Ise Y, Yamaguchi K, Sato K, Yamamura Y, Kitamura F, Tamatani T, Miyasaka M
Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Japan.
Eur J Immunol. 1988 Aug;18(8):1235-44. doi: 10.1002/eji.1830180814.
Large-scale extravasation of lymphocytes takes place in vivo under physiological conditions in lymph nodes at very specialized vascular segments called high endothelial venules (HEV). When circulating lymphocytes leave the blood, they first bind to endothelial cells of HEV (HE cells) and subsequently enter lymph nodes by crossing the endothelial lining of HEV. Although the lymphocyte-HEV interaction has recently been the subject of intense research by many laboratories, it has been studied almost exclusively by the use of the lymphocyte-binding assay in which lymphocyte binding is examined on nonviable HEV present on frozen sections and, hence, no dynamic interaction between HE cells and lymphocytes has been studied. We report herein that endothelial cells of rat HEV can be grown in vitro and that the lymphocyte-HEV interaction can be studied dynamically using viable cells in culture vessels. The identification of the cultured line, termed Ax, as HE cells was based on their phenotypic, morphological, cytochemical and biochemical characteristics, and most importantly on its in vitro behavior, particularly in terms of its specific ability to interact with mature lymphocytes. Phenotypic analysis demonstrated that not only did monoclonal antibodies, known to react with HE cells, recognize the Ax but also a monoclonal antibody raised against the Ax specifically recognized HE cells in vivo, as determined by an immunoperoxidase staining of frozen sections, supporting the notion that the cell strain, Ax, is derived from HEV. This Ax, even after long-term culture (greater than 50 passages), allowed mature, but not immature, lymphocytes to bind to the cell surface and subsequently transport bound cells underneath their cytoplasm. This phenomenon was inhibited in a dose-dependent manner by various reagents known to inhibit lymphocyte recirculation in vivo. The cultured line derived from HE cells should provide a means to investigate the biochemical nature of lymphocyte-HEV interaction, and to understand the molecular mechanisms underlying the large-scale lymphocyte traffic taking place in vivo.
在生理条件下，淋巴细胞会在淋巴结中一种被称为高内皮微静脉（HEV）的非常特殊的血管段发生大规模的血管外渗。当循环淋巴细胞离开血液时，它们首先与HEV的内皮细胞（HE细胞）结合，随后通过穿过HEV的内皮进入淋巴结。尽管淋巴细胞与HEV的相互作用最近已成为许多实验室深入研究的课题，但几乎完全是通过淋巴细胞结合试验进行研究的，该试验是在冷冻切片上存在的无活力HEV上检测淋巴细胞结合，因此，尚未研究HE细胞与淋巴细胞之间的动态相互作用。我们在此报告，大鼠HEV的内皮细胞可以在体外培养，并且可以使用培养容器中的活细胞动态研究淋巴细胞与HEV的相互作用。将培养的细胞系Ax鉴定为HE细胞是基于其表型、形态、细胞化学和生化特征，最重要的是基于其体外行为，特别是其与成熟淋巴细胞相互作用的特定能力。表型分析表明，不仅已知与HE细胞反应的单克隆抗体能识别Ax，而且通过冷冻切片的免疫过氧化物酶染色确定，针对Ax产生的单克隆抗体在体内能特异性识别HE细胞，这支持了细胞系Ax源自HEV的观点。这种Ax即使经过长期培养（超过50代），也能使成熟而非未成熟的淋巴细胞结合到细胞表面，并随后将结合的细胞运输到其细胞质下方。这种现象被已知在体内抑制淋巴细胞再循环的各种试剂以剂量依赖的方式抑制。源自HE细胞的培养细胞系应该为研究淋巴细胞与HEV相互作用的生化性质以及理解体内发生的大规模淋巴细胞运输的分子机制提供一种手段。
