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Articles

Vitamin A Prevents the Decline in Immunoglobulin A and Th2 Cytokine Levels in Small Intestinal Mucosa of Protein-Malnourished Mice¹

Takeshi Nikawa^*,2, Kenji Odahara^*, Hiroyuki Koizumi^*, Yasuhiro Kido, Shigetada Teshima^*, Kazuhito Rokutan^* and Kyoichi Kishi^*

^* Department of Nutrition, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan; and Department of Food Sciences and Nutritional Health, Faculty of Human Environment, Kyoto Prefectural University, Shimogamo, Kyoto 606-8522, Japan

² To whom correspondence should be addressed at Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan.

	ABSTRACT

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We examined whether vitamin A improved mucosal immune depression in mice with wasting protein deficiency. In male C3H/HeN mice fed a semi-purified 1% protein diet for 2 wk, plasma retinol and immunoglobulin A (IgA) concentrations in the small intestinal mucosa were 50 and 55%, respectively, of those in mice fed a semi-purified 20% protein diet, (P < 0.05). Daily supplementation of 0.3 mg of retinyl acetate to protein-deficient mice for 2 wk increased the plasma retinol level to the value in the protein-sufficient mice. However, 1 mg/d of retinyl acetate was required to prevent the decline of the IgA level caused by the protein deficiency. Mice fed the low-protein diet had lower concentrations of IL-4 and IL-5 in the small intestinal mucosa and fewer IL-4- and IL-5-containing cells in the lamina propria (P < 0.05). Retinyl acetate (1 mg) significantly restored the IL-5 level and the number of IL-4- and IL-5-containing cells. After immunization with 20 µg of cholera toxin (CT), the intestinal mucosa of protein-deficient mice contained significantly less CT-specific IgA than control mice. Treatment with 1 mg of retinyl acetate prevented the decline of anti-CT IgA level in the protein-deficient mice, improving their survival rate after an exposure to 0.1 mg of CT. These results suggest that large oral supplements of vitamin A may preserve mucosal IgA level during protein malnutrition, possibly by stimulating Th2 cytokine production and thereby, inducing resistance against infection.

KEY WORDS: • vitamin A • protein malnutrition • immunoglobulin A • Th2 cytokine • mice

	INTRODUCTION

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The secretory immunoglobulin A (IgA)³ system is the principal arm of the mucosal immune response that protects the upper respiratory and enteric tracts against infection of pathogenic organisms. The IgA response involves local production of IgA by plasma cells at submucosal sites, followed by its transport onto mucosal surfaces or into glandular secretions via the polymeric immunoglobulin receptor (Mestecky et al. 1991 ). Recently, several studies in animal models showed that this mucosal IgA response is impaired by vitamin A deficiency (Semba 1998 , Puengtomwatanakul and Sirisinha 1986 , Wiedermann et al. 1993 ). Therefore, to lessen mortality from infectious diseases, community trials of vitamin A supplementation were occasionally performed. Field trials of vitamin A supplementation in Indonesia (Sommer et al. 1986 ), India (Rahmathullah et al. 1990 ) and Nepal (Keith et al. 1991 ) reported annual reductions in preschool child mortality by 34, 54, and 30%, respectively. However, another trial in India (Vijayaraghavan et al. 1990 ) failed to confirm these results, raising concern about the potential impact of vitamin A supplementation on child survival in different places.

Protein malnutrition is a determinative risk factor for the morbidity and mortality of infectious disease (Woodward 1998 ). Several studies demonstrated that protein malnutrition impairs host immune defense, including cell-mediated immunity (Edelman et al. 1973 , Smythe et al. 1971 ) and secretory IgA production (Ha and Woodward 1997 , McGee and McMurray 1988 ). Protein malnutrition often coexists with deficiencies of specific micronutrients, such as zinc (Hansen et al. 1982 ), iron (Shobaki and Rummel 1978 ), and vitamin A (Keusch 1990 ), suggesting that the immuno-depressive effects of protein deficiency may be a result of these associated micronutrient deficiencies. It has been reported that vitamin A deficiency aggravated immunodeficiency caused by protein malnutrition, leading to severe atrophy of spleen and thymus and impaired antibody production (Wiedermann et al. 1996 ). However, the interactions of dietary protein and vitamin A on the mucosal immune system were not studied in detail.

Reportedly the total IgA concentration in bile was significantly lower in the vitamin A-deficient rats than in the pair-fed controls, and daily administration of 0.1 mg of retinyl palmitate was necessary to improve the impaired mucosal IgA response (Wiedermann et al. 1993 ). When C3H/HeN mice were fed a semi-purified 1% protein diet for 2 wk or longer, plasma retinol level and the IgA concentration in the small intestinal mucosa were significantly lower than those of mice fed a protein-sufficient diet. Therefore, we examined whether large oral supplements of vitamin A (0.3 or 1 mg per mouse) could improve indices of humoral immune competence in the small intestine of the protein-malnourished mice.

	Methods

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Diets, administration of vitamin A and immunization with cholera toxin

Male C3H/HeN mice (Japan SLC, Shizuoka, Japan), 4-wk-old, were housed in a room maintained at 23°C on a 12-h light/dark cycle and were allowed free access to standard nonpurified MF diets⁴ (Oriental Yeast, Osaka, Japan) and water. When their body weight reached 25 g (d 0), either a semi-purified 1% protein diet (a low protein diet) or a semi-purified 20% protein diet (a control protein diet) was consumed ad libitum for 7 d (n = 5), 14 d (n = 5) or 21 d (n = 5). Egg white, a kind gift from Q. P. Co., Tokyo, Japan, was used as a protein source, and detailed compositions of these diets are shown in Table 1. The two diets were made isocaloric by adjusting carbohydrate contents, but all of the other nutrients were identical. Both diets contained ~1.5 mg (5,000 IU) of retinyl acetate per kg.

To assess an antigen specific IgA response, mice were immunized with cholera toxin (CT)³ from vibrio cholera (Sigma, St. Louis, MO). Mice (n = 20) were randomly assigned to two groups and given free access to the low or control protein diet for 32 d. During feeding the experimental diets, corn oil or 1 mg of retinyl acetate was administered to the half (n = 5) of each group, as described above. All mice were first immunized by intragastric treatment with 20 µg of CT in 200 µL of 0.1 mol/L sodium bicarbonate, pH 8.1, on d 0. Booster doses of 10 µg CT were given on d 10, 17 and 24.

To examine the protective effect of vitamin A against the enterotoxin, the survival rate of the mice was monitored after a single administration of 0.1 mg of CT. Mice (n = 24) fed the low-protein diet were intragastrically administered 100 µL of vehicle or 1 mg of retinyl acetate in 100 µL of corn oil every day. They were first immunized by intragastric treatment with 20 µg of CT on d 0. The high dose (0.1 mg) of CT was intragastrically administered to the mice on d 14, and the number of surviving mice was counted each day. Vehicle-treated, protein-sufficient mice were also exposed to the high dose of CT in the same manner.

The present experiments were performed according to the Guide for the Care and Use of Laboratory Animals (1985) of the National Institute of Health and were allowed by The Committee of The University of Tokushima for Experimental Animals.

Preparation of plasma and intestinal mucosa

Mice were killed by cervical dislocation. Blood was collected from inferior vena cava in the presence of heparin, and plasma was separated. Small intestinal mucosa was prepared as previously described (Nikawa et al. 1998 ). Briefly, the small intestine was removed, washed with cold phosphate-buffered saline (PBS), and weighed. After the intestine was longitudinally opened, total mucosa was scraped with a slide glass on ice and weighed. The sample (~50 mg) was placed into a microcentrifuge tube. Cold PBS or enzyme-linked immunosorbent assay (ELISA) buffer (1 mL) [PBS, containing 10 g/L of bovine serum albumin (BSA) (KPL, Gaithersburg, MD), 1 µmol/L of pepstatin A (Peptide Inc., Osaka, Japan), 10 µmol/L of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (Peptide Inc.), 100 µmol/L of leupeptin (Peptide Inc.) and 1 mmol/L of phenylmethysulphonyl fluoride (Boehringer Mannheim, Indianapolis, IN)] were added. These samples were homogenized on ice for 1 min using a Teflon microhomogenizer. The supernatant, obtained by centrifugation at 17,500 x g for 15 min at 4°C, was subjected to analyses.

Measurement of vitamin A concentration

Vitamin A concentrations were determined with reverse-phase HPLC according to the method of Chaudhary and Nelson (1984) . Mice were killed 12 h after the last administration of retinyl acetate, and plasma and homogenate of small intestinal mucosa were prepared, as described above. Plasma or homogenate (50 µL) was mixed with 50 µL of ethanol containing 0.002% butylhydroxytoluene (BHT), and the mixture was mixed with a vortex for 10 s. After addition of 300 µL of n-hexane, samples were centrifuged at 800 x g for 5 min, and then lipids were extracted twice with n-hexane. n-Hexane was evaporated with nitrogen gas, and precipitates were dissolved in 2-propanol containing 0.002% BHT. Sample (10 µL) was subjected to reverse-phase HPLC (Shimadzu, Kyoto, Japan) on TSKgel ODS-80Ts (4.5 x 250 mm; Tosoh, Tokyo, Japan) equilibrated with 95% ethanol. Retinoids were detected with a fluorescence spectrophotometer (ex = 340 nm, em = 460 nm; Shimadzu RF-10A_XL). The retention times of retinol, retinyl acetate and retinyl palmitate were at 4.5, 5.4 and 18.1 min, respectively. All procedures were done in a dark room under dim yellow light at room temperature.

Determination of IgA concentration

Total and anti-CT IgA levels were measured with an ELISA according to the method of Wiedermann et al. (1993) with minor modifications. For measurement of total IgA, 50 µL of the appropriately diluted sample from plasma or intestinal mucosa was placed in each well of microtiter plates (Falcon Japan, Tokyo). The samples were kept overnight at 4°C. After washing twice with PBS-T (PBS with 0.05% Tween-20), 200 µL of ELISA buffer per well was added, and the plate was incubated for 2 h at 37°C. After washing four times with PBS-T, 0.2 µg of a rat anti-mouse IgA monoclonal antibody (Vector Lab., Burlingame, CA) in 100 µL of PBS was added for 1 h at 37°C. Bound antibodies were detected with a three-stage indirect immunoperoxidase kit (Vectastain^TM ABC kit, Vector Lab., Burkingame, CA), using 2, 2'-azino-di[3-ethyl-benzthiazoline sulfate] (ABTS) as a substrate (Lavanchy et al. 1990 ). Thirty minutes later, the plates were placed in a spectrophotometer (ImmunoMini-NJ2300; Nalge Nunc International Japan, Tokyo), and absorbance at 405 nm was measured. The amount of IgA was calculated using mouse IgA, purified from mouse myeloma cells (Zymed, South San Francisco, CA), as a standard and expressed as mg/g wet mucosa or as g/L serum. The lower detection limit in this system was 50 µg/L.

To measure the amount of anti-CT IgA by ELISA, plates were coated overnight with 0.1 µg CT in 50 µL PBS per well at 4°C. After washing twice with PBS-T and blocking with ELISA buffer, 100 µL of the diluted sample from intestinal mucosa was added to each well. Plates were incubated for 1 h at 37°C. After washing four times with PBS-T, bound antibodies were detected with a Vectastain^TM ABC kit. The absorbance of each well at 405 nm was determined as described above. The concentration of the IgA specific for CT was calculated from a standard IgA titration curve and expressed as µg/g wet mucosa.

Cytokine measurements in intestinal mucosa

The levels of interleukin (IL)-4, IL-5 and IL-6 in the supernatant of small intestinal mucosa were measured according to the method of Alkan et al. (1996) . Mouse standard cytokines (IL-4, IL-5 and IL-6), and their capture and biotinylated-detection antibodies from Pharmingen (San Diego, CA) were used. Anti-IL-4 and anti-IL-6 capture antibodies were dissolved in 0.1 mol/L Na₂HPO₄, pH 9.0, at the concentration of 2 mg/L, and an anti-IL-5 capture antibody was dissolved in 0.1 mol/L NaHCO₃, pH 8.2, at the same concentration. Plates were coated with each capture antibody by incubating overnight with 50 µL/well of one of the antibody solutions. They were washed twice with PBS-T and then incubated with the ELISA buffer at 37°C for 2 h. After being washed twice with PBS-T, 100 µL/well of the supernatant of intestinal mucosa was incubated overnight at 4°C. After washing four times with PBS-T, 100 µL of each detection antibody (10 mg/L in PBS) was added to each well at 37°C for 1 h. After washing six times with PBS-T, biotinylated-detection antibodies were visualized and detected, as described above. Cytokine levels were expressed as ng/g wet mucosa. The lower detection limits for IL-4, IL-5 and IL-6 in our ELISA system were 40, 80 and 160 ng/L, respectively.

Immunohistochemical analysis for IgA or cytokine-containing cells

Small intestine at the middle portion was fixed with formalin in PBS for 6 h at room temperature. The tissue was dehydrated and embedded in paraffin wax. Section (4 µm) was cut and air-dried for 2 h at room temperature. After dewaxing with xylene, the section was washed with ethanol and rehydrated with distilled water. The section was incubated in 0.3% hydrogen peroxide in PBS for 30 min. After blocking nonspecific binding sites with rabbit sera (Vector Lab.) for 20 min in a humidified chamber, the section was incubated in a 1:40 dilution of a rat monoclonal antibody against mouse IgA (Vector Lab.), IL-4, IL-5 or IL-6 (Pharmingen) for 1 h at room temperature. Bound antibodies were detected with a Vectastain^TM ABC kit, using 3, 3'-diaminobenzidine (Sigma) as a substrate. Following a brief rinse in PBS, the sections were counterstained with hematoxylin.

Assessments for immunohistochemically positive cells were performed by two observers who were blinded to sample identity. The number of positive cells within the lamina propria on duplicate sections was counted with an eyepiece graticule under x400 magnification. The eyepiece graticule consisted of 100 squares, which enabled the villous lamina propria to be counted as a fraction of a complete grid. The area of lamina propria per villus was similar to that of 5 squares of the eyepiece graticule. At least three complete villi were counted per section. The results were expressed as the mean ± SD of positive cells in the lamina propria per villus [= 5 squares (0.05 mm²) of the eyepiece graticule].

Protein concentration

Plasma protein concentration was determined by the biuret method (Itzhaki and Gill 1964 ) with BSA as a standard. Other protein concentrations were measured according to the method of Lowry et al. (1951) .

Statistical analysis

The experimental data were expressed as means ± SD for 3–12 rats per group and statistically evaluated by analysis of variance (ANOVA) with the SPSS computer programs (release 6.1; SPSS Japan Inc., Tokyo). One-way ANOVA was used to determine the significant effects of dietary protein or vitamin A supplement on the variable measured, such as vitamin A status and numbers of IgA- and cytokine-containing cells. Two-way ANOVA was also performed to determine the effects and the possible interactions of dietary protein and vitamin A supplement. Individual differences between groups were assessed using Duncan's multiple range test. Differences in the survival rates of mice were compared using ²-test. Differences were considered significant at P < 0.05.

	Results

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Effects of a 1% protein diet on plasma protein and mucosal IgA levels

Plasma protein concentration in the protein-deficient mice was significantly less on d 7, 14 and 21, compared with that in the protein-sufficient mice, and a significantly lower mucosal IgA content first appeared on d 14 (Fig. 1 ). Based on these results, mice fed the low-protein diet for 2 wk were used to examine whether vitamin A supplementation could prevent the mucosal immunosuppression caused by protein malnutrition.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of a low-protein diet on levels of (A) plasma protein and (B) mucosal immunoglobulin A (IgA) in mice. Male 4-wk-old mice were given free access to a low or control protein diet for 0, 7, 14, or 21 d. They were killed by cervical dislocation on the indicated days, and levels of plasma protein and mucosal IgA were measured. Values are mean ± SD (n = 5). Means with different superscripts are significantly different by one-way analysis of variance followed by Duncan's multiple range test (P < 0.05).

Mice, given free access to a low or control protein diet, were treated with vehicle, or 0.05, 0.3 and 1 mg of vitamin A for 2 wk. In vehicle-treated mice, food intake of the protein-deficient group was significantly greater than that of the protein-sufficient group (Table 2 ).However, mice fed the protein-deficient diet lost body weight. The hematocrit and plasma protein concentration in the protein-deficient mice were significantly less than those in the protein-sufficient mice. Protein concentration in the small intestinal mucosa of the protein-deficient mice was not different from that of the protein-sufficient mice, although the wet weight of the small intestinal mucosa was about 60% of the control value (P < 0.05).

Vitamin A status after administration of retinyl acetate to protein-malnourished mice

Retinoid levels in the plasma and small intestinal mucosa of protein-malnourished mice treated with vitamin A were compared with those of vehicle-treated, protein-sufficient mice. Food intake was greater in mice fed the low-protein diet (Table 2) ; therefore, vitamin A intake was not low in protein-deficient mice. However, plasma retinol concentration in protein-deficient mice treated with vehicle was 50% of the control value (Table 3 ).In contrast, protein malnutrition did not affect mucosal retinol or plasma and mucosal retinyl palmitate.

Effect of vitamin A on total IgA level in mouse small intestine under protein malnutrition

The mucosal IgA level in the vehicle-treated, protein-deficient mice was 55% of that in vehicle-treated, protein-sufficient mice (P < 0.05). Supplementation with 0.05 or 0.3 mg of retinyl acetate did not affect the IgA level in the protein-malnourished mice (Fig. 2 ),while 1 mg of retinyl acetate restored the IgA concentration to the level of the vehicle-treated, protein-sufficient group. A similar effect of vitamin A supplementation on the mucosal IgA level was observed in the mice fed the control protein diet (Fig. 2) .

View larger version (36K): [in this window] [in a new window]   Figure 2. Effects of Vitamin A (retinyl acetate) administration on immunoglobulin A (IgA) concentration in small intestinal mucosa of mice fed a (A) low or (B) control protein diet. Mice were given free access to a low or control protein diet. Retinyl acetate (0.05, 0.3 or 1 mg) in 100 µL of corn oil was intragastrically administered to a mouse for 2 wk after starting the experimental diet (d 0). Vehicle group was treated with 100 µL of corn oil. The sample was prepared from small intestinal mucosa, and the mucosal IgA content was measured. Values are mean ± SD (n = 5). Means with different superscripts are significantly different by two-way analysis of variance followed by Duncan's multiple range test (P < 0.05). Vehi, vehicle; Vit A, vitamin A.

Effects of vitamin A on cytokine levels in the mouse small intestine

In protein-sufficient mice treated with vehicle, the concentrations of IL-4, IL-5 and IL-6 were 31.3 ± 5.3, 138.4 ± 22.1 and 189.0 ± 23.5 ng/g mucosa (n = 5), respectively. Concentrations of IL-4 and IL-5 were lower in protein-deficient mice treated with vehicle, but IL-6 was not different (Fig. 3 ).The difference in IL-5 was the most remarkable: a 1% protein diet decreased this level to 17% of the control value. Administration of 1 mg retinyl acetate to protein-deficient mice significantly increased the IL-5 level about 3.1-fold. In protein-sufficient mice, retinyl acetate significantly increased all of the mucosal cytokine concentrations (Fig. 3) .

View larger version (26K): [in this window] [in a new window]   Figure 3. Vitamin A administration increased (A) interleukin (IL)-4, (B) IL-5 and (C) IL-6 levels in mouse small intestinal mucosa. Mice were given free access to a low or control protein diet. Retinyl acetate (1 mg) in 100 µL of corn oil was intragastrically administered to a mouse for 2 wk after starting the experimental diet (d 0). Vehicle group was treated with 100 µL of corn oil. The sample was prepared from small intestinal mucosa and subjected to the ELISA for (A) IL-4, (B) IL-5 and (C) IL-6. Values are mean ± SD (n = 5). Means with different superscripts are significantly different by two-way analysis of variance followed by Duncan's multiple range test (P < 0.05).

In protein-sufficient mice, a number of IgA-positive cells were present in lamina propria of the intestinal mucosa, whereas a significantly lower number of IgA-positive cells was observed in the protein-deficient mice (Table 4 ).Treatment of protein-deficient mice with 1 mg retinyl acetate normalized the number of IgA-positive cells (Table 4) , suggesting that the vitamin A supplementation increased the mucosal IgA level at least in part by increasing the number of IgA-producing cells.

Effect of vitamin A on specific anti-cholera toxin immunity

When protein-deficient mice administered vehicle were immunized with CT, the anti-CT IgA content in small intestinal mucosa was only 17% of that of protein-sufficient mice treated with vehicle (Fig. 4 A).Treatment of protein-deficient mice with 1 mg retinyl acetate resulted in an anti-CT IgA content not different from the level in vehicle-treated control mice. Furthermore, 1 mg retinyl acetate significantly increased the ratio of CT-specific IgA to total IgA level from 0.8 to 2.1% in mice fed the low-protein diet (Fig. 4B) . In this case, vitamin A supplementation significantly upregulated the mucosal IL-5 concentration in CT-treated mice fed the low-protein diet from 161 ± 23 (n = 6) to 381 ± 91 ng/g mucosa (n = 5). Similar effects of vitamin A on the anti-CT IgA level and the ratio of CT-specific IgA to total IgA level were observed in protein-sufficient mice (Fig. 4) .

View larger version (31K): [in this window] [in a new window]   Figure 4. Vitamin A on cholera toxin (CT)-specific immunoglobulin A (IgA) content in intestinal mucosa. Mice fed a low or control protein diet were immunized with CT. The first immunization was done by intragastrically administrating 20 µg CT with a stainless feeding tube on d 0. Booster doses of 10 µg of CT each were given on d 10, 17 and 24. These mice were treated with vehicle or 1 mg retinyl acetate every day from d 0. They were killed by cervical dislocation on d 32. The sample was prepared from small intestinal mucosa and subjected to the ELISA for CT-specific (A) and total IgA measurements. The ratio of CT-specific IgA to total IgA level was calculated (B). Values are mean ± SD (n = 5). Means with different superscripts are significantly different by two-way analysis of variance followed by Duncan's multiple range test (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 5. Vitamin A improved the survival rate of protein-deficient mice exposed to a high dose of cholera toxin (CT). Mice (n = 12/group) were given free access to a low or control protein diet and were intragastrically treated with vehicle or 1 mg retinyl acetate every day. All mice were immunized with 20 µg CT on d 0, and then a high dose (0.1 mg) of CT was administered to mice on d 14. The survival of mice was monitored every day.

	Discussion

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There are some similarities of mucosal immune dysfunction in vitamin A deficiency and protein malnutrition, including the decrease in antigen-specific immunoglobulin production. It was proposed that the suppressive effects of protein malnutrition on the immune system may be a result of associated micronutrient deficiencies, since protein deficiency often coexists with micronutritional deficiencies (Hansen et al. 1982 , Keusch 1990 , Shobaki and Rummel 1978 ). In fact, wasting protein deficiency decreased plasma retinol concentration to 50% of the control level. Several studies showed that protein malnutrition decreases the concentrations of specific plasma proteins, such as albumin, transferrin, and retinol-binding proteins (Ingenbleek et al. 1975 , McFarlane et al. 1969 ). The protein diet employed in this study might decrease plasma retinol-binding proteins as well as plasma total protein. Based on the average food intake, mice consumed about 0.07 mg of vitamin A per day from the experimental diets. Daily supplementation of a 40-times larger dose (0.3 mg) of retinyl acetate was required to normalize the plasma retinol concentration in the protein-deficient mice. However, even with such a high dose, the mucosal IgA level was not normal. Therefore, we tried a larger dose of retinyl acetate (1 mg) and found that daily supplementation of this dose could normalize the mucosal IgA level in the protein-deficient mice. These results suggest that vitamin A may have acted as a drug, rather than as a nutrient, in our experiments.

The use of vitamin A capsules was recently recognized as one of the most cost-effective interventions besides vaccination and oral rehydration therapy to improve public health in developing countries. A daily dose of about 60 mg retinol equivalents per subject was usually used in field trials (Keith et al. 1991 and Sommer et al. 1986 ). However, the potential impact of vitamin A supplementation on morbidity and mortality of infectious diseases varies with the type and severity of malnutrition in different populations. Our results suggest that large oral supplementation doses (12–40 mg/kg body weight) of vitamin A might be necessary where protein malnutrition is a serious public health problem. However, such excessive doses of retinyl acetate cannot be applied to humans because of its toxicity. Vitamin A exerts multiple biological actions through binding its nuclear receptors, such as retinoic acid receptors and retinoid X receptors. Therefore, more potent vitamin A agonists, which selectively bind distinct subtypes of the retinoid receptors and stimulate humoral immune competence, should be developed to safely and effectively improve the mucosal immune depression of protein-malnourished humans.

Recently, Alkan et al. (1996) demonstrated that the measurement of cytokine levels in various tissues by ELISA can be used to assess the cytokine responses to immunogenic stimuli in vivo. To study the mechanism of the effect of protein malnutrition or vitamin A supplementation on the mucosal IgA production, the B cell-stimulatory cytokines (IL-4, IL-5 and IL-6) in the small intestinal mucosa were quantified by ELISA. The sensitivity and quantitativeness in this assay were acceptable to determine the steady-state levels in the mucosa of mice fed the low-protein diet and to assess the responses to vitamin A or CT administration. Several in vitro investigations suggested that dietary protein and vitamin A regulate the functions of Th2 cells. For example, Zhang and Petro (1997) reported that splenic T cells prepared from protein-deficient mice released less IL-4. In vitamin A-deficient mice, Th2 cells were present in abnormally low numbers in the small intestine, and the production of IL-4 and IL-5 from the cultured Th2 cells was insufficient to drive B cell clonal expansion (Carman et al. 1992 and 1989 ). Retinoic acid can induce IL-5 synthesis by lipopolysuccharide-stimulated splenocytes (Tokuyama and Tokuyama 1995 ). Our results suggest that dietary protein or vitamin A intake also regulates the levels of the Th2 cytokines in small intestinal mucosa in vivo.

In normally nourished mice, administration of a high dose of retinyl acetate (1 mg per mouse) significantly increased the levels of IL-4, IL-5 and IL-6 in small intestinal mucosa, and these elevations were associated with the increase in the total IgA content. Therefore, we tested whether this stimulatory action of vitamin A on the B-cell-stimulating cytokines also operated in protein-malnourished mice. Among the cytokines examined, IL-5 was not only the most sensitive to a low-protein diet, but also the most responsive to vitamin A supplementation, which was confirmed by measurement of IL-5 level by ELISA and by immunohistochemical analysis of IL-5-positive cells in the small intestinal mucosa. The IL-4 concentration in small intestinal mucosa was significantly decreased by protein malnutrition, but the IL-6 level was not. A significant increase of IL-4 and IL-6 due to vitamin A supplementation was not detected by ELISA.

For maturation of B cells into IgA-producing cells, IL-4, IL-5 and IL-6 synergistically and differentiation-dependently stimulate the proliferation and maturation of B cells, and multistep signals besides these cytokines are also required for this process. Therefore, it is difficult to determine which cytokine is primarily responsible for the modification of IgA production by dietary protein and vitamin A supplementation. Recently, it was reported that targeted disruption of the mouse IL-4 or IL-6 gene caused the marked reduction of IgA-producing cells in mucosal tissues and the disappearance of mucosal antibody responses after antigen challenges (Kuhn et al. 1991 , Ramsay et al. 1994 ). Since the present experiments suggested that IL-5 might be the critical factor in the regulation of IgA production by dietary protein or vitamin A supplementation, experiments using IL-5 receptor chain-deficient mice are being conducted to address this issue.

Finally, we showed that vitamin A could induce resistance of mice fed a low-protein diet against toxicity caused by a challenge with a high dose of CT. Vitamin A was also shown to increase the mucosal barrier functions by stimulating the mucin production from goblet cells and differentiation of intestinal epithelial cells (De Luca et al. 1969 , Rojanapo et al. 1980 ). However, a CT-specific IgA response in the mucosa was also impaired in protein-malnourished mice, and treatment with retinyl acetate significantly restored the amount of CT-specific IgA under protein malnutrition. Therefore, vitamin A may induce resistance against infection during protein malnutrition, at least in part by stimulating the mucosal IgA system.

	ACKNOWLEDGMENTS

 
We thank T. Kitano, Fujisawa Pharmaceutical Co. (Osaka, Japan) for the kind gifts of cytokines and antibodies against mouse cytokines. We are also grateful to Q. P. Co. (Tokyo, Japan) for the kind gift of egg white protein. We acknowledge M. Ohnaka, Division of Nutritional for Special Physiological Needs, The University of Tokushima, Japan, for his assistance in statistical analyses. We also thank J. Terao, Division of Food Science, The University of Tokushima, Japan, for his technical advice in analysis of vitamin A using an HPLC.

	FOOTNOTES

 
¹ Financial support: This work was supported by a Grant-in Aid for Scientific Research from The Japanese Ministry of Education, Science and Culture (to T. N.) and Kiei Kai Foundation (Tokyo) (to K. K.).

³ Abbreviations used: ABTS, 2, 2'-azino-di[3-ethyl-benzthiazoline sulfate]; AIN, American Institute of Nutrition; ANOVA, analysis of variance; BHT, butylhydroxytoluene; BSA, bovine serum albumin; CT, cholera toxin; ELISA, enzyme-linked immunosorbent assay; IgA, immunoglobulin A; IL, interleukin; PBS-T; phosphate-buffered saline with 0.05% Tween-20.

⁴ Standard nonpurified MF^TM diet (Oriental Yeast) contains the following nutrients per 1 kg: water, 7.6 g; protein, 246 g; lipid 56 g; carbohydrate 528 g; minerals (AIN-76, 1977), 63 g; vitamins (AIN-76, 1977), 7 g.

Manuscript received September 14, 1998. Initial review completed November 18, 1998. Revision accepted February 5, 1999.

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