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Decreased mitogen response of splenocytes in senescence accelerated mice (SAM-P1) is not restored by high vitamin E diet
Nutrition Research, Vol. 19, No. 9, pp. 1393-1400. 1999 Copyright 0 1999 Elsevier Science Inc. Printed III the USA. All nghts reserved 0271-53 17/99/$-see front matter
PIISO271-5317(9!9)000!96-2 DECREASED MITOGEN RESPONSE OF SPLENOCYTES IN SENEXENCEACCEL ERATED MICE ( SAM-P1 ) IS NOT RESTORED BY HIGH VITAMIN E DIET
-
Mikako Muraga, M.S.i,a, Keizo Umegaki, Ph.D.2, Shigeru Yamamoto, Ph.D.3 and Satoru Moriguchi, Ph.D.1 1Department of Nutrition, Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi, 753-8502, Japan , 2 The National Institute of Health and Nutrition, Tokyo, 162-8636, Japan, and 3 Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan
ABSTRACT
We have previously found that vitamin E (VE) has an ability to restore the decreased mitogen response of splenocytes in spontaneously hypertensive rats (SHR) as a model of aging. The aim of this study is to investigate whether high VE diet can restore the decreased mitogen response of splenocytes in SAM-PI as mouse model of aging. Male SAM-PI and -Rl as control, 9 months old, were fed control (50 mg VE/kg diet ) or high VE diet (585 mg VE/kg diet ). Male SAM-RI, 4 weeks old, were used as young control. After 4 weeks, they were killed underanesthesia and theircellularimmunefunctions were assayed. There was no significant differense in serum VE concentration between old SAM-RI and -Pl. In addition, high VE diet induced the similar increase of serum VE concentration in both old SAM-RI and -PI. Mitogen response of splenic lymphocytes with concanavalin A (Con A) was remarkably decreased in SAM-P1 compared with those of young and old SAM-Rl. High VE diet did not have the ability to restore the decreased mitogen response of splenic lymphocytes in SAM-Pl. The percentage of adherent cells, mainly macrophages (Ma), in splenocytes was also remarkably decreased in old SAM-PI. Furthermore, MB isolated from splenocytes of SAM-PI did not have the enhancing effect on proliferation of splenic lymphocytes from young SAM-R 1. These results suggest that the decreased mitogen response of splenic lymphocytes in SAM-PI is not restored by high VE diet and may be associated to the decreases of both number and function of Mo. 0 ,999E,sev,a science Inc KEY WORDS: Senescence Accelerated Mouse (SAM), Vitamin E, Splenic Lymphocytes, Mitogenesis, Macrophages
INTRODUCIION We have previously found and reported that SHR are in marginal VE deficiency, which is closely associated with decreased mitogen response in their splenic lymphocytes (1). In fact, VE (a - tocopheryl acetate) supplementation could restore the decreased mitogen response in splenic lymphocytes of SHR (2). This result suggests that VE has an ability to restore the decrease of cellular immune function with aging. In mice, there is a SAM-PI as a mutine model of accelerated senescence. In SAM-PI, spontaneous age-associated, systemic amyloidosis is very frequent and severe compared with control SAM-RI with normal aging characteristics (3). It is known that this mouse strain shows a marked decrease in splenic lAddress reprint requests to: M. Muraga, Department of Nutrition, Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi 753-8502, Japan. Tel. 0839-28-2402, Fax. 0839-28-2251, E-mail: [email protected]. 1393
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lymphocyte proliferation with mitogens from 6 months old (4). In this experiment, we have tried to investigate whether the decreased mitogen response of splenic lymphocytes in SAM-PI is associated with the nutritional status of VE and whether high VE diet can restore the decreased mitogen response of splenic lymphocytes in SAM-PI.
MATERlALSANDMETHODS Animals andexperimen&Uets Nine-month-old SAM-RI and -PI, born and reared in our laboratory under conventional conditions, were used. They were divided into two groups of 8 mice each: control group (50 mg VE/kg diet) and high VE group (585 mg VE/kg diet). The composition of the diets is shown in Table 1. To prevent the oxidation of VE in the diets, the diets were stored at -25 “C until used. Each diet was fed for 4 weeks. Food and water were given with free access. Their body weight and food intake were measured daily during the experiment. They were sacrificed under anesthesia with sodium pentobarbital(O.1 ml/100 g BW, Abbot Laboratories, North Chicago, 1L). After the blood of each animal was collected, the spleens of these animals were aseptically removed and weighed.
The spleen from each animal was minced with scissors and passed through a stainless steel mesh in RPM1 1640 culture medium (Gibco Laboratories, Grand Island, NY). The number of splenocytes was counted microscopically and used for the following assay as reported previously (5).
The response of splenic lymphocytes to concanavalin A (Con A; 5 &ml) was determined as described previously (6). Briefly, single-cell suspensions (1 x 10 6 cells/ml) werepreparedin RPM1 1640 culture medium supplemented with 25 mM4(2-hydroxyethyl)-1-ethanesulfonic acid (HEPES; Sigma Chemical, St. Louis, MO), 50 pM 2-mercaptoethanol(2-ME; Sigma Chemical) and 2 mM L-glutamine, penicillin (100 units/ml ), streptomycin (lOO&ml) and 5% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY), respectively. Splenocytes with and without Con A were plated in %-well microtiter plates, incubated at 37 “C in a humidified incubator with 5% CO* and 95% air for 72 h, and then pulsed with [3H] thymidine (specific activity 25 Ci/mmol, New England Nuclear, Boston, MA). After 20 h, the cells were harvested by an automated sample harvester (Flow Laboratory, Rockville, MD). Radioactivity was determined by a liquid scintillation counter (LSC-703, Aloka Corp., Tokyo). .
.
After whole splenocytes were dissociated by passing through a stainless steel mesh in RPMI 1640 culture medium, their suspension was cultured for 1 h at 37 “C in a petri dish. Then, the nonadherent cells were collected by washing the dish three times with the culture medium. Adherent cells were scraped off from the dish by a rubber polishman. M@ were identified by nonspecific esterase staining. As its result, over 84% of the adherent cells were M@. The viability of isolated splenic Me was > 90% by trypan blue dye exclusion. The Me were adjusted to 1 x 10 5 cells/ml in RPM1 1640 culture medium. Measuring the effect of M# on the proliferation of splenic lymphocytes, Me isolated from each group were cultured with splenic lymphocytes from young SAM-RI for 72 h. In this experiment, the ratio of lymphocytes and Mg was 10 : 1. and lymphocyte proliferation was measured by the same way as described in “Prol@mtion of whole splenocytes. ”
VE AND IMMUNODEPRESSION IN SAM
1395
Table 1 Composition of the basal diet
Ingredients
Concentration
Vitamin-free casein * Sucrose Cornstarch Stripped corn oil # Mineral mixture** Vitamin mixture # Cellulose DL-Methionine Sodium selenite
g I kg diet 200 100 550 80 40 10 20 0.3 0.001
*Oriental Yeast, Tokyo, Japan; 0Gti Pharmaceutical Factory Tokyo, Japan; **AINMineral mixture: nrAIN-76 Vitamin mixture. This basal diet contains 50 mg all-rac- a -tocopheryl acetate/kg diet. The high vitamin E diet was prepared by adding 535 mg all-rac- a -tocopheryl acetate per kg diet to this basal diet.
Analysis_of VE inserum VE content in the serum of each mouse was measured by the method of Murata and colleagues (7). The serum of each mouse was extracted with hexane and analyzed by highperformance liquid chromatography (HPLC) with an electrochemical detector (ECD) (IRICA, Kyoto). In this assay we used not in instrinsic standard but external standard. The conditions were as follows: column, Capcell Pack Cl8 UG120 (4.6 mm X 150 mm, Shiseido, Tokyo); column temperature, 35 “C; mobile phase, 50 mM sodium perchlorate in 5% acetonitrile and 95 % methanol; flow rate, 1.2 ml/min ; applied potential of ECD, +600 mv. Statitic&Mlysis Data are means f SD. The statistical significance of differences between control and high vitamin E groups was evaluated by analysis of variance (ANOVA) with Duncan’s Multiple Range Test using a statistical analysis program (Systat, Inc., Evanston, IL). p value < 0.05 was regarded as significant.
The final body weight in old SAM-P1 was not significantly different from that of old SAMRl. The daily food intake of old SAM-RI or -Pl were not significantly different between control and high VE groups throughout the experiment. The serum VE level was not significantly different between old SAM-RI and -Pl (Fig. 1). In addition, high VE diet
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induced significant increases of serum VE level in both old SAM-RI and -Pl to the same degree. Spleen weight was not also significantly different between old SAM-RI and -Pl. Number of splenocytes was slightly decreased in old SAM-R1 and -Pl compared with that of young SAM-RI, whereas there was no significant difference in splenocyte number between old SAM-R1 and -PI ( 9.2 f 3.4 and 8.8 f 3.3 x 10 ‘/ O.lg spleen for control group, 8.9 f 3.3 and 8.5 f 2.8 x 10 7 / 0.1 g spleen for high VE group, respectively.)
The proliferation of splenocytes with Con A was much lower in old SAM-P1 fed control diet than that of young SAM-RI (p < 0.05) (Fig. 2 ). Althougth high VE diet induced a significant increase of splenocyte proliferation with Con A in old SAM-RI, the splenocyte proliferation in SAM-P1 was not restored by high VE diet.
Percent of adherent cells in splenocytes was remarkably decreased in both old SAM-R1 and -Pl compared with that of young SAM-RI (Fig. 3). Percent of adherent cells in splenocytes of old SAM-P1 was much lower than that of old SAM-Rl(p < 0.001). Furthermore, although high VEdiet significantly increased percent of adherent cells in splenocytes of old SAM-Rl, the percent of adherent cells in splenocytes of old SAM-PI was not improved by high VE diet.
The in vitro effect of MW isolated from splenocytes of old SAM-R1 or -PI, on the proliferation of splenic lymphocytes from young SAM-R1 was investigated under Con A stimulation. Although MO isolated from splenocytes of old SAM-R1 increased proliferation of splenic lymphocytes with Con A at the same degree as compared with that of young SAM-Rl, M@isolated from splenocytes of old SAM-P1 did have no effect on the proliferation of splenic lymphocytes from young SAM-RI (Fig. 4). In addition, high VE diet could not improve the decreased splenic Mg function in old SAM-Pl.
-
-vR SAM-P1
comd
Fig. 1 Vitamin E concentration in serum of old SAM-R1 and -PI fed control or the high vitamin E diet for 4 weeks. Values are means * SD. ***p < 0.001 ( vs Control ).
VE AND IMMUNODEPRESSION
IN SAM
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Old SAM-P1
Fig. 2 Effect of the high vitamin E diet on proliferation of splenocytes from old SAM-RI and -Pl with Con A. Values are means f SD. *p < 0.05 ( vs Young SAM-R1 ). ## p < 0.01 ( vs Old SAMR 1 fed control diet ).
Y
E
5!10 % *
c
h
5
0
ContNll HlghvE
Young SAM-Ill
.otd SAM-R1
control
Ht$hvE
Old SAM-P1
Fig. 3 Percent of adherent cells in splenocytes. Values are means f SD. **p c 0.01, ***p < 0.001 ( vs Young SAM-R1 ). W##p< 0.001 ( vs Old SAM-R1 fed control diet ).
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SAM-?1
Fig. 4 Effect of the high vitamine E diet on proliferation of splenic lymphocytes of young SAM-RI following in vitro incubation with Mo isolated from splenocytes of old SAM-RI and -Pl. *p c 0.05 ( vs Young SAM-RI ). “p < 0.05 ( vs Old SAM-R1 fed control diet ).
In the present study we demonstrated that (a) old SAM-PI were not marginally deficient in VE; (b) old SAM-PI showed an increase of serum VE concentration similar to old SAM-RI following intake of the high VE diet for 4 weeks; (c) proliferation of splenic lymphocyte with Con A in old SAM-P1 was significantly decreased compared to young SAM-RI; (d) proliferation of splenic lymphocyte with Con A in old SAM-RI was significantly enhanced by the high VE diet, whereas high VE diet did not have any effect on proliferation of splenic lymphocyte in old SAM-PI. Since old SAM-PI showed the marked decrease of splenic lymphocyte proliferation at 10 months old as shown in Fig. 2, they appear to be immunologically a model for aging. We have previously found that SHR as a rat model for aging showed a significant decrease of serum VE concentration and that VE supplementation restored both the decreased serum VE level and the decreased mitogen response in their splenocytes (8). However, old SAM-PI did not show any decrease of serum VE level and the decreased proliferation of splenocytes with Con A was not improved by the high VE diet for 4 weeks. Since we have previously found that MB play an important role in proliferation of splenic lymphocyte with Con A (6). the decreased proliferation of splenic lymphocyte in old SAM-PI may be associated with the decreased number and/or function of M+Bin splenic lymphocytes. In fact, the percent of adherent cells in splenocytes was significantly decreased in old SAM-P1 compared to both young and old SAM-Rl. This result suggests that the decreased proliferation of splenic lymphocyte in old SAM-P1 may be due to the decreased number of M0 in splenocytes. In contrast, although the percent of adherent cells, mainly Me in splenocytes was also significantly decreased in old SAM-Rl, high VE diet improved their decreased number of Met to the level of young SAM-Rl. This result suggests that VE may have an ability to enhance the number of M0 in splenocytes. As we have previously reported that VE diet has an ability to enhance T-cell differentiation and maturation in rat thymus (5,9), VE may also have the ability
VE AND IMMUNODEPRESSION
IN SAM
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enhance T-cell differentiation and maturation in rat thymus (5,9), VE may also have the ability to enhance the differentiation of monocytes to M#. However, it is unknown the mechanism by which high VE diet improves the decreased number of Mo in splenocytes of old SAM-RI but not in old SAM-PI. Since VE enhances lymphocyte proliferation via Mo activation (6), which is known to be associated with the decreased production of prostaglandin Ez (PC&) (lO,ll), the enhanced proliferation of splenic lymphocytes in old SAM-RI fed the high VE diet may be, in part, due to the enhancement of Mo function. However, Mo isolated from splenocytes of old SAM-RI fed control and the high VE diets had the similar ability to enhance the proliferation of splenic lymphocytes from young SAM-R1 (Fig. 4). This result suggests that Mo isolated from splenocytes of old SAM-RI have the similar ability to enhance proliferation of splenic lymphocytes compared to Mo from young SAM-RI. In short, the decreased proliferation of splenic lymphocytes in old SAM-RI appears to be due not to the decreased function of M@ but the decreased number of Mo in splenocytes. In contrast, the decreased proliferation of splenic lymphocytes in old SAM-P1 was not imploved by the high VE diet (Fig. 2). Since serum VE concentration in old SAM-PI was not different from that in old SAM-RI, the decreased proliferation of splenic lymphocytes in old SAM-PI fed the high VE diet is not due to the increased production of PGE2. MB are also known to produce nitric oxide (NO) from arginine as a substrate (12). NO is a potent inhibitor for lymphocyte proliferation (13- 15). Lee (16) has reported that the leukocytes from the diabetes-prone BB rats show reduced [3H] thymidine uptake and increased release of NO compared to the control rats. In this experment, Ma from splenocytes of old SAM-PI may produce high levels of NO. As a result, the proliferation of splenocytes in old SAM-PI might be suppressed. In fact, Inada et al. have found that nitric oxide (NO) synthase (NOS) activity was increeased in the cerebral cortex of aged SAM compared with that of young adult SAM (17). As shown in Fig. 4, when the number of Ma was adjusted to the same number in each culture, MI from splenocytes of old SAM-P1 induced lower proliferation of splenic lymphocytes. This result suggests that Mo from splenocytes of old SAM-PI may have the less ability to enhance lymphocyte proliferation. Rossi et al. have found that NO may not be involved in the macrophage-mediated inhibition of splenocyte proliferation, which may be associated with the reducing ability of lymphocytes to secrete interleukin-2 (IL-2) (18). However, this explanation does not Iit with the results in this experiment because the splenic lymphocytes from young SAM-RI have well-ability to secrete IL-2. Further study needs to clarify the mechanism of the decreased lymphocyte proliferation in SAM-PI. In summary, the present study has comtirmed that SAM-PI, showing a significant decrease of splenic lymphocyte proliferation, is immunologically a model for aging, which is associated with not VE status but the decreased number and function of Mo in their splenocytes.
REFERENCES I. Moriguchi S, Maekawa K, Miwa H, Kishino Y. Effect of vitamin E supplementation on cellular immune functions deccreased with aging in spontaneously hypertensive rats. Nutr Res 1993; 13: 1039-1051. 2. Moriguchi S, Maekawa K, Okamura M, Oonishi K, Kishino Y. Vitamin E prevents the decrease of cellular immune function with aging in spontaneously hypertensive rats. Nutr Res 1995; 15: 401-414. 3. Takeda T, Hosokawa M, Takeshita S, Irino M, Matsushita T, Tomita Y, Yasuhira K, Hamamoto H, Shimizu K, Ishii M, Yamamuro T. A new murine model of accelerated senescence. Mech Ageing Dev 1981; 17: 183-194. 4. Umezawa M, Hanada K, Naiki H, Chen WH, Hosokawa M, Hosono M, Hosokawa T,Takeda T. Effects of dietary restriction on age-related immune dysfunction in the
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senescence accelerated mouse (SAM). J Nutr 1990, 120: 1393-1400. 5. Moriguchi S, Kobayashi N, Kishino Y. High dietary intakes of vitamin E and cellular immune functions in rats. J Nutr 1990; 120: 1096- 1102.
6. Oonishi K, Moriguchi S, Kishino Y. The role of macrophages in increased mitogen response of rat splenic lymphocytes following in vitro incubation with vitamin E. J Nutr Sci Vitamin01 1995; 41: 445-453. 7. Murata T, Tamai H, Morinobu T, Manago M, Takenaka A, Mino M. Determination carotene in plasma, blood ceils and buccal mocosa by electrochemical. Lipid 1992; 27: 840-843.
of fl-
8. Sakai S, Moriguchi S. Long-term feeding of high vitamin E diet improves the decreased mitogen response of rat splenic lymphocytes with aging. J Nutr Sci Vitamin01 1997; 43: 113-122. 9. Moriguchi S, Miwa H, Okamura M, Maekawa K, Kishino Y, Maeda K. Vitamin E is an important factor in T cell differentiation in thymus of F344 rats. J Nutr Sci Vitamin01 1993; 39: 451463. IO. Moriguchi S, ItohT. Vitamin Eenhances T cell differentiation cell function in rat thymus. Nutr Res 1997,5: 873-883.
through increased epithelial
11. Sakamoto W, Fujie K, Hanada H, Nishihara J, Mino M. Vitamin E inhibits PGb and Ozproductions in rat peritoneal macrophages. B&him Biophys Acta 1991; 1074: 251-255. 12. Meydani SN, Meydani M, Verdon CP, Shapiro AC, Blumberg JB, Hayes KC. Vitamin E supplementation suppresses prostaglandin b synthesis and enhances the immune response in aged mice. Mech Ageing Dev 1986; 34: 191-201. 13. Kumar A, Singh SM. Effect of tumor growth on the blastogenic response of splenocytes: a role of macrophage-derived nitric oxide. Immunol Invest 19%; 25.413-423. 14. Ahvazi BC, Jacobs P, Stevenson MM. Role of macrophage-derived nitric oxide in suppression of lymphocyte proliferation during blood-stage malaria. J Leukoc Biol 1995; 58: 23-3 1. 15. Krenger W, Falzarano G, Delmonte J Jr, Snyder KM, Byon JC, Ferrara JL. Interferongamma suppressesT-cell proliferation to mitogen via the nitric oxide pathway during experimental acute graft-versus-host disease. Blood 19%; 88: 1113-l 121. 16. Lee KU. Nitric oxide produced by macrophages mediates suppression of Con A-induced proliferative responses of splenic leukocytes in the diabetes-prone BB rat. Diabetes 1994,43: 1218-1220. 17. Rossi GR, Cervi LA, Sastre DA, Masih DT. Lack of involvement of nitric oxide in the macrophage-mediated inhibition of spleen cell proliferation during experimental cryptococcosis. Clin Immunol Immunopatholl998; 86: 1626. 18. Inada K, Yokoi I, Kabuto H, Habu H, Mori A, Ogawa N. Age-related increase in nitric oxide synthase activity in senescence accelerated mouse brain and the effect of long-term administration of superoxide radical scavenger. Mech Ageing Dev 19%. 89: 95-102. Accepted
for
publication
February
11,
1999
PIISO271-5317(9!9)000!96-2 DECREASED MITOGEN RESPONSE OF SPLENOCYTES IN SENEXENCEACCEL ERATED MICE ( SAM-P1 ) IS NOT RESTORED BY HIGH VITAMIN E DIET
-
Mikako Muraga, M.S.i,a, Keizo Umegaki, Ph.D.2, Shigeru Yamamoto, Ph.D.3 and Satoru Moriguchi, Ph.D.1 1Department of Nutrition, Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi, 753-8502, Japan , 2 The National Institute of Health and Nutrition, Tokyo, 162-8636, Japan, and 3 Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan
ABSTRACT
We have previously found that vitamin E (VE) has an ability to restore the decreased mitogen response of splenocytes in spontaneously hypertensive rats (SHR) as a model of aging. The aim of this study is to investigate whether high VE diet can restore the decreased mitogen response of splenocytes in SAM-PI as mouse model of aging. Male SAM-PI and -Rl as control, 9 months old, were fed control (50 mg VE/kg diet ) or high VE diet (585 mg VE/kg diet ). Male SAM-RI, 4 weeks old, were used as young control. After 4 weeks, they were killed underanesthesia and theircellularimmunefunctions were assayed. There was no significant differense in serum VE concentration between old SAM-RI and -Pl. In addition, high VE diet induced the similar increase of serum VE concentration in both old SAM-RI and -PI. Mitogen response of splenic lymphocytes with concanavalin A (Con A) was remarkably decreased in SAM-P1 compared with those of young and old SAM-Rl. High VE diet did not have the ability to restore the decreased mitogen response of splenic lymphocytes in SAM-Pl. The percentage of adherent cells, mainly macrophages (Ma), in splenocytes was also remarkably decreased in old SAM-PI. Furthermore, MB isolated from splenocytes of SAM-PI did not have the enhancing effect on proliferation of splenic lymphocytes from young SAM-R 1. These results suggest that the decreased mitogen response of splenic lymphocytes in SAM-PI is not restored by high VE diet and may be associated to the decreases of both number and function of Mo. 0 ,999E,sev,a science Inc KEY WORDS: Senescence Accelerated Mouse (SAM), Vitamin E, Splenic Lymphocytes, Mitogenesis, Macrophages
INTRODUCIION We have previously found and reported that SHR are in marginal VE deficiency, which is closely associated with decreased mitogen response in their splenic lymphocytes (1). In fact, VE (a - tocopheryl acetate) supplementation could restore the decreased mitogen response in splenic lymphocytes of SHR (2). This result suggests that VE has an ability to restore the decrease of cellular immune function with aging. In mice, there is a SAM-PI as a mutine model of accelerated senescence. In SAM-PI, spontaneous age-associated, systemic amyloidosis is very frequent and severe compared with control SAM-RI with normal aging characteristics (3). It is known that this mouse strain shows a marked decrease in splenic lAddress reprint requests to: M. Muraga, Department of Nutrition, Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi 753-8502, Japan. Tel. 0839-28-2402, Fax. 0839-28-2251, E-mail: [email protected]. 1393
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lymphocyte proliferation with mitogens from 6 months old (4). In this experiment, we have tried to investigate whether the decreased mitogen response of splenic lymphocytes in SAM-PI is associated with the nutritional status of VE and whether high VE diet can restore the decreased mitogen response of splenic lymphocytes in SAM-PI.
MATERlALSANDMETHODS Animals andexperimen&Uets Nine-month-old SAM-RI and -PI, born and reared in our laboratory under conventional conditions, were used. They were divided into two groups of 8 mice each: control group (50 mg VE/kg diet) and high VE group (585 mg VE/kg diet). The composition of the diets is shown in Table 1. To prevent the oxidation of VE in the diets, the diets were stored at -25 “C until used. Each diet was fed for 4 weeks. Food and water were given with free access. Their body weight and food intake were measured daily during the experiment. They were sacrificed under anesthesia with sodium pentobarbital(O.1 ml/100 g BW, Abbot Laboratories, North Chicago, 1L). After the blood of each animal was collected, the spleens of these animals were aseptically removed and weighed.
The spleen from each animal was minced with scissors and passed through a stainless steel mesh in RPM1 1640 culture medium (Gibco Laboratories, Grand Island, NY). The number of splenocytes was counted microscopically and used for the following assay as reported previously (5).
The response of splenic lymphocytes to concanavalin A (Con A; 5 &ml) was determined as described previously (6). Briefly, single-cell suspensions (1 x 10 6 cells/ml) werepreparedin RPM1 1640 culture medium supplemented with 25 mM4(2-hydroxyethyl)-1-ethanesulfonic acid (HEPES; Sigma Chemical, St. Louis, MO), 50 pM 2-mercaptoethanol(2-ME; Sigma Chemical) and 2 mM L-glutamine, penicillin (100 units/ml ), streptomycin (lOO&ml) and 5% heat-inactivated fetal bovine serum (FBS; GIBCO, Grand Island, NY), respectively. Splenocytes with and without Con A were plated in %-well microtiter plates, incubated at 37 “C in a humidified incubator with 5% CO* and 95% air for 72 h, and then pulsed with [3H] thymidine (specific activity 25 Ci/mmol, New England Nuclear, Boston, MA). After 20 h, the cells were harvested by an automated sample harvester (Flow Laboratory, Rockville, MD). Radioactivity was determined by a liquid scintillation counter (LSC-703, Aloka Corp., Tokyo). .
.
After whole splenocytes were dissociated by passing through a stainless steel mesh in RPMI 1640 culture medium, their suspension was cultured for 1 h at 37 “C in a petri dish. Then, the nonadherent cells were collected by washing the dish three times with the culture medium. Adherent cells were scraped off from the dish by a rubber polishman. M@ were identified by nonspecific esterase staining. As its result, over 84% of the adherent cells were M@. The viability of isolated splenic Me was > 90% by trypan blue dye exclusion. The Me were adjusted to 1 x 10 5 cells/ml in RPM1 1640 culture medium. Measuring the effect of M# on the proliferation of splenic lymphocytes, Me isolated from each group were cultured with splenic lymphocytes from young SAM-RI for 72 h. In this experiment, the ratio of lymphocytes and Mg was 10 : 1. and lymphocyte proliferation was measured by the same way as described in “Prol@mtion of whole splenocytes. ”
VE AND IMMUNODEPRESSION IN SAM
1395
Table 1 Composition of the basal diet
Ingredients
Concentration
Vitamin-free casein * Sucrose Cornstarch Stripped corn oil # Mineral mixture** Vitamin mixture # Cellulose DL-Methionine Sodium selenite
g I kg diet 200 100 550 80 40 10 20 0.3 0.001
*Oriental Yeast, Tokyo, Japan; 0Gti Pharmaceutical Factory Tokyo, Japan; **AINMineral mixture: nrAIN-76 Vitamin mixture. This basal diet contains 50 mg all-rac- a -tocopheryl acetate/kg diet. The high vitamin E diet was prepared by adding 535 mg all-rac- a -tocopheryl acetate per kg diet to this basal diet.
Analysis_of VE inserum VE content in the serum of each mouse was measured by the method of Murata and colleagues (7). The serum of each mouse was extracted with hexane and analyzed by highperformance liquid chromatography (HPLC) with an electrochemical detector (ECD) (IRICA, Kyoto). In this assay we used not in instrinsic standard but external standard. The conditions were as follows: column, Capcell Pack Cl8 UG120 (4.6 mm X 150 mm, Shiseido, Tokyo); column temperature, 35 “C; mobile phase, 50 mM sodium perchlorate in 5% acetonitrile and 95 % methanol; flow rate, 1.2 ml/min ; applied potential of ECD, +600 mv. Statitic&Mlysis Data are means f SD. The statistical significance of differences between control and high vitamin E groups was evaluated by analysis of variance (ANOVA) with Duncan’s Multiple Range Test using a statistical analysis program (Systat, Inc., Evanston, IL). p value < 0.05 was regarded as significant.
The final body weight in old SAM-P1 was not significantly different from that of old SAMRl. The daily food intake of old SAM-RI or -Pl were not significantly different between control and high VE groups throughout the experiment. The serum VE level was not significantly different between old SAM-RI and -Pl (Fig. 1). In addition, high VE diet
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induced significant increases of serum VE level in both old SAM-RI and -Pl to the same degree. Spleen weight was not also significantly different between old SAM-RI and -Pl. Number of splenocytes was slightly decreased in old SAM-R1 and -Pl compared with that of young SAM-RI, whereas there was no significant difference in splenocyte number between old SAM-R1 and -PI ( 9.2 f 3.4 and 8.8 f 3.3 x 10 ‘/ O.lg spleen for control group, 8.9 f 3.3 and 8.5 f 2.8 x 10 7 / 0.1 g spleen for high VE group, respectively.)
The proliferation of splenocytes with Con A was much lower in old SAM-P1 fed control diet than that of young SAM-RI (p < 0.05) (Fig. 2 ). Althougth high VE diet induced a significant increase of splenocyte proliferation with Con A in old SAM-RI, the splenocyte proliferation in SAM-P1 was not restored by high VE diet.
Percent of adherent cells in splenocytes was remarkably decreased in both old SAM-R1 and -Pl compared with that of young SAM-RI (Fig. 3). Percent of adherent cells in splenocytes of old SAM-P1 was much lower than that of old SAM-Rl(p < 0.001). Furthermore, although high VEdiet significantly increased percent of adherent cells in splenocytes of old SAM-Rl, the percent of adherent cells in splenocytes of old SAM-PI was not improved by high VE diet.
The in vitro effect of MW isolated from splenocytes of old SAM-R1 or -PI, on the proliferation of splenic lymphocytes from young SAM-R1 was investigated under Con A stimulation. Although MO isolated from splenocytes of old SAM-R1 increased proliferation of splenic lymphocytes with Con A at the same degree as compared with that of young SAM-Rl, M@isolated from splenocytes of old SAM-P1 did have no effect on the proliferation of splenic lymphocytes from young SAM-RI (Fig. 4). In addition, high VE diet could not improve the decreased splenic Mg function in old SAM-Pl.
-
-vR SAM-P1
comd
Fig. 1 Vitamin E concentration in serum of old SAM-R1 and -PI fed control or the high vitamin E diet for 4 weeks. Values are means * SD. ***p < 0.001 ( vs Control ).
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Old SAM-P1
Fig. 2 Effect of the high vitamin E diet on proliferation of splenocytes from old SAM-RI and -Pl with Con A. Values are means f SD. *p < 0.05 ( vs Young SAM-R1 ). ## p < 0.01 ( vs Old SAMR 1 fed control diet ).
Y
E
5!10 % *
c
h
5
0
ContNll HlghvE
Young SAM-Ill
.otd SAM-R1
control
Ht$hvE
Old SAM-P1
Fig. 3 Percent of adherent cells in splenocytes. Values are means f SD. **p c 0.01, ***p < 0.001 ( vs Young SAM-R1 ). W##p< 0.001 ( vs Old SAM-R1 fed control diet ).
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SAM-?1
Fig. 4 Effect of the high vitamine E diet on proliferation of splenic lymphocytes of young SAM-RI following in vitro incubation with Mo isolated from splenocytes of old SAM-RI and -Pl. *p c 0.05 ( vs Young SAM-RI ). “p < 0.05 ( vs Old SAM-R1 fed control diet ).
In the present study we demonstrated that (a) old SAM-PI were not marginally deficient in VE; (b) old SAM-PI showed an increase of serum VE concentration similar to old SAM-RI following intake of the high VE diet for 4 weeks; (c) proliferation of splenic lymphocyte with Con A in old SAM-P1 was significantly decreased compared to young SAM-RI; (d) proliferation of splenic lymphocyte with Con A in old SAM-RI was significantly enhanced by the high VE diet, whereas high VE diet did not have any effect on proliferation of splenic lymphocyte in old SAM-PI. Since old SAM-PI showed the marked decrease of splenic lymphocyte proliferation at 10 months old as shown in Fig. 2, they appear to be immunologically a model for aging. We have previously found that SHR as a rat model for aging showed a significant decrease of serum VE concentration and that VE supplementation restored both the decreased serum VE level and the decreased mitogen response in their splenocytes (8). However, old SAM-PI did not show any decrease of serum VE level and the decreased proliferation of splenocytes with Con A was not improved by the high VE diet for 4 weeks. Since we have previously found that MB play an important role in proliferation of splenic lymphocyte with Con A (6). the decreased proliferation of splenic lymphocyte in old SAM-PI may be associated with the decreased number and/or function of M+Bin splenic lymphocytes. In fact, the percent of adherent cells in splenocytes was significantly decreased in old SAM-P1 compared to both young and old SAM-Rl. This result suggests that the decreased proliferation of splenic lymphocyte in old SAM-P1 may be due to the decreased number of M0 in splenocytes. In contrast, although the percent of adherent cells, mainly Me in splenocytes was also significantly decreased in old SAM-Rl, high VE diet improved their decreased number of Met to the level of young SAM-Rl. This result suggests that VE may have an ability to enhance the number of M0 in splenocytes. As we have previously reported that VE diet has an ability to enhance T-cell differentiation and maturation in rat thymus (5,9), VE may also have the ability
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enhance T-cell differentiation and maturation in rat thymus (5,9), VE may also have the ability to enhance the differentiation of monocytes to M#. However, it is unknown the mechanism by which high VE diet improves the decreased number of Mo in splenocytes of old SAM-RI but not in old SAM-PI. Since VE enhances lymphocyte proliferation via Mo activation (6), which is known to be associated with the decreased production of prostaglandin Ez (PC&) (lO,ll), the enhanced proliferation of splenic lymphocytes in old SAM-RI fed the high VE diet may be, in part, due to the enhancement of Mo function. However, Mo isolated from splenocytes of old SAM-RI fed control and the high VE diets had the similar ability to enhance the proliferation of splenic lymphocytes from young SAM-R1 (Fig. 4). This result suggests that Mo isolated from splenocytes of old SAM-RI have the similar ability to enhance proliferation of splenic lymphocytes compared to Mo from young SAM-RI. In short, the decreased proliferation of splenic lymphocytes in old SAM-RI appears to be due not to the decreased function of M@ but the decreased number of Mo in splenocytes. In contrast, the decreased proliferation of splenic lymphocytes in old SAM-P1 was not imploved by the high VE diet (Fig. 2). Since serum VE concentration in old SAM-PI was not different from that in old SAM-RI, the decreased proliferation of splenic lymphocytes in old SAM-PI fed the high VE diet is not due to the increased production of PGE2. MB are also known to produce nitric oxide (NO) from arginine as a substrate (12). NO is a potent inhibitor for lymphocyte proliferation (13- 15). Lee (16) has reported that the leukocytes from the diabetes-prone BB rats show reduced [3H] thymidine uptake and increased release of NO compared to the control rats. In this experment, Ma from splenocytes of old SAM-PI may produce high levels of NO. As a result, the proliferation of splenocytes in old SAM-PI might be suppressed. In fact, Inada et al. have found that nitric oxide (NO) synthase (NOS) activity was increeased in the cerebral cortex of aged SAM compared with that of young adult SAM (17). As shown in Fig. 4, when the number of Ma was adjusted to the same number in each culture, MI from splenocytes of old SAM-P1 induced lower proliferation of splenic lymphocytes. This result suggests that Mo from splenocytes of old SAM-PI may have the less ability to enhance lymphocyte proliferation. Rossi et al. have found that NO may not be involved in the macrophage-mediated inhibition of splenocyte proliferation, which may be associated with the reducing ability of lymphocytes to secrete interleukin-2 (IL-2) (18). However, this explanation does not Iit with the results in this experiment because the splenic lymphocytes from young SAM-RI have well-ability to secrete IL-2. Further study needs to clarify the mechanism of the decreased lymphocyte proliferation in SAM-PI. In summary, the present study has comtirmed that SAM-PI, showing a significant decrease of splenic lymphocyte proliferation, is immunologically a model for aging, which is associated with not VE status but the decreased number and function of Mo in their splenocytes.
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for
publication
February
11,
1999