Dick A D, Ford A L, Forrester J V, Sedgwick J D
Centenary Institute of Cancer Medicine and Cell Biology, Sydney, Australia.
Br J Ophthalmol. 1995 Sep;79(9):834-40. doi: 10.1136/bjo.79.9.834.
This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry.
Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia.
Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+.
Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.
本研究旨在通过流式细胞术分离并分类正常大鼠视网膜中小胶质细胞的细胞表面表型，以期识别视网膜内假定的抗原呈递细胞（APC），而迄今为止通过免疫组织化学尚无法做到这一点。
采用改良的分离技术，利用梯度 Percoll 密度梯度细胞分离法及用于中枢神经系统小胶质细胞的流式细胞术表型标准，对正常大鼠视网膜小胶质细胞进行分离和分类。
视网膜小胶质细胞可通过流式细胞术依据其 CD45lowCD11b/c+CD4low 细胞表面表达来定义。正常大鼠视网膜中组成型 MHC II 类分子的表达几乎完全局限于极少数既不表达低水平（小胶质细胞）也不表达高水平 CD45 的细胞群体。三色流式细胞术分析证实这些 MHC II 类分子阳性细胞为 ED2+。
使用这种灵敏的分离技术，我们已确定了分支状、驻留小胶质细胞的细胞表面特征，并发现它们不组成型表达 MHC II 类分子。然而，在表型不同的细胞群体（CD45low/highED2+）上存在组成型 MHC II 类分子表达。我们推测这些细胞是中枢神经系统中发现的血管周围巨噬细胞的对应物，可将抗原呈递给渗出的 T 细胞，尽管其在视网膜中的确切位置只能通过免疫组织化学分析来确认。实质小胶质细胞作为 APC 的作用仍不明确。未来使用该技术分离小胶质细胞和假定的血管周围细胞将有助于确定这些细胞在视网膜内免疫反应的启动和持续过程中所起的作用。
