Vitamin A affects many aspects of T lymphocyte development and function. The vitamin A metabolites all-trans- and 9-cis-retinoic acid regulate gene expression by binding to the retinoic acid receptor (RAR), while 9-cis-retinoic acid also binds to the retinoid X receptor (RXR). Naive DO11.10 T lymphocytes expressed mRNA and protein for RAR-α, RXR-α, and RXR-β. DNA microarray analysis was used to identify RXR-responsive genes in naive DO11.10 T lymphocytes treated with the RXR agonist AGN194204. A total of 128 genes was differentially expressed, including 16 (15%) involved in cell growth or apoptosis. Among these was Bcl2a1, an antiapoptotic Bcl2 family member. Quantitative real-time PCR analysis confirmed this finding and demonstrated that Bcl2a1 mRNA expression was significantly greater in nonapoptotic than in apoptotic T lymphocytes. The RXR agonist 9-cis-retinoic acid also increased Bcl2a1 expression, although all-trans-retinoic acid and ligands for other RXR partner receptors did not. Treatment with AGN194204 and 9-cis-retinoic acid significantly decreased apoptosis measured by annexin V staining but did not affect expression of Bcl2 and Bcl-xL. Bcl2a1 promoter activity was examined using a luciferase promoter construct. Both AGN194204 and 9-cis-retinoic acid significantly increased luciferase activity. In summary, these data demonstrate that RXR agonists increase Bcl2a1 promoter activity and increase expression of Bcl2a1 in naive T lymphocytes but do not affect Bcl2 and Bcl-xL expression in naive T lymphocytes. Thus, this effect on Bcl2a1 expression may account for the decreased apoptosis seen in naive T lymphocytes treated with RXR agonists.
Vitamin A has long been called the “the anti-infective vitamin” (1). This designation is appropriate because vitamin A deficiency impairs both innate and adaptive immunity and increases the risk of death from infectious disease (2). Vitamin A (retinol) is converted to its active metabolites all-trans- and 9-cis-retinoic acid by specific enzymes (3). Retinoic acid then regulates gene expression by binding to two nuclear receptors, the retinoic acid receptor (RAR-α),3 -β, and -γ, also referred to as NR1B1, NR1B2, and NR1B3, respectively, and the retinoid X receptor (RXR-α), -β, and -γ, or NR2B1, NR2B2, and NR2B3, respectively (4). All-trans-retinoic acid binds with high affinity to RAR while 9-cis-retinoic acid binds with high affinity to both RAR and RXR (5, 6). RXR homodimers or heterodimers formed with RAR or other partner receptors bind to response elements in the promoter-enhancer region of target genes to regulate transcription (4).
Nuclear receptors, including RAR and RXR, play an important role in the immune system, including T lymphocyte development, function, and survival (7, 8, 9, 10, 11). For example, treatment of naive T lymphocytes with 9-cis-retinoic acid and the RXR-selective agonist AGN194204 enhance in vitro development of Th2 memory cells (12). All-trans-retinoic acid has similar effects (13, 14).
Apoptosis is an important regulator of the innate and adaptive immune response (15). For example, apoptosis is induced during thymic selection to minimize the development of self-reactive T lymphocytes, and protection of “appropriately” stimulated thymocytes from apoptosis helps ensure generation of a naive T lymphocyte repertoire adequate for defense against pathogenic organisms (16). In the periphery, naive T lymphocytes induce expression of the antiapoptotic proteins Bcl2a1 and Bcl-xL following stimulation via the TCR to ensure survival during the first few days following antigenic stimulation. During the same time period, cytokines such as IL-2 and IL-4 also enhance survival by stimulating Bcl2 expression (17). In contrast, apoptosis may be induced by overstimulation of the TCR (activation-induced cell death, mediated by Fas-ligand engagement on targeted lymphocytes) and by cytokine withdrawal (death by neglect, which can be reversed by cytokines such as IL-2, IL-4, and IL-7) (18). This balance of survival and apoptosis helps maintain protective immune responses and ensures their successful termination and also protects the host from damaging autoimmune responses.
Little is known about the effect of retinoids on the survival of naive or memory T lymphocytes. However, retinoids are known to affect the survival of thymocytes, T cell hybridomas, and T lymphoma cell lines. For example, retinoic acid and RAR-selective ligands can inhibit Fas-mediated apoptosis by diminishing cell surface Fas ligand expression in T cell hybridomas activated via the TCR (19, 20, 21, 22). This antiapoptotic effect is mediated via RAR-α/RXR heterodimers (23), whereas RAR-γ agonists can up-regulate FAS-ligand and thus facilitate apoptosis in T cell hybridomas (24) as well as thymocytes (25). Thymocyte apoptosis has also been reduced by retinoic acid treatment (26). However, in T lymphoma cells, retinoic acid has been shown to induce apoptosis, although the mechanism is not known (27). To our knowledge, the effects of retinoids on apoptosis have not been examined in primary T lymphocytes.
In the present study, we have identified genes that are differentially regulated by the RXR agonist AGN194204 during primary antigenic stimulation of naive CD4+ T lymphocytes from RAG1−/− DO11.10 TCR-transgenic mice (28). Many genes playing a role in apoptosis were identified. One of these was Bcl2a1, which encodes the antiapoptotic Bcl2 family member A1 (also called Bfl-1 and Hbpa1) (29). Bcl2a1 is expressed in many cells of the immune system, including myeloid progenitor cells (30), macrophages (31), neutrophils (32), thymocytes (13), T lymphocytes (33), and B lymphocytes (34). In T lymphocytes, Bcl2a1 expression is induced by engagement of the TCR (17) via activation of NF-κB (17, 34, 35). Our data suggest that RXR agonists enhance survival of naive T lymphocytes by increasing Bcl2a1 gene expression and thereby decreasing apoptosis.
Materials and Methods
RAG1−/− DO11.10 mice were bred in our facility. They were a gift from Dr. C. London (University of California, Davis, CA). To confirm their identity, peripheral blood from these mice was screened by two-color flow cytometric analysis using anti-CD4 Ab and the TCR clonotypic mAb KJ1–26 (36). BALB/c mice were purchased from Charles River Laboratories.
Cell lines and reagents
The Th1 and Th2 cell cultures were derived from DO11.10 mice as described (37) and were a gift of Dr. C. Weaver (University of Alabama, Birmingham, AL). The RAR and RXR plasmids used as controls in Fig. 1⇓ were a gift from Dr. M. Satre of (University of California, Davis, CA). Abs used in cell culture were purchased from BD Pharmingen and included neutralizing rat mAbs for murine IL-4 (clone BVD40-1D11, IgG2b isotype), IL-12 (p40/p70, clone C17.8, IgG2a isotype) and IFN-γ (clone R4-6A2, clone R4-6A2). Isotype control Abs for IgG1 (clone 11010D), IgG2a (clone 11020D), and IgG2b (clone R35-38) were also used. Recombinant purified IL-4 was also purchased from BD Pharmingen. Abs for FACS analysis included PE-labeled anti-CD4 (clone L3T4; BD Pharmingen) and FITC-labeled KJ1–26 (Caltag Laboratories). The following Abs (identified by product number) were used for Western blot analysis and were purchased from Santa Cruz Biotechnology: RAR-α, SC551; RAR-β, SC552; RAR-γ, SC773; RXR-α, SC553; and RXR-γ, SC555. The Ab for RXR-β (PA1-815) was purchased from Affinity BioReagents. The secondary goat anti-rabbit Ab for Western blots was purchased from Jackson ImmunoResearch Laboratories.
The RXR-selective agonist AGN194204 (38) and other nuclear receptor agonists were diluted in cell culture-grade DMSO (Sigma-Aldrich) and stored at −70°C. Aliquots were thawed and diluted into medium immediately before addition to lymphocyte cultures. The final concentration of DMSO in the cell culture medium was 0.1%. The peroxisome proliferator-activated receptor (PPAR)-γ agonist ciglitazone was purchased from Cayman Chemical, whereas 9-cis-retinoic acid, all-trans-retinoic acid, 1α,25-dihydroxyvitamin D3, 3,3′,5-triiodo-l-thyronine, chenodeoxycholic acid, and 22(R)-hydroxycholesterol were purchased from Sigma-Aldrich.
For reporter gene analysis, the CAT reporter plasmid A1/l-CAT, containing 2 kb of genomic DNA cloned from immediately upstream of the Bcl2a1a gene (39), was provided by S. Gerondakis (The Royal Melbourne Hospital, Parkville, Victoria, Australia) and cloned in a luciferase reporter plasmid (pGL3/Bcl2a1). The heat shock protein 70 (HSP70)-β-galactosidase reporter plasmid was provided by R. Modlin (University of California, Los Angeles, CA). The expression plasmid for pCMX/RXRα and pCMX were provided by D. Mangelsdorf (Southwestern Medical Center, Dallas, TX).
3T3 cells (a murine fibroblast cell line, ATCC CRL1658) and EL4 cells (a T lymphoma cell line, ATCC TIB-39) were obtained from the American Type Culture Collection. Raw 264.7 cells (a murine monocytic cell line, ATCC TIB-71) were a gift from Dr. D. Hwang (U.S. Department of Agriculture Western Human Nutrition Research Center, University of California, Davis, CA).
For microarray analysis, T lymphocytes were isolated and stimulated as described in two independent experiments (12). Briefly, CD4+ cells were purified from RAG1−/− DO11.10 mice by positive selection using CD4-specific magnetic beads (Dynal Biotech). These cells were stimulated with OVA323–339 peptide (Alpha Diagnostics International) and irradiated splenocytes from BALB/c mice as APCs. Cultures were treated with the RXR agonist AGN194204 (10 nM in 0.1% DMSO) or 0.1% DMSO (vehicle control). For RNA extraction and microarray analysis CD4+/KJ1–26+ T lymphocytes were sorted to >96% purity using a MoFlo (DakoCytomation) high-speed cell sorter.
Three independent experiments were done to confirm the microarray results and to determine whether cytokines regulating Th1/Th2 development affected expression of putative RXR-responsive genes identified in the microarray experiments. These experiments were done as just described with the following treatments: 1) AGN194204 (10 nM + 0.1% DMSO) plus neutralizing Abs for IL-4, IFN-γ, and IL-12 (5 μg/ml of each Ab); 2) AGN194204 plus isotype control Abs (5 μg/ml of each Ab); 3) vehicle control (0.1% DMSO) plus neutralizing Abs; 4) vehicle control plus isotype control Abs; and 5) IL-4 (10 ng/ml + 0.1% DMSO).
In other experiments, CD4+ cells were purified from draining lymph nodes and spleens of RAG1−/− DO11.10 mice by positive selection using CD4-specific magnetic beads and then stimulated with plate-bound anti-CD3 (1 μg/ml; clone 145-2C11; BD Pharmingen) and anti-CD28 added to the medium (5 μg/ml; clone 37.51; BD Pharmingen). Culture conditions have been described previously (12).
The Th1 and Th2 cell cultures were stimulated every 2 wk with OVA323–339 peptide Ag and irradiated splenocytes plus added IL-2 (50 U/ml). Culture conditions have been described previously (12).
DNA microarray analysis.
Microarray analysis was done twice, using RNA from two independent experiments. RNA quality and integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies) and absorbance at A260/A280. Only high-quality RNA, having a 28S/18S rRNA ratio of 1.5:2 and an A260/280 ratio of 1.8:2, was used for further experimentation. RNA was converted to double-stranded cDNA and to biotin-labeled cRNA by in vitro transcription labeling with a HighYield BioArray RNA Transcript Labeling kit (Enzo Biochem). The quality of in vitro transcription and fragmentation products was assessed using the Agilent 2100 Bioanalyzer.
We used the U74Av2 murine oligonucleotide arrays (Affymetrix) that contain 12,488 probes representing 5,890 genes. Fifteen micrograms of fragmented, biotin-labeled cRNA was hybridized at 45°C overnight as defined in the Affymetrix7 expression analysis protocol. The hybridization buffer contained 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20, four eukaryotic hybridization controls (1.5 pM BioB, 5 pM BioC, 25 pM BioD, and 100 pM cre), 0.1 mg/ml herring sperm DNA (Promega), and 0.5 mg/ml acetylated BSA. After hybridization, the arrays were washed and stained with an Affymetrix fluidic station following the Ab Amplification Washing and Staining Protocol (Affymetrix). Hybridization was detected with streptavidin-PE and a confocal laser scanner (Affymetrix).
Qualitative PCR analysis to confirm RAR and RXR expression, and quantitative PCR (qRT-PCR) analysis to compare levels of gene expression were done as described previously (12). Primers are described in Table I⇓ and were developed using GCG software (Genetics Computer Group). Primers used for Bcl2 for were purchased from BioSource International. qRT-PCR was performed using a LightCycler rapid thermal cycler system with SYBR Green I dye (Roche Diagnostic Systems). RNA quality for qRT-PCR analysis was assessed as described for microarray analysis. Melting curve analysis was used to confirm specificity of the products. Products were cloned and sequenced during assay development to confirm specificity.
Apoptotic cells were identified using flow cytometric analysis of cells stained with PE-labeled annexin V to identify apoptotic cells and 7-aminoactinomycin D (7-AAD) to label permeable (dead) cells (BD Pharmingen), and staining was done according to the manufacturer’s instructions using a FACSCalibur flow cytometer (BD Pharmingen). Apoptosis assays were performed on CD4-selected naive T lymphocytes stimulated with anti-CD3 and anti-CD28 Ab. In one experiment, these reagents were used to sort viable, nonapoptotic cells (annexin V−/7-AAD−) from viable, apoptotic cells (annexin V+/7-AAD−) using a MoFlo high-speed cell sorter. To determine the purity after sorting, some cells were collected into Annexin V Binding buffer (BD Pharmingen) to retain binding of annexin V. Sorted cells were >90% pure.
Cells were washed in cold PBS containing 0.5 mM PMSF before protein extraction. Whole-cell protein extracts were prepared as follows: washed cells were resuspended in lysis buffer (1% Nonidet P-40, 30 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 150 mM NaCl, 10% glycerol, 0.5 mM PMSF, and 1/500 dilution of protease inhibitor solution) and were incubated for 5 min on ice. The protease inhibitor solution consisted of one complete protease inhibitor tablet (Roche Diagnostic Systems) dissolved in 250 μl of water. A total of 5 M NaCl was added to a final concentration of 400 mM, and cells were incubated for an additional 10 min on ice. Cells were then passed through a Qiashredder (Qiagen) by centrifugation for 10 min at 20,800 × g at 4°C. Nuclear extracts were prepared as follows: washed cells were resuspended in 100 μl of solution A (0.5% Nonidet P-40, 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, and 10 mM KCl) per million cells and incubated for 5 min on ice. Samples were then pelleted by centrifugation at 6800 × g at 4°C for 5 min. The pellet was resuspended in 25 μl of solution B (20 mM HEPES (pH 7.9), 25% glycerol, 400 mM NaCl, 1.5 mM MgCl2, 0.5 mM EDTA (pH 8), 0.2 mM PMSF, 0.5 mM DTT, and 1/500 dilution of protease inhibitor solution) per million cells. Samples were frozen and thawed three times and passed through a Qiashredder as described above.
Proteins were resolved by SDS-PAGE on 4–12% gradient gels (Novex) and transferred to nitrocellulose (Invitrogen Life Technologies). Blots were blocked using 10% skim milk powder in PBS containing 0.1% polyoxyethylenesorbitan monolaurate (Sigma-Aldrich) (PBST) overnight at 4°C. Blots were washed three times with PBST and were blocked again in 5% goat serum (Jackson ImmunoResearch Laboratories) in PBST overnight at room temperature. Each blot was then probed using appropriate Abs diluted at 1/500 at room temperature for 1.5 h and washed three times with PBST. After washing, the secondary Ab diluted at 1/20,000 was added and incubation lasted another 1.5 h at room temperature. Finally, the blots were washed three times with PBST and developed using the ECL kit (Amersham Biosciences).
Reporter gene analysis
Cell lines were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS (FCS) (Invitrogen Life Technologies) and 1× Antibioticum/Antimycoticum (Invitrogen Life Technologies) at 37°C in a 5% CO2/air environment. Cells for reporter assays were seeded in 24-well plates at a density of 1.5–2.0 × 105 cells/well 1 day prior transient cotransfections. Before transfection, the growth medium was replaced with DMEM, supplemented with 10% charcoal/dextran-treated FBS. Per well, 3T3 cells were transfected with 500 ng of pCMX or pCMX/RXRα, 400 ng of pGL3-basic/Bcl2a1, and 200 ng of HSP70-β-galactosidase reporter plasmid using LipofectAMINE 2000 (Invitrogen Life Technologies), following the manufacturer’s instructions; Raw 264.2 cells were transfected in the same way with 500 ng of pCMX or pCMX/RXRα, 800 ng of pGL3-basic/Bcl2a1, and 400 ng of HSP70-β-galactosidase reporter plasmid. Twenty-four hours after transfections, the cells were treated with DMSO, 9-cis-RA, or AGN 194204 at 1 μM or at a range from 1 nM to 10 μM. Twenty-four hours after treatment, the cells were harvested in reporter lysis buffer (Promega). Luciferase and β-galactosidase enzyme activities were determined using Luciferase Assay System and β-galactosidase Enzyme Systems (Promega) following the manufacturer’s instructions. Luciferase activity was determined by a TD-20/20 Luminometer (Turner Designs); β-galactosidase activity was assayed using an ELX800 Universal Microplate Reader (Bio-Tek Instruments).
Microarray Suite 5.0 (Affymetrix) was used to determine the probe intensities and to compare expression among different arrays; the average intensity for each array was normalized by scaling to a target median intensity value of 125. The gene expression values were log transformed (log base 2). Genes were ranked on t test scores, p values (p < 0.05), and fold changes computed as actual expression values. A particular transcript was considered significantly differentially expressed between the groups if it had a fold change > 1.29 (RXR agonist vs vehicle control or vice versa) and a value of p < 0.05, and this finding was concordant in the two independent experiments. The cutoff of >1.29 was selected because previous quantitative RT-PCR analysis (12) showed that changes of this magnitude in Th2-enhancing genes (IL-4 and GATA-3) were both statistically significant and predicted later development of a Th2 phenotype. The annotation of the differentially expressed genes was performed using information from the Affymetrix and National Center for Biotechnology Information EntrezGene web sites.
Other statistical analysis.
Statistical analysis was performed using SigmaStat for Windows 2.03 or 3.1 (SPSS). Multiple comparisons of RNA levels determined by RT-PCR and percentage of apoptotic cells were made using several methods. Paired Student’s t tests were used to control for experiment or time within an experiment when comparing retinoid treatments. One-way ANOVA was used to compare retinoid treatments within a particular experiment. Two-way ANOVA was used to compare retinoid treatments while controlling for the experiment when results from multiple experiments were pooled for analysis. Three-way ANOVA was also used to control for experiment when comparing two treatment variables, such as retinoid treatment and partner ligand treatment, or cytokine treatment and retinoid treatment. Two-way repeated-measure ANOVA was used to compare retinoid treatments when controlling for time and experiment. Duplicates to quadruplicate measurements were made within each experiment, and experiments were repeated at least three times. A value of p < 0.05 using a two-tailed test was considered statistically significant.
Expression of RAR and RXR in CD4+ T lymphocytes
Treatment of naive CD4+ T lymphocytes from DO11.10 mice with RAR and RXR agonists affects Th1/Th2 memory cell development (12, 40, 41). Therefore, we wished to determine which RAR and RXR receptors are expressed in naive or memory CD4+ T lymphocytes from these mice. Using qualitative PCR analysis of cDNA, we detected a single band of appropriate size for RAR-α, RAR-γ, RXR-α, and RXR-β in naive DO11.10 CD4+ T lymphocytes (Fig. 1⇑, top panel) and in Th1 and Th2 cells derived from DO11.10 mice (data not shown). The cDNA sequences of these products were essentially identical to the published sequences for these receptors. Western blot analysis of nuclear extracts found that RAR-α, RXR-α, and RXR-β but not RAR-β, RAR-γ, or RXR-γ were present in naive CD4+ T lymphocytes as well as in the Th1 and Th2 cultures (Fig. 1⇑, bottom panel). Although RAR-γ mRNA was found at levels similar to the other receptors, protein was not seen in either whole cell or nuclear extracts.
RAR and RXR expression following primary stimulation of naive CD4+ T lymphocytes
We previously reported that treatment of naive DO11.10 T lymphocytes with the RXR agonist AGN194204 increases Th2 development and early expression of Th2-promoting genes (12). To determine whether expression of RAR and RXR were also affected by this treatment, samples from these experiments were examined by qRT-PCR. RNA levels for both RAR-α and RAR-γ decreased after antigenic stimulation (Fig. 2⇓). No differences among the three treatments (RXR agonist, vehicle control, IL-4) were seen for RAR-γ, but RAR-α RNA levels were higher in the IL-4 treatment than in the vehicle control (Fig. 2⇓). Although both RXR-α and RXR-β mRNA levels were higher in the IL-4 treatment than in the vehicle control after stimulation (Fig. 2⇓), treatment with the RXR agonist did not have a consistent effect on RAR or RXR expression.
DNA microarray analysis following primary stimulation of naive CD4+ T lymphocytes
Comparison of cultures treated with the RXR agonist AGN194204 (100 nM) or vehicle control (0.1% DMSO) revealed differential expression of 128 genes (Table II⇓⇓⇓), 108 of which have a known function. When examined by functional category, regulation of cell growth and apoptosis encompassed the greatest number of genes (16 of 108 = 15%), followed by regulation of transcription (10 of 108 = 9%). Other functional groups (Table II⇓⇓⇓) were represented at <7%/group.
Several genes known to be directly regulated by RXR homo- or heterodimers were identified. For example, mRNA levels of the stearoyl-CoA desaturase 1 (Scd1) gene are increased by both RAR and RXR agonists (42). In this analysis, the mean fold-increases for Scd1 RNA was 17.1 (Table II⇑⇑⇑). The cellular retinoic acid binding protein 2 (Crabp2) gene, which contains prototypical direct repeat (DR)1 and DR2 response elements and is up-regulated by both RAR and RXR agonists (43), showed the greatest increase of all genes 24-fold (Table II⇑⇑⇑ and Fig. 3⇓). These observations confirm that genes regulated by RAR and RXR can be identified from these experiments.
This microarray analysis also confirmed our previous observations (12) that RNA levels for Th2-enhancing genes are increased by treatment with the RXR agonist. In this analysis, these included genes for IL-4 (1.9-fold), GATA3 (1.5-fold), IL-4Rα-chain (1.4-fold), and growth factor independent (GFI-1) (1.3-fold), although mRNA for IL-13 was decreased (−2.8-fold). mRNA levels for genes involved in Th1 growth and development were decreased by treatment with the RXR agonist, including IL-2 (−1.4-fold), IL-2Rα-chain (−1.4-fold), and IL-12Rβ1- (−1.4-fold) and IL-12R-β2 chains (−1.8-fold).
The high percentage of cell growth and apoptosis genes identified in this analysis was of interest for two reasons. First, retinoids are known to regulate apoptosis in T lymphocytes, thymocytes, and T lymphoma cells (7, 21, 44). Second, in addition to promoting Th2 development, treatment with the RXR agonist AGN194204 during primary antigenic stimulation reproducibly caused a greater accumulation of viable cells after 1 wk of culture than did treatment with the vehicle control. In eight independent experiments the number of viable cells (±SD) in the RXR agonist (10−7 or 10−8 M), IL-4 (10 ng/ml) and vehicle control (0.1% DMSO) treatments were 3.80 ± 2.78, 3.73 ± 1.50, and 2.36 ± 1.91 × 106 cells/ml, respectively. Cell counts in the RXR and IL-4 treatment groups were both significantly greater than the counts in the vehicle control (p = 0.032 and p = 0.024, respectively, by paired Student’s t test), while the RXR agonist and IL-4 treatments did not differ from one another (p = 0.92). The mean fold increases for the RXR and IL-4 treatments were 2.24 and 2.37, respectively.
Because treatment with the RXR agonist increases cell number, we examined differential expression of cell growth and apoptosis genes in more detail. The expression of Bcl2a1 mRNA (Table II⇑⇑⇑; Fig. 3⇑) was significantly greater in cultures treated with AGN194204 than in the vehicle control cultures (∼2-fold). Bcl2a1 also stood out because of the high signal intensity for the two Bcl2a1 probe sets (third and fourth highest of all differentially expressed genes). These two probe sets represent the genes Bcl2a1b and Bcl2a1d, two members of a family of four genes (also including Bcl2a1a and Bcl2a1c, although the latter gene is not expressed) that code for the B cell leukemia/lymphoma 2-related protein A1 (45). These genes have high sequence identity and are Bcl2 family members. As with Bcl2, the Bcl2a1 protein acts at the mitochondrial level to prevent apoptosis and is known to be expressed and protective against apoptosis in naive T lymphocytes undergoing primary Ag stimulation (33).
RXR-stimulated increase in Bcl2a1 is independent of IL-4, IL-12, and IFN-γ stimulation
To confirm the microarray results, we examined Bcl2a1 gene expression by qRT-PCR using primers that detect all Bcl2a1 family members. In addition, because treatment with AGN194204 and other retinoids affect Th1/Th2 cytokine production (12), neutralizing Abs for IL-4, IL-12, and IFN-γ were included in some cultures to determine whether RXR-mediated increases in Bcl2a1 expression were independent of effects on these cytokines (Fig. 4⇓). The mean Bcl2a1 mRNA levels in the AGN194204 treatment groups was greater than in the vehicle control groups either in the presence (p < 0.001, fold increase = 1.83) or in the absence (p < 0.001, fold increase = 2.38) of the cytokine-neutralizing Abs (Fig. 4⇓). Treatment of cultures with IL-4 did not enhance Bcl2a1 expression (Fig. 4⇓). These results are consistent with the findings from the microarray studies and also indicate that RXR-induced increases in Bcl2a1 mRNA occur independent of IL-4, IL-12, and IFN-γ stimulation.
Crabp2 mRNA levels were also analyzed in this experiment to confirm transcriptional activity of the RXR agonist (Fig. 4⇑). Mean Crabp2 RNA levels following treatment were also higher compared with the levels of DMSO-treated cells either in the presence (p < 0.001, fold increase = 4.11) or in the absence (p < 0.001; fold increase = 5.58) of the cytokine-neutralizing Abs. Treatment with the RXR agonist produced no consistent, statistically significant differences in hypoxanthine guanine phosphoribosyl transferase (Hprt) mRNA levels either in the presence or absence of neutralizing Ab (Fig. 4⇑). This result was also consistent with our microarray studies (Fig. 3⇑).
Time course of Bcl2a1 RNA expression: effect of AGN194204 and 9-cis-retinoic acid
The expression of Bcl2a1 is induced in T lymphocytes by stimulation via the TCR complex. Expression peaks before 24 h and then decreases by 48 h (17, 46). Purified CD4+ DO11.10 T lymphocytes were stimulated with anti-CD3 plus anti-CD28 Ab in the presence of AGN194204, the natural RXR agonist 9-cis-retinoic acid (100 nM), or vehicle control (0.1% DMSO). Bcl2a1 mRNA levels were greatest at 20 h in cells treated with vehicle only (Fig. 5⇓). The pattern was similar following retinoid treatments. AGN194204 significantly increased Bcl2a1 expression at both 20 h (41% higher) and 44 h (38% higher). The effect of 9-cis-retinoic acid on Bcl2a1 expression was similar to that of AGN194204 (Fig. 5⇓), although the magnitude of the effect was less. At 20 and 44 h following 9-cis-retinoic acid treatment, the increases in Bcl2a1 expression over the vehicle control were 23 and 42%, respectively (p = 0.027, paired t test). Crabp2 RNA expression was examined as a positive control for the retinoid treatments. Both treatments significantly increased Crabp2 expression over levels seen in the vehicle control (Fig. 5⇓).
Effect of RXR partner ligands on Bcl2a1 RNA expression
RXR may regulate Bcl2a1 expression by forming a heterodimer with a permissive partner receptor. Such receptors include the vitamin D receptor (VDR, NR1I1), thyroid hormone receptor (TR, NR2C), farnesoid X receptor (FXR, NR1H4), liver X receptor (LXR, NR1H), RAR (NR1B), PPAR (NR1C) (47). Thus, treatment of T lymphocytes with the partner ligand alone might increase Bcl2a1 expression. To test this hypothesis, cultures of naive CD4+ DO11.10 T lymphocytes were stimulated with anti-CD3 and anti-CD28 Abs and treated with partner-receptor ligands (100 nM 1,25(OH)2 vitamin D3, 100 nM 3,3′,5-tri-iodothyronine, 50 μM chenodeoxycholic acid, 5 μM 22(R)-hydroxycholesterol, 100 nM all-trans-retinoic acid, and 100 nM ciglitazone) in the presence the RXR agonist AGN194204 (100 nM) or vehicle control (0.1% DMSO). In four independent experiments, treatment with AGN194204 alone significantly increased Bcl2a1 expression above the level seen with the vehicle control, as was expected, but none of the partner ligands used alone significantly increased Bcl2a1 expression (data not shown). Treatment with each partner ligand in combination with the RXR agonist also significantly increased Bcl2a1 expression above the level seen with the vehicle control, but none of these combinations significantly increased Bcl2a1 expression above the level seen with AGN194204 alone (data not shown). Thus, these ligands for VDR, TR, FXR, LXR, RAR, and PPAR-γ do not increase Bcl2a1 mRNA under these experimental conditions.
RXR agonist decreases T lymphocyte apoptosis following primary stimulation
Because Bcl2a1 inhibits apoptotic death of naive T lymphocytes following antigenic stimulation, we anticipated that treatment of stimulated T lymphocytes with AGN194204 would also decrease apoptosis. To test this hypothesis we treated CD4+ DO11.10 T lymphocytes (stimulated with anti-CD3 and anti-CD28 Ab) with the RXR agonist AGN194204 or vehicle control (0.1% DMSO), stained cells with PE-labeled annexin V and 7-AAD, and analyzed cells by flow cytometry. The percentage of cells positive for both annexin V and 7-AAD was lower in the RXR agonist-treated cultures at all time points. These differences were statistically significant by two-way ANOVA at 24 h (p < 0.001, n = 3 experiments) and 48 h (p < 0.001, n = 3 experiments) but not at 72 h (n = 2 experiments) (Fig. 6⇓, top panel). The percentage of cells earlier in apoptosis, which stained only positive for annexin V but not for 7-AAD, did not differ at 24 h, but there was significantly less apoptosis in cultures treated with the RXR agonist at both 48 and 72 h (p < 0.001) (Fig. 6⇓, bottom panel). Thus, treatment with the RXR agonist decreased apoptosis of naive T lymphocytes during initial stimulation via the TCR with CD28-mediated costimulation.
Growth factor deficiency may contribute to apoptosis of T lymphocytes following antigenic stimulation. To determine whether growth factor deficiency was a factor in these experiments, we measured the effect of AGN194204 and 9-cis-retinoic acid on apoptosis 48 h after stimulation in the presence of IL-2 or IL-4 or without cytokine. The percentage of cells undergoing apoptosis was highest without cytokine (43.6%), significantly lower with IL-2 treatment (41.4%) and (significantly) lower still with IL-4 treatment (37.2%) (Fig. 7⇓). Both AGN194204 and 9-cis-retinoic acid significantly decreased the percentage of cells that were positive for both annexin V and 7-AAD with our without cytokine treatment (Fig. 7⇓). Thus, the effect of these RXR agonists on protection from apoptosis is independent of treatment with IL-2 and IL-4.
Effect of AGN194204 and 9-cis-retinoic acid on Bcl2 and Bcl-xL expression
Because Bcl2 and Bcl-xL are antiapoptotic genes that act in a manner similar to Bcl2a1, we examined the effect of retinoid treatment on their expression in stimulated T lymphocyte cultures (Fig. 5⇑). Bcl2 expression was highest at 2 h and decreased steadily through 20 and 44 h, but the RXR agonists AGN194204 and 9-cis-retinoic acid had no significant effects on Bcl2 expression. Bcl-xL expression was lowest at 2 h, increased by 20 h, and then decreased slightly by 44 h. Neither RXR agonist significantly affected Bcl-xL expression (Fig. 5⇑). Thus, these RXR agonists did not affect expression of the antiapoptotic genes Bcl2 and Bcl-xL.
Bcl2a1 RNA expression is higher in nonapoptotic than apoptotic T lymphocytes
Given the direct effect of Bcl2a1 on protection from apoptosis in naive T lymphocytes (17), we anticipated that Bcl2a1 mRNA levels would be higher in nonapoptotic than in apoptotic cells (i.e., the ratio in sorted cells would be >1.0). To confirm this expectation, we stimulated naive DO11.10 T lymphocytes with anti-CD3 and anti-CD28 Abs, separated viable, nonapoptotic cells (annexin V−/7-AAD−) from viable, apoptotic cells (annexin V+/7-AAD−) by cell sorting, and measured Bcl2a1 expression by qRT-PCR 24, 48, and 72 h after stimulation. Cultures were treated with AGN194204 (100 nM) or vehicle control (0.1% DMSO), and one sample from each treatment was sorted and analyzed at each time point. The overall mean ratio (±SD) of Bcl2a1 mRNA levels in nonapoptotic vs apoptotic cells was 3.97 ± 1.20 (n = 6), significantly greater than 1.0 (p = 0.002 by paired Student’s t test controlling for time point). The ratios increased over time (2.8, 4.4, and 4.7 at 24, 48, and 72 h) but did not differ by retinoid treatment. Thus, nonapoptotic cells from both treatments had higher Bcl2a1 expression than apoptotic cells, consistent with the known antiapoptotic activity of Bcl2a1. Crabp2 mRNA levels were examined and no differences in expression ratios were found between nonapoptotic and apoptotic cells (mean ratio 1.06 ± 0.44, p = 0.74), as would be expected for a gene not known to be involved in apoptosis.
AGN194204 and 9-cis-retinoic acid increase transcription from the Bcl2a1 promoter
Because both AGN194204 and 9-cis-retinoic acid increased Bcl2a1 mRNA levels in stimulated T lymphocytes, we wished to determine whether the 5′-regulatory region of Bcl2a1 responds directly to treatment with these RXR agonists. Therefore, we examined the effects of AGN194204 and 9-cis-retinoic acid on the ability of 2.0 kb of genomic DNA found immediately upstream of the Bcl2a1 structural gene to regulate expression of a luciferase reporter gene. This region contains several transcription factor binding sites or importance to regulation of Bcl2a1 gene expression (39). Because the effect of retinoids on transcription may vary by cell type, we initially attempted to transfect this reporter construct into primary T lymphocytes and a T lymphocyte line (EL4). Efficiencies were very low (data not shown) so we used murine macrophage (Raw) and fibroblast (3T3) cell lines.
In both Raw and 3T3 cells, we found that luciferase activity was increased significantly by treatment with either AGN194204 or 9-cis-retinoic acid. As shown in Fig. 8⇓, 9-cis-retinoic acid increased luciferase activity by 103% in Raw cells and 91% in 3T3 cells. When an expression plasmid for RXR-α was cotransfected with the reporter plasmid, basal expression in the Raw and 3T3 cells was 70 and 416% higher than without the expression plasmid. Addition of 9-cis-retinoic acid again increased luciferase activity in the presence of the reporter plasmid by 136% in Raw cells and by 103% in 3T3 cells. The effect of different concentrations of 9-cis-retinoic acid and AGN194204 on transcriptional activity was assessed in 3T3 cells. Both retinoids showed an ability to increase luciferase activity at low concentrations (1 nM for 9-cis-retinoic acid and 10 nM for AGN194204) and a dose-response effect was seen at higher concentrations, with the maximum increase being 94% for 9-cis-retinoic acid and 52% for AGN194204. Thus, the Bcl2a1 promoter is responsive to retinoids at physiologically relevant concentrations.
Many nuclear receptors are expressed in T lymphocytes, including RAR, RXR, PPAR, VDR, ROR-γ (NR1F3), Nur77 (NR4A1), and Nor1 (NR4A3) (7, 10, 48, 49). In TCR-transgenic DO11.10 T lymphocytes, we detected mRNA for RAR-α, RAR-γ, RXR-α, and RXR-β. Protein was also detected for three of these four receptors, with the exception of RAR-γ. It is possible that our Western blot analysis was not sensitive enough to detect RAR-γ, although RNA levels for RAR-α and RAR-γ were similar. Other investigators report the presence of RAR-γ mRNA in T lymphocytes and biological activity for RAR-γ-selective ligands (24). To our knowledge, RAR-γ protein has not been identified in T lymphocytes by Western blot analysis, thus the significance of RAR-γ RNA levels in these cells remains uncertain.
RAR mRNA levels decreased after antigenic stimulation while RXR mRNA levels increased (with IL-4 treatment) or remained stable, suggesting a requirement for RXR-mediated signaling in developing memory T lymphocytes. Increased RXR-α expression has also been reported following TCR stimulation in proliferating human T lymphocytes (50), although stimulation of resting human T lymphocytes decreases RXR-α mRNA and protein levels (51). The latter situation is more analogous to the present study, although our murine, TCR-transgenic T lymphocytes were activated with APCs and specific Ag, rather than the polyclonal stimulation used in the studies of resting, human peripheral blood T lymphocytes. This difference in stimulation, or the many differences between these murine and human culture systems, may account for the differences in RXR-α expression seen between the two studies.
Our microarray analysis demonstrated that the expression of many genes involved in Th1/Th2 development, cell proliferation, and cell survival were regulated by RXR in naive T lymphocytes during primary antigenic stimulation. Regulation of these genes may occur directly or indirectly. Many genes directly regulated by RXR agonists, including Crabp2 and Scd1, were identified in these experiments. Although many microarray studies use cell lines, which may not adequately represent the same cell type in vivo, or use tissues from treated animals, which contain many cell types, our study used primary cells that were purified to >96% homogeneity before RNA extraction. Thus, we have a high degree of confidence that the genes identified by this method are expressed in primary T lymphocytes and that differences in mRNA levels reflect actual differences in gene expression in these cells, rather than differences in frequencies of cells in a mixed population.
One of the genes induced by AGN194204 in our microarray studies was Bcl2a1. This gene is induced by TCR stimulation (33) and treatment with AGN194204 enhanced Bcl2a1 expression above levels seen by TCR stimulation alone, using either APCs and peptide Ag or ant-CD3 plus anti-CD28 Abs. RAR and RXR agonists can also affect expression of IL-12 by APCs and of IL-4 and IFN-γ by T lymphocytes (2). However, neutralizing Abs were used to rule out an effect of these cytokines on Bcl2a1 expression in our experiments. Because AGN194204 is a synthetic agonist, we also tested the naturally occurring RXR agonist 9-cis-retinoic acid for the ability to increase Bcl2a1 RNA expression. 9-cis-retinoic acid also increased Bcl2a1 mRNA levels, although the magnitude of the response was not as great as seen with equal concentrations of AGN194204. This may be because the binding affinity and transactivation activity (EC50 concentration) are 10-fold lower for AGN194204 than for 9-cis-retinoic acid (38), making AGN194204 a more potent agonist. These results confirm that Bcl2a1 mRNA levels are greater in primary T lymphocyte cultures treated with RXR agonists, and are consistent with an RXR-mediated increase in transcription.
To determine whether AGN194204 and 9-cis-retinoic acid directly regulate transcription from the Bcl2a1 promoter, we conducted experiments using a luciferase reporter construct containing 2.0 kb of cloned genomic DNA from the 5′ noncoding region of the Bcl2a1a gene (39). These experiments confirmed that both AGN194204 and 9-cis-retinoic acid increased luciferase activity in 3T3 and Raw cells. To our knowledge, these are the first data to directly indicate that retinoids regulate Bcl2a1 gene expression. Additional experiments will be needed to determine how these agonists affect transcription. It is possible that an RXR homodimer or heterodimer binds to an appropriate response element in the Bcl2a1 promoter and thereby enhance transcription (52). Preliminary examination of the Bcl2a1 promoter revealed a nearly canonical DR4 element (data not shown), but agonists for two nuclear receptors that typically act via a DR4 element, TR and LXR, did not significantly enhance Bcl2a1 expression in the present study, nor did the ligands for PPAR-γ, FXR, RAR, or VDR enhance Bcl2a1 expression. These negative results suggest that these partner receptors are not involved in regulation of Bcl2a1 expression. However, the results are not definitive because these experiments were conducted using cell culture medium containing FBS, which may contain partner ligands (e.g., all-trans-retinoic acid or thyroid hormone) that could obscure enhancement of Bcl2a1 expression by added partner ligands.
Rather than bind to a response element, RXR could interact directly with transcription factors, such as NF-κB, and thereby affect transcription (53). This is plausible because Bcl2a1 transcription is regulated by NF-κB (35), but such interactions often result in decreased rather than increased transcription as a result of competition among different transcription factors for access to the transcription complex. However, a recent study revealed that retinoids induced apoptosis via an RAR-α/RXR pathway in immature dendritic cells. But when dendritic cells were also stimulated with inflammatory signals cell survival, maturation, and Ag presentation were enhanced. The latter effects depended on RXR were independent of RAR and appeared to be mediated by increased nuclear translocation of NF-κB (54). Because Bcl2a1 transcription is increased by NF-κB binding to its promoter following TCR engagement (39), the same RXR-mediated enhancement of NF-κB activity could be responsible for increased Bcl2a1 expression in the present experiments. Further work is needed to address this hypothesis.
Previous studies have found that all-trans-retinoic acid increases Bcl2a1 mRNA levels and decreases apoptosis in the NB4 and PBL985 acute promyelocytic leukemia cell lines (55, 56, 57, 58). In a more recent study with PBL985 cells, 9-cis-retinoic acid increased Bcl2a1 expression but the RXR-selective agonist SR11237 did not (59). These results are consistent with our findings that 9-cis-retinoic acid increased Bcl2a1 mRNA levels in both the leukemia cell lines and in primary T lymphocytes. However, in the present study, all-trans-retinoic acid did not increase Bcl2a1 mRNA without coadministration of AGN194204. It is possible that RAR was not expressed at a sufficient level to mediate these effects in our experiments as expression decreased after antigenic stimulation. In addition, the profile of coactivator and corepressor proteins undoubtedly differs between promyelocytic leukemia cells and primary CD4+ T lymphocytes. Such differences could account for the disparate results with RAR and RXR agonists between these two sets of experiments.
Different mechanisms regulate T lymphocyte survival and loss during the different phases of the immune response. During the postactivation phase, stimulated T lymphocytes are susceptible to activation-induced cell death by restimulation via the TCR. Apoptosis can then be induced by binding of Fas ligand to Fas expressed on the surface of susceptible cells. T cell hybridomas are used as models of activation-induced cell death (18). In this system, retinoic acid inhibits Fas-mediated apoptosis by decreasing cell surface Fas ligand expression (19, 20, 21, 22). However, this mechanism is of limited importance following primary antigenic stimulation as naive T lymphocytes are resistant to activation-induced cell death (18, 46).
Survival of naive T lymphocytes during the first few days following primary stimulation is enhanced by several factors, including the activity of growth-promoting cytokines such as IL-2 and IL-4 (15, 18). Although cytokine withdrawal can induce apoptosis and retinoids are known to affect IL-2 production and receptor expression (60, 61), addition of IL-2 and IL-4 to cultures in the present studies did not alter the antiapoptotic effects of AGN194204 and 9-cis-retinoic acid. This result was not unexpected because these cytokines do not induce Bcl2a1 expression in naive T lymphocytes, although IL-4 can induce Bcl2 expression (17).
Stimulation of naive T lymphocytes via the TCR, with appropriate costimulation, induces expression of prosurvival Bcl2 family members Bcl2a1 and Bcl-xL (17, 46, 62). Bcl2a1 mRNA expression peaks in the first 24 h following stimulation than decreases to basal levels within 3 days, while Bcl2a1 protein is detectable on days 1 through 3, with a peak on day 2 (17, 46). Transgenic expression of Bcl2a1a using the Lck promoter (which is stimulated by activation of the TCR) enhances survival of stimulated T lymphocytes for at least 3 days (46). These findings are similar to our observations that RXR agonists prolong Bcl2a1 expression 2 and 3 days after stimulation, with corresponding decreases in apoptosis. Our observation that expression of RNA for Bcl2 and Bcl-xL was not increased by retinoid treatment supports our hypothesis that RXR agonists diminish apoptosis by increasing Bcl2a1 expression. Additional experiments to block Bcl2a1 expression or function following RXR stimulation will be needed to confirm this hypothesis.
In conclusion, we have demonstrated that RXR agonists increase Bcl2a1 expression, decrease apoptosis, and increase cell number following primary antigenic stimulation of naive CD4+ T lymphocytes. In addition, these agonists induce the transcriptional activity of Bcl2a1 using a luciferase reporter gene. Thus, RXR appears to promote the survival of T lymphocytes during initial exposure to Ag. Retinoids may be pro- or antiapoptotic, depending on the type of cell involved, with proapoptotic effects often being seen in transformed cells (63), including T cell lymphomas (27). In the present experiments, RXR ligands play an antiapoptotic role, as has been seen recently for RXR and LXR in macrophages during antibacterial responses (64). Thus, ligands for RXR appear to promote cell survival during the initiation and effector phases of both innate and adaptive immune responses. This role should be of benefit in fighting infectious diseases but may be a mixed blessing during chronic inflammatory or autoimmune disease when increased cell survival may mean more severe disease. Further work is needed on the specific mechanisms of action of RXR and its partner receptors to understand how the immune response may be modified by dietary and pharmacologic ligands for these receptors.
We thank Xiaowen Jiang and Alina Wettstein for technical assistance, Ling Zhao for sharing specific primer sequences for BCL-xL, and Martin Privalsky for commenting on the manuscript. We also thank Daniel Hwang for helpful discussions and comments on these experiments.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by USDA National Research Initiative Grant 97-35200-4229, USDA cooperative agreements 585306-0-233 and 58-5306-1-451, and National Institutes of Health Grant 1 R01 AI 50863.
↵2 Address correspondence and reprint requests to Dr. Charles B. Stephensen, Nutrition Department, 3243 Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616. E-mail address:
↵3 Abbreviations used in this paper: RAR, retinoic acid receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; HSP70, heat shock protein 70; qRT-PCR, quantitative RT-PCR; 7-AAD, 7-aminoactinomycin D; Crabp2, cellular retinoic acid binding protein 2; DR, direct repeat; Hprt, hypoxanthine guanine phosphoribosyl transferase; VDR, vitamin D receptor; TR, thyroid hormone receptor; FXR, farnesoid X receptor; LXR, liver X receptor.
- Received July 8, 2005.
- Accepted September 13, 2005.
- Copyright © 2005 by The American Association of Immunologists