Cellular Kinetics of Villous Epithelial Cells and M Cells in Rabbit Small Intestine

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1 FULL PAPER Anatomy Cellular Kinetics of Villous Epithelial Cells and M Cells in Rabbit Small Intestine Takashi TAKEUCHI 1) and Tatsuo GONDA 1) 1) Institute of Experimental Animals, Shimane Medical University, Izumo , Japan (Received 25 July 2003/Accepted 19 January 2004) ABSTRACT. The cellular kinetics of villous columnar epithelial cells and M cells in the rabbit small intestine were determined by the use of 5-bromo-2 -deoxyuridine (BrdU) as a tracer. To identify M cells, vimentin antibody was used. The BrdU-labeled nuclei of col umnar epithelial cells reached the base of intestinal villi in all portions at 1 day after BrdU administration. Thereafter, BrdU-labeled cells migrated toward the villous tip, but they did not move at a uniform speed. The epithelial cells which existed in intestinal vil li on circular folds moved faster than those on mucosa other than circular folds. At 7 days after BrdU administration, the leading edge of BrdU-labeled epithelial cells already disappeared from the villous tip in all portions of the small intestine. In the ileal Peyer s patch, t he BrdU-labeled nuclei of microvillous epithelial cells and vimentin-positive M cells appeared near the intestinal crypt orifice at 1 day after BrdU administration, and then migrated toward the luminal surface of the follicle-associated epithelium (FAE). As they moved toward the upper portion of FAE, the number of BrdU-labeled M cells on the side of the dome decreased simultaneously. The leading edge of BrdUlabeled epithelial cells disappeared from the top of the FAE within 7 days. These results suggest that M cells may differentiate from the undifferentiated cells in intestinal crypts within 1 day and disappear from the top of the FAE after the change of their form from M cells into microvillous epithelial cells. KEY WORDS: BrdU, FAE, kinetic, M cell, rabbit. J. Vet. Med. Sci. 66(6): , 2004 The cellular kinetics has been studied mainly on villous columnar epithelial cells of mammalian intestinal tracts. The turnover time of mouse intestinal epithelial cells was estimated to be 3 and 4 days in small and large intestines, respectively [5, 6]. However, the specialized luminal antigen sampling epithelial cells, which are called M cells, also exist in the follicle-associated epithelium (FAE) on the Peyer s patches [4, 8, 11]. Whereas the morphology of M cells has been minutely investigated in various species [4, 8, 11, 12, 14, 16], only a few reports regarding their derivation and cellular kinetics in the mouse and chicken intestines have been made [4, 21, 22]. Up to the present, 2 main hypotheses about the derivation of M cells have been put forth, and these are mostly based on their distribution within the FAE. The first hypothesis is that M cells transform from mature microvillous epithelial cells of the FAE [3, 17 19]. The second is that M cells differentiate directly from undifferentiated cells in the crypt [4, 8 10]. In the rabbit intestine, the cellular kinetics of villous columnar epithelial cells has not yet been clarified, although the morphology and distribution of M cells have been reported in detail [8, 10]. In this study, the cellular kinetics of villous columnar epithelial cells and follicle-associated epithelial cells in the small intestine of adult rabbits were investigated by the use of 5-bromo-2 -deoxyuridine (BrdU) as a tracer. MATERIALS AND METHODS Animals: Twenty adult Japanese white rabbits (weighing kg, both sexes) were used in this study. The sex of the rabbits did not affect the results. They were reared under 23 ± 2 C, 50 ± 10% conditions and permitted free access to food and water. An artificial light was used between 8:00 a.m and 8:00 p.m. The rabbits were sacrificed by exsanguination under anesthesia with i.v. injection of pentobarbital sodium (100 mg/kg). Cellular kinetics of intestinal epithelial cells: At 1, 3, 5 and 7 days after the i.p. administration of BrdU (B-5002; Sigma, U.S.A.) at a concentration of 50 mg/kg, 5 rabbits were dissected, respectively. Thereafter, the duodenum (5 cm distal from pylorus), jejunum (middle region between pylorus and cecocolic orifice) and ileum (10 cm proximal to cecocolic orifice; containing the ileal Peyer s patch) were immediately immersed in 70% ethanol for 24 hr at 4 C and embedded in paraffin. The sections of 4 µm in thickness were pretreated with 1N-HCl for 4 min at 60 C. After endogenous peroxidase was blocked with H 2 O 2 for 30 min, the sections were treated with 1% normal goat serum albumin (CL1200; Cedar Lane, Canada) for 1 hr. They were then incubated with anti-brdu mouse monoclonal antibody at a 1:100 dilution (M 0744; Dako, Denmark) for 18 hr at 4 C. After the incubation with biotinylated anti-mouse IgG (H+L) goat serum at a 1:100 dilution (BA-9200; Vector, U.S.A.) for 2 hr at 4 C, the sections were incubated with streptavidin-biotin peroxidase complex (K 0355; Dako, Denmark) for 30 min at room temperature. They were finally incubated with 3,3-diaminobenzidine (DAB) containing 0.03% H 2 O 2 and counterstained with hematoxylin. For the estimation of the distribution of BrdU-labeled nuclei of villous columnar epithelial cells, 20 intestinal villi with full length were selected randomly from each intestinal portion of 5 rabbits. The relative positions of the BrdUlabeled nuclei of epithelial cells along the entire length of the villi were calculated as a percentage of the length from the bottom to the top of the villi. The mean height of the villi was also estimated from 20 villi from each intestinal

2 690 T. TAKEUCHI AND T. GONDA portion of 5 rabbits. In the ileal Peyer s patch, 5 lymphoid follicles were selected randomly from 5 rabbits for the detection of BrdU-labeled nuclei of epithelial cells. The measurements were done by means of the Scion image (Scion, U.S.A.). For analysis of the data, a paired Student s t-test was employed, and the data of the heights of the villi were also examined by analysis of variance (ANOVA) followed by the Fisher s PLSD test. All data were expressed as means ± SD, and statistical significance was accepted at the 5% level. Identification of M cells: To identify M cells, vimentin antibody was used [5, 6]. The sections of the Peyer s patch were incubated with a mixture of the monoclonal antivimentin V 9 (V6630; Sigma, U.S.A.) at a 1:10000 dilution and anti-brdu mouse monoclonal antibody for 2 hr at 37 C. Thereafter, the sections were incubated with biotinylated anti-mouse IgG goat serum for 2 hr at 4 C. Finally, they were incubated with streptavidin-biotin peroxidase complex for 30 min at room temperature and visualized with DAB. RESULTS At 1 day after BrdU administration, the leading edge of BrdU-labeled nuclei of the epithelial cells reached the base of intestinal villi in all portions of the small intestine. Thereafter, a row of BrdU-labeled epithelial cells gradually migrated toward the villous tips (Fig. 1). In contrast, the epithelial cells in the villi on the circular folds moved 1 day faster than those in the villi on other regions. This difference in the migrating velocity of epithelial cells was seen commonly in every portion of the small intestine (Fig. 3). Since almost all of the BrdU-labeled epithelial cells disappeared from the villous tips at 7 days after BrdU administration in all portions, their turnover time was estimated to be 5 to 6 days. The mean heights of the villi on the circular folds and other regions were ± 169 µm and ± µm in the duodenum, ± µm and ± µm in the jejunum, and ± 59.2 µm and ± µm in the ileum (n=5: respectively). There was a significant difference between the height of the villi on the circular folds and that on other regions in each small intestine (P<0.01: duodenum and jejunum, P<0.05: ileum). In addition, the height of the villi on the circular folds showed no significant difference among each small intestine, whereas that on other regions showed a significant difference (P<0.01: respectively). In the ileal Peyer s patch, the BrdU-labeled nuclei of microvillous epithelial cells and M cells were seen near the crypt orifice at 1 day after BrdU administration. The M cells expressing a specific immunoreactivity against vimentin antibody were mainly found in the portion from the base to the sides of the dome area of the FAE on the lymphoid follicle (Fig. 2), and no BrdU-labeled M cells were observed at the apex of the dome or in the intestinal crypts. But, some of the vimentin-positive epithelial cells were also seen on the side of the dome area, which lacked the morphological features of M cells, such as thick and sparse microvilli, and pocket-like invaginations of the basal or lateral plasma membrane with both intraepithelial lymphocytes and macrophages. Thereafter, the leading edge of BrdU-labeled epithelial cells migrated toward the apex of the dome and Fig. 1. Mucosa of duodenum at 1 day after BrdU administration. The leading edge (arrows) of the BrdU-labeled nuclei of villous columnar epithelial cells is visible at the base of the intestinal villi. Hematoxylin counter-stained. Bar, 10 µm. Fig. 2. FAE of ileal Peyer s patch at 1 day after BrdU administration. The M cells whose cytoplasm expresses immunoreactivity against vimentin antibody, are seen in the portions from the base to the side of the dome area of the FAE (arrows). BrdU-labeled nucleus of M cell is also seen (arrowhead). Hematoxylin counter-stained. Bar, 10 µm.

3 CELLULAR KINETICS OF INTESTINAL M CELLS 691 Fig. 3. Cellular kinetics of BrdU-labeled villous columnar epithelial cells in the duodenum (a), jejunum (b), ileum (c) and ileal Peyer s patch (d). Relative positions are shown as a percentage of the total length from the bottom of the intestinal crypt to the tip of the intestinal villi. Each dot expresses the mean positions of BrdU-labeled nuclei of epithelial cells at each day after BrdU administration. The white dot indicates the villous epithelial cells on the circular fold. The black dot indicates the villous epithelial cells on other mucosa. The villous epithelial cells on the circular fold move faster (about 1 day) than those on other mucosa. Results are expressed as means ± S.D. Each asterisk indicates a significant difference between black and white dots (P<0.01). disappeared from the FAE within 7 days after BrdU administration, indicating that the turnover time of the FAE is also estimated to be 5 to 6 days. Moreover, the diminution of BrdU-labeled M cells on the side of the dome also occurred simultaneously with the migration of BrdU-labeled epithelial cells toward the apex of the dome. The mean height of the follicular domes was ± 67.0 µm (n=5). DISCUSSION The cellular kinetics of villous columnar epithelial cells has been studied in several species [5, 6, 21]. The turnover time of mouse villous columnar epithelial cells was 3.3 days in the duodenum, 3.4 days in the jejunum, and 3.0 to 4.0 days in the descending colon [5, 6]. In our study, since BrdU-labeled epithelial cells disappeared from intestinal villi within 7 days in every portion of the small intestine, the turnover time was estimated to be 5 to 6 days in the adult rabbit small intestine. On the other hand, epithelial cells showed two different migrating velocities, and the difference correlated with the location of the intestinal villi on the intestinal walls. In addition, the difference in the villous height also depended on the location on the intestinal walls. It has been reported that the bacterial infection stimulates the epithelial cellular turnover in the rat jejunum [20]. Moreover, it has been reported that the existence of luminal microflora affects the turnover time of intestinal epithelial cells. In intestines of germ-free animals, the turnover time is significantly longer than that of conventional ones [1, 2]. These reports indicate that the acceleration of the turnover time may contribute to an exclusion of degenerated epithelial cells influenced by luminal microorganism. In the present results, epithelial cells situated on circular folds seemed to have many opportunities to come into contact

4 692 T. TAKEUCHI AND T. GONDA with many kinds of luminal antigens compared with those on other regions. Therefore, the turnover time of villous columnar epithelial cells on circular folds is thought to be shorter than that on other regions of the rabbit small intestine. Two theories have been put forth with regard to the differentiation of M cells. The first theory is that M cells transform from fully differentiated microvillous epithelial cells on the apices of the dome area. This hypothesis is based on the fact that M cells in the mouse ileal Peyer s patch are distributed throughout the upper region of the dome, but not near the intestinal crypt [17 19]. The second theory is that M cells are derived directly from undifferentiated cells in the intestinal crypt. In the rabbit ileal Peyer s patch and appendix, a majority of M cells at the periphery of the lymphoid follicle are located adjacent to the intestinal crypt [9, 10]. The finding supports the second hypothesis. In our results, the majority of immunoreactive epithelial cells against vimentin were found in the portion from the base to the side of the dome area, and BrdU-labeled and vimentinimmunoreactive cells appeared adjacent to the crypt at 1 day after BrdU administration. On the other hand, our investigation showed that some of the vimentin-positive epithelial cells did not express a close contact with intraepithelial lymphocytes and macrophages. Similarly, in the rabbit Peyer s patch, vimentin-positive cells lying at the base of the dome lacked the morphological features of typical M cells [7]. M cells are morphologically distinguished from neighboring epithelial cells, mainly based on the shape of the microvilli on their apical surface and the pocket-like invagination of the basal and lateral plasma membranes with intraepithelial lymphocytes [8, 11, 14, 15]. Therefore, these cells were recognized as immature M cells, as described in the research on the mouse Peyer s patch [4]. In addition to this finding, it was also noted that the intensity of immunoreactivity of M cells against cytokeratin decreased when they migrated from the base toward the apex of the dome [7]. These findings suggest that M cells are derived directly from undifferentiated cells in the intestinal crypt, and that the immature M cells change their form into mature ones during the migration. The cellular kinetics of M cells has not yet been clarified precisely, although the detailed morphological studies have been reported in various species [4, 8, 11, 12, 14, 16]. In the mouse ileal Peyer s patch, H 3 -thymidine labeled epithelial cells on the follicular dome exfoliated simultaneously with those in the intestinal villi. Therefore, this concomitant disappearance of labeled epithelial cells in the FAE after their disappearance from villi seemed to indicate that the turnover time of M cells was not longer than that of the villous columnar epithelial cell [4]. In the present study, the leading edge of the BrdU-labeled epithelial cells has already disappeared from the FAE by 7 days, as in the villous epithelial cells. Moreover, the diminution of BrdU-labeled M cells on the side of the dome occured simultaneously with the migration of the BrdU-labeled epithelial cells toward the apex of the dome. Interestingly, in the study on the chicken cecal tonsil, the distribution of follicular dome epithelial cells closely related with their location along the dome [13]. Immature M cells predominantly populated the base of the FAE, and shrunken epithelial cells clustered together in the transitional portion from the lateral to the apex of the FAE. These findings suggest that M cells do not disappear by apoptosis in the basal area, but fall into apoptosis near the apex of the dome after differentiation into microvillous epithelial cells. 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