Inducible co-stimulator (ICOS) is upregulated in experimental autoimmune uveoretinitis

Purpose To study the expression of inducible co-stimulator (ICOS) and its association with T cell effector function in experimental autoimmune uveoretinitis (EAU).Methods Eighteen Lewis rats were immunized by retinal S-antigen (50 μg) emulsified in complete Freund’s adjuvant (CFA). Twelve normal rats served as normal controls and 18 receiving injection of CFA and PBS as CFA controls for studying the influence of CFA on the expression of ICOS in CD4⁺CD25⁺ T cells. ICOS expression on cells from the spleens, inguinal nodes and retinae on day 0 (normal rats), 7, 13 and 21 was investigated using fluorescent quantitative real-time-PCR and Western blot. Expression of B7RP-1, an ICOS ligand, was also studied by Western blot. The phenotype of the cells from the aforementioned three tissues was identified with flow cytometry using antibodies to ICOS, CD4 and CD25. ICOS⁺ cells from the lymph nodes, and spleens on day 13 were magnetically sorted and cultured with S-antigen to study the cytokines production with enzyme-linked immunosorbent assay.Result An obvious uveitis was induced in all the immunized rats on day 13 after S-antigen immunization. The mRNA and protein of ICOS were scarcely detectable in normal rat spleens. In EAU rats, an up-regulation of ICOS could be observed on day 7 and was very pronounced on day 13, followed by a decrease on day 21 in the spleens, draining nodes and retinae. Similarly, B7RP-1 expression seemed to be up-regulated during EAU. Flow cytometry showed that ICOS⁺ cells were mostly CD4 positive. Kinetics of ICOS⁺CD4⁺CD25⁺ T cells was similar to that of ICOS⁺ cells. CFA alone was also able to induce increased expression of ICOS in CD4⁺CD25⁺ T cells. IFN-γ was secreted predominantly by ICOS⁺ T cells.Conclusion ICOS expression is upregulated in association with T cell effector capacity in EAU. It is presumed that the ICOS/B7RP-1 costimulatory pathway may play a role in the development of EAU.This study is supported by the Fund for Innovative Research Groups of China (30321004), Specialized Research Fund for the Doctoral Program of Higher Education in China (20030558077).     

Upregulation of neurotrophic factor-related gene expression in retina with experimental autoimmune uveoretinitis by intravitreal injection of tacrolimus (FK506)

AIM: The current study was designed to determine whether intravitreal injection of tacrolimus (FK506) modulates the gene expression of neurotrophic factor-related molecules in the retina from eyes with induced experimental autoimmune uveoretinitis (EAU) in rats. METHODS: Rats were immunised with interphotoreceptor retinoid binding protein peptide (R14) and given intravitreal injection of tacrolimus on day 12 after immunisation. As control, immunised rats received intravitreal injection of vehicle. On day 15 after immunisation, changes in the genetic programme associated with neuroprotection and inflammatory responses in the retinas from both groups were determined by DNA microarray analyses and confirmed by real-time PCR analyses. RESULTS: The gene expression of inflammatory responses was markedly reduced in tacrolimus-treated eyes. Genes for molecules associated with neuroprotection (oestrogen receptor, erythropoietin receptor, gamma-aminobutyric acid receptor, protein kinase C, glial cell line-derived neurotrophic factor receptor, fibroblast growth factor and neuropeptide Y receptor) were upregulated in the retinas from tacrolimus-treated eyes. CONCLUSIONS: Intravitreal injection of tacrolimus modulated the genes related to neuroprotection in the retina during the ongoing process of EAU. This treatment may be useful for the neuroprotection of retina with severe uveitis as well as for immunosuppression in the uveitic eyes.     

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.     

Prevention of experimental autoimmune uveoretinitis by blockade of osteopontin with small interfering RNA

Osteopontin (OPN) is elevated during the progression of experimental autoimmune uveoretinitis (EAU) in C57BL/6 (B6) mice. Furthermore, EAU symptoms are ameliorated in OPN knockout mice or in B6 mice treated with anti-OPN antibody (M5). Recently, OPN has been shown to promote the Th1 response not only in the extracellular space as a secretory protein but also in cytosol as a signaling component. Thus, we attempted to reduce OPN in both compartments by using a small interfering RNA (siRNA) targeting the OPN coding sequence (OPN-siRNA). EAU was induced in B6 mice by immunization with human interphotoreceptor retinoid-binding protein (hIRBP) peptide sequence 1-20. The OPN- or control-siRNA was administered with hydrodynamic methods 24 h before and simultaneously with immunization (prevention regimen). When plasma OPN levels were quantified following siRNA administration with the prevention regimen, the level in the OPN-siRNA-treated group was significantly lower than that in the control-siRNA-treated group. Accordingly, the clinical and histopathological scores of EAU were significantly reduced in B6 mice when siRNA caused OPN blockade. Furthermore, TNF-α, IFN-γ, IL-2, GM-CSF and IL-17 levels in the culture supernatants were markedly suppressed in the OPN-siRNA-treated group, whereas the proliferative responses of T lymphocytes from regional lymph nodes against immunogenic peptides was not significantly reduced. On the other hand, the protection was not significant if the mice received the OPN-siRNA treatment on day 7 and day 8 after immunization when the clinical symptoms appeared overt (reversal regimen). Our results suggest that OPN blockade with OPN-siRNA can be an alternative choice for the usage of anti-OPN antibody and controlling uveoretinitis in the preventive regimen.     

实验性自身免疫性葡萄膜视网膜炎中可诱导的共刺激分子的表达及意义

目的探讨可诱导的共刺激分子（ICOS）在实验性自身免疫性葡萄膜视网膜炎(EAU)中的表达及意义。方法Lewis大鼠28只，用视网膜S抗原（50 μg）和福完全佐剂免疫24只大鼠以诱导EAU模型（免疫组），另外4只作为正常对照。裂隙灯显微镜每日观察大鼠眼部变化。免疫组大鼠分别于免疫后第7、12、15、21天被处死，摘除其脾脏后制作冰冻连续切片并提取组织蛋白。使用多克隆抗ICOS抗体，用免疫组织化学细菌蛋白过氧化物酶（SP）法在上述组织切片上进行免疫组织化学染色，并对提取的蛋白进行Western blotting检测。对照组大鼠在相应各时间点做同样处理。结果正常脾组织中可见少量ICOS阳性细胞；分别在免疫后第7、12 d，脾脏中ICOS阳性细胞数量明显增加，免疫后第15天时阳性细胞数量最多，21 d时阳性细胞数量减少，但仍显著高于正常组；Western blotting检测结果显示，ICOS蛋白的动态变化与免疫组织化学显示的阳性细胞数量变化一致。结论脾脏ICOS表达升高出现于EAU发生之前，随炎症的出现和加重而升高，在EAU消退期其表达有所降低，提示ICOS参与EAU的形成、发展和消退，在EAU发病中有重要意义。(中华眼底病杂志,2005,21:114-117)     

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。     

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）     

ORIGINAL ARTICLE,The Immunomodulator Vasoactive Intestinal Peptide (VIP) Does Not Affect Experimental Autoimmune Uveitis (EAU) in B10.RIII Mice

Purpose: Vasoactive intestinal peptide (VIP) exhibits immunomodulatory activities both in vivo and in vitro, including efficient inhibition of murine experimental arthritis. In this study, we investigated the effects of VIP treatment on the induction of experimental autoimmune uveoretinitis (EAU). Methods: EAU was induced in B10.RIII mice by immunization with interphotoreceptor retinoid-binding protein (IRBP) using routine methods, but without treatment with pertussis toxin (PTX). VIP was injected i.p. at different doses into mice on alternate days. Mice were tested by conventional methods for ocular inflammation, antibody levels, lymphocyte proliferation, and cytokine release by cultured lymphocytes. Results: Treatment with VIP, at different doses, had essentially no effect on the development of EAU or antibody production in the B10.RIII mice. The treatment did have variable effects on the low interferon-γ production by lymphocytes of these mice. Conclusion: Unlike its inhibitory effect in the experimental arthritis system, VIP did not modulate the development of EAU in B10.RIII mice.     

Suppression of actively induced experimental autoimmune uveoretinitis by CD4+ T cells

Background: Helper/inducer T cells that exert an inhibitory effect on disease induction have been recently found in many experimental models. In order to clarify the mechanisms of spontaneous remission of experimental autoimmune uveoretinitis (EAU), we investigated the inhibitory effect and the phenotype of the post-recovery suppressor cells. Methods: In a series of experiments, we separated spleen cells of rats that had recovered from EAU. Three groups of spleen cells, CD4⁺ T, CD8⁺ T and B cells, were each adoptively transferred into naive syngeneic rats before active immunization with retinal soluble antigen (S-Ag) and Freund's complete adjuvant or passive immunization with uveitogenic T cells from donor rats. Inflammation was examined clinically and histologically. Results: The development of EAU could be significantly prevented by adoptive transfer of CD4⁺ T cells, whereas CD8⁺ T cells could not suppress the onset. However, post-recovery CD4⁺ T cells failed to inhibit EAU induced by passive immunization with uveitogenic T cells. Conclusion: These findings indicate that CD4⁺ post-recovery (suppressor) T cells may play an important role in the remission of EAU.     

Inhibition of cellular transfer of experimental autoimmune uveoretinitis by Rapamycin

The crucial role of CD4⁺ T cells in mediating uveitis is well recognized. One treatment strategy of non-infectious uveitis therefore seeks to inhibit T cell function. For that purpose the authors have evaluated the efficacy of Rapamycin (RAPA), an inhibitor of lymphocyte response to growth factors. To reproduce as best as possible the immune system condition during active disease, the adoptive transfer of activated T cells was used to induce experimental autoimmune uveoretinitis (EAU). Treatment with RAPA was delivered by continuous intravenous infusion. The results showed a complete inhibition of EAU transfer at the RAPA dose of 0.1 mg/kg/day. They indicate that RAPA could be a useful immunosuppressant for uveitis therapy.     

Management of submacular hemorrhage with intravitreal versus subretinal injection of recombinant tissue plasminogen activator

Aim  

To compare the efficacy of pars plana vitrectomy (ppV) with intravitreal injection of recombinant tissue plasminogen activator (rtPA) and gas versus ppV with subretinal injection of rtPA and intravitreal injection of gas.     

Experimental autoimmune uveoretinitis (EAU) in rats: abrogation of resistance to the induction and augmentation of the inflammation by pertussis toxin

The effect of pertussis toxin (PT) on experimental autoimmune uveoretinitis (EAU) induced by immunization with S-antigen was examined in rats. Intravenous administration of PT (2 micrograms/rat) initiated the development of EAU in rats that had been made resistant to the induction of EAU by immunization with S-antigen and complete Freund's adjuvant (CFA). The capacity of PT to promote EAU was also demonstrated by a marked augmentation of the inflammation in EAU eyes of rats susceptible to the induction of EAU. PT was most effective when it was given from the day before to the day after immunization with S-antigen. However the induction of EAU was promoted by the injection of PT even 7 days before and 14 days after immunization. The clinical and histopathological findings of the EAU produced by the additional PT treatment were described and the mechanisms by which PT augmented the induction of EAU were discussed.     

Immunohistochemical study on IRBP-induced EAU

We studied the pathogenesis of IRBP-induced experimental autoimmune uveoretinitis (EAU) by immunohistochemical detection of various immune cells using specific monoclonal antibodies to their surface markers and adhesion molecules. The following results were obtained: (1) During each stage of EAU, CD4 positive T cells predominated over CD8 positive T cells in the retina and the uvea. (2) One day prior to the clinical onset of disease, la positive cells began to appear in the ciliary body. (3) LFA-1 and ICAM-1 were expressed on intraocularly infiltrating cells. (4) ICAM-1 was also expressed on endothelial cells of uveal and retinal vessels. In addition, ICAM-1 was expressed on ciliary body epithelium and retinal pigment epithelium, a finding which may be associated with the breakdown of the blood-ocular barrier. In conclusion, the expression of LFA-1 and ICAM-1 in EAU demonstrates that adhesion molecules, such as these, play an important role in inflammatory ocular disease in vivo.     

Murine experimental autoimmune uveoretinitis induced by interphotoreceptor retinoid-binding protein and Klebsiella pneumoniae 03 lipopolysaccharide (K03-LPS): a relation between H-2 haplotype and EAU induction

The pathogenicity of interphotoreceptor retinoid-binding protein (IRBP) in the mouse and H-2 restriction of IRBP-induced experimental autoimmune uveoretinitis (EAU) was tested by repeated immunization using Klebsiella pneumoniae 03 lipopolysaccharide (K03-LPS) as an adjuvant. It was shown that IRBP had a greater capacity to induce EAU than S-antigen. Based on the incidence of EAU induction using B10 congenic mice and other strains, the susceptibility to EAU was, at least in part, controlled by the I-A^k haplotype of the H-2 subregion. The results also indicated that non-major histocompatibility complex (MHC) genes play some role in disease susceptibility.     

Levels of vascular endothelial growth factor and pigment epithelium-derived factor in eyes before and after intravitreal injection of bevacizumab

Purpose  Bevacizumab is a human monoclonal IgG1 antibody that blocks the action of vascular endothelial growth factor (VEGF). The purpose of this study was to determine the level of VEGF and pigment epithelium-derived factor (PEDF) in eyes with proliferative diabetic retinopathy (PDR) before and after an intravitreal injection of bevacizumab. Methods  Eleven eyes of ten patients were studied. Patients were included if they had neovascular glaucoma, rubeosis of the iris with PDR, or aggressive PDR. Samples of aqueous humor were collected just before the injection of bevacizumab and the vitrectomy. The concentrations of VEGF and PEDF in the aqueous humor were measured by enzyme-linked immunosorbent assay, and the effects of bevacizumab on PDR were evaluated. Results  The free VEGF concentration before the injection was 676.5 ± 186.7 pg/ml (mean ± SEM, n = 11). Seven days later, it was significantly reduced to 7.1 ± 7.1 pg/ml (P < 0.005, n = 9). The PEDF concentration before the injection was 2.32 ± 0.49 μg/ml (n = 11), and 7 days later, it was 3.23 ± 0.76 μg/ml (P = 0.33). During the vitrectomy, patients had less intraoperative bleeding when the neovascular tissues were cut. Conclusions  An intravitreal injection of bevacizumab significantly decreased the free VEGF in the aqueous humor by 7 days, indicating that the clinical effects of bevacizumab appear rapidly. However, intravitreal bevacizumab did not affect the level of intraocular PEDF.     

Retinal tissue damage and lipid peroxidation in experimental autoimmune uveoretinitis

Retinal lipid peroxidation products were measured at various stages of experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by retinal S-antigen. Histopathologic examination and measurement of myeloperoxidase, which is a quantitative marker of polymorphonuclear leukocytes (PMNs) in tissue, were also performed. The results showed that lipid peroxidation is closely implicated in the progression of uveoretinitis, and is related to the cellular infiltrate, which consisted primarily of PMNs, and to the retinal tissue damage. These findings may support the theory that oxygen-free radicals liberated from activated PMNs play a role in retinal tissue damage via peroxidation of membrane lipids. To ascertain the tissue damage caused by the lipid peroxidation products generated in the eye, intravitreal injection of an oxidized docosahexaenoic acid, a major polyunsaturated fatty acid in the photoreceptors of the retina, was performed and its toxicity in the retina was proved histologically.     

CCN1在氧诱导小鼠视网膜新生血管形成中的表达及意义

目的:研究富含半胱氨酸蛋白61(CCN1/Cyr61)在氧诱导小鼠视网膜新生血管(retinal neovascularization,RNV)中的表达及意义,探讨特异性抑制CCN1对RNV形成的抑制作用。方法:取C57BL/6J小鼠200只,随机分为对照组、高氧组、高氧对照组和CCN1治疗组,每组各50只。高氧对照组和CCN1治疗组分别玻璃体腔内注射空载体质粒和CCN1siRNA表达质粒。ADP酶视网膜铺片观察视网膜血管形态,HE染色计数突破视网膜内界膜的新生血管内皮细胞核数,免疫组织化学、Western blot和RT-PCR法检测CCN1蛋白及mRNA的表达情况。结果:高氧组和高氧对照组视网膜可见大片无灌注区和大量突破内界膜的新生血管内皮细胞核(25.25±1.26个;23.12±1.16个),CCN1治疗组较高氧组和高氧对照组的无灌注区及新生血管内皮细胞核数(8.47±1.15个)明显减少。高氧组和高氧对照组较对照组相比,CCN1蛋白及mRNA表达显著增高,CCN1治疗组较高氧组和高氧对照组显著减弱,均有统计学意义(均为P<0.05)。结论:CCN1的异常表达可能与RNV形成密切相关,特异性抑制CCN1能有效抑制RNV的形成,为预防和治疗ROP提供新的思路及对策。     

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.