Gerlach H, Lieberman H, Bach R, Godman G, Brett J, Stern D
Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
J Exp Med. 1989 Sep 1;170(3):913-31. doi: 10.1084/jem.170.3.913.
Some in vivo observations have suggested that growing or perturbed endothelium, such as that which occurs during angiogenesis, is more sensitive to the action of cytokines (TNF/cachectin, TNF, or IL-1) than normal quiescent endothelial cells. This led us to examine the responsiveness of endothelium to TNF as a function of the growth/motile state of the cell. TNF-induced modulation of endothelial cell surface coagulant function was half-maximal at a concentration of approximately 0.1 nM in subconfluent cultures, whereas 1-2 nM was required for the same effect in postconfluent cultures. Perturbation of endothelial cell shape/cytoskeleton was similarly more sensitive to TNF in subconfluent cultures. Consistent with these results, radioligand binding studies demonstrated high affinity TNF binding sites, Kd approximately 0.1 nM on subconfluent cultures, whereas only lower affinity sites (Kd approximately 1.8 nM) were detected on postconfluent cultures. The mechanisms underlying this change in the affinity of endothelium for TNF were studied in four settings. Crosslinking experiments with 125I-TNF and endothelium showed additional bands corresponding to Mr approximately 66,000 and approximately 84,000 with subconfluent cultures that were not observed with postconfluent cultures. Experiments with X-irradiated endothelium, whose growth but not motility was blocked, indicated that proliferation was not required for induction of high affinity TNF sites. Postconfluent endothelium, triggered to enter the proliferative cycle by microbutuble poisons, expressed high affinity TNF binding sites together with changes in cell shape/cytoskeleton well before their entry into S phase. Using wounded postconfluent monolayers, cells that migrated into the wound and those close to the wound edge displayed enhanced TNF binding and modulation of coagulant properties. These results suggest a model for targetting TNF action within the vasculature; regulation of high affinity endothelial cell binding sites can direct TNF to activated cells in particular parts of the vascular tree.
一些体内观察结果表明，正在生长或受到扰动的内皮细胞，比如在血管生成过程中出现的内皮细胞，比正常静止的内皮细胞对细胞因子（TNF/恶病质素、TNF或IL-1）的作用更敏感。这促使我们研究内皮细胞对TNF的反应性与细胞生长/运动状态之间的关系。在亚汇合培养物中，TNF诱导的内皮细胞表面凝血功能调节在浓度约为0.1 nM时达到半数最大效应，而在汇合后培养物中产生相同效应则需要1 - 2 nM。在亚汇合培养物中，内皮细胞形状/细胞骨架的扰动对TNF同样更敏感。与这些结果一致，放射性配体结合研究表明存在高亲和力的TNF结合位点，在亚汇合培养物上Kd约为0.1 nM，而在汇合后培养物上仅检测到低亲和力位点（Kd约为1.8 nM）。我们在四种情况下研究了内皮细胞对TNF亲和力变化的潜在机制。用125I - TNF与内皮细胞进行交联实验，在亚汇合培养物中显示出对应于Mr约为66,000和约84,000的额外条带，而在汇合后培养物中未观察到。对经X射线照射的内皮细胞进行的实验表明，其生长但运动未受阻碍，这表明诱导高亲和力TNF位点不需要细胞增殖。汇合后内皮细胞通过微管毒物触发进入增殖周期，在进入S期之前就表达了高亲和力TNF结合位点以及细胞形状/细胞骨架的变化。使用汇合后单层细胞损伤模型，迁移到伤口处以及靠近伤口边缘的细胞显示出增强的TNF结合和凝血特性调节。这些结果提示了一种在脉管系统中靶向TNF作用的模型；高亲和力内皮细胞结合位点的调节可将TNF导向血管树特定部位的活化细胞。
