DNA FRAGMENTATION IS NOT THE PRIMARY EVENT IN GLUCOCORTICOID-INDUCED THYMOCYTE DEATH IN-VIVO

Thymocyte death has been recognized as one of the best models for stud ying apoptosis. Our recent study, however, indicated that most thymocy tes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling-positive (TU NEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, i mmunohistochemistry, and transmission electron microscopy as well as f low cytometry to examine in vivo the effect of glucocorticoid (GC), a well-known agent for inducing thymocyte apoptosis in vitro, on thymocy te death to determine whether or not DNA fragmentation was the first e vent 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 showe d that the TUNEL+ thymocytes were phagocytosed by acid phosphatase(+) and Mac-2(+) macrophages. An ultrastructural study indicated that a fa r greater number of small pyknotic thymocytes were present in the cort ex 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+ t hymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These resu lts indicate that, consistent with our previous findings with normal t hymocyte death and B cell death in the germinal centers, DNA fragmenta tion is not involved in the cell death process of the GC-induced rapid thymocyte death in vivo.