In vitro studies of spontaneous and corticosteroid-induced apoptosis of lymphocyte populations from metamorphosing frogs/RU486 inhibition.


Metamorphosis in the South African clawed frog, Xenopus laevis, results in significant changes in the immune system. It is characterized by a striking involution of the thymus and spleen followed by lymphocyte expansion in the postmetamorphic period. While thyroid hormones are generally thought to be the most important mediators of morphological changes during metamorphosis, corticosteroid hormones (CH) have also been shown to accelerate metamorphic changes. We have been studying the possible role of CH as effectors of changes in the immune system at metamorphosis. Because CH induce apoptosis of developing murine thymocytes, we examined in vitro levels of spontaneous and CH-inducible apoptosis of lymphocyte populations removed from the thymus and spleen of tadpoles before metamorphosis, during the period of naturally elevated corticosteroids at the climax of metamorphosis, and from postmetamorphic adults. We show here that spontaneous apoptosis of splenocytes or thymocytes measurable at the time of sacrifice or after culture for 24 h at 4 degrees C is very low at all stages of development and is not increased at metamorphosis. Apoptosis induced by culture of lymphocytes for 24 h at 26 degrees C in 10 nM corticosterone (well below the peak level of 70 nM found at climax of metamorphosis) ranges from about 30-50% in the splenocyte population and 55-70% in the thymocyte population. Using the corticosteroid hormone receptor antagonist, RU486, we separated the CH-dependent component of apoptosis from apoptosis due to other factors. In the spleen, about 12-23% of lymphocytes are susceptible to corticosteroid-induced apoptosis at all larval stages as well as during climax of metamorphosis as measured by this short term culture assay. Another approximately 15% of cells undergo spontaneous apoptosis which is independent of CH. Although dissociated thymocytes exhibit very high levels of apoptosis (55-75%) during culture at 26 degrees C for 24 h, most of the apoptosis is independent of CH and may result from loss of "survival signals" due to the disruption of the thymic microenvironment. These studies support the hypothesis that naturally elevated levels of endogenous free CH delete a significant proportion of the larval splenocyte population during climax of metamorphosis by induction of apoptosis. This clearing of lymphocytes may prevent destructive autoimmune responses to the new set of adult-specific antigens that emerges at metamorphosis.