Reijerkerk Arie, Kooij Gijs, van der Pol Susanne M A, Khazen Shadi, Dijkstra Christine D, de Vries Helga E
Neuroimmunology Research Group, Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands.
FASEB J. 2006 Dec;20(14):2550-2. doi: 10.1096/fj.06-6099fje. Epub 2006 Oct 25.
The blood-brain barrier (BBB), a selective barrier formed by endothelial cells and dependent on the presence of tight junctions, is compromised during neuroinflammation. A detailed study of tight junction dynamics during transendothelial migration of leukocytes has been lacking. Therefore, we retrovirally expressed green fluorescent protein (GFP) fused to the N-terminus of the tight junction protein occludin in the rat brain endothelial cell line GP8/3.9. Confocal microscopy analyses revealed that GFP-occludin colocalized with the intracellular tight junction protein, ZO-1, localized at intercellular connections, and was absent at cell borders lacking apposing cells. Using live cell imaging we found that monocytes scroll over the brain endothelial cell surface toward cell-cell contacts, induce gap formation, which is associated with local disappearance of GFP-occludin, and subsequently traverse the endothelium paracellularly. Immunoblot analyses indicated that loss of occludin was due to protein degradation. The broad spectrum matrix metalloproteinase (MMP) inhibitor BB-3103 significantly inhibited endothelial gap formation, occludin loss, and the ability of monocytes to pass the endothelium. Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack.
血脑屏障（BBB）是一种由内皮细胞形成且依赖紧密连接存在的选择性屏障，在神经炎症期间会受到损害。目前尚缺乏对白细胞跨内皮迁移过程中紧密连接动态变化的详细研究。因此，我们通过逆转录病毒在大鼠脑内皮细胞系GP8/3.9中表达了与紧密连接蛋白闭合蛋白N端融合的绿色荧光蛋白（GFP）。共聚焦显微镜分析显示，GFP-闭合蛋白与细胞内紧密连接蛋白ZO-1共定位，ZO-1定位于细胞间连接，在缺乏相邻细胞的细胞边界处则不存在。通过活细胞成像，我们发现单核细胞在脑内皮细胞表面朝着细胞间接触部位滚动，诱导间隙形成，这与GFP-闭合蛋白的局部消失相关，随后单核细胞通过细胞旁途径穿过内皮。免疫印迹分析表明，闭合蛋白的丢失是由于蛋白质降解所致。广谱基质金属蛋白酶（MMP）抑制剂BB-3103显著抑制内皮间隙形成、闭合蛋白丢失以及单核细胞穿过内皮的能力。我们的研究结果为白细胞穿越血脑屏障的机制提供了新的见解，并表明旨在稳定紧密连接的治疗方法可能有助于抵抗神经炎症攻击。
