Functional Role of Endosomes in Transcytosis of Immunoglobulin G

为了解大鼠卵黄囊上皮细胞获得被动免疫的机制，采用共聚焦激光扫描显微镜（CLSM）荧光比例法和免疫细胞化学方法研究了免疫球蛋白G （IgG）在大鼠卵黄囊上皮细胞中的选择性胞吞作用。用荧光标记的葡聚糖进行内吃标记，然后用CLSM进行双荧光比例法测定分选内小体的内部pH值，pH值为6.1，与之前发现的体外形成igg - fc受体复合物（IgG-FcR）的最佳pH值6.0很好地吻合。检测转铁蛋白（Tf）和IgG的胞内转运途径，并用免疫细胞化学方法与gtr结合蛋白rab4、rab5a和rab8的定位进行比较。除rab8外的rabb蛋白与Tf和/或IgG在分选和/或再循环内体中共定位。3. 在抗生素brefeltin - a （10muM）存在下，追踪Tf和IgG 15- 30min后重复免疫细胞化学检查。Tf在再循环核内体（rab4阳性）中积累，IgG在分选核内体（rab4/rab5A阳性）和携带IgG的胞囊（rab4/rab5A阳性，rab8阴性）中发现。brefeltin - a对igG的胞吞作用影响不大。这些结果证实了IgG-FcR通过温和酸性核内体（包括分选核内体和携带IgG-FcR的囊泡）进行转酶。rab4和rab5A参与了分选核内体的融合/裂变事件的调控。还提出了一种不依赖于网格蛋白和蛋白质螯合蛋白的IgG-FcR分选和转运机制。

To understand the mechanism of acquiring passive immunity, selective transcytosis of immunoglobulin G (IgG) in the rat yolk sac epithelial cell was studied by the fluorescence ratiometry and immunocyto-chemistry with using a confocal laser scanning microscope (CLSM).1.The internal pH of the sorting endosome was measured after endocytic labeling with fluorescently labeled dextran followed by the dual-fluorescence ratiometry using CLSM,The pH was 6.1 in good coincidence with the pH 6.0 previously found to be optimalfor the formation of IgG-Fcreceptor complex (IgG-FcR) in vitro.2. The intracellular transport pathways of transferrin (Tf) and IgG were examined and immunocytochemically compared with the localization of GTR-binding proteins rab4, rab 5A and rab8. The rab proteins except for rab8 were colocalized with Tf and/or IgG in the sorting and/or recycling endosomes. 3. The immunocytochemical examination was repeated after chasing Tf and IgG for 15-30 min in the Presence of an antibiotics brefeldin-A (10muM). Tf was accumulated to recycling endosomes (rab4 positive), while IgG was found in sorting endosomes (rab4/rab5A positive) and IgG-carrying transcytotic vesicles (rab4/rab5A positive, rab8 negative). Transcytosis of igG was apparently little affected by brefeldin-A.These results confirm the idea that IgG-FcR is transcytosed through the mildly acidic endosomes including sorting endosomes and IgG-FcR carrying vesicles. It is suggested that rab4 and rab5A are involved in regulation of fusion/fission events on the sorting endosome. A clathrin-and COPs-independent mechanism of sorting and transport of IgG-FcR is also suggested.