Pfau S, Leitenberg D, Rinder H, Smith B R, Pardi R, Bender J R
Division of Cardiovascular Medicine and Molecular Cardiobiology, Yale University School of Medicine, New Haven, Connecticut 06536-0812.
J Cell Biol. 1995 Mar;128(5):969-78. doi: 10.1083/jcb.128.5.969.
Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3-labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino-phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion-dependent EC signals. mAb studies indicate that the beta 2 integrin-intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1-mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.
血管内皮细胞（ECs）可因体液和细胞刺激而发生显著的表型和功能改变。这些变化通过主动募集免疫效应细胞、增加血管通透性以及改变血管张力，促进内皮细胞参与炎症反应。为了确定淋巴细胞介导的内皮细胞信号传导的早期事件，我们使用ACAS交互式激光细胞仪，研究了单个用Fluo-3标记的人脐静脉内皮细胞（HUVECs）中游离钙离子（Ca2+）的变化。在所有测试的淋巴细胞亚群中，同种异体CD3-、CD56+自然杀伤（NK）细胞在细胞因子（白细胞介素[IL]-1或肿瘤坏死因子[TNF]）处理的内皮细胞中独特地引发振荡性内皮细胞Ca2+信号。这些信号的诱导需要强烈的细胞间黏附，包括Ca2+动员和细胞外内流，并与内皮细胞肌醇磷酸（IP）生成有关。使用双色荧光检测同时记录NK和内皮细胞Ca2+信号显示，黏附时，NK细胞先于内皮细胞发生钙离子流动。用1,2-双-5-甲基氨基苯氧基乙烷-N,N,N'-四乙酸甲酯（MAPTAM）缓冲淋巴细胞Ca2+表明，淋巴细胞流动实际上是黏附依赖性内皮细胞信号的先决条件。单克隆抗体研究表明，β2整合素-细胞间黏附分子（ICAM）-1黏附途径至关重要。然而，ICAM-1反义寡核苷酸抑制IL-1介导的ICAM-1过度诱导对淋巴细胞-内皮细胞结合物中的内皮细胞Ca2+信号传导没有影响，表明还需要其他细胞因子诱导的内皮细胞改变。这些实验结合了淋巴细胞-内皮细胞相互作用、细胞间黏附、内皮细胞细胞因子激活和跨膜信号传导的特征。结果表明IP/Ca2+第二信使途径参与细胞毒性淋巴细胞诱导的内皮细胞外向信号传导，并表明这些信号可能在淋巴细胞黏附引起的内皮细胞改变中起作用。
