Although glucocorticoid has been considered to cause thymocyte apoptosis in vitro, few studies have presented its in vivo effect. We report here on kinetics of glucocorticoid-induced murine thymocyte death in vivo by the TUNEL method. TUNEL-positive cells were observed as early as at 2 h after intraperitoneal injection of glucocorticoid. Most TUNEL-positive thymocytes were phagocytosed by acid phosphatase positive macrophages. 'Free' (not phagocytosed) TUNEL-positive cells were not detected at early stages (by 4 h). At 6 to 8 h after the injection, the number of phagocytosed thymocytes per individual macrophage had reached its maximum, and at 8 to 12 h many ruptured macrophages ingesting too many dying thymocytes became noticeable. During the process, no additional macrophages appeared to be mobilized to the thymus. At 6 to 8 h after the injection, however, coincidentally with the fact that macrophages had become unable to further ingest dying lymphocytes, dead cells were left unphagocytosed, and ultimately became 'free' positive cells, probably due to some proteolytic process ongoing within the thymus. As late as at 12 h, morphological examination revealed that epithelial cells seemed to begin engulfing thymocytes, almost simultaneously with the start of rupture of the macrophages due to the ingestion of too many thymocytes. Epithelial cells were readily identified by desmosomes and tonofilaments, in addition to euchromatic nuclei. Altogether, these results suggest that: 1) even though thymocytes were exposed to glucocorticoid in vivo, most of them were not TUNEL-positive unless they were phagocytosed; 2) even after most macrophages had ingested too many cells at later stages, macrophages in other locations did not migrate to the thymus; and finally, 3) deletion of damaged thymocytes was also carried out by thymic epithelial cells, though not frequently, at around 12 h and later.
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