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LI Zhi-Jie, PENG Guang-Hua, FENG Zheng, LI Chen
Department of Ophthalmology, Institute of Tissue Transplantation & Immunology,
Jinan University, Guangzhou (510632)
AbstractAIM：To determine whether the vitreous cavity (VC) is capable of supporting the induction of deviant immune response to retinal soluble (S) antigen and to observe the influence of interleukin-1 (IL-1) on the immunologic properties of the VC.METHODS： Retinal S antigen was inoculated into the anterior chamber (AC) and VC in Wistar rats. Seven days after antigen inoculation, the recipient animals were immunized with S antigen and complete Freund's adjuvant. Delayed-type hypersensitivity (DTH) was then assessed by footpad challenge. To alter systemic immune conditions, IL-1 was administrated by intraperitoneal injection.RESULTS： Antigen-specific DTH did not develop in rats in which S antigen was injected into the AC and the VC. In contrast, strong DTH was elicited by S antigen injected into the AC and VC if IL-1 was administrated systemically for 7 consecutive days after the antigen challenge.CONCLUSION： The VC is capable of supporting immune deviation to soluble antigen by actively suppressing antigen-specific DTH. Systemic administration of exogenous IL-1 eliminates the capacity of the VC to support immune deviation inducing by soluble antigen injected locally.
MeSH Immunity; Antigens; Vitreous body; Retina;Interleukin-1
摘要 目的：确立玻璃体腔(VC)是否具有支持针对视网膜可溶性抗原(S抗原)刺激诱导偏离式免疫反应的能力，并观察白细胞介素-1(IL-1)对玻璃体腔免疫特性的影响。方法：将视网膜S抗原接种于Wistar大鼠的眼前房和玻璃体腔。抗原接种后7 d，使用S抗原和完全福氏佐剂免疫受主动物。然后，通过足部刺激评估迟发型超敏反应(DTH)。通过腹腔注射IL-1改变全身的免疫状态。结果：前房和玻璃体腔注射S抗原的动物没有发生抗原特异性DTH。与此相反，当全身给予IL-1时，注射于前房和玻璃体腔的S抗原则激发剧烈的DTH。结论：玻璃体腔具有通过抑制抗原特异性DTH，支持针对可溶性抗原的免疫偏离的诱导能力。全身使用外源性IL-1可以消除玻璃体腔针对局部接种可溶性抗原诱导免疫偏离的能力。
Antigenic material introduced into the anterior chamber (AC) is not ignored by the systemic immune apparatus. Instead, intraocular antigen elicits a stereotypic, systemic immune response that is selectively deficient in T cells that mediate delayed-type hypersensitivity (DTH). At the same time, other types of T cells are activated, including regulatory T cells and precursors of cytotoxic T cells. This deviant form of systemic immunity was termed anterior chamber-associated immune deviation (ACAID). Current evidence indicates that ACAID is the central feature of ocular immune privilege. It is only within the latest 25 years that ACAID has been studied in detail［1］. So far, however only a few studies concerning immune deviation elicited by antigens injected into the posterior part of the eye have been reported［2,3］. The aim of present study is to determine whether a deviant immune response similar to ACAID may also be induced by injection of soluble antigens into the vitreous cavity (VC). In addition, we scheduled to observe effect of interleukin-1 (IL-1) on the capacity of the VC to induce antigen-specific immune deviation because IL-1 is a key immunomodulator shown to trigger many events of the inflammatory response.
MATERIALS AND METHEODS
Animals： Adult male Wistar rats were used throughout this study. Inoculations, injections and clinical examinations were conducted in animals under general anesthesia induced by intramuscular injection of 25mg/kg ketamine and 25mg/kg chlorpromazine.
Retinal soluble antigen： S antigen was prepared from bovine retinas by hypotonic buffer extraction followed by 50% ammonium sulfate precipitation, gel filtration, and ion exchange chromatography as described previously ［4］.
Intraocular antigen inoculation： For AC injection, an oblique transcorneal paracentesis was performed with a microinjector under the microscope, and 100 μg S of antigen in 10 μL of phosphate-buffered saline (PBS) were injected. For VC injection, an paracentesis was made at the pars plana and 100 μg S of antigen in 10 μL of PBS were introduced into the center of the vitreous cavity［5］.
DTH assay： Seven days after the intraocular inoculation of S antigen, animals were immunized subcutaneously with 100 μg of S antigen and complete Freund's adjuvant (CFA, Sigma). Footpad-swelling analysis was performed 7 days later. DTH was measured as previously described［5］. Briefly, 100μg of S antigen in 10 μL of PBS solution were injected into the left posterior footpad of the rat. The right posterior footpad serving 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 the animals.
Exogenous IL-1 administration： A single daily dose of 10 units of human recombinant IL-1(hrIL-1) in 10 μL of PBS was delivered to each animal by intraperitoneal injection for 7 consecutive days after VC inoculation of antigen. Control animals received each a daily intraperitoneal injection of 10 μL of PBS.
Statistics： The Student t-test was used to test for the statistical validity of the data.
RESULTS
Immune deviation induced by soluble antigen in the anterior chamber and the vitreous cavity：After injection of S antigen into the AC and the VC, animals were immunized with S antigen and CFA at day 7. Evaluation by footpad challenge was performed 7 days later. No DTH was detectable in animals that had been given intraocular injection (AC or VC) of S antigen. By contrast, animals immunized with S antigen-CFA without previous intraocular injection of S antigen displayed intense DTH (Fig 1).
fig 1 Delayed-type hypersensitivity measurements after inoculation of S antigen into different ocular sites. Footpad swelling analysis was performed 24 hours after footpad challenge with S antigen in animals with inoculation of S antigen into the anterior chamber (AC) or the vitreous cavity (VC). Negative control animals (Naive) were not immunized. Positive control animals (P)were immunized only. * P＜0.05,vs positive control animals.
Effect of IL-1 on the immune deviation induced by S antigen in the anterior chamber and the vitreous cavity： As reported by Benson et al［6］, IL-1 administration compromised the capability of the AC to support immune deviation by soluble antigen injected locally. Thus, our assumption is that IL-1 may also abrogate the immune deviation induced by antigen inoculated into the vitreous cavity. To determine whether IL-1 has a disruptive effect on the immune privilege of the VC, IL-1 was administrated by intraperitoneal injection for 7 days after S antigen had been inoculated into the VC. When S antigen was injected into the eyes of the rats treated with IL-1, the recipients mounted strong DTH response; that is, they displayed no immune deviation (Fig 2).
