The glucocorticoid receptor 1A3 promoter correlates with high sensitivity to glucocorticoid-induced apoptosis in human lymphocytes.

Glucocorticoids (GCs) are powerful inhibitors of inflammation and immunity. Although glucocorticoid-induced cell death (GICD) is an important part of GCs actions, the cell types and molecular mechanisms involved are not well understood. Untranslated exon 1A3 of the human glucocorticoid receptor (GR) gene is a major determinant of GICD in GICD-sensitive human cancer cell lines, operating to dynamically upregulate GR levels in response to GCs. We measured the GICD sensitivity of freshly isolated peripheral blood mononuclear cells and thymocytes to dexamethasone in vitro, relating this to GR exon 1A3 expression. A clear GICD sensitivity hierarchy was detected: B cells>thymocytes/natural killer (NK) cells>peripheral T cells. Within thymocyte populations, GICD sensitivity decreased with maturation. Interestingly, NK cell subsets were differentially sensitive to GICD, with CD16(+)CD56(int) (cytotoxic) NK cells being highly resistant to GICD, whereas CD16(-)CD56(hi) (cytokine producing) NK cells were highly sensitive (similar to B cells). B-cell GICD was rescued by co-culture with interleukin-4. Strikingly, although no significant increases in GR protein were observed during 48 h of culture of GICD-sensitive and -resistant cells alike, GR 1A3 expression was increased over pre-culture levels in a manner directly proportional to the GICD sensitivity of each cell type. Accordingly, this is the first evidence that the GR exon 1A3 promoter is differentially regulated during thymic development and maturation of human T cells. Furthermore, human peripheral blood B cells are exquisitely GICD-sensitive in vitro, giving new insight into how GCs may downregulate immunity. Collectively, these data show that GR 1A3 expression is tied with GICD sensitivity in human lymphocytes, underscoring the potential for GR 1A3 expression to be used as a biomarker for sensitivity to GICD.

Glucocorticoid-mediated repression of T-cell receptor signalling is impaired in glucocorticoid receptor exon 2-disrupted mice.

Studies using glucocorticoid receptor exon 2-disrupted knockout (GR2KO) mice provided strong evidence against an obligatory role for glucocorticoid receptor (GR) signalling in T-cell selection. These mice express a truncated form of the GR that is incapable of transmitting a range of glucocorticoid (GC)-induced signals, including GC-induced thymocyte death. However, one study that suggested that truncated GR function is preserved in the context of GR-mediated repression of T-cell activation-induced genes, challenged earlier conclusions derived from the use of these mice. Because GR versus T-cell receptor (TCR) signalling cross-talk is the means by which GCs are hypothesized to have a role in T-cell selection, we reassessed the utility of GR2KO mice to study the role of the GR in this process. Here, we show that GR-mediated repression of TCR signalling is impaired in GR2KO T cells in terms of TCR-induced activation, proliferation and cytokine production. GC-induced apoptosis was largely abolished in peripheral T cells, and induction of the GC-responsive molecule, interleukin-7 receptor, was also severely reduced in GR2KO thymocytes. Together, these data strongly re-affirm conclusions derived from earlier studies of these mice that the GR is not obligatory for normal T-cell selection.

Nfil3 is a glucocorticoid-regulated gene required for glucocorticoid-induced apoptosis in male murine T cells.

Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4(+)CD8(+) double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development.

Glucocorticoids play a vital role in fetal respiratory development and act via the intracellular glucocorticoid receptor (GR) to regulate transcription of key target genes. GR-null mice die at birth due to respiratory dysfunction associated with hypercellularity and atelectasis. To identify events associated with this lung phenotype we examined perinatal cellular proliferation rates and apoptotic indices. We demonstrate that compared to wild-type controls, day 18.5 postcoitum (p.c.) GR-null mouse lungs display significantly increased cell proliferation rates (1.8-fold P < 0.05) and no change in apoptosis. To examine underlying molecular mechanisms, we compared whole genome expression profiles by microarray analysis at 18.5 days p.c. Pathways relating to cell proliferation, division and cell cycle were significantly down-regulated while pathways relating to carbohydrate metabolism, kinase activities and immune responses were significantly up-regulated. Differential levels of gene expression were verified by quantitative-RT-PCR and/or Northern analysis. Key regulators of proliferation differentially expressed in the lung of 18.5 p.c. GR-null lungs included p21 CIP1 (decreased 2.9-fold, P < 0.05), a negative regulator of the cell cycle, and Mdk (increased 6.0-fold, P < 0.05), a lung growth factor. The more under-expressed genes in 18.5 p.c. GR-null lungs included Chi3l3 (11-fold, P < 0.05), a macrophage inflammatory response gene and Ela1 (9.4-fold, P < 0.05), an extracellular matrix remodeling enzyme. Our results demonstrate that GR affects the transcriptional status of a number of regulatory processes during late fetal lung development. Amongst these processes is cell proliferation whereby GR induces expression of cell cycle repressors while suppressing induction of a well characterized cell cycle stimulator.

Expression of the glucocorticoid receptor from the 1A promoter correlates with T lymphocyte sensitivity to glucocorticoid-induced cell death.

Glucocorticoid (GC) hormones cause pronounced T cell apoptosis, particularly in immature thymic T cells. This is possibly due to tissue-specific regulation of the glucocorticoid receptor (GR) gene. In mice the GR gene is transcribed from five separate promoters designated: 1A, 1B, 1C, 1D, and 1E. Nearly all cells express GR from promoters 1B-1E, but the activity of the 1A promoter has only been reported in the whole thymus or lymphocyte cell lines. To directly assess the role of GR promoter use in sensitivity to glucocorticoid-induced cell death, we have compared the activity of the GR 1A promoter with GC sensitivity in different mouse lymphocyte populations. We report that GR 1A promoter activity is restricted to thymocyte and peripheral lymphocyte populations and the cortex of the brain. The relative level of expression of the 1A promoter to the 1B-1E promoters within a lymphocyte population was found to directly correlate with susceptibility to GC-induced cell death, with the extremely GC-sensitive CD4+CD8+ thymocytes having the highest levels of GR 1A promoter activity, and the relatively GC-resistant alphabetaTCR+CD24(int/low) thymocytes and peripheral T cells having the lowest levels. DNA sequencing of the mouse GR 1A promoter revealed a putative glucocorticoid-response element. Furthermore, GR 1A promoter use and GR protein levels were increased by GC treatment in thymocytes, but not in splenocytes. These data suggest that tissue-specific differences in GR promoter use determine T cell sensitivity to glucocorticoid-induced cell death.

TCR-mediated activation promotes GITR upregulation in T cells and resistance to glucocorticoid-induced death.

T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.

Analysis of the mobility of signaling molecules in lymphocytes using fluorescence photobleaching techniques.

The earliest biochemical events at the plasma membrane that lead to gene activation appear to depend not only on the local concentration of signaling molecules, but also on the mobility of these molecules at the site of signaling. To elucidate the process of signal transduction after receptor engagement in the immune system, it is important to analyze the mobility of signaling molecules in living lymphocytes. Current knowledge of the changes in intracellular localization and dynamic movements of signaling molecules during lymphocyte activation is limited. Here, we describe a method for known as fluorescence recovery after photobleaching, used to measure the diffusion mobility of a signaling molecule in a T cell line after T cell receptor stimulation. This method is a useful tool in studies of spatiotemporal regulation in immunoreceptor signaling.

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

Glucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice.

The involvement of glucocorticoid receptor (GR) signaling in T cell development is highly controversial, with several studies for and against. We have previously demonstrated that GR(-/-) mice, which usually die at birth because of impaired lung development, exhibit normal T cell development, at least in embryonic mice and in fetal thymus organ cultures. To directly investigate the role of GR signaling in adult T cell development, we analyzed the few GR(-/-) mice that occasionally survive birth, and irradiated mice reconstituted with GR(-/-) fetal liver precursors. All thymic and peripheral T cells, as well as other leukocyte lineages, developed and were maintained at normal levels. Anti-CD3-induced cell death of thymocytes in vitro, T cell repertoire heterogeneity and T cell proliferation in response to anti-CD3 stimulation were normal in the absence of GR signaling. Finally, we show that metyrapone, an inhibitor of glucocorticoid synthesis (commonly used to demonstrate a role for glucocorticoids in T cell development), impaired thymocyte development regardless of GR genotype indicating that this reagent inhibits thymocyte development in a glucocorticoid-independent fashion. These data demonstrate that GR signaling is not required for either normal T cell development or peripheral maintenance in embryonic or adult mice.