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EFFECTS OF DIETARY NUCLEOTIDES ON SERUM ANTIBODY AND SPLENIC CYTOKINE PRODUCTION IN MICE
NumitionReswch, Vol.17,No.7,pp. 1163-1174,1997 Copyright631997Ekvier ScienceInc. Printedin theUSA. .411rightsreserved cr271-5317/97 $17.00+.00 ELSEVIER
PII S0271-5317(97)00086-9
EFFECTS OF DIETARYNUCLEOTIDES ON SERUM ANTIBODY AND SPLENIC CYTOKINE PRODUCTIONIN MICE Shinya Nagafuchi, M.SC.,Tomoko Katayanagi, B.SC.,Emiko Nakagawa, B.SC., Takeshi Takahashi, Ph.D.l, Takaji Yajima, Ph.D., Akie Yonekubo, Ph.D., and Tamotsu Kuwata, Ph.D, Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co,, Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo, Japan.
ABSTRACT We examined the effects of dietary nucleotides on the immune response balance in two subtypes of T helper cells, type 1 (Thl) and type 2 (Th2) cells, In experiment 1, BALB/c mice were maintained on a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucieotides (NT(+) diet) from 4 weeks prior to mating and throughout the experiments. The second generations of these mice (Fl) were maintained on the same diet as the respective dams. Male F1-generation mice were used to determine the serum immunoglobulin levels. The serum lgE levels were significantly decreased in mice fed the NT(+) diet (p <0.05) compared with ones fed the NT(-) diet. The serum lgGl and lgG2a levels were not significantly different between the two dietary groups. However, the lgGl :lgG2a level ratio was significantly decreased in mice fed the NT(+) diet (p <0.05). In experiment 2, BALB/c weanling mice were placed on the NT(-) diet or NT(+) diet for 10 weeks. The production of splenic interleukin-4, a cytokine produced by Th2, was decreased in mice fed the NT(+) diet compared with ones fed the NT(-) diet. On the other hand, the production of splenic interferon-y, a cytokine produced by Thl, was increased in mice fed the NT(+) diet. These results suggest that dietary nucleotides may up-regulate the Thl immune response in systemic immunity. @1997 Ek3iuSciraceIn C. Key words: Dietary nucleotides, T helper cells, Serum antibody response, Cytokine production, Mice
1 Address correspondence and reprint requests to: Takeshi Takahashi, Ph.D., Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co,, Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo 189, Japan. Tel: 0423-97-5670. Fax: 0423-95-1829. 1163
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S. NAGAFUCHIet al. INTRODUCTION
Dietary nucleotides are generally thought to be nonessential for humans, because they are synthesized de novo in the body (l). In the developing neonate, however, dietary nucleotides are semi-essential nutrients in early infancy, because the de novo synthesis of nucleotides does not satisfy the body’s requirement (2). Indeed, human milk contains a considerably large amount of nucleotides that satisfy the dietary needs of newborn infants (3). In addition to their nutritional significance, dietary nucleotides have an influence on the intestinal flora, lipoprotein metabolism and the immune function in infants (4, 5, 6). Dietary nucieotides had a protective effect against diarrhoeal disease in infants (7). With respect to the role of dietary nucleotides in the immune system, much work has been performed in animals. A nucleotide-supplemented diet increased the delayed-type hypersensitivity response (8), interleukin-2 (IL-2) production (9), natural killer cell activity (10), and host resistance to bacterial infection and translocation (11, 12) in mice. Furthermore, dietary nucleotides influenced T-cell responses rather than B-cell responses (13), and regulated T cell activation through antigen-presenting cells (14). Recently, T helper (Th) cells have been divided into at least two subtypes, Thl and Th2 cells (15). Thl cells preferentially secrete Thl cytokines, interleukin-2 (IL-2) and interferon-y (iFN-y), and induce lgG2a antibody production, whereas Th2 cells preferentially secrete Th2 cytokines, IL-4, -5 and -6, and induce lgGl and lgE antibody production. Thl and Th2 cells influence each other, since IFN-y and IL-4 down-regulate Th2 and Thl activities, respectively. The enhancement of the delayed-type hypersensitivity response and IL-2 production by dietary nucleotides suggest that dietary nucleotides may induce the Thl immune response. However, it has not yet been determined whether nucleotide supplementation influences the Thl -Th2 immune response balance in young animals, which grow rapidly and have a poor nucleotide pool in their bodies. The aim of this study is to determine whether dietary nucleotides modulate the Tcell immune balance or not. We investigated the effects of dietary nucleotides on the serum immunoglobulin (lgGl, lgG2a and lgE) levels and splenic cytokine synthesis patterns in mice fed a nucleotide-supplemented diet or a nucleotide-free diet.
MATERIALS AND METHODS Animals All procedures received prior review and approval by our Institute’s Committee for Research on Experimental Animals, and was conducted in accordance with the NRC Guide for the Care and Use of Laboratory Animals. Male and female BALB/c mice were purchased from JapanSLC (Shizuoka,
NUCLEOTIDES ANDIMMUNERESPONSE
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Japan), and were maintained in our animal facilities or those of Japan SLC with controlled temperature (25 t2’T), humidity (40-60%), and light (lights on 0700-1900 h). The mice were maintained on a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with nucleotides (NT(+) diet) for two generations (experiment 1) or for 10 weeks (experiment 2). The nucleotide and diet compositions are shown in Tables 1 and 2, respectively. The supplemented nucleotides were obtained from Yamasa Co and whey protein isolate was obtained from Davisco International Inc. (Le Sueur, MN), The nucleotide composition comprised the similar proportions of nucleotides to those in human milk at 12 weeks of lactation (16), Nucleotide 5’-diphosphates were replaced by nucleotide 5’-monophosphates. Adenosine 5’-monophosphate was also replaced by inosine 5’-monophosphate because it is not lawfully permitted to use adenosine 5’monophosphate as a food additive in Japan.
Table 1 Composition of Dietary Nucleotides in the NT(+) Diet, Nucleotides
glkg
Cytidine monophosphate
1.62
Guanosine monophosphate
0.57
Inosine monophosphate
1.1
Uridine monophosphate
0.71
Treatment of Animals In experiment 1,20 BALB/c female mice (7 weeks old) were randomly assigned to two groups; NT(-) diet and NT(+) diet groups. The two groups of mice,l 0-11 weeks old, were mated, and were constantly kept on the NT(-) diet and NT(+) diet, respectively, for the duration of pregnancy and lactation in the animal facilities of Japan SLC. At the time of weaning, their pups were transferred to our animal facilities and kept there. The diets were identical to those of the respective dams. The male pups (NT(-) diet, n=17; NT(+) diet, n=15) from more than three litters of mice were used at 7 weeks of age to obtain blood samples for antibody determinations. After centrifuging the blood, the sera were collected and stored at -70t until analysis. In experiment 2, BALB/c female mice (n=5), 3 weeks old, were maintained on the NT(-) diet or NT(+) diet for 10 weeks in our animal facilities. Thereafter they were killed, and then spleen cells pooled from 5 mice were examined for in vitro IFN-Yand IL-4 production. Immunoalobulin Assavs The serum concentrations of lgGl, lgG2a and lgE in experiment 1 were determined by means of a sandwich enzyme-linked immunosorbent assay (ELISA).
1166
S. NAGAFUCHIet al. Table 2 Compositions of the Experimental Diets (%) Ingredient
NT(-) diet
NT(+) diet
Whey protein isolate
22.0
22.0
5.0
5.0
Sucrose Starch
60.0
59.6
Cellulose
3.0
3.0
Soybean oil
5.0
5.0
Vitamins *
1.0
1.0
Mineralsw
4.0
4.0
Nucleotides
0.4
* The composition was as follows: (in mg/kg) vitamin A, 201U;7-dehydrocholesterol, 20001U; a -tocopheryl acetate, 50; menadione, 5; choline chloride, 2000; p aminobenzoic acid, 100; inositol, 100; niacin, 40; calcium pantothenate, 40; riboflavin, 8; thiamine HCI, 5; pyridoxine HCI, 5; folic acid, 2; D-biotin, 0.4; cyanocobalamine, 0.03. H The composition was as follows: (in mg/kg) NaCl, 5572; Kl, 31.6; KHPP04,15560;
MgS04, 2292; CaCOs, 15256; FeSOq7H20, 1080; MnSOqHpO, 160.4; ZnSOQ7H20, 0.053; CLJS045HP0,19.1; COCIP6HZ0,0.92.
For ELISA of the total lgGl and lgG2a levels, rabbit antibodies to mouse lgG (Zymed Lab., San Francisco, CA), peroxidase conjugated rabbit anti-mouse lgGl (Zymed) and lgG2a (Zymed), and purified mouse lgGl (Zymed) and lgG2a (Zymed) were used. The primary rabbit anti-mouse antibody (5 pg/ml) diluted in 0.05M carbonate-bicarbonate buffer, pH9.6 (CBB), was added to each well of microtitreplates (Nunc, Roskilde,Denmark), followed by incubation for 2 h at room temperature. The wells were then washed with 0.01M phosphate-buffered saline (PBS), PH7.4, containing 0.1Y. Tween-20 (PBS-O.1%Tween), and blocked with CBB containing 1.5Y0 gelatin for 30 min at room temperature. The plates were washed again and then the serum samples or the purified standard diluted with PBS-O.1%Tween containing 3’XO polyethylene glycol 6000 (Nacalai Tesque Inc., Kyoto, Japan) was added, followed by incubation for 2 h. After washing, the peroxidase-conjugated anti-mouse lgGl or lgG2a, diluted with PBS-O.1%Tween containing 3% polyethylene glycol 6000 at 1/20000, was added to each well, followed by standing for 2 h. The plates were washed and then o-phenylenediamine substrate reagent (1mg of o-phenylenediamine and 0.2 PI of 307. HZOZin 1ml of 0.1M citrate-phosphate buffer, pH5.0) was added to each well. The plates were then incubated for 30 min at room temperature and the reaction was terminated by the addition of 2N H2S04 The absorbance was measured optically at 492nm. The lgGl or lgG2a content was calculated by means of a standard curve.
NUCLEOTIDES ANDIMMUNERESPONSE
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For determination of total lgE levels, microtitre-plates were coated with rat antibodies to mouse lgE (Yamasa Co., Chiba, Japan) (1O pg/ml) diluted in 0.15M PBS, pH7.4. The plates were then incubated for 2 h at room temperature. The wells were then washed with PBS-O.1%Tween, blocked with 0,15M PBS, pH7.4, containing 1% ovalbumin (Seikagaku Co., Tokyo, Japan) for 30 min at room temperature, and washed again. Various dilutions of mouse sera or the purified standard in 0.15M PBS (pH7,4) containing 0.05% Tween-20 were added to the wells, followed by further incubation for 2 h. Following washing, a 1:1000 dilution of peroxidase-conjugated anti-mouse lgE (Kanto Chemicals, Tokyo, Japan) in 0.15M PBS (PH7.4) containing 0.05% Tween-20 was added to each well followed by standing for 2 h. The plates were then rinsed and a o-phenylenediamine substrate solution (0.4mg of o-phenylenediamine and 0.4 PIof 30% HP02in 1 ml of 0.2M citrate-phosphate buffer, pH5.0) was added to each well, The reaction was stopped by adding 2N H2S04. The OD at 492nm of each well was then measured. The concentrations of lgE were calculated by means of a standard curve, Cell Culture and Sdenocvte Stimulation Spleens were aseptically removed from the mice and a single cell suspension was prepared by teasing spleens apart in RPMI-1640 medium (GIBCO, Grand Island, NY). The cells were treated with 0.16 mol ammonium chloride/L to Iyse erythrocytes, The spleen cells (5X1OS)were then resuspended in 0.2 ml of RPMI-1640 medium, which was supplemented with 1YO autologous normal mouse serum, 0.05 mmol/L 2-mercaptoethanol, penicillin (l Xl OSU/L),and streptomycin (1X1OSKg/L). The cells were then incubated under a humidified atmosphere of 5’%. COPat 37°C. The cells were cultured with or without a mitogen [concanavalin A (Con A) or pokeweed mitogen (PWM) (Sigma, St Louis, MO)] for 18 to 72 h. Splenocyte stimulation was measured uptake of splenocytes. Each assay was performed in a set of SiX Wells. by 3H-thymidine Cvtokine Assavs For investigation of IFN-Yproduction, cells were incubated for 18 h in RPMI 1640 medium containing Con A at the concentration of 1 pg/ml. After incubation, culture supernatants were collected by centrifugation and stored at -70~. An ELlSA kit (Bio Source Inter., Camarillo, CA) was used to determine IFN-ycontents. The IFN-ylevels in the samples were calculated by means of a standard curve derived with recombinant mouse IFN-Y. For determination of IL-4 levels, cells were incubated for 72 h in RPMI 1640 medium containing PWM at the concentration of 10 pg/ml (17). The supernatant was collected and stored at -70”C.An ELlSA kit (Endogen Inc., Cambridge, MA) was used to measure the levels of mouse IL-4 in the splenocyte supernatant fractions. The IL-4 contents of the supernatants were calculated by means of a standard curve obtained with recombinant mouse IL-4. Splenocytes exhibited a maximal proliferative response in each concentration of Con A or PWM (data not shown).
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S. NAGAFUCHIet al.
Statistics The results were expressed as means or means with standard errors. The computer program, Stat View, for a Macintosh computer was used for statistical analysis. Statistical analysis of differences was performed using the unpaired Student’s t test, Differences of p <0.05 were considered to be significant.
RESULTS
Ex~eriment 1 In experiment 1, we examined the serum immunoglobulin levels in mice. We used male mice fed a NT(-) or NT(+) diet for two generations, because dietary nucleotides are particularly essential for animals during pregnancy and early infancy, and the effect of dietary nucleotides was expected to be more apparent (2). No statistically significant differences were observed in the growth curve for body weight between the two dietary groups (data not shown). The serum lgE levels significantly decreased in mice fed the NT(+) diet (p <0.05) compared with ones fed the NT(-) diet (Fig. 1). On the other hand, the serum lgGl and lgG2a levels were not significantly different between the two dietary groups (Fig. 2). However, the lgGl :lgG2a concentration ratio significantly decreased in mice fed the NT(+) diet compared to ones fed the NT(-) diet (Fig. 3). There were no differences in the serum level of total immunoglobulin (lgA+lgG+lgM) between the two dietary groups
(data not shown). Similar results were obtained when female mice were used (data not shown).
0.6, 0.5 0.4 0.3
❑
NT(-)
❑
NT(+)
r *
0.2 0.1 0L FIG. 1. Effects of dietarv nucleotides on serum laE levels in F1generation BALB/c mice”at 7 weeks of age born lo BALB/c mice (FO) fed the NT(+) or NT(-) diet. Each column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors. The asterisk (*) indicates a significant difference at p <0.05 between the two groups.
NUCLEOTIDES ANDIMMUNERESPONSE 120 = E s ~
1oo-
a g
40-
❑ ❑
8060-
1169
NT(-) NT(+) I
T
200-
lgGl
lgG2a
FIG. 2. Effects of dietary nucleotides on the serum lgGl and lgG2a levels in F1-generation BALB/c mice at 7 weeks of age krn to BALB/c m~ce (FO) fed the NT(+) or NT(-) diet. Ea~h column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors.
FIG. 3. Effects of dietary nucleotides on the serum lgGl and lgG2a level (lgG1/lgG2a) ratio in F1 -generation BALB/c mice at 7 weeks of age born to BALB/c mice (FO) fed the NT(+) or NT(-) diet. Each column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors. The asterisk (*) indicates a significant difference at p <0.05 between the two groups.
Ex~eriment 2 In experiment 2, we examined the pattern of cytokine production of spleen cells from BALB/c mice fed the NT(-) or NT(+) diet from 3 to 13 weeks of age. Decreased IL-4 production was observed in mice fed the NT(+) diet compared with ones fed the NT(-) diet (Fig. 4). In contrast, increased IFN-Yproduction was observed in the NT(+) diet group (Fig. 5). When spleen cells were cultured without a mitogen for 18 h or 72 h, no differences were observed between the two dietary groups, and the background response was under the detection limit (data not shown).
1170
S. NAGAFUCHIet al.
n —
I
❑
NT(-)
FIG. 4. lnterleukin-4 (IL-4) production in vitro bv spleen cells from
mice fed the NT(+) d’iet or”NT(-) diet from 3 to 13 weeks of age. Spleen cells were pooled from five mice of each dietary group. The cell cultures were carried out in the presence of pokeweed mitogen (PWM) at the concentration of 10 @/ml. This is the result of a representative experiment of three experiments. Each column represents the average IL-4 level for six wells.
FIG. 5. Interferon-y (lFN-y) production in vitro by spleen cells from mice fed the NT(+) diet or NT(-) diet from 3 to 13 weeks of age. Spleen cells were pooled from five mice of each dietary group. The cell cultures were carried out in the presence of concanavalin A (Con A) at the concentration of 1 @ml, This is the result of a representative experiment of three experiments. Each column represents the average IFN-y level for six wells.
DISCUSSION
The impact of nutrients on the immune system has been demonstrated in numerous investigations, for example, a deficiency of certain nutrients, such as zinc and vitamin A, is known to lead to profound immune dysfunction (18). It was recently found that dietary nucleotides are also some of the nutrients that are able to cause deficiency diseases under certain conditions. The need for dietary nucleotides
NUCLEOTIDESAND IMMUNE RESPONSE
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increases in patients recovering from tissue injury, major surgery, systemic infection or extensive burn injury, or in periods of rapid growth such as infancy or adolescence (19). Therefore, dietary nucleotides are regarded as conditionally essential nutrients. Thus, we used weanling mice in our study to examine the significance of dietary nucleotides for the immune function. We used a similar nucleotide profile to that of human milk in this study, because human milk is rich in dietary nucleotides, and its nucleotide profile is thought to be a good model for study of the importance of dietary nucleotides in infant nutrition, With regard to this nucleotide profile, we confirmed that nucleotide-fed mice exhibited a higher response to Con A (T-cell response), but a similar response to Iipopolysaccharide (LPS) (B-cell response) compared to nucleotide-deficient mice in a preliminary study (data not shown), This effect of dietary nucleotides is in agreement with an earlier report (13). It has been shown that dietary nucleotides influence T-cell mediated immune responses, increasing the delayed-type hypersensitivity response, T-cell mitogenic response, production of IL-2, and expression of IL-2 receptors and Iyt-1 surface markers (20). Moreover, humoral immune responses were enhanced by nucleotide supplementation. Jyonouchi (14) observed nonspecific increases in antibody and immunoglobulin production when murine spleen cells were briefly incubated with polynucleotides before the antigen in priming culture. However, the details of the modulation of antibody isotype responses by dietary nucleotides remain to be determined. In our study, the serum lgE levels were significantly low in mice fed the NT(+) diet as compared to ones fed the NT(-) diet, while the serum lgG2a levels were high, although not significantly, in mice fed the NT(+) diet. In addition, the lgGl :lgG2a ratio was significantly lower in nucleotide-fed mice than in nucleotide-deficient ones. These results suggest that dietary nucleotides may influence the level of serum antibody isotypes. Since lgE and lgG2a antibody production is induced by Th2 and Thl cells, respectively (21), dietary nucleotides seem to down-regulate the Th2 immune response and up-regulate the Thl immune response. This is supported by the fact that the production of IFN-Y(a cytokine produced by Thl) by Con A-stimulated spleen cells was higher in the nucleotide-supplemented group than in the unsupplemented one, whereas the production of IL-4 (a cytokine produced by Th2) by PWM-stimulated spleen cells was higher in the unsupplemented group than in the nucieotide-supplemented one. We are now further examining IFN-Y production at the cell population level by immunofluorescence staining techniques. Although the nucleotide profile used in this study was not completely consistent with that of human milk, we speculate that the nucleotides in human milk may play a possible role in immune regulation of the early development of Thl vs Th2 responses. Interestingly, vitamin A in human milk has also been shown to modulate the Thl Th2 immune response balance (22). For instance, the serum lgE antibody response to the T-celi dependent antigen was significantly lower in vitamin A-deficient rats than in control ones (23). Furthermore, IL-2 and IFN-y production increased in the vitamin A-deficient rats. It is suggested, therefore, that vitamin A has a different effect on the immune system to dietary nucleotides. Thus, it may be postulated that human milk contains different types of substances which contribute to the well-balanced development of the T-cell function in early infancy.
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It iS Well known that COW’Smilk contains a much lower quantity of nucleotides than human milk (24). Thus, infant formulae based on cow’s milk are usually lacking in nucleotides. This could be one of the reasons for the fact that formula-fed infants are more susceptible to food allergies and have higher serum lgE levels than breast-fed infants (25), because dietary nucleotides in human milk can up-regulate the Thl immune responses such as IFN-yproduction, resulting in down-regulation of the serum lgE response, as shown in this study. In other words, it is possible that by supplementing infant formulas with dietary nucleotides down-regulation of the Th2 immune responses to milk protein allergens in formula-fed infants may occur. In order to confirm this hypothesis, we are now investigating the serum lgE antibody response to cow’s milk proteins, such as ~ -Iactoglobulin, in mice fed the NT(-) or NT(+) diet.
On the other hand, it has been reported that human milk contains food allergens such as cow’s milk and egg proteins derived from the mother’s diet, which in some cases can sensitize the infant to cow’s milk and eggs (26). Thus, food allergies may develop in exclusively breast-fed infants. However, even if a food allergy has developed, it is possible that dietary nucleotides in human milk may decrease the lgE-mediated allergenic response by modulating the Thl -Th2 immune response balance. Therefore, the abundance of dietary nucleotides in human milk could be the fail-safe mechanism preventing infants from developing lgE-mediated allergies, In conclusion, we have provided evidence that dietary nucleotides can influence the serum antibody response by regulating the delicate balance between the Thl and Th2 activities, We believe that our results provide useful information for the development of a milk formula adapted better to human milk.
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Quan R. Dietary nucleotides: Potential for immune enhancement. In: PaubertBraquet M, Dupont C, Paoletti R, eds. Foods, Nutrition and Immunity. Dyn Nutr Res.Vol.l Basel: Karger, 1992: 13-21.
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Uauy R,Quan R.Significance of nucleic acids, nucleotides, and related compounds in infant nutrition. In: Raiha NCR, ed. Protein Metabolism During Infancy. Nestle Nutrition Workshop Series. Vol. 33, New York: Vevey/Raven Press, 1994: 197-210,
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Barness LA. Dietary sources of nucleotides from breast milk to weaning. J Nutr 1994; 124:128S-130S.
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Gil A, Corrai E, Martinez A, Molina JA. Effects of the addition of nucleotides to an adapted milk formula on the microbial pattern of faeces in at term newborn infants. J Clin Nutr Gastroenterol 1986; 1:127-132.
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Sanchez-Pozo A, Morillas J, Molto L, Robles R,Gil A. Dietary nucleotides influence lipoprotein metabolism in newborn infants. Pediatr Res 1994; 35:112116.
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Carver JD, Pimentel B, Cox Wl, Barness LA, Dietary nucleotide effects upon immune function in infants, Pediatrics 1991; 88:359-363.
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Brunser O, Espinoza J, Araya M, Cruchet S, Gil A. Effect of dietary nucleotide supplementation on diarrhoeal disease in infants. Acta Paediatr 1994; 83:188191.
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Kulkarni AD, Fanslow WC, Rudolph FB,Van Buren CT, Modulation of delayed hypersensitivity in mice by dietary nucleotide restriction. Transplantation 1987; 44:847-849.
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Van Buren CT, Kulkarni AD, Fanslow WC, Rudolph FB. Dietary nucleotides, a requirement for helper/inducer T lymphocytes.Transplantation 1985; 40:694-697.
10. Carver JD, Cox Wl, Barness LA. Dietary nucleotide effects upon murine natural killer cell activity and microphage activation, J Parenter Enteral Nutr 1990; 14:1822. 11. Kulkarni AD, Fanslow WC, Rudolph FB,Van Buren CT. Effectof dietary nucleotides on response to bacterial infections. J Parenter Enteral Nutr 1986; 10: 169-171. 12. Adjei AA, Yamamoto S. A dietary nucleoside-nucleotide mixture inhibits endotoxin-induced bacterial translocation in mice fed protein-free diet. J Nutr 1995;125:42-48. 13.
Kulkarni SS, Bhateley DC, Zander AR, Van Buren CT, Rudolph FB, Dicke KA, Kulkarni AD. Functional impairment of T-lymphocytes in mouse radiation chimeras by a nucleotide-free diet. Exp Hematol 1984; 12:694-699.
14. Jyonouchi H. Nucleotide actions on humoral immune responses. J Nutr 1994; 124:138S-143S. 15.
Mosmann TR, Coffman RL. Thl and Th2 cells: different patterns of Iymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7:145173.
16. Janas LM, Picciano MF. The nucleotide profile of human milk. Pediatr Res 1982; 16: 659-662. 17. Bottomly K. A functional dichotomy in CD4+ T lymphocytes. Immunology Today 1988; 9:268-274. 18. Bernadette MH. Diet and immune function. Br J of Biomed Sci 1994; 51:252-259. 19. Adjei M, Yamamoto S, Kulkarni AD. Nucleic acids and/or their components: A possible role in immune function. J Nutr Sci Vitaminol 1995; 41:1-16.
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Carver JD, Walker WA. The role of nucleotides in human nutrition. Nutritional Biochemistry 1995; 6:58-72.
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Carter LL, Dutton RW. Type 1 and type 2: a fundamental dichotomy for all T-cell subsets. Current Opinion in Immunology 1996; 8:336-342.
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Hanson LA, Hahn-Zoric M, Wiedermann U, Lundin S, Dahlman-Hoglund A, Saalman R, Erling V, Dahlgren U, Telemo E. Early dietary influence on later immunocompetence. Nutrition Rev 1996; 54:S23-S30.
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Wiedermann U, Hanson IA, Kahu H, Dahlgren U1.Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats. Immunology 1993; 80:581-586.
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Gil A, Uauy R. Nucleotides and related compounds in human and bovine milks. In: Gordon JR, ed. Handbook of Milk Composition, San Diego: Academic Press Inc., 1995: 436-464.
25.
Vandenplas Y, Deneyer M, Sacre L, Loeb H. Influence of feeding breast milk, adapted milk formula, and a new hypoallergenic formula on allergic manifestations in infants: a field study. In: Schmidt E, ed. Food Allergy. Nestle Nutrition Workshop Series. Vol. 17, New York: Vevey/Raven Press Ltd., 1988: 257-264.
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Accepted for publicationApril 8, 1997.
PII S0271-5317(97)00086-9
EFFECTS OF DIETARYNUCLEOTIDES ON SERUM ANTIBODY AND SPLENIC CYTOKINE PRODUCTIONIN MICE Shinya Nagafuchi, M.SC.,Tomoko Katayanagi, B.SC.,Emiko Nakagawa, B.SC., Takeshi Takahashi, Ph.D.l, Takaji Yajima, Ph.D., Akie Yonekubo, Ph.D., and Tamotsu Kuwata, Ph.D, Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co,, Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo, Japan.
ABSTRACT We examined the effects of dietary nucleotides on the immune response balance in two subtypes of T helper cells, type 1 (Thl) and type 2 (Th2) cells, In experiment 1, BALB/c mice were maintained on a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucieotides (NT(+) diet) from 4 weeks prior to mating and throughout the experiments. The second generations of these mice (Fl) were maintained on the same diet as the respective dams. Male F1-generation mice were used to determine the serum immunoglobulin levels. The serum lgE levels were significantly decreased in mice fed the NT(+) diet (p <0.05) compared with ones fed the NT(-) diet. The serum lgGl and lgG2a levels were not significantly different between the two dietary groups. However, the lgGl :lgG2a level ratio was significantly decreased in mice fed the NT(+) diet (p <0.05). In experiment 2, BALB/c weanling mice were placed on the NT(-) diet or NT(+) diet for 10 weeks. The production of splenic interleukin-4, a cytokine produced by Th2, was decreased in mice fed the NT(+) diet compared with ones fed the NT(-) diet. On the other hand, the production of splenic interferon-y, a cytokine produced by Thl, was increased in mice fed the NT(+) diet. These results suggest that dietary nucleotides may up-regulate the Thl immune response in systemic immunity. @1997 Ek3iuSciraceIn C. Key words: Dietary nucleotides, T helper cells, Serum antibody response, Cytokine production, Mice
1 Address correspondence and reprint requests to: Takeshi Takahashi, Ph.D., Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co,, Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo 189, Japan. Tel: 0423-97-5670. Fax: 0423-95-1829. 1163
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S. NAGAFUCHIet al. INTRODUCTION
Dietary nucleotides are generally thought to be nonessential for humans, because they are synthesized de novo in the body (l). In the developing neonate, however, dietary nucleotides are semi-essential nutrients in early infancy, because the de novo synthesis of nucleotides does not satisfy the body’s requirement (2). Indeed, human milk contains a considerably large amount of nucleotides that satisfy the dietary needs of newborn infants (3). In addition to their nutritional significance, dietary nucleotides have an influence on the intestinal flora, lipoprotein metabolism and the immune function in infants (4, 5, 6). Dietary nucieotides had a protective effect against diarrhoeal disease in infants (7). With respect to the role of dietary nucleotides in the immune system, much work has been performed in animals. A nucleotide-supplemented diet increased the delayed-type hypersensitivity response (8), interleukin-2 (IL-2) production (9), natural killer cell activity (10), and host resistance to bacterial infection and translocation (11, 12) in mice. Furthermore, dietary nucleotides influenced T-cell responses rather than B-cell responses (13), and regulated T cell activation through antigen-presenting cells (14). Recently, T helper (Th) cells have been divided into at least two subtypes, Thl and Th2 cells (15). Thl cells preferentially secrete Thl cytokines, interleukin-2 (IL-2) and interferon-y (iFN-y), and induce lgG2a antibody production, whereas Th2 cells preferentially secrete Th2 cytokines, IL-4, -5 and -6, and induce lgGl and lgE antibody production. Thl and Th2 cells influence each other, since IFN-y and IL-4 down-regulate Th2 and Thl activities, respectively. The enhancement of the delayed-type hypersensitivity response and IL-2 production by dietary nucleotides suggest that dietary nucleotides may induce the Thl immune response. However, it has not yet been determined whether nucleotide supplementation influences the Thl -Th2 immune response balance in young animals, which grow rapidly and have a poor nucleotide pool in their bodies. The aim of this study is to determine whether dietary nucleotides modulate the Tcell immune balance or not. We investigated the effects of dietary nucleotides on the serum immunoglobulin (lgGl, lgG2a and lgE) levels and splenic cytokine synthesis patterns in mice fed a nucleotide-supplemented diet or a nucleotide-free diet.
MATERIALS AND METHODS Animals All procedures received prior review and approval by our Institute’s Committee for Research on Experimental Animals, and was conducted in accordance with the NRC Guide for the Care and Use of Laboratory Animals. Male and female BALB/c mice were purchased from JapanSLC (Shizuoka,
NUCLEOTIDES ANDIMMUNERESPONSE
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Japan), and were maintained in our animal facilities or those of Japan SLC with controlled temperature (25 t2’T), humidity (40-60%), and light (lights on 0700-1900 h). The mice were maintained on a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with nucleotides (NT(+) diet) for two generations (experiment 1) or for 10 weeks (experiment 2). The nucleotide and diet compositions are shown in Tables 1 and 2, respectively. The supplemented nucleotides were obtained from Yamasa Co and whey protein isolate was obtained from Davisco International Inc. (Le Sueur, MN), The nucleotide composition comprised the similar proportions of nucleotides to those in human milk at 12 weeks of lactation (16), Nucleotide 5’-diphosphates were replaced by nucleotide 5’-monophosphates. Adenosine 5’-monophosphate was also replaced by inosine 5’-monophosphate because it is not lawfully permitted to use adenosine 5’monophosphate as a food additive in Japan.
Table 1 Composition of Dietary Nucleotides in the NT(+) Diet, Nucleotides
glkg
Cytidine monophosphate
1.62
Guanosine monophosphate
0.57
Inosine monophosphate
1.1
Uridine monophosphate
0.71
Treatment of Animals In experiment 1,20 BALB/c female mice (7 weeks old) were randomly assigned to two groups; NT(-) diet and NT(+) diet groups. The two groups of mice,l 0-11 weeks old, were mated, and were constantly kept on the NT(-) diet and NT(+) diet, respectively, for the duration of pregnancy and lactation in the animal facilities of Japan SLC. At the time of weaning, their pups were transferred to our animal facilities and kept there. The diets were identical to those of the respective dams. The male pups (NT(-) diet, n=17; NT(+) diet, n=15) from more than three litters of mice were used at 7 weeks of age to obtain blood samples for antibody determinations. After centrifuging the blood, the sera were collected and stored at -70t until analysis. In experiment 2, BALB/c female mice (n=5), 3 weeks old, were maintained on the NT(-) diet or NT(+) diet for 10 weeks in our animal facilities. Thereafter they were killed, and then spleen cells pooled from 5 mice were examined for in vitro IFN-Yand IL-4 production. Immunoalobulin Assavs The serum concentrations of lgGl, lgG2a and lgE in experiment 1 were determined by means of a sandwich enzyme-linked immunosorbent assay (ELISA).
1166
S. NAGAFUCHIet al. Table 2 Compositions of the Experimental Diets (%) Ingredient
NT(-) diet
NT(+) diet
Whey protein isolate
22.0
22.0
5.0
5.0
Sucrose Starch
60.0
59.6
Cellulose
3.0
3.0
Soybean oil
5.0
5.0
Vitamins *
1.0
1.0
Mineralsw
4.0
4.0
Nucleotides
0.4
* The composition was as follows: (in mg/kg) vitamin A, 201U;7-dehydrocholesterol, 20001U; a -tocopheryl acetate, 50; menadione, 5; choline chloride, 2000; p aminobenzoic acid, 100; inositol, 100; niacin, 40; calcium pantothenate, 40; riboflavin, 8; thiamine HCI, 5; pyridoxine HCI, 5; folic acid, 2; D-biotin, 0.4; cyanocobalamine, 0.03. H The composition was as follows: (in mg/kg) NaCl, 5572; Kl, 31.6; KHPP04,15560;
MgS04, 2292; CaCOs, 15256; FeSOq7H20, 1080; MnSOqHpO, 160.4; ZnSOQ7H20, 0.053; CLJS045HP0,19.1; COCIP6HZ0,0.92.
For ELISA of the total lgGl and lgG2a levels, rabbit antibodies to mouse lgG (Zymed Lab., San Francisco, CA), peroxidase conjugated rabbit anti-mouse lgGl (Zymed) and lgG2a (Zymed), and purified mouse lgGl (Zymed) and lgG2a (Zymed) were used. The primary rabbit anti-mouse antibody (5 pg/ml) diluted in 0.05M carbonate-bicarbonate buffer, pH9.6 (CBB), was added to each well of microtitreplates (Nunc, Roskilde,Denmark), followed by incubation for 2 h at room temperature. The wells were then washed with 0.01M phosphate-buffered saline (PBS), PH7.4, containing 0.1Y. Tween-20 (PBS-O.1%Tween), and blocked with CBB containing 1.5Y0 gelatin for 30 min at room temperature. The plates were washed again and then the serum samples or the purified standard diluted with PBS-O.1%Tween containing 3’XO polyethylene glycol 6000 (Nacalai Tesque Inc., Kyoto, Japan) was added, followed by incubation for 2 h. After washing, the peroxidase-conjugated anti-mouse lgGl or lgG2a, diluted with PBS-O.1%Tween containing 3% polyethylene glycol 6000 at 1/20000, was added to each well, followed by standing for 2 h. The plates were washed and then o-phenylenediamine substrate reagent (1mg of o-phenylenediamine and 0.2 PI of 307. HZOZin 1ml of 0.1M citrate-phosphate buffer, pH5.0) was added to each well. The plates were then incubated for 30 min at room temperature and the reaction was terminated by the addition of 2N H2S04 The absorbance was measured optically at 492nm. The lgGl or lgG2a content was calculated by means of a standard curve.
NUCLEOTIDES ANDIMMUNERESPONSE
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For determination of total lgE levels, microtitre-plates were coated with rat antibodies to mouse lgE (Yamasa Co., Chiba, Japan) (1O pg/ml) diluted in 0.15M PBS, pH7.4. The plates were then incubated for 2 h at room temperature. The wells were then washed with PBS-O.1%Tween, blocked with 0,15M PBS, pH7.4, containing 1% ovalbumin (Seikagaku Co., Tokyo, Japan) for 30 min at room temperature, and washed again. Various dilutions of mouse sera or the purified standard in 0.15M PBS (pH7,4) containing 0.05% Tween-20 were added to the wells, followed by further incubation for 2 h. Following washing, a 1:1000 dilution of peroxidase-conjugated anti-mouse lgE (Kanto Chemicals, Tokyo, Japan) in 0.15M PBS (PH7.4) containing 0.05% Tween-20 was added to each well followed by standing for 2 h. The plates were then rinsed and a o-phenylenediamine substrate solution (0.4mg of o-phenylenediamine and 0.4 PIof 30% HP02in 1 ml of 0.2M citrate-phosphate buffer, pH5.0) was added to each well, The reaction was stopped by adding 2N H2S04. The OD at 492nm of each well was then measured. The concentrations of lgE were calculated by means of a standard curve, Cell Culture and Sdenocvte Stimulation Spleens were aseptically removed from the mice and a single cell suspension was prepared by teasing spleens apart in RPMI-1640 medium (GIBCO, Grand Island, NY). The cells were treated with 0.16 mol ammonium chloride/L to Iyse erythrocytes, The spleen cells (5X1OS)were then resuspended in 0.2 ml of RPMI-1640 medium, which was supplemented with 1YO autologous normal mouse serum, 0.05 mmol/L 2-mercaptoethanol, penicillin (l Xl OSU/L),and streptomycin (1X1OSKg/L). The cells were then incubated under a humidified atmosphere of 5’%. COPat 37°C. The cells were cultured with or without a mitogen [concanavalin A (Con A) or pokeweed mitogen (PWM) (Sigma, St Louis, MO)] for 18 to 72 h. Splenocyte stimulation was measured uptake of splenocytes. Each assay was performed in a set of SiX Wells. by 3H-thymidine Cvtokine Assavs For investigation of IFN-Yproduction, cells were incubated for 18 h in RPMI 1640 medium containing Con A at the concentration of 1 pg/ml. After incubation, culture supernatants were collected by centrifugation and stored at -70~. An ELlSA kit (Bio Source Inter., Camarillo, CA) was used to determine IFN-ycontents. The IFN-ylevels in the samples were calculated by means of a standard curve derived with recombinant mouse IFN-Y. For determination of IL-4 levels, cells were incubated for 72 h in RPMI 1640 medium containing PWM at the concentration of 10 pg/ml (17). The supernatant was collected and stored at -70”C.An ELlSA kit (Endogen Inc., Cambridge, MA) was used to measure the levels of mouse IL-4 in the splenocyte supernatant fractions. The IL-4 contents of the supernatants were calculated by means of a standard curve obtained with recombinant mouse IL-4. Splenocytes exhibited a maximal proliferative response in each concentration of Con A or PWM (data not shown).
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S. NAGAFUCHIet al.
Statistics The results were expressed as means or means with standard errors. The computer program, Stat View, for a Macintosh computer was used for statistical analysis. Statistical analysis of differences was performed using the unpaired Student’s t test, Differences of p <0.05 were considered to be significant.
RESULTS
Ex~eriment 1 In experiment 1, we examined the serum immunoglobulin levels in mice. We used male mice fed a NT(-) or NT(+) diet for two generations, because dietary nucleotides are particularly essential for animals during pregnancy and early infancy, and the effect of dietary nucleotides was expected to be more apparent (2). No statistically significant differences were observed in the growth curve for body weight between the two dietary groups (data not shown). The serum lgE levels significantly decreased in mice fed the NT(+) diet (p <0.05) compared with ones fed the NT(-) diet (Fig. 1). On the other hand, the serum lgGl and lgG2a levels were not significantly different between the two dietary groups (Fig. 2). However, the lgGl :lgG2a concentration ratio significantly decreased in mice fed the NT(+) diet compared to ones fed the NT(-) diet (Fig. 3). There were no differences in the serum level of total immunoglobulin (lgA+lgG+lgM) between the two dietary groups
(data not shown). Similar results were obtained when female mice were used (data not shown).
0.6, 0.5 0.4 0.3
❑
NT(-)
❑
NT(+)
r *
0.2 0.1 0L FIG. 1. Effects of dietarv nucleotides on serum laE levels in F1generation BALB/c mice”at 7 weeks of age born lo BALB/c mice (FO) fed the NT(+) or NT(-) diet. Each column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors. The asterisk (*) indicates a significant difference at p <0.05 between the two groups.
NUCLEOTIDES ANDIMMUNERESPONSE 120 = E s ~
1oo-
a g
40-
❑ ❑
8060-
1169
NT(-) NT(+) I
T
200-
lgGl
lgG2a
FIG. 2. Effects of dietary nucleotides on the serum lgGl and lgG2a levels in F1-generation BALB/c mice at 7 weeks of age krn to BALB/c m~ce (FO) fed the NT(+) or NT(-) diet. Ea~h column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors.
FIG. 3. Effects of dietary nucleotides on the serum lgGl and lgG2a level (lgG1/lgG2a) ratio in F1 -generation BALB/c mice at 7 weeks of age born to BALB/c mice (FO) fed the NT(+) or NT(-) diet. Each column represents the mean of 15 to 17 individual mice from more than three litters. Error bars indicate standard errors. The asterisk (*) indicates a significant difference at p <0.05 between the two groups.
Ex~eriment 2 In experiment 2, we examined the pattern of cytokine production of spleen cells from BALB/c mice fed the NT(-) or NT(+) diet from 3 to 13 weeks of age. Decreased IL-4 production was observed in mice fed the NT(+) diet compared with ones fed the NT(-) diet (Fig. 4). In contrast, increased IFN-Yproduction was observed in the NT(+) diet group (Fig. 5). When spleen cells were cultured without a mitogen for 18 h or 72 h, no differences were observed between the two dietary groups, and the background response was under the detection limit (data not shown).
1170
S. NAGAFUCHIet al.
n —
I
❑
NT(-)
FIG. 4. lnterleukin-4 (IL-4) production in vitro bv spleen cells from
mice fed the NT(+) d’iet or”NT(-) diet from 3 to 13 weeks of age. Spleen cells were pooled from five mice of each dietary group. The cell cultures were carried out in the presence of pokeweed mitogen (PWM) at the concentration of 10 @/ml. This is the result of a representative experiment of three experiments. Each column represents the average IL-4 level for six wells.
FIG. 5. Interferon-y (lFN-y) production in vitro by spleen cells from mice fed the NT(+) diet or NT(-) diet from 3 to 13 weeks of age. Spleen cells were pooled from five mice of each dietary group. The cell cultures were carried out in the presence of concanavalin A (Con A) at the concentration of 1 @ml, This is the result of a representative experiment of three experiments. Each column represents the average IFN-y level for six wells.
DISCUSSION
The impact of nutrients on the immune system has been demonstrated in numerous investigations, for example, a deficiency of certain nutrients, such as zinc and vitamin A, is known to lead to profound immune dysfunction (18). It was recently found that dietary nucleotides are also some of the nutrients that are able to cause deficiency diseases under certain conditions. The need for dietary nucleotides
NUCLEOTIDESAND IMMUNE RESPONSE
1171
increases in patients recovering from tissue injury, major surgery, systemic infection or extensive burn injury, or in periods of rapid growth such as infancy or adolescence (19). Therefore, dietary nucleotides are regarded as conditionally essential nutrients. Thus, we used weanling mice in our study to examine the significance of dietary nucleotides for the immune function. We used a similar nucleotide profile to that of human milk in this study, because human milk is rich in dietary nucleotides, and its nucleotide profile is thought to be a good model for study of the importance of dietary nucleotides in infant nutrition, With regard to this nucleotide profile, we confirmed that nucleotide-fed mice exhibited a higher response to Con A (T-cell response), but a similar response to Iipopolysaccharide (LPS) (B-cell response) compared to nucleotide-deficient mice in a preliminary study (data not shown), This effect of dietary nucleotides is in agreement with an earlier report (13). It has been shown that dietary nucleotides influence T-cell mediated immune responses, increasing the delayed-type hypersensitivity response, T-cell mitogenic response, production of IL-2, and expression of IL-2 receptors and Iyt-1 surface markers (20). Moreover, humoral immune responses were enhanced by nucleotide supplementation. Jyonouchi (14) observed nonspecific increases in antibody and immunoglobulin production when murine spleen cells were briefly incubated with polynucleotides before the antigen in priming culture. However, the details of the modulation of antibody isotype responses by dietary nucleotides remain to be determined. In our study, the serum lgE levels were significantly low in mice fed the NT(+) diet as compared to ones fed the NT(-) diet, while the serum lgG2a levels were high, although not significantly, in mice fed the NT(+) diet. In addition, the lgGl :lgG2a ratio was significantly lower in nucleotide-fed mice than in nucleotide-deficient ones. These results suggest that dietary nucleotides may influence the level of serum antibody isotypes. Since lgE and lgG2a antibody production is induced by Th2 and Thl cells, respectively (21), dietary nucleotides seem to down-regulate the Th2 immune response and up-regulate the Thl immune response. This is supported by the fact that the production of IFN-Y(a cytokine produced by Thl) by Con A-stimulated spleen cells was higher in the nucleotide-supplemented group than in the unsupplemented one, whereas the production of IL-4 (a cytokine produced by Th2) by PWM-stimulated spleen cells was higher in the unsupplemented group than in the nucieotide-supplemented one. We are now further examining IFN-Y production at the cell population level by immunofluorescence staining techniques. Although the nucleotide profile used in this study was not completely consistent with that of human milk, we speculate that the nucleotides in human milk may play a possible role in immune regulation of the early development of Thl vs Th2 responses. Interestingly, vitamin A in human milk has also been shown to modulate the Thl Th2 immune response balance (22). For instance, the serum lgE antibody response to the T-celi dependent antigen was significantly lower in vitamin A-deficient rats than in control ones (23). Furthermore, IL-2 and IFN-y production increased in the vitamin A-deficient rats. It is suggested, therefore, that vitamin A has a different effect on the immune system to dietary nucleotides. Thus, it may be postulated that human milk contains different types of substances which contribute to the well-balanced development of the T-cell function in early infancy.
1172
S. NAGAFUCHIet al.
It iS Well known that COW’Smilk contains a much lower quantity of nucleotides than human milk (24). Thus, infant formulae based on cow’s milk are usually lacking in nucleotides. This could be one of the reasons for the fact that formula-fed infants are more susceptible to food allergies and have higher serum lgE levels than breast-fed infants (25), because dietary nucleotides in human milk can up-regulate the Thl immune responses such as IFN-yproduction, resulting in down-regulation of the serum lgE response, as shown in this study. In other words, it is possible that by supplementing infant formulas with dietary nucleotides down-regulation of the Th2 immune responses to milk protein allergens in formula-fed infants may occur. In order to confirm this hypothesis, we are now investigating the serum lgE antibody response to cow’s milk proteins, such as ~ -Iactoglobulin, in mice fed the NT(-) or NT(+) diet.
On the other hand, it has been reported that human milk contains food allergens such as cow’s milk and egg proteins derived from the mother’s diet, which in some cases can sensitize the infant to cow’s milk and eggs (26). Thus, food allergies may develop in exclusively breast-fed infants. However, even if a food allergy has developed, it is possible that dietary nucleotides in human milk may decrease the lgE-mediated allergenic response by modulating the Thl -Th2 immune response balance. Therefore, the abundance of dietary nucleotides in human milk could be the fail-safe mechanism preventing infants from developing lgE-mediated allergies, In conclusion, we have provided evidence that dietary nucleotides can influence the serum antibody response by regulating the delicate balance between the Thl and Th2 activities, We believe that our results provide useful information for the development of a milk formula adapted better to human milk.
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Mosmann TR, Coffman RL. Thl and Th2 cells: different patterns of Iymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7:145173.
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Carver JD, Walker WA. The role of nucleotides in human nutrition. Nutritional Biochemistry 1995; 6:58-72.
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Carter LL, Dutton RW. Type 1 and type 2: a fundamental dichotomy for all T-cell subsets. Current Opinion in Immunology 1996; 8:336-342.
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Hanson LA, Hahn-Zoric M, Wiedermann U, Lundin S, Dahlman-Hoglund A, Saalman R, Erling V, Dahlgren U, Telemo E. Early dietary influence on later immunocompetence. Nutrition Rev 1996; 54:S23-S30.
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Wiedermann U, Hanson IA, Kahu H, Dahlgren U1.Aberrant T-cell function in vitro and impaired T-cell dependent antibody response in vivo in vitamin A-deficient rats. Immunology 1993; 80:581-586.
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Gil A, Uauy R. Nucleotides and related compounds in human and bovine milks. In: Gordon JR, ed. Handbook of Milk Composition, San Diego: Academic Press Inc., 1995: 436-464.
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Vandenplas Y, Deneyer M, Sacre L, Loeb H. Influence of feeding breast milk, adapted milk formula, and a new hypoallergenic formula on allergic manifestations in infants: a field study. In: Schmidt E, ed. Food Allergy. Nestle Nutrition Workshop Series. Vol. 17, New York: Vevey/Raven Press Ltd., 1988: 257-264.
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Accepted for publicationApril 8, 1997.