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The relationship between the kinetics of villous columnar epithelial cells and the expansion of colonies of indigenous bacteria from the narrow apical portions of intestinal villi was immunohistochemically and histoplanimetrically investigated in the small intestine of bromodeoxyuridine administred Wistar rats. As a result, the lifespan of villous columnar epithelial cells was slightly shorter in the distal ileum than in other portions of small intestine, accompanying the minimum height of the intestinal villi of the distal ileum in the small intestine. The migration speed of villous columnar epithelial cells was significantly decreased toward the distal small intestine. The migration speed in the distal ileum was about one-fourth of that in the duodenum. The migration speed of the villous columnar epithelial cells was greater and their lifespans were shorter in the sites with wide expansion of the indigenous bacterial colony from the narrow apical portions of the intestinal villi than that in sites with no or less expansion. Additionally, the expansion of the indigenous bacterial colony from narrow villous apices also immediately shortened the heights of the intestinal villi. These findings suggest that the migration speed of villous columnar epithelial cells might contribute to the regulation of the settlement of bacteria at the villous apices and the inevitable proliferation of indigenous bacteria at the intervillous spaces in the rat small intestine.

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.

Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)(2-4), detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and beta-D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.

Previously, the specific antibody-mediated persorption of antigenic molecules and particulates from the small-intestinal lumen into the peripheral blood was clarified in rats, but the intermediation of the receptor for the specific antibodies was not. In this study, the existence of receptor for the specific antibody was experimentally examined in the rat small intestine. Glutaraldehyde-fixed sheep erythrocytes (SEs) coated by Fc-fragments of IgG (IgG-Fc), (Fab')(2)-fragments of IgG (IgG-Fab) or bovine serum albumin (BSA), were injected into 3 jejunal loops each 2 cm in length in non-orally pre-immunized rats, respectively. Thirty minutes after the injection, IgG-Fc-coated SEs were significantly more engulfed by villous columnar epithelial cells than Fab- or BSA-coated SEs. The most frequent absorption sites were the intestinal villous apices. The IgG-Fc-coated SEs were adhered to the striated borders and were engulfed by villous columnar epithelial cells. IgG-Fc-coated SEs passing through the epithelial cells were also detected in the subepithelial blood capillaries just beneath the villous epithelium, but not in the connective tissue and the lymph vessels. These findings suggest that the absorption of luminal antigenic particulates is probably mediated by the Fc-receptor, and that the absorbed antigenic particulates are directly transferred to the hepatic portal blood by passing through the endothelium of the subepithelial blood capillaries.

The aim of this study was to clarify the regulatory effects of epithelial kinetics on indigenous bacterial proliferation in the large intestine. The lifespan, migration speed and proliferation rate of crypt epithelial cells in the initial 20% of the colon (proximal colon) and the 50% of the colon (middle colon) in bromodeoxyuridine-administrated rats were histoplanimetrically and chronologically compared. The proximal colon possessed well-developed mucosal folds and a large amount of indigenous bacteria which filled the crypt lumen, whereas no folds or bacteria were found to occupy the crypt lumen in the middle colon. The cell lifespans were 32.2, 42.5 and 33.6 hr in the apical and the basal parts of the mucosal folds of the proximal colon, and in the middle colon, respectively. The migration speeds were 4.2, 2.1 and 3.3 microm/hr, respectively, while the appearance frequencies of proliferating cell nuclear antigen (PCNA)-positive crypt epithelial cells were 35.0, 24.6 and 33.8%. These findings suggest that the lifespan was shortened and the migration speed increased in the most luminal mucosa of colon, contributing to the elimination of the adhered bacteria from the most luminal mucosa. By contrast, the elongation of the lifespan and deceleration of the migration of epithelial cells in the basal parts of the mucosal folds might contribute to reliable settlement of indigenous bacteria, resulting in the maintenance of a large amount of indigenous bacteria in the lumen of the proximal colon.

The possibility of persorption of bovine serum albumin (BSA) molecules from mucous epithelial cells and its mechanism were investigated in rats orally pre-immunized by BSA for 14 consecutive days. In the small and large intestines, both the BSA antigen (BSA-Ag) and its specific antibody (SpAb) were absorbed by the epithelial cells at the late apoptotic stage (ApoEp), and were subsequently transcytosed by membranes of the small vesicles. The basal cytoplasms containing highly-concentrated BSA-Ag and SpAb were occasionally fragmented into small cytoplasmic droplets that were secreted into the lamina propria. In Peyer's patches, both BSA-Ag and SpAb were more actively absorbed and transcytosed toward the dome area by the ApoEp of the dome apex than by the M cells. BSA-Ag and SpAb were finally persorbed into the portal blood and lymph, but were never secreted into the bile. They were also engulfed by macrophage-like cells in the villous lamina propria, mesenteric lymph node and spleen, and by hepatocytes in the liver. These findings suggest that sensitized soluble luminal antigens are taken up by ApoEp in the small intestine and are finally persorbed into the peripheral blood. The uptake of luminal antigen might be mediated by its luminal SpAb.

To clarify the relationship between M cells and intestinal microflora, histoplanimetrical investigation into the bacterial colonization and the differentiation to M cells was carried out in rat Peyer's patch under physiological conditions. The follicle-associated epithelium (FAE), except for the narrow area of apical region, was closely covered with both neighboring intestinal villi and a thick mucous layer, the latter of which also filled the intervillous spaces as well as the space between the FAE and the neighboring intestinal villi. Indigenous bacteria adhered almost constantly to the narrow areas of apical regions of both intestinal villi and the FAE. Bacterial colonies were occasionally located on the basal to middle region of FAE, where M cells also appeared, forming large pockets. When bacterial colonies were located on the basal to middle region of FAE, bacteria with the same morphological characteristics also proliferated in the intervillous spaces neighboring the Peyer's patch. In cases with no bacterial colonies on the basal to middle region of FAE, however, M cells were rare in the FAE. Histoplanimetrical analysis showed the similar distribution pattern of bacterial colonies on the FAE and M cells in the FAE. M cells ultrastructurally engulfed indigenous bacteria, which were then transported to the pockets. These results suggest that indigenous bacterial colonization on the FAE stimulates the differentiation of M cells in the FAE under physiological conditions. The uptake of bacteria by M cells might contribute the regulation of the development of indigenous bacterial colonies in the small intestine.