Borisenko Grigory G, Matsura Tatsuya, Liu Shang-Xi, Tyurin Vladimir A, Jianfei Jiang, Serinkan Fatma B, Kagan Valerian E
Department of Environmental and Occupational Health, University of Pittsburgh, 3343 Forbes Avenue, PA 15260, USA.
Arch Biochem Biophys. 2003 May 1;413(1):41-52. doi: 10.1016/s0003-9861(03)00083-3.
Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.
磷脂酰丝氨酸（PS）在细胞中主要局限于质膜的内小叶，但在细胞凋亡过程中会外化到细胞表面。这种外化的PS是巨噬细胞有效吞噬凋亡细胞所必需的。由于PS跨膜不对称性在不同类型的非凋亡细胞中并非绝对，我们推测外化PS的量可能是巨噬细胞区分凋亡细胞和非凋亡细胞的关键。我们开发了一种灵敏的电子顺磁共振方法，基于顺磁性膜联蛋白V-微珠缀合物与细胞表面PS的特异性结合来定量外化PS的量。使用该技术，我们发现非凋亡的Jurkat细胞外化0.9皮摩尔内源性PS/10⁶个Jurkat细胞。对于表面整合有不同量外源性PS的细胞，当PS水平<5皮摩尔/10⁶个Jurkat细胞时未观察到吞噬反应；在较高的PS浓度下，吞噬作用以浓度依赖的方式增加。Jurkat细胞凋亡导致约240皮摩尔PS/10⁶个Jurkat细胞外化；这些外化PS的量比吞噬所需的PS阈值量高许多倍。因此，巨噬细胞对细胞表面外化的PS有一个敏感性阈值，这使得巨噬细胞能够可靠地识别和区分外化PS含量低的正常细胞和PS水平显著升高的凋亡细胞。
