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*
Department of Basic Gerontology, National Institute for Longevity Sciences, Obu, Japan;
Department of Medical Zoology and Immunolgy, Nagasaki University School of Medicine, Nagasaki, Japan; and
Department of Experimental Animal Research, The Institute of Physical and Chemical Research, Tsukuba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been reported that the immune responses declines with aging in
mammals (6, 7, 8, 9, 10). In particular, much of the age-related
decline in protective immune responses is thought to be induced by
changes in the T cell composition. The accumulation of memory-phenotype
(CD44high CD45RBlow)
CD4+ T cells in the place of naive-phenotype
(CD44low CD45RBhigh)
CD4+ T cells (7, 8) has been linked
to the reduced proliferative response (9) and decreased
production of IL-2 by T cells (10) with aging. Some
cytokines such as IL-4 and IL-10, which are preferentially secreted by
the memory T cells, are reported to be produced in greater
concentrations by T cells from aged mice (8, 11). Reports
concerning other cytokines typically produced by memory T cells, such
as IFN-
, are still inconsistent (12).
In contrast, the incidence of malignant tumors, which are a major cause of death, frequently increases with aging. The decline in the activity of the immune system and the increased risk for almost all forms of tumors with aging suggests that the immune system provides, at least in part, protection against tumors. Although p53-deficient (p53-/-)3 mice develop different types of tumors, the most frequently observed tumor is malignant lymphoma, which occurs in >60% of these animals. The high incidence of lymphoma was originally ascribed to the genetic background of the mice, which is 75% in C57BL/6 mice and 25% in 129/Sv mice (13). It is known, for example, that the incidence of lymphoma in normal C57BL/6 mice is relatively high, but the average age of onset is about 27 mo (14). Presumably, the increased incidence of lymphoma in p53-/- mice is also potentially related to the age-related changes in the internal milieu of the animal, in addition to its relationship to the genetic background of the mice. In short, the effects of aging are potentially accelerated by p53 deficiency.
Here, we describe an immunosenescence-like phenomenon in p53-/- mice. Acceleration of immunosenescence by p53 deficiency is discussed from the viewpoint of the functions of p53.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBA/N background, p53-/- (15) and p53+/+ male mice were purchased from Oriental Cobo Industrial (Tokyo, Japan). p53-/- mice between 5 and 20 wk old and p53+/+ mice between 5 and 60 wk old were used. These animals were bred under pathogen-free conditions, and any mice obviously suffering from lymphoma or tumors were eliminated from the study. Lymphoma in the spleen was recognized by the size vs granularity of the cells, determined by the forward scatter (FS) vs side scatter (SS) field in flow cytometry, and the type was confirmed by microscopic examination of Giemsa-stained sections.
Flow cytometry
For analysis of naive/memory CD4+ T cells, spleen cells suspended in PBS were stained with a mixture of FITC-conjugated CD44-specific mAb (Sigma, St. Louis, MO), PE-conjugated CD45RB-specific mAb (PharMingen, San Diego, CA), and Tricolor-conjugated CD4-specific mAb (Caltag, San Francisco, CA). For analysis of T cells/B cells, samples were stained with a mixture of PE-conjugated B220-specific mAb (Caltag) and FITC-conjugated Thy-1.2-specific mAb (Caltag). For analysis of NK cells/NK T cells, samples were stained with a mixture of PE-conjugated NK1.1-specific mAb (PharMingen) and FITC-conjugated Thy-1.2-specific mAb (Caltag). Three-color or two-color analysis was conducted by flow cytometry (FACSCaliber, Nippon Becton Dickinson, Tokyo, Japan).
Cellular culture
T cell enriched spleen cells, prepared by passing the cells through a nylon wool column, were cultured at a density of 1 x 106 cells/200 µl in RPMI 1640 medium supplemented with 5% FCS in 96-well microplates. For stimulation, CD3-specific mAb (1:1000, KT3) (Bio Source International-Tago Prodocts, Camarillo, CA) or Con A (5 µg/ml) (Sigma) or PMA (10 nM) (Sigma) was added to the culture. For the proliferation assay, after 48 h incubation at 37°C under 5% CO2, the cells in each well were labeled with 18.5 kBq of [3H]thymidine for 4 h and harvested onto filter mats.
Assay for Ab-forming cells in vivo
SRBC were purchased from Nippon Seibutu Zairyo Center (Tokyo, Japan). On day 4 after i.p. injection of 10% (v/v) SRBC (0.2 ml) into mice, the spleen cells were collected and the number of plaque-forming cells (PFC)/spleen was determined for estimation of the number of anti-SRBC Ab-forming cells.
RT-PCR
Total cellular RNA from the spleen was extracted by the acid
guanidium thiocyanate phenol-chloroform method. RNA was
retrotranscribed after annealing with 1 µg/20 µl oligo(dT) primers
(Life Technologies, Rockville, MD) in the presence of 100 U of reverse
transcriptase (Superscript II RT; Life Technologies), 10 mM DTT (Life
Technologies), and 500 nM of each deoxynucleotide in a total volume of
20 µl for 1 h at 37°C. Aliquots of the mixture were then
amplified in a thermocycler in a volume of 20 µl with 20 U/ml of
Taq DNA polymerase (Takara, Tokyo, Japan), 200 µM of each
deoxynucleotide, and 200 nM each of the two (forward and reverse)
primers. The sequences of the primers for a amplification
of the cytokine genes are as follows: IFN- forward,
5'-TGAACGCTACACACTGCATCTTGG-3'; IFN- reverse,
5'-CGACTCCTTTTCCGCTTCCTGAG-3'; IL-2 forward, 5'-AACAGCGCACCCACTTCAA-3';
IL-2 reverse, 5'-TTGAGATGATGCTTTGACA-3'; IL-4 forward,
5'-TAGTTGTCATCCTGCTCTT-3'; IL-4 reverse, 5'-CTACGAGTAATCCATTTGC-3';
IL-6 forward, 5'-GAACAACGATGATGCACTTGCAG-3'; IL-6 reverse,
5'-CCTTAGCCACTCCTTCTGTGAC-3'; IL-10 forward,
5'-TCAAACAAAGGACCAGCTGGACAACATACTG-3'; IL-10 reverse,
5'-CTGTCTAGGTCCTGGAGTCCAGCAGACTCAA-3'; GATA3 forward,
5'-GAAGGCATCCAGACCCGAAAC-3'; GATA3 reverse, 5'-ACCCATGGCGGTGACCATGC-3';
GAPDH forward, 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3'; GAPDH reverse,
5'-CATGTAGGCCATGAGGTCCACCAC-3'.
Samples were amplified in 32 cycles (denaturation at 94°C for 30 s, annealing at 55°C for 50 s, and extension at 72°C for 80 s). The PCR products were visualized after electrophoresis through 2% agarose gels by staining with ethidum bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the G2-gated lymphomatous cells are
larger in size and granularity than the normal
G1-gated normal lymphocytes (Fig. 1
a), the nonlymphomatous cells
in the mice could be easilly identified. Then, the type of lymphoma
or the absence of lymphoma in the spleen was determined by microscopic
examination of Giemsa-stained sections (data not shown). The field of
CD4+ cells was gated for analysis of CD44 vs
CD45RB expression levels. Most cells from lymphomatous spleens was
contained in a major population of CD44+
CD45RB+, and the composition was apparently
different from that of the population of nonlymphomatous cells (Fig. 1b). The population of G2 field in
"Mix type" was as same as the population of
G2 field in "Majority type" (data not
shown).
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We analyzed the percentage increase in the number of memory T
cells with aging in p53-/- mice without
lymphoma or malignant tumors. The accumulation of memory T cells was
strongly accelerated in the spleen of p53-/-
mice (Fig. 2a). In contrast to
the increase in the percentage of memory T cells in
p53+/+ mice from 10.54 ± 2.55% (5–6 wk)
to 31.5 ± 3.12% (18–20 wk), that in
p53-/- mice increased dramatically from
22.18 ± 6.09% (5–6 wk) to 58.42 ± 8.98% (18–20 wk). The
percentage of memory T cells in adult (18–20 wk)
p53-/- mice was similar to that in elderly (60
wk) p53+/+ mice (Fig. 2b).
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Next, we examined the T cell-dependent Ab responses to SRBC. Young
p53-/- mice (5–6 wk) showed 
3-fold stronger
responses than p53+/+ mice of the same age
(PFC/spleen; p53+/+, 72,680 ± 21,472;
p53-/-, 209,330 ± 68,962). Although the
percentage of memory T cells was greatly increased in both
p53+/+ mice and in p53-/-
mice following Ag (SRBC) stimulation, the increase was more marked in
p53-/- mice (Fig. 3a). In contrast, because NK T
cells (NK1.1+ Thy-1+) also
have a similar percentage of CD44high
CD45RBlow CD4+ memory
phenotype cells (16), we examined whether the increased
population of memory phenotype cells contained any NK T cells. Although
the memory phenotype cells were determined not to be NK T cells, a
substantial number of cells (NK1.1+
Thy-1-) appeared in the spleen of
p53-/- mice after day 5 of SRBC immunization;
furthermore, the number of NK cells in p53-/-
mice was comparatively higher after day 15 of SRBC immunization (Fig. 3b). Then, we performed a comparative analysis of cytokine
production in the two SRBC-injected groups of mice. High Th2-type
cytokine (IL-4, IL-6, IL-10) production by splenic T cells from
p53-/- mice was noted (Fig. 4). The expression level of the
transcriptional factor GATA-3 necessary for expression of the gene for
Th2-type cytokines (17) was also significantly increased
in p53-/- mice (Fig. 4).
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The T cell proliferative response to Con A (5 µg/ml) in the
spleen of young p53-/- mice (5–6 wk) was 1.43
times stronger than that in p53+/+ mice of the
same age (Fig. 5a), and the
response to anti-CD3 mAb (1: 1000) in
p53-/- mice was 1.25 times stronger than that
in p53+/+ mice of the same age (5–6 wk) (Fig. 5b). These differences were not significant. However, the T
cell proliferative responses in adult p53-/-
mice (17–18 wk) were weaker than those in p53+/+
mice of the same age; the response in p53-/-
mice to Con A was 0.39 times and to anti-CD3 mAb was 0.42 times
that in p53+/+ mice. In contrast, analysis of
cytokine production in adult p53-/- mice
(17–18 wk), measured by RT-PCR (Fig. 6),
revealed that the production of IFN-, IL-4, IL-6, and IL-10 in
response to anti-CD3 mAb stimulation in vitro was significantly
higher than that in p53+/+ mice of the same age;
however, the IL-2 expression levels were almost the same in both
p53-/- mice and p53+/+
mice of the same age. The response was remarkably up-regulated by
combined stimulation with anti-CD3 mAb and PMA (10 nM), an
activator of protein kinase C. IL-10, which had been considered to be
an immunosuppressive cytokine for the anergic state (18),
was frequently secreted in adult p53-/-
mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a and b). p53 deficiency was thus
presumed to accelerate immunosenescence. Some researchers report that
age-related accumulation of memory T cells is related to the poor
proliferative responses of T cells (6, 7, 8, 9). The
proliferative response of T cells to anti-CD3 mAb or Con A, which
was previously reported as being depressed in aged mice (7, 9), was depressed in adult p53-/- mice
(Fig. 5, a and b). Furthermore, our data conform,
in part, to the report of Con A-induced T cell proliferation being
suppressed following inhibition of the p53 gene expression by
p53-specific antisense oligodeoxynuleotides (19). In these
studies, it will be concluded that the observed immune changes in
p53-/- mice are not secondary to lymphomatous
development, because we carefully selected out the mice that had no
lymphoma by flow cytometry (Fig. 1).
Development and aging lead to the accumulation of memory phenotype T
cells, and some of the cytokines produced by the accumulated memory T
cells potentially induce Th1/Th2 imbalance; accordingly, the
predominance of memory phenotype cells in T cell populations is
considered to induce much of the decline in the protective immune
responses. Although age-related alterations in the production of
cytokines (high IL-4, IL-10, IFN-, and low IL-2) have been reported
in 24-mo-old mice (10, 11, 12), examination of cytokine
production in p53-/- mice (17–18 wk) revealed
a high level of expression of IL-4, IL-6, IL-10, and IFN- following
TCR stimulation, but the expression of IL-2 in
p53-/- mice did not change as much as that in
p53+/+ mice (Fig. 6). Considering the reports of
the promoters of IL-2, IL-4, IL-6, and other cytokines being repressed
by p53 (20, 21), it is suggested that the negative
regulation of the transcription of various cytokines is lost in the
presence of p53 deficiency. Furthermore, after Ag (SRBC) stimulation,
the stronger Ab production and Th2-type cytokine production in
p53-/- mice might be caused by the absence of the
negative regulation of cytokines transcription (Fig. 4). After day 5 of
Ag stimulation, an increase in the number of NK cells, largely of
splenic origin, was seen in p53-/- mice (Fig. 3b). Studies on NK cells in senescence have shown that
whereas the number of NK cells increases, NK cell activity decreases
with aging (22). In a recent report, the expression of
Ly-49A receptors, which down-regulate NK cell activation after physical
interaction with target cell MHC class I molecules (23),
increases with aging (24). Age-related down-regulation of
NK cell activity is probably induced by the increase in expression of
Ly-49A receptors with aging. NK clones from
p53-/- mice did not express Ly-49A receptors on
the cell surface, and NK cell activity was independent of MHC class I
molecule expression (25). Therefore, the mechanisms
underlying the alterations in the number/activity of NK cells in
p53-/- mice may be different from those
underlying NK cell alterations with aging.
Much attention has been focused on the etiology of oxidative damage in cells and organisms with aging (26, 27); a recent report strongly suggested a relationship between oxidative stress and aging. It was indicated that an increase in superoxide levels leads to oxygen hypersensitivity and premature aging in mev-1 (kn1) mutant nematodes (28). Evidence has also been presented for p53 being required for cellular apoptosis in response to oxidative stress by hydrogen peroxide (29). Chromosomal damage is potentially accelerated by oxidative stress in p53-/- mice. Extensive evidence has been accumulated that suggests that T cell senescence is due to the acceleration of chromosomal damage and their limited proliferative lifespan. The most significant consequences of these impairments are dysregulation of DNA repair and apoptosis (30). The evidence that T cell senescence was greatly reduced in CD2-Fas transgenic mice was accounted for by the speculation that immunosenescence results from the increase in the number of memory T cells with aging (31). We suggest that the regulation of cytokine production and cell cycle is perturbed by p53 deficiency and that the cellular differentiation induced by the high level of expression of cytokines and the loss of cell cycle arrest cause the increase in the number of memory T cells in p53-/- mice. Additionally, the loss of both DNA repair and apoptosis by p53 deficiency in response to oxidative stress potentially leads to the accumulation of memory T cells, which escaped from cell death in p53-/- mice. The phenomenon of immunosenescence in p53-/- mice might explain, in part, the mechanism of aging.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ken-ichi Isobe, Department of Basic Gerontology, National Institute for Longevity Sciences, Gengo 36-3, Morioka-cho, Obu 474-0031, Japan. E-mail address:
3 Abbreviations used in this paper: p53-/-mice, p53-deficient mice; p53+/+mice, wild-type mice; PFC, plaque-forming cells; FS, forward scatter; SS, side scatter.
Received for publication January 7, 1999. Accepted for publication June 9, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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