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。     

Alternative routes of immunization for the induction of experimental autoimmune uveoretinitis (EAU) in rodents: a comparison

Experimental autoimmune uveoretinitis (EAU) in rodents is a widely used model of ocular autoimmunity. EAU has traditionally been elicited by injecting the uveitogenic protein in complete Freund's adjuvant (CFA) into the footpad(s) (FP). Because this route of immunization causes severe arthritis and inflammation, it is being banned by many institutions and investigators are switching to the subcutaneous (SC) route. However, there are no studies that systematically compare the outcome of these two immunization routes using defined clinical, histopathological and immunological criteria. We therefore undertook to compare the FP and SC routes of immunization in the Lewis rat and in the B 10. A mouse models of EAU. Animals were immunized with interphotoreceptor retinoid-binding protein (IRBP) or the retinal soluble antigen (S-Ag) in CFA, either by the traditional FP route or by the SC route. The parameters studied were kinetics and severity of EAU by clinical observation and by histopathology, respectively, as well as immunological responses by delayed-type hypersensitivity (DTH), serum antibody titers and lymphocyte proliferation to the uveitogen. In mice immunized with graded doses of IRBP, development of disease induced by the FP and SC methods had essentially identical kinetics. However, the SC method resulted in a somewhat higher incidence and severity of disease as well as higher DTH at the lower antigen doses. Antibody titers tended to be higher with FP immunization. In rats immunized with S-Ag, kinetics and severity of disease, DTH, proliferative responses of draining lymph node cells to the immunizing antigen, and serum antibody titers induced by FP and SC methods were similar. In rats immunized with IRBP, SC immunization resulted in somewhat higher responses across the board than FP. We conclude that at higher doses of antigen disease scores and immunological responses in animals immunized SC are comparable to those of FP-immunized animals. At limiting doses of antigen, however, the SC route appears to result in more severe disease than the traditional FP method.     

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.     

Participation of sodium iodate in the induction of experimental autoimmune uveoretinitis (EAU)]

It is known that Brown Norway (BN) rats show resistance to the development of experimental autoimmune uveoretinitis (EAU). Although BN rats don't develop EAU easily when they were immunized with S antigen containing emulsified complete Freund's adjuvant, this paper reports on the development of EAU at the rate of 40-60% in BN rats when immunization is preceded by an injection of more than 0.5 mg (1.79 mg/kg of body wt) of sodium iodate which leads to the destruction of retinal pigment epithelium (RPE). It was thought that the destruction of RPE participated in the induction of EAU. Therefore, it is considered that the existence of RPE may play an important role in the induction of EAU.     

Total dose and frequency of administration critically affect success of nasal mucosal tolerance induction

AIMS: Nasal tolerance induction with autoantigens can effectively protect against a variety of experimental models of autoimmune disease. The aims of this study were to characterise the dosage and kinetics of inhibition of experimental autoimmune uveoretinitis (EAU) via intranasal administration of the uveitogenic antigen interphotoreceptor retinal binding protein (IRBP) in the murine model of IRBP induced EAU. METHODS: B10RIII mice were tolerised by intranasal administration of IRBP either with a long term multiple low dose or a short term/high dosing regimen before subcutaneous immunisation with IRBP in complete Freund's adjuvant (CFA). On day 15 post-immunisation, mice were killed and eyes were removed for histological examination and quantification of inflammatory cell infiltration and degree of target organ (rod outer segment, ROS) destruction. RESULTS: Nasal administration of multiple low doses of IRBP (1 microg or 3 microg IRBP per mouse per day for 10 days) significantly protected mice from IRBP induced EAU. Short term/high dose regimens were only effective when given either as a single or, at most, as two consecutive doses (40 microg per dose). Multiple doses in the range of 45-120 microg over 3 days afforded no protection. CONCLUSIONS: These results indicate that both dose and frequency of intranasal antigen administration are pivotal to tolerance induction and subsequent suppression of T cell mediated autoimmune disease.     

The purification of bovine IRBP and its uveitogenic action]

The interphotoreceptor retinoid-binding protein, IRBP, shuttles the retinoid between photoreceptor cells and the pigment epithelium. It also induces experimental autoimmune uveoretinitis (EAU). The authors first purified IRBP in China by Con A Sepharose affinity chromatography in conjunction with the purification of bovine retinal S-antigen by ion-exchange chromatography. EAU was successfully induced by injection of emulsified IRBP 50 micrograms with Freund's complete adjuvant into the footpad of Lewis rats. It was characterized by panophthalmia with severe damage to the posterior retina, and lymphocytes predominated the inflammatory infiltration that included mononuclear and polymorphonuclear cells.     

Differentiation of Th1 and Th2 cells in lymph nodes and spleens of mice during experimental autoimmune uveoretinitis

PURPOSE: Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that can be elicited in susceptible rodent strains by immunization with a retinal autoantigen, such as interphotoreceptor retinoid-binding protein (IRBP). In this study, we investigated whether there is a correlation between inflammation in the eye and T-helper (Th)1- and Th2-type responses in the lymph nodes and the spleen after immunization of B10.A mice with IRBP. METHODS: B10.A mice were immunized with IRBP emulsified with complete Freund's adjuvant (CFA), and eyes were then enucleated for histological examination of EAU at 1, 2, 4, 6, or 8 weeks after immunization. In addition, lymph node cells and spleen cells were collected, and cultured with IRBP to measure T-cell proliferation responses and Th1-type (interleukin [IL]-2, interferon [IFN]-gamma), Th2-type (IL-4, IL-10) cytokine production. RESULTS: Pathologically, severe ocular inflammation occurred 2 weeks after IRBP immunization, persisted for 2 weeks, and then gradually resolved. Interleukin-2 and IFN-gamma production were observed in draining lymph node cells at 1 and 2 weeks after IRBP immunization. Those responses then diminished, whereas IFN-gamma production by spleen cells was observed from week 1, peaked at week 4, and gradually decreased. Alternatively, significant production of IL-4 or IL-10 by draining lymph node cells was not detected at any time point. Both IL-4 and IL-10 production by spleen cells was observed at week 6. CONCLUSIONS: Th1-type responses were observed early in draining lymph nodes, then in the spleen after IRBP immunization. The levels of IFN-gamma production by spleen cells reflected the severity of EAU, confirming their pathogenic role in this disease. Th2-type responses were generated in the spleen only as the disease receded, suggesting a role for Th2 cells in the spontaneous termination of EAU.     

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.     

The onset mechanism of experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein]

In order to analyze the onset mechanism of experimental autoimmune uveoretinitis (EAU), two experimental models were used; one was EAU induced by one injection of purified bovine interphotoreceptor retinoid-binding protein (IRBP) with complete Freund's adjuvant in Lewis rat, and the other was an IRBP-induced autoimmune uveoretinitis that occurred spontaneously in nude (nu/nu) mice at 4 weeks of age reconstituted by the grafting of rat embryonic thymus (TG nude mouse). EAU develops when the IRBP-reactive lymphocytes in the regional lymph-nodes are activated. Activation begins when the T lymphocyte recognizes the peptide for the epitope bound to a major histocompatibility complex (MHC) molecule in the antigen-presenting cell by way of the T-cell receptor (TCR). In EAU, ten peptide residues p1182-1191 of the IRBP amino acid sequence, were revealed to be sufficiently capable of lymphocyte activation for EAU, and it was also shown that amino acid positions 1182W (tryptophane), 1185G (glycine), 1186V (valine) and 1188P (proline) of IRBP play important roles as the epitopes or agretopes in developing EAU. On the other hand, two amino acids of IRBP, amino acid positions 1182W (tryptophane) and 1194P (proline) were shown to be the agretopes inducing autoimmune uveoretinitis in the TG nude mouse. A study of the variable region of the TCR with a residual p1182-1194 specific T-cell line from the TG nude mouse revealed that as many as 96% utilized the T-cell receptor V beta 6 gene and that the peptide-MHC molecule complex was recognized by restricted receptors. Adhesion molecules such as ICAM-1 and LFA-1 were also found to play an important role as cofactors in activation of lymphocytes in the antigen-recognition process of EAU. Uveoretinitis seemed to result from an immune reaction in the eye occurring when the T lymphocyte arrives there, activating the immunological process. ICAM-1 and LFA-1 were also found to be involved in the infiltration process of inflammatory cells: our immunohistological examination revealed that ICAM-1 was present in the retinal pigment epithelium and epithelium of the ciliary body composing the blood-ocular barrier. In contrast, LFA-1 was expressed in the infiltrating cells. Finally, the tolerance of IRBP was discussed and it was experimentally demonstrated that the absence of IRBP-induced uveoretinitis in human beings and certain experimental animals resulted from endogenous IRBP serving as a tolerogen; we assumed that the breakdown of this self-tolerance would induce EAU due to thymic dysfunction or IRBP antigen injection.     

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.     

实验性自身免疫性葡萄膜炎眼球中自然杀伤细胞的致炎作用及其机制

背景 实验性自身免疫性葡萄膜炎(EAU)是葡萄膜炎常见的动物模型,自然杀伤(NK)细胞是一种强烈的致炎细胞,但其在EAU中的作用及其机制仍有待研究. 目的 探讨EAU模型大鼠不同发病阶段视网膜组织中NK细胞的定位和分布及其机制.方法 将36只SPF级Lewis大鼠应用随机数字表法随机分成对照组和造模后第6、9、12、16、21天组,每组各6只.造模后第6、9、12、16、21天组大鼠采用光感受器间维生素A类结合蛋白(IRBP)联合5 mg/ml结核杆菌和完全福氏佐剂(CFA)乳化液双后足垫皮下注射,然后腹腔内注射400 ng百日咳毒素免疫大鼠建立EAU大鼠模型.对照组大鼠双后足垫皮下注射生理盐水与等容量CFA乳化液,然后腹腔内注射400 ng百日咳毒素.造模后每天用裂隙灯显微镜观察大鼠眼前段炎症反应过程,根据Caspi方法进行眼部炎症症状评分.分别于造模后第6、9、12、16、21天摘取各组大鼠眼球,采用苏木精-伊红染色法检测各组大鼠视网膜炎症反应和组织结构形态学改变;采用免疫荧光双标法检测大鼠视网膜中NK细胞的分布及浸润情况.另取25只Lewis大鼠应用随机数字表法随机分为造模后第0、3、6、9和12天组,每组5只大鼠,电动匀浆机破裂组织并匀浆为眼内液,采用实时荧光定量PCR法检测大鼠眼内液中NK细胞趋化因子CXCL10 mRNA和CXCL12 mRNA的表达. 结果 对照组大鼠眼前节未发现炎症反应.造模后第6天组大鼠虹膜血管扩张,随着造模后时间延长虹膜血管扩张明显,前房逐渐出现渗出或积脓,造模后第12天炎症反应达峰.视网膜组织病理学检查显示,对照组大鼠视网膜组织结构排列整齐,各EAU模型组大鼠造模后随时间延长均出现不同程度的视网膜结构排列紊乱,外核层细胞分离,层间组织松散,视细胞水肿且有炎性细胞浸润,以造模后第12天组最为严重.免疫荧光双标记显示对照组仅见蓝色标记的细胞核,视网膜各层细胞排列规则;造模后第6天组开始可见大鼠视网膜内层大量NK细胞浸润,呈红色荧光,随时间延长逐渐增加,造模后第9天组NK细胞浸润达峰.造模后第9天组大鼠视网膜中CXCL10 mRNA相对表达量为34.298±16.689,明显高于造模后第3、6、12天组的1.390±0.660、3.359±2.581和4.711±1.387,差异均有统计学意义(均P＜0.01);各组大鼠视网膜中CXCL12 mRNA相对表达量的总体比较差异无统计学意义(F=2.851,P＞0.05). 结论 EAU大鼠发病早期视网膜中NK细胞浸润,其严重程度和视网膜中CXCL10的表达动态与EAU炎症发展过程相吻合,提示NK细胞在EAU的早期炎症过程中发挥重要作用,CXCL10是NK细胞的主要趋化因子.     

The requirement for pertussis to induce EAU is strain-dependent: B10.RIII, but not B10.A mice, develop EAU and Th1 responses to IRBP without pertussis treatment.

PURPOSE: Experimental autoimmune uveoretinitis (EAU) in mice is an important model for elucidating basic mechanisms in autoimmune eye disease. The need for pertussis toxin (PTX) as an additional adjuvant to elicit EAU has limited the usefulness of this model in some types of studies by introducing a pleiotropic factor with confounding effects on the immune response. METHODS: In the present study the authors examined the ability of B10.RIII mice, the most susceptible strain known so far, to develop EAU in response to the retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), and to a major uveitogenic epitope of IRBP, peptide (p)161-180, in the absence of PTX treatment. RESULTS: The data indicate that high disease scores in response to IRBP and p161-180 were found in B10.RIII mice, without the need for PTX as part of the immunization protocol. Unlike the B10.A strain in which appreciable disease did not develop without PTX, B10.RIII mice mounted a high IFN-gamma response to IRBP in the absence of PTX treatment. Interestingly, and unlike the effect with IRBP, in vitro recall response to p161-180 was low in IFN-gamma, despite good EAU scores. CONCLUSIONS: The data indicate that an important mechanism through which PTX facilitates induction of cell-mediated autoimmunity is by promoting a Th1 polarization of the immune response. The propensity of B10.RIII mice to mount a more polarized Th1 response to IRBP than other strains may contribute to their ability to develop EAU without pertussis adjuvant. Nevertheless, the induction of EAU by p161-180 in the context of a relatively limited IFN-gamma production indicates that non-Th1- and Th-related mechanisms are likely to act in concert to determine the outcome of disease.     

Evaluation of in vivo cytokine expression in EAU-susceptible and resistant rats: a role for IL-10 in resistance?

Messenger RNAs for six cytokines (IL-12p40, IFN-gamma, IL-10, IL-4, TNF-alpha and TGF-beta1) expressed in vivo during development of experimental autoimmune uveitis (EAU) were quantitated by PCR in (uncultured) peripheral lymphoid cells and in the eyes of EAU-susceptible Lewis and EAU resistant F344 rats. Disease was induced by immunization with the R16 peptide of IRBP (in RT1B haplotype rats) or with whole IRBP (in all haplotypes). In the periphery, both Lewis and F 344 expressed similar cytokine patterns. In ocular tissues, however, only Lewis expressed elevated type 1 and inflammatory cytokines (IL-12p40, IFN-gamma and TNF-alpha), coincident with onset and peak of disease. Interestingly, naive F344 rats expressed higher basal levels of IL-10 mRNA in the eyes. To examine the possible involvement of this phenomenon in resistance, basal levels of IL-10 vs susceptibility to IRBP were compared in Lewis, BN, DA. F344 and ACI strains. Lewis, BN and DA were susceptible and had low levels of IL-10 mRNA in eyes. F344 and ACI were resistant and expressed high basal levels of IL-10 mRNA. In an in vitro study, recombinant rat IL-10 (but not human or mouse IL-10) suppressed lymphocyte proliferation and IFN-gamma production by primed lymph node cells of R16 immunized rats, but did not suppress uveitogenic long-term T-cell lines polarized to the Thl phenotype, suggesting that mature effector lymphocytes in the rat may lose their ability to be suppressed by IL-10. We propose that higher expression of the IL-10 gene in ocular tissues in some rat strains may represent a mechanism that contributes to a higher threshold of resistance to EAU, but this threshold may be overcome by a more mature Thl effector with a reduced sensitivity to IL-10.     

Interphotoreceptor retinoid binding protein is a potent tolerogen in Lewis rat: suppression of experimental autoimmune uveoretinitis is retinal antigen specific

AIMS—Administration of unfractionated retinal antigen(s) (retinal extract, RE) suppresses RE induced experimental autoimmune uveoretinitis (EAU) and offers a potential therapeutic alternative to non-specific immunosuppressive therapies for posterior uveitis and autoimmune diseases. S-Ag and interphotoreceptor retinoid binding protein (IRBP) are two major autoantigens within soluble RE. It was aimed to assess, firstly, as has previously been shown with S-Ag, if IRBP can induce intranasal tolerance and, secondly, the contribution of both these major autoantigens to tolerance induction by whole RE.
METHODS—Animals were tolerised by intranasal administration with S-Ag or IRBP, either alone or in combination, or RE before immunisation with either IRBP or RE. Control animals were administered nasally either PBS or MBP. Daily clinical responses were recorded biomicroscopically and histological grades were obtained using a semiquantitative scoring system. Weekly serum antibody levels to retinal antigens were measured by ELISA and delayed hypersensitivity responses (DTH) were assessed by skin reactivity to intradermal inoculation with retinal or non-specific antigens.
RESULTS—Microgram doses of IRBP successfully suppressed both clinically and histologically IRBP induced EAU. This suppression was accompanied by reduced antigen specific DTH reactivity but maintained T cell dependent (IgG2a) antibody responses. Furthermore, combined S-Ag and IRBP administration afforded equal suppression of RE induced EAU when compared with RE therapy alone. Suppression of RE induced EAU was not achieved with administration of a non-retinal specific autoantigen, MBP. Although individually, both S-Ag and IRBP suppressed RE induced EAU, whole RE was unable to protect against IRBP induced disease.
CONCLUSIONS—Intranasal administration of IRBP suppressed IRBP induced EAU in the Lewis rat. S-Ag and IRBP are the major contributors to the tolerogenicity within RE, despite the known uveogenicity of other retinal antigens within RE and induction of tolerance was retinal antigen specific. Furthermore, suppression induced by single antigen administration is antigen specific although concomitant bystander suppression may also play a role. RE was unable to protect against IRBP induced disease despite tolerogenic levels of antigen within RE. Although this may be due in part to a dose effect of either tolerising or immunising antigen, further investigation into the possible antigen dominance of IRBP or mucosal processing of combinations of antigens is necessary so that the full efficacy of mucosal tolerance therapy can be assessed.

     

大鼠自身免疫性葡萄膜视网膜炎辅助T细胞的类细胞因子水平表达

目的观察实验性自身免疫性葡萄膜视网膜炎（experimental autoimmune uveoretinitis，EAU）大鼠血清以及脾细胞培养上清液中辅助T细胞1／辅助T细胞2（helper T cell 1／helper T cell2，Th1／Th2）类细胞因子水平。方法用Fmoc法合成光感受器间维生素A类结合蛋白R16多肽片段，联合免疫佐剂诱导EAU动物模型。在EAU高峰期取大鼠血清，分离脾细胞，培养后取上清液，应用酶联免疫吸附法检测血清以及脾细胞培养上清液中Th1类细胞因子（interferon-γ，IFN-γ、interleukin-2，IL-2）和Th2类细胞因子（IL-4、IL-10）水平。结果EAU组大鼠血清中IFN-1和IL-2的浓度分别为（33．8±5．2）μg／L，（52．5±7．9）μg/L，显著高于空白对照组[（6．2±1．4）μg/L，（3．7±0．8）μg/L和弗氏完全佐剂对照组[（complete Freund＇s adjuvant，CFA）；（9．2±1．9）μg/L，（5．1±1．1）μg/L]（P〈0．05）；而IL-4、IL-10的浓度与空白对照组和CFA组相比差异无统计学意义。EAU组大鼠脾细胞培养上清液中IFN-γ、IL-2的浓度分别为（1105．3±197．5）μg/L，（45．0±16．2）μg/L，显著高于空白对照组（5．24±1．7）μg/L，（4．1±1．3）μg/L和CFA组（25．14±5．9）μg/L，（5．1±1．9）μg/L（P〈0．01），IL4、IL-10的浓度与空白对照组和CFA组相比差异无统计学意义；CFA组大鼠脾细胞培养上清液中IFN-1的浓度明显高于空白对照组（P〈0.05），IL-2、IL-4、IL-10的浓度与空白对照组相比差异无统计学意义。结论在EAU高峰期，Th1类细胞因子水平显著升高，提示EAU是Th1细胞诱导的疾病（中国眼耳鼻喉科杂志，2008，8：280-282）     

Suppression of interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis by pretreatment with anti-idiotypic monoclonal antibody]

In an experimental model of interphotoreceptor retinoid binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) in Lewis rats, EAU onset was suppressed by pretreatment with anti-idiotypic antibody (TRD3). In the pretreated group, the titers of anti-IRBP antibody and idiotypic antibody were lower and the titer of anti-idiotypic antibody was higher than in the control group, and the skin test (DTH) against IRBP was also suppressed. Histopathological findings of the control group showed destruction of retinal outer granular layer, loss of the outer segment and lymphocytic infiltration, although no definite inflammatory signs appeared in the pretreated group. We suppose that the mechanism of idiotypic suppression is via induction of idiotype-specific or idiotype-related suppressor T cells.     

Residues 1-20 of IRBP and whole IRBP elicit different uveitogenic and immunological responses in interferon gamma deficient mice

Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease induced by immunization with uveitogenic retinal antigens, or by the adoptive transfer of uveitogenic T-cells of the Th-1-like phenotype. We have previously shown that IFN-gamma-deficient mice (GKO) on the C57BL/6 background are equally susceptible to interphotoreceptor retinoid binding protein (IRBP)-induced EAU as the wild type (WT). In the present study, we evaluated EAU induction in GKO mice by the newly described H-2(b)epitope contained in residues 1-20 of human IRBP, and compared it to the response to the whole IRBP molecule. Similarly to previous observations with IRBP-induced EAU, delayed type hypersensitivity (DTH) and lymphocyte proliferation responses were elevated in GKO mice, as was production of IL-5 and TNF-alpha. However, unlike the responses induced by whole IRBP, there was no detectable IL-10 production to the peptide. Histopathology on day 21 after immunization, revealed that both GKO and WT mice developed retinal lesions, including damage to the photoreceptor cell layer, vasculitis and inflammatory cellular infiltration, but disease scores were significantly higher in GKO, and retinal detachment was observed only in GKO mice. In contrast to the wild type, the cellular infiltrate in eyes of GKO mice contained a prominent component of eosinophils, although of lower proportion in peptide-induced than in IRBP-induced EAU. We conclude that the cytokine and inflammatory responses to human peptide 1-20 differ perceptibly from the responses to whole bovine IRBP, and may explain the elevated EAU scores of GKO mice compared to wild type.     

Translational research with experimental autoimmune uveoretinitis (EAU)

Experimental autoimmune uveoretinitis (EAU) induced by immunization with retinal antigen (Santigen or interphotoreceptor retinoid-binding protein; IRBP) serves as an animal model of human uveoretinitis. As the first stage, we demonstrated the similarities between EAU and ocular inflammation in Beh?et's disease by investigating anti-retinal antibodies, leukocyte migration inhibition by retinal antigen, immunogenic antigens, aberrant functions of neutrophils, and dominant Th1 lymphocyte reaction. From these findings, we verified that EAU, which is not associated with the systemic disorders observed in Beh?et's disease, is an appropriate model for translational research targeting ocular inflammation. In the second stage, we set 3 therapeutic strategies for uveitis in Beh?et's disease to be conducted in the translational research: (1) intraocular administration of an immunosuppressive drug; (2) inhibition of Th1 lymphocytes; and (3) activation of immunoregulatory cells. In strategy 1, our studies indicated that intravitreal injection of 10 microg of tacrolimus (FK 506) was not harmful to the retina and was predominantly effective in suppressing ongoing EAU in rats. In strategy 2, two approaches were adopted to prevent differentiation of Thl cells. One is anti-cytokine antibody therapy using anti-IL-12 monoclonal antibodies(mAb). The other is blockade of co-stimulatory signals, especially the ICOS-B7RP-1 pathway. Administration of anti-IL-12 mAb at the time of IRBP immunization completely inhibited development of EAU, and antagonistic anti B7RP-1 mAb suppressed the severity of EAU even when administered after development of EAU. In strategy 3, adoptive transfer of antigen presenting cells treated with a neuropeptide (vasoactive intestinal peptide or calcitonin gene-related peptide) or CD 4+ CD 25+ regulatory T cells suppressed EAU. We look forward to the day when therapies that are being developed in our translational research using EAU will become available for treating intraocular inflammation in Beh?et's disease.     

A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis.

The induction of T cell mediated disease models in animals is usually dependent on the use of complete Freund's adjuvant (CFA). In order to avoid the painful side effects of CFA on the animals, we tested the capacity to induce experimental autoimmune uveoretinitis (EAU) with Hunter's adjuvant (HA). This new adjuvant makes use of nonionic copolymer surfactants, and does not cause deleterious effects to animals. We have found that EAU could be efficiently induced in rats with low doses of S-Ag (10 micrograms) in very small quantity of HA (10 microliters). The biologic parameters of EAU induction showed a potent stimulation of lymphocytes proliferation maximal 11 days after immunization, as well as high levels of antibody production.     

Oral administration of interferon-β suppresses experimental autoimmune uveoretinitis

BACKGROUND: The oral administration of type I interferons (IFNs) have been reported to reduce severity of inflammation in several animal models of autoimmune disease. This study examined whether oral administration of IFN-beta is capable of modulating inflammation in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) emulsified in complete Freund's adjuvant. Rats were treated with either varying doses (10(2), 10(3), 10(4) or 10(5)IU) of mouse recombinant IFN-beta or phosphate-buffered saline for control, via direct oropharyngeal application once a day for 28 days starting 7 days before IRBP immunization. Intraocular inflammation was assessed by slit-lamp biomicroscopy and histopathological examination. Spleen cell proliferation response and cytokine production under IRBP stimulation were assessed. Spleen cell subpopulations were also measured. RESULTS: IFN-beta at doses of either 10(4) or 10(5) IU significantly reduced both the clinical and histopathological severity of EAU. Spleen cell proliferation and IFN-gamma production from rats treated with 10(4) IU IFN-beta were significantly decreased compared with controls. Furthermore, the proportion of both NK cells and NKT cells in the spleen of rats treated with IFN-beta was increased compared with controls. CONCLUSION: These results suggest that the oral administration of IFN-beta reduces inflammation in IRBP-mediated EAU and that the mechanism of this action may involve NK cells and NKT cells.