通过前房相关免疫偏离阻止实验性自身免疫前葡萄膜炎

目的 实验性自身免疫性前葡萄膜炎（ＥＡＡＵ）是研究人前葡萄膜炎的有用模型。本研究旨在观察前房相关免疫偏离（ＡＣＡＩＤ）能否阻止ＥＡＡＵ发生。方法 将溶于磷酸盐缓冲液（ＰＢＳ）的牛黑色素蛋白（ＢＭＰ）注射于大鼠右眼前房;对照组动物仅注射ＰＢＳ。７d后使用且加完全福氏佐剂（ＣＦＡ）的ＢＭＰ和百日咳毒素免疫所有动物。通过临床 观察和组织病理学检测葡萄膜炎的严重程度和发病率。通过由注射ＢＭＰ刺激的足垫水肿反     

Induction of anterior chamber associated immune deviation in rats receiving intracameral injections of retinal S antigen

The injection of a variety of antigens, including transplantation antigens, haptens, and viral glycoproteins, into the anterior chamber of eyes of mice produces a characteristic spectrum of immune responses in which suppressed cell mediated immunity dominates. This phenomenon is called Anterior Chamber Associated Immune Deviation (ACAID). Having recently demonstrated that soluble antigens also can induce ACAID in mice, we examined the possibility that an important soluble ocular antigen, retinal S antigen (S Ag) may be capable of inducing ACAID in rats. We have found that injection of S Ag with adjuvant into the anterior chamber of eyes of adult Lewis rats evokes neither a humoral nor a cell mediated immune response that can be detected. However, animals that received S Ag intracamerally in this manner did respond to a subsequent immunogenic dose of S Ag in CFA injected into the hind foot pads, by producing serum anti S Ag antibodies. Importantly, these rats failed to display antigen-specific delayed hypersensitivity. Moreover, lymphoid cells from anterior chamber pre-treated animals, when adoptively transferred, inhibited the development of S Ag-specific delayed hypersensitivity in naive recipients indicating that an active suppression mechanism had been generated. Thus, a soluble antigen injected intracamerally in adult Lewis rats can generate ACAID. The possibility that ACAID may be relevant to the development of S Ag induced uveitis is discussed.     

Effect of local macrophage depletion on cellular immunity and tolerance evoked by corneal allografts

Corneal graft rejection can be prevented by local macrophage depletion, via subconjunctival injections with clodronate liposomes. To unravel the underlying immunological mechanism responsible for prolonged graft survival in this circumstance, the effect of this regimen on induction of donor-specific delayed type hypersensitivity (DTH) and anterior chamber associated immune deviation (ACAID) was determined. The study showed that although subconjunctival clodronate liposome-treatment failed to alter systemically induced DTH and ACAID, both types of immune response were absent in clodronate liposome-treated rats after corneal transplantation. Thus, elimination of macrophages from the corneal transplant site renders corneal allografts immunologically "invisible" to the recipient. PURPOSE: To determine whether local macrophage depletion, via administration of clodronate liposomes, alters delayed type hypersensitivity (DTH) responses and induction of anterior chamber associated immune deviation (ACAID) after corneal allotransplantation. METHODS: Clodronate liposome-treated and untreated rats received orthotopic corneal allografts and were tested for DTH responses and induction of ACAID towards donor antigens. Also in subconjunctivally treated and untreated rats, DTH responses were measured after subcutaneous immunization or induction of ACAID with allogeneic spleen cells. RESULTS: Subconjunctival injected clodronate liposomes prevented grafted rats from developing donor-specific DTH as well as ACAID. By contrast, subconjunctivally injected clodronate liposomes had no effect on donor-specific DTH responses after systemic immunization or on the induction of ACAID with allogeneic cells. CONCLUSIONS: Depletion of macrophages at the time of corneal allografting seems to render the grafts immunologically invisible to the recipients. This could explain why these grafts survive "indefinitely" without any other form of therapy.     

脾脏在不同种类动物前房相关免疫偏离诱导和维持中的作用

目的 脾脏在小鼠前房相关免疫偏离（ａｎｊｔｅｒｉｏｒ ｃｈａｍｂｅｒ－ａｓｓｏｃｉａｔｅｄ ｉｍｍｕｎｅ ｄｅｖｉａｔｉｏｎ,ＡＣＡＩＤ）的诱导中起着重要的作用。在前房接种抗原４天内摘除脾脏可以消除免疫偏离而导致免疫反应。本研究旨在观察脾脏在其他种类动物ＡＣＡＩＤ的诱导和维持中的作用。方法 在脾脏切除和假性脾脏切除后,使用牛血清白蛋白（ｂｏｖｉｎｅ ｓｅｒｕｍ ａｌｂｕｍｉｎ,ＢＳＡ）作为可溶性抗     

The complement system plays a critical role in the development of experimental autoimmune anterior uveitis

PURPOSE: The role of complement in ocular autoimmunity was explored in a experimental autoimmune anterior uveitis (EAAU) animal model. METHODS: EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen. Complement activation in the eye was monitored by Western blot for iC3b. The importance of complement to the development of EAAU was studied by comparing the course of intraocular inflammation in normal Lewis rats (complement-sufficient) with cobra venom factor-treated rats (complement-depleted). Eyes were harvested from both complement-sufficient and complement-depleted rats for mRNA and protein analysis for IFN-gamma, IL-10, and interferon-inducible protein (IP)-10. Intracellular adhesion molecule (ICAM)-1 and leukocyte-endothelial cell adhesion molecule (LECAM)-1 were detected by immunofluorescent staining. OX-42 was used to investigate the importance of iC3b and CR3 interaction in EAAU. RESULTS: There was a correlation between ocular complement activation and disease progression in EAAU. The incidence, duration, and severity of disease were dramatically reduced after active immunization in complement-depleted rats. Complement depletion also completely suppressed adoptive transfer EAAU. The presence of complement was critical for local production of cytokines (IFN-gamma and IL-10), chemokines (IP-10), and adhesion molecules (ICAM-1 and LECAM-1) during EAAU. Furthermore, intraocular complement activation, specifically iC3b production and engagement of complement receptor 3 (CR3), had a significant impact on disease activity in EAAU. CONCLUSIONS: The study provided the novel finding that complement activation plays a central role in the pathogenesis of ocular autoimmunity and may serve as a potential target for therapeutic intervention.     

Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis. I. Induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium

The uveitogenicity of several protein fractions of the bovine retinal pigment epithelium (RPE) was studied in Lewis rats, and a major pathogenic fraction was selected. Fresh RPE cells were carefully isolated and purified in order to minimize the presence of rod outer segments (ROS). The buffer-insoluble part of the cells was extracted by Triton X-100. Most uveitogenicity was found in the Triton-insoluble pigment and cytoskeleton-containing fraction of RPE (RPE-TI). The S-antigen and opsin contents of RPE-TI were too low to induce an inflammatory response, while transducin, IRBP and cGMP-phosphodiesterase were absent. Hence, a hitherto unknown uveitogenic RPE protein, called PEP-X, evoked the pathogenic response. A typical dose-dependent experimental autoimmune anterior uveitis (EAAU) developed when the rats were immunized with RPE-TI. Initially, mononuclear cells infiltrated the anterior segment. In subsequent severe stages polymorphonuclear cells predominated in the anterior chamber. EAAU differed in particular from the known forms of EAU induced by photoreceptor proteins in that the inflammation remained exclusively anterior and the photoreceptor cells and the pineal gland were not affected. In immunized rats the immune responses to ROS proteins were very low. In contrast, there were consistently high cellular and humoral immune responses to RPE-TI. As in experimental autoimmune (uveo)retinitis (EAU), the development of EAAU could be inhibited by cyclosporin treatment indicating T-cell-dependency. A combination of histopathological, immunological and biochemical results indicates that PEP-X is an intrinsic RPE protein that is highly pathogenic. In view of its characteristics, EAAU may be a valuable model for human acute anterior uveitis, the most prevalent form of uveitis.     

Anti-inflammatory effects of specific cyclooxygenase 2,5-lipoxygenase, and inducible nitric oxide synthase inhibitors on experimental autoimmune anterior uveitis (EAAU)

PURPOSE: Inflammation, in general, causes the release of a variety of inflammatory mediators that in turn induce cyclooxygenase (COX) 2, nitric oxide synthase (iNOS) and 5-lipoxygense (LP) synthesis, producing large amounts of inflammatory prostaglandins (PG), nitric oxide (NO), and leukotriene (LT) B4. Therefore, inhibition of these enzymes may abrogate intraocular inflammation in experimental autoimmune anterior uveitis (EAAU). METHODS: Lewis rats were immunized with melanin-associated antigen (MAA) isolated from bovine iris and ciliary body. These animals were divided into three groups. The first group of rats received subcutaneous injection of COX 2 inhibitor CS 236 at different time points. The second and third groups of animals received subcutaneous aminoguanidine (AG), an iNOS inhibitor, and nordihydroguaiaretic acid (NDGA), a 5-LP inhibitor, respectively. Control animals received vehicle. Rat eyes were examined daily by slit-lamp biomicroscopy from Day 7 to 30 post injection for uveitis. Animals were also sacrificed at various time points for histologic analysis. RESULTS: Control animals developed severe EAAU in both eyes. The disease started in these animals on Day 12 post immunization and lasted for ten days. Interestingly, CS 236, a potent COX 2 inhibitor, completely abrogated EAAU when the animals were treated daily from the Day 0 to 14 or Day 0 to 20 after MAA injection. Furthermore, daily CS 236 treatment after the onset of EAAU (Day 14-20) significantly reduced the severity (both clinical and histologic) of EAAU and shortened the duration of disease. iNOS inhibitor (AG) and 5-LP inhibitor (NDGA) partially attenuated EAAU. CONCLUSIONS: Our results show that EAAU was partially attenuated by AG and NDGA. Interestingly, CS 236, a potent COX 2 inhibitor, completely inhibited EAAU in male Lewis rats most likely by inhibiting the initial phase and onset of the disease.     

视网膜下腔相关的免疫偏离

目的 确立视网膜下腔是不足具有支持针对视网可沉吟性抗原（Ｓ抗原）刺激诱导偏离式免疫反应的能力。方法 将视网膜Ｓ抗接种于Ｗｉｓｔａｒ大鼠的眼前房和视网腊膜下腔。抗原接种后７天,使用Ｓ抗原和安全福氏佐剂免疫受主动物。然后,通过足部刺激评估迟发型超原接种后７天,使用Ｓ抗原和完全福氏佐剂免疫受主动物。然后,通过足部刺激评估这发型超敏反应（ＤＴＨ）。结果 前房和视网膜睛腔注射Ｓ抗原的运行动物发生抗原特异性Ｄ     

Effect of anterior chamber-associated immune deviation (ACAID) on rat islet allograft rejection

Anterior chamber-associated immune deviation (ACAID) is characterized by the systemic inhibition of delayed type hypersensitivity (DTH) reactions to antigens which have previously been placed into the anterior chamber of the eye. Since its initial characterization, ACAID has been elicited to a wide variety of antigens, including alloantigens, and has been shown to be due to the immune deviating effects of factors such as transforming growth factor beta (TGF-beta) in the aqueous humor on ocular antigen-presenting cells (APCs). ACAID can also be induced by injecting animals with nonocular APCs, such as peritoneal exudate cells (PECs), which have been precultured with TGF-beta and antigen in vitro. The objective of this study was to determine whether alloantigenic ACAID can be effective in preventing the rejection of rat islet allografts. The notion that islet allograft rejection can be inhibited by ACAID stems from an early study showing an ACAID-induced delay in the rejection of skin grafts. Furthermore, the immune cells mediating a DTH reaction are similar to those implicated in islet allograft rejection, suggesting that they, too, may be subject to inhibition by ACAID. Our results showed that in spite of successful ACAID induction to islet allografts, recipient rats consistently rejected their grafts. Cytotoxic T cell activity (which is not inhibited by ACAID) directed against donor alloantigens was high in these animals and may have accounted, in part, for graft failure.     

Interleukin-1 abrogates anterior chamber-associated immune deviation.

Alloantigens placed into the anterior chamber of the eye elicit antigen-specific suppression of systemic delayed-type hypersensitivity (DTH) responses and severe impairment of skin allograft rejection. This pattern of immunologic alteration has been termed anterior chamber-associated immune deviation (ACAID) and is the underlying mechanism responsible for immunologic privilege within the anterior chamber of the eye. Previous studies indicate that the immunologic privilege associated with the anterior chamber of the eye might be the result of a deficiency of interleukin-2 (IL-2) during antigen presentation. The present study examined the role of IL-1 in the induction of ACAID. It is well known that intracameral (IC) inoculation of DBA/2 mastocytoma cells (P815) into allogeneic BALB/c recipients results in antigen-specific suppression of DTH responses and progressive tumor growth. The authors found, however, that sublines of P388D1 (DBA/2 monocyte/macrophage tumor) that produce IL-1 not only do not grow progressively in the anterior chamber, but also they can prevent the suppression of DTH (P less than or equal to 0.01). The role of IL-1 in the abolition of ACAID was confirmed in studies with IC-inoculated P815 cells. Systemic administration of exogenous IL-1 (by subcutaneous miniosmotic pumps) prevented the induction of ACAID in hosts that received IC inocula of P815 cells (P less than or equal to 0.01). These results indicate that induction of ACAID and perhaps the immune-privileged character of the anterior chamber is dependent on an IL-1 deficiency during the processing of IC alloantigens.     

Loss of anterior chamber-associated immune deviation (ACAID) in aged retinal degeneration (rd) mice

PURPOSE: To determine whether the capacity to induce ACAID by antigen injection into the anterior chamber is altered in animals with genetically determined retinal degeneration and increased age. METHODS: Anterior chamber-associated immune deviation (ACAID) induced by injection of ovalbumin into the anterior chamber of the eye was studied in three rodent strains with different forms of hereditary retinal degeneration (Royal College of Surgeon [RCS] rats, retinal degeneration [rd] mice, and Norrie-Disease [ND] mice) and in different age groups (age range, 1-23 months). The data were compared with those of age-matched controls. Aqueous humors of rd mice, RCS rats, and age-matched congenic controls were investigated for concentrations of transforming growth factor-beta2 (TGF-beta2) using enzyme-linked immunosorbent assay. RESULTS: ACAID was readily induced in RCS rats and ND mice irrespective of amount of retinal degeneration or aging. In rd mice ACAID could be induced in young animals but not in animals more than 12 months of age. In old rd mice, loss of ACAID was accompanied by a marked reduction in total TGF-beta2 levels in aqueous humor. CONCLUSIONS: Rd mice more than 1 year of age lose the capacity of the anterior chamber to support the induction of ACAID by intracameral injection of soluble protein antigen. Because loss of ACAID correlated with a decrease in TGF-beta2 concentration in aqueous humor, it is proposed that eyes of rd mice are unable to maintain an immunosuppressive microenvironment necessary for ACAID.     

Experimental autoimmune anterior uveitis (EAAU). II. Dose-dependent induction and adoptive transfer using a melanin-bound antigen of the retinal pigment epithelium.

Retinal pigment epithelial cell fractions have been investigated for their capacity to induce experimental uveitis. Cells of the dark (melanotic) and light areas of the bovine RPE have subsequently been extracted by buffer, Triton X-100, sodium dodecyl sulfate (SDS), and treated with various reagents in order to study some characteristics of the antigen. The SDS-insoluble melanotic fraction, consisting of spindle-shaped, mature melanin granules, proved to be the most uveitogenic preparation. Using pertussis toxin as coadjuvant, 1 microgram of melanin-protein (3.4 x 10(6) granules) was able to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats. The pathogenic activity of the responsible pathogen (PEP-X) was not diminished by SDS, nor eliminated by mildly alkaline SDS or formic acid treatment. However, HCl-deproteinized granules were not uveitogenic. The results show that PEP-X is a highly stable melano-antigen that is probably covalently bound to the granule surface. This is the first time that a melanin-bound antigen has been demonstrated to evoke specific autoaggressive activity. EAAU could adoptively be transferred by sensitized and in vitro stimulated CD4 T-lymphocytes. The evoked inflammation started 3-4 days after injection, was similar to those induced by immunization, and consisted mainly of severe iridocyclitis accompanied by dense flare and cells in the anterior chamber. Choroiditis developed in severe cases of EAAU but no inflammation was detected in the retina, pineal gland or other organs of these rats. EAAU could not be transferred by serum. Immunized PVG rats and guinea-pigs did not develop ocular inflammation. In monkeys a high dose of antigen evoked a very mild EAAU accompanied by choroiditis. In view of its characteristics, EAAU may be a new model for human anterior uveitis.     

Crucial role of apoptosis in the resolution of experimental autoimmune anterior uveitis

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) serves as an animal model of human idiopathic anterior uveitis. This study was undertaken to investigate the role of apoptosis in the resolution of EAAU. METHODS: EAAU was induced in Lewis rats by bovine melanin-associated antigen (MAA). Animals were killed at different time points during EAAU, and apoptosis of the inflammatory cells within the eye was monitored. RESULTS: Flow cytometry, TUNEL staining, and light microscopy demonstrated that CD11b/c(+) and CD4(+) T cells undergo apoptosis during EAAU. Electron microscopic analysis demonstrated that the macrophages remove these apoptotic infiltrating cells from the eye by phagocytosis. Caspase-3 levels peaked during the resolution of EAAU, and the upregulation of caspase-8 and -9 preceded that of caspase-3, suggesting that both extrinsic and intrinsic pathways of apoptosis are involved. There was an inverse relationship between the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 during EAAU. Cytochrome c was present in the cytoplasm of the infiltrating cells undergoing apoptosis. CONCLUSIONS: These results demonstrate that extrinsic and intrinsic pathways of apoptosis are involved in the resolution of EAAU. They further suggest that apoptosis followed by phagocytosis plays a critical role in the clearance of infiltrating cells from eyes with uveitis and leads to the resolution of EAAU.     

Requirement of B7-mediated costimulation in the induction of experimental autoimmune anterior uveitis

PURPOSE: To study the role of costimulatory signaling through the CD28-B7 interaction in experimental autoimmune anterior uveitis (EAAU). METHODS: Naive Lewis rats were immunized with insoluble melanin-associated antigen (MAA) derived from bovine iris and ciliary body. CTLA4-Fc, a recombinant protein comprised of the extracellular domain of human CTLA4 bound to mouse IgG2a Fc, was used to block the CD28-B7 interaction. A mutant version (CTLA4-Fc-mutant) was used as a control. The effect of CTLA4-Fc on the in vivo induction of disease with MAA was studied. Subsequently, the mechanism by which CTLA4-Fc blocked the interaction of CD28 and B7 was investigated in vivo, using the adoptive transfer of T cells derived from CTLA4-Fc-treated rats, and in vitro, using the proliferative response and cytokine production of MAA-T cells in the presence of CTLA4-Fc. RESULTS: CTLA4-Fc markedly reduced the incidence and severity of EAAU in Lewis rats after sensitization with MAA. The adoptive transfer of sensitized T cells from CTLA4-Fc-treated donors did not induce EAAU in naive recipients. CTLA4-Fc inhibited the expansion of antigen-specific MAA-T cells and the production of TNF-alpha. CONCLUSIONS: The costimulatory signal delivered through CD28-B7 is required for the induction and pathogenesis of EAAU. In the absence of this signal, antigen-specific expansion of MAA reactive T cells as well as production of TNF-alpha is inhibited. Abrogation of this costimulatory signal may be an important therapeutic option for EAAU.     

前房相关性免疫偏离研究进展

前房相关性免疫偏离(anterior chamber-associated immune deviation．ACAID)是一种机体主动的下调性免疫反应，它有以下几个特点：(1)抑制抗原特异性迟发型超敏反应；(2)抑制补体结合性抗体的分泌；(3)产生抗原特异性致敏的细胞毒T淋巴细胞；(4)产生抗原特异性非补体结合性抗体。其临床意义在于：(1)通过诱导ACAID提高角膜、视网膜等眼组织移植的成功率；(2)通过诱导ACAID防治葡萄膜视网膜炎。避免发生疱疹性角膜基质炎；(3)通过消除ACAID避免其不利影响。     

诱导供体特异性前房相关免疫偏离对高危角膜移植排斥反应的影响

目的:探讨诱导供体特异性的前房相关免疫偏离(anterior chamber-associated immune deviation,ACAID)对高危角膜移植排斥反应的影响。方法:采用新西兰白兔眼角膜建立碱烧伤眼模型,实验动物随机分为4组,A:正常角膜常规角膜移植组(正常对照组);B:碱烧伤常规角膜移植组(碱烧伤对照组);C:正常角膜诱导ACAID的角膜移植组(正常诱导组)。D:碱烧伤后诱导ACAID的角膜移植组(碱烧伤诱导组)。B,D组进行左眼碱烧伤,烧伤后1mo进行角膜移植。C,D组在角膜移植前2wk于右眼前房注入可溶性抗原以预先诱导供体特异性ACAID。A,B组右眼前房注等量平衡盐溶液。术后记录植片存活时间;对角膜新生血管(corneal neovascularization,CNV)生长情况进行评分;记录移植排斥指数(rejection index,RI);角膜固定、包埋后制作切片行HE染色。结果:A、C组角膜植片长期存活,碱烧伤对照组植片平均存活25.13±0.64d,碱烧伤诱导组角膜植片平均存活时间为38.25±1.28d。与碱烧伤对照组相比,碱烧伤诱导ACAID组角膜植片平均存活时间显著延长,两组植片存活时间的差异具有统计学意义(P=0.00)。结论:诱导供体特异性ACAID可以延长碱烧伤高危眼角膜植片的存活时间。     

Splenic B cells act as antigen presenting cells for the induction of anterior chamber-associated immune deviation

PURPOSE: To characterize the role of B cells in the induction of anterior chamber-associated immune deviation (ACAID). METHODS: An in vitro model of the ACAID spleen was used to recapitulate the events that occur when antigen is introduced into the anterior chamber of the eye and culminates in the appearance of antigen-specific, CD8(+) suppressor cells. RESULTS: In vitro-generated suppressor cells mimicked those produced by anterior chamber injection of antigen, as shown by their antigen specificity, surface expression of CD8, and capacity to suppress DTH, which is mediated by previously immunized T cells. B cells were found to be necessary for suppressor cell development. The B cell receptor (BCR) was necessary for the induction of ACAID and conveyed antigen specificity to the suppressor T cells. Lysosomal acidification of internalized antigen was necessary for B cells to induce ACAID; however, transporter of antigen processing (TAP) was not required for the generation of ACAID. CONCLUSIONS: The results suggest that B cells use the BCR to capture and internalize antigen from ACAID-inducing macrophages. Lysosomal acidification of the captured antigen is essential for the processing of the ACAID antigen before TAP-independent presentation to suppressor cells.     

Abstracts of the 187th Meeting of the Netherlands Ophthalmological Society (NOG) Utrecht,The Netherlands,March 17–19,1993

Injection of an antigen into the anterior chamber induces an immune response in which antibody production is normal while delayed hypersensitivity reactivity is depressed. Several antigens have been used to induce this response which has been termed anterior chamber associated immune deviation. We have demonstrated that allogeneic lymphocytes injected into the anterior chamber of Lewis rats increase the success rate of subsequent corneal grafts derived from the lymphocyte donor strain. To begin understanding the antigenic requirements of this phenomenon, Wistar/Furth lymphocytes were partially purified into B- and T-cell populations by panning on anti-immunoglobulin coated petri-dishes. These enriched populations were injected separately into the anterior chamber of Lewis rats. Two weeks later these Lewis rats received a full-thickness corneal graft derived from Wistar/Furth donors. Grafts were scored for opacity and neovascularization over the subsequent 8–10 weeks. In control animals injected with balanced salt solution, 20% of the grafts cleared sufficiently to be judged successful. Grafts placed on rats injected with unseparated splenic lymphocytes were judged successful in 75% of the cases. Comparable percentages for grafts on animals injected with B-cell enriched and T-cell enriched populations were 85 and 50 respectively. These results suggest that B cells, which express both class I and class II major histocompatibility antigens are more efficient at inducing anterior chamber associated immune deviation than are T cells, the majority of which express only class I major histocompatibility antigens.Abbreviations AC anterior chamber - ACAID anterior chamber associated immune deviation - W/F Wistar/Furth - MHC major histocompatibility - CPM CPM of experimental — CPM of control - SI stimulation index - IC intracamerally - BSS balanced salt solution - TNP trinitrophenol