Lu Ying, He Shirley, Jia Lin, Khan Naheed W, Heckenlively John R
Department of Ophthalmology and Visual Sciences Kellogg Eye Center, University of Michigan, Ann Arbor, MI, 48105, USA.
Mol Vis. 2010 Oct 2;16:1936-48.
Recoverin has been demonstrated to be one of the main causative antigenic retinal proteins common in many cases of autoimmune retinopathy (AIR). Strategies for producing two different AIR mouse models associated with anti-recoverin antibodies were tested.
(1) Six-week-old female B6.MRL-Fal(lpr)/J mice (LPR) mice were immunized with recombinant recoverin three times at 2-4 week intervals. (2) Five-month-old Balb/cJ mice were injected with hybridoma cells designed to produce recoverin monoclonal antibodies. Anti-recoverin antibodies were analyzed by immunoblot and enzyme-linked immunosorbent assay (ELISA). Electroretinograms (ERG), histopathologic examination, and flow cytometric analysis were assessed.
High anti-recoverin antibody levels were achieved in both models, accompanied by significantly reduced scotopic and photopic responses on the ERGs. Retinal histology showed swollen cell bodies in the inner nuclear layer in recoverin-immunized LPR mice, while photoreceptor and outer nuclear layer swelling was observed in recoverin hybridoma cells injected into balb/cJ mice. Glial fibrillary acidic protein (GFAP) staining detected a marked increase of Müller cells and astrocyte reactive gliosis in both mouse models. Rhodopsin and S-opsin staining was similar to controls, while decreased numbers of bipolar cells were observed in both models. Complement component C1q and C3 deposits increased upon immunohistopathologic retinal staining in both models, while increased numbers of CD4+ and CD68+ cells from retinas were found upon flow cytometric analysis.
These two models had similar pathology in the retina, indicating the retinal antigens to recoverin antibody set off pathologic events that include leukocyte invasion, complement deposition, reactive gliosis in the retina, and selective retinal degeneration of inner nuclear layer neurons. These two AIR mouse models will allow for detailed pathologic investigation and testing of protein antigens associated with human AIR and can be used to test treatments. It is important to note that, since most AIR patients have multiple anti-retinal antibodies, it will be possible to study which antibodies are pathologic and which have no retinal pathologic effects. These models can also serve as an important research resource for studying the pathophysiology of specific retinal proteins by creating autoantibodies, which potentially will give a better understanding of retinal protein interactions.
恢复蛋白已被证明是许多自身免疫性视网膜病（AIR）病例中常见的主要致病性视网膜抗原蛋白之一。测试了产生两种与抗恢复蛋白抗体相关的不同AIR小鼠模型的策略。
（1）对6周龄雌性B6.MRL-Fal(lpr)/J小鼠（LPR小鼠）每隔2-4周用重组恢复蛋白免疫3次。（2）对5月龄的Balb/cJ小鼠注射旨在产生恢复蛋白单克隆抗体的杂交瘤细胞。通过免疫印迹和酶联免疫吸附测定（ELISA）分析抗恢复蛋白抗体。评估视网膜电图（ERG）、组织病理学检查和流式细胞术分析。
两种模型均产生了高抗恢复蛋白抗体水平，同时ERG上的暗视和明视反应显著降低。视网膜组织学显示，在接受恢复蛋白免疫的LPR小鼠中，内核层细胞体肿胀，而在注射到Balb/cJ小鼠体内的恢复蛋白杂交瘤细胞中观察到光感受器和外核层肿胀。胶质纤维酸性蛋白（GFAP）染色检测到两种小鼠模型中Müller细胞和星形胶质细胞反应性胶质增生均显著增加。视紫红质和S-视蛋白染色与对照组相似，而两种模型中双极细胞数量均减少。两种模型的视网膜免疫组织病理学染色显示补体成分C1q和C3沉积增加，而流式细胞术分析发现视网膜中CD4+和CD68+细胞数量增加。
这两种模型在视网膜中具有相似的病理学表现，表明针对恢复蛋白抗体的视网膜抗原引发了包括白细胞浸润、补体沉积、视网膜反应性胶质增生以及内核层神经元选择性视网膜变性在内的病理事件。这两种AIR小鼠模型将有助于对与人类AIR相关的蛋白质抗原进行详细的病理学研究和测试，并可用于测试治疗方法。需要注意的是，由于大多数AIR患者有多种抗视网膜抗体，因此有可能研究哪些抗体具有致病性，哪些没有视网膜病理效应。这些模型还可作为通过产生自身抗体研究特定视网膜蛋白病理生理学的重要研究资源，这可能会更好地理解视网膜蛋白相互作用。
