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ORIGINAL RESEARCH ARTICLE

Fermentable Dietary Fiber Potentiates the Localization of Immune Cells in the Rat Large Intestinal Crypts

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

¹To whom requests for reprints should be addressed at Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. E-mail: zuka{at}chem.agr.hokudai.ac.jp

	Abstract

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Intestinal crypts are composed of a well-defined hierarchy of epithelial cells, and proliferating epithelial cells reside close to the bottom of the crypts—even in the large intestine. We investigated whether CD8⁺and CD4⁺intraepithelial lymphocytes (IELs) and CD161⁺ natural killer (NK) cells localized in proliferating or differentiated epithelial region of cecum and colon. Both proliferating epithelial layer cells and the immune cells along the longitudinal crypt axis of the large intestine were measured histochemically. Dietary intervention revealed that the physiological localization of the immune cells in the longitudinal crypt axis depended on the immune cell type. CD8⁺ IELs were preferentially located among differentiated epithelial cells. In contrast, CD161⁺ NK cells were located adjacent to the epithelial cells at the bottom of crypt. Cecal crypts contained significantly larger numbers of CD8⁺ IELs than did colonic crypts. However, there was only a minor population of CD4⁺ IEL in the cecal and colonic epithelia. Some dietary fibers increased the densities of CD8⁺ IELs and CD161⁺ NK cells in the cecum, with the magnitude of response varying among the types of fiber. There was a significant relationship between SCFA and the localization of immune cells, especially CD8⁺ IEL and CD161⁺ NK cells, which are considered to be involved in the maintenance of epithelial homeostasis.

Key Words: crypt • dietary fiber • CD161⁺ natural killer cell • CD8⁺ intraepithelial lymphocytes

	Introduction

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Intraepithelial lymphocytes (IELs) play a significant role in epithelial homeostasis. For example, abrogation of gammadelta IELs reduces epithelial cell turnover (1) and enhances azoxymethane-induced colorectal carcinogenesis and aberrant crypt foci (2), which are a preneoplastic biomarker for colorectal carcinogenesis (3). To investigate the relationship between colorectal carcinogenesis and the immune system, we focused on the large intestine and found that a reduction in CD8⁺ IELs by treatment with anti-asialo GM1 is accompanied by an increase in the number of aberrant crypts in the colorectum and that ingestion of sugar beet fiber (SBF) partially restores the number of CD8⁺ IELs in the colonic epthelium (4). Thus, the accumulation of CD8⁺IELs is modified by dietary intervention. Cytotoxicity mediated by natural killer (NK) cells is considered to play an important role in anticancer defense mechanisms (5). Recently, the CD3^–NKR-P1⁺ (CD161⁺) subset was found in the isolated IEL fraction from the rat small intestine, and the CD161⁺ cells were found to have strong NK activity (6, 7). The CD161⁺ NK cells may play a role in mucosal immune surveillance; nevertheless, there is very little information regarding the physiological localization of these immune cells around the crypt.

The intestinal epithelium is a highly proliferative, hierarchical organ in which stem cell position is well defined in terms of the spatial arrangement, even within the large intestinal crypt (8). Potten and colleagues extensively studied the spatial distribution of epithelial cells that undergo apoptosis, mitosis, proliferation, and expression of p53 and p21 protein along the longitudinal crypt axis of the intestinal epithelium. In general, proliferating epithelial cells reside close to the bottom of the crypt. Primarily CD8⁺ IELs are scattered among the epithelial cells, but their distribution is not well defined along the longitudinal crypt axis. A spatial arrangement of immune cells along the longitudinal crypt axis should be observed, as with the proliferating epithelial cells, if the localization of the immune cells is important for epithelial homeostasis. Dietary intervention may influence such localization of immune cells in a physiological condition.

Microbiota affect not only host mammalian nutritional status (9) but also interaction between epithelia and IELs (10). Bacterides thetaiotaomicron in the luminal contents induces angiogenesis in the intestinal villi in a gnotobiotic study (11), and it is widely accepted that ingesting dietary fiber changes the concentration of organic acids in luminal contents of the large intestine (12). Some dietary fibers have the ability to promote production of such organic acids in the luminal contents, and the fermentation profile depends on the source of the dietary fiber ingested. Of these organic acids, short-chain fatty acids (SCFAs) are known to affect the large intestine. For instance, butyrate is used as a primary energy source in colonic epithelial cells (13, 14) and stimulates normal epithelial cell proliferation (15). Indeed, we demonstrated that ingestion of SBF, a fermentable fiber, promotes a greater physiological accumulation of CD8⁺IELs in the crypts of the rat large intestine than does relatively unfermentable fiber (16). The CD8⁺IEL population is greatest in the cecal mucosa and gradually declines toward the distal large intestine.

In this study, we applied cell-positional analysis, which was originally established for epithelial cells (8), to the distribution of immune cells to evaluate the physiological localization of IELs and NK cells along the longitudinal crypt axis in the rat large intestine. Furthermore, we analyzed whether ingestion of dietary fiber modulates the density and distribution of IELs and NK cells on a cell-position basis in the crypt, accompanied with SCFA analysis of the cecal contents.

	Materials and Methods

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Animals and Diets.
This study was approved by the Hokkaido University Animal Use Committee, and the animals were maintained under the guidelines of Hokkaido University for the care and use of laboratory animals. Thirty male WKAH/HkmSlc rats (5 weeks old, Japan SLC) were housed in individual cages in a temperature-controlled room under a 12-hour photoperiod (light: 0800–2000 hours). The fiber-free (FF) diet contained sucrose, casein, corn oil, choline bitartrate, a mineral mixture, and a vitamin mixture according to AIN-93 formulation (17, 18). Rats were allowed free access to food and water over the experimental period. All rats were fed the FF diet for 7 days. After the acclimation period, they were divided into 5 dietary groups fed FF or dietary fiber (50 g/kg diet)–containing diets at the expense of sucrose. The dietary fibers used in this experiment were as follows: cellulose (CE; microcrystalline cellulose PH-102, Asahi Kasei Co.), SBF (Nippon Beet Sugar MFG), guar-gum hydrolysate (GGH; Meiji Seika Kaisha Ltd.), and water-soluble soybean fiber (WSF; Fuji Oil Co.). After 3 weeks of feeding the rats with the test diets, the rats were sacrificed (at 1000–1300 hours) by decapitation under anesthesia with diethylether, and the cecum and distal colon were removed. Rats were injected with bromodeoxyuridine (BrdU) solution at 1 h before the sacrifice, as previously reported (19). Segments of the large intestine (cecum and distal colon) were flushed with saline, embedded into Optimal Cutting Temperature compound (Sakura Finetechnical Co., Ltd.) in liquid nitrogen, and frozen at –80°C. The cecal contents were stored at –40°C until analysis.

Immunohistochemical Analysis.
Antibodies used in this study were as follows: mouse anti-CD4 (clone W3/25, Serotec), mouse anti-CD8 (clone OX-8, Cedarlane), mouse anti-NKR-P1 (CD161; clone 10-78, Cederlane), and anti-BrdU (clone OS94.6, Calbiochem). Frozen sections from these samples were fixed at 4°C with Zamboni’s fixative for CD4 and CD8 staining or with periodatelysine-paraformaldehyde for CD161 detection (16). After being fixed, these sections were immersed in 3% hydrogen peroxide in methanol to reduce endogenous peroxidase activity. They were incubated for 1 hour with 10% normal rabbit serum to block nonspecific bindings. After incubation with the primary antibody, sections were incubated with rabbit anti-mouse IgG conjugated with biotin. They were then incubated with peroxidase-conjugated streptavidin. For the chromogen, we used 3,3'-diaminobenzidine tetrahydro-chloride. All sections were counterstained with hematox-ylin. To verify the specificity of the signals produced, we confirmed the negative control by omission of each primary antibody, but with inclusion of all the other steps and reagents. The number of CD4⁺, CD8⁺, CD161⁺, and BrdU⁺ cells in the epithelial layer cells (epithelial cells and adjacent to the epithelial cells) in the well-shaped crypt section was scored for 50 half crypts in each part of the large intestine. Cell position means the location of certain epithelial cells from the bottom in the well-shaped crypt section. We then calculated the ratio of CD4⁺, CD8⁺, and CD161⁺ cells against 100 epithelial layer cells and the number of these immune cells located at cell positions 1–17 and >17.

Organic Acid Concentration in Cecal Contents.
The concentration of organic acids (succinate, lactate, acetate, propoinate, iso-butyrate, butyrate, iso-valerate, and valerate) in the cecal contents was measured by ion-exclusion chromatography using a high-performance liquid chromatography system equipped with a solvent delivery system (SCL-10AVP, Shimadzu), a double ion-exchange column (Shim-pack SCR-102H, 8 x 300 mm, Shimadzu), and an electroconductivity detector (CDD-6A, Shimadzu) according to the method of Hoshi et al. (20). Briefly, sodium hydroxide aqueous solution containing crotonic acid as an internal standard was added to the cecal contents. After centrifugation, the fat-soluble substance in the supernatant was removed by extraction with chloroform. The aqueous phase was filtered through a membrane filter and applied to high-performance liquid chromatography. The mobile phase consisted of 5 mM p-toluenesulfonic acid.

Statistical Analysis.
All results are expressed as means ± SEM. The data were analyzed by two-way ANOVA (site and diet) for distribution of BrdU⁺ epithelial cells. Statistical comparisons among dietary groups were performed using Tukey-Kramer’s test at P < 0.05. The number of IELs and NK cells between the lower (1–17) and upper (>17) cell positions in crypts was analyzed by Student’s t-test at P < 0.05. We also analyzed linear regression using the least squares method between the number of immune cells and concentration of each organic acid, and the number of CD8⁺IELs at cell position >17 and the number of CD161⁺ NK cells at cell positions 1–17.

	Results

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Representative Immunohistochemistry of BrdU⁺ Epithelial Cells, CD8⁺IEL, CD4⁺IEL, and CD161⁺ NK Cells Around the Large Intestinal Crypt.
There were no significant differences in final body weight after the experimental period regardless of the dietary fiber ingestion. Figure 1 shows representative photographs of BrdU⁺ (Fig. 1A), CD8⁺ (Fig. 1B), CD4⁺ (Fig. 1C), and CD161⁺ (Fig. 1D) cells in the rat large intestine. Many BrdU⁺ epithelial cells and CD8⁺ IELs were observed in the mucosa. In contrast, the number of CD4⁺ IELs was very low, although CD4⁺ cells were often observed in the lamina propria. CD161⁺ cells appeared to locate both in the lamina propria and adjacent to the epithelial cells near the crypt bottom. In this study, we scored the CD161⁺ NK cells as epithelial layer cells like CD8⁺ or CD4⁺ IELs. CD8⁺ IELs were observed mainly near differentiated epithelial cells of the crypts. No pathological accumulation of immune cells was observed in all rats.

View larger version (157K): [in this window] [in a new window]   Figure 1. Representative photographs showing BrdU+ epithelial cells, CD8+ intraepithelial lymphocytes, CD4+ intraepithelial lymphocytes, and CD161+ natural killer cells in the rat large intestinal crypt. BrdU+ epithelial cells located at the bottom of the crypt (A). In contrast, CD8+ intraepithelial lymphocytes are located in the upper region of the lamina propria and the intraepithelial region (B). Very few CD4+ intraepithelial lymphocytes were found (C). A certain number of CD161+ natural killer cells were observed in the lamina propria, and some CD161+ natural killer cells were located adjacent to the epithelial cells in the bottom of the crypt; natural killer clusters are also often observed (D).

View larger version (19K): [in this window] [in a new window]   Figure 2. Number of CD8+ (A) and CD4+ (B) intraepithelial lymphocytes, and CD161+ natural killer cells (C) per 100 epithelial layer cells in the half-crypt section in cecal (open bars) and distal colonic (filled bars) crypts of rats fed various dietary fibers. The data were shown as the means ± SEMs (n = 6). Bars with different small letters show significant differences (P < 0.05) in the cecum among dietary groups. Bars with different capital letters show significant differences (P < 0.05) in the distal colon among dietary groups. Asterisks show significant difference (P < 0.05) from the frequency in the cecum. P-values from two-way ANOVA (diet x site) in CD8+ intraepithelial lymphocytes were <0.0001 for diet, site, and diet x site. P-value from two-way ANOVA in CD4+ intraepithelial lymphocytes was 0.0122 for site. P-values from two-way ANOVA in CD161+ were 0.0050 for diet, <0.0001 for site, and 0.0125 for diet x site.

View larger version (16K): [in this window] [in a new window]   Figure 3. BrdU-incorporation of epithelial-lining cells in the cecum (A) and distal colon (B) in rats fed various dietary fibers (n = 6). Almost all BrdU+ epithelial layer cells were located at the base of the crypt (cell positions 1–17), both in the cecum and colon. At cell position >17, the percentage of BrdU+ cells was below 10% in each cell position.

View larger version (31K): [in this window] [in a new window]   Figure 4. Number of CD8+ (A, D) and CD4+ (B, E) intraepithelial lymphocytes and CD161+ natural killer cells (C, F) in the proliferating region (open bars) and differentiated region (slashed bars) in the crypts of the large intestine of rats fed various dietary fibers. The data were shown as the means ± SEMs (n = 6). Bars with different small letters shows significant differences (P < 0.05) in the proliferating cell position (1–17) among dietary groups. Bars with different capital letters show significant differences (P < 0.05) in differentiated cell position (>17) among dietary groups. Asterisks show significant difference (P < 0.05) from the number in the proliferating cell position (1–17).

View larger version (15K): [in this window] [in a new window]   Figure 5. Relationship between the number of CD8+ intraepithelial lymphocytes located at cell position >17 and the number of CD161+ natural killer cells at cell positions 1–17 in the half-crypt section of the cecum of rats fed various dietary fibers. The scatter plot contains individual values from rats fed fiber-free, cellulose, sugar beet fiber, guar-gum hydrolysate, and water-soluble soybean fiber. Symbols are same as those in Figure 3. Significant regression was observed between the two (r 2 = 0.56, P < 0.0001).

	Discussion

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We investigated the relationship between the localization of immune cells in pericryptal region and that of proliferating epithelial cells, because CD8⁺IELs appeared at a glance to locate at a relatively higher cell position from the crypt base. We found that CD8⁺ IELs clearly exist in the epithelial lining of the crypt, whereas CD161⁺ NK cells did not seem to locate in the intraepithelium. However, many CD161⁺NK cells appeared around the crypt base. Our study indicates that some of the CD161⁺ population locates adjacent to the epithelial cells in the pericryptal region and that the isolated IEL fraction contains such periepithelial immune cells, as well as IELs.

Previous reports have shown that the isolated rat IEL fraction contains CD3^–NKR-P1⁺ (CD161⁺) cells (6, 7). These cells are mostly restricted to the CD4^–CD8^–CD3^– population and have the ability to kill NK-sensitive targets. Some reports show the existence of NKT cells in the IEL fraction. CD161⁺ T cells are shown to be abundant in human intestinal epithelium (22), and CD3⁺NKR-P1⁺ (CD161⁺) cells transiently increase within a few days after infection of Listeria monocytogenes in rats (23). These results indicate a significance of NKT cells in the maintenance of epithelial homeostasis. However, it has also been reported that DX5⁺NK1.1⁺CD4^–CD8^–cells in the T-cell-receptor-negative fraction of the IELs have significant cytotoxic activity in mice (24). In addition, our results showed that the CD161⁺ NK cells were not totally identical to CD8⁺ IELs because of the characteristic localization.

This is, however, the first report showing a spatial arrangement of IELs and NK cells along the longitudinal crypt axis. We do not exclude the possibility that some CD161⁺NK cells have CD8 molecules on the surface; these cells may play a role in the maintenance of epithelial homeostasis in the large intestine in pathogenic conditions. It is especially interesting to clarify the mucosal network between the cells residing at the intestine. Our study showed that CD161⁺ NK cells located as some clusters in cecum (Fig. 1D) rather than as single cells in the large intestine, but their function remains unclear.

The frequency of CD8⁺ IELs (Fig. 2A) decreased toward the distal part of the large intestine. This result is consistent with our previous report using same strain of rats (16). In humans, similar results have been observed for CD3⁺, CD8⁺, and CD103⁺ IELs in the large intestine, although the earlier study does not confirm the longitudinal localization of these immune cells (25). It is notable that ingestion of dietary fibers potentiated such localization of the CD8⁺ IELs and CD161⁺ NK cells. Physiological accumulation of CD8⁺ IELs has been enhanced in crypts of rats fed fermentable dietary fibers (SBF, GGH, and WSF), whereas the level was lower in CE-fed rats (Fig. 2A). This indicates that luminal fermentation pattern is an important factor in the characteristic localization of the immune cells. In contrast, the frequency of CD4⁺ IELs tended to increase toward the distal large intestine but was not changed by modulating the diet.

Almost all proliferating epithelial cells were located at cell positions 1–17 of the crypt in both the cecum (Fig. 3A) and distal colon (Fig. 3B). We further analyzed the distribution of immune cells in the proliferating epithelial area at cell positions 1–17 and in the differentiated epithelial area at cell position >17. Our results indicate that a variation in distribution depends on the type of immune cells in the pericryptal region of the large intestine. The cell-positional analysis revealed evidence that all fermentable dietary fibers used in this study potentiated the frequency of CD8⁺ IELs located among differentiated epithelial cells, whereas the CD161⁺ NK cells were located at the bottom of the crypt. This indicates that the chemoattractant from the proliferating epithelium may be different than those from the differentiated epithelium.

The fermentation profile of the cecal contents in the rats varied according to the dietary fiber ingested (Table 2). In general, fermentation generates SCFAs that are known to affect the activities of the large intestine. For example, butyrate both is used as a primary energy source in colonic epithelial cells (13, 14) and stimulates normal epithelial cell proliferation (15). The dietary fibers used in this study have various cecal fermentation profiles (Table 2). We also found significant relationships between cecal concentration of organic acids and accumulation of IELs and NK cells, although the correlations were not quite strong (Table 3). Sanderson and colleagues showed a regulation of chemo-attractant expression by sodium butyrate using cell culture systems (26). Of dietary fibers used in this study, SBF-fed rats had significantly larger amounts of butyrate in cecal contents than others (Table 2), and the accumulation of CD8⁺ IELs and CD161⁺ NK cells was promoted (Figs. 4A and C) without an increase in the epithelial proliferation (Fig. 3A) in the cecum. Intestinal epithelial cells are also known to produce a variety of chemokines on occasion (28–30).

Recently, it has been reported that butyrate modulates the production of MCP-1 and IL-8 in human intestinal epithelial cells in a different manner (26, 31). The chemo-attractants and cytokines secreted from the epithelial cells may differ between proliferating and differentiated epithelial cells and may also be modulated by the ingestion of fermentable dietary fibers. These results indicate that SCFA produced from dietary fibers is one of the factors modulating localization of these IELs and NK cells. There also seems to be another factor involved, such as bacterial cell components. Naik et al. (27) reported that intestinal epithelial cells have Toll-like receptors for bacterial products in the luminal contents. It is possible that the bacterial cell components themselves modify the expression of a variety of IEL and NK chemoattractants in the epithelial cells.

This study showed that the accumulation of CD8⁺ IELs in the differentiated area in the crypt was accompanied by an increase in CD161⁺ NK cells at the bottom of the crypt (Fig. 5). SBF did not induce an increase in proliferating epithelial cells in the crypt of cecum despite their high concentration of butyrate in the contents (Fig. 3A; Tables 1 and 2), but they did promote both CD8⁺ IEL and CD161⁺ NK accumulation (Figs. 2 and 4). These results indicate that the proliferation of epithelial cells was not directly related to the accumulation of IELs and NK cells in the large intestine. A combination of the organic acid pattern, together with bacterial profile in the luminal contents, seems to alter the pattern of chemoattractants released from the intestinal epithelium for certain IELs or NK cells (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Figure 6. Differences in physiological localization of CD8+ intra-epithelial lymphocytes and CD161+ natural killer cells along the crypt axis of the large intestine.

In conclusion, we demonstrated a spatial arrangement of CD8⁺ IELs and CD161⁺ NK cells along the longitudinal crypt axis in the crypts of the large intestine. Ingestion of fermentable dietary fiber, especially SBF, potentiated the accumulation of CD8⁺ IELs and CD161⁺ NK cells, probably via fermentation in the luminal contents.

	Footnotes

 
This work was supported in part by a grant from Japan Society for the Promotion of Science.

Received for publication December 12, 2003. Accepted for publication June 7, 2004.

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