IN-VIVO RESISTANCE TO GLUCOCORTICOID-INDUCED APOPTOSIS IN RAT THYMOCYTES WITH NORMAL STEROID-RECEPTOR FUNCTION IN-VITRO

Previous studies have shown that although the majority of rat thymic l ymphocytes are sensitive to glucocorticoid-induced apoptosis in vivo, a small population of mature thymic lymphocytes remains even after hig h dose steroid administration. Here, we describe experiments that were performed to understand the molecular basis of the resistance of thes e cells to glucocorticoid-induced apoptosis. Adrenalectomized rats wer e treated for 72 h with a bolus dose (5 mg/kg body weight) of dexameth asone to produce a population of thymocytes that survived glucocortico id administration. Reinjection of these animals with equivalent doses of dexamethasone failed to induce further thymic regression or apoptos is in these cells. Glucocorticoid receptor number and receptor binding affinity for dexamethasone were similar in control and resistant thym ocytes. Western blot analysis using epitope-purified antiglucocorticoi d receptor antibodies confirmed this observation. To delineate the mec hanism of resistance, we evaluated whether cells resistant to dexameth asone in. vivo showed any response to this glucocorticoid in vitro. Th e ability of glucocorticoid to inhibit [H-3]lysine incorporation into protein in cells treated with dexamethasone in vitro was equivalent to control cells, indicating that glucocorticoid receptor function was n ormal in both populations. To evaluate whether in vivo glucocorticoid- resistant thymocytes retain any capacity to undergo apoptosis, in vitr o studies were performed on these cells using the calcium ionophore A2 3187 to induce programmed cell death. Cleavage of chromatin into 30- t o 60-kilobase fragments or oligonucleosomal fragments characteristic o f apoptosis was observed in both sensitive and resistant thymocytes tr eated in vitro with A23187. Cells resistant to glucocorticoid in vivo unexpectedly exhibited internucleosomal cleavage of chromatin and apop tosis in response to dexamethasone in vitro. We examined the levels of the apoptosis suppressor Bcl-2 in thymocytes isolated from control an d 72 h dexamethasone-treated rats to determine whether increased expre ssion of this protein could explain the resistance to glucocorticoid-i nduced apoptosis that we observed. Both glucocorticoid-sensitive and - resistant thymocytes expressed similar levels of Bcl-2. Together, thes e data indicate that resistance to glucocorticoid in vivo is not due t o alteration of the glucocorticoid receptor or to expression of Bcl-2, but rather to some endogenous thymic factor and/or cell-to-cell conta ct that probably alters glucocorticoid receptor signaling.