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* Graduate Program in Nutrition and Department of Nutrition, The Pennsylvania State University, University Park, PA 16802
2To whom correspondence should be addressed at Department of Nutrition, The Pennsylvania State University, 126-S Henderson Building, University Park, PA 16802-5400. Phone: (814) 865-4721; FAX: 814 865-4723
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated proliferative response,
and decreases in the CD4/CD8 T-cell ratio and splenic cell
interleukin-2 production. These differences and the reciprocal changes
observed for NKT cells vs. T- and classical NK cells in aging
VA-marginal rats suggest that low VA status during aging may
increase the risk of infectious or neoplastic diseases that require a
normal balance of T-cell or NK-cell responses.
KEY WORDS: • T-cells • natural T-cell • immunocompetence • vitamin A supplementation • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Pawlec et al. 1998
). The possible mechanisms for this decline may include:
(1) a decrease in T-cell numbers (hematopoiesis, maturation or
distribution); (2) changes in T-cell phenotype, such as a reduction
in the ratio of CD43 to CD8 T-cells or of "naive"
to "memory" helper T-cells; (3) alterations in cytokine production
such as reduced production of interleukin (IL) -2 and interferon-
;
and/or (4) decreased expression or function of cell-surface
molecules such as CD3 and CD28 involved in T-cell signaling
(Miller 1995
, Pawelec et al. 1998
).
Changes in one or more of these parameters were related to
susceptibility to infectious diseases, or disease severity, in aging
rodents or humans. In aging mice, an early onset of morbidity or
mortality was predicted by an increase in "memory" helper
T-cells or in CD8+ T-cells (see Miller 1995
, Pawelec et al. 1998
, and references
therein). In humans, factors that were predictive of either morbidity
or early mortality from infectious disease during aging included a
decline in CD4+ T-cells and an increase in
CD3+/CD28- cells; a reduced in vivo
delayed-type hypersensitivity reaction to various antigens; or a
reduction in a cluster of immune functions [e.g., Concanavalin A (Con
A)-induced T-cell proliferation, CD4/CD8 ratio and B cell numbers
(Miller 1995
, Pawelec et al. 1998
)].
The effects of vitamin A
(VA)4
status on T-cell function during aging are largely unknown. In
young animals, changes in VA status or the administration of natural or
synthetic retinoids were shown to significantly affect T-cell
functions (reviewed in Ross and Hämmerling 1994
).
However, epidemiological evidence is scarce regarding a relationship
between VA status and T-cell function in the elderly. A single
report suggested a positive association between plasma retinol
concentration and delayed-type hypersensitivity in the elderly
(Chavance et al. 1985
), while other studies did not find
any relationship between plasma retinol levels and T-cell function
(Gardner et al. 1997
, Goodwin and Garry 1988
). A small number of studies described attempts to modify
T-cell responses in aged humans through direct supplementation with
ß-carotene or multivitamin and mineral supplements which included
ß-carotene or preformed VA (Pike and Chandra 1995
,
Santos et al. 1997
). Because these studies used
supplements that included other nutrients that may modify immune
responses, it is not possible to ascribe any of the immune system
changes directly to VA.
A very limited number of animal and human studies were conducted to
directly assess the impact of VA status on the aging immune system. In
one study, antigen-stimulated splenocytes from aging mice fed
supplemental VA produced more biologically-active IL-2 and
interferon than controls (Forni et al. 1986
). In a study
on the effects of supplemental VA on T-cell responses of elderly
nursing home residents, those given a daily supplement containing 700
µg RE for 3 mo had a lower number of total T-cells and
CD4+ T cells (Fortes et al. 1998
). Neither
VA deficiency nor marginal VA status was examined in either of these
studies.
Although these data are limited, they allow us to hypothesize on the
impact of diets high and low in VA on T-cell function during aging.
In the current study, we hypothesized that changes in T-cell number
and function that are known to occur with aging, such as reduced cell
numbers, proliferative capacity, and IL-2 production, are likely to be
further reduced by marginal VA status. The effect of lifetime
supplementation with VA (at nontoxic levels) on T-cell phenotype
and function are difficult to predict but may also be
immunosuppressive. With these hypotheses in mind, a unique
long-term study was conducted in which rats were fed diets
throughout life to produce chronic marginal VA status or to maintain a
high level of plasma and tissue VA characteristic of chronic VA
supplementation. The effects of these diets on plasma retinol, physical
and biochemical parameters, and natural killer (NK) cells are described
in Dawson et al. (1999)
. In the present communication,
we report on the percentage, number and function of T-cells and a
recently identified lymphocyte population bearing both T-cell and
NK cell markers (which were referred to variously as "natural"
T-cells, natural killer T-cells, or NKT cells).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animals, diets and experimental design are described in detail by
Dawson et al. (1999)
. Briefly, the effect of VA status
and aging on T-cell number and function was assessed using a 3
x 3 factorial design in which rats were fed AIN-93M rodent diet
(Reeves et al. 1993
) modified to contain VA at 0.35, 4.0
or 50.0 mg of RE (in the form of retinyl palmitate)/kg of diet. These
levels of VA were designated marginal, control and supplemented,
respectively. Rats were raised on these diets until they were 2–3,
8–10 or 20–22 mo of age. These ages were designated young,
middle-aged and old, respectively.
Cellular subsets and surface antigen number determination by flow cytometry.
Tissue collection and determination of peripheral white blood cell
counts, differential counts, and staining procedures were as previously
described (Dawson et al. 1999
). The monoclonal
antibodies used and the cell types with which they react are listed in
Table 1.
PE (phycoerythrin)-labeled streptavidin was obtained from Biosource
International (Camarillo, CA). As controls, anti-trinitrophenol
(TNP) antibodies of the same isotype and same label were used.
Monoclonal antibodies (Table 1)
G4.18, OX-19 JJ319 and OX-1 were
purchased from Pharmingen (San Diego, CA), monoclonal antibodies W3/25,
OX-8, OX-22, ED-1 and OX-12 were purchased from Serotec (Raleigh, NC),
and monoclonal antibody 3.2.3, as murine ascites, was kindly provided
by William Chambers, Pittsburgh Cancer Institute, Pittsburgh, PA, and
was labeled as described by Dawson et al. (1999)
.
|
),
percentage of T lymphocytes, or as the absolute number of
cells/mm3 of blood (WBC count x % of lymphocytes
from the differential count x % of cell type in the lymphocyte
population determined from flow cytometry data). For selected markers, relative cell size, cell granularity, and antigen number were also determined. Determination of the size and fluorescence of dual-labeled (FITC and PE) control beads (Rainbow Brite beads, Spherotec Inc., Libertyville, IL) showed that the daily coefficient of variation for multiple fluorescence channels of both labels, and the size of the beads was always <1%, and the linearity (R2) of the measurement of FITC and PE labels was >0.998.
Cell proliferation assays.
Azide-free anti-CD3 (G4.18, mIgG3) was purchased from
Pharmingen, Con A from Sigma Chemical Co. (St. Louis, MO), and
3H-thymidine from Amersham Life Sciences (Arlington
Heights, IL). The optimal conditions for the proliferative response to
anti-CD3 and Con A were determined beforehand by dose-response
titration over various time points and defined as the lowest dilution
of monoclonal antibody or Con A which provided the maximal
proliferative response. Purified PBMCs or splenocytes were suspended at
2 x 109 cells/L in mitogen media (RPMI 1640
containing 0.1 µmol/L of 2-mercaptoethanol, 10 mg/L of gentamycin, 5
mmolL of glutamine, and 5% heat-inactivated fetal bovine serum
(FBS). Quadruplicate samples of 200 µL for each stimulant as well as
unstimulated control cells were added to 96-well round-bottom
plates. To assess CD3-induced stimulation, 96-well round-bottom
plates were pre-coated with 2 mg/L of anti-CD3 overnight at
4°C. Con A (5 µg/L) was added directly to cultures. Cells
stimulated with CD3 or Con A were incubated at 37°C in an atmosphere
of air/5% CO2 for 48 or 72 h, respectively.
Immediately before pulsing with 3H-thymidine, 100 µL of
supernatant from each well of Con A-stimulated and control cells
was removed for cytokine analysis and replaced with an equal volume of
fresh mitogen media. After addition of 3H-thymidine, cells
were incubated for 4 h at 37°C, then harvested (Series 2800
Harvester; Cambridge Technology Inc., Watertown, MA) onto glass fiber
filter mats and counted by liquid scintillation spectrophotometry.
Cytokine ELISA.
Polyclonal rabbit anti-rat IL-2, biotinylated anti-rat IL-2
(A38-3, mIgG1) and streptavidin-conjugated horseradish
peroxidase were purchased from Pharmingen. Recombinant rat IL-2 and
IL-4 and polyclonal goat anti-rat IL-4 were purchased from R&D
Systems (Minneapolis, MN). Monoclonal anti-rat IL-4 (OX-81,
mIgG1) was purchased from Serotec. Recombinant rat
interferon-, polyclonal mouse anti-rat interferon-, and
anti-rat interferon- (DB-1, mIgG1) were purchased
from Biosource International. The monoclonal antibodies DB-1 and OX-81
were conjugated with biotin using sulfo-NHS-LC-biotin as per the
manufacturer's instructions (Pierce Chemical Co., Rockville, IN).
MaxisorpTM plates (Nunc, Naperville, IL) were coated with 50 µL of
the appropriate concentration of polyclonal capture antibody (8.0 mg/L
for IL-2 and 1.0 mg/L for IL-4 and interferon-) in binding buffer
(0.1 mmol/L Na2HPO4 buffer, pH 9.0) and
incubated overnight at 4°C. After this and each following step, the
plates were washed extensively and nonspecific binding was inhibited.
Samples in duplicate or recombinant cytokine standards (6.1 to 2560
ng/L) and blank wells were loaded on each plate. After overnight
incubation at 4°C, biotinylated detection antibody was added to each
well and, after incubation for 1 h at room temperature and
washing, color was read at 405 nm at 5 min intervals. Data from the
cytokine ELISA are expressed as ng/L of cytokine in culture
supernatants.
Statistical analysis.
Data are expressed as the mean and standard error for each group. The
analysis of data by F-test, two-factor ANOVA, Tukey-Kramer
post-hoc analysis and least-squares means test is described in
Dawson et al. (1999)
. A P value <0.05 was
considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both the percentage and number of total CD3+ T cells in
blood were reduced as rats aged (Table 2
).VA supplementation was associated with a reduced percentage, but not
number, of T-cells. There was a significant effect of VA status,
but not of age, on the percentage of CD3+ T-cells in
the spleen (Table 2)
. Similar data were obtained for CD5+
cells (data not shown). The percentages of CD3+ and
CD5+ cells were highly correlated in blood
(R2 = 0.865, P < 0.0001) and spleen (R2 = 0.808, P < 0.0001).
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. The percentage of
CD3+/CD28+ cells was lower in
VA-supplemented than in VA-marginal or control rats. In
contrast, there was no effect of age or VA on the percentage of
CD3+/CD28+ cells in spleen (Table 2)
.
Cells expressing CD3 at a low to intermediate intensity and lacking
expression of CD28 (CD3int/CD28- cells)
increased with age in blood (percentage and number, Table 2
). The
percentage and number of CD3int/CD28- cells in
blood and the percentage in spleen were higher in VA-marginal than
control or VA-supplemented groups.
The percentage and number of CD4+ T-cells in PBMC and
the percentage in spleen decreased with age (Table 3
).VA status had no significant effect on the percentage or number of
CD4+ T-cells. Both the "naive"
(CD45RC+) and "memory" (CD45RC-)
populations of CD4+ cells were reduced with age (data not
shown). The percentage of CD8+ T-cells increased in
PBMC between young and middle-aged groups (Table 3)
. VA status
significantly affected the percentage, but not number, of
CD8+ T-cells in PBMC, which was lower in
VA-supplemented than VA-marginal rats. In spleen, the
percentage of CD8+ T-cells increased with age (young
<old, P < 0.05), but there was no significant effect
of VA status.
|
).Marginal VA status was also associated with a lower CD4/CD8 ratio in
PBMC, but not in spleen (P = 0.44 for VA,
two-way ANOVA). Double-positive
CD4+-CD8+ T-cells were not found in any
samples.
|
CD3int/NKR-P1dim cells increased as a
percentage of CD3+ cells in PBMC (Fig. 2
A) and spleen (not shown) as rats aged. This increase was also observed in
the number of CD3int/NKR-P1dim cells
(Fig. 2
B). VA status affected both the percentage and
number of CD3int/NKR-P1dim cells in blood
[VA-marginal >control or VA-supplemented rats (Fig. 2A
and 2B
)].
Similar effects of age and marginal VA status were observed on the
percentage of CD3int/NKR-P1dim cells in spleen
(not shown).
|
).The percentage of peripheral blood lymphocytes expressing
CD3int/NKR-P1Adim was directly related to the
percentage of granular CD5+ cells in blood (Fig. 3A)
and
spleen (R2 = 0.185, P
< 0.0001, not shown), and to
CD3int/CD28- cells in blood (Fig. 3B)
and
spleen (R2 = 0.185, P
< 0.0005, not shown). Additionally, the percentage of
CD3+/NKR-P1- cells was directly correlated
with the percentage of CD3+/CD28+ cells (blood,
R2 = 0.746, P < 0.0001;
spleen, R2 = 0.833, P < 0.0001, not shown). Conversely, the percentage of peripheral blood
lymphocytes expressing CD3int/NKR-P1Adim was
inversely correlated with the CD4/CD8 ratio in blood (Fig. 3C)
, and
spleen (R2 = 0.192, P
< 0.0001, not shown).
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Proliferation.
With age, the proliferation of peripheral blood mononuclear cell (PBMC)
T-cells stimulated with anti-CD3 was reduced (Table 4
). However, when proliferation was adjusted for the number of
CD3+ cells present in the population assayed, then age was
no longer a factor. Although marginal VA status was associated with
higher proliferative responses, this effect was significant only
relative to VA-supplemented rats. After adjustment for the number
of CD3+ T-cells, the effect of VA status on
proliferation was diminished, although it still was significant
(P < 0.05). Neither age nor VA status
was a significant factor in the proliferative response of
anti-CD3-stimulated splenocytes (Table 4)
. Results for cells stimulated
with both anti-CD3 and anti-CD28 were similar to those of cells
stimulated with anti-CD3 alone (not shown).
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. However, after
correction for the number of CD3+ cells in the assay,
proliferation in middle-aged and old rats did not difffer (Table 4)
. Cytokine production.
The production of IL-2 by Con A-stimulated PBMC declined
significantly with age (Table 5
).However, VA status was not a significant factor. There was no
age-related difference in IL-2 production by spleen cells, but IL-2
production was higher in VA-marginal than VA-supplemented rats.
Neither age nor VA status was a factor in the production of
interferon- by cells from either blood or spleen (Table 5)
. IL-4 was
not detected in the supernatants from either tissue (limit of detection
5–10 ng/L).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VA status affected several of these variables, especially as animals aged. Indeed, if we had examined the effects of VA status exclusively in young rats, very few statistically significant effects of dietary treatment would have been apparent. However, in old rats in which marginal VA status had been maintained throughout their lives, the percentage of CD8+ T was higher and the ratio of CD4/CD8 was lower compared to any other group. Conversely, cells of the CD3int/NKR-P1dim and CD3int/CD28- phenotypes were significantly elevated. As discussed below, these changes, which are indicative of low T-cell and increased NKT cell populations, suggest changes in functional immune capacity.
In contrast to marginal VA deficiency, VA supplementation resulted in fewer significant differences in T-cells during aging. These differences included a reduction in the percentage of CD3+ cells, lower T-cell proliferation (due to fewer T-cells in the population assayed), and a lower IL-2 response by Con A-stimulated cells.
Flow cytometry was used to characterize various populations of
CD3-positive cells and to determine whether they were correlated to
better understanding population changes due to age or induced by
differences in VA status. The age-related reduction of peripheral
blood T cells was reflected in both CD3+ and
CD5+ cells, which were highly correlated in both blood and
spleen. CD5+ B cells were associated with autoimmunity and
were observed in some aging studies in humans and mice (Miller 1995
). However, the lack of an increase in total CD5 expression
in our rats suggests that expansion of autoreactive cells probably did
not occur in this model.
Increasing age was associated with fewer
CD3+/CD28+ cells in blood (Table 2)
. In
contrast, there was an age-related increase in the percentage and
number of CD3int/CD28- cells. To our
knowledge, this is the first definitive observation of CD28-negative
cells in rodents. The exact function of
CD3+/CD28- T-cells is unknown; however,
these cells were identified in in vitro cultures as potent immune
suppressor cells (Freedman et al. 1991
, Jiang et al. 1998
, Lui et al. 1998
). In clinical studies,
the number of CD3+/CD28- T-cells was
predictive of the degree of immunosuppression during such conditions as
AIDS and after bone marrow transplantation (Batliwalia et al. 1996
). In elderly humans, CD3+/CD28-
T-cells were termed the most reliable marker of lymphocyte aging
(Pawelec et al. 1998
). The progressively higher number
and percentage of these cells in middle-aged and old
VA-marginal rats (Table 2)
suggest accelerated lymphocyte aging due
to this marginal dietary deficiency.
In the present study, it is unknown whether
CD3int/CD28- T-cells were 
/ß or
/
T cells. Based on studies of young Lewis rats, it was
previously reported that all /ß T-cells and 90% of /
T-cells from the blood and spleen express CD28 (Tacke et al. 1995
), and that about 2% of blood and spleen T-cells are
/ T-cells. Based on these data, the expected frequency of
CD3int/CD28- cells in the blood of our young
Lewis rats would be 0.1–0.2% of CD3+ cells. A much higher
proportion (2–5%) of these cells among total CD3+ cells
was found in the young rats in our study. An expansion of /
T-cells is an unlikely explanation for the increase in
CD3int/CD28- cells because the expression of
CD5, which is found at low levels on / T-cells
(Lawetsky et al. 1990
), was not affected by age.
Together, these data imply that a large proportion of the
CD3int/CD28- cells was likely to be present
among /ß T-cells. Furthermore, the expansion of
CD3int/CD28- T-cells in our
middle-aged and old rats is quantitatively similar to previous data
on human blood T-cells (Sansoni et al. 1997
). In one
previous study which examined CD3+/CD28-
T-cells in humans infected with HIV, those who had lower plasma
retinol levels compared to healthy controls had greater numbers of
CD3+/CD28- cells (Semba et al. 1996
). However, whether this decreased level of retinol and
increased numbers of CD3+/CD28- cells reflect
the severity of HIV infection or true VA deficiency is unknown.
The ratio of CD4/CD8 T-cell subsets in blood was previously
reported to decline with age in humans and rodent models (Miller 1995
, Pawelec et al. 1998
). In our study, the
number and/or percentage of CD4+ T-cells were reduced
(Table 3)
. However, we did not detect a change in the proportion of
"virgin" to "memory" CD4+ T-cells with age.
This finding may be related to the observation that the expression of
CD45RA, a marker of naive cells by human CD4+ T-cells,
is relatively stable in the period from 30–70 y, only decreasing
significantly after this interval (Cossarizza et al. 1997
). Of interest, a study of very old Swedish men and women
(>86 y old) showed a low CD4/CD8 T-cell ratio, and low mitogen
response, to be associated with higher mortality (Wikby et al. 1998
). Thus, our old rats may not yet have reached a critical
age.
The ratio of CD4 to CD8 T-cells declined with age in both blood and
spleen (Fig. 1)
. Marginal VA status was associated with a significantly
lower CD4/CD8 T-cell ratio in PBMC (marginal <control or
supplemented, Tukey-Kramer test, P < 0.05).
Although most studies in animals did not detect differences in the
CD4/CD8 T-cell ratio due to changes in VA status (reviewed in
Ross and Hämmerling 1994
), this may be because the
methods used did not adequately distinguish the expression of these
markers, particularly CD8, by non-T cells. In one study of children
reported to be VA-deficient, the CD4/CD8 T-cell ratio was low
(Semba et al. 1993
).
Within the past few years, NKT cells have become recognized as a
lymphocyte population with unique phenotypic and functional properties
(see Bendelac et al. 1997
). In our study, these cells
were quantified by two-color flow cytometry as cells expressing an
intermediate level of CD3 and the NK cell marker, NKR-P1 (expressed
dimly on NKT cells as compared to brightly on NK cells). An interesting
finding is the significant age-related increase in
CD3int/NKR-P1dim cells in both PBMC and spleen
(Fig. 2)
. The mean percentage of T-cells expressing NKR-P1 in
control young, middle-aged, and old rats compares well with
analyses of human blood in which the percentage of T-cells
expressing the homologous antigen to rat NKR-P1 ranged from <5% in
infants to 15–40% in adults (Lanier et al. 1994
). In
our study, the percentage of CD3int/NKR-P1dim
NKT cells was correlated positively with the percentage of
CD8+ T-cells, and negatively with the CD4/CD8
T-cell ratio. The positive correlation with CD8+
T-cells was not unexpected because, in rats, almost all
CD3int/NKR-P1dim cells are also
CD8+ (Brissette-Storkus et al. 1994
). Cells
of the CD3int/NKR-P1dim phenotype were also
positively correlated with granular T-cells and
CD3int/CD28- T-cells (Fig. 3)
. The
expression of lower intensity CD3 by
CD3int/NKR-P1dim and
CD3int/CD28- cells was nearly identical (data
not shown) and each population, by itself, accounted for most of the
CD3int T-cells. From these data, we conclude that a
large proportion of rat CD3int/NKR-P1dim cells
(NKT cells) lacks CD28, suggesting that rat NKT cells are not entirely
analogous to CD4+/NK1.1+ mouse NKT cells (which
express CD28, Bendelac et al. 1997
), but instead more
closely resemble human NKT cells which were characterized as
CD3int/CD8+/CD28-/CD57+
(Batliwalia et al. 1996
).
When the percentage and number of CD8+ T cells or the
CD4/CD8 ratio were adjusted for NKT cells, these variables were no
longer affected by VA status. Although there was still a significant
negative effect of age on the CD4/CD8 ratio, it too was reduced after
adjustment. Therefore, these data strongly suggest that the
age-related increase in the percentage of CD8+
T-cells and the decrease in the CD4/CD8 ratio were driven by an
increase in NKT cells of the phenotype
CD3int/CD28-/NKR-P1dim. Aging in
humans and rats appears to be associated with greater expression of
T-cells with a reduced expression of CD3, often lacking CD28, and
having a T-cell receptor repertoire that is highly restricted
(Effros et al. 1994
, Hosono et al. 1995
,
Posnett et al. 1994
, Watanabe et al. 1995
). The expression of NK cell markers on these unusual cells
is likely to confer additional unique functional properties typically
associated with "innate" immunity.
The mechanism of the expansion of NKT cells during aging is unknown. It
was hypothesized that continuous exposure of experimental animals or
humans to extrinsic or autoantigen(s) results in oligoclonal expansion
of CD8+ T cells (Ku et al. 1997
). It is
unlikely that this expansion is solely due to exposure to autoantigen
or extrinsic antigen because several studies found dramatic oligoclonal
CD8+ T-cell expansion in specific pathogen free mice
and in undiseased humans (Ku et al. 1997
). A more likely
explanation is that these T-cells reflect an increase in
extrathymic T-cell differentiation as a result of decreased thymic
T-cell export (Pawelec et al. 1998
). In the present
study, the expansion of granular cells of the
CD3int/CD8+/CD28-/NKR-P1dim
phenotype is likely to have occurred extrathymically because: (1) no
CD3int/CD28- cells were detected in the
thymus; (2) the mature thymocyte CD4/CD8 ratio actually increased
slightly with age; and (3) no increase in granular cells was detected
among thymocyte populations. One consequence of thymic involution, as
occurs with aging, is that extrathymic sites including the liver,
spleen and the gut-associated lymphoid tissue progressively become
important sites of T-cell maturation and generation (Abo 1993
, Mackall and Gress 1997
). Extrathymically
derived T-cells often differ from thymus-derived T-cells in
phenotype and function; for example, they may express a restricted
T-cell receptor repertoire, less CD3, preferentially express CD8
vs. CD4, and display markers usually associated with NK cells. The data
presented in this and other reports (Brissette-Storkus et al. 1994
; Knudsen et al. 1997
) suggest that rat
CD3int/CD28-/NKR-P1dim T-cells
possess all of these characteristics. It was hypothesized that an
increase in extrathymically-derived T-cells (and also of NK cells)
is an active compensatory mechanism in response to
age-related decreases in thymic T-cell number and function
(Franceschi et al. 1995
). The mechanism of expansion of
CD28- cells by marginal VA status is unknown. It is
interesting and possibly quite relevant that a putative retinoic
acid response element was identified in the human CD28 gene
promoter region (Vallejo et al. 1998
).
In the present study, the number of large granular lymphocytes
increased with age regardless of VA status. These cells included NKT
cells and classical NK cells (NKR-P1Abright, see
Dawson et al. 1999
). However, because VA status affected
the number of NK and NKT cells reciprocally, with decreased NK and
increased NKT cells in marginally VA-deficient animals, the ratio
of NKT to NK cells changed markedly (marginal > control > supplemented by Tukey-Kramer test, P < 0.01 for
blood; and marginal > control or supplemented by Tukey-Kramer
test, P < 0.02 for spleen). This reciprocal regulation
of NKT cells and NK cells could be responsible for a significant
proportion of the changes in immune function in this and other studies
by regulating, for example, the efficacy or characteristics of the
immune response to viruses or tumor cells in vivo (Batliwalia et al. 1996
, Chambers et al. 1996
, Tamada et al. 1997
).
Changes in T-cell numbers due to age and diet also help to explain
differences that were observed in T-cell proliferation and IL-2
production. The proliferative capacity of T-cells stimulated with
anti-CD3 (or anti-CD3 and anti-CD28) was significantly reduced with age
(Table 4)
, and marginal VA status was associated with higher
proliferative responses (P < 0.01).
However, after adjustment for the number of CD3+
T-cells present in the assays, the effect of age was
eliminated and the effect of VA status was diminished
(P < 0.05). These results indicate
that the change in the percentage of T-cells that occurred with age
and VA status was the most important contributor to the changes in
proliferative responses. Similarly, IL-2 production by Con
A-stimulated cells was a greatly diminished in peripheral
blood T-cells during aging (Table 5)
. This effect was also partly
related to a reduction in the number of T-cells. Con A-induced
interferon- production was not affected by age or VA status (Table 4)
, nor was it correlated with the degree of induced proliferation or
percentage of NKT cells or CD4+/CD45RC+
T-cells (two populations with the potential to produce high amounts
of interferon-).
In conclusion, several aspects of T-cell function were
diminished in aging rats, independent of level of dietary VA.
Additionally, marginal VA status was associated with higher
CD8+ T-cell numbers, a lower CD4/CD8 ratio, lower
production of IL-2 by PBMC and splenocytes, an increase in NKT cells.
These changes, and the reduction in NK cells in marginally
VA-deficient animals (Dawson et al. 1999
), became
more apparent during aging and suggest a progressive dysfunction of
hematopoiesis and cell differentiation. Although VA supplementation at
the level used in this study produced fewer effects, the percentage of
T-cells and T-cell proliferative responses were each below
those of control rats. If the changes observed in this rat model are
indicative of changes in aging humans, diets either chronically low or
chronically high in VA may impair the maintenance of T and/or NKT
cell-dependent immune responses during aging, which may increase
the risk of infectious and/or neoplastic diseases in the elderly.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Present address: Laboratory of Immunology,
National Institute on Aging, 5600 Nathan Shock Dr., Baltimore, MD
21224-6825.
4 Abbreviations used: CD, cluster of
determination/differentiation; Con A, Concanavalin A; FBS, fetal bovine
serum; FITC, fluorescein isothiocyanate; IL, interleukin; int,
intermediate; NK cell, natural killer cell; NKT, natural killer T cell;
PBMC, peripheral blood mononuclear cells; PE, phycoerythrin; VA,
vitamin A.
Manuscript received March 31, 1999. Initial review completed May 18, 1999. Revision accepted June 25, 1999.
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