摘要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，使用添加完全福氏佐剂(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刺激的足垫水肿反应评估迟发型超敏反应(DTH)。结果与对照组相比，预先眼内注射BMP的大鼠，其EAAU的严重程度和发病率减低，且DTH反应受到显著抑制。结论这些资料提示ACAID可以用来阻止EAAU的发生。

Prevention of experimental autoimmune anterior uveitis

by anterior chamber-associated immune deviation

LI Zhi-Jie,MD, PENG Guang-Hua,MD, FENG Zheng,

LI Chen,MD

Key wordsimmune privilege; immune deviation; uveitis; autoimmune; bovine melanin protein

AbstractObjectiveExperimental autoimmune anterior uveitis (EAAU) is a useful model for human anterior uveitis. The purpose of this study was to observe whether EAAU development could be prevented by anterior chamber-associated immune deviation (ACAID). MethodsBovine melanin protein (BMP) in phosphate-buffered saline (PBS) was injected into the anterior chamber of the right eye of rats; control animals were injected with PBS alone. Seven days later, all animals were immunized with BMP in complete Freund's adjuvant (CFA) and pertussis toxin. The severity and incidence of uveitis was monitored by clinical examination and histopathology. The delayed-type hypersensitivity (DTH) responses were evaluated by footpad swelling elicited by injection of BMP.ResultsRats intraocularly injected with BMP showed a reduced severity and incidence of EAAU, and significantly suppressed DTH response compared to control rats.ConclusionThese data suggest that the ACAID procedure can be utilized to prevent the development of EAAU.

Certain antigens injected into anterior chamber of the eye elicit uniquely a deviant form of immune response. This spectrum of immunity has been termed anterior chamber-associated immune deviation (ACAID), characterized by impaired delayed-type hypersensitivity (DTH), suppression of complement-fixing antibodies production and intact generation of non-complement fixing antibodies and cytotoxic T cells^［1,2］.It has previously been demonstrated that injection of bovine retinal S antigen or bovine interphotoreceptor retinoid binding protein (IRBP) into the AC of Lewis rat and mice eyes reduced the incidences of experimental autoimmune uveitis^［3～5］ (EAU).In a recent series of elegant studies, Broekhuyse and associates^［6～9］described a new experimental autoimmune disease that affects mainly the anterior segment, and named this model experimental autoimmune anterior uveitis (EAAU). In the experiments to be described, EAAU-inducing antigen, bovine melanin protein (BMP) purified from the retinal pigment epithelium, the iris and choroid, has been injected intraocularly into rats to test the hypothesis that induction of ACAID can prevent the development and expression of EAAU.

MATERIALS AND METHODS

AnimalsA total of 60 male Wistar rats, 8 to 10 weeks of age, weighing 150～180g, were purchased from the Experimental Animal Center in Jinan University. All animal procedures were carried out under katamine and chlorpromazine mixtures as anesthetics.

AntigensSodium dodecyl sulfate-insoluble BMP from the choroid and the remnant retinal pigment epithelium (RPE) was obtained as described by Broekhuyse and Kuhlmann^［8］. Briefly, the anterior segment, vitreous, and retina were removed from the fresh pigmented cow eye. The eyecup was rinsed with phosphate-buffered saline (PBS). The choroid and RPE wererubbed in mortar with water. The suspen-sion was filtrated and centrifuged. The sediment was suspended in 20g*L^-1 sodiumdodecyl sulfate, and heated at 75℃. After centrifugation, purified melanin was washed, dried, and stored at -20℃.

Anterior chamber inoculationAnterior chamber (AC) injection of BMP was carried out as described previously^［10］.Briefly, anesthetized rats received AC injections with Hamilton syringe. For each injection, 3μL of air followed by 10μL of antigen were drawn into the tubing. For AC inoculations, an oblique transcorneal paracentesis tunnel was formed with a sharp needle. The blunt needle was inserted through this track, followed by inoculation of 10 μL of antigen and 3μL of air, sealing the injection tract. The AC of the right eye of each mouse received 10μg^．L^-1of antigen.

Induction and assessment of DTHInduction and assessment of DTH was accomplished as previously described^［11］. Briefly, animals were immunized subcutaneously with 100 μg BMP and complete Freund's adjuvant (CFA, Sigma) 7 days after AC inoculation of BMP. Seven days after immunization, 100μg of BMP in 10μL PBS was injected into the left footpad of the rat. The right footpad, served as unchallenged control, was injected with PBS alone. Footpad swelling was measured 24 hours later with an engineer's micrometer. Units of swelling were determined by the difference in thickness between the challenged left footpad and the unchallenged control right footpad of experimental animals. The unpaired Student's t test was performed for statistics analysis.

Induction and assessment of EAAUSeven daye after AC inoculation of BMP or PBS, a 1∶1 mixture of 100 μg BMP in 0.05 mL PBS and CFA (0.05mL) was injected subcutaneously into the base of the tail of rats. At the same time, 50 μg BMP (0.1mL) and Bordetella pertussis 3 ×10⁹ organisms in 0.1mL PBS were injected intraperitoneally. Experimental animals were examined every other day using the slitlamp microscope and the dissecting microscope by three masked investigators. The clinical observations were graded on a scale of 0 to 4, based on the following criteria^［12］: 0 = no inflammation; 0.5⁺ = mild dilatation of iris vessels and/or flare in the anterior chamber; 1⁺ = cells in the anterior chamber and/or fibrin at the pupillary margin; 2⁺ = fibrin in the anterior chamber and/or synechiae; 3⁺ = fibrin clots in the anterior chamber and/or corneal edema; and 4⁺ = hypopyon, hyphema, and/or loss of re reflex.

Histopathologic studiesThe eyes of rats were fixed in 40g*L^-1 glutaraldehyde for 30 minutes and then transferred into 100g^．L^-1buffered formalin. After appropriate fixation, the tissues were embedded in methacrylate and sectioned for routine histopathology.

RESULTS

Induction of ACAID with BMPBMP and PBS (positive control) were inoculated into the AC of the right eye of rats seven days prior to immunization of all rats with BMP in CFA subcutaneously. Assay for DTH response was performed by injection of BMP in the right footpad of rats seven days after immunization. Footpad swelling was measured 48 hours later. The results are shown in Figure 1. An impaired BMP-specific DTH response was found in the AC-BMP pretreated group of rats, whereas the PBS pretreated group made vigorous DTH responses.

Figure 1Induction of ACAID with BMP. Rats were immunized SC with 5