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Departments of Applied Biological Chemistry and * Veterinary Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) and tumor necrosis factor-
(TNF-), were greater in the
food-restricted group before tumor inoculation (P
< 0.05). Furthermore, the food-restricted mice had
significantly higher plasma levels of IFN- and TNF- after tumor
inoculation, although the treatment significantly increased these
cytokine levels in both groups. Splenic natural killer cell
cytotoxicity was also higher in the tumor-bearing food-restricted
mice than in controls (P < 0.05).
Food-restricted mice have strong antitumor immunity, and as a
result, tumor growth is suppressed and survival time is prolonged in
these mice.
KEY WORDS: • food restriction • mice • antitumor immunity • interferon- • tumor necrosis factor- • natural killer cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Comfort 1963
, Ross 1961
, Weindruch and Walford 1988
). In addition, FR of rodents results in a reduced
incidence and delayed onset of tumors and other age-associated
diseases (Weindruch 1989
, Yu et al. 1982
).
FR results in the activation of cellular immunity, altered subsets of T
cells (Gartner et al. 1992
), enhanced responses of T
cells to mitogens and interleukin-2 (Hishinuma et al. 1990
), and altered production of antibodies and interleukin-2
(Spear-Hartley and Sherman 1994
). It has been proposed
that host immune surveillance mechanisms effectively suppress the
incidence of tumors in FR animals (Konno et al. 1991
). FR also has been shown to greatly reduce white blood
cell (WBC) number, particularly the lymphocyte count (Kubo et al. 1984
). Although mechanisms by which FR reduces WBC number
have not yet been clarified, it has been reported that the incidence of
leukemia is lowered in FR mice due to the reduction of leukocytes
(Volk et al. 1994
). Moreover, strong immune responses
and a high resistance to developing spontaneous tumors in FR mice are
thought to be closely related to the reduction of lymphocyte number
(Weindruch and Walford 1988
).
Effects of FR on immune functions in tumor-bearing animals have not yet been well elucidated so we inoculated L1210 tumor cells intradermally into 60% FR mice, and studied host immune functions after the tumor inoculation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male BDF1 mice (5-wk-old) were obtained from Clea (Osaka, Japan). These
mice had free access to water and to a diet reported by Konno et al. (1991)
, which was composed of 20% casein, 64% potato starch, 5%
soybean oil, 5% cellulose powder, 4% salt mixture and 2% vitamin
mixture.3
These mice were kept at controlled temperature (23 ± 2°C),
humidity (60 ± 10%) and lighting (12-h light and 12-h dark
cycle), and housed individually. When 8-wk-old, mice were randomly
divided into control and 60% FR groups. Mice in the control group were
fed 5.0 g of the diet daily (71 kJ/d), whereas 60% FR mice
received 3.0 g (43 kJ/d). All experimental procedures involving
laboratory animals were approved by the Animal Care and Use Committee
of Osaka Prefecture University.
Inoculation of tumor cells.
L1210 leukemia cells (America Type Culture Collection, CCL219), obtained from Riken Cell Bank (Tsukuba, Japan), were maintained in Dulbecco’s modified Eagle’s medium, supplemented with 5% heat-inactivated fetal calf serum and 10 mmol/L HEPES. At 12 wk of age (4 wk after the initiation of FR), mice in both the FR and control groups were inoculated with L1210 by intradermal injection of ~10 6 cells. For determination of tumor growth, 2, 3 and 3.5 wk after the inoculation these mice (seven mice in each group at each time point) were anesthetized with ethyl ether, and tumors in these mice were isolated and weighed.
Determination of leukocyte, lymphocyte, neutrophil and monocyte counts.
Mice (five mice in each group) were anesthetized with ethyl ether, and
blood was collected by cardiac puncture. Blood samples (10% in an EDTA
solution) were incubated on ice until measured with an automatic blood
corpuscle count apparatus (Sysmex K-1000; Toua Iyou Electric, Kobe,
Japan). For investigation of differential counts, blood smears were
prepared on microscope slides, and the slides were air-dried and
stained by a modified Giemsa method (Seki et al. 1981
).
Leukocytes (over 200 cells) on the slides were examined and the
percentages of lymphocytes, neutrophils and monocytes were determined.
Determination of plasma cytokine levels.
Blood was collected by cardiac puncture, and plasma interferon-
(IFN-) and tumor necrosis factor- (TNF-) levels were
determined by enzyme-linked immunosorbent assay methods using
commercial kits (QuantikineTM M Mouse IFN- Immunoassay
and QuantikineTM M Mouse TNF- Immunoassay; R&D Systems,
Minneapolis, MN).
Cytotoxicity assay for natural killer (NK) cells.
Spleen cells were prepared according to a published method
(Weindruch et al. 1983
). NK-sensitive YAC-1 cells
were used as target cells for the assay of NK cell cytotoxicity. Spleen
and target cells were co-cultured in a 96-well microtiter plate at
the effector to target ratio of 100:1 for 4 h at 37°C in 5%
CO2, and NK cell cytotoxicity was measured by a lactate
dehydrogenase-release method using a commercial kit (CytoTox
96® Non-Radioactive Cytotoxicity Assay; Promega,
Madison, WI) according to manufacturer’s instructions.
Statistical analysis.
Statistical analyses were performed with GB-Stat 5.4 (Dynamic
Microsystems, Sliver Spring, MD). Body weights were compared between
the FR and control groups during the experiment by two-way ANOVA
for repeated measures, and post-hoc analyses were done by Tukey’s
method. Mean survival time after the tumor inoculation, and lymphocyte,
neutrophil and monocyte counts were compared between the two groups by
one-way ANOVA followed by Scheffé post-hoc test. Effects of
FR on tumor growth, WBC number and NK cell activity were evaluated by
two-way ANOVA, and multiple comparisons were done by Tukey’s test.
For IFN- and TNF- data, values were logarithmically transformed
to improve normality and to compensate for unequal variance, and were
analyzed by two-way ANOVA followed by Tukey’s test. All data are
shown as means ± SD, and statistical significance is
defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The FR-induced body weight change observed in this experiment is
consistent with previous reports (Hishinuma et al. 1988
,
Konno et al. 1991
). Tumors appeared at the site of the
inoculation in both the FR and control groups by 2 wk after the
treatment (at 12 wk), and the tumors progressively grew thereafter.
However, the rate of the tumor growth was significantly slower in the
FR group, and 3.5 wk (25 d) after the inoculation, tumor weight in the
FR mice was < 30% (P < 0.05) of that in
controls (Fig. 2A
). A significant difference was also seen in survival curves between
the FR and control groups (Fig. 2B)
. Mean survival time after tumor
inoculation in the control group was 30.7 d, and all control mice
died by 35 d. In contrast, in the FR group, the mean survival time
was prolonged to 38.5 d (P < 0.05), and the final
mouse survived until 54 d.
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), as reported previously (Kubo et al. 1984
). Tumor cell
inoculation did not affect WBC number in either group and the number
was significantly lower in the FR mice even 3.5 wk after inoculation.
Lymphocytes and neutrophils were reduced in the FR group, compared to
the controls, in the tumor-bearing state(P < 0.05) (Table 1).
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concentration in the FR mice
was higher than that in the control (P < 0.05). When
L1210 tumor cells were implanted, a significant elevation in the plasma
IFN- level occurred which peaked at 3 wk in both groups (Fig. 4A
). However, the FR mice had a higher plasma IFN- concentration even
after the tumor inoculation, and at the peak, the level in the FR mice
was about 3.5-fold greater than that in the controls (P
< 0.05). The plasma TNF- concentration also was significantly
augmented by FR in the nontumor-bearing mice (Fig. 4B)
.
Furthermore, 3.5 wk after tumor inoculation, plasma TNF-
concentration in the FR mice was also higher (about 2.5-fold greater)
than that in the control (P < 0.05), although the
tumor inoculation significantly increased the TNF- level even in the
control group.
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). However, tumor inoculation significantly augmented NK cell
cytotoxicity in the FR mice, but not in the control mice. Thus, at 3.5
wk after inoculation, NK cell cytotoxicity was greater in FR mice than
in control mice (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kolaja et al. 1996
, Yoshida et al. 1997
). In addition, it has been proposed that these effects of
FR on the onset of tumors are due to the activation of host immune
surveillance mechanisms (Konno et al. 1991
,
Weindruch et al. 1986
). Our present data obtained in
mice inoculated with L1210 tumor cells show that the growth of the
implanted tumor is significantly suppressed in 60% FR mice, and
survival time is longer in these mice (Fig. 2)
.
In the tumor-bearing state, it has been shown that tumor-primed
CD4+ T cells are activated to produce IFN-
(Yamamoto et al. 1995
). IFN- possesses an
antiproliferative activity against many transformed cell lines in
addition to its antiviral and immunoregulatory functions (Abe et al. 1998
, Giovarelli et al. 1986
, Stark et al. 1998
). TNF-, which was originally defined by its
antitumor activity in vitro and in vivo (Carswell et al. 1975
, Feinman et al. 1987
, Sugarman et al. 1985
), has been reported to be mainly produced by
macrophages after stimulation with IFN- (Collart et al. 1986
, Celada and Maki 1991
, Han et al. 1990
). L1210 leukemia cells, which were used in this
experiment, are insensitive to TNF- cytolysis in vitro (Leu et al. 1991
), whereas the IFN- receptor is expressed in this
cell line (Wietzerbin et al. 1986
). It has also been
reported that macrophage-mediated cytotoxicity of L1210 cells is
augmented by IFN- in synergy with interleukin-2 or
lipopolysaccharide in vitro, and TNF- participates in the cytotoxic
mechanism to produce nitric oxide by an autocrine mechanism in
macrophages (Jiang et al. 1992
). Our data (Fig. 4)
indicate that in the
normal state, IFN- and TNF- levels are significantly greater in
the 60% FR group compared to the control. Furthermore, the FR mice
have greater abilities to produce IFN- and TNF- even after
inoculation of L1210 tumor cells, although this treatment increases
cytokine production in both FR and control groups. We contend the
activated production of IFN- and TNF- in FR mice is related
closely to the effective suppression of tumor growth. However, detailed
mechanisms by which IFN- and TNF- production are stimulated by FR
are unclear. It has been reported that the CD4+ T
cell subpopulation is augmented in FR mice in both the normal and
tumor-bearing states (Gartner et al. 1992
,
Volk et al. 1994
).
NK cells are important antitumor effectors both in vitro and in vivo
(Herberman 1985
, Ortaldo and Herberman 1984
), and high NK cell cytotoxicity has been associated with
reductions in tumor development (Reisenfeld et al. 1980
). In the 60% FR group, but not in the control group, NK
cell cytotoxicity was significantly augmented by the tumor inoculation
(Fig. 5)
, suggesting that NK cells improve antitumor immunity in the FR
mice. Since activated NK cells can produce IFN- and TNF-
(Perussia 1991
), it is thought that NK cells participate
in the active production of IFN- and TNF- in the
tumor-bearing FR mice.
It has been reported that FR reduces leukocyte number particularly
lymphocytes (Kubo et al. 1984
, Weindruch and Walford 1988
). In our study, tumor inoculation did not
significantly change total WBC number in either the 60% FR or control
groups, and lymphocyte number in the FR mice was significantly lower
even in the tumor-bearing state. It has been shown that FR induces
apoptosis, and immune organs, such as spleen and thymus, are
comparatively more sensitive to apop-tosis than other organs
(Keusch et al. 1983
). Thus, the low lymphocyte count in
FR mice may be closely related to apop-tosis in spleen and thymus.
Energy restriction has been suggested to enhance T cell function in
aged mice by maintaining apoptosis at levels found in younger mice,
thereby removing non- or poorly functioning T cells (Spaulding et al. 1997
). It is thus thought that immunologically less
effective T cells are eliminated in the FR mice, presumably by
apoptosis, even in the tumor-bearing state. Since
CD4+ T cells participate in the production of
IFN- in tumor-bearing mice (Yamamoto et al. 1995
), the elimination of nonfunctional T cells may be one of
the important factors in improving antitumor immunity in the FR mice.
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FOOTNOTES |
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, interferon-; NK, natural killer; TNF-,
tumor necrosis factor-; WBC, white blood cell. 
3 Vitamin mixture contains 46,000 IU vitamin A
acetate, 23,300 IU cholecalciferol, 1,200 mg vitamin E acetate, 6 mg
vitamin K3, 59 mg thiamin HCl salt, 59 mg riboflavin, 29 mg
vitamin B6 HCl salt, 0.2 mg vitamin B12, 588 mg
vitamin C, 1 mg D-biotin, 2 mg folic acid, 235 mg pantothenic acid
Ca salt, 294 mg nicotinic acid, and 1,176 mg inositol in 100 g
with a balance with lactose. Mineral mixture contains 0.43 g
CaHPO4 · 2H2O, 34.31 g
KH2PO4, 25.06 g NaCl, 0.623 g Fe-citrate
(Fe 17%), 4.8764 g MgSO4, 0.02 g ZnCl2,
0.121 g MnSO4 · 4–5H2O, 0.156 g
CuSO4 · 5H2O, 0.0005 g KI, 29.29 g
CaCO3, and 0.0025 g
(NH4)6Mo7O24 ·
4H2O in 100 g with a balance with cellulose powder.
Manuscript received June 14, 1999. Initial review completed July 7, 1999. Revision accepted September 30, 1999.
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