Site differences of Toll-like receptor expression in the mucous epithelium of rat small intestine

Toll-like receptors (TLRs) are known to recognize pathogen-associated molecular patterns and might function as receptors to detect microbes. In this study, the distribution of TLR-2, -4 and -9 were immunohistochemically investigated in the rat small intestine. As a result, TLR-2 was detected in the striated borders of villous columnar epithelial cells throughout the small intestine, except for the apices of a small number of intestinal villi. TLR-4 and -9 were detected in the striated borders of the villous columnar epithelial cells only in the duodenum. TLR-4-immunopositive minute granules were found in the apical cytoplasms of epithelial cells, subepithelial spaces and blood capillary lumina. TLR-2 and -4 were detected in the striated borders of undifferentiated epithelial cells and in the luminal substances of the intestinal crypts throughout the small intestine, but TLR-9 was not detected in the crypts throughout the small intestine. Only TLR-4 was detected in the secretory granules of Paneth cells in both the jejunal and ileal intestinal crypts. These findings suggest that duodenal TLRs might monitor indigenous bacteria proliferation in the upper alimentary tract, that TLR-2 might also monitor the proliferation of colonized indigenous bacteria throughout the small intestine, that the lack of TLR-2 at the villous apices might contribute to the settlement of indigenous bacteria, and that TLR-2 and -4 are secreted from intestinal crypts.     

Pathomorphology of the Intestinal Mucosa in Diarrheic Calves

The intestinal mucosa was examined in twelve 2–5-week-old calves with a spontaneous intestinal disorder, 8 with diarrhea and 4 convalescents. The calves were fed a defined milk replacer. Light microscopy including morphometry, showed villous atrophy and crypt elongation. Villous epithelial cells had decreased height, and epithelial cells of the posterior small intestine contained an increased amount of fat droplets. Accumulation of neutrophils in crypts was frequent. Scanning electron microscopy revealed blunt villi with increased numbers of necrotic cells in the extrusion zone at the tips of the villi. The convalescents had generally milder changes, particularly in the anterior small intestine. The probable etiological factors included a rotavirus and chlamydial infection, and it is concluded that these agents together with other possible noxious influences were responsible for the increased necrobiosis of apical senescent villous epithelial cells, resulting in villous atrophy and crypt hyperplasia.     

Developmental changes in the inner surface structure of the bovine large intestine

The developmental pattern of the bovine fetal large intestine was studied with particular reference to the appearance and decline of the intestinal villi during the fetal period. In the bovine large intestine, the first rudimentary villus and goblet cells were seen in the rectum in a fetus estimated to be 3 months old. By 5-6 months, the goblet cells, absorptive cells in the intestinal crypts, and vacuolated cells in the villi were present along all segments of the large intestine. By 8-9 months, the villi have disappeared from the colon and rectum, epithelial cells no longer contain vacuoles, and absorptive and goblet cell populations are emerging from the crypts. These histological results suggest that development in the bovine large intestine follows a recto-cecal gradient and the most distinct turning point during the fetal period is the first disappearance of fetal villi in the rectum of fetuses estimated to be 7 months old. After this stage, the mucous membrane of the colon and rectum matured rapidly before birth. In contrast, the cecum may seem to require further development in perinatal life.     

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.     

RNA-binding protein HuR promotes growth of small intestinal mucosa by activating the Wnt signaling pathway

Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor–related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3′-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

DIFFERENTIAL LOCALIZATION OF CELL SURFACE AND SECRETORY COMPONENTS IN RAT INTESTINAL EPITHELIUM BY USE OF LECTINS

Sections through various levels of small intestine from adult male rats were examined by fluorescence microscopy after treatment with fluorescein isothiocyanate-labeled lectins from Dolichos biflorus, Lotus tetragonolobus, Ricinus communis, and Triticum vulgare (wheat germ). The latter three lectins reacted with the microvillar portion of the epithelial cells lining the crypts and villi in sections of intestine adjacent to the pylorus. This pattern of reactivity was sharply altered along the first 15 cm of intestine so that in sections distal to this point the luminal surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with the L. tetragonolobus and R. communis lectins, whereas the wheat germ lectin reacted with the surfaces of the cells lining the villi. In sections from the distal end of the small intestine, all three lectins reacted with the surfaces of cells only at the base of the villi and in the crypts. These results show a difference in surface components in cells at various portions on the villi and the dependence of these differences on the region of intestine. The D. biflorus lectin reacted with approximately 25% of the goblet cells at each level of intestine studied whereas the reactivities of the goblet cells with the other three lectins were dependent upon the region of intestine.     

Hypersensitivity reactions in the small intestine. III. The effects of allograft rejection and of graft-versus-host disease on epithelial cell kinetics.

Allograft rejection of fetal intestine and graft-versus-host (GvH) disease have been used to study the effects of cell-mediated immune reactions on epithelial cell kinetics in mouse small intestine. In heterotopically transplanted isografts the cell production rate per crypt was similar to that in normally sited small intestine of the same age. However there was a six-fold increase in the rate of cell production per crypt during allograft rejection and a three-fold increase during GvH disease. Furthermore animals with GvH disease developed villous atrophy and had fewer crypts per villus that littermate controls. At the age of 19 days cell production per villus per hour was 97-5 in animals with GvH disease compared with 54-6 in controls. These results indicate that the pathological entity of 'partial villous atrophy' evolves in two distinct phases. Phase 1, a state of increased cell turnover with crypt hyperplasia but villi of normal length precedes the development of Phase 2, true villous atrophy.     

The distribution of endocrine cells along the mouse intestine: A quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30–60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the botom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3–4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

Pathogenesis of murine astrovirus in experimentally infected mice

Astroviruses are often associated with gastrointestinal diseases in mammals and birds. Murine astrovirus (MuAstV) is frequently detected in laboratory mice. Previous studies on MuAstV in mice did not report any symptoms or lesions. However, little information is available regarding its pathogenicity in immunodeficient mice. Therefore, in this study, we experimentally infected germ-free NOD.Cg-Prkdc^scidIl2rg^tm1Sug/ShiJic (NOG) mice, which are severely immunodeficient, with MuAstV. Germ-free mice were used for experimental infection to eliminate the effects of intestinal bacteria. Mice in each group were then necropsied and subjected to PCR for MuAstV detection, MuAstV RNA quantification in each organ, and histopathological examination at 4 and 28 days post inoculation (DPI). Tissue samples from the small intestine were examined by transmission electron microscopy. No symptoms or abnormalities were detected in any mice during necropsy. The MuAstV concentration was highest in the lower small intestine, where it increased approximately 8-fold from 4 to 28 DPI. Transmission electron microscopy revealed circular virus particles of approximately 25 nm in diameter in the cytoplasm of the villous epithelial cells of the lower small intestine. Histopathological examination did not reveal any abnormalities, such as atrophy, in the intestinal villi. Our results suggest that MuAstV proliferates in the villous epithelial cells of the lower small intestine and has weak pathogenicity.     

HYPERSENSITIVITY REACTIONS IN THE SMALL INTESTINE

Allograft rejection of fetal intestine and graft-versus-host (GvH) disease have been used to study the effects of cell-mediated immune reactions on epithelial cell kinetics in mouse small intestine. In heterotopically transplanted isografts the cell production rate per crypt was similar to that in normally sited small intestine of the same age. However there was a six-fold increase in the rate of cell production per crypt during allograft rejection and a three-fold increase during GvH disease. Furthermore animals with GvH disease developed villous atrophy and had fewer crypts per villus than littermate controls. At the age of 19 days cell production per villus per hour was 97.5 in animals with GvH disease compared with 54.6 in controls. These results indicate that the pathological entity of ‘partial villous atrophy’evolves in two distinct phases. Phase 1, a state of increased cell turnover with crypt hyperplasia but villi of normal length precedes the development of Phase 2, true villous atrophy.     

Transit time of epithelial cells in the small intestines of germfree mice and ex-germfree mice associated with indigenous microorganisms.

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were tested for the rate at which the epithelial cells transited from the crypts of Lieberkuhn to the tips of the villi in their small intestines. The method for estimating the rate of transit of epithelial cells involved the use of liquid scintillation counting to determine the amount of radioactivity entering the cells while the animals were being injected with [3H]thymidine and statistical analysis of th data with a computer program developed for the purpose. As estimated by that method, the cells transited from the crypts to the villous tips in germfree mice in about 115 h and in the associated animals in about 53 h. In monoassociated mice, a strain of a Lactobacillus sp. had no effect on the transit time of the epithelial cells. A strain of Torulopsis pintolopesii stimulated uptake of 3[H]thymidine by the small bowel mucosae in mice monoassociated with the organisms for 5 weeks. In animals monoassociated with the yeast fo 3, 4, and 6 weeks, however, the radioactive compound was incorporated into the bowel mucosae to the same extent as the mucosae of germfree mice. Therefore, similarly to the Lactobacillus strain, T. pintolopesii has no obvious influence on the transit rate of small bowel epithelial cells.     

Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi

Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca(2+) measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca(2+) concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine.     

Use of fetal intestinal isografts from normal and transgenic mice to study the programming of positional information along the duodenal-to-colonic axis.

The four principal cellular constituents of the mouse intestinal epithelium are all derived from a multipotent stem cell functionally anchored near the base of its crypts. Differentiation of enterocytes, enteroendocrine, and goblet cells occurs during an orderly upward migration from monoclonal crypts supplied by a single active stem cell to adjacent polyclonal small intestinal villi or to their colonic homologs, the surface epithelial cuffs. Paneth cells differentiate as they descend to the base of crypts. This epithelium undergoes rapid and perpetual renewal yet is able to maintain cephalocaudal (duodenal-to-colonic) differences in the differentiation programs of its four cell types from the time of its initial cytodifferentiation in late fetal life (embryonic (E) days 16-17). Rat liver fatty acid-binding protein/human growth hormone transgenes (Fabpl/hGH) have been used as novel phenotypic markers to describe the biological properties of gut stem cells and the differentiation programs of their enterocytic and enteroendocrine lineages. To determine whether the multipotent stem cell is able to retain a "positional" address in the absence of luminal signals, we prepared isografts from the proximal small intestine or distal small intestine and colon of E15-E16 Fabpl/hGH transgenic mice and their normal littermates and implanted them into the subcutaneous tissues of young, adult male CBY/B6 nude mice. Immunocytochemical and histochemical studies indicate that appropriate position-specific differences in the differentiation programs of each of the four principal cell lineages are present along the cephalocaudal and crypt-to-villus (or crypt-to-epithelial cuff) axes of isografts harvested 4-6 weeks after implantation. This suggests that the gut stem cell can be characterized not only by its multipotency and enormous capacity for self-renewal but also by its ability to be programmed (? imprinted) with positional information. Transgene expression is reduced in a number of enteroendocrine subpopulations in small intestinal and colonic isografts compared to the intact gut. Moreover, the decision to express the Fabpl/hGH transgene appears to be coordinated between adjacent crypts as evidenced by (i) the presence of multicrypt patches of wholly reporter (hGH)-positive or reporter-negative cells in the intact colon and in colonic isografts and (ii) by the presence of coherent bands of reporter-positive or -negative cells that emanate from adjacent monophenotypic crypts and extend to the apical extrusion zone of distal small intestinal villi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mosaic analysis of small intestinal development using the<Emphasis Type="Italic"> spf</Emphasis><Superscript><Emphasis Type="Italic">ash</Emphasis></Superscript>-heterozygous female mouse

Mosaic analysis using the spf(ash)-heterozygous female mouse was performed to clarify the cell lineage and cell behavior during small intestinal development with special attention given to the villus and crypt formation. The spf(ash) mutation, located on the X-chromosome, causes ornithine transcarbamylase (OTC) deficiency, which leads to mosaic expression of this enzyme in the small intestine of the heterozygous female mouse. In the small intestine in heterozygous fetuses, very small patches, which were aggregates of OTC-positive cells or negative cells, with no definite orientation to the villus structures were observed. In the neonatal small intestine, the intervillus region (the presumptive crypts) was polyclonal, and the majority of crypts were comprised exclusively cells of either genotype in 2-week-old small intestine. These results suggest that extensive migration and cell mixing of small intestinal epithelial cells, which have no definite correlation with the villus formation, occur in fetal stages of development, and that the crypt morphogenesis commences after birth independently of the monoclonality of the epithelial cells. Our data with the mosaic mice also reconfirmed the monoclonality of the adult small intestinal crypts demonstrated in mouse aggregation chimeras.