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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370
4To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05 for each age). Across
all ages, the mean differences in popliteal lymph node weights were 1.9
± 0.3 vs. 1.0 ± 0.3 mg with the arginine and isonitrogenous
diets, respectively (P 0.05). Only the 33-mo-old
mice fed the isonitrogenous diet did not respond to this immune
challenge. These findings suggest that arginine supplementation may
enhance in vivo immune responses and/or reverse age-associated
changes.
KEY WORDS: • mice • arginine • aged • immune function • delayed-type hypersensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When supplemented at
pharmacologic levels, arginine prevents thymic involution and increases
mitogen-induced thymocyte proliferation in young-adult rodent
models (Barbul et al. 1980a
and 1980b
). In healthy
humans, dietary arginine supplementation enhances mitogen-induced
peripheral blood lymphocyte proliferation (Barbul et al. 1981a
). Under conditions of stress, arginine supplementation
enhances in vivo delayed-type hypersensitivity (DTH) and in vitro
mitogen-induced lymphocyte proliferation, and prevents
injury-induced atrophy of the thymus (Barbul et al. 1980a
, 1981band 1985
, Kennedy et al. 1994
,
Nirgiotis et al. 1991
, Saito et al. 1987
). Although arginine supplementation appears to benefit in vitro immune responses in healthy adult populations, there is little, if any knowledge of the effect of arginine supplementation on in vivo immune function or on immune responses across ages. Arginine may be of benefit to immune function in the extremes of life. In the young, the immune system is immature, and in the elderly, immune function is impaired.
The age-associated decline in immune function increases
susceptibility to infection (Roberts-Thomson et al. 1974
, Wick and Grubeck-Loebenstein 1997
).
Depressed DTH responses measured in elderly nursing home residents are
associated with increased mortality (Cohn et al. 1983
).
This study evaluated DTH and popliteal lymph node weights after an
immune challenge in mice of three different ages previously
supplemented with a 2% arginine diet. The efficacy of arginine
supplementation on reversing the age-associated changes in these
immune responses was also evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) for aged animal models, hybrid,
specific pathogen–free mice were obtained for this study. Animals and Diets.
Recently weaned 1-mo-old and 10- and 33-mo-old male CB6F1 (BALB/c
x C57BL/6) mice (n = 52) were purchased from
the National Institute of Aging Colonies at Charles River Laboratories
(Stone Ridge, NY). Mice of similar age were housed in groups of two or
three in an environmentally controlled room with constant temperature
(22°C) and 12-h light:dark cycle. Mice were randomly assigned to
either an arginine diet (20 g/kg) or a diet made isonitrogenous to the
arginine diet with the addition of alanine. The 1-mo-old mice received
an AIN 93 G diet (Reeves et al. 1993
) supplemented with
arginine or alanine and the 10- and 33-mo-old mice received an AIN 93 M
diet supplemented with arginine or alanine. The isonitrogenous AIN 93 G
and AIN 93 M diets provided 6.6 and 4.6 g/kg arginine, respectively.
All diets were made by Harlan Teklad (Table 1
; Madison, WI). After a 7- to 10-d acclimation period, the mice were
weighed and given free access to the diets for 2 wk. Free access to
water was given throughout the study.
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On d 9 of the experiment, the mice were anesthetized with methoxyflurane. To sensitize the mice, 50 µL of 5 mL/L 2,4-dinitrofluorobenzene (DNFB) in a 4:1 acetone/olive oil solution was applied to their shaved abdomens. On d 10, the mice were then resensitized to DNFB by again applying 50 µL of the above 5 mL/L solution. On d 14, the mice were challenged with DNFB by applying 20 µL of 2 mL/L DNFB in a 4:1 acetone/olive oil solution to the left ear and 20 µL of the 4:1 acetone/olive oil solution to the right ear to serve as an unchallenged control. The mice were reanesthetized 24 h later and ear swelling was measured with a micrometer by the first author who was unaware of diet group.
Popliteal lymph node weight.
While the mice were anesthetized on d 9 for the DTH protocol, 1.0 x 108 sheep red blood cells (Sigma Chemical, St. Louis, MO) in 50 µL of sterile PBS were injected into the left hind foot pad of the mice and 50 µL of PBS was injected into the right hind foot pad to serve as an unchallenged control. On d 15, the mice were anesthetized and killed by cervical dislocation. The lymph nodes were removed from each leg and weighed.
Statistical analysis.
Beginning animal weights and the percentage of weight change were
compared using two-way ANOVA with a regular factorial design.
Preplanned comparisons were made among means using Fisher’s least
significant difference tests. DTH responses and popliteal lymph node
weights across ages and diets were analyzed using a
repeated-measures ANOVA design with two between-subject
variables and one within-subject variable. Contrast and profile
transformations were made for repeated measures. Within each age group,
comparisons were made using univariate repeated-measures ANOVA to
test for significance between challenged and unchallenged measures with
each diet. To account for experimentwise error, the Bonferroni
procedure was used. All comparisons were made using Statistical
Analysis Systems (version 6.12, SAS Institute, Cary, NC). A
P-level 0.05 was considered significant. Unless
otherwise stated, all data are reported as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial body weights of mice of similar ages were not different between diet groups. After 14 d of diet treatment, the change in weight was not significantly different between age or diet groups (data not shown).
Delayed-type hypersensitivity.
When diet, age and DTH response were considered in a single statistical
model, the DTH response was affected by diet (P 0.05). Across all ages, there was a mean difference of 9.5 ± 0.8
µm in ear thickness between the DNFB-challenged vs.
the unchallenged ear in mice fed the arginine diet. This was
significantly greater than the 2.3 ± 0.7 µm difference in ear
thickness in mice fed the isonitrogenous diet (P 0.05). When data were analyzed within each age group, ear thickness was
significantly greater with DNFB challenge in the arginine-fed mice
at every age (P 0.05). Only the 10-mo-old mice fed
the isonitrogenous diet responded to the DNFB challenge; however, the
response was significantly greater in the arginine-fed mice
(P 0.0001) (Fig. 1
).
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0.05).
Because younger animals have thinner ears, there was a significant
effect of age on all ear measurements that could be attributed to the
size of the mouse. Popliteal lymph node weight.
When diet, age and popliteal lymph node weight were considered in a
single statistical model, there was an effect only of diet on the lymph
node weights from the challenged and unchallenged legs (P
0.05). Across all ages, there was a mean difference of 1.9
± 0.3 mg in popliteal lymph node weights between the leg
challenged with red blood cells and the unchallenged leg in the
arginine-fed mice. This was significantly greater than the 1.0
± 0.3 mg difference in mice fed the isonitrogenous diet
(P 0.05). (Fig. 2
) When data were analyzed within each age group, popliteal lymph node
weights increased after the sheep red blood cell challenge in all diet
and age groups except the 33-mo-old mice fed the isonitrogenous diet.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Early studies by Hori et al. (1973)
demonstrated that
mitogen-induced murine splenocyte proliferation increased from 2 wk
of age until 8 mo of age when functional maturity appeared to be
maximal. After 8 mo of age, the proliferative response decreased with
advancing age. By 36 mo of age, the proliferative response was 2.5% of
maximal (Hori et al. 1973
). Aging of adult animals is
associated with a progressive decline in immune competence with the
major age-related changes attributed to altered T-lymphocyte
function. Such changes include thymic involution and decreased
mitogen-induced proliferation and DTH responses (Goodwin et al. 1982
, Goya et al. 1992
, Hori et al. 1973
, Waldorf et al. 1968
). Changes in the
T-cell subsets, determined by cytokine expression, have also been
observed with aging. There is a decrease in the
TH1-associated cytokines and a parallel increase
in the TH2-associated cytokine secretion
(Hobbs et al. 1991
, Kubo and Cinader 1990
). A shift in T-cell subsets would account for the
decrease in interleukin-2 secretion observed in an aged population.
The DTH response decreases with age (Bender and Tallman 1992
, Goodwin et al. 1982
, Waldorf et al. 1968
), and no DTH response (anergy) is associated with
increased morbidity and mortality (Cohn et al. 1983
,
Meakins et al. 1977
). Our experiments reported here are
consistent with previously published data in that there was no observed
DTH response in the 33-mo-old mice fed the isonitrogenous diet. At the
other extreme of age, young mice fed the isonitrogenous diet were also
unable to mount a DTH response. The mean percentage of increase in ear
thickness in challenged vs. unchallenged ears was 7, 12 and 0% with
the isonitrogenous diet and 27, 35 and 24% with the
arginine-supplemented diet in the 1-, 10-, and 33-mo-old mice,
respectively. (Fig. 1)
. This suggests that arginine supplementation
increased the DTH response in adult mice and reversed
age-associated changes in DTH response in young and aged mice. To
our knowledge, this is the first study to report that arginine
supplementation enhances the DTH response in normal, healthy mice of
any age.
Saito et al. (1987)
demonstrated an enhanced DTH
response with arginine supplementation in a stressed rodent model.
Guinea pigs with burns representing 30% of their total body surface
area were fed intragastrically a solution supplemented with arginine at
0, 1, 2 and 4% of total energy intake. Ear thickness in response to
DNFB challenge was greatest in the group supplemented with 2% of their
total intake as arginine. Additionally, the mortality rates were 56,
29, 22 and 56% in the groups supplemented with 0, 1, 2 and 4%
arginine, respectively (Saito et al. 1987
). This
suggests that arginine supplementation may be beneficial to a point,
after which toxicity may occur. We used a 2 g/100 g arginine diet. This
amount of arginine would represent ~2% of total energy of the growth
and maintenance diets.
Changes in popliteal lymph node weights in response to antigenic
challenge were also measured in this study. Sheep red blood cells
injected into the hind leg foot pad enter the lymphatic vessels and
become trapped in the popliteal lymph node. T lymphocytes within the
node are activated and begin proliferating (Papadimitriou et al. 1983
). Lymphocytes from the blood also migrate into the area
resulting in visible swelling of the nodes.
TH1-type memory T cells preferentially localize
into peripheral lymph nodes (Premier et al. 1996
).
Popliteal lymph node weights from hind legs injected with sheep red
blood cells were significantly increased over control lymph node
weights in young and adult mice fed the isonitrogenous diet
(P 0.05). Aged mice fed the isonitrogenous diet did
not respond to the immune challenge. However, aged mice fed the
arginine diet before immune challenge did respond (P 0.05).
Popliteal lymph node data were also analyzed in a single statistical
model to determine the overall effect of diet and age. Regardless of
age, popliteal lymph node weights were significantly greater in mice
fed the arginine diet than in mice fed the isonitrogenous diet
(P 0.05) (Fig. 2)
. These data support the DTH
findings in that popliteal lymph node weights and the DTH response
across ages were greater in mice fed the arginine diet and that the
response to immune challenge in aged mice was evident only after
arginine supplementation.
Although T lymphocytes may be the effector cells responsible for
directly mediating the observed effects of arginine supplementation
(Barbul et al. 1977
, Kennedy et al. 1994
,
Kirk et al. 1992
, Reynolds et al. 1988
),
the mechanism by which arginine may influence immune function is via
its ability to increase growth hormone secretion (Merimee et al. 1967
). In animal studies, the thymotrophic effects of arginine
after injury are abolished by hypophysectomy. Additionally, arginine
supplementation has no thymotrophic effect in hypophysectomized animals
given growth hormone replacement therapy (Barbul et al. 1983
). In animals and humans, aging is associated with
decreases in growth hormone secretion and a reduction in thymus weight
and function (Goya et al. 1992
, Rudman et al. 1981
). Growth hormone treatment increases the proliferative
response of thymocytes to the T-cell mitogen concanavalin A by
~57 and 328% in middle-aged and old mice, respectively
(Goya et al. 1992
). Although arginine may exert some of
its trophic effect through the action of growth hormone on the thymus,
an intact thymus is not a requirement. Kirk et al. (1992)
demonstrated an increase in mitogen-induced
splenocyte proliferation and DTH response in athymic nude mice
supplemented with arginine. Therefore, growth hormone may stimulate
immune function directly in that many immune cells possess receptors
for growth hormone and its principal mediator, insulin-like growth
factor-I (Eshet et al. 1975
, Geffner et al. 1990
).
These experiments showed no DTH response in young and aged animals and no increase in popliteal lymph node weight after immune challenge in aged animals fed a control diet. Supplementation of diets with pharmacologic levels of arginine enhanced the DTH response in adult mice and reversed age-associated changes in young and aged mice. Aged mice supplemented with arginine also showed an increase in lymph node weight in response to immune challenge. Because an increase in a specific immune variable does not correlate necessarily with a positive clinical outcome, future studies will examine the effect of arginine supplementation on morbidity and mortality after infectious challenge.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by a grant from the North American
Branch of the International Life Sciences Institute (ILSI N.A.). The
opinions expressed herein are those of the authors and do not
necessarily represent the views of ILSI N.A.
3 This is Florida Agricultural Experiment Station, Journal Series No. R-07006.
Manuscript received February 17, 2000. Revision accepted February 23, 2000.
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