Kokura S, Wolf R E, Yoshikawa T, Ichikawa H, Granger D N, Aw T Y
Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
Microcirculation. 2000 Feb;7(1):13-23.
The objectives of this study were to 1) determine the time-course of T-lymphocyte adhesion to monolayers of human umbilical vein endothelial cell (HUVEC) that were exposed to 60 min of anoxia followed by 24 h of reoxygenation, and 2) define the mechanisms responsible for the hyperadhesivity of postanoxic HUVEC to human T-lymphocytes.
Human peripheral blood mononuclear leukocytes were isolated from heparinized peripheral blood. T-lymphocytes were obtained by negative selection using a MACS column. HUVEC monolayers were exposed to anoxia/reoxygenation (A/R), and then reacted with 51Cr -labeled T-lymphocytes in adhesion assays.
A/R leads to an increased adhesion of T-lymphocytes to HUVEC monolayers, with peak responses occurring at 8 h after reoxygenation. This adhesion response was largely attributed to the CD4+ T-cell subset. The hyperadhesivity of A/R-exposed HUVEC was inhibited by monoclonal antibodies directed against either LFA-1, VLA-4, ICAM-1, or VCAM-1, indicating a contribution of these adhesion molecules and their ligands. Moreover, T-cell hyperadhesivity was attenuated by anti- IL-8. consistent with a role for this chemokine in the adhesion response. Protein synthesis inhibitors (actinomycin D and cycloheximide) as well as chemical inhibitors of (and binding ds-oligonucleotides to) NFkappaB and AP-1 significantly attenuated the A/R-induced T-lymphocyte adhesion responses. The kinetics of VCAM-1 on post-anoxic HUVEC correlated with the T-lymphocyte adhesion response.
A/R elicits a T-lymphocyte-endothelial cell adhesion response that involves transcription-dependent surface expression of VCAM-1.
本研究的目的是：1）确定暴露于60分钟缺氧后再复氧24小时的人脐静脉内皮细胞（HUVEC）单层上T淋巴细胞黏附的时间进程，以及2）确定缺氧后HUVEC对人T淋巴细胞高黏附性的机制。
从肝素化外周血中分离人外周血单个核白细胞。使用MACS柱通过阴性选择获得T淋巴细胞。将HUVEC单层暴露于缺氧/复氧（A/R），然后在黏附试验中与51Cr标记的T淋巴细胞反应。
A/R导致T淋巴细胞对HUVEC单层的黏附增加，在复氧后8小时出现峰值反应。这种黏附反应主要归因于CD4+T细胞亚群。针对LFA-1、VLA-4、ICAM-1或VCAM-1的单克隆抗体可抑制A/R暴露的HUVEC的高黏附性，表明这些黏附分子及其配体起作用。此外，抗IL-8可减弱T细胞高黏附性，这与该趋化因子在黏附反应中的作用一致。蛋白质合成抑制剂（放线菌素D和环己酰亚胺）以及NFκB和AP-1的化学抑制剂（和与双链寡核苷酸结合）显著减弱了A/R诱导的T淋巴细胞黏附反应。缺氧后HUVEC上VCAM-1的动力学与T淋巴细胞黏附反应相关。
A/R引发T淋巴细胞-内皮细胞黏附反应，该反应涉及VCAM-1的转录依赖性表面表达。
