Journal of Nutrition Animal Diets/Enrichment Products...

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lim, B. O.
Right arrow Articles by Sugano, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lim, B. O.
Right arrow Articles by Sugano, M.

The Journal of Nutrition Vol. 127 No. 5 May 1997, pp. 663-667
Copyright ©1997 by the American Society for Nutritional Sciences

Dietary Fibers Modulate Indices of Intestinal Immune Function in Rats1

Beong Ou Lim, Koji Yamada, Michiko Nonaka, Yuichiro Kuramoto, Pham Hung, and Michihiro Sugano

Laboratory of Food Science, Kyushu University School of Agriculture, Higashi-ku, Fukuoka 812-81, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
LITERATURE CITED

ABSTRACT

To explore the immunological roles of dietary fiber, male 4-wk-old Sprague-Dawley rats were fed for 2 wk cellulose (water-insoluble), konjak mannan (water-soluble), pectin (water-soluble) or chitosan (acid-soluble) at 5 g/100 g diet. Serum IgE concentrations in rats fed konjak mannan, pectin and chitosan were significantly lower than in those fed cellulose (mean ± SEM: 5.0 ± 1.1, 3.6 ± 1.3, 3.0 ± 1.2 and 9.6 ± 1.9 µg/L, respectively). Rats fed pectin had significantly higher serum IgA and IgG concentrations (358 ± 38 and 424 ± 36 mg/L for IgA and IgG, respectively) than those fed cellulose (240 ± 31 and 337 ± 25 mg/L) or chitosan (176 ± 22 and 379 ± 23 mg/L), while the IgM concentration did not differ among the groups. Concentrations of IgA, IgG and IgM in mesenteric lymph node (MLN) lymphocytes generally were greater, while IgE concentration was lower, in rats fed pectin and chitosan than in those fed cellulose. The proportion of CD4+ T-cells in MLN lymphocytes was also dietary fiber-dependent, and the CD4+/CD8+ ratio was significantly higher in the pectin fed group than in all other groups. Under certain experimental conditions, MLN lymphocytes from rats fed pectin had markedly greater interferon-gamma concentration than cells from other groups, while the effect on tumor necrosis factor-alpha concentration was less marked. Thus, dietary fiber may have an immunoregulatory effect on the intestinal immune system of rats.

KEY WORDS: dietary fiber · immunoglobulin · mesenteric lymph node · lymphocytes · cytokine · rats

INTRODUCTION

There has been great interest for many years in the physiological and pharmacological effects of dietary fibers (Edwards 1995, Mackeown-Eyssen and Bright-See 1984, Schneeman and Tinker 1995). Because of their diverse effects on various metabolic pathways, it is possible that dietary fibers also influence immune function as a consequence of changes in mucosal structure and gut microflora as well. Fermentation by intestinal bacteria of dietary fibers leads to a more acidic environment in the intestine, and this situation inhibits the conversion of primary bile acids to their secondary counterparts (Jacobs 1988). Bile acids enhance immunoglobulin (Ig2) E production by mesenteric lymph node (MLN) lymphocytes and suppress the production of IgA, IgG and IgM at relatively high serum concentrations, 400-500 µmol/L, that can be encountered in disease states (Lim et al. 1994). Similar class-specific regulation of immunoglobulin production is induced by unsaturated fatty acids (Yamada et al. 1996). Although consequent changes in intestinal microflora may influence immune functions, available information on immunoregulatory activity of dietary fiber is inadequate. Since class-specific regulation of Ig production is also effected by cytokines (Pene et al. 1988), the effect of dietary fibers on the production of Ig and cytokines in the MLN lymphocytes deserves further study.

Several dietary components other than dietary fiber have been shown to modify Ig production (Herald et al. 1994, Lim et al. 1994, 1995, 1996, Pene et al. 1988, Yamada et al. 1993, 1996). The modification of Ig production may affect the incidence of various diseases through induction of hypersensitivity (Vollenweider et al. 1991) and immunosuppression (Newble et al. 1975). Since the physiological functions of dietary fibers depend at least on their physical characteristics, i.e. solubility, we used water-insoluble (cellulose), water-soluble (pectin and konjak mannan) and acid-soluble (chitosan) fibers in this study. Chitosan, a glucosamine polymer, is a dietary fiber of animal origin, prepared from crustacean chitin, and is known to have a solubility-dependent hypocholesterolemic activity possibly through binding with bile acids in the gut (Sugano et al. 1988). This report shows that dietary fiber plays an important role in typical immune indices such as T-cell population, cytokine production and Ig production in rat MLN lymphocytes.

MATERIALS AND METHODS

Materials. Concanavalin A (Con A) was purchased from Vector Labs (Burlingame, CA) and dissolved in phosphate buffered saline (PBS; 8.0 g NaCl, 0.2 g KH2PO4, 0.2 g KCl, and 1.15 g Na2HPO4/L, pH 7.5). Enzyme-linked immunosorbent (ELISA) kits for determination of interferon-gamma (IFN-gamma ) and tumor necrosis factor-alpha (TNF-alpha ) were purchased from Biosource International Co. (Camarillo, CA). Fetal bovine serum (FBS) was the product of Intergen Co., NY. For enzyme-linked immunosorbent assay (ELISA) of rat Ig, 0.5 mL Tween 20 per L PBS (TPBS) was used for rinsing, and Block Ace (Dainihon Pharmaceutical Co., Osaka) for blocking and antibody dilution, as described previously (Lim et al. 1994).

Animals and diets. The animal experiment adhered to the Kyushu University guide for the care and use of laboratory animals. Male, 4-wk-old Sprague-Dawley rats were obtained from Seiwa Experimental Animals (Fukuoka, Japan) and housed individually in a room with controlled temperature and light (20-23°C and lights on 0800-2000 h). After acclimation for 4 d, rats were divided into four groups of five rats which were given free access to experimental diets. The diets were prepared according to the recommendation of the American Institute of Nutrition (AIN 93G diet) (Reeves et al. 1993). The basal diet contained following ingredients, in g/100 g diet: cornstarch, 39.8; casein, 20.0; dextrinized cornstarch, 13.2; sucrose, 10.0; soybean oil, 7.0; AIN-93G mineral mixture, 3.5; AIN-93 vitamin mixture, 1.0; L-cystine, 0.3; choline bitartrate, 0.25; tert-butylhydroquinone, 0.0014; and fiber, 5.0. Cellulose (Seiwa Experimental Animals, Fukuoka), konjak mannan (Shimizu Co., Tokyo), pectin (Snow Brand Food Co., Tokyo) and chitosan (Katakura Chikkarin, Tsukuba) were used as dietary fiber. Body weight and food intake were recorded every other day. After 2 wk of feeding, rats were killed by withdrawing blood from the abdominal aorta under light diethyl ether anesthesia, and serum Ig concentration was measured by ELISA (Engvall and Perlman 1971).

T-cell population analysis. MLN lymphocytes were suspended in RPMI 1640 medium (Nissui Pharmaceutical Co., Tokyo, Japan) and incubated at 37°C for 30 min to remove fibroblasts (Lim et al. 1994). Then, 5 mL of the cell suspension was layered on 4 mL of Lympholytes-Rat (Cedarlane, Hornby, Canada) and centrifuged at 1500 × g for 30 min. The lymphocyte band at the interface was recovered, and the cells were rinsed three times with RPMI 1640 medium. The lymphocytes were then resuspended in PBS containing 100 mL FBS/L. Viability was determined by trypan blue staining and was above 90%. After adjusting the cell concentration to 1 x 109 cells/L, 5 µL of fluorescecein-labeled mouse anti-CD4 (W3/25, mouse IgG1) or phycoerythrin-labeled mouse anti-CD8 (MRC OX-8, mouse IgG1) was added to 100 mL of the cell suspension (both from Serotec Ltd., Kidlington, Oxford, UK). After incubation for 30 min at 4°C, lymphocytes were rinsed three times with 100 mL FBS/L PBS and centrifuged at 700 × g for 5 min. The stained lymphocytes were fixed with 2% paraformaldehyde and analyzed with the EPICS PLOFILE II flowcytometer (Coulter Electronics Ltd., Luto, UK) (Osada et al. 1995). Each analysis, including those of negative control samples, was based on at least 104 events after dead cells and residual erythrocytes were eliminated by gating on the basis of forward angle light scatter.

Cells and cell culture. The MLN lymphocytes isolated from each dietary group were pooled and cultured in 100 mL FBS/L RPMI 1640 medium. The MLN lymphocytes were adjusted to 2 × 109 cells/L in 24-well microtiter plates (Becton Dickinson & Co., Franklin Lakes, NJ) in a final volume of 1 mL. Cells were incubated at 37°C for 24 h or 72 h in the absence or presence of 25 mg/L Con A, the concentration at which it exerts the highest IgE production (Lim et al. 1994). The concentrations of IgE, IgA, IgM and IgG were measured by ELISA (Lim et al. 1994). The concentrations of IFN-gamma and TNF-alpha were also measured on incubated samples using ELISA kits. In these measurements, pooled samples were used for immunoglobulin and cytokine assays due to the limited amount of MLN lymphocytes available.

Statistical analyses. Data, except from cell culture experiments that used pooled lymphocytes, were examined for normal distribution by F-test, and were then analyzed by one-way ANOVA followed by Duncan's new multiple-range test to identify significant differences (Duncan 1955). Values in the text are means ± SEM.

RESULTS

Growth variables. Weight gain of rats (initial weight of all rats, mean ± SEM, 127 ± 5 g) in the chitosan-fed group (96 ± 3 g/2 wk) was significantly lower (P < 0.05) than that of rats fed konjak mannan or pectin (115 ± 5 and 112 ± 5 g/2 wk, respectively), while that in the cellulose-fed group was intermediate (106 ± 5 g/2 wk). Food intake did not differ among groups (overall mean, 18.3 ± 0.5 g/d).

Serum immunoglobulin concentrations. Serum IgA concentration of the pectin-fed group was significantly higher than that of the cellulose- and chitosan-fed groups, and not different than that of rats fed konjak mannan. It was significantly lower in the chitosan-fed group than in the pectin- and konjak mannan-fed groups (Fig. 1). IgE concentration of rats fed konjak mannan, pectin and chitosan were significantly lower than that of the cellulose diet group. Compared to rats fed cellulose, IgG concentration in rats fed pectin was significantly higher (P < 0.05). The IgM concentration did not differ among groups.
Fig. 1. Serum immunoglobulin (Ig) concentration in Sprague-Dawley rats fed different dietary fibers. Values are means ± SEM, n = 5. Values with different letters are significantly different, P < 0.05.
[View Larger Version of this Image (55K GIF file)]

Immunoglobulin concentrations of MLN lymphocytes. Data were not analyzed statistically due to the use of pooled samples. In the absence of Con A, IgA concentration after 24-h incubation of MLN lymphocytes was higher in rats fed pectin and chitosan than in those fed cellulose. Con A treatment increased the concentration of IgA in cells from the cellulose- and pectin-fed groups, and the value was markedly high in the latter (Table 1). In the konjak mannan and chitosan groups, Con A did not increase IgA concentration. On the contrary, IgE concentration in MLN lymphocytes from rats fed pectin and chitosan was lower than in those fed cellulose when Con A was absent. However, the difference disappeared in the presence of Con A, although the concentration apparently increased in all groups. IgG concentration in MLN lymphocytes was also affected markedly by the type of dietary fibers. IgG concentrations in MLN lymphocytes from the pectin- and chitosan fed groups were higher than in cells from the cellulose-fed group. Con A did not affect IgG concentration. IgM concentration was greater in cells from the konjak mannan- and pectin-fed groups than in cells from the cellulose- and chitosan-fed groups, and this difference generally was maintained even when Con A was added, although the addition of Con A lowered the concentration of IgM in all groups. When the cells were cultured for 72 h with or without Con A, similar response patterns were observed.

Table 1. Immunoglobulin concentration in mesenteric lymph node lymphocytes of rats fed different dietary fibers1

[View Table]

T-cell populations of MLN lymphocytes. The proportion of CD4+ and CD8+ T-cells in MLN lymphocytes was measured in relation to the changes in Ig concentration (Table 2, Fig. 2). The proportion of CD4+ cells was higher in the pectin-fed group and lowest in the chitosan-fed group. On the other hand, the proportion of CD8+ cells was higher in the cellulose group than in the other groups. As a result, the ratio of CD4+/CD8+ cells was higher in rats fed pectin than in all other groups.

Table 2. Spleen T lymphocyte populations in rats fed different dietary fibers1

[View Table]

Fig. 2. Flow cytometer histograms illustrating expression of CD4+ and CD8+ by mesenteric lymph nood (MLN) lymphocytes of Sprague-Dawley rats. Cells were stained with fluorececein-labeled anti-CD4 and with phycoerythrin (PE) labeled anti-CD8. Negative control histogram is that observed with cells not stained for the CD surface markers. Histograms were obtained from the population of the red fluorescence of PE-stained cells such that all events represent either CD4+ and CD8+ T cells. Each histogram was obtained from analyses of at least 104 cells after removing dead cells and erythrocytes according to the sizing with forward angle scatter. Each panel depicts results from one rat fed the cellulose diet.
[View Larger Version of this Image (14K GIF file)]

Cytokine concentrations in MLN lymphocytes. Data were not analyzed statistically due to the use of pooled samples. Various types of lymphokines specifically regulate Ig production by class (Pene et al. 1988). When lymphocytes were cultivated for 24 h without Con A, the concentrations of IFN-gamma and TNF-alpha were below the detection limit (Table 3). In the presence of Con A, the cells from rats fed dietary fibers other than cellulose had detectable concentrations of IFN-gamma , while TNF-alpha was detected at low concentration even in the cellulose fed group. The concentrations of these cytokines were the highest in cells from the pectin-fed group, but the effect on TNF-alpha was less marked. After 72-h cell culture, cytokines were detectable only in cells from the pectin-fed group in the absence of Con A. In the presence of Con A, IFN-gamma concentrations were comparable in all groups.

Table 3. Cytokine concentrations in mesenteric lymph node lymphocytes of rats fed different dietary fibers1

[View Table]

DISCUSSION

The effect of dietary fiber on immunological function is not fully understood, although it is plausible that dietary fiber may influence immune functions through a change in intestinal microflora (Finegold et al. 1974, Rao 1995). We chose a dietary fiber level of 50 g/kg according to the AIN recommendation (Reeves et al. 1993). Since the hypocholesterolemic effect of dietary fibers is usually apparent when rats are fed for 2 wk (Sugano et al. 1989), we chose this feeding period. Feeding periods of 2 wk may be short, but our experiment showed that even during this period of time, dietary fibers exerted different effects. The type of dietary fiber can influence the composition of short-chain fatty acids (SCFA) produced in the gut (Brydon 1995). For example, the soluble fiber pectin is fermented to acetic acid (approximately 80% of total SCFA) and only minimal amounts of butyric acid. In addition, dietary SCFA increased T-cell numbers in the gastrointestinal system of rats, suggesting a possible mechanism for the immune regulatory influences of dietary fiber (Tappenden et al. 1995). Recently we reported that bile acids influence Ig production by spleen or MLN lymphocytes (Lim et al. 1994 and 1995, Yamada et al. 1993). Although this effect was seen at relatively high concentrations of bile acids, 400-500 µmol/L (Lim et al. 1994, Yamada et al. 1993), the level was within that encountered in the pathological situation (Pennington et al. 1977). Thus, interaction of dietary fiber, SCFA and bile acids could at least in part be an important determinant for the currently observed responses.

Lymphocytes in the intestinal mucosa first interact with antigens in the organized lymphoid tissues (Peyer's patches and lymphoid follicles in the colon) and further differentiate and mature in the germinal centers of the lymphoid follicles. Thereafter, they rapidly leave the mucosa and migrate through the MLN and the thoracic duct to reach the systemic circulation (Stephen and Martin 1994). Among the dietary fibers examined, pectin enhanced IgA concentration in MLN lymphocytes and most markedly elevated the serum IgA concentration. Since IgA plays an important role in the prevention of allergic reactions through inhibition of allergen absorption (Metcalfe 1991), this effect is interesting. In addition, konjak mannan, pectin and chitosan lowered serum IgE concentration compared to cellulose, and IgE production in MLN lymphocytes from rats fed these fibers tended to be low. Therefore, they may increase the immune response induced by helper T-cells (Mosmann and Moore 1991). The serum concentrations of IgG and IgM were not greatly affected by the type of dietary fibers. On the other hand, their concentrations in MLN lymphocytes was dietary fiber-dependent, suggesting that the effect of dietary fiber on Ig production is limited to the gut immune system.

In addition to Con A, several T-cell mitogens such as anti-CD3 antibodies and phytohemagglutinin have been used in immunologic studies. Because some of these mitogens may stimulate selected lymphocyte populations, in this study we used Con A, which stimulates IgE production by MLN lymphocytes in culture (Lim et al. 1994) and an inducer of the production of those cytokines which enhance IgE production (Konno et al. 1993). Of the dietary fibers examined, pectin stimulated IFN-gamma and TNF-alpha concentration to the greatest extent. Because we measured cytokine production at a Con A concentration of 25 mg/L, the concentration at which it exerts its highest stimulatory effect on IgE production, it is not clear whether this concentration elicits maximal cytokine production in the experimental conditions we employed here. Further study is needed on this point.

Dietary fiber moderately modified the proportion of CD4+ and CD8+ T-cells in the MLN lymphocytes. CD8+ T-cells may regulate the development of CD4+ helper T- (Th) cells by producing IFN-gamma or other regulatory cytokines which suppress the development of Th2 cells and favor Th1 cell growth (Cher and Mosmann 1987). Th1 cells produce IL-2, IFN-gamma and lymphotoxin, whereas Th2 cells produce IL-4 and IL-5 (Fiorentino et al. 1989). Pectin increased IFN-gamma concentration in cell cultures without Con A at 72 h, but the effect on the concentration of TNF-alpha was marginal. In rats fed pectin, a higher proportion of CD4+ cells and a lower proportion of CD8+ cells were observed in addition to a higher IFN-gamma compared to those fed other dietary fibers. Therefore, the effect of pectin could be mediated, at least in part, through an influence on the differentiation of T-cells to become Th1 cells. Because of the lack of the absolute cellular numbers in this experiment, there is a possibility that dietary fiber type might influence cell numbers without affecting the relative number of cells within major lymphocyte subset.

When one considers the response of IgA and IgE, it is likely that dietary fibers other than cellulose, in particular pectin, can alleviate the type I allergic reaction. Although the mechanism by which dietary fiber modifies immune indices is not apparent at present, the current observations opened a new aspect of the physiological role of dietary fiber.

FOOTNOTES

1   The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
2   Abbreviations used: Con A, concanavalin A; ELISA, enzyme linked immunosorbent assay; FBS, fetal bovine serum; Ig, immunoglobulin; IL, interleukin; INF, interferon; MLN, mesenteric lymph node; Th, helper T-cell; TNF, tumor necrosis factor.

Manuscript received 22 April 1996. Initial reviews completed 23 May 1996. Revision accepted 11 December 1996.

LITERATURE CITED

0022-3166/97 $3.00 ©1997 American Society for Nutritional Sciences



This article has been cited by other articles:


Home page
 J. Am. Coll. Nutr.Home page
A. D. Azezli, T. Bayraktaroglu, and Y. Orhan
The Use of Konjac Glucomannan to Lower Serum Thyroid Hormones in Hyperthyroidism
J. Am. Coll. Nutr., December 1, 2007; 26(6): 663 - 668.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
M. S. Gomez-Conde, J. Garcia, S. Chamorro, P. Eiras, P. G. Rebollar, A. Perez de Rozas, I. Badiola, C. de Blas, and R. Carabano
Neutral detergent-soluble fiber improves gut barrier function in twenty-five-day-old weaned rabbits
J Anim Sci, December 1, 2007; 85(12): 3313 - 3321.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
C. Porporatto, I. D. Bianco, and S. G. Correa
Local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration
J. Leukoc. Biol., July 1, 2005; 78(1): 62 - 69.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
K. K. Buddington, J. B. Donahoo, and R. K. Buddington
Dietary Oligofructose and Inulin Protect Mice from Enteric and Systemic Pathogens and Tumor Inducers
J. Nutr., March 1, 2002; 132(3): 472 - 477.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
B. O. Lim, C. A. Jolly, K. Zaman, and G. Fernandes
Dietary (n-6) and (n-3) Fatty Acids and Energy Restriction Modulate Mesenteric Lymph Node Lymphocyte Function in Autoimmune-Prone (NZB NZW)F1 Mice
J. Nutr., July 1, 2000; 130(7): 1657 - 1664.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lim, B. O.
Right arrow Articles by Sugano, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lim, B. O.
Right arrow Articles by Sugano, M.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]