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Effect of dietary choline deficiency on immunocompetence in Wistar rats
Nutrition Research 23 (2003) 519 –526 www.elsevier.com/locate/nutres
Effect of dietary choline deficiency on immunocompetence in Wistar rats Maria C. Courre`gesa,*, Fabian Benenciab,1, Ana Ucedaa, Alberto J. Monserrata,1 a
Experimental Pathology, Department of Pathology, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina b Laboratory of Immunochemistry, Department of Biological Chemistry, School of Sciences, University of Buenos Aires, Buenos Aires, Argentina Received 3 August 2002; received in revised form 15 November 2002; accepted 29 November 2002
Abstract In order to study the effect of dietary choline deficiency on immunity, adult Wistar rats were fed for two months with choline supplemented (CS, 0.35% choline chloride) or deficient (CD) diets and different parameters of immune response against sheep blood red cells or ovoalbumin (OVA) were evaluated. We found a significant reduction of haemagglutinating (HA) antibodies in sera (day 5 post-immunization: CD, 110 ⫾ 12 HAU vs. CS 320 ⫾ 40 HAU, p ⬍ 0.05) and only a slight difference in anti-OVA antibodies in CD group. Delayed type hypersensitivity was significantly impaired in CD group (footpad swelling in mm: CD, 0.97 ⫾ 0.51 vs. CS, 2.32 ⫾ 0.53, p ⬍ 0.05). In vitro, concanavalin A (Con A) stimulated proliferation of lymphocytes from spleen or lymph nodes of CD rats were diminished to 1:3 of control values while no difference was observed when lipopolisaccharide (LPS) was used as stimulus. So, we can conclude that choline dietary deficiency affects immune response in rats reducing host response to antigens. © 2003 Elsevier Inc. All rights reserved. Keywords: Choline deficiency; Rats; Immunocompetence
* Corresponding author. Tel.: ⫹1-215-746-5137; fax: ⫹1-215-573-7627. E-mail address: [email protected] (M.C. Courreges). 1 Members of the Research Career of the National Council of Research of Argentina. 0271-5317/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0271-5317(02)00544-4
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1. Introduction The important role of methyl metabolism in experimental animals as well as in human beings is widely and increasingly recognized. Nutrients involved in this metabolism are also known as lipotropic factors and are supplied in the diet mainly as methionine and choline, though folate and B12 are necessary for a normal metabolism. Choline contributes to the structural integrity of cells membranes, signaling functions and is involved in cholinergic neurotransmission, lipid transport and metabolism [1– 4]. Rats fed a diet deficient in lipotropic factors may develop liver (steatosis, cellular death, cirrhosis and cancer) [1,2,5,6], heart (steatosis and necrosis) and renal pathology [7–10]. It has been reported that, in addition to already mentioned pathologies the methyl deficiency was associated with enlargement of the spleen and regression of the thymus [10]. Despite these interesting observations, the effects of methyl deficiency on immunocompetence were apparently ignored for a long time. In the 70’s, Newberne et al. [11–14] published results of studies indicating a role of lipotropes in resistance to infection. In particular, they established the rats deficient in B12 were more susceptible to Salmonella infection as well as pups coming from mothers fed during gestation with diets severely or moderately deficient in lipotropes. In literature, there are several reports about the effect of deprivation of one-carbon metabolites on immune response but none of them involves exclusively choline. The mentioned metabolites regulate the activity of 5-methyltetrahydrofolate homocysteine methyltransferase that participates in methionine salvage in immunocompetent cells [15]. As they share a common metabolic pathway, it is difficult to elucidate the specific role of each one [16]. The purpose of the experiments we are reporting here was to investigate the effect of choline deficiency on different parameters of immune response in adult rats.
2. Materials and methods 2.1. Animals Female Wistar rats, 2 month old, from the Bioterio of the Department of Pathology of the School of Medicine of the University of Buenos Aires were used in the experiments. All animals had free access to one of two diets described below and drinking water. They were individually housed in suspended wire bottomed cages in an air conditioned room and were exposed to light from 7 am to 7 pm. All conditions for handling of animals followed NIH Guidelines for the Care and Use of Laboratory Animals. Body weight and food intake were measured daily. 2.2. Diets Rats were given free access to semi-purified diets in powered form during 2 months before immunization and up to the end of experiment. The diets met the National Council Nutrition
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Table 1 Composition of the diets (g/100g) Diet Composition a
Soy bean protein Sucrose Celluloseb Vitamin mixture (without B12 and choline)c Salts (W)d L-cystinee Hidrogenated vegetable oilf Corn oilg Choline chlorideh
Group CD
CS
20 49.5 4 4 2 0.5 14.3 5.7 0
20 49.15 4 4 2 0.5 14.3 5.7 0.35
CD ⫽ choline deficient, CS ⫽ choline supplemented. Soybean protein grade II. U.S. Biochemical Corp., USA b Celufil. Non-nutritive Bulk, U.S. Biochemical Corp., USA c Vitamin Diet Fortification Mixture. U.S. Biochemical Corp., USA d Salt Mixture Wesson Modification, U.S. Biochemical Corp., USA e U.S. Biochemical Corp., USA f Flora Danica, Buenos Aires, Argentina g Mazola. Refinerias de Maiz, Buenos Aires, Argentina h Sigma Chemical Co, St Louis, USA a
Requirements and varied in choline chloride content. Choline deficient (CD) or choline supplemented (CS) diets compositions are described in Table 1. 2.3. Antibody response to sheep red blood cells (SRBC) Groups of 10 rats, fed with CD or CS diets, were intraperitoneally (ip) immunized with 2 ⫻ 108 SRBC. Haemagglutinating (HA) antibodies in sera were measured 5 and 21 days post-immunization (pi.) by a standard haemagglutination microtechnique [17]. Results were expressed as the reciprocal of the highest dilution of serum that produced evident agglutination. 1 HAU is considered as the reciprocal of the highest dilution of serum with evident agglutination. 2.4. Determination of anti-ovoalbumin (OVA) antibody levels Groups of ten rats fed with CD or CS diets, were subcutaneously (sc) immunized with 1 mg of OVA (Sigma, Chemical Co, Saint Louis MO) in complete Freund’s adjuvant (Difco, Surrey ND). Anti-OVA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Briefly, polystyrene microtitre plates (Corning, New York NY) were coated with OVA solution in carbonate buffer at a concentration of 20 g/ml and incubated overnight at 4°C. Residual protein binding sites were blocked with carbonate buffer containing 8% defatted milk. Sera were analyzed at dilutions 1/600 and 1/10000. Samples were incubated for 1 h at
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37°C. Anti OVA antibodies were detected with affinity purified goat anti-rat total IgG, IgG1 or IgG2a class specific antisera (Bethyl Laboratories Inc, Montgomery TX) followed by IgG affinity purified rabbit anti-goat immunoglobulin horseradish peroxidase conjugate (Sigma Chemical Co, Saint Louis MO) The microtitre plates were washed five times with phosphate buffered saline (PBS)-0.05% Tween between each step. O-Phenylenediamine, 1 mg/ml with 0.025% H2O2 was finally added to the wells, the reaction was stopped with SO4H2 2N and absorbance at 492 nm (A492) was read using an automatic plate reader (Tosoh). Every time, a reference sample was simultaneously run with the unknown samples. Anti-OVA levels in sera were expressed as A492 when 1/600 dilution of sera was used. 2.5. Measurement for delayed type hypersensitivity (DTH) reaction in rats Rats fed CD or CS diets were primed on day 0, with 1 ⫻ 105 SRBC administered ip. or with 1 mg OVA in complete Freund’s adjuvant administered sc. The DTH reaction was elicited by injecting 1 ⫻ 108 SRBC in a volume of 250 l of PBS or 200 g of OVA into the right hind footpad of each animal and 250 l of PBS into the left footpad as a control, 6 or 21 days pi. The footpad swelling was measured with a caliper 24 h later and the response was expressed as the difference in thickness between the SRBC or OVA and PBS injected footpads. 2.6. Cell proliferation assay Rats of each experimental group were sacrificed by ether anesthesia and the spleens or lymph nodes were aseptically removed. Cell suspension were prepared by mincing and tapping the spleen fragments on a stainless 200 mesh in RPMI 1640 medium (Life Technologies, Grand Island NY). Cells were washed three times with the same media and suspended in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum, 50 g/ml of gentamicine and 10⫺5 M 2mercaptoethanol. 2 ⫻ 105 cells/well were incubated in the presence of 4 g/ml Con A (Sigma Chemical Co, Saint Louis MO) or 10 g/ml LPS (Sigma Chemical Co, Saint Louis MO) in a 96 well flat bottomed microplate at 37°C in a 5% CO2 humified atmosphere. Proliferation was measured using a colorimetric method as previously described [18]. Briefly, after 72 h at 37°C, 100 l of a 1 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide (MTT, Sigma Chemical Co, Saint Louis MO) in PBS were added to the cultures and incubated three additional hours. Then, the microplate was centrifuged (5 min, 800 ⫻ g) and untransformed MTT removed. Ethanol was added to each well. The microplate was vigorously shaken to ensure solubilization of blue formazan. The optical density of each well was measured using and automatic plate reader (Tosoh) with a 560 nm test wavelength and 690 nm reference wavelength. The results were expressed as the mean of ten determinations minus the background reading obtained using cells incubated with medium alone. 2.7. Statistics The statistical significance of data was determined by the Student’s t test. A P value less than 0.05 was taken as significant.
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Fig. 1. Effect of choline deficiency on HA antibodies in sera of rats fed CD or CS diets. At day 5 (filled column) and 21 pi. (open column), animals were bled and sera obtained in order to determine the presence of anti-SRBC antibodies. HA antibodies were measured by a standard haemagglutination microtechnique. 1 HAU is considered the reciprocal of the highest dilution of serum with evident agglutination. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
3. Results In order to study the effect of dietary choline deficiency on immunity, adult Wistar rats were fed during 2 months with a diet with or without 0.35% choline chloride and different parameters of immune response against SRBC or OVA were evaluated. As it is shown in Fig. 1, haemagglutinating antibodies in sera collected 5 days pi were significantly (p ⬍ 0.05) reduced in CD group but 21 days pi, only a slight difference was observed. In order to better characterize the response we evaluated by ELISA total IgG and IgG isotype ratios as a marker of the Th responses when OVA was used as antigen. As it is shown in Table 2, CD group exhibited a lower relation IgG1/IgG2a indicating that response in CD group is more shifted toward Th1 than in supplemented counterpart. When DTH response against OVA was measured, we found a significant (p ⬍ 0.05) impairment of the paw edema in CD group (Fig. 2). Similar results were obtained when SRBC was used as antigen (data not shown). Table 2 Effect of choline deficiency on anti-OVA antibody response Diet
Total IgG
IgG1
IgG2a
IgG1/IgG2a
CS CD
0.550 ⫾ 0.140 0.690 ⫾ 0.180
0.5999 ⫾ 0.070 0.760 ⫾ 0.090
0.230 ⫾ 0.020 0.347 ⫾ 0.060
2.60 2.19
Sera were collected 21 days pi and studied by ELISA. Antibody levels are expressed as A492 of a 1/600 dilution of each serum. Values represent mean ⫾ SD of ten independent determinations.
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Fig. 2. Effect of choline deficiency on DTH response in rats. Groups of ten animals were sensitized on day 0 with OVA. Twenty-one days pi., the DTH reaction was elicited by injecting the antigen into the right hind footpad of each animal and PBS into the left footpad as a control. Twenty-four hours later, footpad swelling was measured and the response was expressed as the difference in thickness between the OVA and PBS injected footpads. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
Finally, we decided to investigate the response of splenic lymphocytes coming from CD or CS rats after in vitro stimulation with LPS and Con A. As it is shown in Fig. 3, there was a significant diminution of proliferation in cell cultures coming from CD group with Con A while no difference was observed when LPS was used as stimulus. Similar results were obtained when lymphocytes coming from lymph nodes were used (data not shown).
4. Discussion Griffith et al. [10] reported for the first time the effects on immune response of dietary deficiency in methyl-donor compounds. Newberne et al. performed the following advances on this field and found that folate deficiency was accompanied by an increased susceptibility to infection [11–14]. Herewith, we have demonstrated that choline dietary deficiency produces an impairment of the immune response in rats. As it is shown in our work, we found impaired antibody response to SRBC together with a diminished DTH response to SRBC and OVA. Secondary antibody responses to SRBC and OVA were slightly impaired. When in vitro response to mitogens was tested, we found a strongly inhibition of Con A dependent proliferation and a slight diminution in response to LPS in spleens and lymph nodes coming from CD rats. Alterations in the balance IgG1/IgG2a usually are accompanied by promotion of T or B cell response. In our case, we found that dietary choline deficiency is accompanied by a diminution of Th2/Th1 response and so it is not surprising that antibody responses were
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Fig. 3. Effect of dietary choline deficiency on in vitro lymphocyte proliferation. 2 ⫻ 105 cells/well were incubated in the presence of 4 g/ml Con A (filled column) or 10 g/ml LPS (open column) in a 96 well flat-bottomed microplate at 37°C in a 5% CO2 humified atmosphere. After 72 h incubation at 37°C, 100 l of a 1 mg/ml solution of MTT in PBS were added to the cultures and incubated three additional hours. Then, the microplate was centrifuged (5 min, 800 ⫻ g) and untransformed MTT removed. Ethanol was added to each well. The microplate was vigorously shaken to ensure solubilization of blue formazan. The optical density of each well was measured using an automatic plate reader (Tosoh) with a 560 nm test wavelength and 690 nm reference wavelength. The results were expressed as the mean of ten determinations minus the background reading obtained using cells incubated with medium alone. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
impaired. Antibody production is dependent of T cell collaboration in the immune response to most antigens. Taking into account the previously mentioned facts, we can speculate that impaired antibody response could be an indirect consequence of an impaired T cell response. Mother rats fed diets either severely or marginally deficient in dietary lipotropes, littered pups significantly more susceptible to infection in postnatal life, compared with controls. In addition, postnatal supplementation did not correct the effects of maternal deficits in methyldeficient groups [10,11]. These observations were confirmed by Newberne et al. who identified the target tissues for the depressing effect of lipotrope deficiency as the T-cell components of that system in agreement with our findings [13,14]. The study of cell-mediated immunity of human patients with different types of anemia revealed that cell mediated immunity was depressed in megaloblastic anemia due to folate deficiency and that it was reversed by folate supplementation. On the other hand, iron deficiency was not accompanied by T cell impairment [19]. Complementary studies in folate deficiency rats and mice confirmed these results [16,20,21]. Cellularity in thymus and thymic dependent areas of spleen and lymph nodes was decreased together with cytotoxicity after stimulation with T-cell mitogens. Additional investigations have shown that the age at which the deficiency is studied, have a marked effect on the observed changes being the earlier the worse [22]. Our results have demonstrated that not only choline dietary deficiency in critic moments of life such as gestation, lactancy or weanling can affect immune response in rats but also its deficiency in adulthood is enough to reduce host response to antigens.
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Acknowledgments This work was partially supported by grants from the National Council of Research of Argentina and from the University of Buenos Aires, Argentina. References [1] Best C. The lipotropic agents in the protection of the liver, kidney, heart and other organs of experimental animals. Proc Royal Soc 1956;145:11–169. [2] Griffith WH, Wade NJ. Effects of deficiency. In: Sebrell WH Jr, Harris RS, editors. The vitamins: chemistry, physiology and pathology. 2nd ed. New York: Academic Press, 1971. p. 81–122. [3] Zeisel SH, Blusztajn JK. Choline and human nutrition. Annu Rev Nutr 1994;14:269 –96. [4] Zeisel SH, Szuhaj BF. Choline, phospholipids and disease. Champaign: AOCS Press, 1998. [5] Goshal AK, Farber E. Liver biochemical pathology of choline deficiency and methyl group deficiency: a new orientation and assessment. Histol Histopathol 1995;10:457– 62. [6] Shin OH, Mar MH, Allbright CD, Citarella MT, da Costa KA, Zeisel SH. Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency. J Cell Biochem 1997;64:196 –208. [7] Arienti de Garcia I, Perazzo JC, Monserrat AJ. Necrosis cardiaca y deficiencia de factores lipotropicos. Medicina (Bs. Aires) 1981;41:556 – 64. [8] Monserrat AJ, Musso AM, Tartas N, Nicastro M, Konopta HF, Arienti de Garcia I, Sanchez Avalos JC. Consumption coagulopathy in acute renal failure induced by hypolipotropic diets. Nephron 1981;28:276 – 84. [9] Wigram GF, Hartroft WS, Best CH. Dietary choline and the maintenance of the cardiovascular system in rats. Br Med J 1954;II:1–5. [10] Griffith WH, Wade NJ. Choline metabolism I. The occurrence and prevention of hemorrhagic degeneration in young rats on a low choline diet. J Biol Chem 1939;131:567– 637. [11] Newberne PM, Wison RB, Williams G. Effects of severe and marginal maternal lipotrope deficiency on response of postnatal rats to infection. Brit J Exp Pathol 1970;51:229 –35. [12] Newberne PM, Wilson RB. Prenatal, malnutrition and postnatal responses to infection. Nutr Rpts Internat 1972;5:151– 8. [13] Newberne PM, Wison RB. Pre and postnatal malnutrition and response to infection. Nutr Rpts Internat 1973;7:407–20. [14] Gebbart BM, Newberne PM. T-cell function in the offspring of lipotrope and protein deficient rats. Immunology 1974;26:489 –95. [15] Finkelstein JD, Kyle WE, Harris BJ. Methionine metabolism in mammals. Regulation of homocysteine methyltransferases in rat tissue. Arch Biochem Biophys 1971;146:84. [16] Williams EAI, Gebbart BM, Norton B, Newberne PM. Effects of early marginal methionine-choline deprivation on the development of the immune system in the rat. Am J Clin Nutr 1979;32:1214 –23. [17] Yoshikai Y, Miake S, Matsumoto T, Nomoto K, Takeya K. Effect of stimulation and blockade of mononuclear phagocyte system on the delayed footpad reaction to SRBC in mice. Immunology 1979;38:571– 83. [18] Sudo K, Komo K, Yokota T, Shigeta S. A sensitive assay system for screening of antiviral compounds against herpes simplex virus type 1 and 2. Virol Methods 1994;49:168 –78. [19] Gross RL, Reid JVO, Newberne PM. Depressed cell mediated immunity in megaloblastic anemia due to folic acid deficiency. Am J Clin Nutr 1975;28:225–32. [20] Williams EAJ, Gross RL, Newberne PM. Effect of folate deficiency on the cell mediated immune response in rats. Nutr Rpts Internat 1975;12:137– 48. [21] Williams EAJ, Gebbart BM, Yee H, Newberne PM. Immunological consequences of choline vitamin B12 deprivation. Nutr Rpts Internat 1978;17:279 –92. [22] Nauss KM, Connor AM, Kavanaugh A, Newberne PM. Alterations in immune function in rats caused by dietary lipotrope deficiency effect of age. J Nutr 1982;112:2333– 41.
Effect of dietary choline deficiency on immunocompetence in Wistar rats Maria C. Courre`gesa,*, Fabian Benenciab,1, Ana Ucedaa, Alberto J. Monserrata,1 a
Experimental Pathology, Department of Pathology, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina b Laboratory of Immunochemistry, Department of Biological Chemistry, School of Sciences, University of Buenos Aires, Buenos Aires, Argentina Received 3 August 2002; received in revised form 15 November 2002; accepted 29 November 2002
Abstract In order to study the effect of dietary choline deficiency on immunity, adult Wistar rats were fed for two months with choline supplemented (CS, 0.35% choline chloride) or deficient (CD) diets and different parameters of immune response against sheep blood red cells or ovoalbumin (OVA) were evaluated. We found a significant reduction of haemagglutinating (HA) antibodies in sera (day 5 post-immunization: CD, 110 ⫾ 12 HAU vs. CS 320 ⫾ 40 HAU, p ⬍ 0.05) and only a slight difference in anti-OVA antibodies in CD group. Delayed type hypersensitivity was significantly impaired in CD group (footpad swelling in mm: CD, 0.97 ⫾ 0.51 vs. CS, 2.32 ⫾ 0.53, p ⬍ 0.05). In vitro, concanavalin A (Con A) stimulated proliferation of lymphocytes from spleen or lymph nodes of CD rats were diminished to 1:3 of control values while no difference was observed when lipopolisaccharide (LPS) was used as stimulus. So, we can conclude that choline dietary deficiency affects immune response in rats reducing host response to antigens. © 2003 Elsevier Inc. All rights reserved. Keywords: Choline deficiency; Rats; Immunocompetence
* Corresponding author. Tel.: ⫹1-215-746-5137; fax: ⫹1-215-573-7627. E-mail address: [email protected] (M.C. Courreges). 1 Members of the Research Career of the National Council of Research of Argentina. 0271-5317/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0271-5317(02)00544-4
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1. Introduction The important role of methyl metabolism in experimental animals as well as in human beings is widely and increasingly recognized. Nutrients involved in this metabolism are also known as lipotropic factors and are supplied in the diet mainly as methionine and choline, though folate and B12 are necessary for a normal metabolism. Choline contributes to the structural integrity of cells membranes, signaling functions and is involved in cholinergic neurotransmission, lipid transport and metabolism [1– 4]. Rats fed a diet deficient in lipotropic factors may develop liver (steatosis, cellular death, cirrhosis and cancer) [1,2,5,6], heart (steatosis and necrosis) and renal pathology [7–10]. It has been reported that, in addition to already mentioned pathologies the methyl deficiency was associated with enlargement of the spleen and regression of the thymus [10]. Despite these interesting observations, the effects of methyl deficiency on immunocompetence were apparently ignored for a long time. In the 70’s, Newberne et al. [11–14] published results of studies indicating a role of lipotropes in resistance to infection. In particular, they established the rats deficient in B12 were more susceptible to Salmonella infection as well as pups coming from mothers fed during gestation with diets severely or moderately deficient in lipotropes. In literature, there are several reports about the effect of deprivation of one-carbon metabolites on immune response but none of them involves exclusively choline. The mentioned metabolites regulate the activity of 5-methyltetrahydrofolate homocysteine methyltransferase that participates in methionine salvage in immunocompetent cells [15]. As they share a common metabolic pathway, it is difficult to elucidate the specific role of each one [16]. The purpose of the experiments we are reporting here was to investigate the effect of choline deficiency on different parameters of immune response in adult rats.
2. Materials and methods 2.1. Animals Female Wistar rats, 2 month old, from the Bioterio of the Department of Pathology of the School of Medicine of the University of Buenos Aires were used in the experiments. All animals had free access to one of two diets described below and drinking water. They were individually housed in suspended wire bottomed cages in an air conditioned room and were exposed to light from 7 am to 7 pm. All conditions for handling of animals followed NIH Guidelines for the Care and Use of Laboratory Animals. Body weight and food intake were measured daily. 2.2. Diets Rats were given free access to semi-purified diets in powered form during 2 months before immunization and up to the end of experiment. The diets met the National Council Nutrition
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Table 1 Composition of the diets (g/100g) Diet Composition a
Soy bean protein Sucrose Celluloseb Vitamin mixture (without B12 and choline)c Salts (W)d L-cystinee Hidrogenated vegetable oilf Corn oilg Choline chlorideh
Group CD
CS
20 49.5 4 4 2 0.5 14.3 5.7 0
20 49.15 4 4 2 0.5 14.3 5.7 0.35
CD ⫽ choline deficient, CS ⫽ choline supplemented. Soybean protein grade II. U.S. Biochemical Corp., USA b Celufil. Non-nutritive Bulk, U.S. Biochemical Corp., USA c Vitamin Diet Fortification Mixture. U.S. Biochemical Corp., USA d Salt Mixture Wesson Modification, U.S. Biochemical Corp., USA e U.S. Biochemical Corp., USA f Flora Danica, Buenos Aires, Argentina g Mazola. Refinerias de Maiz, Buenos Aires, Argentina h Sigma Chemical Co, St Louis, USA a
Requirements and varied in choline chloride content. Choline deficient (CD) or choline supplemented (CS) diets compositions are described in Table 1. 2.3. Antibody response to sheep red blood cells (SRBC) Groups of 10 rats, fed with CD or CS diets, were intraperitoneally (ip) immunized with 2 ⫻ 108 SRBC. Haemagglutinating (HA) antibodies in sera were measured 5 and 21 days post-immunization (pi.) by a standard haemagglutination microtechnique [17]. Results were expressed as the reciprocal of the highest dilution of serum that produced evident agglutination. 1 HAU is considered as the reciprocal of the highest dilution of serum with evident agglutination. 2.4. Determination of anti-ovoalbumin (OVA) antibody levels Groups of ten rats fed with CD or CS diets, were subcutaneously (sc) immunized with 1 mg of OVA (Sigma, Chemical Co, Saint Louis MO) in complete Freund’s adjuvant (Difco, Surrey ND). Anti-OVA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Briefly, polystyrene microtitre plates (Corning, New York NY) were coated with OVA solution in carbonate buffer at a concentration of 20 g/ml and incubated overnight at 4°C. Residual protein binding sites were blocked with carbonate buffer containing 8% defatted milk. Sera were analyzed at dilutions 1/600 and 1/10000. Samples were incubated for 1 h at
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37°C. Anti OVA antibodies were detected with affinity purified goat anti-rat total IgG, IgG1 or IgG2a class specific antisera (Bethyl Laboratories Inc, Montgomery TX) followed by IgG affinity purified rabbit anti-goat immunoglobulin horseradish peroxidase conjugate (Sigma Chemical Co, Saint Louis MO) The microtitre plates were washed five times with phosphate buffered saline (PBS)-0.05% Tween between each step. O-Phenylenediamine, 1 mg/ml with 0.025% H2O2 was finally added to the wells, the reaction was stopped with SO4H2 2N and absorbance at 492 nm (A492) was read using an automatic plate reader (Tosoh). Every time, a reference sample was simultaneously run with the unknown samples. Anti-OVA levels in sera were expressed as A492 when 1/600 dilution of sera was used. 2.5. Measurement for delayed type hypersensitivity (DTH) reaction in rats Rats fed CD or CS diets were primed on day 0, with 1 ⫻ 105 SRBC administered ip. or with 1 mg OVA in complete Freund’s adjuvant administered sc. The DTH reaction was elicited by injecting 1 ⫻ 108 SRBC in a volume of 250 l of PBS or 200 g of OVA into the right hind footpad of each animal and 250 l of PBS into the left footpad as a control, 6 or 21 days pi. The footpad swelling was measured with a caliper 24 h later and the response was expressed as the difference in thickness between the SRBC or OVA and PBS injected footpads. 2.6. Cell proliferation assay Rats of each experimental group were sacrificed by ether anesthesia and the spleens or lymph nodes were aseptically removed. Cell suspension were prepared by mincing and tapping the spleen fragments on a stainless 200 mesh in RPMI 1640 medium (Life Technologies, Grand Island NY). Cells were washed three times with the same media and suspended in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum, 50 g/ml of gentamicine and 10⫺5 M 2mercaptoethanol. 2 ⫻ 105 cells/well were incubated in the presence of 4 g/ml Con A (Sigma Chemical Co, Saint Louis MO) or 10 g/ml LPS (Sigma Chemical Co, Saint Louis MO) in a 96 well flat bottomed microplate at 37°C in a 5% CO2 humified atmosphere. Proliferation was measured using a colorimetric method as previously described [18]. Briefly, after 72 h at 37°C, 100 l of a 1 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide (MTT, Sigma Chemical Co, Saint Louis MO) in PBS were added to the cultures and incubated three additional hours. Then, the microplate was centrifuged (5 min, 800 ⫻ g) and untransformed MTT removed. Ethanol was added to each well. The microplate was vigorously shaken to ensure solubilization of blue formazan. The optical density of each well was measured using and automatic plate reader (Tosoh) with a 560 nm test wavelength and 690 nm reference wavelength. The results were expressed as the mean of ten determinations minus the background reading obtained using cells incubated with medium alone. 2.7. Statistics The statistical significance of data was determined by the Student’s t test. A P value less than 0.05 was taken as significant.
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Fig. 1. Effect of choline deficiency on HA antibodies in sera of rats fed CD or CS diets. At day 5 (filled column) and 21 pi. (open column), animals were bled and sera obtained in order to determine the presence of anti-SRBC antibodies. HA antibodies were measured by a standard haemagglutination microtechnique. 1 HAU is considered the reciprocal of the highest dilution of serum with evident agglutination. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
3. Results In order to study the effect of dietary choline deficiency on immunity, adult Wistar rats were fed during 2 months with a diet with or without 0.35% choline chloride and different parameters of immune response against SRBC or OVA were evaluated. As it is shown in Fig. 1, haemagglutinating antibodies in sera collected 5 days pi were significantly (p ⬍ 0.05) reduced in CD group but 21 days pi, only a slight difference was observed. In order to better characterize the response we evaluated by ELISA total IgG and IgG isotype ratios as a marker of the Th responses when OVA was used as antigen. As it is shown in Table 2, CD group exhibited a lower relation IgG1/IgG2a indicating that response in CD group is more shifted toward Th1 than in supplemented counterpart. When DTH response against OVA was measured, we found a significant (p ⬍ 0.05) impairment of the paw edema in CD group (Fig. 2). Similar results were obtained when SRBC was used as antigen (data not shown). Table 2 Effect of choline deficiency on anti-OVA antibody response Diet
Total IgG
IgG1
IgG2a
IgG1/IgG2a
CS CD
0.550 ⫾ 0.140 0.690 ⫾ 0.180
0.5999 ⫾ 0.070 0.760 ⫾ 0.090
0.230 ⫾ 0.020 0.347 ⫾ 0.060
2.60 2.19
Sera were collected 21 days pi and studied by ELISA. Antibody levels are expressed as A492 of a 1/600 dilution of each serum. Values represent mean ⫾ SD of ten independent determinations.
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Fig. 2. Effect of choline deficiency on DTH response in rats. Groups of ten animals were sensitized on day 0 with OVA. Twenty-one days pi., the DTH reaction was elicited by injecting the antigen into the right hind footpad of each animal and PBS into the left footpad as a control. Twenty-four hours later, footpad swelling was measured and the response was expressed as the difference in thickness between the OVA and PBS injected footpads. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
Finally, we decided to investigate the response of splenic lymphocytes coming from CD or CS rats after in vitro stimulation with LPS and Con A. As it is shown in Fig. 3, there was a significant diminution of proliferation in cell cultures coming from CD group with Con A while no difference was observed when LPS was used as stimulus. Similar results were obtained when lymphocytes coming from lymph nodes were used (data not shown).
4. Discussion Griffith et al. [10] reported for the first time the effects on immune response of dietary deficiency in methyl-donor compounds. Newberne et al. performed the following advances on this field and found that folate deficiency was accompanied by an increased susceptibility to infection [11–14]. Herewith, we have demonstrated that choline dietary deficiency produces an impairment of the immune response in rats. As it is shown in our work, we found impaired antibody response to SRBC together with a diminished DTH response to SRBC and OVA. Secondary antibody responses to SRBC and OVA were slightly impaired. When in vitro response to mitogens was tested, we found a strongly inhibition of Con A dependent proliferation and a slight diminution in response to LPS in spleens and lymph nodes coming from CD rats. Alterations in the balance IgG1/IgG2a usually are accompanied by promotion of T or B cell response. In our case, we found that dietary choline deficiency is accompanied by a diminution of Th2/Th1 response and so it is not surprising that antibody responses were
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Fig. 3. Effect of dietary choline deficiency on in vitro lymphocyte proliferation. 2 ⫻ 105 cells/well were incubated in the presence of 4 g/ml Con A (filled column) or 10 g/ml LPS (open column) in a 96 well flat-bottomed microplate at 37°C in a 5% CO2 humified atmosphere. After 72 h incubation at 37°C, 100 l of a 1 mg/ml solution of MTT in PBS were added to the cultures and incubated three additional hours. Then, the microplate was centrifuged (5 min, 800 ⫻ g) and untransformed MTT removed. Ethanol was added to each well. The microplate was vigorously shaken to ensure solubilization of blue formazan. The optical density of each well was measured using an automatic plate reader (Tosoh) with a 560 nm test wavelength and 690 nm reference wavelength. The results were expressed as the mean of ten determinations minus the background reading obtained using cells incubated with medium alone. Data are expressed as mean ⫾ SD of 10 independent determinations. *p ⬍ 0.05.
impaired. Antibody production is dependent of T cell collaboration in the immune response to most antigens. Taking into account the previously mentioned facts, we can speculate that impaired antibody response could be an indirect consequence of an impaired T cell response. Mother rats fed diets either severely or marginally deficient in dietary lipotropes, littered pups significantly more susceptible to infection in postnatal life, compared with controls. In addition, postnatal supplementation did not correct the effects of maternal deficits in methyldeficient groups [10,11]. These observations were confirmed by Newberne et al. who identified the target tissues for the depressing effect of lipotrope deficiency as the T-cell components of that system in agreement with our findings [13,14]. The study of cell-mediated immunity of human patients with different types of anemia revealed that cell mediated immunity was depressed in megaloblastic anemia due to folate deficiency and that it was reversed by folate supplementation. On the other hand, iron deficiency was not accompanied by T cell impairment [19]. Complementary studies in folate deficiency rats and mice confirmed these results [16,20,21]. Cellularity in thymus and thymic dependent areas of spleen and lymph nodes was decreased together with cytotoxicity after stimulation with T-cell mitogens. Additional investigations have shown that the age at which the deficiency is studied, have a marked effect on the observed changes being the earlier the worse [22]. Our results have demonstrated that not only choline dietary deficiency in critic moments of life such as gestation, lactancy or weanling can affect immune response in rats but also its deficiency in adulthood is enough to reduce host response to antigens.
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