Immunology Letters 98 (2005) 63–72
Mega-dose Vitamin C modulates T cell functions in Balb/c mice only
when administered during T cell activation
Kahwa Noha,b, Hyunja Lima,b, Sung-kyu Moona,b, Jae Seung Kanga,b, Wang Jae Leea,b,
Dongsup Leeb, Young-il Hwanga,b,∗
a Tumor Immunity Medical Research Center, Seoul National University College of Medicine, Seoul 110-799, South Korea
b Department of Anatomy, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu,
Seoul 110-799, South Korea
Received 5 August 2004; received in revised form 20 October 2004; accepted 21 October 2004
Abstract
Previously we reported that a mega-dose of Vitamin C enhanced the initial stage of delayed-type hypersensitivity reaction in Balb/c mice.
In this study its effects were further evaluated as follows. Mice were administered Vitamin C intraperitoneally at 0.625 mg/day or at 5 mg/day
for variable days before, during, or after being sensitized with DNFB. T cells were isolated in each group and examined. When stimulated
antigen-specifically or non-specifically in vitro, mice showed elevated thymidine uptake and a shift of cytokine secretion profiles toward Th1,
i.e., elevated levels IL-2, TNF-, and IFN-, and lowered level of the Th2 cytokine IL-4, only when Vitamin C was administered during
sensitization. T cells from those mice administered Vitamin C before sensitization or after challenge showed the same T cell properties as those
from PBS-treated mice. Mice were also given 0.625 mg/day of Vitamin C during primary and/or secondary immunizations with KLH and
secondary specific antibody titers in sera were measured. The total specific antibody titer was lowered in Vitamin C-treated animals whenever
treatments were administered, and this was entirely attributed to decreased levels of IgG1 and IgE antibodies. Based on these results, we
suggest that an exogenously administered mega-dose of Vitamin C shifts immunity in Balb/c mouse toward Th1 and that these affects occur
only when Vitamin C is administered during T cell activation.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Mega-dose; Vitamin C; Th1 polarization; Delayed type hypersensitivity; T cell activation; Isotype
1. Introduction
Vitamin C acts as an electron donor for many kinds of human
enzymes, facilitates iron transport, and is regarded as one
of the most important physiological antioxidants [1,2].In addition,
VitaminCexerts several diverse effects on the immune
system. It increases neutrophil motility [3,4] and phagocytic
function [5] in human. Macrophage functions in mice such as
chemotaxis, phagocytosis, and superoxide anion production
are enhanced by several antioxidants including Vitamin C
[6]. Increased proliferation of T cells [7,8], and inhibition of
∗ Corresponding author. Tel.: +82 2 740 8209; fax: +82 2 745 9528.
E-mail address: hyi830@snu.ac.kr (Y.-i. Hwang).
various forms of T cell death [9] and Fas-induced apoptosis
of monocytes [10] by Vitamin C have also been reported. The
increased cytotoxic activity of natural killer cells in humans
is another example of an effect ofVitamin C supplementation
[11,12].This biologically important micronutrient should be
exogenously administered in diets or as tablets to those, including
human, that lack terminal enzyme l-gulonolactone
oxidase in the Vitamin C synthetic pathway [13], and thus
cannot make Vitamin C themselves.
The recent recommended daily allowance (RDA) for
ascorbic acid by National Academy of Sciences is 75 mg and
90 mg per day for women and men, respectively. Nevertheless,
several authors have claimed that this dose is inadequate
and have suggested that the RDA be increased. For example,
0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.imlet.2004.10.012
64 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Levine et al. [14] suggested 200 mg/day to maintain optimal
blood concentrations. Considering that low concentrations of
serum ascorbate are related to increased mortality [15,16], the
recent RDA could be below that actually needed to maintain
health.
Meanwhile, despite many controversies about its beneficial
effects, the usage of even higher doses of Vitamin C at
the level of grams per day has been suggested for both preventative
and therapeutic purposes in a number of diseases,
including cancer [17], atherosclerosis [18], viral infections
[19], H. pylori infection [20], and acute pancreatitis [21]. In
addition, supplement users regularly consume more than 1 g
of Vitamin C per day [22]. However, the effects and mechanisms
of such ‘mega-doses’ of Vitamin C have not been well
defined.
Previously we reported that exogenously given megadose
of Vitamin C augment the initial stage of delayedtype
hypersensitivity (DTH) response against 2,4-dinitro-1-
fluorobenzene (DNFB) in Balb/c mice [23]. In this study, we
further analyzed the ways in which mega-dose Vitamin C
modifies the functions of T cells in mice. When mega-dose
Vitamin C was administered during sensitization, activated
and memory T cells were rendered more vulnerable to both
ex vivo antigen-specific and non-specific stimuli and their
cytokine secretion profiles were shifted to Th1. Furthermore,
vitamin C also affected humoral immune response against
keyhole limpet hemagglutinin (KLH), by critically lowering
specific serum IgG1 and IgE levels.
2. Materials and methods
2.1. Mice
Seven-week-old male Balb/c mice were purchased from
BioLink (Seoul, Korea) and housed in animal facilities at
Seoul National University, College of Medicine (Seoul, Korea).
They were kept under a 12 h light–dark cycle and all
the animals were given mice chow and tap water ad libitum
through the experimental period.
Experimental groups were administered Vitamin C
(Sigma, St. Louis, MO) intraperritoneally at a daily dose
of 0.625 mg or 5mg freshly dissolved in 200l of PBS.
The durations of the injections varied in each experimental
group. An equal volume of PBS was given to control
animals.
2.2. Induction of delayed type hypersensitivity (DTH)
reaction
To induce DTH response, animals were shaved on the
dorsum (approximately 1 cm×1 cm). Sensitizationwas done
for two consecutive days by applying 25 l of 0.5% DNFB
solution gradually onto the shaved skin. DNFB (Sigma, St.
Louis, MO) solutionwas freshly prepared in acetone:olive oil
(4:1) just before use. On variable days after the last sensitization,
DTH was induced by the topical application of 20l
of 0.2% DNFB solution on the dorsal surface of the right
pinna [24]. The interval between sensitization and challenge
varied depending on the experiments. The left pinna was left
untreated or treated with vehicle only. The thickness of both
pinnae were measured with a constant-loading micrometer
(Mitutoyo, Japan) once just before sensitization (designated
as basal thickness) and for 7–10 days after challenge at the
same time of the day. The magnitude of DTH response was
expressed as the percentage of pinna swelling compared to
its basal thickness.
2.3. T lymphocyte proliferation assay
To assess the effect of mega-dose Vitamin C on na¨ıve,
act¨ıvated, and memory T cells, mice were administered
0.625 mg of Vitamin C for various days before, during, or
after sensitization with DNFB. Splenocytes were isolated
using Ficoll-Hypaque (Amersham Pharmacia Biotech, Sweden)
density gradient and incubated at 37 ◦C for 2 h to remove
adherent monocytes and macrophages, and then in
a dish coated with 10 ml of 2 g/ml anti-NK1.1 antibody
(Pharmingen, San Diego, CA) for another 2 h to eliminate
NK cells. T cells were further purified by negative selection
using Dynabeads® Mouse pan B (Dynal Biotech, Oslo, Norway)
by following the manufacturer’s instructions. Briefly,
4×107 Dynabeads were added to 107 cells in a 1.5 ml microcentrifuge
tube, mixed well, and incubated at 4 ◦C for 40 min.
The tubes were then placed in a Dynal MPC (magnetic particle
concentrator) for 2 min. Un-bound cells were collected
by washing with PBS containing 0.1% fetal bovine serum.
The purity of T cells was assessed by FACS analysis using
PE-conjugated hamster anti-CD3 antibody (Pharmingen, San
Diego, CA).
Non-specific T cell activation was determined using hamster
anti-CD3 antibody (Pharmingen, San Diego, CA) or
PMA/ionomycin (Calbiochem, San Diego, CA). Ninety sixwell
plates were coated with 100l of 1g/ml anti-CD3
antibody in each well at 4 ◦C for 24 h. T cells were plated
in coated wells (at 2×105/well), or were cultured in the
presence of 100 ng/ml PMA and 500 ng/ml ionomycin. Incubation
was carried out for 24 h in Dulbeco’s minimum
essential medium (DMEM) supplemented with 10% fetal
bovine serum (Gibco BRL, Carlsbad, CA), 100 U/ml
of penicillin and 100g/ml of streptomycin (Gibco BRL,
Carlsbad, CA) at 37 ◦C in an atmosphere supplemented
with 5% CO2. DNFB-specific T cells were also stimulated.
T cells, 2×105/well, were co-cultured in 96-well
plates with 5×105 mitomycin-treated syngeneic spleen
cells pulsed with 2,4-dinitrobenzenesulfonic acid (DNBS;
Aldrich, Milwaukee, WI), a water-soluble DNFB analogue
[25]. After 24 h, cells were pulsed with 1Ci/well of [3H]
thymidine (Amersham Pharmacia Biotech, Oslo, Norway)
for 18 h, and then harvested using a cell harvester (Skatron
Instrument, Lier, Norway) on glass-fiber filters. Radioactivity
was counted in a scintillation counter (WalK.
Noh et al. / Immunology Letters 98 (2005) 63–72 65
lac, Fort Wayne, IN) and all samples were prepared in
triplicate.
2.4. Cytokine detection
To determine the effect of a mega-dose Vitamin C on
cytokine secretion profiles, T cells were purified as described
above. T cells, 3×106/well, were plated in six-well plates
and stimulated with anti-CD3 antibody, PMA/ionomycin,
or DNBS, as described above. After 24 h incubation,
supernatants were collected. Cytokines in the cell-free
supernatants were detected using commercial quantitative
sandwich immunoassay kits for IL-2, IFN-, TNF- (R&D
Systems, Minneapolis, MN), and IL-4 (Biosource, Nivelles,
Belgium) by following the manufacturer’s manual.
2.5. Immunization with keyhole limpet hemocyanin
(KLH) and specific antibody titration
To assess the effects of Vitamin C on humoral immune
response, we immunized mice twice, on days 1 and 21, with
an intraperitoneal injection of 100g KLH (Calbiochem, La
Jolla, CA) in 200l PBS. Control mice were injected with
PBS only. Blood samples were drawn from the orbital plexus
on days 11 and 31, sera were obtained and stored at 4 ◦C until
used.
To titrate KLH-specific antibodies in sera, 96 well-ELISA
plates (Nunc, Rochester, NY) were coated with 100l of
4g/ml KLH/well for 2 days at 4 ◦C. Plates were then briefly
washed and blocked with 1% skim milk in PBS for 1 h at
room temperature. Serum samples were diluted 1:100 in 1%
skim milk/PBS containing 0.05% Tween 20, applied to the
first row of ELISA plate, and were four-fold serially diluted.
All samples were prepared in duplicate. After incubation
for 2 h at room temperature, plates were washed with
a PBS-0.05% Tween 20 mixture, and alkaline phosphataseconjugated
secondary antibodies for each isotype were added
and incubated for 60 min at room temperature. Plates were
washed three times, and p-nitrophenyl phosphate substrate
(Amresco, Solon, OH) solution was added. OD values were
measured at 405 nm. The secondary antibodies used were
as follows; goat anti-mouse polyvalent immunoglobulins
(1:1000 diluted, Sigma), goat anti-mouse IgM, IgG1, IgG2a,
IgG2b, IgG3, and IgE antibodies (1:1000 diluted, Southern
Biotech, Birmingham, AL). KLH-specific anti-sera obtained
from other experiments were collected in a tube and used as
a standard serum in every ELISA plate. Titers are expressed
as relative to standard serum values.
2.6. Statistical analysis
Statistical analysis was performed by one-way ANOVA
with Student–Newman–Keuls post-hoc analysis using
PRISM software (GraphPad, San Diego, CA). Significance
was set at p < 0.05.
3. Results
3.1. Vitamin C pre-treatment before challenge enhances
the initial stage of DTH response, but suppresses overall
inflammation accompanying DTH response
Mice were treated intraperitoneally with a daily dose of
0.625 mg or 5mg of Vitamin C, or 200 l of PBS for 26 days,
during which sensitization (on days 4 and 5) and challenge
(on day 16) with DNFB were done. Each group consisted
of eight mice. After inducing DTH response, pinna thickness
was measured and the % increment calculated versus
basal thickness (Fig. 1A). Because, in pilot tests, ears treated
with vehicle only (acetone:olive oil) showed just slight and
transient thickness increases (data not shown), we left con-
Fig. 1. The effect of Vitamin C injected during or after the induction of
skin DTH response against DNFB. Mice were intraperitoneally injected
with daily doses of 0.625 or 5mg of Vitamin C for whole 26 days of experiment
(A) or only after challenge, (B) respectively. Sensitization was
done with DNFB on days 11 and 12. Control groups were injected with
200l of PBS. The thickness of pinna was measured after challenge as
indicated, and the percentage thickness increment vs. basal thickness was
calculated. When Vitamin C was administered all through the experimental
days (A), the pinnae of experimental groups on the first day after challenge
were dose-dependently thicker than those of the control groups (p < 0.001).
Subsequently, from the second day, this situation reversed. When Vitamin C
was administered only after challenge (B), the profile was nearly the same
as that shown by Panel A, except on the first day, when the values do not
reveal statistically significant differences. Data represent mean ear swelling
(±S.D.) of eight mice per group.
66 K. Noh et al. / Immunology Letters 98 (2005) 63–72
trol pinnae untreated (even by vehicle). Pinna thickness in all
groups peaked 2 days after challenge (32.8, 24.4, and 18.6%
increment in control, 0.625 mg-, and 5 mg-treated groups,
respectively) and then gradually decreased; control group
values were highest and those of the 5 mg-treated group
lowest. However, the values were reverse 1 day after challenge,
the initial stage of DTH response. The control group
showed the lowest increment (12.7%) and the 5 mg-treated
group showed the highest (17.9%). These differences between
the three groups were statistically significant. This pattern
was exactly the same to as that reported previously [23].
Thus, Vitamin C treatment seemed to accentuate the initial
stage of DTH response on the one hand, and to exert antiinflammatory
effects on an initiated DTH response on the
other.
Considering that intraperitoneally injected exogenous vitamin
C accumulates in several tissues of the body [23], the
observed initial accentuation of DTH response by Vitamin
C treatment could be the result of accumulated tissue Vitamin
C, or the result of any alterations of immune components
before challenge. To clarify this issue, we induced DTH response
in another set of experimental groups, which were
injected with a daily dose of 0.625 mg or 5mg of Vitamin C,
or 200l of PBS for 10 days from the day of challenge, and
then measured the pinna thickness (Fig. 1B). The overall profile
was similar to the results of the groups in Fig. 1A, except
that the increment on the first day after challenge was lowest
in the 5 mg-treated group (12.4 compared to 14.4% both in
the control and in the 0.625 mg-treated groups) even though
thiswas not statistically significant. These results suggest that
pre-treating with Vitamin C before and during sensitization
affects those immune components that induce an increase
in the early DTH response. Of course, the inflammatory response
itself seemed to be suppressed by the presence of
exogenous Vitamin C alone when the inflammation was proceeding.
3.2. Vitamin C treatment only during sensitization
enhanced the initial stage of DTH
We further divided the experimental group in Fig. 1A into
“before sensitization” and “during sensitization” subgroups.
In this case, challenge was done at 38 days, instead of at 6
days after sensitization to allow the activated immune cells to
become quiescent. Thus, four groups were given a daily dose
of 0.625 mg of Vitamin C, (1) during all experimental days
(60 days; () in Fig. 2), (2) for 7 days before sensitization
(() in Fig. 2), (3) for 10 days during sensitization (() in
Fig. 2), or (4) for 10 days after challenge ((♦) in Fig. 2). The
control group received PBS on all experimental days (() in
Fig. 2). As is shown in Fig. 2, two groups injected during the
sensitization period showed a greater pinna thickness increment
on the first day after challenge than the other groups,
including the control group (p < 0.01). Injection of Vitamin C
before sensitization or after challenge failed to augment the
initial DTH response.
Fig. 2. The effect of Vitamin C treatment before sensitization, during sensitization,
and after challenge on DTH response was further analyzed. Mice
were intraperitoneally injected with 0.625 mg of Vitamin C for all days of
the experiment (), for 7 days before sensitization (), for 10 days during
sensitization (), for 10 days after challenge (♦), or injected with 200 l
PBS during the experiment (). DTH response was induced with DNFB 38
days after sensitization to allow the T cells activated during sensitization
to become quiescent. Only the groups administered with Vitamin C during
sensitization (and on the graph) showed an initial accentuation of DTH
response as shown in Fig. 1A. The data represent mean ear swelling (±S.D.)
of eight mice per group.
3.3. Vitamin C enhanced T cell proliferation
The next task was to identify which of the immune components
was most probably affected by Vitamin C during
sensitization, and responsible for the accentuation of the initial
stage of DTH response. We thought that T cells were
the most probable candidate, because T cells, especially Th1
cells, are the prime responders during sensitization and the
initiation of DTH response by antigen challenge [26]. Therefore,
we evaluated the functional differences of T cells from
mice treated and not treated with Vitamin C.
Mice were administrated 0.625 mg of Vitamin C or 200 l
of PBS (A) for 10 days without sensitization (na¨ıve T cells;
equivalent to () in Fig. 2), or (B) for 16 days with sensitization
at days 11 and 12 (activated T cells; equivalent to ()
in Fig. 2), or (C) for 54 days with sensitization on days 10
and 11 (memory T cells; equivalent to () in Fig. 2). At the
end of the Vitamin C administration, mice were sacrificed
and T cells were obtained as described in Section 2. Purity of
isolated T cells was over 90% as assessed by FACS analysis
(data not shown).
Na¨ıve T cells (Fig. 3A) proliferated in the presence of
anti-CD3 antibody and PMA/I, but were not proliferated by
DNBS and without stimulation (“control” in the graph). Furthermore,
the magnitude of thymidine incorporation was the
same in both the Vitamin C-treated and PBS-treated groups.
These results imply the absence of both active T cells and
DNFB-specific memory T cells. After sensitization (Fig. 3B),
T cells proliferated in the absence of any kind of proliferative
stimulus (‘control’ in the graph) implying T cell activation in
K. Noh et al. / Immunology Letters 98 (2005) 63–72 67
Fig. 3. The effect of exogenously administered mega-dose Vitamin C on
T cell proliferative activities. Mice were treated with 0.625 mg of Vitamin
C for 10 days without sensitization (A), for 16 days with sensitization on
days 11 and 12 (B), or for 54 days with sensitization on days 11 and 12 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with a DNFB-specific or non-specific stimuli (anti-CD3
antibody or PMA/I). Tritiated thymidine was pulsed for an additional 18 h
and radioactivities were measured. The filled bar () and the unfilled bar ()
represent the Vitamin C-treated and PBS-treated groups, respectively. The
proliferation of T cells from Vitamin C-treated mice exceeded that of the
control groups only when the mice were sensitized. Values are the average
cpm±S.D. of triplicates.
vivo by sensitization. When stimulated with anti-CD3 antibody
or PMA/I, thymidine uptake was elevated more in the
Vitamin C-treated group than in the PBS-treated group. To
be noticed in these groups is that DNBS also enhanced T cell
proliferation in contrast to the na¨ıve T cell groups, implying
the presence of DNFB-specific activated T cells. T cells
from mice 42 days after sensitization (Fig. 3C) revealed similar
results to those in Fig. 3B. However, the control group
showed minimal uptake indicating the absence of activated T
cells. Meanwhile, proliferation was induced by DNBS both
in Vitamin C-treated and PBS-treated groups, which implies
the presence of DNFB-specific memory T cells. However,
the magnitude of thymidine uptake in the Vitamin C-treated
group was double that of the PBS-treated group.
Besides the status of the T cells isolated, another possible
factor that could elicit differences among groups in Fig. 3
is the duration of Vitamin C treatment (10, 16, and 54 days,
respectively). Therefore, we repeated the T cell proliferation
assay with some modification and examined the effect
of the duration of Vitamin C treatment. Mice were treated
with a daily dose of 0.625 mg Vitamin C (A) for 43 days
with sensitization on days 4 and 5, or (B) for 43 days without
sensitization, or (C) treated with Vitamin C only for 10 days
during sensitization 38 days before T cell isolation. Thymidine
uptake assays were done in the same way as in Fig. 3;
the results are shown in Fig. 4. Results for the 43 day-treated
and sensitized groups (Fig. 4A) were the same as those of
the groups in Fig. 3C, which were treated for 54 days and
sensitized. Despite long-term treatment of Vitamin (for 43
days), if sensitization was not carried out during Vitamin
C treatment (Fig. 4B, similar to Fig. 3A), T cells showed
unaltered thymidine uptake compared to the corresponding
PBS-treated group. Meanwhile, those treated with Vitamin
C only during sensitization (Fig. 4C) elicited similar results
to the sensitized 43-day Vitamin C-treated group (Fig. 4A),
suggesting that sensitization in the presence of exogenously
administered mega-dose Vitamin C results in an increase
in T cell response to both antigen-specific and non-specific
stimuli.
3.4. T cells treated with Vitamin C shifted their cytokine
secretion profiles from Th2 to Th1
The enhanced initial stage of DTH response could be regarded
as an outcome of the shift of general immune response
from Th2 to Th1 [27]. Thus, changes in the cytokine secretion
profiles of T cells could be expected. Mice were treated with
Vitamin C or PBS and T cells were isolated as in Fig. 3. After
the ex vivo stimulation of T cells for 24 h, supernatants were
collected and titers of Th1 cytokines including IL-2, TNF-
, and IFN- and the Th2 cytokine IL-4 were measured by
ELISA.
Without sensitization (left column in Fig. 5), Vitamin C
treatment did not alter the cytokine secretion profiles of T
cells versus those of PBS-treated mice, at least with respect
to the four cytokines tested. T cells not-stimulated or stimulated
with DNBS secreted no detectable cytokines. In contrast,
T cells from sensitized mice, regardless of their being
activated (middle column in Fig. 5) or memory (right column
in Fig. 5) cells, showed elevated IL-2, TNF-, and IFN- secretions
in Vitamin C-treated mice, whereas the secretion of
IL-4 decreased.
68 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 4. The effect of the duration of Vitamin C treatment on T cell proliferative
activities. Mice were injected with 0.625 mg of Vitamin C for 43 days
with sensitization on days 4 and 5 (A), for 43 days without sensitization (B),
or for the first 10 days of the 43 days with sensitization on days 4 and 5 (C).
Control groups were injected with 200l of PBS. T cells were isolated and
cultured for 24 h with DNFB specific or non-specific stimuli (anti-CD3 antibody
or PMA/I). Tritiated thymidine was pulsed for an additional 18 h, and
radioactivities were measured. Filled bar () and unfilled bar () represent
Vitamin C-treated and PBS-treated groups, respectively. Even though mice
were treated with Vitamin C for 43 days, T cells show no difference in their
proliferative activity vs. those of PBS-treated mice if they were not sensitized
during Vitamin C treatment (B). In contrast, only a 10-day Vitamin C
treatment affected T cell proliferation when the treatment was done during
sensitization (C). Values are the average cpm±S.D. of triplicates.
3.5. Vitamin C decreased anti-KLH IgG1 and IgE
antibody responses in vivo
Because cytokine profiles were shifted toward Th1, it
could be predicted that humoral response would be attenuated
[27]. To verify this, we injected mice with Vitamin C
or PBS, immunized them withKLHtwice, and then measured
the serum KLH specific antibody titers by ELISA. A daily
dose of 0.625 mg Vitamin C was intraperitoneally administered;
(1) on all experimental days, or (2) during the primary
immunization for 7 days, or (3) during the secondary immunization
for 7 days. The control group was treated with PBS
on all experimental days.
The results shown in Fig. 6 are for secondary immune
sera. Whole specific titer (IgM, IgG, and IgA) was reduced
in Vitamin C-treated mice regardless of treatment time, and
this seemed to be mainly due to reduced IgG1 and IgE titer,
which are typical Th2-driven isotypes in mice. Other isotypes
were unaffected in terms of KLH specific titer.
4. Discussion
In this study, we examined the effects of Vitamin C on
immune response in Balb/c mice. Previous results that an
exogenously administered mega-dose of Vitamin C accentuated
the initial stage of DTH response [23] were reproduced
in the present experiment. When sensitization was carried
out with the administration of exogenous mega-dose Vitamin
C, the resulting in vivo activated and memory T cells
showed increased proliferative activities in response to ex
vivo antigen-specific and non-specific stimuli, and shifted
cytokine secretion profiles from Th2 to Th1. When immunizationwas
done in the presence of an exogenous mega-dose
of Vitamin C, the titer of whole antigen-specific antibodies in
secondary sera was reduced, and this reduction was mainly
attributed to decreased titers of the Th2-driven isotypes, IgG1
and IgE.
DTH response to skin sensitizer molecules such as DNFB
was used as an experimental model to evaluate cell-mediated
immune function [24]. When memory T cells against an antigen
are exposed to the same antigen again (challenge), they
are reactivated (initial stage of DTH response) to secrete cytokines
like IL-2 and IFN-, which in turn activate tissue
macrophages (cognition and activation). These events occur
within 24 h [28]. Activated macrophages again secrete inflammatory
cytokines and reactive oxygen radicals to propagate
and maintain inflammation [29].
UV irradiation [30,31] and burn stress [32] suppress DTH
response in skin by producing reactive oxygen species (ROS).
Topically or orally administered Vitamin C protects against
this suppression, probably by acting as an antioxidant. In this
experiment, we examined the effect of Vitamin C on DTH
response in normal mice, not from the aspect of protection
against DTH suppression. When injected intraperitoneally,
Vitamin C showed biphasic effect on DTH response. The
thickness of pinnae on the first day increased in a Vitamin C
dose-dependent manner. However, this thickness increment
was inversely related to the amount of Vitamin C administered
from the second day on (Fig. 1). We thought that the
Vitamin C altered T cell function and thus augmented initial,
and cognition and activation stages of DTH response, whereK.
Noh et al. / Immunology Letters 98 (2005) 63–72 69
Fig. 5. The effect of Vitamin C on cytokine secretion by T cells. Mice were treated with 0.625 mg of Vitamin C for 10 days without sensitization (left column),
for 16 days with sensitization on days 11 and 12 (middle column), and for 54 days with sensitization on days 11 and 12 (right column). Control groups were
injected with 200l of PBS. T cells were isolated and cultured for 24 h with DNFB specific or non-specific stimuli. ELISA was done on the supernatants for
the cytokines. In general, Vitamin C elevated the secretion of Th cytokines (IL-2, TNF-, IFN-) and lowered Th2 cytokine (IL-4) secretion by T cells. Filled
bars () and unfilled bars () represent Vitamin C-treated and PBS-treated groups, respectively. Values are the average±S.D. of triplicates.
upon it performed a role as an anti-inflammatory agent from
the second day on.
The anti-inflammatory effects of Vitamin C have already
been suggested. It exerts these effects by scavenging ROS
that is produced by macrophages, destroys tissues and activates
macrophages and neutrophils to progress inflammation
[33,34]. It inhibits the activation of transcription factor NF-
B, which plays a critical role in the production of inflammatory
cytokines such as TNF-, IL-1, and IL-6 [35–37].
Actually, patients with cystic fibrosis with high plasma Vitamin
C levels had lower inflammation indexes than those with
low Vitamin C levels [34]. Based on such results, we suggest
that the dose-dependent suppression of ear thickening in Vitamin
C-treated mice is due to the anti-inflammatory effect
of Vitamin C. This explanation is further supported by the
finding in Fig. 2. Challenged ears were thinner from day 2
after challenge in groups which were administeredVitamin C
during active inflammation (() and () in Fig. 2) than in the
other groups not givenVitamin C at the time of inflammation.
Even though we measured changes in ear thickness for 10
days, we focused on the first day of DTH response. To explain
the augmentation of the initial stage of DTH response,
70 K. Noh et al. / Immunology Letters 98 (2005) 63–72
Fig. 6. The effect of Vitamin C treatment upon humoral immune response.
Mice were treated with 0.625 mg of Vitamin C for 41 days during primary
and secondary immunization ( ), for 10 days after primary immunization
( ), or for 10 days after secondary immunization (). Control group mice
were injected with 200 l of PBS during the experiment (). Ten days after
secondary immunization, sera were obtained and ELISA was performed
for whole and each isotype of KLH-specific antibody. Titers are expressed
relative to standard serum. In all experimental groups, whole KLH-specific
antibody titers were reduced vs. the control group. Among isotypes, the titers
of IgG1 and IgE were markedly reduced in all experimental groups, and thus,
contributed to the reduction in whole antibody titer. Each group consisted of
eight mice. Values are the average±S.D. **p < 0.001 vs. control. *p < 0.01
vs. control.
we analyzed the effect of Vitamin C on T cells, the initial
effector cells in this response.We found that an exogenously
administered Vitamin C mega-dose affected T cell functions,
such as proliferative activity and cytokine secretion profiles.
Furthermore, these effects occurred only when Vitamin C
was given during the sensitization period, i.e., when na¨ıve T
cells become activated and memory T cells begin to emerge.
It is notable that the duration of Vitamin C treatment did
not significantly influence these effects. For example, though
mice were treated with Vitamin C for as much as 43 days, no
change in T cell proliferation was observed in the absence of
sensitization (Fig. 4B). On the other hand, when mice were
treated withVitamin C for only10 days, changes in T cell proliferative
activity was observed if the treatment was applied
during sensitization (Fig. 4C).
T cells obtained 43 days (Fig. 3C) and 38 days (Fig. 4A
and C) after sensitization showed minimal thymidine uptake
without any ex vivo stimulation. This implies that almost
all T cells activated through sensitization were deactivated.
Actually, in vivo activated T cells cease proliferation within
1 week of antigen challenge [38,39]. However, when nonspecifically
stimulated with anti-CD3 antibody or PMA/I,
Vitamin C-treated T cells showed more uptake of thymidine
than PBS-treated T cells, a different feature from the na¨ıve
T cells in which there was no difference of thymidine uptake
between the Vitamin C-treated and the PBS-treated groups
(Fig. 3A and B). T cells in Figs. 3 and 4A and C were composed
of not only na¨ıve T cells but memory T cells, and it
can be presumed that the difference between the Vitamin Ctreated
and the PBS-treated groups is due to the activation
of memory T cells. Indeed, when these cell compartments
were stimulated with DNBS, a vivid difference in thymidine
uptake was observed between the Vitamin C-treated and the
PBS-treated groups.We do not know whether this difference
be the result of either a difference in the number of memory
cells or a difference in the vulnerability of memory T cells to
proliferative stimuli or both. Since it has been reported that
Vitamin C inhibits the apoptosis of activated T cells [9] and
increases the number of plasma T cells in elderly patients
[40], an increased number of DNFB-specific memory T cells
in theVitamin C-treated group can be expected. However, the
increased number of memory cells cannot simply explain the
increased thymidine uptake in Vitamin C-treated T cells in
Figs. 3 and 4A and C by non-specific stimulations, which are
presumably independent of specific memory T cells. Further
studies are needed to solve this issue.
The cytokine secretion profiles of T cells were also
changed in addition to and in parallel with changes in proliferative
activities (Fig. 5); that is, the increased secretion of
Th1 cytokines (IL-2, IFN- and TNF-) and the decreased
secretion of Th2 cytokine (IL-4). These results are similar to
the increased IL-2 and IFN- production of T cells by supplementary
Vitamin E in patients with advanced colorectal
cancer [41]. Since the elevated Th1 cytokines are involved
in cell-mediated immune responses [42,43], these results explain,
in part, the augmented initial stage of DTH response
observed in the present study.
Another point is that, since IL-4 is involved in humoral
immunity [42,43], we expected to observe reduced serum
immunoglobulin levels, and in fact this was the case. In Vitamin
C-treated groups during sensitization, total KLH-specific
antibody titers were reduced. In particular, specific IgG1 and
IgE levels were less than a tenth of those of the PBS-treated
group, markedly contributing to a reduced total specific antibody
titer. These results are in accordance with lower IL-4
secretion by Vitamin C-treated T cells, since IL-4 is known
to induce isotype switching to IgG1 and IgE [44].
Some contradictory results have been reported with respect
to the effects of Vitamin C on humoral response. Longterm
treatment of Vitamin C increased serum IgA and IgM,
but not IgG levels in humans [45]. In guinea pigs, Vitamin
C administration during immunization enhanced specific humoral
responses [46]. Many others have reported unaltered
humoral responses in Balb/c mice [8], humans [47], and in
guinea pigs [48]. These discrepancies could be the results of
differences in the Vitamin C dose and administration routes,
the co-administration of other nutrients, and species used, or
on the presence of T cell stimulation. Meanwhile, in situations
such as chronic granulomatous disease, in which we
could expect the persistent activation of T cells in vivo, longterm
treatment of Vitamin C lowered plasma Ig levels [3].
Vitamin C also reduced serum total IgE levels in asthma patients
[49].
It remains to be elucidated how the two groups treated
with Vitamin C, i.e., during primary or secondary immunization,
had the same secondary antibody secretion profiles.
Those treated only during secondary immunization would
K. Noh et al. / Immunology Letters 98 (2005) 63–72 71
have the same repertoire of memory B cells as the control
group, we would expect a higher titer of IgG1 and IgE
in this group than in the group treated during primary immunization.
However, the titers of both groups showed no
differences. The possibilities are that the isotype-switched
memory B cells during the primary response either underwent
apoptosis or isotype-switched once again during the
secondary response probably due to decreased IL-4 production
by T cells. In this experiment we obtained no data on
this issue, and a search of the literatures provided no clear
suggestions.
To summarize, mega-dose Vitamin C altered the proliferative
capacity of T cells against specific and non-specific
stimuli, and changed cytokine profiles toward Th1, and finally
resulted in an increase in the onset of DTH response and a
decrease in the total specific antibody titer in serum. This
Th1 polarization of immune response by Vitamin C has been
mentioned of its possibility [50]. What is important is that
these effects of exogenous Vitamin C occurred only when it
was administered during sensitization. Thus, we suggest that,
only activated T cells are vulnerable to changes by exogenous
mega-dose Vitamin C. It is not known whether this effect occurs
due to the intracellular accumulation of Vitamin C or
to the ligation of putative Vitamin C membrane receptors,
which have not yet been identified.
Acknowledgement
This work was supported by the Korea Science & Engineering
Foundation (KOSEF) through the Tumor Immunity
Medical Research Center (TIMRC) at Seoul National University,
College of Medicine.
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