Shimizu K, Wu G S, Sultana C, Kalra V K, Rao N A
Doheny Eye Institute, Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles 90033-1088, USA.
Invest Ophthalmol Vis Sci. 1999 Dec;40(13):3215-23.
In previous work, it has been shown that in experimental autoimmune uveitis, the peroxynitrite-mediated protein nitration product nitrotyrosine was localized in the degenerating photoreceptors. Subsequently, phagocyte-generated inducible nitric oxide synthase (iNOS) was also found to localize, primarily in the outer retina and to a lesser extent in the anterior segments. This study was intended to determine whether retinal soluble proteins such as S-antigen and interphotoreceptor retinoid-binding protein (IRBP) play a role in the induction of *NO and superoxide by a macrophage cell line and by rat and rabbit peritoneal macrophages.
Cells from the murine macrophage cell line RAW 264.7 and rat and rabbit peritoneal macrophages were incubated in the presence of retinal soluble proteins. The nitrite level in the cultured supernatant was evaluated for *NO production using the Griess reaction. Activation of nuclear transcription factor kappaB (NF-kappaB) was determined by electrophoretic mobility shift assay. Superoxide production was measured by superoxide dismutase-inhibitable reduction of cytochrome C.
Both S-antigen and IRBP induced significant, dose-dependent nitrite production in RAW 264.7 and rat peritoneal macrophages. Induction of iNOS by retinal proteins was inhibited by the iNOS-specific inhibitor aminoguanidine and the tyrosine kinase inhibitor genistein. This iNOS induction was accompanied by the activation of NF-kappaB. S-antigen also induced superoxide production in rabbit peritoneal macrophages, but not in RAW 264.7.
These results show that soluble retinal proteins significantly induce *NO and superoxide production by macrophages. Increased production of reactive oxygen species by macrophages in the presence of these soluble retinal proteins in vivo may accelerate photoreceptor degeneration in uveitis.
在先前的研究中已经表明，在实验性自身免疫性葡萄膜炎中，过氧亚硝酸盐介导的蛋白质硝化产物硝基酪氨酸定位于退化的光感受器中。随后，还发现吞噬细胞产生的诱导型一氧化氮合酶（iNOS）主要定位于视网膜外层，在前房段中定位程度较低。本研究旨在确定视网膜可溶性蛋白，如S抗原和光感受器间类视黄醇结合蛋白（IRBP），是否在巨噬细胞系以及大鼠和兔腹膜巨噬细胞诱导一氧化氮（NO）和超氧化物的过程中发挥作用。
将小鼠巨噬细胞系RAW 264.7以及大鼠和兔腹膜巨噬细胞与视网膜可溶性蛋白一起孵育。使用格里斯反应评估培养上清液中的亚硝酸盐水平以检测NO的产生。通过电泳迁移率变动分析确定核转录因子κB（NF-κB）的激活情况。通过超氧化物歧化酶抑制的细胞色素C还原法测量超氧化物的产生。
S抗原和IRBP均在RAW 264.7和大鼠腹膜巨噬细胞中诱导了显著的、剂量依赖性的亚硝酸盐产生。视网膜蛋白对iNOS的诱导被iNOS特异性抑制剂氨基胍和酪氨酸激酶抑制剂金雀异黄素所抑制。这种iNOS的诱导伴随着NF-κB的激活。S抗原还在兔腹膜巨噬细胞中诱导了超氧化物的产生，但在RAW 264.7中未诱导。
这些结果表明，视网膜可溶性蛋白可显著诱导巨噬细胞产生NO和超氧化物。在体内存在这些可溶性视网膜蛋白的情况下，巨噬细胞活性氧产生的增加可能会加速葡萄膜炎中光感受器的退化。
