DNA fragmentation is not the primary event in glucocorticoid‐induced thymocyte death in vivo

Abstract

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.

Glucocorticoid-Induced Thymocyte Death in the Murine Thymus: The Effect at Later Stages.

“…4). Flow cytometric analysis that revealed virtually no TUNEL-positive cells in the glucocorticoid-treated thymus even 4h after the injection was consistent with findings on the early stage effects of glucocorticoid on murine thymocyte viability as described elsewhere (NAKAMURA et al, 1997), as well as in the normal untreated thymus (Fig. 5a, b) (NAKAMURA et al, 1995).…”

Phosphatidylserine, a death knell

“…Indeed, the bulk of autodigestion occurs after apoptotic cells have been safely segregated and are already being digested within a phagolysosome. 6,7 Autodigestion alone, without phagocytosis, can leave corpses lying about. 8 In organisms such as mammals, where morphological, neural and immunological development depends heavily on the elimination of unwanted cells, and where tissue inflammation is a risk of uncontrolled cell lysis, the mechanisms of apoptotic cell recognition and removal are of critical physiological importance.…”

Decreased Levels of Recent Thymic Emigrants in Peripheral Blood of Simian Immunodeficiency Virus-Infected Macaques Correlate with Alterations within the Thymus

“…However, it is likely that the macrophages in the thymus may be becoming TUNEL-positive due to their role as scavengers responsible for removal of the apoptotic thymocytes. In mice, thymic macrophages have been demonstrated to acquire low-molecularweight DNA from the apoptotic thymocytes and become TUNEL positive (35,57). The increased level of apoptotic T cells within the thymic cortex is unlikely to be directly due to viral infection.…”

“…9B). These macro- (35,57). Although many of the TUNEL-positive cells were located among the immature thymocytes in the cortex, apoptotic cells were also observed within the thymic medulla.…”