In Vivo Resistance to Glucocorticoid-Induced Apoptosis in Rat Thymocytes with Normal Steroid Receptor Function in Vitro

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Abstract
Previous studies have shown that although the majority of rat thymic lymphocytes are sensitive to glucocorticoid-induced apoptosis in vivo, a small population of mature thymic lymphocytes remains even after high dose steroid administration. Here, we describe ex- periments that were performed to understand the molecular basis of the resistance of these cells to glucocorticoid-induced apoptosis. Adre- nalectomized rats were treated for 72 h with a bolus dose (5 mg/kg body weight) of dexamethasone to produce a population of thymocytes that survived glucocorticoid administration. Reinjection of these an- imalswithequivalentdosesofdexamethasonefailedtoinducefurther thymic regression or apoptosis in these cells. Glucocorticoid receptor numberandreceptorbindingaffinityfordexamethasoneweresimilar in control and resistant thymocytes. Western blot analysis using epitope-purified antiglucocorticoid receptor antibodies confirmed this observation. To delineate the mechanism of resistance, we evaluated whether cells resistant to dexamethasone in vivo showed any re- sponse to this glucocorticoid in vitro. The ability of glucocorticoid to inhibit (3H)lysine incorporation into protein in cells treated with dexamethasoneinvitrowasequivalenttocontrolcells,indicatingthat glucocorticoid receptor function was normal in both populations. To evaluate whether in vivo glucocorticoid-resistant thymocytes retain any capacity to undergo apoptosis,in vitrostudies were performed on these cells using the calcium ionophore A23187 to induce pro- grammed cell death. Cleavage of chromatin into 30- to 50-kilobase fragments or oligonucleosomal fragments characteristic of apoptosis was observed in both sensitive and resistant thymocytes treated in vitro with A23187. Cells resistant to glucocorticoid in vivo unexpect- edly exhibited internucleosomal cleavage of chromatin and apoptosis in response to dexamethasonein vitro. We examined the levels of the apoptosis suppressor Bcl-2 in thymocytes isolated from control and 72 h dexamethasone-treated rats to determine whether increased expression of this protein could explain the resistance to glucocorti- coid-induced apoptosis that we observed. Both glucocorticoid-sensi- tive and -resistant thymocytes expressed similar levels of Bcl-2. To- gether, these data indicate that resistance to glucocorticoid in vivo is not due to alteration of the glucocorticoid receptor or to expression of Bcl-2, but rather to some endogenous thymic factor and/or cell-to-cell contact that probably alters glucocorticoid receptor signaling. (En- docrinology 138: 810-818, 1997)

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