Berg M, James S P
Mucosal Immunity Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Blood. 1990 Dec 1;76(11):2381-8.
The Leu-8 molecule, the human homologue of the murine MEL-14 peripheral lymph node homing receptor, is expressed on neutrophils in both species and may be important in localization of cells to sites of inflammation. Most circulating human neutrophils express the Leu-8 molecule, and activation of neutrophils with phorbol myristate acetate causes a rapid decline in Leu-8 membrane fluorescence staining within 15 minutes. Northern blot analysis of total cellular RNA from neutrophils demonstrated two species of Leu-8 messenger RNA, a major one of 2.4 kb and a minor one of 1.9 kb. Because two different Leu-8 cDNA clones were obtained from human lymphocytes that were predicted to encode both transmembrane and phosphatidylinositol (PI)-anchored forms of the molecule, experiments were conducted to determine whether Leu-8 is anchored to neutrophils by a PI-anchor. There was a slight decrease in expression of Leu-8 on neutrophils when they were treated with PI-specific phospholipase C (PI-PLC). However, Leu-8 was abundant on neutrophils obtained from a patient with paroxysmal nocturnal hemoglobinuria. To determine the fate of the Leu-8 molecule during cell activation, neutrophils were labeled with 125I-anti-Leu-8. During activation antibody was rapidly lost from the cell surface and was not internalized, suggesting that Leu-8 is released from the cell membrane during cell activation. When cell extracts of neutrophils were compared with extracts of lymphoid cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, the Leu-8 species expressed on neutrophils had a significantly higher and more variable relative mobility (70 to 120 Kd for neutrophils v 70 Kd for Jurkat T cells). In addition, Leu-8 molecules were detected in the supernatants of activated neutrophils. These results indicate that human neutrophils express a high-molecular-weight form of the Leu-8 molecule that has a conventional transmembrane anchor and is rapidly released from the membrane during activation. The loss of the Leu-8 membrane glycoprotein during activation may be a mechanism for rapid alteration of neutrophil adhesion characteristics.
亮氨酸-8分子是小鼠MEL-14外周淋巴结归巢受体的人类同源物，在两种物种的中性粒细胞上均有表达，可能在细胞定位到炎症部位中起重要作用。大多数循环中的人类中性粒细胞表达亮氨酸-8分子，用佛波酯肉豆蔻酸酯乙酸盐激活中性粒细胞会导致15分钟内亮氨酸-8膜荧光染色迅速下降。对中性粒细胞总细胞RNA的Northern印迹分析显示有两种亮氨酸-8信使RNA，一种主要的为2.4kb，一种次要的为1.9kb。由于从人类淋巴细胞中获得了两个不同的亮氨酸-8 cDNA克隆，预计它们编码该分子的跨膜和磷脂酰肌醇（PI）锚定形式，因此进行了实验以确定亮氨酸-8是否通过PI锚定在中性粒细胞上。用PI特异性磷脂酶C（PI-PLC）处理中性粒细胞时，亮氨酸-8的表达略有下降。然而，在一名阵发性夜间血红蛋白尿患者的中性粒细胞上亮氨酸-8含量丰富。为了确定细胞激活过程中亮氨酸-8分子的命运，用125I-抗亮氨酸-8标记中性粒细胞。在激活过程中，抗体迅速从细胞表面丢失且未被内化，这表明亮氨酸-8在细胞激活过程中从细胞膜释放。当中性粒细胞的细胞提取物与淋巴细胞提取物通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹进行比较时，中性粒细胞上表达的亮氨酸-8种类具有明显更高且更可变的相对迁移率（中性粒细胞为70至120Kd，而Jurkat T细胞为70Kd）。此外，在活化中性粒细胞的上清液中检测到了亮氨酸-8分子。这些结果表明，人类中性粒细胞表达一种高分子量形式的亮氨酸-8分子，其具有传统的跨膜锚定，并且在激活过程中迅速从膜上释放。激活过程中亮氨酸-8膜糖蛋白的丢失可能是中性粒细胞黏附特性快速改变的一种机制。
