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The Journal of Immunology, 1999, 162: 314-322.
Copyright © 1999 by The American Association of Immunologists

A NF-{kappa}B/c-myc-Dependent Survival Pathway Is Targeted by Corticosteroids in Immature Thymocytes1

Weihong Wang*, Joanna Wykrzykowska{dagger}, Todd Johnson{dagger}, Ranjan Sen* and Jyoti Sen2,{dagger}

* Rosenstiel Research Center and Department of Biology, Brandeis University, Waltham, MA 02254; and {dagger} Dana-Farber Cancer Institute, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid hormones modulate T cell maturation in vivo. While low levels of hormones are required for appropriate T cell development, high levels of glucocorticoid hormones target immature developing thymocytes for cell death during systemic stress. In this report, we propose a molecular mechanism for the induction of apoptosis in CD4+CD8+ double-positive thymocytes by dexamethasone in vivo. Dexamethasone injection induced the expression of I{kappa}B{alpha} and I{kappa}Bß in thymocytes and down-regulated NF-{kappa}B DNA binding activated by intrathymic signals. Down-regulation of NF-{kappa}B DNA binding preceded cell death, suggesting that NF-{kappa}B may be important for the survival of immature thymocytes. In addition, ex vivo treatment of thymocyte single-cell suspension with dexamethasone accelerated p65/RelA down-regulation and cell death. Conversely, NF-{kappa}B induction diminished dexamethasone-induced death. Expression of the c-myc proto-oncogene, a NF-{kappa}B target, was also reduced in thymocytes of dexamethasone-treated animals, and ectopic transgenic expression of c-myc in mice provided partial rescue of double-positive thymocytes from dexamethasone mediated cell death. These observations suggest that viability of CD4+CD8+ thymocytes may be maintained by an NF-{kappa}B/c-myc-dependent pathway in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of T cells occurs in the inductive environment of the thymus. Signals provided by the thymic microenvironment can be broadly divided into two categories: developmental signals and survival signals. Developmental signals are those that result in the differentiation of the cell from one stage to the next. For example, the most immature precursor cells have the potential to differentiate into B or T lymphocytes. However, this multipotency is lost soon after migration to the thymus. Thus, one of the earliest developmental signals that thymocytes receive commit them to further differentiate along the T lineage. Subsequently, functional rearrangement of TCR ß-chain genes induces differentiation to the CD4+CD8+ double-positive (DP)3 stage of T cell development followed by rearrangement and expression of TCR {alpha}-chain. DP thymocytes expressing TCR-{alpha}ß are subsequently subject to positive and negative selection signals that are likely to be provided by interaction of thymocytes with stromal cells in the thymus (1, 2). Survival signals are those that keep thymocytes alive for a defined time period to receive developmental signals. At the end of this period, the bulk of thymocytes die due to neglect. Clearly, it is critical to coordinate both kinds of signals for appropriate T cell development.

The signaling pathways and the molecules that regulate thymocyte survival have not been systematically studied, but are likely to result from interactions of thymocytes with stromal cells and/or locally produced cytokines. We have previously shown that several transcription factors, which are normally induced in response to T cell activation, are present in the nucleus of freshly isolated DP thymocytes. These include NF-{kappa}B and NF of activated T cells (NF-AT) family members and proteins that bind to AP-1 probes (3, 4). Presence of such factors in the nucleus suggests an "activated" state of the DP cells. For these cells, the activated state could reflect survival and/or developmental signals received from the thymic environment. We further showed that disruption of the thymic microenvironment led to loss of DNA binding by these factors. One interpretation of these observations is that intrathymic signals induce and maintain these factors in a nuclear, DNA-binding form, and disruption of such signals in vitro leads to the down modulation of DNA binding.

An activated phenotype of freshly isolated DP thymocytes was proposed several years ago by Nakayama et al. (5) when they found that tyrosine phosphorylation of several proteins, including TCR-associated {zeta}-chain, was constitutively high in these cells. Interestingly, {zeta}-chain phosphorylation was reduced upon incubation of thymocytes in vitro, similar to our observation with NF. More recently, we have found that the p38 mitogen-activated protein (MAP) kinase pathway is also activated in thymocytes by intrathymic signals and is down-regulated upon disruption of these signals in vitro (6). These observations suggest that specific cytoplasmic signaling pathways and inducible NF mediate intrathymic signals in thymocytes. However, these studies do not provide an indication as to the function of these pathways and factors in the thymus.

A classical observation in T cell biology has been the extreme susceptibility of immature DP thymocytes to corticosteroid-induced cell death (7, 8). Indeed, thymocyte apoptosis occurs even when endogenous glucocorticoid levels are elevated during systemic stress (9). Recent studies have further highlighted the importance of corticosteroids in the thymus. Ashwell and colleagues showed that thymic glucocorticoid production is essential for appropriate T cell development (10, 11) and that down-modulation of glucocorticoid receptor (GR) activity in developing T cells significantly reduced thymic cellularity (12). They proposed that increased apoptosis as well as decreased generation of DP cells contributed to the resulting phenotype. Thus, glucocorticoids significantly alter the development and maintenance of the immune response of an individual. However, the mechanism of action and the basis for the observed thymocyte subset-specificity of glucocorticoid action are not known.

We noted several apparently disparate observations that could explain the observed sensitivity of DP thymocytes to corticosteroids. First, NF-{kappa}B proteins have been shown to protect cells from death induced by TNF-{alpha}, chemotherapeutic agents (13, 14, 15), and oncogenic ras (16). Furthermore, in the immature B cell line, WEHI 231, anti-Ig-induced apoptosis correlated with decreased NF-{kappa}B activity after prolonged stimulation (17, 18). Second, glucocorticoids have been shown to decrease NF-{kappa}B function in several cell lines. This is the result of reduced NF-{kappa}B induction (19, 20) as well as inhibition of NF-{kappa}B function directly by the GR (21). Third, we observed that DP thymocytes contain NF-{kappa}B that is apparently activated by intrathymic signals. We reasoned that if intrathymically activated NF-{kappa}B was a part of the DP, but not CD4-CD8- double-negative (DN) thymocytes or the CD4+ or CD8+ single-positive (SP) cell viability program, then corticosteroids may target this subpopulation by down-regulating NF-{kappa}B in vivo.

In experiments reported in this paper, we tested the predictions of this hypothesis. We found that dexamethasone injection down-regulated intrathymically activated NF-{kappa}B. Down-regulation of NF-{kappa}B DNA binding preceded significant cell death in the DP thymocyte population and was accompanied by increased expression of I{kappa}B{alpha} and I{kappa}Bß. Ex vivo treatment of thymocyte single-cell suspension with dexamethasone accelerated p65/RelA down-regulation and cell death, whereas NF-{kappa}B activation by phorbol ester (PMA) diminished dexamethasone-induced death. As a possible NF-{kappa}B target in thymocytes, we examined the expression of the c-myc proto-oncogene, which has been shown to protect WEHI 231 cells from anti-Ig-induced cell death (17, 18). c-myc is expressed at high levels in DN thymocytes, at lower levels in DP thymocytes and at intermediate levels in SP thymocytes (22). c-Myc protein expression was further down-regulated by dexamethasone treatment, with a time course slower than that observed for NF-{kappa}B. A role for c-myc was further substantiated because DP thymocytes from c-myc transgenic mice were less susceptible to dexamethasone-induced death. Lastly, we showed that dexamethasone-induced effects circumvented, but did not inhibit several known signaling pathways operative in thymocytes. Our observations suggest that NF-{kappa}B/c-myc-dependent pathway maintains DP cell viability, and that disruption of these signals by corticosteroids contribute to the extreme sensitivity of these cells to dexamethasone-induced death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c mice were bred and maintained at the pathogen-free facility at the Dana-Farber Cancer Institute (Boston, MA). BALB/c mice carrying the mouse mammary tumor virus (MMTV)-c-myc transgene (23) were purchased from Charles River Laboratories (Wilmington, MA).

Wild-type or transgenic mice were injected i.v. with 2.5 mg dexamethasone (Sigma, St. Louis, MO) in PBS or PBS alone, as indicated. Thymuses were removed at various times after injection and processed for flow cytometry or extract preparation as necessary.

Flow cytometry

Thymocyte single-cell suspension (4) were stained with anti-CD4 (PharMingen, San Diego, CA) and anti-CD8 Abs coupled to fluorescein and phycoerythrin. Cell death was analyzed by propidium iodide (PI) exclusion (5 µg/ml) and by staining with annexin coupled to fluorescein.

Extracts and protein assays

Electrophoretic mobility-shift assay (EMSA). Thymocyte nuclear extracts were prepared as previously described (3). Probes used for EMSA were the H2K {kappa}B element, and the serum response factor (SRF) and SP-1 sites from the IL-2R {alpha}-chain gene promoter. All probes and conditions for EMSA have been previously described (4).

Immunoblotting. For I{kappa}B Western blots, 107 cells were lysed with 30 µl TNT buffer (20 mM Tris-HCl 7.5, 200 mM NaCL, 1% Triton X-100, 1 mM PMSF, and protease inhibitors leupeptin, aprotinin, pepstatin A, chymostatin, and antipain each at a final concentration of 10 µg/ml). Then, 20 µg protein from each sample was loaded onto the gel. c-myc and SP-1 westerns were done as previously described (18). For detection of phosphoproteins, 3 x 107 cells were lysed with 50 µl lysis buffer (50 mM Tris-HCl 7.6, 150 mM NaCL, 1% Triton X-100, 2 mM PMSF, and NaF, 120 mM Na pyrophosphate, 1 mM Na orthovanadate, 3 µg/ml leupeptin, and 3 µg/ml aprotinin). Then, 60 µg protein was loaded onto the gel. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes and probed with various Abs at the following concentrations. I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA; SC371), 1:1000; I{kappa}Bß (Santa Cruz; SC945), 1:1000; and SP-1 (Santa Cruz; SC59), 1:250. Chemiluminescent detection was conducted using supersignal substrate (Pierce, Rockford, IL) according to the manufacturer’s specification.

mRNA analysis

Total RNA was isolated from thymocytes from normal (BALB/c) or MMTV-c-myc transgenic mice thymocytes using Ultraspec (Biotecx Laboratories, Houston, TX). A total of 3 µg RNA was reverse transcribed (2'-deoxynucleoside 5'-triphosphates 1.5 mM, 20 units avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim)) using random hexanucleotide primers. c-myc-specific sequences were amplified using primers 5'-GCTGGTGCTGTCTTTGCG-3' and 5'-GGC TGG ATT TCC TTT GGG-3'. PCR contained 0.2 mM 2'-deoxynucleoside 5'-triphosphate and Vent polymerase (New England Biolabs, Beverly, MA) in a final volume of 100 µl. We used 25 cycles of 1-min denaturation at 94°C, 1.5-min annealing at 53°C, and 1.5-min elongation. Then, 20 µl of the reaction was electrophoresed through 1% agarose gels and blotted on to nylon membrane (ICN, Aurora, OH), which was probed with a c-myc probe (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of NF-{kappa}B DNA binding in thymocytes by dexamethasone

To investigate the molecular mechanism of dexamethasone-induced death of immature thymocytes, we first characterized the time course of this effect. Thymocytes isolated from uninjected mice or mice injected with dexamethasone, or PBS as control, were labeled with annexin and PI and examined by flow cytometry. Staining thymocytes with annexin (Fig. 1GoA, left panel) suggested that the number of cells targeted for death increased with time, reaching up to 50% by 6 h posttreatment with dexamethasone. However, PI exclusion (Fig. 1GoA, right panel) showed that the majority of these cells were still viable. As expected, 48 h after dexamethasone treatment the thymus contained primarily immature DN thymocytes and mature CD3high SP thymocytes, the immature CD4+CD8+ DP cells having succumbed to the drug (Fig. 1GoB). Based on this time course, we decided to use 6 h as the maximum treatment time for further molecular studies, so as to minimize confusion in the interpretation of the results with events occurring after cell death.



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  		FIGURE 1. Thymocyte loss in dexamethasone-injected mice. A, Time course of dexamethasone-induced thymocyte death. Thymocytes from PBS-injected or dexamethasone-injected mice were stained with annexin and PI as a measure of dead cells. Percentage of cells staining with annexin (left) and PI and annexin and (right) is shown at different time points after PBS or dexamethasone injection. B, Four- to 6-wk-old BALB/c mice were injected with PBS or dexamethasone. After 48 h, thymocyte single-cell suspensions were assayed by flow cytometry after staining with anti-CD3, anti-CD4, and anti-CD8 Abs.

 
We have previously proposed that several members of the NF-{kappa}B family are maintained in an active form in thymocytes by intrathymic signals. To investigate if dexamethasone affected NF-{kappa}B DNA binding, thymocyte nuclear extracts were prepared from mice that had been injected with PBS (as control) or dexamethasone for varying times and examined by EMSA. As expected, thymocyte extracts from PBS-treated mice produced several nucleoprotein complexes with a DNA probe containing a NF-{kappa}B binding site (Fig. 2GoA, lane 1). We have previously shown that all major {kappa}B binding complexes represent sequence-specific DNA/protein interactions by competition assays (3). Thymocyte extracts from mice that had been exposed to dexamethasone for 2 h show a pattern similar to the control (Fig. 2GoA, lane 2). However, longer periods of dexamethasone treatment showed a time-dependent decrease of proteins binding to the NF-{kappa}B probe (Fig. 2GoA, lanes 3 and 4). Thus, DNA binding by intrathymically induced NF-{kappa}B family proteins was down-regulated in thymocytes from dexamethasone-treated mice.



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  		FIGURE 2. DNA-binding proteins in thymocytes from dexamethasone-treated mice. Mice were injected with PBS for 6 h or with dexamethasone for the times indicated, and freshly isolated thymocytes were used to prepare nuclear extracts as previously described. Extracts were used in EMSA with radiolabeled probes containing binding site for NF-{kappa}B (A, labeled H2K), and SRF and SP-1 (B, labeled SRF/SP-1). Multiple sequence-specific complexes were observed on the H2K probe (thin arrows), among which the position of the p50/p65 heterodimer is indicated by the bold arrow in A. Two sequence-specific complexes on the SRF-SP-1 probe are indicated by arrows in B. Representative data are shown from one experiment of three independent sets.

 
Although the binding data shown in Fig. 2GoA were normalized to protein content, we wished to rule out the possibility that decreased DNA-protein complex formation in extracts from thymocytes from mice treated with dexamethasone for 4 h and 6 h was due to nonspecific protein degradation. Therefore, we assayed DNA binding by two other factors, SRF and SP-1, using the same nuclear extracts. Proteins binding to the SRF/SP-1 probe were not affected by dexamethasone treatment (Fig. 2GoB). These data show that dexamethasone treatment of immature thymocytes interferes specifically with intrathymic signals that induce NF-{kappa}B activity in immature thymocytes.

Recently, dexamethasone has been shown to inhibit NF-{kappa}B induction by TNF-{alpha} or anti-CD3 Ab treatment of T cells (19, 20). One of the mechanisms proposed to mediate this effect was the enhanced synthesis of the inhibitory molecule I{kappa}B{alpha}. To investigate whether down-regulation of thymic NF-{kappa}B was also mediated by the I{kappa}Bs, we assayed I{kappa}B{alpha} and ß expression by immunoblotting. Whole cell extracts prepared from thymocytes obtained from dexamethasone-injected mice were fractionated by SDS-PAGE and the proteins were transferred to nitrocellulose filters that were then probed with anti-I{kappa}B{alpha} or anti-I{kappa}Bß anti-serum. Both I{kappa}B{alpha} and ß levels decreased transiently with 2 h of dexamethasone treatment (Fig. 3Go, compare lanes 1 and 4), and then at 4–6 h increased to levels above those seen in the PBS-injected controls (Fig. 3Go, compare lanes 1 to 2 and 3).



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  		FIGURE 3. I{kappa}B protein expression in thymocytes of dexamethasone-treated mice. Mice were injected with PBS (4 h) or dexamethasone for the times indicated, and 20 µg of protein from whole-cell extracts were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-I{kappa}B{alpha} (lower panel) or anti-I{kappa}Bß (upper panel) Abs. Immunoblots were developed using a chemiluminescence assay. Levels of I{kappa}Bs were quantitated and normalized to the level of each I{kappa}B present in untreated thymocytes; I{kappa}B{alpha}: untreated, 1; 2 h dexamethasone (DEX), 0.53; 4 h, 1.28; 6 h, 1.4. I{kappa}Bß: untreated, 1; 2 h, 0.84; 4 h, 2.62; 6 h, 2.7. Data shown are representative of one of three independent experiments.

 
NF-{kappa}B has been shown to be a positive regulator of I{kappa}B{alpha} transcription (25), and NF-{kappa}B function has been shown to be inhibited directly by dexamethasone (21). Therefore, dexamethasone-dependent loss of NF-{kappa}B function may transiently decrease I{kappa}B{alpha} gene expression, resulting in the decrease of I{kappa}B{alpha} protein at early time points. At later times, dexamethasone-induced signals may override the negative effect of decreased NF-{kappa}B activity, resulting in higher amounts of I{kappa}B{alpha} proteins. The mechanism of I{kappa}Bß regulation in thymocytes by dexamethasone is unclear at present. However, it is interesting to note that both proteins responded similarly to signals mediated by dexamethasone. Irrespective of the mechanism of modulation of I{kappa}B expression, our observations suggest that increased I{kappa}B may contribute to NF-{kappa}B down-regulation, particularly after longer periods of treatment with dexamethasone.

Effects of dexamethasone ex vivo

Incubation of thymocyte single-cell suspension at 37°C results in the down-regulation of NF-{kappa}B induced by intrathymic signals and at later times in cell death. Because dexamethasone treatment of thymocytes enhances cell death, it was possible that dexamethasone also affected NF-{kappa}B down-regulation ex vivo. To test this, thymocytes were incubated with, or without, dexamethasone, and the loss of nuclear NF-{kappa}B was monitored by immunoblotting. Decreased nuclear p65/RelA was evident after 3 h incubation (Fig. 4Go, compare lanes 1, 2, 4, and 6), and this was augmented markedly by dexamethasone treatment (Fig. 4Go, lanes 6 and 7). These observations are consistent with those in vivo and suggest that dexamethasone may modulate DP cell viability by decreasing NF-{kappa}B activity.



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  		FIGURE 4. Nuclear p65/RelA expression in thymocytes treated with dexamethasone ex vivo. Nuclear extracts prepared from thymocyte single-cell suspensions treated as indicated above the lanes were fractionated by SDS-PAGE and transferred to nitrocellulose membranes, which were probed with anti-p65 antiserum. The position of the p65 protein is indicated. Lane 1, Thymocytes kept at 0°C; lane 2–7, Thymocytes incubated at 37°C in the absence, or presence, of dexamethasone for the times indicated. Data shown are representative of one of two independent experiments.

 
If NF-{kappa}B activation provides survival signals, then increasing nuclear NF-{kappa}B should decrease cell death. To test this prediction, we activated NF-{kappa}B in thymocytes with PMA and monitored dexamethasone-induced cell death. Dexamethasone treatment alone induced significant cell death compared with untreated thymocytes, which was reduced considerably when PMA was included together with dexamethasone (Fig. 5GoA). NF-{kappa}B activity in these cells was monitored by immunoblotting of nuclear extracts prepared from thymocytes after 6 h treatment. Nuclear p65 was undetectable in untreated or dexamethasone-treated extracts (Fig. 5GoB, lanes 1 and 3), whereas nuclear p65 was evident in PMA or PMA plus dexamethasone-treated cells (Fig. 5GoB, lanes 2 and 4). Taken together with the cell death assays, these observations support the hypothesis that NF-{kappa}B proteins may protect thymocytes from cell death.



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  		FIGURE 5. Effect of PMA on dexamethasone induced thymocyte cell death in vitro. A, Single-cell thymocyte suspensions were incubated with PMA and dexamethasone as indicated. Cells were assessed for viability at the indicated times by trypan blue uptake. Results shown are derived from one experiment performed in triplicate and are representative of two independent experiments. B, Nuclear p65/RelA levels were determined by immunoblotting in thymocytes treated with PMA and dexamethasone for 6 h. Shown are thymocytes incubated at 37°C with no pharmacologic agents (lane 1) and thymocytes treated with PMA alone (lane 2), dexamethasone alone (lane 3), and PMA plus dexamethasone (lane 4). Results shown are from a representative experiment from two independent experiments.

 
Two additional points need to be noted. First, untreated thymocytes contained much less NF-{kappa}B compared with PMA-treated thymocytes at 6 h (Fig. 5GoB), yet the viability of both sets of cells was very similar (Fig. 5GoA). This may be because, in addition to or subsequent to down-regulation of NF-{kappa}B, additional death-inducing mechanisms need to be activated to kill the cells. Dexamethasone not only augments the loss of NF-{kappa}B from the nucleus, but also activates other pathways that culminate in death. However, if a high level of NF-{kappa}B is maintained in the nucleus, then dexamethasone-mediated cell death is reduced. Second, dexamethasone reduced the level of PMA-induced p65/RelA, which may be expected based on earlier observations that NF-{kappa}B induction is inhibited by corticosteroids. This is probably the reason that PMA does not fully rescue the cells from dexamethasone-induced death.

Regulation of c-myc expression in thymocytes by dexamethasone

NF-{kappa}B has been shown to be a positive regulator of c-myc (26, 27). Studies from Sonenshein and colleagues using WEHI 231 cells as a model for anti-Ig-mediated death of immature B cells have shown that c-myc expression protects from cell death (17, 18). Indeed, by stably expressing the c-myc gene in WEHI 231 cells, they were able to show a causal relationship between c-myc expression and protection of WEHI 231 cells from anti-Ig Ab-mediated cell death. Because DP thymocytes and WEHI 231 cells both represent immature lymphocytes that are particularly sensitive to death, it was possible that c-myc was involved in maintaining DP cell viability. We examined whether dexamethasone-mediated down-regulation of NF-{kappa}B affected c-myc expression in immature thymocytes. Whole cell extracts were prepared from PBS or dexamethasone-treated thymocytes according to the procedure of Wu et al. (17, 18) and were assayed by immunoblotting with anti-c-myc Abs. c-Myc protein was easily detected in PBS-injected controls and after 2 or 4 h dexamethasone treatment (Fig. 6Go, lanes 1–3). However, 6 h after dexamethasone injection the levels of c-myc expression were significantly reduced (Fig. 6Go, lane 4). We confirmed equal loading of proteins by probing the same filter with an anti-SP-1 Ab (Fig. 6Go, lower panel). We conclude that dexamethasone treatment down-regulated c-myc expression in thymocytes with a time course that was consistent with this gene being a downstream target of intrathymically induced NF-{kappa}B.



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  		FIGURE 6. Expression of c-myc in thymocytes of dexamethasone-treated mice. Whole-cell extracts were prepared from thymocytes obtained from PBS-injected (6 h) mice or mice injected with dexamethasone for various times. Western blot analysis was as described in Wu et al. (17) using anti-c-myc anti-serum (top panel) or anti-SP-1 anti-serum (bottom panel) used as the normalizing control. Data shown are representative of two independent experiments.

 
These results are consistent with the model that DP cell viability is maintained by target genes such as c-myc that are activated via the NF-{kappa}B pathway. To directly test this model, the effects of dexamethasone treatment were compared in BALB/c mice and BALB/c mice carrying a c-myc transgene regulated by the glucocorticoid-responsive MMTV promoter/enhancer (23). In these mice, dexamethasone treatment was expected to induce c-myc expression and thereby, perhaps, compensate for the drop in c-myc observed in the thymocytes of wild-type mice. We reasoned that inducible c-myc activation would minimize alteration in the cell populations at the start of the experiment, such as might occur with constitutively high c-myc expression. Indeed, 24 h after dexamethasone injection, greater numbers of DP thymocytes remained in MMTV-c-myc mice compared with normal mice (Fig. 7GoA). To determine the status of c-myc expression in the residual DP cells, we assayed c-myc mRNA expression. In wild-type animals, c-myc mRNA was reduced after dexamethasone treatment (Fig. 7GoB, lanes 1 and 2), whereas in the transgenic mice c-myc mRNA was greatly increased (Fig. 7GoB, lanes 3 and 4). Equivalent RNA levels were established by comparing GAPDH expression in the four samples. It is likely that the mRNA levels closely reflect the levels of c-myc protein as suggested by the close correlation between c-myc mRNA and protein levels in nontransgenic thymocytes. Use of less RNA resulted in reduced RT-PCR signals, indicating that we were not in a saturating range in this assay. Increased c-myc expression in DP cells that survived dexamethasone treatment in the transgenic mice supports a role for this gene in the survival of DP thymocytes. The partial rescue of DP thymocytes observed in MMTV-c-myc mice indicated that c-myc expression was not sufficient to abrogate the effects of dexamethasone. This could be because other NF-{kappa}B target genes also contributed to cell survival. Alternatively, it is also possible that transgenic c-myc was not activated in a timely fashion in all DP cells due to integration-site effects or the induction characteristics of the transgene.



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  		FIGURE 7. Dexamethasone treatment of MMTV-c-myc mice. A, BALB/c mice expressing c-myc transgene or nontransgenic BALB/c mice were injected with dexamethasone. Thymocytes were isolated after 24 h and analyzed by flow cytometry after staining with anti-CD4 and anti-CD8 Abs and with PI. Results from several independent experiments (E1-E7; left panel) and the mean of the data from these several experiments (right panel) are shown. Error bars represent the SD from the mean. B, c-myc expression in thymocytes from BALB/c and BALB/c MMTV-c-myc mice. Total thymocyte RNA isolated from normal and MMTV-c-myc mice before and after treatment with dexamethasone was analyzed by RT-PCR as described in Materials and Methods. c-myc RNA was visualized after fractionation of the PCR reaction and transfer to nylon membranes and probing with a radioactive c-myc probe (upper panel). Note that c-myc RNA in untreated MMTV-c-myc mice is not significantly higher than in untreated normal mice. For normalization, the reverse transcription products were amplified with primers specific for GAPDH (lower panel). PCR products for GAPDH were visualized by ethidium bromide staining.

 
Modulation of other signaling pathways in thymocytes by dexamethasone

Dexamethasone may cause down-regulation of the activated NF by interfering with intrathymic signals required for their induction, or by overriding these signals. Although the signals that induce NF-{kappa}B proteins in thymocytes are not known, several cytoplasmic pathways have been shown to be activated in thymocytes by intrathymic interactions. In particular, tyrosine kinases (5) as well as the p38 MAP kinase pathways (6) have been shown to be active in freshly isolated thymocytes. As an indication of whether dexamethasone treatment inhibited intrathymic signaling pathways, we tested for tyrosine-phosphorylated proteins and p38 MAP kinase activity in thymocytes from PBS or dexamethasone-injected mice. Tyrosine-phosphorylated proteins were assayed by separating total cell proteins by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine Ab. In PBS-injected control animals, or animals injected with dexamethasone for 2, 4, or 6 h, the levels of tyrosine-phosphorylated proteins were comparable (Fig. 8GoA). This observations suggests that dexamethasone did not inhibit the activation of tyrosine kinases.



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  		FIGURE 8. Intrathymic signaling pathways in dexamethasone-treated mice. A, Analysis of tyrosine kinase activity in thymocytes from PBS- or dexamethasone-treated mice. Proteins from whole-cell extracts were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-phospho-tyrosine mAb RC-20. Immunoblot was developed using chemiluminescent reagents. B, Analysis of the p38 MAP kinase pathway in dexamethasone-treated mice. Extracts were prepared from uninjected mice (lanes 1–3), and PBS- or dexamethasone-injected mice (lanes 4–7). Lane 1, Freshly isolated thymocytes. Lane 2, Thymocytes that had been incubated as single-cell suspension at 37°C for 3 h. Lane 3, Single-cell thymocytes suspension that were maintained at 37°C for 2 h 45 min and treated with PMA and calcium ionophore for an additional 15 min. Lane 4, PBS-injected animals (6 h). Lanes 5–7, Fresh thymocytes from mice that had been injected with dexamethasone for 2, 4, and 6 h, respectively. Proteins were fractionated by SDS-PAGE and transferred to nitrocellulose membranes, which were probed with anti-phospho-p38 MAP kinase Ab. Data shown are representative of one of two independent experiments.

 
The p38 MAP kinase is activated by specific tyrosine phosphorylation in the Thr-Gly-Tyr sequence (28). Active p38 MAP kinase activity was assayed by immunoblotting with Abs recognizing only the phosphorylated enzyme. Phospho-p38 MAP kinase protein was present in freshly isolated thymocytes, but not in thymocytes incubated at 37°C (Fig. 8GoB, lanes 1–3). These results corroborate our earlier studies using in vitro kinase assays to measure p38 MAP kinase activity, and validate the use of anti-phospho-p38 MAP kinase Abs to assay kinase activity in thymocytes. We found similar levels of phosphorylated p38 MAP kinase protein in thymocyte extracts from PBS-injected or dexamethasone-injected mice (Fig. 8GoB, lanes 4–7). These observations indicate that two signaling pathways known to be active in DP cells were not affected by dexamethasone, suggesting that the corticosteroids induce death despite the presence of these intrathymic signals. However, we cannot rule out the possibility that dexamethasone inhibits presently unknown signal transduction pathways in thymocytes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that several inducible proteins that bind to the {kappa}B DNA element are activated in freshly isolated immature thymocytes (3). We proposed that these factors were induced in response to signals from the thymic microenvironment. Because disruption of the thymus and incubation of single-cell thymocyte suspension at 37°C down-regulated DNA binding, we suggested that sustained signals from the thymic microenvironment were required to keep these factors in the nucleus in DNA-binding form. Other factors, in particular those binding to a probe containing SRF/SP-1 binding sites, were not down-regulated under these conditions, indicating that only certain NF required intrathymic signals to maintain them in nuclear DNA-binding form (4). In this paper, we show that the NF-{kappa}B proteins, induced in response to intrathymic signals, are down-regulated in vivo in response to dexamethasone treatment. Reduced NF-{kappa}B binding correlated with increased I{kappa}B{alpha} and ß expression and decreased expression of the putative NF-{kappa}B target gene, c-myc. We also found that cytoplasmic signaling pathways known to be active in thymocytes were not affected by dexamethasone, indicating that dexamethasone-mediated signals override those signals. We propose that dexamethasone-induced apoptosis of thymocytes may be mediated, in part, by down-regulation of intrathymically activated {kappa}B-binding proteins that maintain thymocyte viability.

Several features of these studies warrant further discussion. First, dexamethasone-dependent effects, such as NF-{kappa}B suppression, required several hours of treatment. The time course observed, however, was well within the range seen in earlier studies of glucocorticoid-mediated NF-{kappa}B regulation. In Jurkat cells, PMA-induced NF-{kappa}B was down-regulated by dexamethasone after 1 h of pretreatment, but not at earlier time points (19). NF-{kappa}B induced in response to TNF-{alpha} was suppressed in HeLa cell cultures pretreated with dexamethasone for 12 h (20). We further note that in these earlier studies cells were pretreated with dexamethasone to suppress the induction of NF-{kappa}B by several stimuli. In thymocytes, dexamethasone down-regulates preactivated NF-{kappa}B. Thus, dexamethasone activates signals that down-regulate pre-existing NF-{kappa}B.

Second, dexamethasone had several effects on DP thymocytes, which could be measured sequentially, culminating in cell death. I{kappa}B up-regulation and NF-{kappa}B down-regulation occurred within 4 h, while c-myc down-regulation was observed only 6 h after dexamethasone injection. The late turn-off of c-myc expression suggests that this is a more downstream event relative to NF-{kappa}B suppression in a cascade of events initiated by dexamethasone. This is consistent with the proposed role of NF-{kappa}B as a positive regulator of c-myc transcription. That c-myc down-regulation may participate in thymocyte death is suggested by partial rescue of DP thymocytes in c-myc transgenic mice. However, because the protection was not complete, other target genes of NF-{kappa}B may also play an important role in thymocyte survival in vivo.

Increased expression of c-myc protein has been implicated in cell death in several model systems (29), including anti-CD3 Ab-induced apoptosis of T cell hybridomas (30). Our proposal that reduced c-myc contributes to thymocyte cell death appears contrary to this accepted paradigm. We suggest that the apparent contradiction may be resolved by the plausible hypothesis that viability of immature and mature T cells is maintained by different cellular mechanisms. Therefore, the requirements for induction of cell death may also be quite different at the two stages. For example, in both the cases cited above, serum-deprived fibroblasts (29) and anti-CD3-activated T cell hybridomas (30), cell death is mediated by the Fas/Fas ligand pathway, which is not believed to be important in the death of immature thymocytes (31, 32, 33). Down-regulation of NF-{kappa}B/c-myc pathway leading to cell death may be a property of immature thymocytes, while mature T cells may require increased c-myc expression for activation-induced death. An independent line of evidence for the importance of c-myc in maintaining cell viability of immature B cells was recently provided by Sonenshein and colleagues. They demonstrated that activation-induced apoptosis of WEHI 231 correlated with reduced c-myc expression. In their studies, the importance of c-myc was directly demonstrated by ectopic expression of a transfected c-myc gene, which prevented cell death after anti-Ig cross-linking (17, 18).

bcl-x, a homologue of bcl-2, is expressed primarily in DP thymocytes (34), and the viability of these cells to various stimuli in culture is selectively diminished in bcl-x-deficient mice (35, 36). These observations suggest that bcl-x may regulate DP cell survival. However, it is interesting to note that the thymus of bcl-x-/- mice appears normal in terms of cell numbers and subsets. This suggests that DP thymocytes do not require bcl-x for survival in vivo. Additionally, bcl-x is not essential for modulating either negative selection or death by neglect. We found that bcl-x expression was not affected by dexamethasone treatment (data not shown). It is also possible that bcl-x-deficiency is compensated by other means of keeping DP cells alive. Perhaps the intrathymically induced pathway that is reflected in NF-{kappa}B/c-myc activation is one such mechanism. These data suggest that NF-{kappa}B/c-myc and bcl-x are independent regulators of DP cell viability.

As mentioned above, a well-known aspect of glucocorticoid-mediated effects on the thymus is the selective targeting of DP-stage thymocytes for apoptosis. What determines the sensitivity of this cell population to glucocorticoids? Based on our observations and known facts about the thymus, we propose the following model. DP thymocytes survive about 3–4 days in the thymus, during which time they must be positively selected to mature to SP cells. In the absence of positive selection, most of these cells die due to neglect. We have previously shown that NF-{kappa}B expression in DN thymocytes is low and the levels rise considerably as the cells transition to the DP stage (3). We propose that intrathymically activated NF-{kappa}B is a component of the survival signal of DP thymocytes. We speculate that in normal T cell differentiation the lifetime of DP thymocytes may be determined by the maintenance of inducible factors such as NF-{kappa}B. Down-regulation of these would lead to death by neglect when a positively selecting signal is not received. One view may be that the molecular interactions that activate NF-{kappa}B are restricted to specific regions of the thymic cortex. Relocation of maturing cells from this region would lead to down-regulation of NF-{kappa}B and consequent death, unless they were rescued by positive selection signals. Evidence presented in this paper suggests that some of the effects of NF-{kappa}B may be mediated by c-myc. Glucocorticoids interrupt these survival signals and cause death of DP thymocytes.

Though the studies with MMTV-c-myc transgenic mice provide direct evidence in favor of this model, our data connecting NF-{kappa}B to DP cell viability remains largely correlative. Several recent reports describe experiments aimed to modulate NF-{kappa}B levels in thymocytes. Esslinger et al. (37) overexpressed the NF-{kappa}B inhibitor, I{kappa}B{alpha}, in thymocytes in transgenic mice. They found decreased NF-{kappa}B expression and reduced numbers of DP and SP cells in the thymus. These observations are consistent with the model that decreased NF-{kappa}B expression led to premature death of DP thymocytes. In contrast, Boothby et al. (38) did not observe any thymic hypocellularity in mice carrying a dominant negative I{kappa}B transgene. Transgenic overexpression of NF-{kappa}B p65 component in thymocytes also did not reveal a significant thymic phenotype, probably because increased I{kappa}B{alpha} production in these mice kept "active" NF-{kappa}B at normal levels. These observations underscore the complexity of the NF-{kappa}B activation network (due to functional redundancy and feedback regulation) and therefore the difficulty of experimentally modulating NF-{kappa}B levels in vivo.

As mentioned before, the p65/RelA subunit of the NF-{kappa}B family has been shown to be prosurvival in a variety of cell types (13, 14, 15). For example, deficiency in the p65/RelA gene causes death of embryos during gestation due to massive apoptosis of hepatocytes. To study the role of p65/RelA in lymphocytes, fetal liver cells were transferred to irradiated mice and allowed to reconstitute the immune systems. Under these conditions, T and B cells were reconstituted in the peripheral compartments. These observations suggested that p65/RelA may not be required for the development of immune cells. Our proposal that p65 regulates DP thymocyte viability is not at odds with these studies, because it is possible that shorter lifetimes of DP cells will be reflected in more subtle developmental defects in repertoire formation, which were not scored in the earlier studies.

Though excess corticosteroids lead to DP cell death, recent studies of Ashwell and colleagues have demonstrated that low levels of corticosteroid production in the thymus is essential for proper T cell development (10, 11, 12). In particular, they showed that DP cell numbers were significantly reduced in the absence of GR expression in thymocytes or when thymic corticosteroid production was inhibited. Furthermore, the remaining DP cells were highly sensitized to TCR-mediated cell death. These observations suggest that corticosteroids regulate both cell death (as in the case of stress-induced responses) as well as cell viability (as shown with the anti-sense GR transgenic mice). How can the apparently contradictory effects of corticosteroids on DP cells be explained? One possibility is outlined below. Perhaps low levels of thymic steroids "dampen," but do not eliminate, the activated state of DP thymocytes. For example, attenuated NF-{kappa}B function may be reflected in lower levels of gene expression or selective expression of prolife genes such as c-myc. In the absence of thymic steroids, or in anti-sense GR-transgenic mice, a heightened state of activation may make these DP cells more prone to activation-induced cell death (negative selection) that results in decreased DP cell numbers. This would also explain why DP thymocytes in the absence of thymic steroids are more sensitive to death induced by TCR cross-linking. This is most clearly demonstrated by the loss of DP cells by a positive-selecting signal in the absence of GR signals (11). In the presence of excess steroids, induced by stress or by exogenous administration, the balance required to "dampen" the activated state is lost, leading to inactivation of factors such as NF-{kappa}B and their downstream targets such as c-myc, which are required to maintain cell viability. Consequently, these cells die. Thus, regulated attenuation of the activated state is crucial for the transient cell viability required of DP cells and, as noted by Vacchio and Ashwell (11), for distinguishing between positive- and negative-selecting signals.

Why are mature SP thymocytes more resistant to dexamethasone? The simplest interpretation is that the viability of these cells depends on a different set of cellular proteins. In SP cells, NF such as NF-{kappa}B are no longer induced in response to intrathymic signals (3) and are not required for cell viability. Consequently, dexamethasone treatment does not affect these cells as much. Instead, NF-{kappa}B now serves as one of the inducible NF that respond to cellular activation when the mature cell encounters Ag in the peripheral lymphoid organs. An obvious candidate for maintaining mature T cell viability is a bcl-2-dependent pathway. This protein is expressed at higher levels SP thymocytes than in DP thymocytes (39, 40), and in bcl-2-deficient mice the viability of mature cells to various stimuli is significantly diminished (5, 41). The crux of our model is that up-regulation of bcl-2 and down-regulation of NF-{kappa}B occur concomitantly with positive selection, switching thymocytes from one kind of survival mechanism to another.


   Acknowledgments
 
We thank Drs. Steven J. Burakoff and Gail Sonenshein for helpful discussions and Elaine Ames for preparation of the manuscript. J.S. would like to thank Steven J. Burakoff for continued support.


   Footnotes
 
1 This work was supported by a Barr Program Small Grant (Dana-Farber Cancer Institute) to J.S. and National Institutes of Health Grants CA74404 to J.S. and GM43874 to R.S. Back

2 Address correspondence and reprint requests to Dr. Jyoti Sen, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: DP, double-positive; MAP, mitogen-activated protein; DN, double-negative; SP, single-positive; MMTV, mouse mammary tumor virus; EMSA, electorphoretic mobility-shift assay; SRF, serum response factor; PI, propidium iodide; GR, glucocorticoid receptor. Back

Received for publication March 24, 1998. Accepted for publication September 4, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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