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, result s 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 ge nerally thought to be the most important mediators of morphological ch anges during metamorphosis, corticosteroid hormones (CH) have also bee n shown to accelerate metamorphic changes. We have been studying the p ossible role of CH as effecters of changes in the immune system at met amorphosis. Because CH induce apoptosis of developing murine thymocyte s, we examined in vitro levels of spontaneous and CH-inducible apoptos is of lymphocyte populations removed from the thymus and spleen of tad poles before metamorphosis, during the period of naturally elevated co rticosteroids at the climax of metamorphosis, and from postmetamorphic adults. We show here that spontaneous apoptosis of splenocytes or thy mocytes 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 nor inc reased at metamorphosis. Apoptosis induced by culture of lymphocytes f or 24 h at 26 degrees C in 10 nM corticosterone (well below the peak l evel of 70 nM found at climax of metamorphosis) ranges from about 30-5 0% in the splenocyte population and 55-70% in the thymocyte population . Using the corticosteroid hormone receptor antagonist, RU486, we sepa rated the CH-dependent component of apoptosis from apoptosis due to ot her factors. In the spleen, about 12-23% of lymphocytes are susceptibl e 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 apoptosi s 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 map result fr om loss of ''survival signals'' due to the disruption of the thymic mi croenvironment. These studies support the hypothesis that naturally el evated levels of endogenous free CH delete a significant proportion of the larval splenocyte population during climax of metamorphosis by in duction of apoptosis. This clearing of lymphocytes may prevent destruc tive autoimmune responses to the new set of adult-specific antigens th at emerges at metamorphosis. (C) 1997 Academic Press.