Searle S, Bright N A, Roach T I, Atkinson P G, Barton C H, Meloen R H, Blackwell J M
Department of Medicine, University of Cambridge Clinical School, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
J Cell Sci. 1998 Oct;111 ( Pt 19):2855-66. doi: 10.1242/jcs.111.19.2855.
The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.
小鼠天然抗性相关巨噬细胞蛋白Nramp1对巨噬细胞激活具有多种多效性作用，并调节包括利什曼原虫、沙门氏菌和分枝杆菌属在内的细胞内病原体的存活。Nramp1作为一种铁转运蛋白，但其与巨噬细胞激活和/或病原体存活的确切关系仍不清楚。为了深入了解其功能，本文使用抗Nramp1单克隆抗体和多克隆抗体在激活和感染后定位Nramp1。未感染巨噬细胞的共聚焦显微镜分析表明，该蛋白的突变型（感染易感型）和野生型（抗感染型）均定位于细胞内囊泡区室的膜上。金标和电子显微镜更精确地将这些区室定义为电子透亮的晚期内体和电子致密的溶酶体区室，Nramp1在这两个区室中均与Lamp1、组织蛋白酶D和L共定位，在晚期内体中与巨噬涎蛋白共定位，在预加载的溶酶体中与5nm牛血清白蛋白金颗粒共定位。Nramp1在干扰素-γ和脂多糖处理后上调，同时溶酶体中的标记相对于晚期内体增加，且Nramp1阳性囊泡从核周位置沿着微管网络向细胞质周边明显分散。在对照和激活的巨噬细胞中，野生型巨噬细胞中该蛋白的表达比突变型巨噬细胞高3至4倍。在感染利什曼原虫的巨噬细胞中，Nramp1存在于含病原体吞噬体的膜上，这些吞噬体在静息巨噬细胞中保持核周定位。在感染鸟分枝杆菌的静息和激活巨噬细胞中，Nramp1阳性囊泡迁移并聚集，但并不总是与含病原体的吞噬体融合。因此，Nramp1蛋白位于能够对吞噬体融合和病原体微环境产生直接影响的位置，以及在巨噬细胞内体/溶酶体功能的更普遍调节中发挥作用。
