Vimentin-positive cells in the epithelium of rabbit ileal villi represent cup cells but not M-cells.

Membranous (M)-cells are specialized epithelial cells of the Peyer's patch domes that transport antigens from the intestinal lumen to the lymphoid tissue. Vimentin is a reliable marker for M-cells in rabbits. Using immunohistochemistry (IHC), a subpopulation of epithelial cells has recently been identified in ordinary rabbit ileal villi, which are vimentin-positive and share morphological characteristics with the M-cells of the domes. To test the hypothesis that these cells represent M-cells outside the organized lymphoid tissue, lectin labeling and tracer uptake experiments were performed. Lectins specific for N-acetyl-glucosamine oligomers selectively bound to the vimentin-positive villous cells but not to M-cells in the domes. Microbeads instilled into the ileal lumen were taken up by M-cells within 45 min but not by the vimentin-positive cells in the villi. Lectin-gold labeling on ultrathin sections revealed that the lectin binding sites were located in the brush border and in vesicles in the apical cytoplasm. The vimentin/lectin-positive cells shared ultrastructural characteristics with the so-called "cup cells." We conclude (a) that the vimentin-positive cells in ordinary villi represent cup cells but not M-cells, (b) that they are readily detectable by (GlucNAc)(N)-specific lectins, and (c) that they do not transcytose experimental tracers. Although the specific function of cup cells is still obscure, they most probably represent a cell type distinct from M-cells of the domes with respect to both function and expression of the two new markers.     

Changes in basolateral membrane of villus cells in the rat small intestine at low pH

The basolateral membrane of isolated villus cells of rat small intestine was isolated and was used to investigate the structural changes as well as the simultaneous alterations of its protein pattern at low pH-value. For this purpose the alterations, which occur on the membrane under the influence of HCL, were studied in the electron microscope (negative staining) and by SDS-Polyacryl-amid-Gel electrophoresis. The results show firstly a total disintegration of the membrane and the formation of very uniformly shaped fragments and secondly the absence of protein bands in the fragments in comparison to the protein pattern of the intact membrane. Extraction of more extensive protein bands could not be demonstrated. Possible conclusions concerning the structure of the basolateral membrane and parallels to the reaction of Erythrocyte membrane under the same conditions are discussed.     

The kinetics of villus cell populations in the mouse small intestine

Abstract. The cell population kinetics of the villus epithelium of the mouse have been analysed with respect to the size, flux and time. Microdissection methods were employed to measure the villus cell population size and yielded reproducible, precise results. There was a proximodistal negative size gradient in villus cell population and, in those villi of normal morphology, there was a good correlation with the usual morphometric estimators such as height and row count, although correlation was improved by a product variable consisting of a height multiplied by a width parameter.
Flux onto the villus is the product of the crypt cell production rate, which was measured by a metaphase arrest method using vincristine and crypt microdissection, and the crypt:villus ratio; net villus influx was maximum proximally in the bowel, where the largest villi were found, and decreased distally. The distribution of transit times of labelled cells to the crypt: villus junction and to the villus tip was measured, allowing the measurement of the median villus transit time.
Comparison of the measured villus transit time with the theoretical transit time calculated from the villus influx and population size gave results consistent with a steady state hypothesis. It was found, at each level of the small intestine studied, that the number of epithelial cells on the villus was equivalent to the total number of crypt cells associated with the villus.     

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Summary Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.Supported by a research grant from the Medical Research Council of Canada (J.H.)     

The distribution of (Na+ + K+)-ATPase along the villus crypt-axis in the rabbit small intestine

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na+-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive (Na+ + K+)-ATPase in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent Kd of around 10(-7) M. Both enzyme assay and ouabain-binding measurements suggest that a 2-3-fold increase in the number of Na+-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.     

Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A

Immunoglobulin A (IgA) is the primary immune response induced in the intestine by rotavirus infection, but vaccination with virus-like particles induces predominantly IgG, not IgA. To definitively assess the role of IgA in protection from rotavirus infection, IgA knockout mice, which are devoid of serum and secretory IgA, were infected and then rechallenged with murine rotavirus at either 6 weeks or 10 months. Following primary rotavirus infection, IgA knockout mice cleared virus as effectively as IgA normal control mice. Rotavirus-infected IgA knockout mice produced no serum or fecal IgA but did have high levels of antirotavirus serum IgG and IgM and fecal IgG, whereas IgA normal control mice made both serum IgA and IgG and fecal IgA. Both IgA normal and IgA knockout mice were totally protected from rotavirus challenge at 42 days. Ten months following a primary infection, both IgA normal and knockout mice still had high levels of serum and fecal antirotavirus antibody and were totally protected from rotavirus challenge. To determine if compensatory mechanisms other than IgG were responsible for protection from rotavirus infection in IgA knockout mice, mice were depleted of CD4(+) T cells or CD8(+) T cells. No changes in the level of protection were seen in depleted mice. These data show that fecal or systemic IgA is not essential for protection from rotavirus infection and suggest that in the absence of IgA, IgG may play a significant role in protection from mucosal pathogens.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Accessibility of glycolipid and oligosaccharide epitopes on rabbit villus and follicle-associated epithelium

The initial step in many mucosal infections is pathogen attachment to glycoconjugates on the apical surfaces of intestinal epithelial cells. We examined the ability of virus-sized (120-nm) and bacterium-sized (1-microm) particles to adhere to specific glycolipids and protein-linked oligosaccharides on the apical surfaces of rabbit Peyer's patch villus enterocytes, follicle-associated enterocytes, and M cells. Particles coated with the B subunit of cholera toxin, which binds the ubiquitous glycolipid GM1, were unable to adhere to enterocytes or M cells. This confirms that both the filamentous brush border glycocalyx on enterocytes and the thin glycoprotein coat on M cells can function as size-selective barriers. Oligosaccharides containing terminal beta(1,4)-linked galactose were accessible to soluble lectin Ricinus communis type I on all epithelial cells but were not accessible to lectin immobilized on beads. Oligosaccharides containing alpha(2, 3)-linked sialic acid were recognized on all epithelial cells by soluble Maackia amurensis lectin II (Mal II). Mal II coated 120-nm (but not 1-microm) particles adhered to follicle-associated enterocytes and M cells but not to villus enterocytes. The differences in receptor availability observed may explain in part the selective attachment of viruses and bacteria to specific cell types in the intestinal mucosa.     

The development of thermotolerance in hyperthermal injury to the villus compartment of mouse small intestine

The development and decay of thermotolerance in the villus compartment of the intestinal mucosa of mouse was investigated by giving a primary treatment of 41.5 degrees C for 1 hour (subthreshold for thermal injury) at various intervals before a second, test treatment of 43.0 degrees C for 30 min. The test treatment was given 65 hours after an intraperitoneal injection of 3H-thymidine (i.e. at a time when the heavily labelled cells could be seen to have moved from the crypts on to the upper halves of the villi) and thermal damage assessed by loss of radioactive label. A transient tolerance to the second treatment was induced by the primary treatment. This 'thermotolerance' was maximal 3-13 hours after the first treatment and had decayed by 24 hours. Both the extent and time course of expression and decay of thermotolerance in this post-mitotic functional compartment were very similar to those previously reported for damage to the proliferative epithelium as assayed by crypt loss. This suggests either that the kinetics of thermotolerance are not dependent on the proliferative status of the tissue or that there is a common limiting factor in thermotolerance development, despite the apparent difference between the two mucosal compartments in their susceptibilities to thermal injury.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

Endocrine cells in the rabbit colon mucosal epithelium

The epithelium of the rabbit colon was studied by light and electron microscopy. The highest number of endocrinocytes in colon are observed in terminal parts of colon, i.e. in a distal part of appendix (135 +/- 15 cells/mm2) and in rectum (142 +/- 20), to decrease in the ileocaeal region (caecum proxinmal part--39 +/- 9, colon proximal part--56 +/- 9), where the least number of cells was marked. Agrentaffin cells (EC) number the same way, however, with a weaker difference in the number of cells between terminal departments and ileocaeal region. An electron microscope study of mucosal epithelium of the colon enabled us to identify 5 types of endocrinocytes. I-III types: EC-, D- and L-cells. IV and V are seldom met types, the same way as the "mixed" cells have been indentified. Whose cytoplasm simultaneously contained both mucous and endocrine granules. The received data show a certain degree of similarity in the endocrine apparatus of the rabbit with that of humans, although essential differences exists in regards of the appendix pattern.     

Hemomicrocirculatory bed in the rabbit small intestine wall during ontogenesis

Hemomicrocirculatory bed has been studied in fragments of the small intestine wall, obtained from 30 rabbits of various age. During ontogenesis the number and caliber of all components of the hemomicrocirculatory bed increase, as well as density of the capillary network in connection with formation and growth of the small intestine membranes. At the age of two or three years involutive changes are observed in the blood bed of this organ.