Sandusky H, Cilluffo M, Braun J, Gordon L K
Department of Ophthalmology, University of California, Los Angeles 90095, USA.
Ocul Immunol Inflamm. 2001 Mar;9(1):25-34. doi: 10.1076/ocii.9.1.25.3981.
pANCA marker autoantibody is expressed by a subset of patients with anterior uveitis. A recombinantly isolated pANCA monoclonal antibody, Fab 5-3, identifies ocular expression of corresponding pANCA antigens in human ciliary body and retina. In this study, Fab 5-3 was used to explore pANCA antigen expression in ocular tissues of multiple mammalian species and identify the ciliary body cell type expressing the pANCA antigen.
Ocular tissues were obtained from several mammalian species and evaluated for expression of the pANCA (Fab 5-3) antigen(s) using immunohistochemistry and Western analysis of tissue extracts. Additionally, primary cultures of nonpigmented and pigmented rabbit ciliary body epithelium were analyzed for pANCA expression using immunofluorescence and Western analysis.
Ocular pANCA (Fab 5-3) antigen expression was observed by immunohistochemistry only in the cytoplasm of retinal ganglion cells and ciliary body epithelium. Retinal antigen expression was conserved in all species examined. Ciliary body expression was observed in human, rabbit, rat, and mouse, but not in pig or cow. Antigen expression in the rabbit ciliary body was restricted to the nonpigmented layer as defined in primary cultures of nonpigmented and pigmented ciliary body epithelium. Immunoreactive proteins in both the human and rabbit included a 32-33 kDa doublet (histone H1), and novel 80 and 100 kDa proteins.
This study identifies ocular pANCA antigen expression in multiple mammalian species localized to the retinal ganglion cell layer and the non-pigmented ciliary body epithelium. The present study also establishes novel 80 and 100 kDa proteins which may correspond to the cytoplasmic antigens detected in situ and can be further characterized biochemically and immunologically using small animal model systems.
pANCA标记自身抗体在一部分前葡萄膜炎患者中表达。一种重组分离的pANCA单克隆抗体Fab 5-3可识别在人睫状体和视网膜中相应pANCA抗原的眼部表达。在本研究中，Fab 5-3被用于探索多种哺乳动物眼部组织中pANCA抗原的表达，并鉴定表达pANCA抗原的睫状体细胞类型。
从几种哺乳动物获取眼部组织，使用免疫组织化学和组织提取物的蛋白质印迹分析评估pANCA（Fab 5-3）抗原的表达。此外，使用免疫荧光和蛋白质印迹分析对兔非色素和色素睫状体上皮的原代培养物进行pANCA表达分析。
通过免疫组织化学观察到眼部pANCA（Fab 5-3）抗原仅在视网膜神经节细胞和睫状体上皮细胞的细胞质中表达。在所检查的所有物种中视网膜抗原表达是保守的。在人、兔、大鼠和小鼠中观察到睫状体表达，但在猪或牛中未观察到。在兔睫状体中的抗原表达局限于非色素层，这在非色素和色素睫状体上皮的原代培养物中得到证实。人和兔中的免疫反应性蛋白包括一个32 - 33 kDa的双峰（组蛋白H1）以及新的80和100 kDa蛋白。
本研究鉴定了多种哺乳动物眼部pANCA抗原表达定位于视网膜神经节细胞层和非色素睫状体上皮。本研究还确定了新的80和100 kDa蛋白，它们可能与原位检测到的细胞质抗原相对应，并且可以使用小动物模型系统进行进一步的生物化学和免疫学表征。
