Immunohistochemical and histoplanimetrical study on the endothelial receptor involved in transportation of minute chylomicrons into subepithelial portal blood in intestinal villi of the rat jejunum.

A portion of the minute chylomicrons less than 75 nm in diameter are transcytosed from the extravascular tissue into the subepithelial blood capillaries (sBC) in the villous apices of the rat jejunum. However, the details of the transportation mechanism have not been clarified. In this study, the endothelial receptor involved in the transportation of minute chylomicrons into the sBC's lumina was immunohistochemically and histoplanimetrically examined in intestinal villi of the rat jejunum. Immunopositivity for very low density lipoprotein (VLDL) receptor was detected on the luminal and basal surfaces of the endothelial cells of sBC in approximately 68% of those apices of jejunal villi that possessed numerous chylomicrons in the lamina propria, while VLDL receptor was detected on the endothelial cells of sBC in only approximately 8% of intestinal villi that possessed few or no chylomicrons in the lamina propria. No immunopositivity for LDL receptor was detected in the sBC of all intestinal villi. These findings suggest that VLDL receptor is expressed by the endothelial cells of the sBC in conjunction with the filling of the lamina propria of jejunal villi with many chylomicrons produced by the villous columnar epithelial cells and that the VLDL receptor mediates the transportation of minute chylomicrons, maybe VLDL, into the subepithelial portal blood from the extravascular tissue of the rat jejunal villi.

Ultrastructural and histochemical study on the Paneth cells in the rat ascending colon.

Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, ß-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon.

Peculiar composition of epithelial cells in follicle-associated intestinal crypts of Peyer's patches in the rat small intestine.

The epithelial cell composition was investigated in the follicle-associated intestinal crypt (FAIC) of rat Peyer's patches. The epithelium of the FAIC mainly consisted of columnar epithelial cells, goblet cells and Paneth cells. The characteristics of secretory granules in Paneth cells and goblet cells of both the FAIC and ordinary intestinal crypts (IC) were almost the same in periodic acid-Schiff (PAS) reaction, Alcian blue (AB) staining and the immunohistochemical detection of lysozymes and soluble phospholipase A2. Both goblet cells and Paneth cells were markedly less frequent on the follicular sides than on the anti-follicular sides of the FAIC. Goblet cells were also markedly less frequent in the follicle-associated epithelium (FAE) than in the ordinary intestinal villi (IV). Indigenous bacteria were more frequently adhered to FAE than to follicle-associated intestinal villi or IV. These findings suggest that the host defense against indigenous bacteria is inhibited on the follicular sides of FAIC, which might contribute to the preferential settlement of indigenous bacteria on the FAE; they also suggest that differentiation into secretory cells is inhibited in the epithelium of the follicular sides of FAIC, so that differentiation into M cells might be admitted in the FAE of rat Peyer's patches. Furthermore, intermediate cells possessing characteristics of both Paneth cells and goblet cells were rarely found in the FAIC, but not in the IC. This finding suggests that the manner of differentiation into Paneth cells in the FAIC differs from that in the IC.

Ultrastructural demonstration of the absorption and transportation of minute chylomicrons by subepithelial blood capillaries in rat jejunal villi.

Chylomicrons from villous columnar epithelial cells are generally known to be transported only by central lymph vessels (CLV), whereas antigenic particulates derived from the intestinal lumen can also be transported by subepithelial blood capillaries (sBCs) in rat intestinal villi. The possibility of chylomicron absorption by sBCs was histoplanimetrically studied in the rat jejunum under a transmission electron microscope. The chylomicrons more abundantly presented in villous venules than in arterioles. The most frequent size (MFS) of chylomicrons was 75 to 90 nm in diameter in the areas near sBCs, while it was 45 to 60 nm in the epithelial intercellular spaces just above sBCs or the intermediate areas between sBCs. The MFS of chylomicrons was 45 to 60 nm in the intermediate areas between sBCs and in the epithelial intercellular spaces just above these areas. The MFS of chylomicrons in CLV was intermediate between that in the area adjacent to sBCs and that in the intermediate areas between sBCs. Chylomicrons were found in small vesicles in the endothelial cytoplasms of sBCs. No chylomicrons larger than 600 nm were observed in the lamina propria. These findings suggest that some of the chylomicrons smaller than 75 nm, which are probable intestinal very low-density lipoproteins (VLDL), are directly transported to the liver by hepatic portal blood in addition to CLV and that epithelial fat droplets larger than 600 nm are not discharged into lamina propria in rat jejunum under physiological conditions.

Histoplanimetrical study on the relationship between invasion of indigenous bacteria into intestinal crypts and proliferation of epithelial cells in rat ascending colon.

The relationship between the invasion of indigenous bacteria into intestinal crypts and the proliferation of epithelial cells was histoplanimetrically investigated in the rat ascending colon. Indigenous bacteria preferentially adhered to the intestinal superficial epithelial cells in the mesenterium-attached mucosa (MAM) compared to those in the mesenterium-non-attached mucosa (MNM). Intestinal crypts with indigenous bacteria were also significantly more frequently found in MAM than in MNM. Total epithelial cells, columnar epithelial cells and goblet cells were significantly more abundant in the intestinal crypts with no-indigenous bacteria in MAM (MAM-C) than those in MNM (MNM-C), whereas the columnar epithelial cells were less abundant in MAM-C than in the intestinal crypts with indigenous bacteria in MAM (MAM-C-B). Columnar epithelial cells and goblet cells immuno-positive for proliferating cell nuclear antigen (PCNA) in MAM-C were more abundant than those in MNM-C, but less abundant than those in MAM-C-B. Toll-like receptor (TLR)-2, -4 and -9 were immuno-positive in the striated borders of the intestinal superficial epithelial cells, but their positive intensities were weaker in MAM than in MNM. From these findings, indigenous bacteria were confirmed to preferentially settle on the intestinal superficial epithelium of MAM in the rat ascending colon, and low TLRs-expression might contribute to the preferential settlement of indigenous bacteria in MAM. The increase of proliferating epithelial cells is probably induced by the invasion of indigenous bacteria into the intestinal crypts of MAM.

Immunohistochemical detection of toll-like receptor-2, -4 and -9 in exocrine glands associated with rat alimentary tract.

Localization of Toll-like receptors (TLRs) in the exocrine glands associated with the rat alimentary tract was immunohistochemically studied using anti-TLR antibodies. TLR-2, -4 and -9 were detected in the secretory granules of acinar cells or the luminal substances of the gustatory gland, extraorbital lacrimal gland, Harderian gland, proper gastric gland and pancreas. TLR-2 and -9 were also detected in the mucous acinar cells of the sublingual gland. Positivity for all TLRs was found in the striated borders of columnar epithelial cells and the luminal substances of the intestinal crypts throughout the small intestine, and also in the goblet cells throughout the large intestine. Only TLR-4 was detected in the secretory granules of Paneth cells. A reduction of TLR-4-positive secretory granules and the formation of TLR-4-positive vacuoles were found in the ileal Paneth cells under the hyper-proliferation of indigenous bacteria. In the apical to middle intervillous portions of the ileum, Gram-positive bacterial colonies were significantly more abundant than Gram-negative bacterial colonies, whereas this difference disappeared in the basal intervillous portions. These findings suggest that there are distribution differences in the secretory sources of soluble TLRs that possibly neutralize their luminal ligands, in the rat alimentary tract. Therefore, the bacterial ligand-recognition system composed of the membranous TLRs of villous columnar epithelial cells and soluble TLRs from crypt epithelial cells might contribute to host defense mechanisms for the selective elimination of Gram-positive bacteria rather than Gram-negative bacteria in the rat small intestine.

Immunohistochemical and histoplanimetrical study on the spatial relationship between the settlement of indigenous bacteria and the secretion of bactericidal peptides in rat alimentary tract.

To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.

Immunohistochemical study on the secretory host defense system of bactericidal peptides in rat digestive organs.

To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. ß-defensin1 was detected in the gustatory and extraorbital lacrimal glands. ß-defensin2 was detected in the Harderian glands. In the stomach, ß-defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and ß-defensins were detected in the ascending colon, whereas only ß-defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.