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Effect of vitamin E supplementation on cellular immune functions decreased with aging in spontaneously hypertensive rats
NUTRITION RESEARCH, Vol. 13, pp. 1039-1051, 1993 0271-5317/93 $6.00 + .00 Printed in the USA. Copyright (c) 1993 Pergamon Press Ltd. All rights reserved.
EFFECT OF VITAMIN E SUPPLEMENTATIONON CELLULAR IMMUNEFUNCTIONS DECREASED WITH AGING IN SPONTANEOUSLYHYPERTENSIVE RATS Satoru Moriguchi, Ph.D.1, Keisei Maekawa, M.S., Hitomi Miwa, B.S. and Yasuo Kishino, M.D. Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770, Japan
ABSTRACT This study was performed to determine the effect of vitamin E supplementation on cellular immune functions decreased with aging in spontaneously hypertensive rats(SHR). Both Wistar Kyoto rats(WKY) as control rats and SHR, 6 weeks old, were fed a diet supplemented with 50 or 585 mg vitamin E/kg diet for 2 or 6 weeks. SHR fed the control diet were apparently in the vitamin E deficient status. In those SHR, mitogenesis and natural killer cell(NK) activity of splenocytes remarkably declined with aging while alveolar macrophage(AM) showed a higher phagocytic activity compared to that of WKY. Furthermore, high vitamin E diet could restore proliferations of thymocytes and splenocytes with phytohemagglutinin(PHA) and concanavalin A(Con A) in SHR. However, the effect of dietary vitamin E on T cell responses was strongly shown in WKY rather than SHR and in 2-week rather than those in 6-week. NK activity of splenocytes in SHR remained the decreased state even after 6 weeks-feeding of high vitamin E diet. These results suggest that vitamin E supplementation may restore, in part, cellular immune functions decreased with aging in SHR and the effect of high vitamin E diet may be limited in T cell responses.
KEY WORDS: Vitamin E, Aging, Mitogenesis, NK activity, Phagocytosis, Rats
INTRODUCTION Spontaneously hypertensive rats(SHR) were established from Wistar Kyoto rats(WKY) by Okamoto and Aoki and now were widely used an animal model for the study of human essential hypertension (1). In recent years, Takeichi et al. found that SHR showed not only hypertension but also immunodeficiency with the decreases of both number and 1Address reprint requests to: Dr. S. Moriguchi, Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770, Japan. 1039
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S. MORIGUCHI et al.
function of T cells with aging (2,3) . Since SHR represent the accelerated decrease and abnormality of cellular immune functions with aging, they have proposed that SHR is a model for not only the study of human essential hypertension but also the study on the mechanism of aging (4). There are many reports that cellular immune functions decline with aging in human (5,6) and experimental animals (7,8). Previous investigation in our laboratory indicated that high vitamin E diets could enhance both splenic lymphocyte and alveolar macrophage(AM) functions in rats (9). Tengerdy et al. have reported that dietary vitamin E supplementation leads to enhanced humoral immune responses and increases resistance to bacterial infection in mice (10) and chickens (11). Tanaka et al. found that dietary supplementation of vitamin E induced the enhancement of helper T-cell activity in mice (12). Bendich et al. have also studied on dietary vitamin E requirement for optimum immune responses (13). However, there is little information about the effect of vitamin E on cellular immune functions decreased with aging. In the present study we investigated whether dietary supplementation of vitamin E could restore cellular immune functions decreased with aging in SHR.
MATERIALS AND METHODS Animals: Since SHR were established by crossbreeding hypertensive male and female Wistar Kyoto rats (WKY) (1), WKY was used for the control against SHR. SHR and WKY, 6 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan) and were fed a basal diet (Table 1) containing 50 mg vitamin E/kg diet for 3 days. Then, they were randomly divided into two groups and fed a diet with 50 or 585 mg vitamin E(dl-a-tocopheryl acetate; Sigma Chemical, St. Louis, MO)lkg diet, respectively. Each group consisted of 10 rats. Food and water were given ad libitum. 9Body weight and food intake were measured daily. The animals were killed while anesthetized with sodium pentobarbital (5 mg/100g body weight; Abbott Laboratory, North Chicago, IL) after consumption of experimental diets for 2 or 6 weeks. After the spleen of each animal was removed, AM were harvested from their lungs, and then their thymuses were removed. Spleen and thymus were used for the mitogenesis assay after weighing. Cell line: The cell line YAC-1 is a Moloney virus induced T-cell lymphoma (14). YAC1 is a nonadherent cell line used extensively in murine natural killer cell (NK) assay. The cells were grown in RPMI 1640 medium with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (100~ug/ml) and fetal bovine serum (FBS;10%). Culture medium and all additives were purchased from GIBCO, Grand Island, NY. This medium was used in all assays in this experiment. Mitoqenesis of thymocvtes and splenocytes: Thymus and spleen were minced with scissors and passed through a stainless steel sieve. Numbers of thymocytes and splenocytes were counted microscopically. Thymic and splenic T cell responses to mitogens such as phytohemagglutinin (PHA;10pg/ml) and concanavalin A (Con A; 5~ug/ml) were measured as described previously (15). Briefly, single cell suspensions
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TABLE 1 Composition of the basal diet
Ingredient
Concentration % 20 10 57 8 4 1
Vitamin-free casein* Sucrose Cornstarch Stripped corn oil# Mineral mixture** Vitamin mixture## *Oriental Yeast, Tokyo, Japan. #Eisai Pharmaceutical Factory, Tokyo, Japan. **The mineral mixture had the following composition, in mg/100g of mixture: K,420; P,990; Na,250; Mg,74.9; Fe,27.0; Zn,5.1; Mn,2.2; Cu,0.57; 1,0.46. ##The vitamin mixture had the following composition, in mgllO0g of mixture: thiamin, 2.4; riboflavin,8.0; pyridoxine,l.6; cyanocobalamin,0.001; ascorbic acid, 60.0; menadione,10.4; biotin,0.04; folic acid, 0.4; Capantothenate,10.0; p-aminobenzoic acid,10.0; inositol,12.0; niacin,12.0; choline-chloride,400.0 and dl-a-tocophetyl acetate,500.0, the following vitamins were also, in mg/kg diet: retinyl acetate,0.03; cholecalciferol,0.005. The high vitamin E diet was prepared by adding 535 mg dl-atocopheryl acetate per kg diet to this basal diet. (10x106 cells/ml for thymocytes and lx10~ cells/ml for splenocytes) were prepared in the above RPMI 1640 culture medium supplemented with 25 mM 4-(2-hydroxylethyl)-l-piperazine-ethanesulfonic acid (HEPES; Sigma Chemical, St. Louis, MO) and 50pM 2-mercaptoethanol (Sigma Chemical, St. Louis, MO). Thymocytes and splenocytes, with or without mitogens, were plated in 96-well microtiter plates, incubated at 37~ in a humidified incubator with 5% CO2 and 95% air for 72 hr, and then pulsed with [3H] thymidine (specific activity 25 Ci/mmol, New England Nuclear, Boston, MA). After 20 hr, they were harvested by an automated sample harvester (Flow Laboratory, Rockville, MD). Radioactivity was determined by a liquid scintillation counter (LSC-703, Aloka Corp., Tokyo). Data are presented as percent proliferation, which was calculated by assigning 100% to mitogenic activity of thymocytes or splenocytes in WKY fed the control diet and comparing to mitogenic activity of thymocytes or splenocytes of other groups. Preoaration and culture of alveolar macroohaaes (AM): AM were collected aseptically by tracheopulmonary lavage as reported previously (16). Briefly, each rat
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S. MORIGUCHI et al.
was anesthetized with sodium pentobarbital and exsanguinated. The chest cavity was opened, and the trachea was cannulated. The lungs were washed with 5 to 8 ml of 0.9% NaCI at 37~ and this was repeated to obtain a total volume of 50 ml of lavage fluid per rat. The number and viability of nucleated cells were determined by trypan blue staining. More than 95% of the lavaged cells were AM. The AM were collected by centrifugation (20~ 300xg) of the lavage fluid and layered on wells of Multiwell plates (Falcon Plastic, Oxford, CA) containing 1 ml of RPMI 1640 medium with 10% FBS (GIBCO, Grand Island, NY). After 60 min, nonadherent cells were removed by washing the plates with medium. Phagocvtosis assay: Sheep red blood cells (SRBC; Nihon Biotest, Tokyo) were maintained in Alserver's solution (Grand Island Biological, Grand Island, NY) at 4~ Opsonization was accomplished by incubating 10 ml of a 2% v/v SRBC suspension with 0.2 ml of rat anti-SRBC antiserum (heat-inactivated) for 60 rain at 37~ The antiserum was prepared by injecting intraperitoneally 0.2 ml of washed SRBC into rat at weekly intervals (17). Radioactive labeling of the opsonized SRBC was accomplished by incubation with 200 pCi of sodium chromate (Na251CrO4; specific activity 128 mCi/mg, New England Nuclear, Boston, MA) for 1 hr at 37~ The opsonized SRBC were then washed three times with RPMI 1640 medium to remove excess 51Cr, and the final volume was adjusted to give a 0.6% suspension of SRBC. AM (2x105 cells/ml) were incubated with opsonized SRBC labeled with 51Cr. After 2 hr-incubation at 37~ the cultures were rinsed once with distilled water to lyse nonphagocytosed SRBC and then washed twice with 0.1 M phosphate buffer, pH 7.2. All remaining cells were lysed with 0.1 M NaOH, and the radioactivity of the lysate was measured in a gamma counter (ARC-361, Aloka Corp., Tokyo). Assav of natural killer cell (NK) activity: NK-sensitive YAC-1 cells were used as target cells. A mixture of 0.05 ml of 51Cr-labeled target cells (2x105 cells/ml) and 0.1 ml of effector cells (2x107 splenocytes/ml) was dispensed in U-bottom microtiter plates. After centrifugation at 20~ and 300xg for 5 rain, they were incubated for 4 hr at 37~ in a 5% CO2 incubator. Then, the plates were centrifuged again at 20~ and 300xg for 5 rain, and 0.1 ml of the supernate was collected from each well. The radioactivity released from target cells was determined by using a gamma counter (ARC-361, Aloka Corp., Tokyo). The percent lysis was calculated as follows: Experimental 51Cr release - Spontaneous 51Cr release % lysis= X 100 Maximum 51Cr release - Spontaneous 51Cr release Spontaneous 51Cr release was determined from target cells incubated with medium alone. Maximum 51Cr release was determined from target cells incubated with 0.1 ml of 1 M NaOH. Blood samples: In this experiment heparinized blood was collected from the inferior vena cava of WKY and SHR fed the control or high vitamin E diet. Plasma mtocopherol level was determined by high performance liquid chromatography (HPLC) (18).
IMMUNOMODULATION BY VITAMIN E Statistical (ANOVA) statistical regarded
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analvsis: Data were evaluated statistically by analysis of variance with separation of treatment means by Duncan's Multiple Range Test using a analysis program (Systat Systat, Inc., Evanston, IL). P value < 0.05 was as significant.
RESULTS Vi~tamin E levels in serum, thymus and spleen of WKY or SHR: The serum vitamin E level in SHR fed the control diet was lower than that of WKY fed the control diet. Although the vitamin E level in serum of SHR was increased by feeding the high vitamin E diet, it was still lower than that of WKY fed the high vitamin E diet (Table 2). Vitamin E level in thymus was also lower in SHR than that of WKY. High vitamin E diet provided a restoration of vitamin E level in both thymus and spleen in SHR to the level of WKY fed the high vitamin E diet. Food intake, and bodv and organ weights: Daily food intake was not different between control and high vitamin E groups in both WKY and SHR, but the body weight of SHR at the same age was lower than that of WKY (Fig. 1). The weight of spleen per 100g body weight was not different between WKY and SHR, and between control and high vitamin E groups (Fig. 2A). Although the weight of thymus also showed no difference among dietary groups, the weights of thymus in both WKY and SHR at 6week were remarkably decreased in comparison with those of both WKY and SHR at 2-week (Fig. 2B). N_umbers of thymocytes, splenocytes and alveolar macrophages (AM): As shown in Table 3, the number of thymocytes in both WKY and SHR decreased with aging, but the difference between WKY and SHR at 2- and 6-week were not significant. The number of splenocytes per 0.1g spleen was higher in SHR than in WKY, but the difference was not significant. The number of AM was not different between WKY and SHR at 2-week after the onset of experiment. At 6-week AM number of WKY per 100g body weight was remarkably lower, which was restored by feeding the high vitamin E diet. Proliferation of thvmocvtes and splenoc_vtes: Proliferation of thymocytes with PHA and Con A in WKY was increased in high vitamin E group at both 2- and 6-week. In SHR the proliferation of thymocytes with PHA and Con A was markedly lower than those of WKY (Fig. 3A and 3B). At 2-week the proliferation of thymocytes with Con A in SHR was completely restored to the level of WKY fed the control diet, but the high vitamin E diet could no longer restore the proliferation of thymocytes with Con A at 6-week. Proliferation of splenocytes with PHA and Con A in SHR was slightly decreased at 2-week and considerably decreased at 6-week. At 6-week proliferation of splenocytes with PHA in SHR was restored by high vitamin E diet compared to that of SHR fed the control diet, but its degree was very small and the value was much less than that with PHA in WKY (Fig. 4A).
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S. MORIGUCHI et al. TABLE 2 Vitamin E levels in serum, thymus and spleen in WKY and SHR fed the control and high vitamin E diets for 6 weeks Dietaly vitamin E Serum Thymus* Spleen* Groups
(mg/kg diet)
WKY
(l~g/ml)
50 585
(pg/g)
4.6 + 0.4 # 17.9 + 1.4 a
(IJg/g)
14.2 + 1.2 64.6 + 3.6 34.6 + 3.4 a 162.8 + 20.4 a
SIR
50 2.3 + 0.3 a 6.8 • 0.6 a 31.6 • 2.8 a 585 11.3• b,c 33.4• c 168.3+25.4 c *The vitamin E levels in homogenates of thymus and spleen were measured by HPLC as described in Materials and Methods. #Values are means + SD. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from WKY fed the high vitamin E diet by P<0.01. cSignificantly different from SHR fed the control diet by P<0.05.
300
A
o~ 200 1,=
"o 100 0 II1
0
I
I
0
1
,I
I
I
I
2 3 4 5 Experimental period (weeks)
Fig. 1 Changes of body weights of WKY and SHR fed for 6 weeks. 0 0 WKY fed the control diet, e---e [] [] SHR fed the control diet, II- - 4 Values are means + SD. The body weights of (P<0.05) from those of WKY at all ages.
I
6
the control or the high vitamin E diet WKY fed the high vitamin E diet SHR fed the high vitamin E diet SHR were significantly different
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TABLE 3 Numbers of thymocytes, splenocytes and alveolar macrophages (AM) in WKY and SHR fed the control and high vitamin E diets for 6 weeks Dietaty vitamin E Groups WHY
SHR
Thymocytes * Splenocytes *
(mg/kg diet)
AM#
(X10"8)
(X10"8)
(X10 "6)
50 585
2.12 + 0.61 2.72:1:0.22 a
1.08 + 0.28 1.24:1:0.31
2.61:1:0.35 4.12+0.28
50 585
2.26 + 0.28 2.43 + 0.26
1.35 + 0.20 1.41 + 0.23
4.85 + 0.92 a 5.97 -t- 1.89 b
*The numbers of thymocytes and splenoctes represent values per 0.1g of thymus or spleen. #The number of AM represents value per 100g body weight. **Values are means + SD. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from WKY fed the high vitamin E diet by P
=
"~ 0.2
Z
O m
~0.1
(B) ~" 0.4
"0
0 m
~
0.2
V.E
C
V.E
WKY SHR 2-week
C
V.E
C
V.E
WKY SHR 6-week
Fig. 2 Weights of thymus (A) and spleen (B) of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD.
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S. MORIGUCHI et al.
200
(A)
0 i
200
(B)
lOO
##
a.
o
C V.E WKY
C V.E SHR
C V.E WKY
2-week
C V.E SHR
o
C
V,E
C
WKY
6-week
V.E
SHR
2-week
C
V,E
WKY
C
V,E
SHR
6-week
Fig. 3 Proliferation of thymocytes with PHA (A) and Con A (B) in WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD and repesent as percent proliferation as described in Materials and Methods. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from SHR fed the control diet by P<0.05. (%) 150
100 "6
~ so
~ C V,E WKY
C V,E SHR
2-week
(A)
150
**
(B)
100
5O
C V.E WKY
C V.E SHR
0
C V.E WKY
C V.E SHR
2-week
6-week
C V.E WKY
C V.E SHR
6-week
Fig. 4 Proliferation of splenocytes with PHA (A) and Con A (B) in WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD and represent as percent proliferation as described in Materials and Methods. aSignificantly different from WKY fed the control diet by P<0,05. bSignificantly different from SHR fed the control diet by P<0.05.
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40
30 "T ,,r >-
"6 o')
20
10
0
C V.E C V.E WKY SHR 2-week
C
V.E WKY
C
V.E SHR
6-week
Fig. 5 Nutural killer cell (NK) activity of splenocytes of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks, aSignificantly different from WKY fed the control diet by P<0.05. Natural killer cell (NK) activity of splenocytes: NK activity of splenocytes in SHR was similar to that of WKY at 2-week. High vitamin E diet did induce higher NK activity in WKY but not in SHR (Fig. 5). At 6-week NK activity in splenocytes of SHR showed a marked decrease compared to that of WKY fed the control diet. Further, high vitamin E diet could not restore the decreased NK activity in splenocytes of SHP,. Phagocytic activity of AM: Phagocytic activity of AM in SHR fed control diet was higher than those of WKY fed the control and high vitamin E diets at both 2- and 6week (Fig. 6). Further, high vitamin E diet induced higher phagocytic activity of AM not in WKY but in SHR at 2-week.
DISCUSSION In the present study we demonstrated that (a) the body weight of SHR was significantly lower than that of WKY at the same age; (b) SHR showed significantly decreased vitamin E levels not only in serum but also in thymus and spleen compared to those of WKY; (c) high vitamin E diet restored the decreased vitamin E levels shown in both thymus and spleen of SHR to the levels of WKY fed the high vitamin E diet; (d) cellular immune functions of SHR except for phagocytic activity of AM decreased with aging; (e) the decreased proliferations of thymocytes and
1048
S. MORIGUCHI et al. (%) 200 n.'
"E
o
D. O
"6
100
uJ
C V.E WKY
CHRV'E S
C
2-week
V.E WK Y
C
V.E SHR
6-week
Fig.
6
Phagocytic activity of AM of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Phagocytic activity of AM was measured by using 51Cr-labeled opsonized sheep red blood cells (SRBC). Phagocytic activity of AM was calculated by assigining 100 % to phagocytic activity of AM in WKY fed the control diet and comparing to phagocytic activity of other groups, aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from SHR fed the control diet by P<0.05.
splenocytes with PHA and Con A in SHR were restored by the high vitamin E diet. Although daily food intake was not significantly different between WKY and SHR, the body weight of SHR was lower compared to that of WKY. Since SHR was a strain derived from WKY, it does not appear that both strains are greatly different. SHR may acquire genes concerning not only hypertension but also constitution of slender frame during the crossbreeding hypertensive male and female WKY. As its result, the body weight of SHR may be lower than that of WKY. Further, although there were not differences in the weights of thymus and spleen, and the numbers of thymocytes and splenocytes between WKY and SHR, the number of AM in SHR was significantly higher than that of WKY fed the control diet at 6-week (Table 3). High vitamin E diet induced the increase of AM number in WKY, whereas the effect of vitamin E diet on AM number was not seen in SHR. As reported previously by Takeichi et al. (2,3), cellular immune functions of SHR decreased with aging. In the present study proliferations of thymocytes and splenocytes, and NK activity of splenocytes decreased in SHR at 6-week. There have been many reports showing that vitamin E deficiency impairs both humoral and cellmediated immunity. Bendich (19) has reported that vitamin E deficiency causes impaired T cell function and moderates impairment of B cell function. Vitamin E deficiency was also shown to decrease mitogenic response of T cells, which was reversible following vitamin E repletion (20). Decreased cellular immune functions in SHR may be evoked by the decrease of vitamin E level as shown in Table 2. Vitamin E deficiency causes increased production of prostaglandin E2 (PGE2) which depresses
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T cell functions via the increase of cellular cAMP level. In fact, the recovery of vitamin E levels in thymus and spleen of SHR to the levels of WKY by feeding the high vitamin E diet induced the improvement of T cell mitogenesis in thymocytes and splenocytes as shown in Fig. 3 and 4. NK activity of splenocytes in SHR was not affected by high vitamin E diet (Fig. 5). Further, phagocytic activity of AM against opsonized SRBC was significantly higher in SHR than that of WKY (Fig. 6). Since it is known that macrophage function is nonspecifically enhanced in athymic, nude mice (21), AM function in SHR showing the decreased functions of T cells may be also enhanced, which may relate to the compensation for the defect in cellular immune system. Possible explanations for the decreased vitamin E levels in serum and some immune tissues of SHR are considered as follows. Since the high vitamin E diet induced the increased levels of vitamin E in serum, thymus and spleen of SHR, the decreased vitamin E level in SHR dose not appear to due to maldigestion and malabsorption of vitamin E. The present study has shown that the number of AM was significantly increased in SHR, and that the phagocytic activity of AM was significantly higher in SHR than that of WKY. These results suggest that AM of SHR were in activated state. Since it is known that activated macrophages produce 02" in large quantities and kill bacterias invaded within body (22), the enhanced phagocytic activity of AM in SHR implies highly production of O2", which may result in the wastage of vitamin E and then cause vitamin E deficient state in SHR. In our previous study vitamin E deficiency caused the higher phagocytic activity of AM rather than the decrease of T cell functions (17). There are many studies showing the decreased T cell functions in vitamin E deficiency (23,24). Thus, compared with the functions of T cells, macrophage functions appear to be well-maintained in vitamin E deficiency, and indicate the enhancement rather than the depression of AM function as shown in Fig. 6. The difference between T cell and AM functions in vitamin E deficiency may be caused by the difference of sensitivity to PGE2, which is highly produced by vitamin E deficiency (25,26) and depresses cellular immune functions. Conversely, T cell functions appear to be greatly affected by vitamin E deficiency. Since we have found that vitamin E plays an important role in T cell differentiation in thymus (Moriguchi et al., submitted for publication), the decreased vitamin E level in thymus of SHR may also cause the abnormality of T cell differentiation in thymus and result in the decreased proliferation of thymocytes. Another possible explanation for the decreased T cell functions in SHR is that the production of natural thymocytotoxic autoantibody (NTA) is increased in SHR with aging (27). The increase of NTA in SHR may cause the impairment of T cell differentiation in thymus of SHR. Further, corn oil used in this experiment contains a considerable quantity of polyunsaturated fatty acid, which may also promote the wastage of vitamin E in SHR. Since essential hypertension causes the abnormalities of humoral and cellular immunities in human (28,29), the increase of blood pressure in SHR with aging may also relate to the decrease of cellular immunity in SHR as shown in this experiment.
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S. MORIGUCHI et al.
In conclusion, we have found that SHR show decreased cellular immune functions except for AM function at early stage of aging, which is closely related to the decrease of vitamin E levels. Further studies are needed to elucidate the precise mechanism of the decreased cellular immune functions in SHR at early stage of aging.
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17. Moriguchi S, Kobayashi N, Kishino Y. Effects of vitamin E deficiency on the functions of splenic lymphocytes and alveolar macrophages. J Nutr Sci Vitaminol 1989; 35:419-430. 18. Kato H, Tanimura H, Hikasa Y. Determination of serum tocopherol by highperformance liquid chromatography. Arch Jpn Chir 1981; 50:740-746. 19. Bendich A. Antioxidant vitamins and immune response. In: Chandra RK, ed. Nutrition and Immunology, New York: Alan R. Liss Inc., 1988:125-148. 20. Jensen M, Fossum C, Ederoth M, Hakkarainen RV. The effect of vitamin E on the cell-mediated immune response in pigs. J Vet Med 1988; 35:549-555. 21. Sharp SK, Colston MJ. Elevated macrophage activity in nude mice. Exp Cell Biol 1984; 52:44-47. 22. Suga M, Ando M, Nishikawa H, Araki T. Release of active oxygen from pulmonary alveolar macrophages, and its significance in bacterial infection. Jpn J Inflammation 1984; 4:542-544. 23. Eskew ML, Schulz RW, Reddy CC, Todhunter DA, Zarkower A. Effects of vitamin E and selenium deficiencies on rat immune function. Immunology 1985; 54: 173-180. 24. Langweiler M, Schultz RD, Sheffy BE. Effect of vitamin E deficiency on the proliferative response of canine lymphocytes. Am J Vet Res 1981; 42: 16181685. 25. Machlin LJ. Vitamin E and prostaglandins. In: De Dure C, Hayaishi O., eds. Tocopherol, Oxygen, and Biomembranes, Amsterdam: Elsevier/North-Holland Biomedical Press, 1978:208-215. 26. Meydani SN, Hayek M. Vitamin E and the immune response. In: Chandra RK., ed. Nutrition and Immunology, Newfoundland, Canada: ARTS Biomedical Publishers and Distributors, 1992:105-128. 27. Takeichi N, Hamada J, Takimoto M, Fujiwara K, Kobayashi H. Depression of T cell-mediated immunity and enhancement of autoantibody production by natural infection with microorganisms in spontaneously hypertensive rats (SHR). Microbiol Immunol.1988; 32:1235-1244. 28. Dzielak DJ. The immune system and hypertension. Hypertension 1992; 19:136-44 29. Svendsen UG. Immunological aspects of hypertension. Int J 1979; 1:81-84. Accepted for publication June 8, 1993.
EFFECT OF VITAMIN E SUPPLEMENTATIONON CELLULAR IMMUNEFUNCTIONS DECREASED WITH AGING IN SPONTANEOUSLYHYPERTENSIVE RATS Satoru Moriguchi, Ph.D.1, Keisei Maekawa, M.S., Hitomi Miwa, B.S. and Yasuo Kishino, M.D. Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770, Japan
ABSTRACT This study was performed to determine the effect of vitamin E supplementation on cellular immune functions decreased with aging in spontaneously hypertensive rats(SHR). Both Wistar Kyoto rats(WKY) as control rats and SHR, 6 weeks old, were fed a diet supplemented with 50 or 585 mg vitamin E/kg diet for 2 or 6 weeks. SHR fed the control diet were apparently in the vitamin E deficient status. In those SHR, mitogenesis and natural killer cell(NK) activity of splenocytes remarkably declined with aging while alveolar macrophage(AM) showed a higher phagocytic activity compared to that of WKY. Furthermore, high vitamin E diet could restore proliferations of thymocytes and splenocytes with phytohemagglutinin(PHA) and concanavalin A(Con A) in SHR. However, the effect of dietary vitamin E on T cell responses was strongly shown in WKY rather than SHR and in 2-week rather than those in 6-week. NK activity of splenocytes in SHR remained the decreased state even after 6 weeks-feeding of high vitamin E diet. These results suggest that vitamin E supplementation may restore, in part, cellular immune functions decreased with aging in SHR and the effect of high vitamin E diet may be limited in T cell responses.
KEY WORDS: Vitamin E, Aging, Mitogenesis, NK activity, Phagocytosis, Rats
INTRODUCTION Spontaneously hypertensive rats(SHR) were established from Wistar Kyoto rats(WKY) by Okamoto and Aoki and now were widely used an animal model for the study of human essential hypertension (1). In recent years, Takeichi et al. found that SHR showed not only hypertension but also immunodeficiency with the decreases of both number and 1Address reprint requests to: Dr. S. Moriguchi, Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770, Japan. 1039
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S. MORIGUCHI et al.
function of T cells with aging (2,3) . Since SHR represent the accelerated decrease and abnormality of cellular immune functions with aging, they have proposed that SHR is a model for not only the study of human essential hypertension but also the study on the mechanism of aging (4). There are many reports that cellular immune functions decline with aging in human (5,6) and experimental animals (7,8). Previous investigation in our laboratory indicated that high vitamin E diets could enhance both splenic lymphocyte and alveolar macrophage(AM) functions in rats (9). Tengerdy et al. have reported that dietary vitamin E supplementation leads to enhanced humoral immune responses and increases resistance to bacterial infection in mice (10) and chickens (11). Tanaka et al. found that dietary supplementation of vitamin E induced the enhancement of helper T-cell activity in mice (12). Bendich et al. have also studied on dietary vitamin E requirement for optimum immune responses (13). However, there is little information about the effect of vitamin E on cellular immune functions decreased with aging. In the present study we investigated whether dietary supplementation of vitamin E could restore cellular immune functions decreased with aging in SHR.
MATERIALS AND METHODS Animals: Since SHR were established by crossbreeding hypertensive male and female Wistar Kyoto rats (WKY) (1), WKY was used for the control against SHR. SHR and WKY, 6 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan) and were fed a basal diet (Table 1) containing 50 mg vitamin E/kg diet for 3 days. Then, they were randomly divided into two groups and fed a diet with 50 or 585 mg vitamin E(dl-a-tocopheryl acetate; Sigma Chemical, St. Louis, MO)lkg diet, respectively. Each group consisted of 10 rats. Food and water were given ad libitum. 9Body weight and food intake were measured daily. The animals were killed while anesthetized with sodium pentobarbital (5 mg/100g body weight; Abbott Laboratory, North Chicago, IL) after consumption of experimental diets for 2 or 6 weeks. After the spleen of each animal was removed, AM were harvested from their lungs, and then their thymuses were removed. Spleen and thymus were used for the mitogenesis assay after weighing. Cell line: The cell line YAC-1 is a Moloney virus induced T-cell lymphoma (14). YAC1 is a nonadherent cell line used extensively in murine natural killer cell (NK) assay. The cells were grown in RPMI 1640 medium with L-glutamine (2mM), penicillin (100 units/ml), streptomycin (100~ug/ml) and fetal bovine serum (FBS;10%). Culture medium and all additives were purchased from GIBCO, Grand Island, NY. This medium was used in all assays in this experiment. Mitoqenesis of thymocvtes and splenocytes: Thymus and spleen were minced with scissors and passed through a stainless steel sieve. Numbers of thymocytes and splenocytes were counted microscopically. Thymic and splenic T cell responses to mitogens such as phytohemagglutinin (PHA;10pg/ml) and concanavalin A (Con A; 5~ug/ml) were measured as described previously (15). Briefly, single cell suspensions
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TABLE 1 Composition of the basal diet
Ingredient
Concentration % 20 10 57 8 4 1
Vitamin-free casein* Sucrose Cornstarch Stripped corn oil# Mineral mixture** Vitamin mixture## *Oriental Yeast, Tokyo, Japan. #Eisai Pharmaceutical Factory, Tokyo, Japan. **The mineral mixture had the following composition, in mg/100g of mixture: K,420; P,990; Na,250; Mg,74.9; Fe,27.0; Zn,5.1; Mn,2.2; Cu,0.57; 1,0.46. ##The vitamin mixture had the following composition, in mgllO0g of mixture: thiamin, 2.4; riboflavin,8.0; pyridoxine,l.6; cyanocobalamin,0.001; ascorbic acid, 60.0; menadione,10.4; biotin,0.04; folic acid, 0.4; Capantothenate,10.0; p-aminobenzoic acid,10.0; inositol,12.0; niacin,12.0; choline-chloride,400.0 and dl-a-tocophetyl acetate,500.0, the following vitamins were also, in mg/kg diet: retinyl acetate,0.03; cholecalciferol,0.005. The high vitamin E diet was prepared by adding 535 mg dl-atocopheryl acetate per kg diet to this basal diet. (10x106 cells/ml for thymocytes and lx10~ cells/ml for splenocytes) were prepared in the above RPMI 1640 culture medium supplemented with 25 mM 4-(2-hydroxylethyl)-l-piperazine-ethanesulfonic acid (HEPES; Sigma Chemical, St. Louis, MO) and 50pM 2-mercaptoethanol (Sigma Chemical, St. Louis, MO). Thymocytes and splenocytes, with or without mitogens, were plated in 96-well microtiter plates, incubated at 37~ in a humidified incubator with 5% CO2 and 95% air for 72 hr, and then pulsed with [3H] thymidine (specific activity 25 Ci/mmol, New England Nuclear, Boston, MA). After 20 hr, they were harvested by an automated sample harvester (Flow Laboratory, Rockville, MD). Radioactivity was determined by a liquid scintillation counter (LSC-703, Aloka Corp., Tokyo). Data are presented as percent proliferation, which was calculated by assigning 100% to mitogenic activity of thymocytes or splenocytes in WKY fed the control diet and comparing to mitogenic activity of thymocytes or splenocytes of other groups. Preoaration and culture of alveolar macroohaaes (AM): AM were collected aseptically by tracheopulmonary lavage as reported previously (16). Briefly, each rat
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S. MORIGUCHI et al.
was anesthetized with sodium pentobarbital and exsanguinated. The chest cavity was opened, and the trachea was cannulated. The lungs were washed with 5 to 8 ml of 0.9% NaCI at 37~ and this was repeated to obtain a total volume of 50 ml of lavage fluid per rat. The number and viability of nucleated cells were determined by trypan blue staining. More than 95% of the lavaged cells were AM. The AM were collected by centrifugation (20~ 300xg) of the lavage fluid and layered on wells of Multiwell plates (Falcon Plastic, Oxford, CA) containing 1 ml of RPMI 1640 medium with 10% FBS (GIBCO, Grand Island, NY). After 60 min, nonadherent cells were removed by washing the plates with medium. Phagocvtosis assay: Sheep red blood cells (SRBC; Nihon Biotest, Tokyo) were maintained in Alserver's solution (Grand Island Biological, Grand Island, NY) at 4~ Opsonization was accomplished by incubating 10 ml of a 2% v/v SRBC suspension with 0.2 ml of rat anti-SRBC antiserum (heat-inactivated) for 60 rain at 37~ The antiserum was prepared by injecting intraperitoneally 0.2 ml of washed SRBC into rat at weekly intervals (17). Radioactive labeling of the opsonized SRBC was accomplished by incubation with 200 pCi of sodium chromate (Na251CrO4; specific activity 128 mCi/mg, New England Nuclear, Boston, MA) for 1 hr at 37~ The opsonized SRBC were then washed three times with RPMI 1640 medium to remove excess 51Cr, and the final volume was adjusted to give a 0.6% suspension of SRBC. AM (2x105 cells/ml) were incubated with opsonized SRBC labeled with 51Cr. After 2 hr-incubation at 37~ the cultures were rinsed once with distilled water to lyse nonphagocytosed SRBC and then washed twice with 0.1 M phosphate buffer, pH 7.2. All remaining cells were lysed with 0.1 M NaOH, and the radioactivity of the lysate was measured in a gamma counter (ARC-361, Aloka Corp., Tokyo). Assav of natural killer cell (NK) activity: NK-sensitive YAC-1 cells were used as target cells. A mixture of 0.05 ml of 51Cr-labeled target cells (2x105 cells/ml) and 0.1 ml of effector cells (2x107 splenocytes/ml) was dispensed in U-bottom microtiter plates. After centrifugation at 20~ and 300xg for 5 rain, they were incubated for 4 hr at 37~ in a 5% CO2 incubator. Then, the plates were centrifuged again at 20~ and 300xg for 5 rain, and 0.1 ml of the supernate was collected from each well. The radioactivity released from target cells was determined by using a gamma counter (ARC-361, Aloka Corp., Tokyo). The percent lysis was calculated as follows: Experimental 51Cr release - Spontaneous 51Cr release % lysis= X 100 Maximum 51Cr release - Spontaneous 51Cr release Spontaneous 51Cr release was determined from target cells incubated with medium alone. Maximum 51Cr release was determined from target cells incubated with 0.1 ml of 1 M NaOH. Blood samples: In this experiment heparinized blood was collected from the inferior vena cava of WKY and SHR fed the control or high vitamin E diet. Plasma mtocopherol level was determined by high performance liquid chromatography (HPLC) (18).
IMMUNOMODULATION BY VITAMIN E Statistical (ANOVA) statistical regarded
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analvsis: Data were evaluated statistically by analysis of variance with separation of treatment means by Duncan's Multiple Range Test using a analysis program (Systat Systat, Inc., Evanston, IL). P value < 0.05 was as significant.
RESULTS Vi~tamin E levels in serum, thymus and spleen of WKY or SHR: The serum vitamin E level in SHR fed the control diet was lower than that of WKY fed the control diet. Although the vitamin E level in serum of SHR was increased by feeding the high vitamin E diet, it was still lower than that of WKY fed the high vitamin E diet (Table 2). Vitamin E level in thymus was also lower in SHR than that of WKY. High vitamin E diet provided a restoration of vitamin E level in both thymus and spleen in SHR to the level of WKY fed the high vitamin E diet. Food intake, and bodv and organ weights: Daily food intake was not different between control and high vitamin E groups in both WKY and SHR, but the body weight of SHR at the same age was lower than that of WKY (Fig. 1). The weight of spleen per 100g body weight was not different between WKY and SHR, and between control and high vitamin E groups (Fig. 2A). Although the weight of thymus also showed no difference among dietary groups, the weights of thymus in both WKY and SHR at 6week were remarkably decreased in comparison with those of both WKY and SHR at 2-week (Fig. 2B). N_umbers of thymocytes, splenocytes and alveolar macrophages (AM): As shown in Table 3, the number of thymocytes in both WKY and SHR decreased with aging, but the difference between WKY and SHR at 2- and 6-week were not significant. The number of splenocytes per 0.1g spleen was higher in SHR than in WKY, but the difference was not significant. The number of AM was not different between WKY and SHR at 2-week after the onset of experiment. At 6-week AM number of WKY per 100g body weight was remarkably lower, which was restored by feeding the high vitamin E diet. Proliferation of thvmocvtes and splenoc_vtes: Proliferation of thymocytes with PHA and Con A in WKY was increased in high vitamin E group at both 2- and 6-week. In SHR the proliferation of thymocytes with PHA and Con A was markedly lower than those of WKY (Fig. 3A and 3B). At 2-week the proliferation of thymocytes with Con A in SHR was completely restored to the level of WKY fed the control diet, but the high vitamin E diet could no longer restore the proliferation of thymocytes with Con A at 6-week. Proliferation of splenocytes with PHA and Con A in SHR was slightly decreased at 2-week and considerably decreased at 6-week. At 6-week proliferation of splenocytes with PHA in SHR was restored by high vitamin E diet compared to that of SHR fed the control diet, but its degree was very small and the value was much less than that with PHA in WKY (Fig. 4A).
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S. MORIGUCHI et al. TABLE 2 Vitamin E levels in serum, thymus and spleen in WKY and SHR fed the control and high vitamin E diets for 6 weeks Dietaly vitamin E Serum Thymus* Spleen* Groups
(mg/kg diet)
WKY
(l~g/ml)
50 585
(pg/g)
4.6 + 0.4 # 17.9 + 1.4 a
(IJg/g)
14.2 + 1.2 64.6 + 3.6 34.6 + 3.4 a 162.8 + 20.4 a
SIR
50 2.3 + 0.3 a 6.8 • 0.6 a 31.6 • 2.8 a 585 11.3• b,c 33.4• c 168.3+25.4 c *The vitamin E levels in homogenates of thymus and spleen were measured by HPLC as described in Materials and Methods. #Values are means + SD. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from WKY fed the high vitamin E diet by P<0.01. cSignificantly different from SHR fed the control diet by P<0.05.
300
A
o~ 200 1,=
"o 100 0 II1
0
I
I
0
1
,I
I
I
I
2 3 4 5 Experimental period (weeks)
Fig. 1 Changes of body weights of WKY and SHR fed for 6 weeks. 0 0 WKY fed the control diet, e---e [] [] SHR fed the control diet, II- - 4 Values are means + SD. The body weights of (P<0.05) from those of WKY at all ages.
I
6
the control or the high vitamin E diet WKY fed the high vitamin E diet SHR fed the high vitamin E diet SHR were significantly different
IMMUNOMODULATION BY VITAMIN E
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TABLE 3 Numbers of thymocytes, splenocytes and alveolar macrophages (AM) in WKY and SHR fed the control and high vitamin E diets for 6 weeks Dietaty vitamin E Groups WHY
SHR
Thymocytes * Splenocytes *
(mg/kg diet)
AM#
(X10"8)
(X10"8)
(X10 "6)
50 585
2.12 + 0.61 2.72:1:0.22 a
1.08 + 0.28 1.24:1:0.31
2.61:1:0.35 4.12+0.28
50 585
2.26 + 0.28 2.43 + 0.26
1.35 + 0.20 1.41 + 0.23
4.85 + 0.92 a 5.97 -t- 1.89 b
*The numbers of thymocytes and splenoctes represent values per 0.1g of thymus or spleen. #The number of AM represents value per 100g body weight. **Values are means + SD. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from WKY fed the high vitamin E diet by P
=
"~ 0.2
Z
O m
~0.1
(B) ~" 0.4
"0
0 m
~
0.2
V.E
C
V.E
WKY SHR 2-week
C
V.E
C
V.E
WKY SHR 6-week
Fig. 2 Weights of thymus (A) and spleen (B) of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD.
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S. MORIGUCHI et al.
200
(A)
0 i
200
(B)
lOO
##
a.
o
C V.E WKY
C V.E SHR
C V.E WKY
2-week
C V.E SHR
o
C
V,E
C
WKY
6-week
V.E
SHR
2-week
C
V,E
WKY
C
V,E
SHR
6-week
Fig. 3 Proliferation of thymocytes with PHA (A) and Con A (B) in WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD and repesent as percent proliferation as described in Materials and Methods. aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from SHR fed the control diet by P<0.05. (%) 150
100 "6
~ so
~ C V,E WKY
C V,E SHR
2-week
(A)
150
**
(B)
100
5O
C V.E WKY
C V.E SHR
0
C V.E WKY
C V.E SHR
2-week
6-week
C V.E WKY
C V.E SHR
6-week
Fig. 4 Proliferation of splenocytes with PHA (A) and Con A (B) in WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Values are means + SD and represent as percent proliferation as described in Materials and Methods. aSignificantly different from WKY fed the control diet by P<0,05. bSignificantly different from SHR fed the control diet by P<0.05.
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40
30 "T ,,r >-
"6 o')
20
10
0
C V.E C V.E WKY SHR 2-week
C
V.E WKY
C
V.E SHR
6-week
Fig. 5 Nutural killer cell (NK) activity of splenocytes of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks, aSignificantly different from WKY fed the control diet by P<0.05. Natural killer cell (NK) activity of splenocytes: NK activity of splenocytes in SHR was similar to that of WKY at 2-week. High vitamin E diet did induce higher NK activity in WKY but not in SHR (Fig. 5). At 6-week NK activity in splenocytes of SHR showed a marked decrease compared to that of WKY fed the control diet. Further, high vitamin E diet could not restore the decreased NK activity in splenocytes of SHP,. Phagocytic activity of AM: Phagocytic activity of AM in SHR fed control diet was higher than those of WKY fed the control and high vitamin E diets at both 2- and 6week (Fig. 6). Further, high vitamin E diet induced higher phagocytic activity of AM not in WKY but in SHR at 2-week.
DISCUSSION In the present study we demonstrated that (a) the body weight of SHR was significantly lower than that of WKY at the same age; (b) SHR showed significantly decreased vitamin E levels not only in serum but also in thymus and spleen compared to those of WKY; (c) high vitamin E diet restored the decreased vitamin E levels shown in both thymus and spleen of SHR to the levels of WKY fed the high vitamin E diet; (d) cellular immune functions of SHR except for phagocytic activity of AM decreased with aging; (e) the decreased proliferations of thymocytes and
1048
S. MORIGUCHI et al. (%) 200 n.'
"E
o
D. O
"6
100
uJ
C V.E WKY
CHRV'E S
C
2-week
V.E WK Y
C
V.E SHR
6-week
Fig.
6
Phagocytic activity of AM of WKY and SHR fed the control and high vitamin E diets for 2 or 6 weeks. Phagocytic activity of AM was measured by using 51Cr-labeled opsonized sheep red blood cells (SRBC). Phagocytic activity of AM was calculated by assigining 100 % to phagocytic activity of AM in WKY fed the control diet and comparing to phagocytic activity of other groups, aSignificantly different from WKY fed the control diet by P<0.05. bSignificantly different from SHR fed the control diet by P<0.05.
splenocytes with PHA and Con A in SHR were restored by the high vitamin E diet. Although daily food intake was not significantly different between WKY and SHR, the body weight of SHR was lower compared to that of WKY. Since SHR was a strain derived from WKY, it does not appear that both strains are greatly different. SHR may acquire genes concerning not only hypertension but also constitution of slender frame during the crossbreeding hypertensive male and female WKY. As its result, the body weight of SHR may be lower than that of WKY. Further, although there were not differences in the weights of thymus and spleen, and the numbers of thymocytes and splenocytes between WKY and SHR, the number of AM in SHR was significantly higher than that of WKY fed the control diet at 6-week (Table 3). High vitamin E diet induced the increase of AM number in WKY, whereas the effect of vitamin E diet on AM number was not seen in SHR. As reported previously by Takeichi et al. (2,3), cellular immune functions of SHR decreased with aging. In the present study proliferations of thymocytes and splenocytes, and NK activity of splenocytes decreased in SHR at 6-week. There have been many reports showing that vitamin E deficiency impairs both humoral and cellmediated immunity. Bendich (19) has reported that vitamin E deficiency causes impaired T cell function and moderates impairment of B cell function. Vitamin E deficiency was also shown to decrease mitogenic response of T cells, which was reversible following vitamin E repletion (20). Decreased cellular immune functions in SHR may be evoked by the decrease of vitamin E level as shown in Table 2. Vitamin E deficiency causes increased production of prostaglandin E2 (PGE2) which depresses
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T cell functions via the increase of cellular cAMP level. In fact, the recovery of vitamin E levels in thymus and spleen of SHR to the levels of WKY by feeding the high vitamin E diet induced the improvement of T cell mitogenesis in thymocytes and splenocytes as shown in Fig. 3 and 4. NK activity of splenocytes in SHR was not affected by high vitamin E diet (Fig. 5). Further, phagocytic activity of AM against opsonized SRBC was significantly higher in SHR than that of WKY (Fig. 6). Since it is known that macrophage function is nonspecifically enhanced in athymic, nude mice (21), AM function in SHR showing the decreased functions of T cells may be also enhanced, which may relate to the compensation for the defect in cellular immune system. Possible explanations for the decreased vitamin E levels in serum and some immune tissues of SHR are considered as follows. Since the high vitamin E diet induced the increased levels of vitamin E in serum, thymus and spleen of SHR, the decreased vitamin E level in SHR dose not appear to due to maldigestion and malabsorption of vitamin E. The present study has shown that the number of AM was significantly increased in SHR, and that the phagocytic activity of AM was significantly higher in SHR than that of WKY. These results suggest that AM of SHR were in activated state. Since it is known that activated macrophages produce 02" in large quantities and kill bacterias invaded within body (22), the enhanced phagocytic activity of AM in SHR implies highly production of O2", which may result in the wastage of vitamin E and then cause vitamin E deficient state in SHR. In our previous study vitamin E deficiency caused the higher phagocytic activity of AM rather than the decrease of T cell functions (17). There are many studies showing the decreased T cell functions in vitamin E deficiency (23,24). Thus, compared with the functions of T cells, macrophage functions appear to be well-maintained in vitamin E deficiency, and indicate the enhancement rather than the depression of AM function as shown in Fig. 6. The difference between T cell and AM functions in vitamin E deficiency may be caused by the difference of sensitivity to PGE2, which is highly produced by vitamin E deficiency (25,26) and depresses cellular immune functions. Conversely, T cell functions appear to be greatly affected by vitamin E deficiency. Since we have found that vitamin E plays an important role in T cell differentiation in thymus (Moriguchi et al., submitted for publication), the decreased vitamin E level in thymus of SHR may also cause the abnormality of T cell differentiation in thymus and result in the decreased proliferation of thymocytes. Another possible explanation for the decreased T cell functions in SHR is that the production of natural thymocytotoxic autoantibody (NTA) is increased in SHR with aging (27). The increase of NTA in SHR may cause the impairment of T cell differentiation in thymus of SHR. Further, corn oil used in this experiment contains a considerable quantity of polyunsaturated fatty acid, which may also promote the wastage of vitamin E in SHR. Since essential hypertension causes the abnormalities of humoral and cellular immunities in human (28,29), the increase of blood pressure in SHR with aging may also relate to the decrease of cellular immunity in SHR as shown in this experiment.
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S. MORIGUCHI et al.
In conclusion, we have found that SHR show decreased cellular immune functions except for AM function at early stage of aging, which is closely related to the decrease of vitamin E levels. Further studies are needed to elucidate the precise mechanism of the decreased cellular immune functions in SHR at early stage of aging.
REFERENCES 1. Okamoto K, Aoki K. Development of a strain of spontaneously hypertensive rats. Jpn Circ J 1963; 27:287-293. 2. Takeichi N, Suzuki K, Kobayashi H. Characterization of immunological depression in spontaneously hypertensive rats. Eur J Immunol 1981; 11:483-487. 3. Tekeichi N, Suzuki K, Okayasu T, Kobayashi H. Immunological depression in spontaneously hypertensive rats. Clin exp Immunol 1980; 40:120-126. 4. Takeichi N, Ba D, Suzuki K, Kobayashi H. The arrest of T cell maturation in spontaneously hypertensive rats with a deficient production of thymic factors. Acta Pathol Jpn 1985; 35:351-360. 5. Gillis S, Kozak R, Durante M, Weksler ME. Immunological studies of aging. Decrease production of and response to T cell growth factor by lymphocytes from aged human. J Clin Invest 1981; 67:937-942. 6. Makinodan T, Kay MB. Age influence of the immune system. Adv Immunol 1980; 2:287-330. 7. Menon M, Jaroslow BN, Koesterer R. The decline of cell-mediated immunity in aging mice. J Gerontol 1974; 29:499-505. 8. Hori Y, Perkins EH, Halsall MK. Decline in phytohemagglutinin responsiveness of spleen cells from aging mice. Proc Exp Biol Med 1973; 144:48-53. 9. Moriguchi S, Kobayashi N, Kishino Y. High dietary intakes of vitamin E and cellular immune functions in rats. J Nutr 1990; 120:1096-1102. 10. Tengerdy RP, Heinzerling RH, Brown GL, Mathias MM. Enhancement of humoral immune response by vitamin E. Int Arch Allergy Appl Immunol 1973; 44:221-232. 11. Tengerdy RP, Heinzerling RH, Nockels CF. The effect of vitamin E on the immune response of hydroxic normal chickens. Infec Immunity 1972; 5:987-989. 12. Tanaka J, Fujiwara H, Torisu M. Vitamin E and immune response, enhancement of helper T-cell activity by dietary supplementation of vitamin E in mice. Immunology 1979; 38:727-734. 13. Bendich A, Gabriel E, Machlin LJ. Dietary vitamin E requirement for optimum immune response in the rat. J Nutr 1986; 116:675-681. 14. Moriguchi S, Werner L, Watson RR. High dietary vitamin A(retinyl palmitate) and cellular immune functions in mice. Immunology 1985; 56:169-177. 15. Moriguchi S, Toba M, Kishino Y. Effect of dietary restriction on cellular immunity in rats. J Nutr Sci Vitaminol 1989; 35:49-59. 16. Moriguchi S, Sone S, Kishino Y. Changes of alveolar macrophages in proteindeficient rats. J Nutr 1983; 113:40-46.
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