von Asmuth E J, Smeets E F, Ginsel L A, Onderwater J J, Leeuwenberg J F, Buurman W A
Department of Surgery, University of Limburg, Maastricht, The Netherlands.
Eur J Immunol. 1992 Oct;22(10):2519-26. doi: 10.1002/eji.1830221009.
E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.
E选择素是内皮细胞上一种可诱导的黏附分子。在肿瘤坏死因子（TNF）激活的培养人脐静脉内皮细胞（HUVEC）上研究了这种糖蛋白的内化过程。在平行实验中研究了细胞间黏附分子-1（ICAM-1）的动力学。在内化研究中，使用放射性碘化抗体进行酸洗脱内吞试验，并通过免疫组织学进行研究；两种方法得到的结果相当。[125I]ENA1是一种对E选择素特异的单克隆抗体（mAb），以每分钟约1.7%的膜结合[125I]mAb的速率内化。相比之下，每分钟内化的膜结合[125I]RR1/1（一种对ICAM-1特异的mAb）不到0.1%。对TNF激活的HUVEC进行免疫染色，并通过光学显微镜（LM）和电子显微镜（EM）检查。LM显示，在用这些mAb孵育30分钟后，ENA1存在于细胞质囊泡中，但RR1/1不存在，EM将这些囊泡鉴定为多囊泡体。在内皮细胞未预先接触mAb的情况下，在相同的细胞器中检测到大量与胶体金复合的E选择素和牛血清白蛋白，胶体金用作液相内化的标志物，因此反对mAb相互作用诱导的E选择素内化。此外，E选择素表面表达的量不受持续存在的mAb的影响，这也反对mAb对正常E选择素动力学的干扰。综上所述，这些结果表明TNF激活的HUVEC组成性地内化E选择素。E选择素内化在调节E选择素膜表达以及从循环中清除E选择素配体方面的生理意义需要进一步研究。
