Newman W, Beall L D, Carson C W, Hunder G G, Graben N, Randhawa Z I, Gopal T V, Wiener-Kronish J, Matthay M A
Department of Endothelial Cell Biology, Otsuka America Pharmaceutical, Inc., Rockville, MD 20850.
J Immunol. 1993 Jan 15;150(2):644-54.
A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active.
已开发出一种用于检测液相中E选择素（可溶性E选择素，sEs）的定量夹心酶联免疫吸附测定法，其灵敏度可达100 pg/ml。该测定法对L选择素或P选择素均无反应性。我们已利用此方法来确定细胞表面表达后E选择素的去向，并测试体内测得的水平是否可能代表内皮细胞活化状态。E选择素在IL-1刺激的内皮细胞上清液中于24小时首次可检测到，并缓慢增加直至72小时。然而，在此时间段内，系统中可检测到的总E选择素（细胞加上清液）急剧下降。培养24小时的125I表面标记内皮细胞在上清液中显示出分子量降低的E选择素，表明该分子从表面脱落。根据免疫沉淀和分子筛研究确定，脱落形式似乎也比完整的膜形式略小。此外，通过与针对细胞质结构域的抗肽抗体反应性丧失确定，在活化内皮细胞上清液和血清中发现的该分子的细胞质结构域不完整。我们检测了71名正常个体的血清。无一例外，血清中sEs的含量在0.13至2.8 ng/ml之间，这表明即使在没有明显炎症过程的情况下，E选择素也在合成并释放到血液中。此外，有低血压的菌血症患者，而非无低血压的患者，其sEs值显著升高。通过细胞结合研究确定，血液来源的E选择素具有生物活性。
