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Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, and
Department of Pathology, Sackler Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The significance of IL-2 for cellular immunity is evident by its role in mediating clonal expansion of activated T cells (7). The induction of IL-2 production by activated T cells results in marked up-regulation of high-affinity IL-2R on T cells and, thus, responsiveness to IL-2 and progression through the cell cycle, as well as differentiation of naive T cells into effectors. Therefore, it has been suggested that alteration in IL-2 production with age significantly contributes to the age-dependent dysregulation of cellular immunity (8).
Some of the age-related changes in T cell function may involve free radical damage to critical cell components and other age-related changes accumulated over the life span of the organism. In T cells, the intracellular balance between oxidizing and reducing conditions is closely linked to the cellular proliferative capacity. Production of reactive oxygen species (ROS) by activated macrophages during an inflammatory response as well as cellular depletion of reduced glutathione (GSH), a major cellular reducing agent, has been shown to depress T cell responses (9, 10, 11). The effect of ROS on T cell function has been found to be different between naive and memory T cells. Lohmiller et al. (12) reported that memory T cells had higher levels of GSH compared with naive T cells. Exposure to ROS had a greater suppressive effect on functional measures of naive T cells compared with that of memory T cells.
Vitamin E is the most biologically active fat-soluble antioxidant capable of protecting unsaturated fatty acids in cellular membranes from per-oxidation and thus contributing to membrane stability (13). Both human clinical trials and animal studies have shown a beneficial effect of supplemental vitamin E on immune function in the aged. Meydani et al. (14) reported that when old mice were fed 500 ppm of vitamin E, macrophage production of the T cell-suppressive lipid mediator PGE2 was lowered. In addition, measures of cell-mediated immune function, such as delayed-type hypersensitivity (DTH) response, in vitro T cell proliferation and IL-2 production in response to the T cell mitogen Con A were increased compared with those fed 30 ppm (an adequate dietary level) of vitamin E. A dietary supplement of 800 mg of vitamin E for 30 days to men and woman over 60 years of age, increased DTH, mitogenic response to Con A, and the production of IL-2 (15). A subsequent randomized double-blind, placebo-controlled trial by Meydani et al. (16) found that vitamin E supplementation improved DTH response in a group of healthy elderly subjects. In addition, Ab titer against T cell-dependent hepatitis B vaccine was significantly increased in subjects receiving supplemental vitamin E. Previous studies from our laboratory show that vitamin E exerts its effect in part by reducing macrophage PGE2 production (14, 17). However, preliminary results also indicated a direct effect of vitamin E on T cells independent of its effect on macrophage PGE2 production. Therefore, the purpose of the present study was first to determine whether vitamin E can enhance T cell function in old mice, independently of its enhancing effects mediated through the reduction of macrophage PGE2 production, and, second, to determine the mechanism of the direct enhancing effect of vitamin E on T cell function of old mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Young (6 mo) and old (26 mo) male C57BL/6 mice fed autoclaved Purina (Ralston, St. Louis, MO) mouse chow and water ad libitum were housed in filtered cages maintained at a constant temperature (23°C) with a 12-h light-dark cycle. Mice were euthanized via CO2 asphyxiation and spleens were aseptically removed and placed in sterile, endotoxin-free RPMI 1640 (BioWhittaker, Walkersville, MD) medium supplemented with 5% heat-inactivated FBS, 25 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY; complete RPMI). All handling and animal conditions were approved by the Animal Care and Use Committee of the Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University. Mice exhibiting tumors, splenomegaly, grossly visible skin lesions, or significant pathology were excluded from the study.
Purification of CD3+ spleen cells
Single-cell suspensions were obtained by disrupting spleens between two sterile frosted glass slides. RBCs were lysed using a hemolytic ammonium chloride-based Gey’s solution. Splenocytes depleted of RBCs were washed and resuspended in degassed buffer consisting of PBS, 2 mM EDTA, and 0.5% BSA (Sigma, St. Louis, MO). The cell suspension was then incubated with a mixture of mAb (anti-Mac-1, anti-MHC class II, and anti-NK) directly conjugated to immunomagnetic beads (Miltenyi Biotec, Auburn, CA). Following a 15-min incubation at 4°C, cells were washed and T cells negatively selected using a magnetic-activated cell sorter separation column (Miltenyi Biotec) equipped with a flow restrictor. To assess T cell enrichment, aliquots of unseparated and negatively selected cells were stained with PE-conjugated anti-CD3 and FITC-conjugated anti-CD19 (both from BD PharMingen, San Diego, CA) or FITC-conjugated anti-Mac-1 (Caltag Laboratories, Burlingame, CA) mAb. Cytofluorographic analysis was performed on a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) using standard acquisition and analysis software.
T cell supplementation with vitamin E
A stock solution of natural vitamin E was prepared by dissolving
RRR-
-tocopherol (Henkel, Gulph Mills, PA) in absolute ethanol to a
final concentration of 37 mg/ml. To optimize cellular uptake, a
solution of vitamin E was then mixed in FBS at 1 mg/ml and incubated in
a 37°C water bath for 1 h in the dark with intermittent
vortexing. Purified T cells, adjusted to a concentration of 2 x
106 cells/ml, were preincubated in complete RPMI
supplemented with vitamin E (46 µM) in a 37°C and 5%
CO2 incubator for 4 h. This level of vitamin
E represents the average plasma -tocopherol levels of humans
taking a daily vitamin E supplement of 200 IU (18). T
cells at the same concentration were also suspended in complete RPMI
with 0.06% ethanol as vehicle control. Vitamin E-loaded T cells were
washed twice in serum-free PBS before continuing. To compare the uptake
of vitamin E by T cells from young and old mice, 5 x
106 T cells incubated with or without vitamin E
as above were analyzed by HPLC with electrochemical detection as
described by Wu et al. (19).
Proliferation assay
For the assessment of T cell proliferative capacity, 96-well
round-bottom cell culture plates (Nunc, Roskilde, Denmark) were coated
with 50 µl of anti-CD3
mAb (hamster anti-mouse, clone
145-2C11; anti-CD3i; BD PharMingen) at 5 µg/ml in sterile PBS. Plates
were incubated at 37°C for 90 min and then washed twice with PBS
before cells were plated. In addition to anti-CD3i, cells were also
activated with soluble anti-CD28 mAb (hamster anti-mouse, clone
37.51; BD PharMingen) at 2 µg/ml (anti-CD3i/CD28). Cells were
plated in triplicate for each treatment at 2 x
105 cells/well for 64 h, pulsed with 0.5
µCi of [3H]TdR (New England Nuclear, Boston,
MA) in a 20-µl volume of RPMI, and then incubated for an additional
8 h. The cells were harvested onto glass filter paper and
proliferation was quantified as the amount of
[3H]TdR incorporated into DNA, as determined by
liquid scintillation counting (Beckman Coulter, Fullerton, CA). Data
are expressed as absolute counts per minute.
Secreted and intracellular IL-2 determination
Purified T cells were plated at 1 x 106 cells/well of a 24-well tissue culture plate following preincubation with vitamin E or vehicle control. The cells were then subjected to various stimulation times with anti-CD3i/CD28 or for initial experiments with anti-CD3i only. Cell-free supernatants were collected and stored at -70°C for later analysis. Secreted IL-2 concentration was determined by an ELISA according to the manufacturer’s instructions (BD PharMingen) using reference standard curves prepared with known amounts of mouse rIL-2 (BD PharMingen). For intracellular staining of IL-2, monensin (Sigma) at 2 µM was added to each well for the last 5 h of a 16-h activation period. The cells were collected and incubated with purified anti-CD16/CD32 mAb (Fc block; BD PharMingen) for 5 min and then with PE-conjugated anti-Thy1.2 mAb (Caltag Laboratories) in staining buffer containing 0.1% NaN3 (Sigma) and 1% FBS in PBS for 30 min at 4°C. All staining procedures were performed at 4°C in the dark. The cells were then washed with staining buffer and fixed in 2% formaldehyde for 20 min. Fixed cells were washed again in staining buffer, followed by an incubation with allophycocyanin-conjugated anti-IL-2 mAb (BD PharMingen) or allophycocyanin-conjugated anti-IgG2b (BD PharMingen) isotype control in permeabilization buffer containing 0.1% NaN3, 0.1% saponin (Sigma), and 1% FBS in PBS for 30 min. For the detection of intracellular IL-2 in naive and memory T cells separately, a final concentration of 2 µM monensin was added to each well for the last 10 h of a 48-h activation period. The cells were harvested and incubated with Fc block as before and then with PE-conjugated anti-phagocytic gp1 (anti-CD44) mAb (BD PharMingen). Intracellular staining of IL-2 was performed as described above. The cells were then washed in permeabilization buffer and resuspended in staining buffer for immediate analysis on a FACSCalibur flow cytometer (BD Biosciences) as previously described.
PGE2 production
Purified T cells were plated at 4 x 105 cells/well of a 96-well tissue culture plate following preincubation with vitamin E or vehicle control. After 48 h of stimulation with anti-CD3i or anti-CD3i/CD28, the plates were centrifuged and cell-free supernatants collected and stored at -70°C for later analysis. PGE2 was analyzed by RIA as described by Wu et al. (20). To rule out any possible involvement of PGE2, T cells from young and old mice were also activated and plated with the cyclooxygenase inhibitor indomethacin (Sigma) at 4 µg/ml, and then assayed for proliferation and IL-2 production by ELISA as described above.
RNA preparation and RNase protection assay
RNA was obtained from 1 x 107 T
cells/sample following activation for 0, 6, 16, and 26 h as
described above using a guanidinium thiocyanate-phenol-chloroform-based
extraction procedure according to manufacturer’s instructions (Ambion,
Austin, TX). Following ethanol precipitation, the RNA pellet was
reconstituted in 10–30 µl of RNase-free water. Total RNA
purity and quantity were determined by measuring absorbance at 260 and
280 nm on a UV spectrophotometer (Shimadzu, Columbia, MD). For relative
quantitation of IL-2 mRNA transcripts, an RNase protection assay was
conducted. Briefly, the 32P-labeled antisense
single-stranded IL-2 and L32 RNA probes were synthesized by using an in
vitro transcription kit (Ambion) with a T7 RNA polymerize-directed
synthesis. Linearized single-stranded 204-nt mIL-2 and 141-nt mL32 DNA
fragments were used as templates (BD PharMingen) with
[-32P]UTP (800 Ci/mmol; New England Nuclear)
as the labeled nucleotide. The transcribed probes were purified by
extraction with phenol-chloroform and precipitated with ammonium
acetate and ethanol. Before hybridization, RNA samples were evaporated
in a SpeedVac concentrator. The overnight hybridization and digestion
reaction were conducted using 8 µg of total sample RNA per 5.75
x 105 cpm labeled probe and a combination of
RNase T1 and A (Ambion), respectively. For the separation of protected
dsRNA fragments, the denatured samples were electrophoresed in standard
5% acrylamide/8 M urea gels. A set of RNA markers (Ambion) were
transcribed as described above and run in parallel with samples. Gels
were exposed to BioMax MS film (Kodak, Rochester, NY) at -70°C and
autoradiographs were analyzed by densitometry (Alpha Innotech, San
Leandro, CA).
Cell division analysis of naive and memory T cells
For cell division analysis, purified T cells were incubated with 0.2 µM of the cell tracing compound CFSE (Molecular Probes, Eugene, OR) for 7 min in PBS at room temperature. Staining was stopped with an equal volume of FBS and the cells were washed twice in complete RPMI containing 5% FBS. CFSE-loaded T cells were then activated for 48 h. Upon harvesting, cells were stained with PE-conjugated anti-CD44 mAb or a PE-conjugated IgG2b isotype control Ab (both from BD PharMingen) as described above. In addition, CFSE and CD44 fluorescence was measured in resting T cells. The expression pattern of the CD44 Ag and cell division analysis was determined on a FACSCalibur flow cytometer (BD Biosciences). For the proportion of naive and memory cells going through cell division cycles during the 48-h culture period, each two-dimensional dot plot region representing zero, one, and two cell division cycles was evaluated for low and high expression patterns of the CD44 Ag as described above. The zero cell division cycle histogram peaks and dot plot regions were identified by the peak CFSE fluorescence of unactivated resting T cells.
Statistics
Data were analyzed by a two-way ANOVA with significant differences evaluated by a Tukey’s honestly significant difference (HSD) post hoc procedure using Systat 9 statistical software (Systat, Evanston, IL). Data are reported as mean ± SEM. Significance was set at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-tocopherol by T cells from young and old mice
T cells were purified from the spleens of young and old C57BL/6
mice. Cytofluorographic analysis demonstrated that 
94% of the
enriched cell population expressed CD3, an Ag that defines mature T
cells (data not shown). Cells staining positive for macrophages (Mac-1
Ag) or B cells (CD19 Ag) represented <1% of the total flow-through
population. In vitro supplementation of T cells with 46 µM vitamin E
for 4 h increased cellular vitamin E content in T cells from young
and old mice as determined by HPLC analysis (p
< 0.001; data not shown). There was no significant difference in the
ability of T cells from young and old mice to take up vitamin E under
the conditions of this experiment. Therefore, any functional
differences due to vitamin E cannot be contributed to differential
uptake of the vitamin by T cells from young and old mice.
Effect of vitamin E on the age-dependent decline in T cell proliferation and IL-2 production
Proliferative capacity of purified T cells from young and old mice
was compared using immobilized anti-CD3 mAb only (anti-CD3i) or
in a combination with soluble anti-CD28 mAb (anti-CD3i/CD28).
This mimics T cell activation by providing a primary signal via the
TCR-CD3 complex and a secondary signal through the CD28 T cell
coreceptor. The proliferative capacity of T cells from old mice in
response to anti-CD3i or anti-CD3i/CD28 was significantly lower
(p < 0.001) when compared with T cells from
young mice (Fig. 1
). The addition of
anti-CD28 mAb did not increase proliferation of T cells from young
or old mice above what was observed when anti-CD3i was used alone.
Anti-CD28 mAb activation alone did not induce proliferation above
resting T cells. When T cells were supplemented with vitamin E-enriched
medium, anti-CD3i- and anti-CD3i/CD28-induced proliferative
capacity of T cells from old mice was significantly enhanced (>4-fold
increase; p < 0.05), whereas young T cells did not
show a significant increase. The positive effect of vitamin E on the
proliferative response of T cells in a mixed splenocyte culture
originating from old mice has previously been reported
(17). This was, in part, due to vitamin E-induced
reduction in T cell-suppressive lipid mediator
PGE2, the production of which by macrophages is
increased with age. Purified T cells were used to exclude macrophages,
a major cellular source of PGE2. To rule out any
possible role of PGE2 originating from T cells or
contaminating macrophages, we evaluated whether purified T cells
originating from young and old mice could be stimulated to produce
PGE2. No PGE2 was detected
in any of the samples collected from activated T cells of young or old
mice (data not shown). Furthermore, T cell activation in the presence
of the cyclooxygenase inhibitor, indomethacin, did not alter the
vitamin E-induced effects in old T cells (data not shown). Thus, the
enhancing effect of vitamin E on the proliferation of purified T cells
is not mediated through PGE2.
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). T cells from old mice produced
significantly less IL-2 compared with T cells from young mice, both
when activated with anti-CD3i (p < 0.01)
or with anti-CD3i/CD28 (p < 0.01). To
evaluate the effect of vitamin E on the ability of T cells from young
and old mice to produce IL-2, T cells supplemented with vitamin E
were activated with anti-CD3i or anti-CD3i/CD28. Preincubation
with vitamin E significantly increased both anti-CD3i- and
anti-CD3i/CD28-induced IL-2 secretion by activated T cells from old
mice (p < 0.05). However,
activation-induced secretion of IL-2 by young T cells was not increased
by vitamin E supplementation. Vitamin E restored IL-2 secretion by T
cells from old mice to a level that was not significantly different
from that observed by T cells from young mice. As was observed with
proliferation, indomethacin did not have an effect on IL-2 production
or the ability of vitamin E to increase IL-2 production by old T cells
(data not shown).
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A). In addition,
IL-2-specific fluorescence of IL-2+ T cells from
old mice was less (p < 0.05) compared with
that of young mice, indicating lower activation-induced intracellular
accumulation of IL-2 per T cell from old mice (Fig. 3B).
Supplementing T cells from old mice with vitamin E resulted in an
increased number of activation-induced IL-2+
cells (p < 0.01; Fig. 3A) as well
as an increased level of IL-2 accumulated per cell
(p < 0.05; Fig. 3B). No effect of
vitamin E on T cells from young mice was observed. Thus, for T cells
originating from old mice, supplementation with vitamin E increased the
number of IL-2+ T cells as well as intracellular
IL-2 protein levels, resulting in higher total secreted IL-2 comparable
to levels observed for T cells originating from young mice.
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One major regulatory site for IL-2 production is at the
transcriptional level. Therefore, we next investigated whether the
effect of age and vitamin E on IL-2 was at a pre- or a
posttranscriptional level. A time course study for IL-2 mRNA expression
showed that maximum IL-2 mRNA was obtained following 6 h of
activation for T cells from both young and old mice (data not shown). T
cells from additional young and old mice were next activated with
anti-CD3i/28 for 6 h, and total RNA was extracted and analyzed
for levels of IL-2 mRNA transcripts. Despite lower IL-2 protein levels
at 48 h, old T cells had significantly higher IL-2 mRNA expression
levels at 6 h compared with that of young T cells
(p < 0.05; Fig. 4). Preincubation with vitamin E did not
have an effect on IL-2 mRNA expression in young or old T cells. This
indicated that the age-related defect in IL-2 production and the effect
of vitamin E are posttranscriptional events. Alternatively, T cells
originating from old mice may have responded faster to activation than
young T cells, and vitamin E could have mediated its effect on a slow
responding T cell subpopulation. This prompted us to determine whether
the lower IL-2 protein levels in T cells from old mice at 48 h and
its reversal by vitamin E would also be observed at earlier time
points.
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To better characterize the effect of age and vitamin E on
anti-CD3i/CD28-induced IL-2 secretion, supernatants were collected
at five different time points of activation: 0, 6, 16, 26, and 48
h. As previously observed, at 48 h T cell cultures from young mice
had produced significantly greater amounts of IL-2 protein compared
with those of the old mice (p < 0.05; Fig. 5). In agreement with IL-2 mRNA
expression, at earlier time points of 6 and 16 h, T cells from old
mice produced more IL-2 protein compared with T cells from young mice
(p < 0.05). At 26 h, IL-2 protein
secretion by T cells from young and old mice was comparable.
Supplementation with vitamin E increased the IL-2 protein level by old
T cells only at 48 h (p < 0.05), the time
point at which IL-2 secreted by old T cells was lower than that by
young T cells. There was no indication of an effect of vitamin E at any
of the earlier time points. As was previously seen, the secretion of
IL-2 at 48 h by old T cells preincubated with vitamin E was not
significantly different from that of young controls at the same time
point. These results demonstrate that T cells from young and old mice
have a different time course pattern for IL-2 secretion. Furthermore,
they suggest that the lower overall IL-2 production and proliferation
by T cells from old mice was due to the inability of an IL-2-producing
T cell population in old mice to maintain proliferation and IL-2
production. In addition, these results suggest that vitamin E increased
IL-2 production by preserving the proliferative and/or IL-2-producing
ability of this particular T cell population from the old
mice.
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A major change which occurs in the T cell compartment of the immune system with age is a gradual decrease in the proportion of naive T cells with a corresponding increase in the proportion of Ag-experienced memory T cells (2, 3). Naive T cells have different response kinetics to Ag challenge than memory T cells, with Ag-experienced memory cells responding faster and to a lower Ag dose than naive T cells do (4). This led us to speculate that the higher production of IL-2 and higher IL-2 mRNA expression at an earlier time point from old mice was due to a higher proportion of memory cells among old T cells. On the other hand, the lower IL-2 production at a later time point reflected either the inability of memory T cells from old mice to maintain IL-2 production or naive T cells from old mice to initiate proliferation and IL-2 production. We hypothesized that vitamin E increases IL-2 production by maintaining the function of memory T cells or by initiating cell division of naive T cells from old mice. Thus, we investigated the expression patterns of CD44, a T cell surface marker expressed at high levels on memory T cells (CD44high) and at low levels on naive T cells (CD44low).
As previously reported by others (1, 2), resting T cells
from old mice were predominantly memory T cells of the
CD44high phenotype, whereas T cells from young
mice were predominantly naive T cells of the
CD44low phenotype (p <
0.05; Fig. 6). Vitamin E did not have an
effect on the percent resting naive or memory T cells. To investigate
the progression of naive and memory T cells through cell division
cycles separately, cells were labeled with CFSE cell-tracing reagent
before activation and then stained for CD44 expression following
48 h of activation. Compared with naive T cells from young mice,
naive T cells from old mice were less able to progress through
one and two cell division cycles
(p < 0.05; Figs. 7 and 8). Vitamin E increased the ability of
naive T cells from old mice to progress through one as well as two cell
division cycles (p < 0.05). This effect of
vitamin E was not observed for memory T cells from old mice. This
confirmed that it is the naive but not the memory T cell population
from the old mice that fails to divide when activated. In addition,
higher production of IL-2 at an early time point is a reflection of the
memory-rich population of T cells from old mice, which responds with
faster kinetics. However, at a later time point, naive T cells from old
mice failed to respond, thus resulting in lower overall IL-2 production
by old T cells. Furthermore, vitamin E mediates its effect by
increasing the ability of naive T cells to respond to stimuli by
proliferating and producing IL-2.
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To further confirm that the age-related defect in IL-2 production
was due to a change in the proliferative capability of naive T cells
and that the effect of vitamin E on IL-2 production was specifically
mediated through this population, T cells were activated for 48 h
and then stained for CD44 expression and for intracellular IL-2. This
allowed us to determine IL-2 production by naive and memory T cells
separately. In agreement with the data obtained for cell proliferation,
naive T cells from old mice failed to produce IL-2 at the same level as
young naive T cells. Both the proportion of IL-2+
T cells (Fig. 9A) and the
amount of IL-2 produced per cell (Fig. 9B) was less for
naive T cells from old compared with that of young
(p < 0.05). Vitamin E increased both the
proportion of IL-2+ naive T cells
(p < 0.05) and the amount of IL-2 produced per
naive T cell (p < 0.05). More memory T cells
from old mice were IL-2+
(p < 0.05), but there was no age difference in
the amount of IL-2 produced per memory T cell. Vitamin E did not have
an effect on IL-2 production by memory T cells from young or old mice.
Thus, it is the naive T cell population that exhibits the greatest
age-related defect in IL-2 production, and supplemental vitamin E is
able to specifically enhance the functional properties of this
population of T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The age-related shift in the proportions of T cells from mainly those that have not encountered Ag (naive) to those that have (memory) is a major change that influences T cell-mediated immunity in the aged (2, 3). This, in part, results from decreased output of positively selected immature naive T cells from the aging thymus. Naive T cells have different response kinetics to Ag challenge than memory T cells, with Ag-experienced memory cells responding faster and to a lower Ag dose than naive T cells (4, 21). In addition, the requirements for cell activation have been shown to be different between naive and memory T cells, with naive T cells more dependent on costimulatory signals provided by APCs through the CD28 Ag expressed on T cells (21). Therefore, the anti-CD3i/CD28 stimulation protocol used in the present study should have not compromised naive T cell activation.
The age-related decrease in T cell function has been reported for both the memory (1, 22, 23) as well as the naive (5, 6, 24) T cell subpopulations. The critical role IL-2 plays in the T cell response is reflected in its ability to up-regulate high-affinity IL-2R by T cells and, therefore, responsiveness to IL-2 (7). IL-2 is also critical for T cells to progress through the cell cycle and for the differentiation of naive T cells into effectors. The age-related defect in IL-2 production is an important contributing factor to the decline in T cell function with age (8). Reduced output of IL-2 by naive T cells may indeed influence the generation of functional memory T cells, as responsive but IL-2-negative naive T cells have been shown to become unresponsive memory T cells (25). Using transgenic mice with naive T cells specific for PCC, Linton et al. (5) reported that naive Ag-inexperienced transgene-positive T cells isolated from old mice had lower proliferative and IL-2-producing capacity when compared with naive transgene-positive T cells from young mice. This was supported by Garcia and Miller (6), who found that naive PCC-specific T cells from old mice had at least two age-related functional defects in the early stages of T cell activation: they were defective in translocating signaling proteins to the cell membrane and forming an immunologic synapse, and those which did form a synapse had less nuclear translocation of NFAT, a transcription factor critical for IL-2 production.
We used the anti-phagocytic gp1 Ag (the murine equivalent of CD44) and flow cytometry to separate naive and memory T cell subsets based on low and high expression of this Ag, respectively (26). The age-related defect in activation induced IL-2 production and proliferation at 48 h was mainly observed in naive (CD44low) T cells when anti-CD3i/CD28 mAb was used, an activation protocol known to be particularly effective for stimulating naive T cells (21). We hypothesized that vitamin E increased the total IL-2 production by old T cells by either preserving the proliferation of naive T cells and/or by boosting memory T cell function. When young and old T cells supplemented with vitamin E were examined for their ability to divide in response to activation over a 48-h period, vitamin E significantly increased the ability of naive T cells from old mice to progress through one as well as two cell division cycles. This effect of vitamin E was not observed for memory T cells from old mice. Furthermore, vitamin E supplementation increased IL-2 production by naive T cells from old mice, while there was no effect on IL-2 production by memory T cells.
During the inflammatory response, lymphocytes are exposed to increased
levels of intra- and extracellular ROS. This initial increase in ROS
has an important effect on a subsequent proliferative response as well
as on other cell functions. As Harman (27) first proposed
in 1956, oxygen-derived free radicals cause damage to cells, which
leads to age-associated impairments in functions at organ and cellular
levels. This is supported by studies indicating that aging is
associated with increased free radical burden with resulting damage to
cellular proteins and lipid components (28, 29). The
exposure of T cells to oxidative stress results in decreased IL-2
production, as a consequence of reduced intracellular calcium response,
and altered activity of transcription factors required for IL-2
transcription (30, 31). These alterations are similar to
changes which have been reported to occur in aged T cells
(31, 32, 33). There is ample evidence demonstrating that ROS
and oxidative stress play an integral role in T cell activation
(11, 30, 34, 35, 36, 37). The expression of the TCR/CD3
chain
was reported to decrease in response to
H2O2 or diamide, which
resulted in the suppression of the Ag-specific T cell response
(11). Treatment with N-acetylcysteine, a
glutathione precursor known to increase cellular GSH, prevented this
effect. Other signaling components may respond to oxidative stress due
to the presence of redox-sensitive amino acid residues
(35). Thus, oxidative stress may have various effects on T
cell function by influencing multiple signaling components. Another
factor that may contribute to the age-associated loss of T cell
response is an increase in T cell apoptosis with age. This increase has
been reported for both TNF- and activation-induced apoptosis
(38, 39). Decreased anti-CD3-induced apoptosis of T
cells from old mice has also been reported (40).
Antioxidants may decrease apoptotic events in various cell systems,
particularly when apoptosis is induced by oxidative stress. However, a
recent study showed that vitamin E and catalase failed, while GSH did
prevent apoptosis when human peripheral T cells were induced via Fas-
and CD2-mediated signals (41). This suggests that an
antiapoptotic effect of vitamin E is not likely.
The age-associated decline in T cell response can be partially reversed by dietary (14, 15, 16, 42) or in vitro (10, 17, 19, 43) antioxidant supplementation. Dietary vitamin E supplementation was found to stimulate T helper cell activity in a dose-dependent manner in mice immunized with a hapten-carrier conjugate (44). Sakai and Moriguchi (45) described increased proliferative response and IL-2 production of T cells from old rats in response to Con A when they were fed a high vitamin E diet for 12 mo. It has been reported from work in our laboratory that mitogen-activated macrophages and mixed splenocytes from old mice produce more PGE2 than corresponding cells from young mice (17, 46). The age-related decline in some of the T cell immune parameters such as proliferative response and IL-2 production has been contributed to the age-associated increase in macrophage PGE2 production resulting from increased cyclooxygenase 2 activity in the old mice (15, 46). The effect of supplemental vitamin E on lowering macrophage PGE2 production and the resulting enhancement of cell-mediated immunity have previously been described both for mice and for human subjects (15, 16, 17, 46). However, other effects of supplemental vitamin E on T cell immunity that may not involve PGE2 have been suggested (17).
In the present study, we demonstrate that vitamin E can also enhance the function of T cells from old mice directly, independent from its macrophage PGE2-lowering effect. The effect of vitamin E is mediated through increasing IL-2 production by naive T cells from old mice. This leads to greater cell dividing capacity by naive T cells without an effect on the memory T cell population. The effect of age on the naive T cell population and the preferential effect of vitamin E on naive but not memory T cells from old mice may be due to an underlying difference in the susceptibility of naive and memory T cells to oxidative stress-induced damage. This is supported by a recent report that memory T cells from old mice are more resistant to oxidative injury than naive T cells (12).
In summary, as reported by others, we show here that aging is
characterized by decreased T cell proliferation and IL-2 production. We
also show that vitamin E increases the proliferative capacity of
purified T cells from old mice. This effect of vitamin E is mediated
through increasing the production of IL-2, the major auto- and
paracrine growth factor of activated T cells. Furthermore, T cells from
old mice responded faster to activation than T cells from young mice,
with greater IL-2 protein and mRNA expression at an early time point.
At a later time point though, young T cells proliferated more
vigorously and secreted more IL-2 than old T cells. The faster response
of old T cells was in part due to the higher proportion of fast
responding memory T cells in old mice. However, it was the naive T cell
subpopulation that showed the age-associated defect in progressing
through cell division and producing IL-2, and thus failed to match the
overall response of the young T cells in IL-2 production at a later
time point. Furthermore, our data demonstrate that vitamin E
significantly increased proliferation and IL-2 production of T cells
from old mice. The effect of vitamin E was mediated through increasing
the cell dividing- and IL-2-producing capacity of the naive T cell
subpopulation. Thus, in addition to its inhibition of
PGE2 production by macrophages, vitamin E
increases the function of T cells from old mice through a direct effect
on T cells (Fig. 10). This enhancing
effect is mediated through increased IL-2 production and cell
dividing-capacity of naive T cells. This is the first demonstration of
a nutrient having a direct enhancing effect on naive T cells, a
population of T cells that exhibits the greatest age-related defect.
These findings will have important implications for developing
strategies to reverse age-associated defects in T cell-mediated immune
function. Further studies are needed to elucidate the mechanisms by
which vitamin E specifically enhances the function of naive T cells in
old mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Simin Nikbin Meydani, Nutritional Immunology Laboratory, Jean Mayer Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111. E-mail address: smeydani{at}hnrc.tufts.edu
3 Abbreviations used in this paper: PCC, pigeon cytochrome c; ROS, reactive oxygen species; GSH, reduced glutathione; DTH, delayed-type hypersensitivity; HSD, honestly significant difference.
Received for publication May 30, 2001. Accepted for publication August 9, 2001.
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  T. Ahmed, S. K. Das, J. K. Golden, E. Saltzman, S. B. Roberts, and S. N. Meydani Calorie Restriction Enhances T-Cell-Mediated Immune Response in Adult Overweight Men and Women J Gerontol A Biol Sci Med Sci, November 1, 2009; 64A(11): 1107 - 1113. [Abstract] [Full Text] [PDF]
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M. G. Marko, H.-J. E. Pang, Z. Ren, A. Azzi, B. T. Huber, S. C. Bunnell, and S. N. Meydani Vitamin E Reverses Impaired Linker for Activation of T Cells Activation in T Cells from Aged C57BL/6 Mice J. Nutr., June 1, 2009; 139(6): 1192 - 1197. [Abstract] [Full Text] [PDF]
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Z. Ren, Z. Guo, S. N. Meydani, and D. Wu White Button Mushroom Enhances Maturation of Bone Marrow-Derived Dendritic Cells and Their Antigen Presenting Function in Mice J. Nutr., March 1, 2008; 138(3): 544 - 550. [Abstract] [Full Text] [PDF]
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M. G. Marko, T. Ahmed, S. C. Bunnell, D. Wu, H. Chung, B. T. Huber, and S. N. Meydani Age-Associated Decline in Effective Immune Synapse Formation of CD4+ T Cells Is Reversed by Vitamin E Supplementation J. Immunol., February 1, 2007; 178(3): 1443 - 1449. [Abstract] [Full Text] [PDF]
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S. N. Han, O. Adolfsson, C.-K. Lee, T. A. Prolla, J. Ordovas, and S. N. Meydani Age and Vitamin E-Induced Changes in Gene Expression Profiles of T Cells J. Immunol., November 1, 2006; 177(9): 6052 - 6061. [Abstract] [Full Text] [PDF]
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S. N. Meydani Are Fat-Soluble Vitamins Effective in Enhancing Tumoricidal Cell Activity? J. Nutr., December 1, 2005; 135(12): 2918S - 2918S. [Full Text] [PDF]
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A. Smith, K. B. Madden, K. J. A. Yeung, A. Zhao, J. Elfrey, F. Finkelman, O. Levander, T. Shea-Donohue, and J. F. Urban Jr. Deficiencies in Selenium and/or Vitamin E Lower the Resistance of Mice to Heligmosomoides polygyrus Infections J. Nutr., April 1, 2005; 135(4): 830 - 836. [Abstract] [Full Text] [PDF]
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T. P. Plackett, E. D. Boehmer, D. E. Faunce, and E. J. Kovacs Aging and innate immune cells J. Leukoc. Biol., August 1, 2004; 76(2): 291 - 299. [Abstract] [Full Text] [PDF]
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