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Original Articles–Liver and Nutrition

Dietary Nucleotides Accelerate Changes in Intestinal Lymphocyte Maturation in Weanling Mice

Manzano, Manuel*; Abadía-Molina, Ana Clara; Olivares, Enrique-García; Gil, Angel; Rueda, Ricardo*

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Journal of Pediatric Gastroenterology and Nutrition: October 2003 - Volume 37 - Issue 4 - p 453-461
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Abstract

INTRODUCTION

The gastrointestinal tract must adapt to extrauterine life immediately after birth. This maturation process entails a series of developmental changes, including structural and functional modifications, which depend on the timing and dose of antigens encountered in the diet and the establishment of a normal luminal bacterial flora (1).

The gut associated lymphoid tissue (GALT) is a secondary lymphoid organ responsible for processing antigens that interact with the intestinal mucosa and for developing appropriate immune responses. Antigens present in the intestinal lumen are transported into the Peyer's patches through M cells, which are located between the enterocytes in the epithelium. Once in the Peyer's patches, antigens interact with antigen-presenting cells, mainly macrophages and dendritic cells, which present antigens to immature B and T lymphocytes in the germinal centers and in the interfollicular regions. After the B and T cells are activated by antigens, they enter the regional lymph nodes and migrate through the thoracic duct to the bloodstream. Finally, after circulating for several days, they differentiate into mature effector cells, migrating back to the lamina propria and the intestinal epithelium. As result of this process, we can distinguish three main intestinal lymphocyte populations: Peyer's patch lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL) (2).

Nutrients in the gastrointestinal tract can influence the maturation of both T and B cells, especially at weaning. Dietary nucleotides have been reported to influence the development of the small intestine and the systemic immune response in early life in both humans and animals (3–6). Nucleotides participate in different biochemical processes and can be synthesized by the organism through two main pathways: de novo, from low–molecular-weight precursors; and salvage, from nucleosides or nucleobases in the diet or derived from the catabolism of endogenous nucleotides (7–9). Some cells, such as lymphocytes and enterocytes have little ability to synthesize nucleotides by the de novo pathway. As a consequence, an external supply of nucleosides seems to be needed for optimal function. Thus, nucleotides may be considered as conditionally essential nutrients, especially during the neonatal period.

Nucleotides are present in milk, and their profile is species specific. Different biologic functions have been proposed for dietary nucleotides, such as increasing iron bioavailability, modulation of intestinal microflora, regulation of lipid metabolism, promotion of liver and intestinal maturation, and injury repair (10). They are also able to reduce the incidence and duration of episodes of diarrhea in infants (11–12), probably through stimulating the development of the intestinal immune system. During the last 20 years, nucleotides have been reported to modulate various immunologic mechanisms, including lymphocyte activation and proliferation, natural killer cell and macrophage activation, delayed hypersensitivity, and allograft and tumor responses (10,13–16). Most of the studied immunomodulatory effects of dietary nucleotides have to do with systemic immunity. Little is known about their potential effects on intestinal immunity (3).

The main objective of this study was to evaluate the influence of dietary nucleotides on the expression of characteristic antigens of the different lymphocyte subpopulations (PPL, IEL and LPL) in the intestine of mice at weaning.

MATERIALS AND METHODS

Animals

One hundred weanling (3-weeks old) Balb/C mice (Iffa Credo, France) were housed in groups of five animals per cage, under 12-hour cycles of light and dark at 22°C and were given free access to food and water. The animals were divided into two groups: the first group was fed a semipurified nucleotide-free diet following the American Institute of Nutrition (AIN-76) guidelines (17), and the second group received a control diet supplemented with 3 g/kg of each of the following nucleotides: adenosine monophosphate, cytosine monophosphate, guanosine monophosphate, and uridine monophosphate (FN group). Mice (n = 10) were killed at 0, 4, 7, 12, and 18 days after the beginning of the experiment. The Animal Welfare Committee of the University of Granada approved the study protocol.

Sample preparation

Intestinal lymphocytes were isolated following the procedure of Gautreaux et al. (18) as modified by us. The small intestine from the Treitz ligament to the ileocecal valve was isolated, and the luminal content was flushed with cold Hanks balanced salt solution (HBSS; Sigma, St. Louis, MO). Visible Peyer patches were excised, and the intestine was opened longitudinally and cut into small pieces. To isolate the small intestinal epithelium, those pieces were incubated for 15 minutes at 37°C in 25 mL of HBSS with 5 mM dithiothreitol (DTT; Roche Molecular Biochemicals, Indianapolis, IN), 2 mM EDTA (Sigma) and 25 mM Tris buffer (Sigma) in a shaking water bath (100 strokes per min); the supernatant was discarded, fresh HBSS-DTT-EDTA-Tris was added, and the incubation procedure was repeated. Supernatants containing the epithelial cells from both incubation steps were pooled, and the cells were washed by centrifugation with RPMI 1640 culture medium containing 5% (v/v) heat-inactivated fetal calf serum (Sigma), 20 mM HEPES (Sigma), 2 mM L-glutamine, 500 U penicillin, and 100 μg/mL streptomycin (Sigma) (complete medium). LPL were liberated from the remaining sediment by placing the intestinal debris in 25 mL of complete medium with collagenase 0.02 U/mL, dispase 0.12 U/mL, and DNase I 200 U/mL (Roche Molecular Biochemicals) for 90 minutes in a 37°C shaking water bath at 100 strokes per minute. Excised Peyer patches were placed in complete medium and dissected with scalpels and were also subjected to enzymatic digestion for 30 minutes.

Cell preparations from the epithelium, the lamina propria, and Peyer patches were subjected to discontinuous Percoll (Pharmacia, Uppsala, Sweden) density-gradient centrifugation to enrich the lymphocyte populations. The commercial Percoll solution was diluted 9:10 with 9% NaCl, yielding an isotonic Percoll solution that was diluted with complete medium to obtain three solutions with several Percoll percentages (75%, 40%, and 30%), which were used in descending order (75% Percoll was overlaid with 2 mL of 40% Percoll and this with 2 mL of 30% Percoll) to form a discontinuous gradient. Cells were resuspended in 2 mL of complete medium and placed over the 30% fractions. After centrifugation at 650 g for 20 minutes, the interfaces between the 75% and the 40% layers were removed, and the cells were washed by centrifugation in 25 mL of complete medium. Cells were then resuspended in 2 mL of 40% Percoll and centrifuged at 650 g. The cell pellets, enriched for lymphocytes (PPL, IEL and LPL), were collected and washed by centrifugation with phosphate-buffer saline (PBS, Sigma).

Staining with monoclonal antibodies and flow cytometry

Lymphocyte preparations (1 × 106 cell/mL, 100 μL) were placed in 3-mL tubes with different concentrations of monoclonal antibodies, previously determined according to the recommendations of the manufacturer (Pharmingen, San Diego, CA), and were incubated for 30 minutes in darkness at 4°C. The following antibodies were used: anti-CD45 FITC (to identify leukocytes), anti-CD3 FITC (to identify T lymphocytes), anti-CD4 FITC (to identify helper T lymphocytes), anti-CD8α FITC (to identify α chain cytotoxic T lymphocytes), anti-CD8β PE (to identify β chain cytotoxic T lymphocytes), anti-TCRαβ PE (to identify αβ chain T-cell receptor), anti-TCRγδ (to identify γδ chain T-cell receptor), anti-CD5 PE (to identify thymus-dependent T lymphocytes and B-1 cells), anti-CD22 PE (to identify mature peripheral B cells) and anti-CD45R/B220 PE (to identify B cells). Cells were pelleted by centrifugation (500 g, 5 minutes), washed with PBS, and resuspended in 100 μL of 4% (v/v) formaldehyde. After an incubation of 20 minutes at 4°C, the cells were washed with PBS and fixed with 300 μL of 1% (v/v) formaldehyde.

Fluorescence-activated cell-sorter (FACS) analysis of cell preparations was performed on an ORTHO-cytoron flow cytometer (Ortho Diagnostic Systems, Westwood, MA), acquiring between 1500 and 3000 gated events, based on the forward light scatter and side light scatter properties of the cell preparations. Nonspecific fluorescence was determined by two controls: anti-IgG1 fluorescein isothiocyanate (FITC) and anti-IgG1 phycoerythrin (PE), prepared for each cell preparation.

Statistical analysis

Total numbers of CD45+ cells were calculated for each population (PPL, IEL and LPL) and specific lymphocyte subsets were expressed as mean percentages of CD45+ cells ± standard deviation of the mean. To evaluate differences attributable to diet and weaning period for the different lymphocyte subsets, we performed two-way ANOVA (SPSS 9.0®, Chicago, IL). Because data from different times and dietary groups did not have similar variances, we normalized those data previous to the ANOVA, weighting by the inverse of the variance. A posteriori comparisons between different times and time 0 were performed by the Dunnet test. Differences between the two dietary groups at a given time were corroborated, checking that the differences were significantly higher than those observed at time 0, using the standard error of the differences as given by the Tukey test correction. Probabilities of less than 0.05 were considered significant. For every intestinal lymphocyte population (PPL, IEL and LPL), we studied the relationship between each pair of lymphocyte subsets for the whole period of the study using simple linear correlation coefficient adjusted over the entire study period and a subsequent F test to determine whether it differed from zero; P < 0.05 was considered significant.

RESULTS

Tables 1, 2, and 3 show the percentages of CD45+ cells expressing different surface antigens in each of the intestinal lymphocyte populations for several feeding times (0, 4, 7, 12, and 18 days) after weaning. The results for the antigens in each lymphocyte population were expressed as differences between the percentage of positive cells at each day of feeding and day 0 (% ti − % t0;Figs. 1, 2, and 3); this representation clearly shows the evolution of the expression of each antigen within every cell population.

T1-10
TABLE 1:
Percentage of total CD45+ cells expressing different surface antigens in Peyer patch lymphocytes (PPL) for several feeding times (0, 4, 7, 12, and 18 days) in weaned mice fed (F) control or (FN) nucleotide-supplemented diet
T2-10
TABLE 2:
Percentage of total CD45+ cells expressing different surface antigens in intraepithelial lymphocytes (IEL) for several feeding times (0, 4, 7, 12, and 18 days) in weaned mice fed (F) control or (FN) nucleotide-supplemented diet
T3-10
TABLE 3:
Percentage of total CD45+ cells expressing different surface antigens in lamina propria lymphocytes (LPL) for several feeding times (0, 4, 7, 12, and 18 days) in weaned mice fed (F) control or (FN) nucleotide-supplemented diet
F1-10
FIG. 1.:
Differences for the percentage of total CD45+ cells expressing different surface antigens in Peyer patch lymphocytes (PPL) in mice at weaning, between each feeding time (4, 7, 12, and 18 days) and time 0 (% ti − % t0); this representation clearly shows the evolution of the expression of each antigen within every cell population. (*) = significant differences with respect to day 0 (P < 0.05); F = semipurified nucleotide-free diet; FN = control diet supplemented with nucleotides
F2-10
FIG. 2.:
Differences for the percentage of total CD45+ cells expressing different surface antigens in intraepithelial lymphocytes (IEL) in mice at weaning, between each feeding time (4, 7, 12, and 18 days) and time 0 (% ti − % t0); this representation clearly shows the evolution of the expression of each antigen within every cell population. (*) = significant differences with respect to day 0 (P < 0.05); F = semipurified nucleotide-free diet; FN = control diet supplemented with nucleotides.
F3-10
FIG. 3.:
Differences for the percentage of total CD45+ cells expressing different surface antigens in lamina propria lymphocytes (LPL) in mice at weaning between each feeding time (4, 7, 12, and 18 days) and time 0 (% ti − % t0); this representation clearly shows the evolution of the expression of each antigen within every cell population. (*) = significant differences with respect to day 0 (P < 0.05); F = semipurified nucleotide-free diet; FN = control diet supplemented with nucleotides.

In Peyer patches, we observed a decrease in the percentage of T cells (CD3+) and an increase in the percentage of B cells (CD22+) for the two experimental diets during the period of study (Fig. 1). The decrease of T cells was mainly attributable to a reduction of the subpopulation TCRαβ+. We also observed that those developmental changes occurred clearly sooner in animals fed the FN diet.

In the epithelium, we observed an increase in the percentage of T cells, as is shown in Figure 2 corresponding to CD3 and CD5+ cells. There was also an increase in the percentage of TCRαβ+ cells and a decrease in the percentage of TCRγδ+ cells during the period of study. As described for Peyer patches, in the epithelium the developmental changes occurred clearly sooner in animals fed the FN diet.

In the lamina propria, the most important result was the pronounced rise in the expression of CD5 lymphocytes in animals fed the FN diet (Fig. 3). We also observed an increase in the percentage of CD4+ cells, which occurred sooner in the animals fed the FN diet. The increase of CD4+ cells was mainly attributable to the TCRαβ subpopulation.

We also established linear correlations between the expressions of different antigens during the whole period of study in the different intestinal lymphocyte populations. To evaluate the specific influence of dietary nucleotides, we analyzed the correlations for the F and FN groups in the three lymphocyte populations. The most representative differences between both groups are shown in Table 4.

T4-10
TABLE 4:
Correlation coefficient (r) between each pair of antigen expression during study in the small intestinal lymphocyte populations (PPL, IEL, and LPL) for mice at weaning fed (F) control diet or (FN) nucleotide-supplemented diet; the most representative differences between both groups are shown

For PPL, there was a positive correlation between CD3 and CD8 expression in the control group that did not exist in the FN group. In addition, CD3 expression was positively correlated with CD4 expression in both groups, F and FN (Table 4). In IEL there was a negative correlation between the expression of CD3 and the B cell markers (CD22 and CD45R/B220) in the F group that was not detected in animals in the FN group (Table 4). Finally in LPL, there were negative correlations between the expression of TCRs and CD22 and between TCRγδ and CD5 in the FN group that was not detected in the F group. Moreover, there was a positive correlation between TCRαβ and CD5 expression in the F group (Table 4).

DISCUSSION

The most important result of this work is that dietary nucleotides accelerated maturational changes in intestinal lymphocytes at weaning, a period of time in which these populations are changing as the intestinal immune system matures (19–22).

Different studies previously have reported the influence of dietary nucleotides on systemic lymphocyte subpopulations. It was observed that mice injected with complete Freund's adjuvant and fed an RNA-enriched diet displayed a higher percentage of CD4+ cells than did those fed a control diet (23). Another study, which used mice immunized with allogenic spleen cells, reported an increased expression of Lyt 1+, Mac1+, and IL-2 receptor cells in the mice fed a diet supplemented with RNA or uracil (24). In another study, in which the influence of nucleotide supplementation of the diet on peripheral blood lymphocyte subpopulations of low–birth-weight newborn infants was evaluated, an increase of the CD4+ lymphocyte subpopulation at 10 days after birth in children fed with nucleotides was detected (5,25).

In the current study we found a selective increase in the expression of certain surface antigens in the different intestinal lymphocyte populations (PPL, IEL and LPL). We have noted that these developmental changes in the expression of some antigens, such as CD3 in PPL and IEL, CD5 and TCRαβ in IEL and LPL, CD22 in PPL, and CD4 in LPL, clearly occurred sooner in mice fed the FN diet. This suggests a clear effect of nucleotides in the process of maturation of these lymphocyte populations. It may be that the process of maturation of intestinal lymphocyte populations involves a number metabolic reactions and cell divisions. Therefore, an external supply of nucleotides is necessary, in addition to the de novo synthesis typically found in rapidly proliferating tissues, such as the intestine or the immune system (26). In animals fed the nucleotide-free diet, the process of intestinal lymphocyte maturation would be slower because of a deficit in the external supply of nucleotides. Consequently, the lymphocyte maturation of these animals must proceed through the slow and energy-expensive de novo synthesis.

We observed the nucleotide influence on maturation was not the same in all lymphocyte populations. In IEL, we observed a negative correlation among CD22+, CD45R/B220+, and CD3+ subpopulations in the control animals that was not detected in animals fed the nucleotide diet. These results, in addition to the results previously shown, suggest that although the B-cell subpopulation is relatively scarce in the epithelium, its development is promoted by the feeding of nucleotides. This nucleotide effect is similar to that reported by other authors, indicating that dietary nucleotides promote B lymphocyte proliferation, maturation, and differentiation (16,27–32).

Nagafuchi et al. (3) recently reported that dietary nucleotides increase the proportion of TCRγδ subset in epithelium of Balb/C mice at weaning. That report is in contrast to our work, which reports that dietary nucleotides promote primarily TCRαβ expression in IEL (Fig. 2) and also in LPL (Fig. 3). There are some differences between the study of Nagafuchi et al. and our study that would partially explain this disagreement. First, the experimental diet used by Nagafuchi et al. was based on a whey protein isolate as the source of protein, whereas our study used a semipurified diet based on AIN-76 recommendations (17), with calcium caseinate as the protein source. Second, there are important differences in the content and profile of nucleotides between the two nucleotide-supplemented diets. In our case, the content of nucleotides was higher than in the study of Nagafuchi et al., especially in the case of uridine monophosphate, one of the most important immunomodulating mononucleotides (33). There are other differences, such as the use of female mice in the study by Nagafuchi et al. and the use of male mice in our study which may have had an impact on the results. In addition, Nagafuchi et al. performed only one determination of TCRγδ in epithelium 2 weeks after feeding nucleotides, whereas we performed several determinations at several times after feeding. They also used fewer animals (n = 4) than we did (n = 10). Our results are in agreement with other reports describing TCRαβ, not TCRγδ in epithelium as requiring antigen stimulation for their development because these cells seem to be directly stimulated by dietary antigens present at weaning (19,34–36). Moreover, Cerf-Bensussan and Guy Grand (37) have reported the predominance of TCRαβ+CD8+ cells in epithelium, which is in agreement with our results.

The positive correlation between CD3 and CD8β in PPL of the F group but not of the FN group suggests that nucleotides promote the development of the CD4+ T cells in Peyer patches. This agrees with previous studies (16,33) that report the modulation T-helper cells influence the immunoglobulin production in infants.

In LPL, the negative correlation between the expression of TCRs and CD22 in the FN group suggests, as other authors have reported for the systemic immune system (16,29–32), that nucleotides might positively modulate the activation and differentiation of B lymphocytes. On the other hand, the positive correlation between TCRαβ and CD5 in the F group and the negative correlation between TCRγδ and CD5 in the FN group suggest that dietary nucleotides stimulate the expression of CD22 and CD5. Taking into account the significant increase in the expression of CD5 by LPL observed in animals fed the FN diet (Fig. 3), we suggest that dietary nucleotides might stimulate the differentiation of B-1 cells. B-1 cells express both CD5 and CD22 and are precursors of a high number of plasma cells producing IgA at the intestinal level and of peripheral plasma cells producing IgA and IgM. We speculate that the modulation of B-1 cells might be one of the potential mechanisms by which nucleotides promote immunoglobulin production. Several authors agree that this effect is the most important effect derived from nucleotides (5,16,29–33).

In summary, dietary nucleotides affect the process of maturation and differentiation of intestinal lymphocytes that usually take place at weaning. This process accelerates the developmental changes of most of the antigens expressed by these lymphocytes. Although nucleotides exert a selective effect on the different lymphocyte populations (PPL, IEL and LPL). In general, they seem to promote the development of T-helper lymphocytes, and consequently the maturation and differentiation of B cells. These effects would partially explain the widely reported positive modulation that nucleotides exert on immunoglobulin production.

Acknowledgments:

The authors thank Mrs. Maria Luisa Jiménez for help and technical assistance and Dr. Juan de Dios Luna for help in statistical treatment of data. The authors also thank to Stacey Garber for her critical review of the manuscript.

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Keywords:

Nucleotides; Intestinal lymphocytes; Epithelium; Lamina propria; Peyer's patches; Mice at weaning

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