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.     

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.     

Lymphocyte migration in the adoptive transfer of EAU

Experimental autoimmune uveoretinitis (EAU) was transferred into naive male Lewis rats using 1 X 10(8) indium-111 labeled lymphocytes from syngeneic donors immunized with S-antigen. The migration of the lymphocytes was monitored by gamma camera imaging and by determining the accumulation of radioactivity in selected organs. The majority of the cells leave the peritoneal cavity within 24 hr and migrate to the liver, spleen, and thymus. Only a small fraction of the labeled cells reach the eye. However, there were significantly more labeled cells present in eyes that developed EAU as compared with controls using lymphocytes sensitized against bovine serum albumin. These results indicate the adoptive transfer of EAU is a complex process in which only a small number of transferred cells actually reach the eye to induce uveoretinitis.     

Effects of FTY720, a novel immunosuppressant, on experimental autoimmune uveoretinitis in rats

The immunosuppressive properties of FTY720, a novel immunosuppressant obtained by structural modification of ISP-I isolated from the fermentation broth of Isaria sinclairii, were studied in experimental autoimmune uveoretinitis (EAU) in rats. Lewis rats were immunized with S-antigen and treated with FTY720 (0. 03, 0.06, 0.1 mg kg(-1)day(-1)) or distilled water for 16 days after the immunization. FTY720 suppressed the incidence and intensity of EAU in a dose-dependent manner as demonstrated by clinical and histological examinations. The drug significantly suppressed the serum levels of antibodies to S-antigen and antigens-specific lymphocyte proliferation. The number of peripheral lymphocytes, but not neutrophils, was markedly reduced by FTY720 treatment. FTY720 also suppressed the intensity of EAU when it was given from the day of EAU onset. These results indicate that FTY720 has intense immunosuppressive effects on EAU in rats and may be a potential candidate for use in the treatment of patients with autoimmune uveitis.     

Involvement of the pineal gland in rats with experimental autoimmune uveitis

Pineal glands of rats with experimentally induced autoimmune uveitis (EAU) were studied histologically. Inflammatory changes, characterized by mononuclear infiltration, were found in the pineal glands of one-third of the Lewis rats that developed EAU by active immunization with S-antigen. No changes in the pineal gland were observed in AVN rats which are "low responders" for EAU and did not develop ocular disease. Frequency and severity of both pineal gland and ocular involvement clearly were elevated by intravenous injection of Bordetella pertussis along with the S-antigen immunization; all B. pertussis-treated rats of both Lewis and AVN strains developed pineal and ocular changes. Inflammatory changes of the pineal gland also were found in rats in which EAU was induced passively by transfer of lymphocytes from S-antigen-immunized donors. The frequency of involvement of the pineal gland was found to be lower than that of the retinas in rats where EAU was induced by active immunization or by adoptive transfer of lymphocytes.     

Experimental autoimmune uveoretinitis in brown Norway rats induced by bovine interphotoreceptor retinoid-binding protein and renal cryosurgery

In Brown Norway (BN) rats, it is known to be difficult to induce experimental autoimmune uveoretinitis (EAU) by the injection of retinal S-antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP) together with complete Freund's adjuvant (CFA), unless intravenous Bordetella Pertussis is used as an additional adjuvant. In the present study it was found that the rate of onset of EAU could be increased in BN rats immunized with IRBP and CFA by simultaneous cryosurgery to the renal cortex. There was no evidence of retinal vasculitis, pinealitis or nephritis in the rats with EAU except for renal inflammatory infiltrates as a reaction to the cryosurgery. Affected eyes eventually showed destruction of most retinal components and prominent infiltration of the retina by macrophages, with the changes being more severe than those previously reported in Lewis rats with EAU induced by IRBP. Data suggesting the existence of an antibody that cross-reacts with the proximal renal tubules and the retinal pigment epithelium were also obtained.     

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.     

Effects of cyclosporine and other immunosuppressive drugs on experimental autoimmune uveoretinitis in rats

Five immunosuppressive and anti-inflammatory agents were tested for their effects on development of experimental autoimmune uveoretinitis (EAU) and immune responses to S-antigen in rats immunized with this retinal antigen. When administered daily from day 0-14 after immunization, cyclosporine at 5-20 mg/Kg was nontoxic and yet effective in inhibiting the development of EAU for at least 30 days. All other tested drugs were found toxic at their immunosuppressive doses. Of these drugs, only cyclophosphamide (at 5-20 mg/Kg) was capable of inhibiting EAU in some of the treated rats for up to 30 days. Other agents, bredinin (40-100 mg/Kg), dexamethasone (0.2-0.4 mg/Kg), or colchicine (0.5 mg/Kg) produced only a delay in the disease onset. Cyclosporine was also unique in its effect on the immune responses of the rats by selectively inhibiting only the specific T-cell-mediated responses to S-antigen (delayed skin response and lymphocyte response in culture), while having no negative effect on antibody production or the lymphocyte response to the polyclonal mitogen, concanavalin A. Other drugs, when effective, inhibited all types of immune response. In addition, cyclosporine was capable of preventing EAU even when given to rats as late as from day 7 after immunization. Only cyclophosphamide (at 20 mg/Kg/day) had a similar effect on 1/3 of the rats, while other drugs only delayed or had no effect on the disease onset when given by this late schedule.     

实验性自身免疫性葡萄膜视网膜炎中T-bet的表达及意义

目的 研究实验性自身免疫性葡萄膜视网膜炎（EAU）中T-bet的表达及意义 方法 Lewis大鼠28只，用视网膜S抗原与Freund完全佐剂免疫24只大鼠以诱导EAU模型，另外4只作为正常对照。免疫组大鼠分别于免疫后7、12、15、21 d被处死，取所有大鼠的眼球和脾脏，固定后制作眼组织平片和眼球及脾脏的石蜡连续切片。使用单克隆抗T-bet 和CD4的抗体，通过免疫组织化学细菌蛋白过氧化物酶法在上述组织平片及切片上进行免疫组织化学单染和双染色，在光学显微镜下观察并计数阳性细胞，数据经SPSS 11.0统计学软件分析。 结果 正常眼和脾组织中均见少量T-bet阳性细胞；免疫后7 d，虹膜、视网膜及脾脏中T-bet的表达增加，免疫后15 d达高峰，免疫后21 d表达降低，但仍高于正常组。免疫组织化学双染色显示，T-bet阳性细胞中多数为CD4+。 结论 T-bet在EAU的发病中起着重要作用，其作用可能是通过激活辅助性T淋巴细胞1而实现的。（中华眼底病杂志，2004，20：172-174）     

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.     

Experimental autoimmune uveitis induced by immunization with retinal pigment epithelium-specific 65-kDa protein peptides

PURPOSE: To investigate the pathogenic potential and sites of retinal pigment epithelium-specific 65-kDa protein (RPE65) for inducing experimental autoimmune uveitis (EAU) in Lewis rats. METHODS: Twenty-six peptides were chemically synthesized based on the amino acid sequences of human RPE65. These peptides spanned the entire RPE65 sequence. Each peptide was injected into a footpad and the peritoneum of Lewis rats. The eyes were examined by slit-lamp biomicroscopy, and the findings were correlated with the histological findings. The serum antibody titer and lymphocyte reactivity against each peptide was also determined by enzyme-liked immunosorbent assay (ELISA) and lymphocyte proliferation assay, respectively. RESULTS: Active immunization of rats resulted in the induction of EAU with 14 (3 severe and 11 mild) of the 26 peptides. The clinical course of the EAU was similar to that induced by the injection of retinal antigens such as S-antigen or inter-photoreceptor retinoid binding protein (IRBP). However, the histopathologic changes differed from the EAU induced by these retinal antigens. The inflammation was induced mainly from the retinal pigment epithelium (RPE) and the choroid, while the retina was relatively well-preserved except for some granulomatous changes adjacent to the RPE. CONCLUSIONS: Active immunization with peptides making up RPE65 will induce EAU. RPE65 has multiple EAU-inducing sites for Lewis rats.     

Effect of Gallium Nitrate on Experimental Autoimmune Uveitis

Gallium nitrate (GN) has been shown to inhibit T cell-mediated inflammatory disease. The purpose of our study was to test the effect of gallium nitrate (GN) on experimental autoimmune uveitis (EAU). Experimental autoimmune uveitis was induced in male Lewis rats immunized with retinal S-antigen. Rats received subcutaneous injections of GN or saline one day prior to immunization and 1, 4, 7, 10, 13, 16, and 19 days after immunization. Ocular inflammation was graded clinically and histologically by masked observers, and in vitro assays of cell-mediated and humoral immunity were performed. GN significantly inhibited the development of EAU graded clinically (P=0.001) and histologically (P=0.002). Treatment with GN also resulted in a small (30–41%) decrease in the lymphocyte responses to retinal S-Antigen and a small (12–37%) reduction in antibody production to S-antigen. These data show that GN suppresses the development of EAU, and inhibits both lymphocyte proliferative responses to antigen and antibody production.     

EAU大鼠视网膜抗氧酶类变化的实验研究

利用兔视网膜S抗原诱发大鼠EAU模型，观察了视网膜抗氧化酶类（超氧化物歧化酶，过氧化氢酶及谷胱甘肽过氧化物酶活性）及中性白细胞标志酶（髓过氧化物酶）的变化。实验结果表明：在EAU的病变过程中，抗氧化酶活性下降，髓过氧化物酶活性升高。由此认为在EAU病变过程中，视网膜抗氧化酶活性下降，自由基介导的脂类过氧化加剧，造成视网膜损伤，如果利用抗氧化剂治疗可减轻EAU的脂类过氧化损伤。     

Effects of a new immunosuppressive agent, FK 506, on the efferent limb of the immune responses.

The effects of a novel immunosuppressant, FK 506, on the efferent limb of the immune response were studied in the experimental autoimmune uveoretinitis (EAU) in the rat, using two different treatment schedules. First, rats actively immunized with S-antigen were treated with FK 506 only after the onset of EAU. FK 506 (1 or 3 mg kg-1 day-1) reduced the intensity of EAU as compared to that of non-treated rats. Especially, a daily dose of 3 mg kg-1 completely suppressed further development of EAU. It is, therefore, suggested that FK 506 treatment is effective in suppressing the ongoing process of the immune response, even after the disease has been initiated. Second, FK 506 (0.3 mg kg-1 day-1) was given only to the recipient rats which received IRBP-sensitized lymphocytes. None of FK 506-treated recipients developed EAU, while all control recipients developed the disease approximately 4 days after the cell transfer. The immune responses of FK 506-treated rats in the two experiments were also significantly suppressed. The antibody levels to S-antigen, the antigen-specific proliferative responses of lymphocytes, and even the proliferative responses to Con A were markedly suppressed in the rats in which FK 506 was given only during the efferent limb of the immune response.     

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.     

Effects of a new immunosuppressive agent, FK506, on experimental autoimmune uveoretinitis in rats

A comparative study was carried out between cyclosporine and a new immunosuppressive agent, FK506, isolated from the Streptomyces organism. This agent has the capacities to suppress the development of S-antigen-induced experimental autoimmune uveoretinitis (EAU) as well as immune responses to S-antigen in rats immunized with the antigen. When administered daily beginning on the day of immunization and for 14 days thereafter, FK506 at doses between 0.1 and 1 mg/kg suppressed EAU in a dose-dependent manner. Complete inhibition of EAU was achieved at doses of 1, 3 and 10 mg/kg. Cyclosporine (1-20 mg/kg) also produced a dose-dependent suppression of EAU and only the highest dose (20 mg/kg) caused complete inhibition of the disease. On the basis of the dose-response study, the capacity of FK506 in preventing EAU induction is 10-30 times more intense than that of cyclosporine. In addition, the FK506 (1 and 3 mg/kg) was found to be effective in preventing EAU even when administered only in the early induction phase (days 0-5) or late effector phase (days 7-12). Similar effects were obtained by cyclosporine at a daily dose of 30 mg/kg. Furthermore, none of the rats immunized with S-antigen and treated with FK506 (1 mg/kg) on days 0-14 developed EAU when reimmunized with S-antigen on day 30. In contrast, similarly treated rats were fully susceptible to the induction of experimental allergic encephalomyelitis, or even to EAU when immunized with another retinal antigen, interphotoreceptor retinoid-binding protein. Therefore, as with cyclosporine, as demonstrated in our previous study, FK506 has the capacity to induce immunological unresponsiveness specific to the S-antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen-specific suppressor cells in experimental autoimmune uveitis.

Anti-I-A antibodies, administered in vivo at the time of S-antigen injection, suppress development of experimental autoimmune uveitis (EAU) in Lewis rats. While the effects of anti-I-A are profound, the exact mechanism for this suppression is unknown. We attempted adoptive transfer of this form of suppression by injecting lymphocytes from anti-I-A-treated animals into syngeneic recipients which were later injected with S-antigen. Histologically, globes of 75% of the anti-I-A-treated animals showed no inflammation while 25% of these animals developed mild uveitis. In the group of animals which were injected with S-antigen and also received spleen cells from anti-I-A-treated rats, only 1 showed mild uveitis while the remaining 7 had no inflammation. The animals undergoing adoptive transfer of spleen cells and which were primed with an irrelevant antigen, readily developed uveitis. Suppression of S-antigen-induced EAU was abrogated by pretreatment of donor animals with cyclophosphamide. In vitro studies revealed that spleen cells of S-antigen-primed, anti-I-A-treated donors specifically suppressed lymphocyte responses to S-antigen. These in vivo and in vitro results suggest that generation of antigen-specific suppressor cells play a role in the anti-I-A immunotherapy of EAU.     

A new method for induction of experimental autoimmune uveoretinitis (EAU) in mice

Experimental autoimmune uveoretinitis (EAU) was induced in two strains of mice by repeated-immunization protocol. SMA mice (H-2 nondefined) and C57BL/6 mice (H-2b) were immunized with S-antigen mixed with Klebsiella 03 lipopolysaccharide (K03 LPS) repeatedly at intervals of 1 to 4 weeks. Following the tertiary immunization, the mice exhibited histopathological changes of EAU as well as significant immune responses to the antigen. The antigen doses required for successful EAU induction were 4 micrograms or more at each immunization time. The histopathology of EAU was characterized by mild infiltration of mononuclear cells in the retina and the choroid, particularly, at the retinal blood vessels and the photoreceptor cell layer. The anterior segment of the eye was not affected by inflammation, and therefore clinical signs of EAU were not detected even under an operating microscope. Since the mouse is a genetically and immunologically well-defined species, this model is useful for study of immunopathogenic mechanisms of EAU.     

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.