Nakamura M, Yagi H, Ishii T, Kayaba S, Soga H, Gotoh T, Ohtsu S, Ogata M, Itoh T
Department of Anatomy, Tohoku University School of Medicine, Sendai, Japan.
Eur J Immunol. 1997 Apr;27(4):999-1004. doi: 10.1002/eji.1830270429.
Thymocyte death has been recognized as one of the best models for studying apoptosis. Our recent study, however, indicated that most thymocytes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling-positive (TUNEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, immunohistochemistry, and transmission electron microscopy as well as flow cytometry to examine in vivo the effect of glucocorticoid (GC), a well-known agent for inducing thymocyte apoptosis in vitro, on thymocyte death to determine whether or not DNA fragmentation was the first event of GC-induced thymocyte death. At 2 h and 4 h after GC injection, a large number of cortical thymocytes were TUNEL+. Most TUNEL+ cells were aggregated to form clusters. Double staining of the section showed that the TUNEL+ thymocytes were phagocytosed by acid phosphatase+ and Mac-2+ macrophages. An ultrastructural study indicated that a far greater number of small pyknotic thymocytes were present in the cortex of the GC-treated thymus than were observed in the control thymus, that all those pyknotic thymocytes were TUNEL-, and moreover, that at the electron microscopic level, TUNEL+ cells were all phagocytosed by macrophages. Flow cytometric analysis did not detect a single TUNEL+ thymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These results indicate that, consistent with our previous findings with normal thymocyte death and B cell death in the germinal centers, DNA fragmentation is not involved in the cell death process of the GC-induced rapid thymocyte death in vivo.
胸腺细胞死亡已被公认为研究细胞凋亡的最佳模型之一。然而，我们最近的研究表明，大多数胸腺细胞在没有DNA片段化的情况下死亡，并且只有在被巨噬细胞吞噬后才会变成末端脱氧核苷酸转移酶（TdT）介导的dUTP-生物素缺口末端标记阳性（TUNEL+）。在本研究中，我们使用TUNEL法、组织化学、免疫组织化学和透射电子显微镜等组织学技术以及流式细胞术，在体内研究糖皮质激素（GC）（一种在体外诱导胸腺细胞凋亡的知名试剂）对胸腺细胞死亡的影响，以确定DNA片段化是否是GC诱导的胸腺细胞死亡的首要事件。在GC注射后2小时和4小时，大量皮质胸腺细胞呈TUNEL+。大多数TUNEL+细胞聚集形成簇。切片的双重染色显示，TUNEL+胸腺细胞被酸性磷酸酶+和Mac-2+巨噬细胞吞噬。超微结构研究表明，与对照胸腺相比，GC处理的胸腺皮质中存在的小固缩胸腺细胞数量要多得多，所有这些固缩胸腺细胞均为TUNEL-，而且在电子显微镜水平上，TUNEL+细胞均被巨噬细胞吞噬。流式细胞术分析即使在GC处理后4小时也未检测到单个TUNEL+胸腺细胞，这表明在DNA片段化后几乎不存在游离的死亡胸腺细胞。这些结果表明，与我们之前关于正常胸腺细胞死亡和生发中心B细胞死亡的发现一致，DNA片段化不参与体内GC诱导的快速胸腺细胞死亡的细胞死亡过程。
