IDENTIFICATION OF A TRANSITIONAL PREAPOPTOTIC POPULATION OF THYMOCYTES

Apoptosis, a major form of cell death in the immune system, is charact erized by cell shrinkage, chromatin condensation, and cleavage into nu cleosomal fragments. Apoptosis may be the mechanism for the eliminatio n of autoreactive and unselected CD4+ CD8+ thymocytes in the thymus. A large number of diverse agents are capable of inducing apoptosis in i mmature thymocytes. Rat thymocytes were treated with etoposide, a DNA topoisomerase II reactive agent, or dexamethasone, a glucocorticoid, a nd separated on discontinuous Percoll gradients. We have identified an d isolated a transitional preapoptotic population of thymocytes that e xhibited early morphologic and biochemical changes associated with apo ptosis. These preapoptotic cells were intermediate in size and density between normal and apoptotic thymocytes and exhibited a decreased sur face expression of both CD4 and CD8 molecules compared to control thym ocytes. On ultrastructural examination, they were shown to possess sha rply defined clumps of condensed chromatin abutting onto the nuclear m embrane. These morphologic changes, the first detectable signs of apop tosis, occurred prior to the internucleosomal cleavage of DNA, often r egarded as the biochemical hallmark of apoptosis. Nucleosomal fragment s of 180 to 200 base pairs or multiples thereof were, however, detecte d following subsequent dramatic changes in the nuclear structure of th ese preapoptotic cells that resulted in morphology typical of apoptosi s. These results suggest that early critical events in apoptosis prece de internucleosomal cleavage of DNA.