Human and Clinical Nutrition

In Vitro Glutathione Supplementation Enhances

Interleukin-2 Production and Mitogenic Response
of Peripheral Blood Mononuclear Cells
from Young and Old Subjects1'2
DAYONG WU, SIMIN /Y. MEYDAHI,3 JUAN SASTRE,* MICHAEL HAYEK AND
MOHSEN MEYDANl*
Nutritional Immunology Laboratory and *Antioxidant Research Laboratory,
U.S. Department of Agriculture Human Nutrition Research Center on Aging
at Tufts university, Boston, MA 02111

et al. 1982, Miller 1989). Upon stimulation, macro

ABSTRACT The effect of in vitro glutathione (GSH)
supplementation on mitogenic response, interleukin-1,
interleukin-2 and prostaglandin £2production, and cel
lular GSH level in peripheral blood mononuclear cells
(PBMC) from healthy young and old human subjects was
studied. In vitro addition of GSH increased cellular GSH
level (P < 0.001). Glutathione supplementation at con
centrations between 2 to 10 mmol/L enhanced lymph
ocyte proliferation but at low concentrations (0.5 and 1
mmol/L) decreased mitogenic response. Glutathione-in-
duced enhancement of lymphocyte proliferation due to
phytohemagglutinin or concanavalin A was greater in the
PBMC from old subjects than in those from young sub
jects. At optimal concentration (5 mmol), GSH in
creased interleukin-2 production (P < 0.05) and
decreased prostaglandin E2 and leukotriene 64
production (P < 0.01) in both age groups. Furthermore,
decreased PBMC mitogenic response by in vitro addition
of prostaglandin £2was reversed by GSH supplemen
tation. Glutathione did not have an effect on ¡nterleukin-
1 production by PBMC from young subjects; however,
GSH supplementation tended (P = 0.08) to increase
interleukin-1 production by PBMC from old subjects. We
conclude that GSH supplementation enhances T cell-
mediated mitogenic response in young and old subjects.
This effect is due at least in part to decreased ei-
cosanoid production. J. Nutr. 124: 655-663, 1994.
INDEXING KEY WORDS:
•glutathione •aging humans
•immune response
The age-associated decline in T cell-mediated
immune response is well documented (Miller 1989).
Qualitative and quantitative changes in T cells as
well as suppressive factors from macrophages con
tribute to the age-associated changes of T cells (Chang
phages release arachidonic acid metabolites that sup
press lymphocyte proliferation and cytokine syn
thesis. Other oxidative metabolites of activated
macrophages, such as hydrogen peroxide, have also
been shown to suppress lymphocyte proliferation
(Metzger et al. 1980). It is interesting to note that
lymphocytes from old humans (Goodwin and Mes
sener 1979) and rats (Franklin et al. 1993) are more
susceptible to the inhibitory effects of suppressive
factors from macrophages, such as prostaglandin
(PG)4 £2-Free radical formation and lipid peroxidation
increase with age, at least in part due to decreases in
the enzymatic and non-enzymatic antioxidant de
fense systems. Along these lines, T cells from aged
animals were shown to have a defective glutathione
peroxidase-dependent detoxification system (Franklin
et al. 1990). Cell membranes are primary targets for
the deleterious effects of free radicals. Cells of the
'Supported in part by USDA contract #53-3K06-l and a gift from
Nutri-Quest Inc. The contents of this article do not necessarily
reflect the views or policies of the U.S. Department of Agriculture,
nor does mention of trade names, commercial products or organiza
tions imply endorsement by the United States government.
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 USC section 1734
solely to indicate this fact.
3To whom correspondence and reprint requests should be ad
dressed.
Abbreviations used: BSO, buthionine sulfoximine; Con A, con
canavalin A; ccpm, corrected counts per minute. CTLL-2, cytotoxic
T lymphocyte line 2; FBS, fetal bovine serum; GSH, glutathione; IL,
interleukin; LT, leukotriene; PBMC, peripheral blood mononuclear
cells; PHA, phytohemagglutinin; PG, prostaglandin.

0022-3166/94 $3.00 ©1994 American Institute of Nutrition.

Manuscript received 8 September 1993. Initial review completed 3 November 1993. Revision accepted 1 December 1993.
655
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 656
immune system have a high content of polyunsatu-
rated fatty acids and thus are highly susceptible to the
harmful effects of products of free radical reactions.
Interventions associated with decreased production of
lipid peroxides, such as vitamin E supplementation
and food restriction, have been shown to enhance the
immune response of the aged (Meydani et al. 1990a
and 1990b).
Glutathione (GSH), a well-known antioxidant and
the most abundant nonprotein thiol in mammalian
cells, has been shown to play an important role in
many biological phenomena, including DNA and
protein synthesis, transport of amino acids, metab
olism of various endogenous compounds (Meister and
Anderson 1983), enzyme activation (Fanger et al.
1970), and protection of cells from oxidative damage
(Reed 1969). In recent years, several studies have em
phasized the importance of intracellular GSH level for
maintenance of the immune function. Essentiality of
GSH for T lymphocyte proliferation has been demon
strated by changing the intracellular GSH level, using
agents that either elevate intracellular GSH [such as
2-oxothiazolidine-4-carboxylate and 2-mercapto-
ethanol (Fidelus et al. 1987)] or deplete intracellular
GSH [such as buthionine sulfoximine (BSO) (Messina
and Lawrence 1989)].
Our previous study revealed that spleens from old
mice have lower GSH levels and T cell-mediated
immune responses than do spleens from young mice.
Dietary GSH supplementation enhanced splenocyte
GSH level, T cell-mediated mitogenic response and
delayed-type hypersensitivity skin response
(Furukawa et al. 1987). The present study was con
ducted to investigate the effect of in vitro GSH sup
plementation on mitogenic response of peripheral
blood mononuclear cells (PBMC) from healthy young
and old subjects and its mechanism of action.
MATERIALS AND METHODS
Peripheral blood mononuclear cells preparation.
Blood was collected from young (25-35 y old) and old
(65-84 y old) fasting male human subjects who were
screened for health status and required to take no
prescribed or over-the-counter medications for 2 wk
before blood collection. The study protocol was ap
proved by the Human Investigation Review Com
mittee of New England Medical Center, Tufts
University. All subjects signed an approved consent
form. Peripheral blood mononuclear cells were sepa
rated from heparinized blood according to the
procedure of Boyum (1968). Peripheral blood
mononuclear cells rather than purified T cells were
used because our hypothesis was that GSH-induced
changes in proliferation and interleukin (IL)-2
production are mediated through a decrease in PGE2
production. Although T cells do not synthesize PGE2,
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WU ET AL.
monocytes are the major source of PGE2 as well as
other oxidants (Goldyne and Stobo 1982). Peripheral
blood mononuclear cells were removed from the in
terface and washed twice in endotoxin-free RPMI
1640 supplemented with 1 x 10s U/L penicillin, 100
mg/L streptomycin, 2 mmol/L L-glutamine and 25
mmol/L HEPES (all from Sigma Chemical, St. Louis,
MO). Endotoxin-free RPMI 1640 was prepared as
described by Schindler and Dinarello (1990). Cell via
bility was assessed using trypan blue exclusion. Cells
were suspended at appropriate concentration in the
medium for measurement of cellular GSH level,
lymphocyte proliferation, and ex vivo interleukin and
eicosanoid production.
Lymphocyte proliferation. Peripheral blood
mononuclear cells at 5 x IO8 cells/L in endotoxin-free
RPMI 1640 with 50 mL/L heat-inactivated fetal
bovine serum (FBS) were cultured in flat-bottomed
96-well microtiter plates (Becton Dickinson, Oxnard,
CA) with or without different concentrations (0.5 to
10 mmol/L) of GSH (Sigma Chemical) and stimulated
by the T cell mitogens concanavalin A (Con A) (0.5 to
10 mg/L) (Sigma Chemical) and phytohemagglutinin
(PHA) (0.5 to 10 mg/L) (Difco, Detroit, MI) for 72 h at
37°C in an atmosphere of 5% CO2 and 95% hu
midity. Mitogenic response was also performed in the
presence of BSO (0.2 mmol/L) (Sigma Chemical) or
PGEj (0.1 fiinol/L) (Cayman, Ann Arbor, MI), alone or
in combination with GSH (5 mmol/L). Cultures were
pulsed by addition of 18.5 Ã-Ã-Bq of [3H]thymidine
(specific radioactivity 247.9 GBq/mol; Du Pont NEN
Products, Boston, MA) in a 20-^iL volume of complete
RPMI 1640 for the final 4 h of incubation. The cells
were harvested onto glass filter paper, and prolifer
ation was quantified as the amount of [3H]thymidine
incorporated into DNA as determined by liquid scin
tillation counting (Beckman Instruments, Palo Alto,
CA). Data are expressed as corrected counts per
minute (ccpm), which is the average cpm of mitogen-
stimulated cultures minus the average cpm of cul
tures without mitogen.
Interleukin-2. Peripheral blood mononuclear cells
at 1 x IO9 cells/L in endotoxin-free RPMI 1640 with
90 mL/L FBS were cultured with or without 5 mmol/
L GSH (optimal concentration) and stimulated by 10
mg/L PHA or Con A (optimal concentration for IL-2
production) for 48 h. Cell-free supernatants were col
lected and stored at -70°C for later analysis of IL-2.
Interleukin-2 activity was measured using the bio-
assay method described by Gillis et al. (1978). Briefly,
the samples at graded dilutions were cultured with
cytotoxic T lymphocyte line 2 (CTLL-2) cells for 24 h.
Cultures were pulsed by adding 18.5 /¿Bq of
[3H]thymidine 6 h before the cells were harvested.
Recombinant human IL-2 (Genzyme Corp., Boston,
MA) was used as standard. One unit per milliliter is
defined as the amount of recombinant IL-2 that
causes a half-maximal incorporation of [3H]thymidine
into 5 x IO3 CTLL-2 cells.
 GLUTATHIONE, AGING AND IMMUNE RESPONSE 657

Interleukin-lft. Peripheral blood mononuclear cells
at 2.5 x IO9 cells/L in endotoxin-free RPMI 1640 with
10 mL/L FBS were cultured with or without 5 mmol/
L GSH and stimulated by 1 /ug/L lipopolysaccharide
(Sigma Chemical) for 24 h. Whole cultures were
stored at -20°C. To measure total IL-1/3 (cell-as
sociated and secreted), the samples were exposed to
three freeze-thaw cycles before IL-1/3 concentration
was determined by RIA (Lisi et al. 1987). Antibody to
IL-10 was a gift from Cistron Biotechnology (Pine
Brook, N}) through J. Cannon of USDA-HNRCA at
Tufts University. 125I-Labeled IL-1/3 was from Du
Pont NEN Products.
Eicosanoids. Peripheral blood mononuclear cells at
1 x IO9 cells/L were preincubated with or without 5
mmol/L GSH at 37°C for 20 min and were then
stimulated with calcium ionophore A23187 (1 mg/L)
for another 20 min. Cell-free supernatant was col
lected by centrifugation at 4°Cand stored at -70"C
for eicosanoid analysis by RIA. The method for PGE2
analysis was based on the procedure described by
McCosh et al. (1976). The details of antibody speci
ficity and crossreactivity have been published
(Meydani and Dupont 1982). Leukotriene (LT) 64 and
LTC4 were analyzed using commercial RIA kits pur
chased from Advanced Magnetic (Cambridge, MA)
and Du Pont NEN Products, respectively, following
manufacturers' instructions.
Cellular glutathione level. Peripheral blood
mononuclear cells at 8 x IO9 cells/L were cultured in
the presence or absence of 5 mmol/L GSH for 20 h
(preliminary studies indicated that cellular GSH level
peaked -20 h after exposure to GSH). At the end of
culture, cells were washed three times with PBS
before being resuspended in 1 mol/L perchloric acid
containing 1 mmol of bathophenanthrolinedisulfonic
acid. The acid extract was sonicated, frozen and
thawed and centrifuged at 2000 x g for 10 min. The
resulting supernatant was derivatized and analyzed by
the HPLC method of Fariss and Reed (1987).
Statistics. The effect of age and GSH supplemen
tation, as well as their interaction, on lymphocyte
proliferation, IL-1, IL-2 and eicosanoid production,
and cellular GSH level was analyzed by two-way
repeated-measure ANOVA using SAS (SAS Institute,
Cary, NC). Multiple comparisons were evaluated
using Tukey's honestly significant difference test
(Daniel 1991). The effect of in vitro addition of BSO
and PGE2 on mitogenic response of lymphocytes in
the presence or absence of GSH was analyzed by
Student's paired î
test, and adjustments for multiple
comparison were made by placing Bonferroni bounds
on probability values. Differences at P < 0.05 were
considered significant. Data are reported as means ±
SEM.
RESULTS
Effect of glutathione supplementation on mito
genic proliferation of PBMC. A wide range of both
GSH (0.5 to 10 mmol/L) and the mitogens Con A and
PHA (0.5 to 10 mg/L) were used in this study. There
was no difference in [3H]thymidine incorporation by
PBMC from young and old subjects in the absence of
mitogen. The optimal concentrations for Con A and
PHA were 2 mg/L in both age groups (Table 1). As
expected, old subjects had a lower mitogenic response
to T cell mitogens. Glutathione supplementation had
a dose-related biphasic effect on lymphocyte prolifer
ation. The summary of statistical analysis is given
first, followed by a more detailed explanation of the

TABLE 1

Mitogenic response of peripheral blood mononuclear cells (PBMC) from young and old subjects to graded levels
of concanavalin A (Con A) and phytohemagglutinin (PHA)1

Mitogen concentration (mg/L]

Con
AYoung
±2717 ±3063 ±3198 ±1724 ± 586
OldPHAYoung 169541,024
8908 ± 270751,683
13,388 ± 278560,530
18,213 ± 234759,413
12,468 ± 99757,264
3568 ±

±4690 ±7558 ±9293 ±9638 ±9740

Old0.519,683 18,304 ±47401.026,458 29,010 ±37572.0ccpm227,457
34,046 ±37815.014,50932,674 ±2761103563 30,699 ±2605
'Peripheral blood mononuclear cells from both young and old subjects were cultured with addition of 0.5-10 mg/L Con A or PHA for 72 h.
Proliferation was measured as described in Materials and Methods. Values are means ±SEMof corrected counts per minute (ccpm), n = 9 for
Con A and n = 8 for PHA in each age group. Repeated-measure ANOVA indicated significant age effect in responses to all levels of PHA (P <
0.02| and to 0.5 and 1 mg/L Con A (P < 0.02 and P < 0.01 for 0.5 and 1 mg/L, respectively). An overall significant mitogen (Con A and PHA) x
age interaction at P < 0.001 and P < 0.0001, respectively, was also observed.
2The cpm of cultures without mitogen were not significantly different from each other (385 ±50 for young vs. 391 ±33 for old subjects).
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 658
40000
0.5 1 25
GSH (mmol/L)
FIGURE 1 Effect of in vitro glutathione (GSH) sup
plementation on concanavalin A-induced peripheral blood
mononuclear cell proliferation. Peripheral blood
mononuclear cells were cultured with optimal concen
tration of concanavalin A (2 mg/L) in presence or absence of
various levels of GSH (0.5 to 10 mmol/L) as described in
Materials and Methods. Values are means ±SEM,n = 9 in
each age group. Values represent corrected counts per
minute (ccpm), which is the cpm of stimulated cultures
minus the cpm of non-stimulated cultures. Repeated-
measure ANOVA indicated significant age (P < 0.001) and
GSH (P < 0.001) effects.
results. Analysis of variance indicated 1} a significant
overall age difference in both Con A- and PHA-in-
duced lymphocyte proliferation (P < 0.05) (Table 1); 2)
a significant biphasic effect of GSH on the mitogenic
response to Con A and PHA (P < 0.001) (Fig. 1); 3) a
significant interaction between GSH and mitogen
level (P < 0.001) (Fig. 2 and 3); and 4) a significant
interaction between age and GSH on mitogenic re
sponse to PHA and Con A (P < 0.05 and P = 0.055, Fig.
3 and 2, respectively). Figure 1 shows the proliferative
response of PBMC induced by optimal concentration
of Con A (2 mg/L) in the presence or absence of
different levels of GSH (0.5-10 mmol/L). Glutathione
did not have an effect on proliferation of unstimu-
lated cultures. In vitro supplementation of PBMC
with GSH, in the presence of mitogens, altered their
proliferation in a biphasic manner. At concentrations
between 2 and 10 mmol/L, GSH enhanced mitogenic
response, with maximal enhancement occurring at 5
mmol/L. At lower concentrations (0.5 and 1.0 mmol/
L), however, GSH decreased mitogenic response. The
pattern of the effect was the same in both age groups.
The magnitude of the GSH effect on mitogenic
response of PBMC differed with the type and dose of
mitogen. Figures 2 and 3 show the percent change in
mitogenic response to different levels of Con A and
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WU ET AL.
110
•young
95 0 old
60
OÃ-
00 65
6
«i
36
20
5-
-10
io 0.5 1 25 10
Con A (mg/L)
FIGURE 2 Effect of in vitro glutathione (GSH) sup
plementation on peripheral blood mononuclear cell (PBMC)
proliferation induced by different levels of concanavalin A
(Con A). Peripheral blood mononuclear cells were cultured
with graded doses of Con A (0.5 to 10 mg/L) in presence of
optimal concentration of GSH (5 mmol/L) for mitogenic
response as described in Materials and Methods. Values
represent percent change compared with medium control
and are means ±SEM, n = 9 in each age group. Percent
change was calculated as:
|ccpm (+GSHI - ccpm (-GSH)]
ccpm (-GSH) x 100.
Two-way repeated measure ANOVA indicated significant
overall GSH effect (P < 0.001). A marginally significant
interaction between GSH and age was observed (P = 0.055).
PHA, respectively, following addition of the optimal
level of GSH (5 mmol/L). Glutathione increased mito
genic response at all concentrations of Con A tested
except for 10 mg/L in young subjects. The largest
increase was observed at suboptimal concentrations
of Con A [0.5 mg/L in young subjects (62%) and 1 mg/
L in old subjects (86%)]. The percent increase caused
by the addition of GSH at Con A concentrations of
0.5 and 1 mg/L was larger in old subjects than in
young subjects. At the optimal concentration (2 mg/L)
of Con A, however, the percent increase in lymph
ocyte proliferation following addition of GSH was not
significantly different between young and old sub
jects. At supraoptimal concentration (10 mg/L) of Con
A, GSH did not increase mitogenic response in young
subjects, whereas a significant enhancement was ob
served in old subjects (Fig. 2). In vitro addition of GSH
(5 mmol/L) increased mitogenic response to 2 mg/L
PHA (optimal concentration) in young subjects and to
2 and 5 mg/L PHA in old subjects (Fig. 3). The percent
increase caused by GSH at 1, 2, 5 and 10 mg/L PHA
was higher in old subjects than in young subjects.
 GLUTATHIONE, AGING AND IMMUNE RESPONSE 659

TABLE 2
Effect of in vitro glutathione (GSH) supplementation on ex vivo interleukin-2 production by peripheral blood mononuclear cells
(PBMC) from young and old subjects1

PHA Con A
Age Control GSH Control GSH
x 10~3 units/L
Young (n = 7) 48.7 ±15.6 145.7 ±30.0* 63.7 ±23.5 155.3 ±34.0*
Old |n = 6) 27.8 ± 6.0 113.0 ±27.6* 22.6 ± 4.4 104.2 ±22.0*
'Peripheral blood mononuclear cells were cultured with or without addition of 5 mmol GSH, and stimulated by 10 mg/L phytohemaglu-
tinin IPHA) or concanavalin A (Con A) for 48 h. Interleukin-2 was measured by bioassay using CTLL-2 cells as described in Materials and
Methods. Values are means ±SEM. *Significantly different from control at P < 0.05.

Effect of glutathione supplementation on ex vivo from old subjects (P = 0.16 and P = 0.08, respectively).
interleukin-2 production by PBMC. Interleukin-2 Addition of 5 mmol/L GSH significantly increased IL-
production was measured in the presence or absence 2 production induced by both mitogens in both age
of optimal level of GSH (5 mmol/L). Glutathione groups (197 and 140% in young subjects vs. 306 and
supplementation did not have an effect on prolifer 372% in old subjects for PHA and Con A, respec
ation of the CTLL-2 cells used for IL-2 assay (data not tively).
shown). As shown in Table 2, both PHA- and Con A- Effect of glutathione supplementation on inter-
induced IL-2 production by PBMC from young sub leukin-lß production by PBMC. Lipopolysaccharide-
jects tended to be higher than production by PBMC induced IL-Ißproduction was not different between
the two age groups. Addition of 5 mmol/L GSH
tended to increase (P < 0.08) total IL-Ißproduction by
PBMC from old subjects only (Table 3).
Effect of glutathione supplementation on ei-
young cosanoid production by PBMC. No significant
old difference was found between the two age groups in
synthesis of PGE2 by PBMC stimulated with calcium
ionophore A23187. With addition of 5 mmol/L GSH,
PGE2 synthesis by PBMC from both age groups was
significantly inhibited (P < 0.01) (Table 4). Leu-
kotriene 64 production was examined in PBMC
samples from four young and six old subjects. As
shown in Table 5, GSH supplementation significantly
suppressed LTB4 production in both young and old
subjects. Leukotriene C4 production (analyzed on

0.5

PHA (mg/L) TABLE 3

Effect of glutathione (GSH) supplementation on ex vivo

interleukin-l$ production by peripheral blood mononuclear
FIGURE 3 Effect of in vitro glutathione (GSH) sup cells from young and old subjects1
plementation on peripheral blood mononuclear cell (PBMC)
proliferation induced by different levels of phytohemag-
Age Control GSH % Change
glutin (PHA). Peripheral blood mononuclear cells were cul
tured with graded doses of PHA (0.5 to 10 mg/L) in presence
of optimal concentration of GSH (5 mmol/L) for mitogenic =
Young 1.17
response as described in Materials and Methods. Values Old (n(n= 10|10)5.45 13.35*
5.90±
±US
0.69115.53
8.63±±0.78
1.511.47 48.51±
±0.93
represent percent change compared with medium control
and are means ±SEM, n = 8 in each age group. Two-way 'Peripheral blood mononuclear cells were cultured with or
repeated-measure ANOVA indicated significant overall without 5 mmol GSH and stimulated by 1 jtg/L lipopolysaccharide
GSH effect (P < 0.001) and interaction between GSH and age for 24 h. Interleukin-10 was measured by RIA. Values are means ±
(P < 0.05). SEM. *P = 0.077.
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 660 WU ET AL.

TABLE 4 TABLE 6

Effect of glutathione (GSH) supplementation on ex vivo Effect of glutathione (GSH) supplementation on cellular GSH
prostaglandin Eg production by peripheral blood mononuclear level of peripheral blood mononuclear cells (PBMC) from
cells (PBMC) from young and old subjects1 young and old subjects1

Age Control GSH % Inhibition AgeYoung GSH\imol/L1.12 Increase153

Young 0.76 ±0.10 2.79 ±0.28 ±16-

Old1.13 1.17± 0.02 0.69±
±nmol/L0.16 0.0931
±0.12 33±±6*7' OldControl 0.92 ±0.08 2.32 ±0.10% 167 ±32'
'Peripheral blood mononuclear cells were pretreated with 5 'Peripheral blood mononuclear cells were cultured with or
mmol/L GSH for 20 min at 37°Cand then stimulated with calcium
without 5 mmol/L GSH for 20 h, and then washed, extracted,
ionophore A23187 (1 mg/L] for another 20 min. Prostaglandin £2 derivatized and analyzed by HPLC as described in Materials and
content was determined by RIA. Values are means ±SEM,n = 9 in Methods. Values are means ±SEM, n = 7 in each age group.
each age group. 'Significant inhibition by GSH at P < 0.01. 'Significant increase at P < 0.001.

samples collected from old subjects only) was not
affected by GSH supplementation.
Cellular glutathione level measurement.
Peripheral blood mononuclear cells were cultured
with or without addition of GSH at 5 mmol/L for 20 h
and measured for cellular GSH level. As shown in
Table 6, cellular GSH level in old subjects tended to
be lower than that in young subjects [P = 0.11). In
vitro supplementation with GSH significantly
creased cellular GSH level in both age groups (P <
0.001).
Effect of in vitro addition of buthionine sulfox-
imine and prostaglandin E¿on concanavalin A-
induced PBMC proliferation. For these studies, the
PBMC from five young and five old subjects were
used. A suboptimal dose of Con A (1 mg/L), at which
maximal effect of GSH can be seen, was used to
activate the cell proliferation. As can be seen in
Figure 4, response of PBMC from old subjects to Con
A was consistently lower in cultures without GSH,
and its enhancement by adding 5 mmol/L GSH was
larger in old subjects than in young subjects. At con
centrations as low as 0.2 mmol/L, buthionine sulfox-
imine (an inhibitor of GSH synthesis) exhibited a
suppressive effect on pHjthymidine incorporation
[42% (P < 0.08) and 49% (P < 0.05) in young and old
subjects, respectively). This inhibition was com
pletely reversed in both age groups by addition of 5
mmol/L GSH. Further increasing the dose of BSO
caused a total inhibition of PBMC proliferation that
was not reversed by addition of GSH (data not
in
shown). At a dose of 0.1 /¿mol/L,PGE2 significantly
inhibited [3H]thymidine incorporation compared with
its vehicle control in old subjects (36% inhibition, P <
0.01). However, PGE2 only tended to inhibit incorpo
ration in cells from young subjects (19% inhibition, P
E2 young
•old

TABLE 5

Effect of glutathione (GSH) supplementation on ex vivo

¡eukotriene (IT) 84 and LTQ production by human
peripheral blood mononuclear cells (PBMC)1
0 10000 20000

Control GSH % Change 3H-Thymidine Incorporation tccpm)

nmol/LLTB4LTB4LTC4(young,(old,
4)- = 0.07±
FIGURE 4 Effect of buthionine sulfoximine (BSO) and
6)-8)0.710.830.54±±±0.030.090.050.470.500.55±
0.05±
prostaglandin (PG) £2 on lymphocyte proliferation in
n(old,
presence
0.04-34-401.8±±±7*3**0.4
and absence of glutathione (GSH). Peripheral blood
nn
mononuclear cells were cultured in presence or absence of
'Peripheral blood mononuclear cells were pretreated with 5 the following compounds: 5 mmol/L GSH, 0.2 mmol/L BSO
mmol/L GSH for 20 min at 37°Cand then stimulated with calcium or 0.1 ftmol/L PGÊ2,and stimulated by concanavalin A (1
ionophore A23187 (1 mg/L) for another 20 min. Leukotriene B4 and mg/L). Results are means ±SEM, n = 5 in each age group. 'P
LTC4 levels were determined using RIA. Values are means ±SEM; < 0.05, "P < 0.01, compared with medium control cultures
'P < 0.01, "P < 0.001 compared with controls. (without GSH); ccpm = corrected counts per minute.
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 GLUTATHIONE,
< 0.07). Addition of 5 mmol/L GSH to culture totally
reversed PGE2-induced inhibition (Fig. 4).
DISCUSSION
Intracellular GSH level has been shown to play an
important role in different aspects of T cell function,
including the binding, internalization and degradation
of IL-2, and DNA synthesis (Fidelus et al. 1987, Liang
et al. 1989, Messina and Lawrence 1989, Suthanthiran
et al. 1990). It has been reported that intracellular
GSH decreases with age and might contribute to age-
associated decline of T cell-mediated function
(Furukawa et al. 1987). Reduced levels of GSH have
also been implicated in other conditions associated
with impaired immune function, such as AIDS (Eck
et al. 1989). In this study, we investigated the effect of
in vitro GSH supplementation on human PBMC cel
lular GSH level, cytokine production and prolifer
ation. Effect of GSH on eicosanoid synthesis was
studied as a possible mechanism of the GSH effect.
No significant difference in PBMC GSH level was
observed between young and old subjects. Peripheral
blood mononuclear cells from old subjects, however,
tended to have lower GSH levels than those from
young subjects (P = 0.11). Lower GSH level, however,
has been reported in spleens from old mice compared
with young mice (Chen et al. 1990, Furukawa et al.
1987). The lack of a significant difference in PBMC
GSH content between young and old subjects might
argue against involvement of GSH in age-associated
decrease of proliferation. Some studies, however, have
indicated differential distribution of GSH in subpopu
lation of lymphocytes (Kavanagh et al. 1990). Thus,
although a significant decrease in total PBMC GSH
content might not occur with aging, GSH content of a
particular subpopulation of lymphocytes that are im
portant in IL-2 production and proliferation might be
affected by aging. On the other hand, the lack of a
significant difference in this study might also rep
resent heterogeneity of age-related changes in humans
compared with inbred mice. Thus, a larger sample
than that used in this study might be required to
reach statistically significant difference in GSH
content between young and old subjects. Addition of
GSH to culture medium increased cellular GSH levels
by 153 and 167% in young and old subjects, respec
tively (Table 6). Meister and Anderson (1983) pro
posed that exogenous GSH can increase cellular GSH
level through breakdown of exogenous GSH by the
action of 7-glutamyl transpeptidase and dipeptidase,
absorption of products formed, and resynthesis of in
tracellular GSH.
Glutathione has been shown to be essential for
lymphocyte activation. In the present experiment,
PBMC from old subjects had consistently lower
proliferative response to Con A than those from
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AGING AND IMMUNE RESPONSE 661
young subjects (Fig. 1). Supplementation with GSH at
high concentrations (2 to 10 mmol/L) enhanced mito-
genie response in young and old subjects,- however,
lower concentrations of GSH (0.5 and 1.0 mmol/L)
decreased mitogenic response. This decrease in
proliferative response by lower concentrations of GSH
is unexpected and difficult to interpret in light of the
previously reported cell-protecting and immune-en
hancing effects of GSH. This phenomenon was highly
consistent and was observed in every subject tested. A
similar result was reported by Franklin et al. (1990).
They demonstrated augmentation of Con A-induced
proliferation of rat splenocytes with the addition of 1
and 10 mmol/L GSH. But when the level of GSH
added to the culture medium was lowered to 0.1
mmol/L, only a significant inhibition of cell prolifer
ation was observed. No explanation for this finding
was given. An observation reported by Rowley and
Halliwell (1982) may help in explaining this
phenomenon. These investigators added various con
centrations of GSH to a hydroxyl radical (OH'| gener
ating system. At high concentrations [i.e., those con
sidered to be at or slightly higher than physiological
levels (up to 5 mmol/L) in most body tissues], GSH
supplementation decreased the amount of OH'
formed. When GSH was added at levels below 1.5
mmol/L, the generation of OH' was higher than that
of controls (without GSH). Because products of free
radical reactions have been shown to suppress mito
genic response (Metzger et al. 1980), one can postulate
that the biphasic effect of GSH on mitogenic response
reflects different levels of free radicals produced at
low and higher levels of GSH. Cyclooxygenase ac
tivity is influenced by peroxide level (Kulmacz et al.
1991). Thus it will be interesting to study the effect of
different GSH concentrations on eicosanoid
production.
The magnitude of GSH effect on PBMC mitogenic
response varied according to the age of the donors and
the type of mitogen. Franklin et al. (1990) reported
that 10 mmol/L GSH caused a greater increase
(eightfold) in Con A-induced proliferative responses of
splenocytes from old rats as compared with those
from young rats (threefold). We observed similar ef
fects in human PBMC. However, the magnitude of
GSH effect and the age differences were smaller in our
study than in the report of Franklin et al. (1990). In
addition, GSH supplementation was more effective in
enhancing Con A-induced mitogenic response than in
enhancing PHA-induced mitogenic response. This is
in agreement with the observation that BSO inhibited
PBMC response to Con A and PHA through reducing
the cellular GSH level, and that Con A response was
more sensitive than the PHA response to inhibition
by BSO (Messina and Lawrence 1989).
The importance of the intracellular GSH level for
the activation of lymphocytes is well established, but
the mechanisms involved are not well understood. In
 662
this study, inhibition of GSH synthesis by BSO at
levels as low as 0.2 mmol/L reduced [3H]thymidine
incorporation to -50% of that of control cultures.
Addition of 5 mmol/L GSH reversed BSO-induced
suppression (Fig. 4). One possible mechanism is
through antioxidant activity of GSH. Reduced
glutathione (GSH) is capable of directly scavenging
radicals and peroxides by being oxidized to either
disulfide glutathione (GSSG) or to a mixed disulfide,
thereby preventing cell membrane lipid peroxidation
and its subsequent deleterious effects on cellular
functions. This antioxidant ability to inhibit free
radical formation may underlie the im-
munostimulatory action of GSH, because both
specific and nonspecific blockers of various stages of
the process of lipid peroxidation have been shown to
enhance mitogenic response of spleen cells (Hoffeld
1981). However, factors other than a reduction in
non-enzymatic free radical formation and lipid peroxi
dation seem to be involved in the immuno-
stimulatory effect of GSH.
Macrophages are known to play an important role
in the regulation of T cell responses. Activated mac
rophages produce Superoxide anión(Oj) and hydrogen
peroxide, as well as prostaglandins and leukotrienes
that can suppress lymphocyte proliferation. Gluta
thione is believed to decrease the level of these
products and thus may enhance lymphocyte response.
Numerous studies have revealed the immunoregula-
tory effect of PGE2- Prostaglandin £2suppresses
lymphocyte proliferation (Metzger et al. 1980), IL-2
production, IL-2 receptor a expression (Anastassiou et
al. 1992) and IL-1 production (Knudsen et al. 1986).
Increased PGE2 production has been found in aged
animals (Meydani et al. 1986) and humans (Meydani
et al. 1990a). We observed in this study that PBMC
pretreated with 5 mmol/L GSH released significantly
less PGE2 and LTB4during 20 min of incubation than
did control cultures. No difference in LTC4
production was observed. To further delineate the
contribution of GSH-induced decrease in PGEj syn
thesis to the GSH-induced increase in lymphocyte
proliferation, 0.1 /¿mol/LPGE2 was added to standard
PBMC culture for lymphocyte proliferation with or
without the addition of 5 mmol/L GSH. Prostaglandin
£2significantly inhibited lymphocyte proliferation by
36% in old subjects, but only a nonsignificant inhi
bition of 19% (P = 0.13) was seen in young subjects,
indicating that PBMC from old subjects are more
sensitive to PGE2- Higher sensitivity of PBMC from
old subjects to PGE2 inhibition was also reported by
Goodwin and Messner (1979), and this higher sensi
tivity to PGE2 inhibition was recently reported by
Franklin et al. (1993) in purified T cells from rats.
This inhibition was reversed by the addition of 5
mmol/L GSH (Fig. 4). Because PGE2 and LTB4 have
been shown to down-regulate lymphocyte prolifer
ation (Metzger et al. 1980, Shapiro et al. 1993), GSH-
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WU ET AL.
induced decrease in PGE2 and LTB4 might contribute
to its enhancement of mitogenic proliferation.
Glutathione supplementation caused the same degree
of decrease in PGE2 production by young and old
subjects but a higher percent increase in lymphocyte
proliferation in old subjects compared with young
subjects. It is possible that a similar GSH-induced
decrease in PGE2 level in young and old subjects
might result in a larger increase of mitogenic response
in cells of old subjects compared with young subjects.
The fact that GSH reduced PGE2 and LTB4
production following a short period of incubation in
dicates that, in addition to its intracellular effects,
GSH may also exert its effect extracellularly on mem
brane-mediated events. Studies are currently being
conducted to determine the contribution of extracel
lular and intracellular GSH to the observed effects.
Interleukin-2 is essential for lymphocyte acti
vation. The effect of GSH on immune response has
been linked to its influence on IL-2 production and
activity. Glutathione has been shown to enhance the
binding of IL-2 to its high affinity receptor and to
increase the internalization and degradation of IL-2
(Liang et al. 1989). In this study, the addition of 5
mmol/L GSH caused a significant increase in IL-2
production in both age groups (Table 2). The increase
in IL-2 production can be due to a GSH-induced de
crease in eicosanoids or to a direct effect of GSH on
IL-2 production via changes in early activation events.
Thus GSH can enhance mitogenic response by
decreasing production of mediators with suppressive
effect (eicosanoids and hydrogen peroxide, etc.) or by
increasing production of mediators with enhancing
effect (IL-1 and IL-2). It can influence proliferation by
influencing the nature of regulatory mediators
secreted from macrophages or by directly affecting T
cell activation. Our current studies are designed to
determine the relative contribution of each cellular
compartment to the GSH effect.
In summary, we have shown that in vitro GSH
supplementation enhances mitogenic response of
PBMC from both young and old subjects. This effect
is associated with increased cellular GSH level and IL-
2 production and decreased PGE2 and LTB4
production. The GSH-induced enhancement of mito
genic response and IL-2 production is more pro
nounced in old subjects than in young subjects.
Further studies are needed to delineate the efficiency
of in vivo GSH supplementation on the immune re
sponse of old and young subjects with compromised
immune response.
ACKNOWLEDGMENTS
The authors wish to thank Antonio Martin for his
help in HPLC analysis, Laura C. Rail for her contri
bution to statistical analysis, Joseph Cannon for
 GLUTATHIONE, AGING AND IMMUNE RESPONSE
critical review of the manuscript, and Jennifer
Munnis for manuscript preparation.
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