The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, S. N.
Right arrow Articles by Meydani, S. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, S. N.
Right arrow Articles by Meydani, S. N.
The Journal of Immunology, 2006, 177: 6052-6061.
Copyright © 2006 by The American Association of Immunologists, Inc.

Age and Vitamin E-Induced Changes in Gene Expression Profiles of T Cells1,2

Sung Nim Han3,*, Oskar Adolfsson4,*, Cheol-Koo Lee{dagger}, Tomas A. Prolla{ddagger}, Jose Ordovas§ and Simin Nikbin Meydani*

* Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111; {dagger} Division of Biotechnology and Genetic Engineering, College of Life and Environmental Sciences, Korea University, Seoul, Korea; {ddagger} Department of Genetics and Medical Genetics, University of Wisconsin, Madison, WI 53706; and § Nutrition and Genomics Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells are vulnerable to age-associated changes. Vitamin E has been shown to improve T cell functions in the old. We studied gene expression profiles of T cells to better understand the underlying mechanisms of age and vitamin E-induced changes in T cell function. Young and old C57BL mice were fed diets containing 30 (control) or 500 (supplemented) ppm of vitamin E for 4 wks. Gene expression profiles of T cells were assessed using microarray analysis with/without anti-CD3/anti-CD28 stimulation. Genes associated with cytokines/chemokines, transcriptional regulation, signal transduction, cell cycle, and apoptosis were significantly up-regulated upon stimulation. Higher SOCS3 and lower growth factor independent 1 (Gfi-1) expression in old T cells may contribute to age-associated decline in proliferation. Higher Gadd45 and lower Bcl2 expression may contribute to increased apoptosis in old T cells. Vitamin E supplementation resulted in higher expression of genes involved in cell cycle regulation (Ccnb2, Cdc2, Cdc6) in old T cells. Vitamin E supplementation resulted in higher up-regulation of IL-2 expression in young and old T cells and lower up-regulation of IL-4 expression in old T cells following stimulation. These findings suggest that aging has significant effects on the expression of genes associated with signal transduction, transcriptional regulation, and apoptosis pathways in T cells, and vitamin E has a significant impact on the expression of genes associated with cell cycle and Th1/Th2 balance in old T cells. Further studies are needed to determine whether these changes are due to the effects of aging at a single-cell level or to the shift in the ratio of naïve:memory T cells with age.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Aging is associated with the dysregulation of immune response, which is believed to contribute to the higher morbidity and mortality from infectious and neoplastic diseases. Among the immune cells, T cells are the most vulnerable to age-associated functional dysregulation as demonstrated by decreased T cell proliferation and IL-2 production, delayed type hypersensitivity, and T cell-dependent Ab responses (1). Although several investigators have conducted studies to determine the underlying mechanisms of age-related defects in T cell function including those in early signal transduction events, it has been difficult to construct a comprehensive picture of molecular changes that lead to functional dysregulation of T cells. This is mainly due to the fact that activation of T cells involves simultaneous up- or down-regulation of numerous signaling pathways with complex interactions. Thus far, only a limited number of studies have investigated the age-associated changes in the gene expression profile of immune cells (2, 3). Mo et al. (2) stimulated purified CD4+ T cells from young and old mice with anti-CD3 and anti-CD28 for 72 h and determined their gene expression profiles. They found significantly higher expression of CCR2, CCR5, and CXCR5 and lower expression of CCR7 in unstimulated CD4+ T cells from old compared with those from young animals. Visala Rao et al. (3) examined the response of lymphocytes purified from young or old individuals to heat shock (1 h at 42°C), and reported that genes associated with signal transduction and mitochondrial respiration were up-regulated and genes associated with heat shock response and cell survival were down-regulated in lymphocytes from old individuals after heat shock treatment.

Vitamin E has been shown to enhance T cell-mediated functions in aged animals and humans. Vitamin E supplementation in animal and human subjects resulted in increased lymphocyte proliferation, IL-2 production, and decreased production of the immunosuppressive factor PGE2 (4, 5). Furthermore, vitamin E supplementation was shown to reduce the risk of acquiring upper respiratory infection in the elderly (6) and influenza infection in old mice (7). Vitamin E exerts its effect on immune function in part by reducing macrophage PGE2 production (4). However, recent studies indicated a direct effect of vitamin E on T cells independent of its effect on macrophages (8, 9). This immunoenhancing effect of vitamin E on T cells was shown to be mediated through increasing the ability of T cells from old mice to progress through cell division cycles and to produce IL-2 (9). However, further studies are needed to elucidate the mechanisms by which vitamin E specifically enhances T cell function in the aged.

In this study, we investigated the gene expression profiles of T cells from young and old mice to address which genes or group of genes in resting and activated T cells are affected by aging and vitamin E supplementation. We have identified several groups of genes, which are influenced by aging and which could explain the lower ability of T cells to proliferate and their increased susceptibility to apoptosis. We also report that vitamin E supplementation significantly alters expression of genes associated with the cell cycle and the Th1/Th2 balance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and diets

Young (4 mo) and old (24 mo) male C57BL/6NIA mice were purchased from Harlan Teklad and kept at the animal care facility of Jean Mayer Human Nutrition Research Center on Aging (HNRCA) at Tufts University for at least 7 days before they were randomly assigned to the experimental diets. Animals were fed modified AIN-93 diet containing 5% fat (vitamin E-stripped soybean oil; Harland Teklad), and 30 ppm or 500 ppm of vitamin E (dl-{alpha}-tocopherol acetate; Harland Teklad) for 4 wk. Four animals per each group (total of four groups) were used. The vitamin E levels were selected based on our previous studies in which 500 ppm vitamin E was shown to significantly improve T cell-mediated function in old mice (4, 7). In addition, this is the level of vitamin E that results in similar fold increases (2- to 2.7-fold) in blood {alpha}-tocopherol levels of mice compared with those observed in humans following consumption of 200–800 mg/day for a short period of time (4, 10, 11). All animal procedures were approved by the HNRCA Animal Care and Use Committee. Mice were euthanized by CO2 gas asphyxiation, and spleens were processed to a single-cell suspension that was used to prepare purified T cells.

Cells and culture conditions

T cells were purified from spleen cells by negative selection using a mixture of mAbs conjugated to immunomagnetic beads (anti-Mac-1, anti-MHC class II, and anti-CD49b; Miltenyi Biotec). This negative selection method using immunomagnetic beads was shown to result in ≥94% of the enriched cell population expressing CD3 and <1% of cells staining positive for macrophages or B cells (9). Purified T cells (1 x 107 cells/sample) from the same animal were cultured with medium (unstimulated sample) or with 5 µg/ml plate-bound anti-CD3{epsilon} mAb (hamster anti-mouse, clone 145-2C11; BD Pharmingen) and 2 µg/ml soluble anti-CD28 mAb (hamster anti-mouse, clone 37.51; BD Pharmingen) (stimulated sample) for 2 h.

RNA preparation and microarray analysis

Cells were collected after 2 h of culture and washed with PBS. T cell pellets were lysed in RNA lysis buffer (Qiagen). RNA was isolated using a RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Five micrograms of total RNA was converted to double-stranded cDNA using SuperScript Choice System (Invitrogen Life Technologies) with an oligo(dT) primer containing a T7 RNA polymerase promoter (Genset). After second-strand synthesis, the reaction mixture was extracted with phenol-chloroform-isoamyl alcohol, and double-stranded cDNA was recovered by ethanol precipitation. Biotin-labeled cRNA was synthesized from double-stranded-cDNA using a BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics) according to the manufacturer’s instructions. Biotin-labeled cRNA was fragmented to 35–200 bases and hybridized to Affymetrix Murine Genome U74Av2 microarray chips for 16 h at 45°C. Chips were stained with streptavidin-PE (Molecular Probes) and scanned with a Gene Array Scanner (Agilent Technologies).

Statistical analysis

Statistical analysis of gene expression was performed using SAS (SAS Institute). Effects of age and vitamin E on gene expressions in unstimulated and stimulated T cells were determined by ANOVA on expression intensities of genes followed by Tukey’s post hoc test. Effect of stimulation was tested using paired Student’s t test on each pair of unstimulated and stimulated T cells from the same animal (four pairs per group). Differences in response to stimulation between groups were tested using ANOVA, followed by Tukey’s post hoc test on differences in expression intensities between stimulated and unstimulated cells. Significance was set at p < 0.05. Fold changes were calculated after normalization using Affymetrix Microarray Suite 5.0. Genes showing fold changes >2 and p values <0.05, and with at least one present call were selected.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Overview of gene expression patterns: effects of age and vitamin E

Of the 12,422 probe sets on the microarray chip, 6,099 (49.1%) and 5,845 (47.1%) probe sets were expressed in unstimulated and stimulated T cells from the old control group; 6,245 (50.3%) and 5,977 (48.1%) probe sets were expressed in unstimulated and stimulated T cells from the old vitamin E group; 5,961 (48.0%) and 5,991 (48.2%) probe sets were expressed in unstimulated and stimulated T cells from the young control group; and 6,322 (50.9%) and 5,997 (48.3%) probe sets were expressed in unstimulated and stimulated T cells from the young vitamin E group. Neither the age of the animals nor the diets fed had a significant effect on the number of genes expressed. The numbers of genes that were significantly affected by age or vitamin E are shown in Table I, and the data suggest that vitamin E has a more pronounced effect on gene expression in old T cells compared with young T cells.


View this table:
[in this window]
[in a new window]
  		Table I. Effect of age and vitamin E on number of genes expressed in unstimulated and stimulated T cells

 
Overview of gene expression patterns: effect of stimulation

Stimulation of T cells did not significantly increase the total number of genes expressed. Upon stimulation of T cells with anti-CD3 and anti-CD28, 103 genes were up-regulated in at least one of the four groups (Old Control, Old Vitamin E, Young Control, and Young Vitamin E), and 16 genes were down-regulated in at least one of the groups. Genes that showed significant up-regulation upon stimulation were cytokine or chemokine genes, genes associated with regulation of transcription, and those involved in signal transduction, cell cycle, and apoptosis. Selected list of genes that showed significant changes in expression levels with stimulation are shown in Table II.


View this table:
[in this window]
[in a new window]
  		Table II. Effect of anti-CD3/CD28 stimulation on gene expression of T cells

 
Many genes that are known to be the targets of NFAT were up-regulated upon stimulation including IL-2, IFN-{gamma}, GM-CSF, TNF-{alpha}, MIP1-{alpha}, IL-4, IL-13, and Egr2 (12). Significant up-regulation of AP-1 transcriptional complex, c-Fos and JunB, were also observed with stimulation.

Aging is associated with lower expression of TCR-associated genes

Several TCR-related genes were expressed at significantly lower levels in T cells from the old compared with those of young mice (Tables III and IV). Expression of CD3{delta} and TCR-beta mRNA was significantly lower in both unstimulated and stimulated old T cells compared with the young T cells, and expression of CD3{zeta} was significantly lower in unstimulated old T cells compared with T cells from the young mice. Decreased expression of CD28 has been reported to contribute to the impairment of T cell activation in the old (13). However, in this study, we did not observe a significant difference in expression of CD28 genes between young and old T cells. Expression of TCR-related genes including CD3 molecules did not change significantly following anti-CD3/CD28 stimulation.


View this table:
[in this window]
[in a new window]
  		Table III. Effect of age on gene expression profile of unstimulated T cells

 

View this table:
[in this window]
[in a new window]
  		Table IV. Effect of age on gene expression profile of anti-CD3/CD28-stimulated T cells

 
Aging is associated with decreased expression of genes associated with lymphocyte proliferation

T cells from old animals show decreased ability to proliferate following stimulation. This result has been attributed to the functional defects in the early stage of T cell activation such as defects in translocating signaling proteins to the cell membrane and forming an immunologic synapse, and translocation of NFAT (14). We observed significantly higher activation-induced expression of SOCS3 (U88328) (FC 2.70 and 1.20 in old and young, respectively) and lower expression of growth factor independent 1 (Gfi-1;5 U58972) (FC 2.11 and 4.74 in old and young, respectively) (Table II). Gfi-1 is a transcriptional repressor that accelerates the S-phase entry of primary T cells in response to antigenic stimulation, and the SOCS3 gene contains multiple Gfi-1 binding sites in its promoter region (15, 16). Activation of T cell is associated with transient down-regulation of SOCS3 mediated by increased expression of Gfi-1. Overexpression of SOCS3 blocks anti-CD3/CD28-mediated proliferation (17). Therefore, higher expression of SOCS3 may contribute to the observed decrease in T cell proliferation with aging; the higher expression of SOCS3 in the old T cells may be due to lower expression of Gfi-1. Increased expression of SOCS3 with aging has been reported in other tissues such as hypothalamus (18).

Aging is associated with higher expression of apoptosis-related genes

The increase in the mRNA expression of antiapoptotic Bcl2 (L31532) was significantly smaller (FC 2.10 and 3.54 in old and young, respectively) and that of apoptotic and growth-suppressive Gadd45b (X54149) (19) was larger (FC 2.76 and 1.68 in old and young, respectively) in old T cells upon stimulation (Table II). The expression of Fasl (U06948) in both unstimulated (1.98-fold higher in old compared with the young) and stimulated T cells (1.92-fold higher in the old compared with the young), and the expression of Prep (prolyl endopeptidase, AB022053) in stimulated T cells were significantly higher in old T cells compared with young T cells (2.41-fold higher) (Tables III and IV). Recently, higher expression of Prep in T cells has been suggested to be associated with higher susceptibility to activation-induced cell death (20). Upon stimulation, T cells from the old control group showed the highest increase in expression of Egr2 (FC 34.09 in old control, 19.66 in old vitamin E, 20.11 in young control, and 16.01 in young vitamin E), which has been shown to induce transcription of Fasl (21).

Aging is associated with higher expression of Ig-associated genes

Stimulation by anti-CD3 and anti-CD28 did not increase the expression of Ig-related genes. However, both unstimulated and simulated T cells from old mice expressed significantly higher levels of many Ig-related genes compared with those from young mice. Expression of anti-DNA Ig genes such as anti-DNA IgM L chain, anti-DNA IgG L chain, and anti-DNA Ig {kappa}-chain were elevated in T cells from the old mice.

In vivo vitamin E supplementation increased expression of cell cycle-related genes

In our previous study, vitamin E was shown to improve the ability of naive T cells from the old mice, but not young, to progress though the cell cycle as determined by FACS analysis of CFSE-labeled cells at 48 h after anti-CD3 and anti-CD28-mediated stimulation (9). The gene expression patterns from this study indicate that vitamin E has a significant effect on expression of genes associated with the cell cycle (Tables V and VI). Expression of many genes involved in the cell cycle were significantly affected by vitamin E in both unstimulated and stimulated T cells from old mice including Ccnb2 (X66032), Cdc2 or Cdk1(M38724), Cdc6 (AJ223087), and Aurka (U80932) (Table V). Vitamin E did not have a significant effect on these cell cycle-related genes in young T cells. Ccnb2 and Cdc2 are important for entry of cells into the M-phase of the cell cycle, and Cdc6 is a key regulator in the early step of DNA replication (22, 23).


View this table:
[in this window]
[in a new window]
  		Table V. Effect of vitamin E on gene expression profile of unstimulated T cells

 

View this table:
[in this window]
[in a new window]
  		Table VI. Effect of vitamin E on gene expression profile of anti-CD/CD28-stimulated T cells

 
Vitamin E supplementation affects transcription of genes associated with the Th1/Th2 balance

Aging has been reported to be associated with dysregulation of Th1/Th2 balance. In our previous studies (7, 9), we observed an increase in IL-2 production and an enhancement of Th1 response with vitamin E supplementation. In this study, we observed a significant increase in expression of IL-2 (87.04 FC in old vitamin E vs 31.66 FC in old control; 62.73FC in young vitamin E vs 23.04 FC in young control) and IL-1R antagonist (IL-1rn) (L32838, 4.48FC in old vitamin E vs 1.82 FC in old control; 2.13 FC in young vitamin E vs 0.39 FC in young control) genes, and a significant decrease in the expression of IL-4 genes (X03532, 18.29 FC in old vitamin E vs 68.52 FC in old control; 140.63 FC in young vitamin E vs 151.13 FC in young control) with vitamin E supplementation. ANOVA indicated a significant interaction between age and vitamin E on IL-4 mRNA expression, which indicates that vitamin E has a differential effect on IL-4 gene expression in young and old T cells. A significant effect of vitamin E on IL-4 gene was observed in T cells from the old, but not in those from the young mice. Recently, IL-1R antagonist has been shown to down-regulate IL-4 responses and enhance IFN-{gamma} responses (24).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We investigated the effect of aging and vitamin E on gene expression profiles of T cells to better understand the mechanisms involved in functional changes associated with aging and the mechanisms by which vitamin E modulates these defects. Results from this study confirmed previously reported changes in gene expression upon stimulation and with aging in T cells. We also observed age- and vitamin E-induced changes in several genes, which have not been previously reported, but might be of significant relevance to age- and vitamin E-induced changes in T cell functions. However, further studies are needed to determine whether the observed age-related changes are due to the effects of aging at a single-cell level or to the shifts in the ratios of naïve:memory T cells with aging.

Stimulation of T cells did not increase the number of genes expressed. Teague et al. (25) also reported that activation of T cells in vivo, by i.v. injection of the Vbeta8.x-specific superantigen, staphylococcal enterotoxin B, did not change the total number of genes expressed. Our results confirmed the anti-CD3/CD28-induced increase in expression of several genes reported by others (26) such as IL-2, IL-4, IL-13, and IFN-{gamma}. Among the genes that showed significant increase in expression level with stimulation, IFN-{gamma}, MIP-1b, GM-CSF, TNF-{alpha}, IL-2, IL-4, and Egr2 exhibited the highest increase in their expression level (>30-fold increases). Many of these genes are known targets of the NFAT transcription factor. Enrichment of NFAT target genes by CD28 costimulation has been reported by Diehn et al. (26). NFAT has been reported to act synergistically with AP-1 to induce several genes such as IL-2 (12). Significant up-regulation of AP-1 transcriptional complex, c-Fos and JunB, were also observed with stimulation in this study.

We observed an age-related change in the expression pattern of several genes associated with apoptosis. Old T cells have been shown to be more susceptible to apoptosis (27, 28). Several factors have been shown to be involved in higher susceptibility of old T cells to apoptosis including increased expression of Fas/Fasl,TRADD (TNF receptor-associated death domain), FADD (Fas-associated death domain), and Bax, and decreased expression of Bcl2, TRAF-2, and TNF-RII (27). An increased expression of Fas, Fasl, and Bax and a decreased expression of Bcl2 expression were observed with aging in human peripheral blood lymphocytes (28), and an increased protein expression of p53, Bax, and caspase-3 with aging was reported in splenocytes from rats (29). We observed significantly higher expression of apoptotic Fasl in both unstimulated and stimulated T cells from the old compared with that of the young. Higher expression of Prep was observed in the stimulated T cells from the old. Odaka et al. (20) recently reported that T cells expressing higher prolyl endopeptidase activity are more susceptible to activation-induced cell death. We observed less increase in expression of antiapoptotic Bcl2 in old T cells compared with the young following stimulation. Lower expression of Bcl2 in old T cells may be due to an age-associated increase in oxidative stress. Reactive oxygen species have been shown to control the expression of Bcl2, and treatment with synthetic antioxidants increased the expression of Bcl2 in activated T cells (21). However, vitamin E did not increase the mRNA expression of Bcl2 in this study.

We also report here that the expression of Ig-related genes are significantly higher in T cells from the old mice. Many genes for Ig H and L chains were up-regulated in the old T cells. This may be due to higher exposure of the old T cells to antigenic challenge than young T cells. Anti-DNA Ab genes were also expressed at higher levels in the old T cells. A higher percentage of elderly humans are reported to have elevated serum levels of autoantibodies. However, it is not certain whether this increase in autoantibodies with aging puts the aged at higher risk of autoimmune diseases or is an indication of subclinical autoimmune disease status (30). Vitamin E supplementation did not have a significant effect on expression of Ig genes or that of anti-DNA Abs. Similarly, we did not find a significant effect of vitamin E on serum Ig levels or anti-DNA Ab levels in elderly humans (31).

In addition, we have identified several significant, previously unreported, differences in the pattern of gene expression between young and old T cells, which could help explain the phenotypic changes observed with aging. Lower proliferative response of T cells is a common functional change observed with aging. Several different mechanisms have been suggested to be responsible for lower proliferative response of T cells such as decreased expression of CD28 (13, 32), decreased ability to form effective synapses at the site of T cell and APC ligation (14), and defects in signaling pathways (33). In this study, we report for the first time that old T cells express significantly higher levels of the negative feedback regulator SOCS3 following stimulation compared with young T cells. SOCS3 is a member of the SOCS family of intracellular cytokine-inducible negative feedback regulators, which has been shown to play an important role in Th cell activation. Increases in proliferative responses and IL-2 production are associated with the inhibition of SOCS3 expression. Furthermore, overexpression of SOCS3 blocks anti-CD3/CD28-mediated proliferation (17). In this study, stimulation with anti-CD3/CD28 resulted in an average of 2.70-fold increase in SOCS3 in old T cells compared with a nonsignificant 1.20-fold change in young T cells. We also report a significant age difference in the fold change observed in Gfi-1 expression levels following stimulation (2.10-fold change and 4.74-fold change following stimulation in old and young mice, respectively). Gfi-1 is a transcriptional repressor, and the SOCS3 gene contains multiple Gfi-1 binding sites in its promoter (16). Thus, Gfi-1 is one of the key mediators responsible for the down-regulation of SOCS3 during T cell activation. Down-regulation of SOCS3 has been shown to correlate with up-regulation of Gfi-1 (17). Less increase in activation-induced Gfi-1 expression in old T cells compared with that of young might result in higher expression of SOCS3, which could contribute to lower proliferative response of old T cells. Higher expression of SOCS3 with aging in rat hypothalamus has been reported (18). Higher expression of SOCS3 with aging in T cells might also have implications for other age-associated defects in T cell function such as the shift in Th1/Th2 balance, because high SOCS3 expression has been reported to skew helper T cell differentiation toward Th2 cells and inhibit IL-12-mediated STAT4 activation in T cells (34). Vitamin E supplementation, however, did not have a significant effect on the expression levels of SOCS3 and Gfi-1. Therefore, regulation of SOCS3 expression does not appear to be the mechanism through which vitamin E supplementation enhances proliferative response of old T cells.

Several TCR-related genes were expressed at a lower level in T cells from the old compared with those of young mice. Expression of CD3{delta} and TCR-beta was significantly lower in both unstimulated and stimulated old T cells compared with those of young T cells, and the expression of CD3{zeta} was significantly lower in unstimulated old T cells compared with that of young mice. The first step in T cell activation is the engagement of an APC with the TCR-CD3 complex. CD3 is important in mediating signal transduction by coupling TCR with intracellular signaling molecules for ultimate activation and proliferation of T cells. However, age-related changes in CD3 expression have not been conclusive. Wakikawa et al. (35) reported a faster re-expression of TCR{alpha}beta and CD3 after mitogenic stimulation in T cells from young than those from old mice. In contrast, Fulop et al. (36) showed that expression of CD3 in T cells, determined by mean fluorescence intensities, did not differ between T cells from young and old humans. Decreased expression of CD28 was also reported to contribute to the impairment of T cell activation in the old (13). In this study, we did not observe a significant difference in the expression of the CD28 gene between young and old T cells. Xu et al. (37) also reported that CD28 expression was not significantly influenced by age. Thus, further studies are needed to determine the role of TCR and associated genes in age-related defects of T cells.

Activation of T cells in vitro did not result in a significant change in the expression of TCR-related genes including CD3 molecules. In agreement with our findings, expression of the CD3 gene was reported to be unaffected in T cells activated in vivo (25).

One of the most significant effects of vitamin E on T cell gene expression was observed in expression of genes associated with the cell cycle. Impairment of cell cycle progression has been suggested to be partially responsible for the decline of T cell proliferation with aging. Quadri et al. (22) attributed the dysregulation of cell cycle with aging in human T lymphocytes to the lower Cdk1 activity and lower protein level of Ccnb1 and Cdk1/Ccnb1 complex compared with those of young. A decline in the mRNA and protein levels of Ccnd2 at 32–36 h after stimulation with anti-CD3 and anti-CD28 has also been proposed to be responsible for decreased proliferative response in aged CD4+ T cells from mice (38). In this study, we did not observe a significant age-related difference in expression levels of these cell cycle-related genes. Vitamin E, however, increased the expression of cell cycle-related molecules in old T cells, but not young T cells. Expression of Ccnb2, Cdc2 (Cdk1), and Cdc6 were significantly higher (3- to 4-fold higher) in T cells from the vitamin E-supplemented old compared with those of the control group. Many cell cycle regulatory proteins seem to be sensitive to vitamin E status. Fischer et al. (39) reported a significant down-regulation of G1/S-specific Ccnd1 in rat liver by selenium and vitamin E deficiency, which was restored by vitamin E supplementation. Increased expression of Ccnb2, Cdc2, and Cdc6 with vitamin E in old T cells may provide the mechanism for our previous observation that vitamin E increased the ability of naive T cells from the old mice, but not the young mice, to progress through cell cycle divisions (9). Cyclin B and Cdc2 are important for entry of cells into the M phase of the cell cycle. Cdc6 is a key regulator in the early step of DNA replication, and reduction of Ccd6 is associated with the loss of proliferative capacity (23).

Previously, we observed that vitamin E supplementation enhances the Th1 response in old animals challenged with influenza infection, by increasing the production of IFN-{gamma} and IL-2 (7). Modulation of the Th1/Th2 response by vitamin E has been reported by others as well (40). The results from this study indicated that vitamin E supplementation had significant effects on several genes associated with regulation of Th1/Th2 response. A larger increase in expression levels of the IL-1R antagonist and IL-2 following anti-CD3/anti-CD28 stimulation was observed in T cells from young and old animals fed the vitamin E diet compared with those from old mice fed the control diet. Expression levels of IL-4, a major Th2 effector cytokine and a key promoter of Th2 development, increased less following stimulation in old T cells from the vitamin E group compared with those from the old control group. The IL-1R antagonist has been reported to down-regulate IL-4 response while enhancing IFN-{gamma} response in vivo (24). Th1:Th2 ratio, as indicated by ratio of expression levels of IFN-{gamma} and IL-4 in stimulated T cells, were 2.1 for the old control group and 9.4 for the old vitamin E group. This vitamin E-induced difference in the Th1:Th2 gene expression ratio in old T cells is mainly driven by a difference in the expression of IL-4 genes, because there was no statistically significant difference between the old control group and the old vitamin E group in their expression levels of IFN-{gamma} in stimulated T cells.

In this study, we evaluated the effect of age and vitamin E on CD3+ T cells. Changes in the percentage of naive and memory T cells have been reported consistently (1, 41). Thus, it is not clear whether the observed age-induced changes in the gene expression profile of T cells simply reflect a shift in the proportion of naive and memory T cells and the difference in gene expression profiles of naive and memory T cells, or whether these are due to specific changes in either naive or memory T cells or both. Future studies are needed to determine these. Vitamin E, however, did not change the percentage of naive or memory T cells, and its effect was specific to IL-2 production and cell cycle division of naive cells and not memory T cells (9). Thus, the vitamin E-induced gene expression changes are likely mediated through its effect on naive T cells.

In conclusion, we have identified several groups of genes that are differentially expressed with aging, and which could contribute to the functional changes observed with aging such as lower ability of T cells to proliferate and their increased susceptibility to apoptosis. Furthermore, we report that vitamin E supplementation significantly alters expression of genes associated with the cell cycle and the Th1/Th2 balance, which could contribute to the improved T cell proliferation and increase in Th1:Th2 ratios observed in old mice supplemented with vitamin E.


   Acknowledgments
 
We thank Dr. Gerard Dallal for help with statistical analysis and Dr. Angelo Azzi for review of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 U.S. Department of Agriculture (under agreement no. 58-1950-9-001), National Institute on Aging Grant 2RO1 AG009140-10A1, and funding from DSM Nutritional Products. Back

2 Any opinion, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. Back

3 Address correspondence and reprint requests to Dr. Sung Nim Han, Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail address: sungnim.han{at}tufts.edu Back

4 Current address: Nestlé Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland. Back

5 Abreviation used in this paper: Gfi1, growth factor independent 1. Back

Received for publication May 8, 2006. Accepted for publication August 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Miller, R. A.. 1996. The aging immune system: primer and prospectus. Science 273: 70-74. [Abstract]
  2. Mo, R., J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, P. A. Lescure, S. Hanash, R. L. Yung. 2003. T cell chemokine receptor expression in aging. J. Immunol. 170: 895-904. [Abstract/Free Full Text]
  3. Visala Rao, D., G. M. Boyle, P. G. Parsons, K. Watson, G. L. Jones. 2003. Influence of ageing, heat shock treatment and in vivo total antioxidant status on gene-expression profile and protein synthesis in human peripheral lymphocytes. Mech. Ageing Dev. 124: 55-69. [Medline]
  4. Meydani, S. N., M. Meydani, C. P. Verdon, A. A. Shapiro, J. B. Blumberg, K. C. Hayes. 1986. Vitamin E supplementation suppresses prostaglandine E2 synthesis and enhances the immune response of aged mice. Mech. Ageing Dev. 34: 191-201. [Medline]
  5. Meydani, S. N., M. P. Barklund, S. Liu, M. Meydani, R. A. Miller, J. G. Cannon, F. D. Morrow, R. Rocklin, J. B. Blumberg. 1990. Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am. J. Clin. Nutr. 52: 557-563. [Abstract/Free Full Text]
  6. Meydani, S. N., L. S. Leka, B. C. Fine, G. E. Dallal, G. T. Keusch, M. F. Singh, D. H. Hamer. 2004. Vitamin E and respiratory tract infections in elderly nursing home residents: a randomized controlled trial. J. Am. Med. Assoc. 292: 828-836. [Abstract/Free Full Text]
  7. Han, S. N., D. Wu, W. K. Ha, A. Beharka, D. E. Smith, B. S. Bender, S. N. Meydani. 2000. Vitamin E supplementation increases T helper 1 cytokine production of old mice infected with influenza virus. Immunology 100: 487-493. [Medline]
  8. Beharka, A. A., D. Wu, S. N. Han, S. N. Meydani. 1997. Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E. Mech. Ageing Dev. 93: 59-77. [Medline]
  9. Adolfsson, O., B. T. Huber, S. N. Meydani. 2001. Vitamin E-enhanced IL-2 production in old mice: naive but not memory T cells show increased cell division cycling and IL-2-producing capacity. J. Immunol. 167: 3809-3817. [Abstract/Free Full Text]
  10. Meydani, S. N., A. C. Shapiro, M. Meydani, J. B. Macauley, J. B. Blumberg. 1987. Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6Nia mice. Lipids 22: 345-350. [Medline]
  11. Meydani, S. N., M. Meydani, J. B. Blumberg, L. S. Leka, G. Siber, R. Loszewski, C. Thompson, M. C. Pedrosa, R. D. Diamond, B. D. Stollar. 1997. Vitamin E supplementation and in vivo immune response in healthy subjects. J. Am. Med. Assoc. 277: 1380-1386. [Abstract]
  12. Macian, F., C. Lopez-Rodriguez, A. Rao. 2001. Partners in transcription: NFAT and AP-1. Oncogene 20: 2476-2489. [Medline]
  13. Vallejo, A. N., A. R. Nestel, M. Schirmer, C. M. Weyand, J. J. Goronzy. 1998. Aging-related deficiency of CD28 expression in CD4+ T cells is associated with the loss of gene-specific nuclear factor binding activity. J. Biol. Chem. 273: 8119-8129. [Abstract/Free Full Text]
  14. Garcia, G. G., R. A. Miller. 2001. Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice. J. Immunol. 166: 3151-3147. [Abstract/Free Full Text]
  15. Karsunky, H., I. Mende, T. Schmidt, T. Moroy. 2002. High levels of the onco-protein Gfi-1 accelerate T-cell proliferation and inhibit activation induced T-cell death in Jurkat T-cells. Oncogene 21: 1571-1579. [Medline]
  16. Jegalian, A. G., H. Wu. 2002. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B: two routes for STAT5 target gene induction by erythropoietin. J. Biol. Chem. 277: 2345-2352. [Abstract/Free Full Text]
  17. Yu, C. R., R. M. Mahdi, S. Ebong, B. P. Vistica, I. Gery, C. E. Egwuagu. 2003. Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells. J. Biol. Chem. 278: 29752-29759. [Abstract/Free Full Text]
  18. Peralta, S., J. M. Carrascosa, N. Gallardo, M. Ros, C. Arribas. 2002. Ageing increases SOCS-3 expression in rat hypothalamus: effects of food restriction. Biochem. Biophys. Res. Commun. 296: 425-428. [Medline]
  19. Yoo, J., M. Ghiassi, L. Jirmanova, A. G. Balliet, B. Hoffman, A. J. Fornace, Jr, D. A. Liebermann, E. P. Bottinger, A. B. Roberts. 2003. Transforming growth factor-beta-induced apoptosis is mediated by Smad-dependent expression of GADD45b through p38 activation. J. Biol. Chem. 278: 43001-43007. [Abstract/Free Full Text]
  20. Odaka, C., T. Mizuochi, T. Shirasawa, P. Morain, F. Checler. 2002. Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death. FEBS Lett. 512: 163-167. [Medline]
  21. Hildeman, D. A., T. Mitchell, J. Kappler, P. Marrack. 2003. T cell apoptosis and reactive oxygen species. J. Clin. Invest. 111: 575-581. [Medline]
  22. Quadri, R. A., A. Arbogast, M. A. Phelouzat, S. Boutet, O. Plastre, J. J. Proust. 1998. Age-associated decline in cdk1 activity delays cell cycle progression of human T lymphocytes. J. Immunol. 161: 5203-5209. [Abstract/Free Full Text]
  23. Pelizon, C.. 2003. Down to the origin: Cdc6 protein and the competence to replicate. Trends Cell Biol. 13: 110-113. [Medline]
  24. Lin, K. W., S. C. Chen, F. H. Chang, J. T. Kung, B. R. Hsu, R. H. Lin. 2002. The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses. J. Biomed. Sci. 9: 26-33. [Medline]
  25. Teague, T. K., D. Hildeman, R. M. Kedl, T. Mitchell, W. Rees, B. C. Schaefer, J. Bender, J. Kappler, P. Marrack. 1999. Activation changes the spectrum but not the diversity of genes expressed by T cells. Proc. Natl. Acad. Sci. USA 96: 12691-12696. [Abstract/Free Full Text]
  26. Diehn, M., A. A. Alizadeh, O. J. Rando, C. L. Liu, K. Stankunas, D. Botstein, G. R. Crabtree, P. O. Brown. 2002. Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation. Proc. Natl. Acad. Sci. USA 99: 11796-11801. [Abstract/Free Full Text]
  27. Pahlavani, M. A., D. A. Vargas. 2001. Aging but not dietary restriction alters the activation-induced apoptosis in rat T cells. FEBS Lett. 491: 114-118. [Medline]
  28. Aggarwal, S., S. Gupta. 1998. Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl-2, and Bax. J. Immunol. 160: 1627-1637. [Abstract/Free Full Text]
  29. Kapasi, A. A., P. C. Singhal. 1999. Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3. Mol. Cell Biol. Res. Commun. 1: 78-81. [Medline]
  30. Weksler, M. E., M. Goodhardt. 2002. Do age-associated changes in ‘physiologic’ autoantibodies contribute to infection, atherosclerosis, and Alzheimer’s disease?. Exp. Gerontol. 37: 971-979. [Medline]
  31. Meydani, S. N., M. Meydani, J. B. Blumberg, L. S. Leka, M. Pedrosa, R. Diamond, E. J. Schaefer. 1998. Assessment of the safety of supplementation with different amounts of vitamin E in healthy older adults. Am. J. Clin. Nutr. 68: 311-318. [Abstract]
  32. Dennett, N. S., R. N. Barcia, J. D. McLeod. 2002. Age associated decline in CD25 and CD28 expression correlate with an increased susceptibility to CD95 mediated apoptosis in T cells. Exp. Gerontol. 37: 271-283. [Medline]
  33. Fulop, T., Jr, A. Larbi, G. Dupuis, G. Pawelec. 2003. Ageing, autoimmunity and arthritis: perturbations of TCR signal transduction pathways with ageing: a biochemical paradigm for the ageing immune system. Arthritis Res. Ther. 5: 290-302. [Medline]
  34. Kubo, M., T. Hanada, A. Yoshimura. 2003. Suppressors of cytokine signaling and immunity. Nat. Immunol. 4: 1169-1176. [Medline]
  35. Wakikawa, A., M. Utsuyama, K. Hirokawa. 1997. Altered expression of various receptors on T cells in young and old mice after mitogenic stimulation: a flow cytometric analysis. Mech. Ageing Dev. 94: 113-122. [Medline]
  36. Fulop, T., Jr, D. Gagne, A. C. Goulet, S. Desgeorges, G. Lacombe, M. Arcand, G. Dupuis. 1999. Age-related impairment of p56lck and ZAP-70 activities in human T lymphocytes activated through the TcR/CD3 complex. Exp. Gerontol. 34: 197-216. [Medline]
  37. Xu, Z., C. George, C. A. Jolly. 2004. CD28 activation does not down-regulate Cbl-b expression in aged rat T-lymphocytes. Mech. Ageing Dev. 125: 595-602. [Medline]
  38. Hale, T. J., B. C. Richardson, L. I. Sweet, D. L. McElligott, J. E. Riggs, E. B. Chu, J. M. Glynn, D. LaFrenz, D. N. Ernst, R. Rochford, M. V. Hobbs. 2002. Age-related changes in mature CD4+ T cells: cell cycle analysis. Cell. Immunol. 220: 51-62. [Medline]
  39. Fischer, A., J. Pallauf, K. Gohil, S. U. Weber, L. Packer, G. Rimbach. 2001. Effect of selenium and vitamin E deficiency on differential gene expression in rat liver. Biochem. Biophys. Res. Commun. 285: 470-475. [Medline]
  40. Wang, Y., D. S. Huang, B. Liang, R. R. Watson. 1994. Nutritional status and immune responses in mice with murine AIDS are normalized by vitamin E supplementation. J. Nutr. 124: 2024-2032. [Abstract/Free Full Text]
  41. Ernst, D. N., M. V. Hobbs, B. E. Torbett, A. L. Glasebrook, M. A. Rehse, K. Bottomly, K. Hayakawa, R. R. Hardy, W. O. Weigle. 1990. Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4+ T cells from young and aged mice. J. Immunol. 145: 1295-1302. [Abstract]



This article has been cited by other articles:


Home page
 J. Nutr.Home page
A. D. Smith, S. Botero, and O. A. Levander
Copper Deficiency Increases the Virulence of Amyocarditic and Myocarditic Strains of Coxsackievirus B3 in Mice
J. Nutr., May 1, 2008; 138(5): 849 - 855.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
P. A. Sheridan and M. A. Beck
The Immune Response to Herpes Simplex Virus Encephalitis in Mice Is Modulated by Dietary Vitamin E
J. Nutr., January 1, 2008; 138(1): 130 - 137.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Han, S. N.
Right arrow Articles by Meydani, S. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, S. N.
Right arrow Articles by Meydani, S. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS