Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis

Evidence is presented for the involvement of immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis, an ocular inflammatory disease induced in Lewis rats by injection of the purified S antigen from bovine retina. Several parameters at various dates after immunization have been studied. In vivo degranulation of choroidal mast cells, in vitro degranulation of peritoneal mast cells in the presence of S antigen and cutaneous anaphylaxis were observed as early as 6–8 days after immunization. Serum antibodies were detected by passive hemagglutination from day 7 on. These phenomena preceded the onset of the ocular inflammation, consisting of a transient polymorphonuclear leukocyte infiltration, a long-lasting lymphoid cell infiltration and extensive damage to the retina and surrounding tissues. Such time course of events may suggest a role for reaginic antibody as a trigger for vascular changes in the target ocular tissue, favoring the development of inflammatory lesions.     

Pathology of experimental autoimmune uveoretinitis in mice

The histopathology and immunopathology of murine experimental autoimmune uveoretinitis (EAU) following active immunization with the interphotoreceptor retinoid-binding protein (IRBP) were studied. The methods used included conventional light microscopy and immunoperoxidase staining. Lesions were located mainly in the uvea and the retina and were characteristically focal. The prominent histopathologic findings in the retina were vasculitis, granuloma, retinal fold, focal serous detachment, and loss of photoreceptors. Granulomas, formation of Dalen-Fuchs nodules, inflammatory cellular infiltration and increase in the thickness of the choroid and ciliary body were frequent findings. Subretinal neovascularization occurred in 10% of the experimental animals. Mild to moderate inflammation was also noted in the vitreous. The predominant infiltrating cells in the retinal and uveal granuloma and the Dalen-Fuchs nodules were macrophages. In contrast, the predominant infiltrating cell types in the vitreous were T helper/inducer lymphocytes. T suppressor/cytotoxic cells were rarely seen. Expression of Ia antigens on the ocular cells was confined to the immediate area of the inflammatory sites. The kinetics of histopathology showed two peaks at the 5th and 10th week after immunization, suggesting a relapsing course of the disease.     

Immunopathology of experimental autoimmune uveoretinitis in primates.

The eyes and pineal glands from 10 monkeys immunized with S-antigen were studied using routine histopathological and immunohistochemical techniques. Seven out of 10 animals developed uveitis between 19 and 33 days after the initial immunization. Histopathology of the eyes harvested 70 days after immunization showed moderate to marked uveoretinitis, subretinal fibrosis, retinal necrosis and gliosis. The pineal glands demonstrated chronic pinealitis. The infiltrating cells were both CD3 and CD19/CD22 lymphocytes with a ratio of 1.4 in the eye and 2.2 in the pineal gland. The ratio of CD4 to CD8 lymphocytes was 1.5:1. MHC Class II antigens and adhesion molecule (ICAM-1) were observed on resident cells. The influx of B lymphocytes and the formation of subretinal fibrosis differentiate the disease in the monkey from that in the rat and mouse. These findings are similar to Vogt-Koyanagi-Harada syndrome and subretinal fibrosis with uveitis syndrome in human.     

Combined anti-interleukin-2 receptor and low-dose cyclosporine therapy in experimental autoimmune uveoretinitis.

The effect of combination treatment with anti-interleukin-2 (IL-2)-receptor monoclonal antibody (ART18) and cyclosporine A (CsA) on the effector stage of experimental autoimmune uveoretinitis (EAU) was examined. Efferent-stage EAU was induced in Lewis rats by adoptive transfer of a T-helper cell line specific to retinal soluble antigen (SAg). Rats were treated with ART18 (0.5 mg/kg/day), low dose CsA (1.5 mg/kg/day), or a combination of both. The results were compared to groups treated with high dose CsA (10 mg/kg/day) and to sham-treated animals, with respect to clinical and histological EAU, lymphocyte proliferative responses to SAg, and the ability to transfer EAU to secondary recipients. Ten-day combination therapy with ART18 and low-dose CsA was more effective than high-dose CsA and almost completely suppressed EAU development. ART18 as sole therapy was partially effective, and was better than low dose CsA as sole therapy. Splenocytes of protected animals did not transfer EAU to secondary recipients, while splenocytes of sham-treated controls did, suggesting that the number of uveitogenic lymphocytes in the treated host was reduced by the therapy. In contrast, this therapy was completely ineffective against EAU induced by active immunization. The possible reasons for this discrepancy between the two respective models of EAU are discussed.     

大鼠实验性自身免疫性葡萄膜视网膜炎模型

目的 :建立大鼠实验性自身免疫性葡萄膜视网膜炎 (EAU)模型 ,为探讨人类葡萄膜炎的发病机制奠定基础。方法 :18只Wistar大鼠用 3只不同剂量牛视网膜可溶性抗原 (S Ag)免疫后 ,每日扩瞳进行EAU临床观察 ;当大鼠出现中度以上EAU临床表现时处死、其他大鼠第 3～ 4w时处死后眼球摘除 ,行组织学观察。结果 :3组不同剂量S Ag免疫大鼠EAU发病率分别为 :10 0 μgS Ag组 6只大鼠 (12只眼 )为 2 12、2 0 0 μgS Ag组 6只大鼠 (12只眼 )为 6 12、30 0 μgS Ag组 6只大鼠 (12只眼 )为8 12 ;组织学炎症评分分别为 :0、16 76± 11 0 2、17 5 6± 9 96。结论 :使用中等纯度的S Ag ,以及中等敏感度的Wistar大鼠 ,可成功诱发出EAU模型     

Systemic and local anti‐C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis

Activation of complement occurs during autoimmune retinal and intraocular inflammatory disease as well as neuroretinal degenerative disorders. The cleavage of C5 into fragments C5a and C5b is a critical event during the complement cascade. C5a is a potent proinflammatory anaphylatoxin capable of inducing cell migration, adhesion and cytokine release, while membrane attack complex C5b‐9 causes cell lysis. Therapeutic approaches to prevent complement‐induced inflammation include the use of blocking monoclonal antibodies (mAb) to prevent C5 cleavage. In these current experiments, the rat anti‐mouse C5 mAb (BB5·1) was utilized to investigate the effects of inhibition of C5 cleavage on disease progression and severity in experimental autoimmune uveoretinitis (EAU), a model of organ‐specific autoimmunity in the eye characterized by structural retinal damage mediated by infiltrating macrophages. Systemic treatment with BB5·1 results in significantly reduced disease scores compared with control groups, while local administration results in an earlier resolution of disease. In vitro, contemporaneous C5a and interferon‐γ signalling enhanced nitric oxide production, accompanied by down‐regulation of the inhibitory myeloid CD200 receptor, contributing to cell activation. These experiments demonstrate that C5 cleavage contributes to the full expression of EAU, and that selective C5 blockade via systemic and local routes of administration can suppress disease. This presents great therapeutic potential to protect against tissue damage during autoimmune responses in the retina or inflammation‐induced degenerative disease.     

Differential roles for IFN-gamma and IL-17 in experimental autoimmune uveoretinitis

IL-17-producing CD4(+) T cells, so called T(h)17 cells, constitute a newly identified inflammatogenic cell population, which is critically involved in some inflammatory diseases. To explore the role of T(h)17 cells in murine experimental autoimmune uveoretinitis (EAU), a model of human autoimmune uveitis where T(h)1 responses predominantly participate in the pathogenesis, IL-17(-/-) mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 for disease induction. Funduscopic examination revealed that EAU was induced in IL-17(-/-) mice just like in wild-type (WT) mice at early phases of the disease. However, at later/maintenance phases, the severity was significantly reduced in IL-17(-/-) mice. Expression of IFN-gamma and MCP-1 was comparable between WT and IL-17(-/-) mice during the time course. In vivo blockade of IFN-gamma and IL-4 resulted in exacerbation of EAU at later phases with augmented IL-17 production. Taken together, our data demonstrated that IL-17/T(h)17 participates in the late phases of EAU and also that T(h)1 and T(h)17 responses are differentially required for EAU.     

Human immunoglobulin preparations for intravenous use prevent experimental autoimmune uveoretinitis

We have evaluated the effect of human Igs for intravenous use(IVIg) on the onset and development of experimental autoimmuneuveoretinitis (EAU), a T cell-dependent autoimmune disease inducedin rats by a single immunization with retinal S-antigen (S-Ag).Five consecutive daily infusions of IVIg, starting on the sameday as S-Ag immunization, protected (Lewis x Brown-Norway) F¹rats against EAU. The prevention of EAU was IVIg-specific, i.e.mediated by pooled human IgG from multiple donors, since neitherinfusions of BSA nor infusions of pooled Ig from only two healthyindividuals were effective. Treatment with IVIg decreased lymphocyteprollferative and antibody responses to S-Ag and the proliferativeresponse to concanavalin A. Lack of proliferation was not dependentupon generation of suppressor cells. Lymph node (LN) cells fromIVIg-treated and S-Ag-immunized animals neither proliferatednor secreted IL-2 in response to S-Ag but proliferated whenco-cultured with LN cells from rats immunized with S-Ag. Ourfindings are compatible with an induction of a state of functionalinactivatlon/anergy of T lymphocytes by infusions of IVIg. Thisfunctional inactivation may be due to the presence in IVIg ofantibodies that bind both in vivo and in vitro to rat lymphocytes.Results from the present study suggest a novel mechanism bywhich IVIg may be beneficial in human autoimmune diseases.     

In vivo lymphokine production in experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) is a well-characterized model of immune-mediated intraocular inflammation. The intraocular infiltrate in EAU consists predominantly of T lymphocytes. The in vivo production of interleukin-2 (IL-2), lymphotoxin and IL-4 by these T cells was investigated by in situ hybridization using cDNA probes to lymphokine mRNA. Localization of lymphokine mRNA was found simultaneous with disease onset in areas of T-cell infiltration. Positive signal was seen over cells in the uveal tract, retina and extraocular region. Less than 10% of the population of T cells defined immunohistochemically had positive localization of mRNA for these lymphokines. The number of positive cells was similar for each of the three probes and increased as the disease progressed. The findings suggest that these lymphokines are produced in vivo in immune-mediated intraocular inflammation and may play a role in the immunopathology seen in these conditions.     

Cytokines and apoptotic molecules in experimental melanin-protein induced uveitis (EMIU) and experimental autoimmune uveoretinitis (EAU).

The cytokine profile and occurrence of apoptosis during experimental melanin-protein induced uveitis (EMIU) were investigated and compared with that of experimental autoimmune uveoretinitis (EAU). EMIU or EAU was induced in Lewis rats. Eyes were collected at different time points after immunization. Cytokine mRNA expression was identified in the inflammatory cells in the uvea of EMIU rats; IL-2, IFN-gamma and IL-12 increased at the peak of the inflammation, and then tapered off as inflammation subsided. IL-4 and IL-10 increased at the peak of ocular inflammation, and persisted with inflammation resolved. Fas and FasL were expressed consistently in ocular resident cells of EMIU, but were elevated in EAU. In EAU, Bcl-2 expression showed a sharp peak in inflammatory cells but not in the resident cells. In EMIU, high levels of Bcl-2 were present and persisted in both ocular resident and inflammatory cells. Expression of Bax was relatively stable in both EAU and EMIU. Cellular DNA fragmentation was detected in the retinal glial cells of EAU and some inflammatory cells of EMIU. In EMIU, the dynamics of Th1 cytokines were consistent with the ocular inflammation, whereas persistent expression of Th2 cytokines was consistent with their known regulatory role. The continuous high expression of Bcl-2 and the high ratio of Bcl-2 to Bax in the eyes of EMIU may possibly contribute to prevention of ocular tissue damage, and of inflammatory cells from undergoing apoptosis, thus resulting in chronic recurrent inflammation.     

Kinetics of intraocular cytokines in the suppression of experimental autoimmune uveoretinitis by type I IFN

The systemic administration of IFN-alpha/beta was previously found to suppress inflammation in rats with experimental autoimmune uveoretinitis (EAU); however, an effect on the systemic immune response was not identified. In order to investigate an immunological basis for suppression at the intraocular level, rats immunized with interphotoreceptor retinoid-binding protein (IRBP) were administered daily intramuscular injections of 10(5) IU IFN-alpha/beta and cytokines were measured by ELISA in intraocular extracts prepared by ultrasonification at various timepoints throughout the course of EAU. In control EAU, intraocular concentrations of IFN-gamma were found to be non-detectable on day 8 before the onset of inflammation, significantly elevated on day 12 at peak inflammation (182+/-106 pg/ml), then non-detectable again on day 16 after inflammation had begun to subside. In contrast, intraocular IFN-gamma in IFN-alpha/beta- treated rats remained non-detectable or low at all timepoints. Measurement of intraocular IL-2 revealed no difference between the two groups of rats. Intraocular IL-4 concentrations were elevated in rats treated with IFN-alpha/beta, although this cytokine was also detected in the same range in controls as well as normal rats. Finally, intraocular IL-10 was non-detectable on day 8, significantly elevated at peak inflammation on day 12 (588+/-139 pg/ml), then decreased to low levels on day 16 in control EAU rats, while remaining non-detectable or low in IFN-alpha/beta-treated rats. These results suggest that acute inflammation in IRBP-induced EAU in rats involves both IFN-gamma and IL- 10 at the local intraocular level, and that systemic administration of IFN-alpha/beta inhibits EAU via a mechanism that involves suppression of both cytokines.      

IL-10 has a protective role in experimental autoimmune uveoretinitis

The role of IL-10 in the regulation of ocular autoimmune disease was studied in experimental autoimmune uveoretinitis (EAU) elicited in mice by immunization with the retinal antigen interphotoreceptor retinoid binding protein. IL-10-deficient mice were susceptible to EAU, indicating that pathogenesis can occur without presence of IL-10. Treatment of normal mice with IL-10 for 5 days after uveitogenic immunization ameliorated subsequent EAU scores, and down-regulated antigen-specific production of tumor necrosis factor-alpha and IFN- gamma. A concomitant treatment with IL-4 further reduced disease, and resulted in emergence of antigen-specific IL-4 and IL-10 production, as well as in enhancement of the IgG1 antibody isotype. IL-4 by itself was not protective. Only IL-10, but not IL-4, was able to inhibit the function of differentiated uveitogenic T cells in culture. Expression of mRNA for Th1 and Th2 cytokines in the eye during the course of EAU showed that while a Th1 pattern predominated early, IL-10 mRNA expression coincided with down-regulation of the Th1 response and resolution of EAU. Systemic neutralization of IL-10 during the expression phase of EAU resulted in elevated disease scores. Our results suggest that endogenous IL-10 limits expression of EAU and may play a role in the natural resolution of disease. The data further suggest that exogenous IL-10 may be useful in therapeutic control of autoimmune uveitis. While IL-10 by itself is sufficient to suppress Th1 effector development and function, a concomitant administration of IL-4 is required to shift the autoimmune response towards a non-pathogenic Th2 pathway.      

Identification of genomic regions controlling experimental autoimmune uveoretinitis in rats

The present study attempts to identify specific genetic locicontributing to experimental autoimmune uveoretinitis (EAU)susceptibility in F₂ progeny of resistant Fischer (F344/N) andsusceptible Lewis (LEW/N) inbred rats. F₂ progeny of F344/Nx LEW/N inbred rats were immunized with the R16 peptide of interphotoreceptorretinoid-binding protein (IRBP). A genome-wide scan was conductedusing 125 simple sequence length polymorphism markers in selectedF₂ animals that developed severe eye disease or remained unaffectedto identify phenotype:genotype co-segregation. The F₂ population(n = 1287) demonstrated a wide range of histologically assessedEAU scores (assessed on a scale of 0–4). The disease incidenceand severity were not consistent with a simple Mendelian inheritancemodel. Of the F₂ hybrid rats, 60% developed EAU, implying theexistence of a potent susceptibility locus with incomplete penetranceassociated with the LEW genome or a more complex polygenic modelof inheritance. Two genomic regions, on chromosomes 4 and 12,showed strong genetic linkage to the EAU phenotype (P < 0.0016),suggesting the presence of susceptibility loci in these chromosomalregions. In conclusion, we have identified two genomic candidateintervals from D4Arb8 to D4Mit17 on chromosome 4 and from thechromosome end to D12Arb8 on chromosome 12, that appear to influenceEAU susceptibility in LEW/F344 rats. Further analysis of thesegenomic regions may lead to identification of the susceptibilitygenes and to characterization of their function.     

The effect of retinal S-antigen-specific monoclonal antibody therapy on experimental autoimmune uveoretinitis (EAU) and experimental autoimmune pinealitis (EAP).

We induced an autoimmune uveitis and pinealitis in Lewis rats by inoculating them with bovine S-antigen. This type of uveitis forms a useful experimental model of human chronic intra-ocular inflammation. Induction of experimental autoimmune uveitis (EAU) but not of experimental autoimmune pinealitis (EAP) could be prevented by the administration of S-antigen-specific monoclonal antibody simultaneously with the S-antigen. Inhibition of EAU was associated with significantly raised levels of anti-S antibodies during the first 2 weeks post-immunisation. Immunocytochemical staining for lymphocyte subsets, monocytes and macrophages showed that eyes of monoclonal antibody treated animals contained no immunocompetent inflammatory cells unless they also had clinical signs of inflammation. In contrast, the inflammatory exudate in the pineal glands of both treated and untreated animals contained equal numbers of infiltrating lymphocytes and monocytes in the same relative proportions. These results indicate that the inhibitory effect of the monoclonal antibody S2.4.C5 may be directed towards the effector arm of the immune-mediated cytotoxic response.     

Inhibition of experimental autoimmune uveoretinitis in rats by S-antigen-specific monoclonal antibodies

Mouse monoclonal antibodies (mAb), of either IgG2a or IgG2b isotypes, specific for the retinal S-autoantigen (S-Ag) or a pool of rat anti-S-Ag sera prevented experimental autoimmune uveoretinitis in Lewis rats when injected i.p. at the time of immunization. Control mAb of the same isotypes, irrelevant to S-Ag, had no inhibitory effect. The humoral response to S-Ag, as studied by enzyme-linked immunosorbent assay using a mouse mAb specific for rat kappa chain, was moderately but significantly reduced in suppressed animals. The rapid disappearance of the injected mAb from rat sera, as measured using a rat mAb specific for mouse kappa chain, could be explained by its complexing with either autologous antigen released from the retina at the site of inflammation, or anti-idiotypic antibodies.     

Cyclosporine-induced specific unresponsiveness to retinal soluble antigen in experimental autoimmune uveoretinitis

Cyclosporine (CsA) was previously reported to effectively suppress the induction of experimental autoimmune uveoretinitis (EAU) in rats immunized with S-antigen. The present study provides information concerning the development of specific unresponsiveness in the CsA-treated rats. The majority of Lewis rats immunized with S-antigen and treated with CsA from Day 0 to Day 14 failed to develop EAU when reimmunized with S-antigen on Day 30. In contrast, similarly treated rats were fully susceptible to induction of experimental allergic encephalomyelitis when immunized with myelin basic protein, or even to EAU when immunized with another retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). The possible involvement of suppressor cells in establishing the unresponsiveness state was indicated by experiments both in vitro and in vivo. The enriched fractions of suppressor cells from rats unresponsive to induction of EAU by S-antigen were found to inhibit the specific mitotic response of lymphocytes to S-antigen, but had no effect on the response to IRBP. In vivo, injection of such enriched suppressor cell fractions to naive syngenic rats inhibited or delayed the development of EAU following immunization with S-antigen. It is proposed, therefore, that specific unresponsiveness plays a role in the suppression of EAU by CsA and that the unresponsiveness is mediated in part by specific suppressor cells.     

Morphometric analysis of T lymphocyte compartmentation in experimental autoimmune uveoretinitis.

Multiple or single halothane exposure of rabbits or guinea pigs induces an antibody reactive with trifluoroacetylated (TFA) proteins. The antigen that initiates this immune response was investigated in halothane-exposed rabbits and guinea pigs for its anatomical location in the liver, the chronology of its expression in situ and exposure conditions which would modulate its expression. Using an immuno-staining technique, binding by an anti-TFA antibody to the antigen was detected in liver tissue from all halothane-exposed rabbits and guinea pigs. Antigen could be detected only in the centrilobular area around the central vein where staining intensity was concentrated in an area seven to nine cells deep. In halothane-exposed rabbits, the appearance of TFA antigen was most predominant on the first and second days following a single exposure. Multiple exposures induced TFA antigen in a larger area around the central vein than did a single exposure. Though maximal expression of TFA antigen occurred following two or three exposures, subsequent exposures did not potentiate antigen expression. In halothane-exposed guinea pigs, exposure to deuterated halothane, which reduces the extent and metabolites of oxidative halothane metabolism, elicited the appearance of TFA antigen around the central veins, although to a lesser extent than during halothane exposure. Halothane-induced antigen was evident in guinea pigs as early as 6 h post-exposure and was still apparent 90 h later. Thus, halothane exposure by inhalation elicits the appearance of TFA protein conjugates which may, in turn, evoke the anti-TFA immune response.     

Type I interferons in host defense

Type I interferons (IFNs) are a family of cytokines specialized to coordinate immunity to viruses and other intracellular infections. In the past several years, many of the receptors and signaling pathways that link pathogen detection to induction of type I IFNs have been identified and characterized. An integrated picture has emerged in which type I IFNs have essential functions in several seemingly disparate processes: they restrict viral spread by engaging machinery that ultimately cripples and kills infected cells, yet they are also positively linked to the activation and expansion of lymphocytes that are important for control of intracellular infections. These advances highlight the context-specific actions of type I IFNs and clarify the multiple points at which they are integrated into both innate and adaptive immunity.