Transient expression of M-cell phenotype by enterocyte-like cells of the follicle-associated epithelium of mouse Peyer's patches

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) over mucosa-associated lymphoid tissues consists of distinct enterocytes and M cells concentrated at its periphery. The basement membrane composition was analyzed to test whether differences account for the distinct differentiation programs along the crypt-villus and crypt-FAE axes. To determine whether the decreased number of M cells in the FAE apex is caused by premature extrusion, we mapped the site where they undergo apoptosis. METHODS: The FAE basal lamina of Peyer's patches from BALB/c mice was analyzed by immunochemistry. M cells were identified using the Ulex europaeus agglutinin lectin. The cell proliferation and apoptotic compartments were characterized using bromodeoxyuridine incorporation and the TUNEL assay. RESULTS: The perlecan and laminin 2 stainings were different in FAE and villi. Myofibroblasts were absent beneath the FAE. The migration kinetics of cells along the FAE was similar to that along the villi. Apoptotic cells were detected exclusively at the apex of the FAE. CONCLUSIONS: FAE and M-cell differentiation is associated with a distinct basal lamina composition. FAE enterocytes express transient M-cell features as they move from the crypts toward the apoptotic compartment. M cells have a highly plastic phenotype that raises interesting questions about the control of intestinal epithelial cell differentiation.     

Chicken cathelicidin-B1, an antimicrobial guardian at the mucosal M cell gateway

Mucosal epithelial M cells provide an efficient portal of entry for microorganisms. Initially defined by their irregular microvilli and abundant transcytotic channels in the avian bursa of Fabricius, M cells also are found in the lymphoid follicle-associated epithelium of the mammalian appendix, Peyer's patches, and other mucosal surface-lymphoid interfaces. We describe here a previously unrecognized cathelicidin gene in chickens, chCATH-B1, that is expressed exclusively in the epithelium of the bursa of Fabricius. Like the mature peptides of previously identified cathelicidins, the carboxyl-terminal peptide of chCATH-B1 has broad antimicrobial activity against Gram-positive and Gram-negative bacteria. chCATH-B1 expression is restricted to the secretory epithelial cell neighbors of the M cells, whereas its mature peptide is transported to become concentrated on the fibrillar network surrounding basolateral surfaces of the M cells that overlie the bursal lymphoid follicles. We conclude that chCATH-B1 is well placed to serve a protective antimicrobial role at the M cell gateway.     

Crypt alterations and collagen deposition in hyperplastic polyps of colorectum

In order to investigate the nature of hyperplastic polyps in the colorectum, 44 longitudinally sectioned crypts from biopsied polyps were analyzed morphometrically and compared with 81 control crypts. Although the crypts in hyperplastic polyps were longer and wider, containing more cells, their cell density was less, particularly in the serrated epithelium. In these crypts, both the tall and short epithelial cells contained cytoplasmic vacuoles, even in the surface epithelium. These cells exhibited increased expression of carcinoembryonic antigen. The subepithelial collagen table was of similar thickness in the polyp and control colonic mucosa, but it extended down along the cryptal wall to a greater depth in the polyp. These and other data indicate an aberrant differentiation of cryptal epithelial cells in the polyp. On upward migration to the surface, these cells appeared to undergo an arrested maturational process. Hence, the hyperplastic polyp may be considered a disease of epithelial cell differentiation.     

Remodeling of the intestine during metamorphosis of Xenopus laevis

Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the Xenopus laevis tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers undergo programmed cell death. We conclude that muscle remodeling and contraction play key roles in the shortening process. Shortening is accompanied by a temporary "heaping" of the epithelial cells into many layers at climax. Cells that face the lumen undergo apoptosis. By the end of metamorphosis, when the epithelium is folded into crypts and villi, the epithelium is a single-cell layer once again. Throughout this remodeling, DNA replication occurs uniformly throughout the epithelium, as do changes in gene expression. The larval epithelial cells as a whole, rather than a subpopulation of stem cells, are the progenitors of the adult epithelial cells.     

Glycoprotein 2 (GP2): grabbing the FimH bacteria into M cells for mucosal immunity

Membranous (M) cells are specialized epithelial antigen-transporting cells scattered in the follicle-associated epithelium covering the gut lymphoid follicles such as Peyer's patches. Although the importance of M cells as a main portal for luminal antigens has long been recognized, molecular mechanisms for M-cell antigen uptake has remained largely elusive. We have recently found that glycoprotein 2 (GP2) is exclusively expressed on M cells among intestinal epithelial cells and serves as an uptake receptor for a subset of commensal and pathogenic bacteria. GP2 interacts with FimH, a major component of the type 1 pilus on the outer membrane of a subset of gram-negative enterobacilli such as E. coli and Salmonella enterica. Furthermore, GP2-FimH interaction is necessary for efficient uptake of FimH(+) bacteria by M cells and subsequent bacteria-specific mucosal immune responses. Pancreatic GP2 may also be involved in innate immunity by 'opsonization' of FimH(+) bacteria to facilitate their egestion in feces as well as translocation across the intestinal epithelium.     

Pathologic findings from intestinal biopsy specimens in human cholera

A histopathologic study of the jejunal biopsy specimens obtained from 6 patients with cholera was carried out. The cultures of their rectal swabs were all positive forVibrio cholerae. These studies demonstrated pathologic desquamation and necrosis of intestinal villi as well as degenerative changes. It was clarified that epithelial desquamation in cholera was not due to postmortem autolysis nor terminal vascular collapse. Abundant mucus was found in the lumens of the crypts, and the intestinal villi were coated with mucus. The authors wish to emphasize that rice watery stool is due to multiple pathologic desquamation and necrosis of intestinal epithelium, as well as to hypersecretion of epithelial cells and excessive production of mucus in the crypts.     

Immunohistochemical localisation of intercellular adhesion molecule-1 in follicle associated epithelium of Peyer's patches.

The epithelium covering domes of lymphoid follicles in Peyer's patches includes membranous cells, which are sites of entry for various macromolecules, absorptive cells, a few goblet cells, and many migrating lymphoid cells. The mechanism of migration of lymphoid cells into the follicle associated epithelium of lymphoid follicles in Peyer's patches is still unknown. This study investigated the relation between localisation of intercellular adhesion molecule-1 (ICAM-1) and the follicle associated epithelium of Peyer's patches immunohistochemically. It was found that subepithelial fibroblasts expressed ICAM beneath the follicle associated epithelium on lymphoid follicles, but not on surrounding villi. The results show that the massive lymphocytic traffic between follicle associated epithelium and lymphoid follicles may be related to ICAM-1 expression.     

Characteristic expression of γ-aminobutyric acid and glutamate decarboxylase in rat jejunum and its relation to differentiation of epithelial cells

AIM: To investigate the expression between γ-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum.METHODS: Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms,GAD65 and GAD67) was investigated in rat jejunum.Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore,evaluation of cell kinetics in jejunum was conducted by 3Hthymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA).RESULTS: The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells.3H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone.CONCLUSION: The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.     

Characteristic expression of γ-aminobutyric acid and glutamate decarboxylase in rat jejunum and its relation to differentiation of epithelial cells

AIM: To investigate the expression between γ-aminobutyric acid (GABA) and glutamate decarboxylase and its relation with differentiation and maturation of jejunal epithelial cells in rat jejunum.METHODS: Immunohistochemical expression of GABA and glutamate decarboxylase (GAD, including two isoforms,GAD65 and GAD67) was investigated in rat jejunum.Meanwhile, double staining was performed with GAD65 immunohistochemistry, followed by lectin histochemistry of fluorescent wheat germ agglutinin. Furthermore,evaluation of cell kinetics in jejunum was conducted by ^3Hthymidine autoradiography and immunohistochemistry using a monoclonal antibody to proliferating cell nuclear antigen (PCNA).RESULTS: The cells showing positive immunoreactivity GABA and GAD65 were mainly distributed in the villi in rat jejunum, while jejunal epithelial cells were negative for GAD67. Positive GABA or GAD65 staining was mainly located in the cytoplasm and along the brush border of epithelial cells in the middle and upper portions. In addition, a few GABA and GAD65 strongly positive cells were scattered in the upper two thirds of jejunal villi. Double staining showed that GAD65 immunoreactivity was not found in goblet cells.^3H-thymidine-labeled nuclei were found in the lower and middle portions of jejunal crypts, which was consistent with PCNA staining. Therefore, GABA and GAD65 were expressed in a maturation or functional zone.CONCLUSION: The characteristic expression of GABA and GAD suggests that GABA might be involved in regulation of differentiation and maturation of epithelial cells in rat jejunum.     

Studies on translocation of immunoglobulins across intestinal epithelium. IV. Evidence for binding of IgA and IgM to secretory component in intestinal epithelium.

We conducted studies concerning the issue of whether secretory component (SC) is a specific receptor on intestinal epithelial cells for IgA and IgM. Initially, frozen sections of human intestinal mucosa were incubated with dimeric monoclonal human IgA, conjugated to horseradish peroxidase; adjacent sections were reacted with peroxidase-conjugated antibodies to SC. The conjugated IgA and anti-SC bound to similar sites in the epithelium, that is to basolateral margins and supranuclear cytoplasm of columnar epithelial cells, principally in gland crypts. In subsequent tests of binding specificity, binding of the dimeric IgA conjugate was inhibited by pretreating the tissues with unconjugated dimeric IgA or 19S IgM, pretreating the tissues with unconjugated antibodies to SC, or preincubating the dimeric IgA conjugate with free SC. Binding was not inhibited or only partially inhibited by pretreating the tissues with monomeric IgA or IgG, pretreating the tissues with antibodies to human or heterologous immunoglobulins, or preincubating the dimeric IgA conjugate with 11S secretory colostral IgA. The findings indicate that dimeric IgA and 19S IgM are capable of binding in vitro to specific sites on intestinal epithelial cells, most likely to SC. This supports the hypothesis that transport of these immunoglobulins into intestinal fluids involves their combination with SC in the epithelium.     

Glycoprotein 2 (GP2)

Membranous (M) cells are specialized epithelial antigen-transporting cells scattered in the follicle-associated epithelium covering the gut lymphoid follicles such as Peyer's patches. Although the importance of M cells as a main portal for luminal antigens has long been recognized, molecular mechanisms for M-cell antigen uptake has remained largely elusive. We have recently found that glycoprotein 2 (GP2) is exclusively expressed on M cells among intestinal epithelial cells and serves as an uptake receptor for a subset of commensal and pathogenic bacteria. GP2 interacts with FimH, a major component of the type 1 pilus on the outer membrane of a subset of gram-negative enterobacilli such as E. coli and Salmonella enterica. Furthermore, GP2-FimH interaction is necessary for efficient uptake of FimH⁺ bacteria by M cells and subsequent bacteria-specific mucosal immune responses. Pancreatic GP2 may also be involved in innate immunity by 'opsonization' of FimH⁺ bacteria to facilitate their egestion in feces as well as translocation across the intestinal epithelium.     

Use of transgenic mice to infer the biological properties of small intestinal stem cells and to examine the lineage relationships of their descendants.

Transgenes, composed of elements of the 5' nontranscribed region of the liver fatty acid-binding protein (L-FABP) gene linked to various reporters, have previously been used to explore the cellular, regional, and temporal differentiation of the mouse intestinal epithelium. In this report, we have analyzed a pedigree of L-FABP/human growth hormone (hGH) transgenic mice that display a stable, heritable, mosaic pattern of reporter expression: wholly hGH-positive or hGH-negative populations of differentiating enterocytes arise from hGH-positive or hGH-negative crypts, respectively, and migrate as vertical coherent bands up the villus producing striped (polyclonal) villi. The ability of enteroendocrine cells within a given villus stripe to support hGH expression coincides with the enterocytic reporter phenotype, suggesting that these two terminally differentiated cells arise from a common multipotent stem cell. hGH-negative crypts are nonrandomly distributed around each villus and their frequency increases along the duodenal-to-ileal axis. Statistical analysis of the observed villus striping pattern suggests that transgene expression is not independently determined in individual crypts but rather in multicrypt "patches." The intact endogenous mouse L-FABP gene (Fabpl) exhibits a similar striped villus pattern of expression in a portion of the distal small intestine. These studies indicate that Fabpl and L-FABP/hGH transgenes represent sensitive markers for exploring the biological properties of gut stem cells and how positional information is encoded in this rapidly and continuously renewing epithelium.     

Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection

Defining molecular interactions that occur at the interface between "normal" and "abnormal" cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to "control" crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.     

Lymphocyte-filled villi: Comparison with other lymphoid aggregations in the mucosa of the human small intestine

Background & Aims: Solitary lymphoid structures that may be sites of primary extrathymic T-cell differentiation have been described recently in murine (cryptopatches) and rat (lymphocyte-filled villi) small intestine. This study tests the hypothesis that similar structures occur in human small intestine. Methods: Normal small intestine was obtained during surgery. Fixed tissue was examined histologically, and frozen sections were examined by an indirect immunoperoxidase technique using a panel of mouse monoclonal antibodies. Results: A new isolated lymphoid structure, with epithelium resembling follicle-associated epithelium of Peyer's patches, is described as a lymphocyte-filled villus. These structures contain major histocompatibility complex (MHC) class II–positive dendritic cells, a majority of memory T cells, a variable B-cell component, and no evidence of immature lymphocytes that express either c-kit or CD1a. Two previously described lymphoid aggregations (isolated lymphoid follicles and submucosal lymphoid aggregations) are components of a single structure. The complete structure contains a B-cell follicle, T cells with mainly memory (CD45RO-positive) phenotype, high endothelial venules, and no detectable population of immature lymphocytes. Conclusions: A new solitary lymphoid structure is described in the human small intestine. Neither these structures nor isolated lymphoid follicles appear to be similar to solitary primary lymphoid structures in rodent intestine.GASTROENTEROLOGY 1998;115:1414-1425     

Morphometric evidence against lymphocyte-induced differentiation of M cells from absorptive cells in mouse Peyer's patches

As the result of our morphometric study of mouse follicle-associated epithelium, we obtained evidence against the hypothesis that absorptive cells of this epithelium, under the influence of contact with intraepithelial lymphoid cells, transform into cells with short and sparse microvilli--M cells. The evidence consists of the following: (a) lack of correlation was demonstrated between the length (or density) of microvilli of absorptive cells and the number of lymphoid cells in contact with a given absorptive cell; (b) no intermediate forms between absorptive cells and M cells were found to exist; (c) the proportion of M cells among the epithelial cells in the follicle-associated epithelium was not significantly reduced in mice housed under specified pathogen-free conditions as compared with that in conventionally housed mice (9% and 11%, respectively), despite almost twofold depletion of the intraepithelial lymphoid cells in specified pathogen-free mice.     

Morphologic changes in ileal reservoir mucosa after long-term exposure to urine. A study in patients with continent urostomy (Kock pouch)

In patients with supravesical urinary diversion, continent ileal reservoirs utilized for urinary collection were examined by endoscopy at intervals of 1 month to 9 years after surgical construction, and biopsy specimens were obtained for light microscopy and morphometry. The gross appearance of the mucosa showed alterations in the shape of the shorter and broader finger-like villi during the first postoperative month to the subsequent very low ridges and convolutions or, in some instances, flat mucosa devoid of individual villi. Starting between 1 and 3 years after the surgical construction, endoscopically smooth areas were encountered in caudal areas of the reservoir, and the areas were mixed with islands of villous mucosa. Microscopically and morphometrically, the specimens from villous areas confirmed reduction in villous height and increase in crypt depth, whereas no changes were seen in the epithelial mitotic frequency. The number of mucus-storing goblet cells increased already within 1 month after construction. Specimens obtained from smooth areas showed alterations in the intestinal structure, with reduction of crypts, decreased height of epithelial cells, and occasional epithelial denudation. No signs of fibrosis, foreign-body reaction, or dysplasia were encountered. The constant exposure to urine leads to adaptive changes of the reservoir mucosa, resulting in a true atrophy of villi, crypts, and individual epithelial cells.     

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.     

Localization of carbonic anhydrase activity in the developing rat colon

Carbonic anhydrase activity was localized histochemically by light and electron microscopy in the proximal and distal colon of developing rats. Fixed tissue was taken for normal morphology and carbonic anhydrase localization from fetal (20-22 days gestation), suckling (1-19 days postnatal), weanling (20-25 days postnatal), and adult rats. The proximal colon had distinct villi at birth which were diminished between days 5 and 11 postnatally. The distal colon lacked villi at birth but had rudimentary crypts (ridges and furrows) which were replaced during the suckling period by a flat mucosa interspersed with true crypts. Carbonic anhydrase first appeared in both proximal and distal colonic epithelial cells on the day of birth (22 days gestation). Goblet cells were nonreactive at each developmental period. In neonatal rats, epithelial cells in the upper half of the villi of the proximal colon and on the surface and upper crypts of the distal colon were positive for carbonic anhydrase throughout the cytoplasm. Cells at the villar base (proximal colon) or in the deep crypt (distal colon) had reaction product in the intercellular spaces but not the cytoplasm. By 11 days postnatal, cytoplasmic reaction product was present in proximal colonic cells in the upper three-fourths of the crypt and was concentrated in a heavy band in the apical cytoplasm. In the distal colon, cytoplasmic positive cells did not extend as deeply into the crypts and the apical banding pattern was weak. Intercellular spaces in the deeper crypt epithelium were positive in both proximal colon and distal colon, suggesting a membrane-bound carbonic anhydrase. It was concluded that carbonic anhydrase appeared suddenly at birth and was continuously present in mid- to upper-crypt (or upper villus in early neonatal proximal colon) non-goblet cells into adulthood. This suggests a functional role for carbonic anhydrase in chloride-bicarbonate exchange across the neonatal and adult colonic mucosa.