CFA+PTX+IRBP对6～10周龄雌性C57BL/6小鼠后肢及尾部皮下进行三点免疫建立EAU模型,于免疫3,7,14,21,28d取外周血行流式细胞术检测。

结果：用特异性抗原IRBP免疫后,EAU疾病在第14d左右产生,在第21d达最高峰,以后开始逐渐缓解。随着EAU疾病的发生,CD4⁺CD25⁺调节性T细胞、CD4⁺CD3⁺辅助T细胞数量均有增加,CD4⁺CD25⁺调节性T细胞增加更为明显,CD4⁺CD25⁺Foxp3⁺调节性T细胞数量第21d达到高峰,第28d开始下降,CD4⁺CD25⁺Foxp3⁺/CD4⁺CD25^-Foxp3⁺比值从第3d开始逐渐增加,到第21d达到高峰,从第28d开始下降。

结论：EAU疾病的发生和转归与CD4⁺CD25^+/-Foxp3⁺Treg细胞密切相关,CD4⁺CD25^+/-Foxp3⁺Treg细胞为阐明EAU的缓解机制、预防和治疗人类葡萄膜炎提供了新思路。     

Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用

背景 C57BL/6及B10RⅢ小鼠是实验性自身免疫性葡萄膜炎(EAU)动物模型常用的小鼠种系,其中C57B L/6小鼠免疫后眼部炎症较轻,而B10RⅢ小鼠免疫后眼部表现为典型的后葡萄膜炎病理改变.目的 观察培养的光感受器间维生素A类结合蛋白(IRBP)抗原特异性Th1、Th17细胞对小鼠视网膜星形细胞的杀伤作用,研究EAU中Th1及Th17细胞的致病机制. 方法 选取C57BL/6小鼠、B10RⅢ小鼠各10只,尾根部及躯干皮下注射200 μl含有200 μg IRBP1-20或IRBP161-180抗原及完全弗氏佐剂(CFA)的乳化液,共均匀注射6个点以免疫动物.流式细胞仪分析B10RⅢ小鼠模型的眼内浸润T细胞类别.分离培养C57BL/6小鼠淋巴结及脾脏IRBP特异性T细胞,分别加入白细胞介素2(IL-2)或IL-23以适于Th1、Th17细胞生长.将培养至第5天的Th1、Th17细胞分别加入到经干扰素-γ(IFN-γ)预处理的单层视网膜星形细胞中,观察细胞间的相互作用,检测肿瘤坏死因子-α( TNF-α)的质量浓度. 结果 B10RⅢ小鼠EAU模型眼球中有大量炎性细胞浸润,流式细胞仪检测证实含有IFN-γ+、IL-17+细胞和CD45+细胞,分别占9.5％、5.1％和41.4％.IRBP1-20刺激后6d,含IL-2和IL-23培养基中IFN-γ+细胞分别为44.0％、8.0％,IL17+细胞分别为1.0％、26.0％.Th1、Th17与视网膜星形细胞相互作用24 h后,可见视网膜星形细胞死亡脱落,Th17较Th1具有更强的杀伤作用.Th1、Th17细胞分别与星形细胞共培养48 h,两种培养基中TNF-α的质量浓度分别为(500±10)、(801±24) μg/L,差异有统计学意义(t=-20.36,P=0.00).结论 Th1、Th17对视网膜星形细胞均有杀伤作用,Th17细胞杀伤作用更强,在葡萄膜炎致病过程中发挥更重要的作用.杀伤过程中既有细胞直接接触作用,又有通过细胞因子介导的间接作用.     

抗肿瘤坏死因子-α单克隆抗体治疗实验性自身免疫性葡萄膜视网膜炎的研究

目的 观察抗肿瘤坏死因子-α单克隆抗体（TNF-α MCAb）对于实验性自身免疫性葡萄膜视网膜炎（EAU）的治疗作用。方法 合成光感受器间维生素A类结合蛋白（IRBP）R16多肽片段，联合免疫佐剂诱导EAU动物模型。按照注射次数不同将大鼠分为2组，分别于IRBP R16免疫后的第6天和第4、6、8天自大鼠尾静脉注入TNF-α MCAb，裂隙灯显微镜观察其眼部临床表现，同时设未治疗组大鼠作为对照。于IRBP R16免疫后第13天测量迟发型超敏反应（DTH），并于第14天处死大鼠，取眼球行组织病理检查。应用酶联免疫吸附试验（ELISA）检测抗体注射后14 d 时大鼠血清中Th1类细胞因子γ-干扰素（IFN-γ）、Th2类细胞因子白细胞介素-4（IL-4）水平以及房水中IFN-γ水平。测量引流淋巴结细胞的抗原特异性淋巴细胞增生反应。结果 TNF-αMCAb治疗组大鼠的眼部炎症较未治疗组明显减轻，病理分级下降；房水和血清中IFN-γ水平降低，血清中IL-4增高；DTH反应下降；引流淋巴结细胞对IRBP R16多肽刺激的增生反应下降，差异均有统计学意义（P＜0.01）。与单次注射相比，3次注射的治疗效果更显著（P＜0.05）。结论 TNF-αMCAb能够有效减轻EAU大鼠眼部的炎症反应和特异性细胞免疫反应，改变Th1/Th2细胞因子平衡；多次应用作用更显著。     

短期高浓度葡萄糖摄入对小鼠实验性自身免疫性葡萄膜炎发展的作用

目的 探索短期高浓度葡萄糖摄入对小鼠实验性自身免疫性葡萄膜炎(EAU)发展的促进作用.方法 选取健康SPF级6～8周龄小鼠38只,随机分为模型对照组(EAU组)和高糖模型组(GLU组),每组19只小鼠.GLU组小鼠从造模前14 d开始给予200 g·L-1葡萄糖溶液饮水直至处死,EAU组小鼠给予正常饮水,同时保证EAU...     

Adoptive transfer of experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein

The immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) were studied. Lymph node (LN) cells or spleen cells were collected from donor rats 14 days after the immunization with IRBP. After the 3-day pre-incubation of these cells with either IRBP or Concanavalin A (Con A), the cells were transferred to naive syngeneic rats intraperitoneally. EAU was successfully transferred by LN cells pre-cultured with IRBP, while EAU was poorly transferred by LN cells pre-cultured with Con A. On the other hand, spleen cells pre-cultured with either IRBP or Con A were quite potent to transfer EAU. In addition, the enriched helper/inducer T-cells repeatedly transferred EAU, while the enriched suppressor/cytotoxic T-cells did not. There also existed the simultaneous involvement of pinealitis. The histopathological features of EAU and pinealitis were generally similar to those in actively immunized rats. The immune responses to IRBP in recipients with EAU were mostly cellular (delayed hypersensitivity type skin response and proliferative responses of lymphocytes), while humoral responses (Arthus type skin response and antibody activities) were quite weak in most of the recipients. Thus, it was confirmed that cellular immunity plays a major role in the adoptive transfer of EAU by IRBP as in S-antigen-induced EAU.     

Antigen-specific suppressor cells induced by FK506 in experimental autoimmune uveoretinitis in the rat.

The authors previously reported that FK506 effectively suppressed the induction of experimental autoimmune uveoretinitis (EAU) in rats with much lower doses than cyclosporine A. This study was aimed at analyzing the immune status of the FK506-treated and EAU-suppressed rats and examining the hypothesis whether the agent could induce antigen-specific suppressor T (Ts) cells. It was found that spleens from S-antigen-immunized and FK506-treated rats contained a population of Ts cells inhibiting the proliferative responses of S-antigen-sensitized lymphocytes to S-antigen, yet these cells did not affect the proliferative responses of interphotoreceptor retinoid-binding protein (IRBP)-sensitized lymphocytes to IRBP. The helper T (Th) cells did not exhibit such suppressor activities. Furthermore, transfer of Ts cells from S-antigen-immunized and FK506-treated rats to naive syngenic rats induced partial inhibition of EAU induction or delay of EAU onset after immunizing the recipient rats with S-antigen. Lymphocytes from the EAU-suppressed recipients showed low proliferative response to S-antigen and low levels of antibody to S-antigen. These data thus indicate that FK506 treatment after S-antigen immunization induces an activation of Ts cells specific to S-antigen and that the Ts cells might contribute, at least in part, to the uniquely prolonged and intensive immunosuppression by FK506.     

Serial adoptive transfer of experimental autoimmune uveoretinitis in rats

Experimental autoimmune uveoretinitis (EAU) and pinealitis induced by an interphotoreceptor retinoid-binding protein (IRBP)-derived peptide (R4) was serially transferred into naive recipient rats, using spleen cells from recipients of previous "orders" of transfer. The cells initiating the disease in recipients of the first order were either lymph node cells from rats immunized against peptide R4, or lymphocytes of a cell line specific toward this peptide. The serial transfer was successfully carried out through as many as four orders of sequential recipients.     

Adoptive transfer of experimental autoimmune uveoretinitis in rats. Immunopathogenic mechanisms and histologic features

In order to learn about the immunopathogenic mechanisms of experimental autoimmune uveoretinitis (EAU), the capacity of lymphocytes to transfer the disease was studied. EAU was transferred to naive syngeneic rats by intraperitoneal injection of spleen or lymph node (LN) cells from S-antigen immunized rats, following their incubation in culture with either S-antigen or concanavalin A (Con A). In contrast, the same cells did not cause inflammatory changes in the recipient eyes when injected intravitreally. The identity of the lymphocytes that transfer EAU was determined by using monoclonal antibody enriched subsets of lymphocytes. EAU was transferred by the subset of helper/inducer T-cells, but not by T-cells of the suppressor/cytotoxic subset. Recipient rats of spleen or LN cells cultured with S-antigen exhibited both humoral and cellular immune responses to S-antigen. On the other hand, recipients of spleen cells cultured with Con A developed only cellular immune response to S-antigen. Yet, both groups of recipients fully developed EAU. The clinical and histologic changes in recipient rats closely resembled those in rats in which EAU was induced by active immunization. Severe tissue damage occurred at the photoreceptor cell layer, but inflammatory infiltration also was found in other ocular tissues. Involvement of polymorphonuclear leukocytes (PMNs) was noted throughout ocular tissues even in the eyes of recipients with no detectable antibodies to S-antigen, suggesting that the ocular PMNs infiltration in rats is not necessarily the result of an Arthus-like inflammatory process.     

Supplementation of CD4+CD25+ regulatory T cells suppresses experimental autoimmune uveoretinitis

AIMS: To investigate whether supplementation of natural CD4+CD25+ regulatory T cells ameliorates mouse experimental autoimmune uveoretinitis (EAU) induced by CD4+ T cell-dependent interphotoreceptor retinoid-binding protein (IRBP). METHODS: C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP(1-20)), and IRBP(1-20)-sensitised T cells were obtained. CD4+CD25+ T cells derived from naive mice were cocultured with IRBP(1-20)-sensitised T cells, and their proliferation responses and cytokine production were measured. In addition, CD4+CD25+ T cells were transferred intravenously into mice 7 or 15 days after immunisation with IRBP(1-20), and the severity of EAU and T cell proliferation responses were evaluated. RESULTS: CD4+CD25+ regulatory T cells effectively inhibited both the proliferation of, and interleukin (IL)2, IL5 and interferon (IFN)gamma production by, IRBP(1-20)-sensitised T cells. Adoptive transfer of CD4+CD25+ regulatory T cells to IRBP(1-20)-immunised mice conferred considerable protection from EAU development and inhibition of T cell proliferation responses to IRBP(1-20). CONCLUSION: These findings show that natural CD4+CD25+ regulatory T cells possess the ability to inhibit activation of IRBP-reactive T cells that have been already sensitised in vivo, and adoptive transfer of these cells ameliorates EAU even in the effector phase. Supplementation of natural CD4+CD25+ regulatory T cells may have therapeutic potential for effective treatment of uveitis.     

IRBPR16多肽的合成及其致葡萄膜视网膜炎活性的探讨

目的 探讨光感受器间维生素A类结合蛋白（IRBP）的R16多肽片段的致葡萄膜视网膜炎活性。设计实验研究。研究对象36只Lewis大鼠。方法应用Fmoc法合成并纯化牛IRBPR16多肽片段,以诱导实验性自身免疫性葡萄膜视网膜炎（EAU）模型,并对该模型进行临床观察和组织学检查。培养EAU大鼠的引流淋巴结细胞,测定淋巴细胞增殖反应。各实验同时建立单纯弗式完全佐剂（CFA）免疫组和空白对照组。主要指标多肽分析,视网膜形态学,淋巴细胞增殖反应。结果合成的IRBPR16多肽片段纯度为95．6％。应用IRBPR16多肽片段作为抗原免疫Lewis大鼠,可成功诱导出EAU模型。EAU的临床分级为（3．33±0．52）级,病理分级为（3．67±0．92）级;CFA组和空白对照组大鼠眼部均无异常改变。EAU组大鼠引流淋巴结中抗原特异性淋巴细胞增殖反应增强,为（33．27±7．24）×10^cpm,显著高于CFA组[（1．91±1．16）×10^3cpm]和空白对照组[（1．23±0．51）×10^3cpm]（P〈0．05）。结论IRBPR16多肽片段具有较强的致葡萄膜视网膜炎活性,引流淋巴结抗原特异性淋巴细胞增殖反应增强。IRBPR16多肽诱导的EAU为研究人类葡萄膜视网膜炎提供了一个重要的动物模型。     

Auxiliary production of antibodies to ocular antigens in experimental autoimmune uveoretinitis

In experimental autoimmune uveoretinitis (EAU), there are concurrent autoreactive humoral and cellular immune responses. Many studies have recently focused on T-cell reactivities in EAU, while analysis of autoantibody responses to uveitogenic epitopes has been less well characterized. In this study, a defined 16-mer uveitogenic interphotoreceptor retinoid binding protein (IRBP) peptide, designated #896, was used to induce EAU. Histological analysis of eyes at day 10 demonstrated extensive leukocyte infiltration of the anterior segment, with mononuclear and plasma cells in the posterior segment. The presence of plasma cells suggests local production of antibodies within the eye. ELISA analyses of serum, aqueous, and vitreous from rats with IRBP-induced, severe EAU revealed the presence of antibodies against peptide #896. There was little difference between the serum and intraocular antibody titers, presumably due to breakdown of the blood-ocular barriers. In addition to the anti-#896-IRBP antibodies, there were detectable antibodies reactive against separate and distinct IRBP epitopes, as well as, against epitopes on retinal-S antigen. These results indicate that in #896-peptide-induced severe EAU, humoral immune responses are induced to the primary immunogen and that there is also auxiliary production of autoreactive antibodies against other epitopes present on intraocular antigens. These auxiliary responses may contribute to the immunopathogenesis of severe EAU.