Na+ transport in jejunal crypt cells.

As enterocytes migrate from crypts to villi they differentiate and mature. To examine the effect of epithelial differentiation on ion transport we studied 22Na+ efflux and (Na+--K+)-adenosine triphosphatase activity in suspensions of epithelial cells selectively isolated from different regions of the villus to compare crypt cells with villous tip cells. Enterocytes were isolated from rat jejunum by a dilation-vibration technique. Thymidine kinase, sucrase, and alkaline phosphatase activities were measured as markers of specific cell populations. Compared to villous cells, cells from the crypt region demonstrated lower (Na"--K+)-adenosine triphosphatase activity, lower total and passive Na+ efflux rate constants, and failure of Na+ transport to respond to an actively transported nonelectrolyte.     

Crypt/villus site of substrate-dependent regulation of mouse intestinal glucose transporters.

The intestinal epithelium is in a constant state of turnover, with cells differentiating at the crypts and then migrating toward the tips of the villi. Does substrate-dependent regulation of intestinal Na+/D-glucose cotransporters occur only in crypt cells, or can transport activity be subsequently reprogrammed in mature enterocytes? We used in situ, glucose-protectable specific phlorizin binding to determine site density of brush border glucose transporters in enterocytes fractionated along the crypt/villus axis of mice that were killed shortly after drastic changes in carbohydrate levels of their diets. Dietary carbohydrate-induced changes in site density of specific phlorizin binding initially appeared only in crypt cells before spreading, over the course of several days, to the villus tips. Thus, only crypt cells perceive the signal for glucose transporter regulation, and the observed time lag of diet-induced changes in intestinal glucose uptake is due mainly to cell migration times.     

The Profile of Antioxidant Systems and Lipid Peroxidation Across the Crypt-Villus Axis in Rat Intestine

The distribution of lipid peroxiation and profile of antioxidant-pro-oxidant enzyme systems have been studied in rat intestinal enterocyte across the length of villi. The MDA levels estimated as a measure of lipid peroxidation, under induced or uninduced in vitro conditions, indicated markedly high levels at villus tip cells compared to that in the crypt base. The activities of glutathione-S-transferase and glutathione reductase were three- to sixfold higher in villus tip cells compared to that in the crypt base. However the levels of catalase and superoxide dismutase showed a reverse pattern, being high in the crypt base and lowest in the villus tip region. Feeding coconut oil, sunflower oil, or groundnut oil did not modify the distribution pattern of these systems across crypt-villus unit in rat intestine. These findings suggest that the large amount of free radicals generated in villus tip cells may be responsible for the release of enterocytes from the villus tip as a consequence of apoptosis.     

Human immunodeficiency virus infection of enterocytes and mononuclear cells in human jejunal mucosa

Intestinal malabsorption is a recognized cause of malnutrition in patients infected with human immunodeficiency virus. However, the relationships among human immunodeficiency virus infection, morphological changes in the intestine, and development of intestinal malabsorption are not well established. Nine patients infected with human immunodeficiency virus underwent tests of intestinal absorption and jejunal biopsies for morphometric measurements, enzyme assays, and virus detection by in situ hybridization. Steatorrhea and low lactase activities were found in more than 85% of the patients. All biopsy specimens were abnormal with reversal of the ratio of villus length to crypt depth in seven and enlarged enterocyte nuclear size in nine. Human immunodeficiency virus was detected in five jejunal biopsy specimens, within villus enterocytes of one patient who had the most severe malabsorption of the group and in four other biopsy specimens in mononuclear infiltrating cells of the lamina propria. These results suggest that human immunodeficiency virus infection of the small intestinal mucosa is an early event that is associated with altered enterocyte differentiation and function.     

Epithelial cell renewal and turnover and relationship to morphologic abnormalities in jejunal mucosa in tropical sprue

A morphometric study confirmed that increasing severity of the jejunal mucosal morphologic lesion is accompanied by increased crypt height and reduced villus length in patients with tropical sprue. Mitotic activity in the crypts was increased. Pulse labeling of jejunal mucosal biopsies cultured in vitro with [3H]thymidine confirmed that there was increased uptake of label in tropical sprue with more rapid migration of labeled cells to the villi. The label was also lost more rapidly. Earlier ultrastructural studies have shown enterocyte damage and extrusion in the crypt and villus epithelium. The present data suggest that in the jejunal mucosa of patients with tropical sprue, the loss of damaged enterocytes leads to villus shortening and increased cell production in the crypts, with hypertrophy of the crypts.     

Characterization of isolated duodenal epithelial cells along a cryptvillus axis in rats fed diets with different iron content

The intestinal mucosa is characterized by cell proliferation, commitment, differentiation, digestion and absorption. These processes occur at specified locations along the crypt to villus axis. A technique is reported for the isolation of cells along this axis which allows the study of any one of these processes in an enriched population of cells. As an example, the uptake of transferrin-bound iron by enterocytes was studied. Rats were fed diets normal, high (3% carbonyl iron) or low in iron for 12 days. Cells from either the duodenum or ileum were isolated by incubating in a Ca²⁺-, Mg²⁺-free, cation chelating solution for varying periods. The incorporation of thymidine into DNA was measured in these cells as a marker of the crypt region, while alkaline phosphatase and sucrase activities marked mature enterocytes. The in vivo uptake of transferrin-bound ⁵⁹Fe was measured in cells isolated either 2 or 4 h after intravenous injection. This procedure resulted in the isolation of 10 fractions of viable cells. Earlier fractions were enriched at least 10-fold in villus cells and the last fractions in crypt cells. Cells in intermediate fractions were at various stages of development. Uptake of transferrin-bound iron into enterocytes was highest with feeding an iron-loaded diet compared with control or iron-deficient diets. However, with all diets uptake was highest in crypt cells and this fell at the crypt-villus junction to be only 25%, as high at the villus tip as the crypt. A technique for the reproducible isolation of viable enterocytes along a crypt-villus axis is described. Transferrin receptor activity changes with maturation of the enterocyte.     

Effects of Protein Malnutrition on Experimental Giardiasis in the Mongolian Gerbil

To determine the effects of protein malnutrition on the severity and duration of infection with Giardia lamblia, Mongolian gerbils were pair-fed a pelleted control (C) diet (20% protein) and a low-protein (.5%; LP) diet for 3 weeks before and after being infected with 100,(MM) cysts orallv. Weight loss, fecal fat, enteropooling. and the duration of cyst excretion were all greater in the infected LP than in the infected C animals. During peak infection the upper intestinal intraepithelial lymphocyte infiltration, crypt enlargement, and villus enterocyte migration were greater in C than in LP animals, as was the villus mast cell number at the end of the infection. It is concluded that in the protein-malnourished host the increased severity of Giardia infection correlates with a reduction in enterocyte production and migration, probably secondary to a reduced lymphocyte infiltration, and the increased infection duration correlates with blunted mast cell migration into affected villi.     

Differentiation status of rat enterocytes after intestinal adaptation to jejunoileal bypass.

The differentiation status of epithelial cells in intestinal adaptation remains unclear. To determine whether enterocytes reach optimum maturity following adaptation after 85% shortening of the rat gut by jejunoileal bypass surgery, activities of two brush border enzymatic markers of differentiation, alkaline phosphatase and sucrase, were examined in subpopulations of epithelial cells isolated sequentially from the villus/crypt axis of normal (sham operated) and hyperplastic mucosa. In jejunal villi, adaptational hyperplasia was associated with an increase in total epithelial alkaline phosphatase, but not total sucrase, activity; alkaline phosphatase activity increased most obviously in cells at the 11-50% position (from the tip) on villi. In hyperplastic ileal villi, total alkaline phosphatase activity fell, although sucrase activity did not change significantly. Specific activity (per mg protein) of sucrase on jejunal villus epithelium was reduced by the adaptational changes to bypass; alkaline phosphatase specific activity remained unchanged. In the ileum, despite adaptational changes to bypass, there was no increase in the normally low specific activities of sucrase and alkaline phosphatase. Bypass surgery did not change the major site of expression of either enzyme on jejunal or ileal villi. In conclusion, enzymatic markers of functional differentiation are not all equally affected by adaptational hyperplasia. Hypertrophy of villi and increased cell proliferation seen in jejunum remaining exposed to luminal contents resulted in an increase in the alkaline phosphatase but not the sucrase content. This is not, therefore, the result of a simple immaturity of villus cells. Morphological adaptation in the ileum, however, is not accompanied by adaptation of brush border enzyme markers of differentiation, confirming a functional immaturity of these cells. Strategies for increasing the expression of these markers may have clinical value.     

High-dose erythropoietin inhibits apoptosis and stimulates proliferation in neonatal rat intestine.

BACKGROUND: Erythropoietin (Epo) receptors are widely expressed in the small bowel of neonatal rats and evidence suggests Epo has important trophic effects in developing bowel. OBJECTIVE: To compliment in vitro data, we directly examine in vivo the hypotheses that systemic Epo treatment can promote cell division and enterocyte migration, and arrest apoptosis in the ileum of neonatal rats. DESIGN: Epo (5000 U/kg s.c.) or vehicle treatments were given to one week old Sprague-Dawley rats (n = 86) along with timed injections of the thymidine analog 5-bromo-2-deoxyuridine (BrdU, 50mg/kg s.c.) to label DNA synthesis and track newly proliferating cells. To characterize the time course of effects, animals were killed at scheduled times from 30 min to 24 h after treatment. BrdU-containing cells were immunostained and counted in intestinal crypts, villi, and muscle wall of ileum. Effects of Epo on apoptosis were analyzed by TUNEL staining. Calibrated measurements were made to determine the density or relative proportion of BrdU- and TUNEL-positive cells. RESULTS: Systemic high-dose Epo promoted cell division in intestinal smooth muscle and enterocytes, stimulated migration of intestinal epithelial cells, and arrested apoptosis of enterocytes at the villous tips. CONCLUSION: These data provide in vivo evidence that Epo functions trophically in developing intestine tissues.     

Alterations in quantitative distribution of Na,K-ATPase activity along crypt-villus axis in animal model of malabsorption characterized by hyperproliferative crypt cytokinetics

The distribution of sodium- and potassium-stimulated ATPase (Na, K-ATPase) along the crypt-villus axis and crypt cytokinetics were examined in an infective model of celiac disease produced by infection of the rat with the nematodeNippostrongylus brasiliensis. In controls, levels of enzyme activity remained stable during enterocyte migration to the villous apex. In the jejunum of infected rats, the structural lesion of villous atrophy and crypt hyperplasia, observed at day 10 of infection, was associated with a three-dimensional expansion of the crypts. Cell cycle time was shortened and this resulted in a markedly increased crypt cell production rate. Enterocytes emerged from the crypts at a faster rate, and this functional immaturity was paralleled by decreased Na, K-ATPase activity. Further decreases in enzym levels were observed during enterocyte migration along the villi. This may reflect enterocyte damage or increased enzyme turnover. In the ileum of these animals, enterocyte maturation was prolonged and enzyme activity was increased at the level of the crypt villus junction with further increases noted during enterocyte transit. These changes in ileal Na, K-ATPase appear to be adaptive.This study was presented in part at the Annual Meeting of the American Gastroenterological Association, Chicago, May 12, 1987) and published in abstract from (Gastroenterology 92:1695, 1987).These studies were supported by a grant from the Medical Research Council of Canada.     

Small intestinal epithelial cell kinetics and protozoal infection in mice

The effects of chronic protozoal infection on small intestinal architecture have been examined in mice, infected with Giardia muris and Hexamita muris. Techniques used were conventional histology, quantitation of intraepithelial lymphocytes, microdissection and measurement of individual villi and crypts, and epithelial cell kinetic studies. The histology of small intestine from infected mice appeared normal apart from the intraepithelial lymphocyte numbers. Mean intraepithelial lymphocyte counts in two groups of uninfected mice were 11.6 and 13.6 per 100 epithelial cells, and in two groups of infected mice were 17.6 and 21.8. Dynamic studies showed that protozoal infection doubled the cell production per crypt per hour from mean values of 6.2, 7.3, and 8.2 in three groups of uninfected animals, to 11.8, 13.4, and 17.1 in groups of chronically infected mice. Cell production per villus was also influenced by protozoal infection, with values of 93, 99, and 101 cells per hr in groups of uninfected animals whereas in infected mice the values were 155, 162, and 180 cells per hr. Although there was no reduction in villus height in the infected animals, radioautography using [3H]thymidine confirmed that the enterocytes moved rapidly up the sides of the villi than was the case for uninfected mice.     

Initial kinetic changes of prostaglandin E2-induced hyperplasia of the rat small intestinal epithelium occur in the villous compartments

This study was performed to further identify the sequence of cell kinetics that occurs in the development of gastric and intestinal epithelial hyperplasia after orally administered prostaglandins of the E series. A high-dose, short-treatment schedule was used to examine the initial effects on kinetic parameters in the rat small intestinal epithelium. Groups of rats were killed after a single dose of oral prostaglandin E2 at 1 h after in vivo labeling with [methyl-3H]thymidine and during continued treatment at 6, 12, 24, 48, 72, and 96 h. As evidenced by autoradiography, the earliest change produced by prostaglandin E2 was an increased cellularity of the villous compartment (p less than 0.05 after 24 h). There was no change of labeling index of the villous compartment or of the leading edge of labeled cells within 24 h. At 48 h, the increased cellularity was accompanied by a significantly elevated labeling index of the villi. Throughout the study period no significant differences were observed between groups in the number of cells or labeling indices in the jejunal crypts, or in cellular input from the crypts to the villi. Epithelial turnover time in the placebo and treatment groups was 69 and 71 h, respectively. To exclude the possibility that prostaglandin E2 initially affects cell birth rate and mean cell cycle time, a metaphase blocker was given after 4 days of treatment in a second study. Animals were killed after 0, 0.5, 1.0, 1.5, and 2.5 h. The rate of entry into mitoses was 8.1% cells/h in controls compared with 8.2% cells/h in treated rats. The distribution of mitoses within crypts was identical in the two groups and the mean cell cycle time was 13.6 and 13.2 h, respectively. Also in this study there were trophic changes of the villi. It is concluded that the hyperplasia produced by oral prostaglandin E2 starts in the villi of the small intestine and is initiated by reduced cell exfoliation from the villous tips. Previously recorded retention of cellular elements in villi and crypts, increased cellularity of the proliferative compartments, and reduced mitotic index are secondary events.     

Ontogenic and phylogenic studies of intestinal, hepatic, and placental alkaline phosphatases. Evidence that intestinal alkaline phosphatase is a late evolutionary development

We have studied the intrauterine development of guinea pig and rat organ phosphatases using biochemical, immunologic, and histologic techniques. In all organs tested the features of the adult phosphatase activity were achieved during the second or third gestational phases. In the rat, the tissue-unspecific phosphatase activity was found by immunoprecipitation with antiserum to rat liver phosphatase in all gestational phases in liver and placenta. The high liver activity in the first phase of gestation corresponded to hematopoietic cells. Hepatocyte phosphatase did not appear until the second gestational phase. The tissue-unspecific phosphatase activity was found in the first and second gestational phase in surface intestinal epithelial cells, even after crypt formation occurred. Once phosphatase appeared in enterocytes on villi, only the intestinal-type enzyme was detected. Alkaline phosphatase was measured in the liver and intestine from animals in various phyla. In fish and reptiles, the intestinal activity had the enzymatic characteristics of the tissue-unspecific enzyme. The appearance of intestinal alkaline phosphatase with unique properties and high specific activity is a characteristic of mammals.     

The effects of glutamine on intestinal epithelial cell proliferation in parenterally fed rats

BACKGROUND: Several papers have indicated that glutamine is a preferred fuel for the enterocyte and that it can increase intestinal epithelial cell proliferation. AIMS: To investigate the effects of glutamine on intestinal epithelial cell proliferation in the parenterally fed rat. METHODS: Five groups of six rats were fed parenterally; a group of orally fed rats was also studied. Crypt cell proliferation was studied after six days using native mitoses in microdissected crypts and bromodeoxyuridine labelling. RESULTS: No effect of treatment was seen on intestinal weight; however, the weights of the small intestine, caecum, and colon were all significantly heavier in the orally fed group than in the total parenteral nutrition groups (p<0.001). There was no effect of any of the glutamine treatments on mitotic activity in the small intestine. In the colon there was a small increase in native mitoses with glutamine (p=0.03). There was also an indication of increased proliferative activity in the first fifth of the small intestine and colon with glutamine. Little effect of glutamine on bromodeoxyuridine labelling in either site was observed, but there was a small but significant reduction in growth fraction of the colon of the glutamine treated group. The labelling distribution curve from sections and the mitotic distribution curve obtained from crypt squashes showed a good correlation. CONCLUSION: Glutamine has a small, but significant effect on mitotic activity but only in the colon. Modest effects on the distribution of labelled cells were also seen.     

Intestinal mucosal injury is associated with mast cell activation and leukotriene generation duringNippostrongylus-induced inflammation in the rat

We examined mucosal injury in the jejunum of the rat during infection with the nematode parasite, Nippostrongylus brasiliensis (Nb). Injury was documented morphologically (increase in crypt length with or without villus atrophy) and biochemically (activities of digestive or proliferative enzymes) and related to mast cell activation and leukotriene generation. At day 4 crypt length and thymidine kinase activity were increased; no changes in villus parameters were recorded. No evidence of mast cell activation was found and leukotriene levels in the mucosa were normal. At day 7, the gut was acutely inflamed and edema was present at the tips of the villi. This progressed to enterocyte detachment, resulting in villus atrophy with decreased activities of brush border enzymes. At this stage mucosal histamine was decreased and rat mast cell protease II (RMCP II) was increased in serum, indicating mast cell activation. In addition, mucosal leukotrienes (LTB4, LTC4, LTE4) were present in significant quantities. Following worm expulsion, the villus abnormalities resolved and serum RMCP II returned to normal. However, the crypt hyperplasia persisted. Our results suggest that during Nb infection at least two components of injury can be identified. One component, epithelial injury at the villus tips, may be related to activation of mucosal mast cells.     

Hypersensitivity reactions in the small intestine. 2. Effects of allograft rejection on mucosal architecture and lymphoid cell infiltrate.

Small intestinal mucosa contains both thymus dependent and thymus independent lymphoid cells and thus has the capacity to act via humoral and cellular mechanisms as a site of local immunity and local hypersensitivity. Allograft rejection of mouse small intestine is a model of a local cell mediated reaction. The effects of this clearly defined, immunologically mediated damage villi, crypts, enterocytes, and lymphoid cell infiltrate have been assessed by comparing the morphology of rejecting allografts with that of isografts and normal small intestine of the same age. In rejection there is infiltration of the lamina propria with lymphocytes, hyperplasia of the crypts of Lieberkuhn, and an eventual sloughing off of the mucosa. Usually, but not always, there is villous atrophy and increased numbers of intraepithelial lymphocytes. However, the morphology of individual enterocytes remains normal throughout rejection and neither plasma cells nor polymorphonuclear leucocytes infiltrate the lamina propria before mucosal ulceration. These results show unequivocally that a local T cell mediated immune response causes villous atrophy and crypt hyperplasia in this animal model, and since there is no evidence of local enterocyte cytotoxicity, a lymphokine may be the link between the activated T cell and the effects on mucosal architecture. We suggest that a local CMI reaction may be the cause of villous atrophy, crypt hyperplasia, and malabsorption in many clinical and experimental conditions, including coeliac disease, food allergy, and intestinal infections.     

Changes in the structure and regeneration mode of the rat small intestinal mucosa following benzalkonium chloride treatment

Tritiated thymidine was administered IP to rats that had been exposed to benzalkonium chloride in the duodenum, jejunum, and ileum, resulting in neuronal ablation. Epithelial cell proliferation and migration were studied 21 and 7 days after treatment. Significant hyperplasia and hypertrophy of the villi and crypts was seen from day 7 on. This was half as pronounced as that of the muscle layer, whose maximal percent increase was not seen until day 21. In the crypt, the proliferation had increased significantly (65% 3H index corrected) and its zone had expanded proportionally to the total crypt depth. After an average of 36 hours in the ileum (48 hours in normal rats), labeled cells reached the tip of the lengthened villi, reflecting significantly accelerated migration. Concerning the distributional pattern of the labeled cells in the crypt, a nonsignificant shift to the lower two thirds of the crypt could be distinguished. From this the author concludes that treatment with benzalkonium chloride influences the proliferation and migration of the epithelial cells in the treated area. These alterations may result from loss of the myenteric plexus, but other factors cannot be excluded.